Disease-specific fibrils and uses thereof

Abstract

Provided herein are compositions, methods, and kits useful for addressing the shortcomings of previous disclosures for the synthesis, evaluation, and uses of disease-specific pathogenic fibrils. In aspects, the disease-specific pathogenic fibril is a disease-specific pathogenic tau fibril.

Description

LVM Ref.72-24WO; 340383 DISEASE-SPECIFIC FIBRILS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No.63 / 683,057, filed August 14, 2024, and U.S. Provisional Patent Application No.63 / 701,989, filed October 1, 2024, each of which is hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number AG056058 awarded by the National Institutes of Health. The government has certain rights in the invention. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] The content of the electronic sequence listing (72-24 WO_Seq_Listing_14August2025; Size: 51,025 bytes; and Date of Creation: August 14, 2025) is herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0004] Synthetically recreating tau fibrils with the secondary, tertiary, and quaternary folds that mimic the conformations seen in tauopathies is a roadblock that has prevented the development of diagnostic and therapeutic tools. Screening for disease- specific antibodies, small molecule inhibitors, and positron emission tomography (PET) agents necessitates a well-defined and representative tau amyloid fibril target. To date, no antibody or antigen that uniquely targets neurofibrillary tangles of tau representing tauopathies have been developed.

[0005] There are few reports of synthetic fibrils that replicate pathogenic tau fibrils, but the lack of mechanistic understanding hinders the reproducible synthesis and widespread application of synthetic tau fibrils for different tauopathies. Goedert and Scheres et al developed experimental conditions for the synthesis of tau fibrilsLVM Ref.72-24WO; 340383 replicating the conformation found in Alzheimer’s disease and CTE patients for use in cryoEM experiments. Goedert, M. et al., Neuron, 8(1), 159–168 (1992). However, their conditions were discovered empirically, and are not replicable in cellular milieu and not transferrable to other tauopathies. Additionally, the mechanistic implication of different aggregation conditions was not investigated, further hindering widespread application. Similarly, peptide-based active seeds were developed by Lashuel et al, but they were not designed and verified as a minimal template that induces the critical fold of tau monomers. Holistically, there is no platform that uses a peptide-based minimal template to induce the critical tau fibril fold in naive tau monomers. Similarly, there have been no methods developed that ensure the molecular level structure of synthetic tau fibrils using real-time spectroscopic methods. Lastly, there are no methods that are amenable to application in living cells, which is critical for tauopathy research.

[0006] Moreover, under physiological conditions, hyperphosphorylation of tau is proposed to play an essential role in regulating tau function and / or a hallmark of pathogenic condition. While the human tau protein isoforms exhibit phosphorylation at up to 40-50 out of 85 sites of serine, threonine, and tyrosine residues under pathological conditions, the post-translational modification (PTM) combinations that co-occur are not currently known. Hyperphosphorylated tau proteins are key constituents of neurofibrillary tangles (NFTs) found under several neurodegenerative disease conditions referred to as tauopathies, including Alzheimer’s Disease (AD), Chronic Traumatic Encephalopathy (CTE), Pick’s Disease (PiD), Corticobasal Degeneration (CBD), Progressive Supranuclear Palsy (PSP), and Argyrophilic Grain Disease (AGD). While the current understanding in biology and neuropathology is that the hyperphosphorylated tau and their fibrillar assemblies are the neurotoxic form of tau, it remains unclear whether hyperphosphorylation is an aggravating factor in, or simply a reflection and consequence of, the process of tau self-assembly into filaments. Indeed, the molecular level, mechanistic, role of this hyperphosphorylation in enhancing or reducing the aggregation propensity of tau is unclear, especially considering that combinatorial phosphorylation of multiple sites can have complex, non-additive, effects on tau protein aggregation.

[0007] Therefore, there is still a need in the art for compositions and methods for the synthesis, stabilization, and analysis of pathogenic fibrils, such as pathogenic tau fibrils.LVM Ref.72-24WO; 340383 SUMMARY OF THE INVENTION

[0008] Aspects disclosed herein include a method for generating a prion-like mini-tau peptide, the method comprising: identifying a filament-core region of a disease-specific tau fibril; selecting a structural motif of the filament-core region, wherein the structural motif contributes to a critical protein fold of the disease-specific fibril; synthesizing a mini-tau peptide comprising an amino acid sequence encoding the structural motif; wherein the mini-tau peptide mimics the critical protein fold of the disease-specific tau fibril, thereby generating a prion-like mini-tau peptide.

[0009] Aspects disclosed herein include a method for generating a prion-like mini-tau fibril, the method comprising: generating a prion-like mini-tau peptide, wherein the generating step comprises: identifying a filament-core region of a disease-specific tau fibril; selecting a structural motif of the filament-core region, wherein the structural motif contributes to a critical protein fold of the disease-specific fibril; synthesizing a prion-like mini-tau peptide comprising an amino acid sequence encoding the structural motif; wherein the prion-like mini-tau peptide mimics the critical protein fold of the disease- specific tau fibril, introducing a plurality of the prion-like mini-tau peptide to a solution, thereby generating the prion-like mini-tau fibril.

[0010] Aspects disclosed herein include a method for generating full-length pathogenic tau fibrils, the method comprising: selecting one or more synthetic mini-tau fibril comprising a plurality synthetic mini-tau peptides wherein at least a portion of the synthetic mini-tau peptides mimic a critical protein fold of a disease-specific tau fibril; providing a first set of tau monomers; and contacting a plurality of the synthetic mini-tau fibrils with the first set of tau monomers in a solution to induce aggregation of the first set of monomers, wherein the synthetic mini-tau fibrils act as templates that stabilize the tau monomers to adopt a disease-specific tau fibril core formation, thereby generating full-length pathogenic tau fibrils.

[0011] Aspects disclosed herein include a kit for generating prion-like mini-tau fibrils, the kit comprising at least one prion-like mini-tau peptide wherein each prion-like mini- tau peptide independtly comprises a sequence having 75% or greater sequence identify of: SEQ ID NO: 2 (DNIKHVLGGGSVQIVYKPV); SEQ ID NO: 11 (DNIKHVLGGGSPhosVQIVYKPV); SEQ ID NO: 12 (DNIKHVLGGGSVQIVYPhosKPV);LVM Ref.72-24WO; 340383 SEQ ID NO: 17 (VQIVYKPGGGNHKLTF); or SEQ ID NO: 18 (KVQIINKPGGGKLTFRE); or any combination thereof.

[0012] Aspects disclosed herein include a kit for the development of tauopathy therapeutics, imaging agents, and / or other technologies for the identification and treatment of tauopathies, the kit comprising: a 0N4R isoform tau protein (UniProt accession number (P10636-8)); a tau protein fragment having 75% or greater sequence identity of SEQ ID NO: 13 (NVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKP VDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKI ETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLAD EVSASLAKQGL); a tau protein fragment having 75% or greater sequence identity of SEQ ID NO: 23 (NVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVLGGGSVQIVYKP VDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKI ETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLAD EVSASLAKQGL); a spin labeled full-length pathogenic tau fibril of any aspect disclosed herein, or a fragment thereof; or an isotope labeled full-length pathogenic tau fibril of any of any aspect disclosed herein, or a fragment thereof; or any combination thereof.

[0013] Aspects disclosed herein include a synthetic phosphorylated amyloid fibril comprising a structurally ordered cluster of peptide monomers, wherein at least a portion, optionally all, of the peptide monomers comprise a site-specific phosphorylated residue, wherein the phosphoryl groups of the site-specific phosphorylated residues are aligned in a linear, stacked conformation along the axis of the synthetic phosphorylate amyloid fibril.

[0014] Aspects disclosed herein include a method for generating a synthetic phosphorylated amyloid fibril comprising: synthesizing a peptide monomer comprising an aggregation-prone domain; performing a post-translational phosphorylation of at least one residue of the peptide monomer; combining a plurality of the peptide monomer to form a mixture of peptide monomers; optionally, tuning at least one condition of the combining step to promote the formation of a structurally ordered cluster of peptide monomers; thereby generating a synthetic phosphorylated amyloid fibril.LVM Ref.72-24WO; 340383

[0015] Aspects disclosed herein include a method for stabilizing synthetic amyloid fibril formation of a plurality of peptide monomers, the method comprising: combining a plurality of peptide monomers, or peptide fragments thereof, in a solution to form a mixture of peptide monomers, wherein at least a portion, optionally all, of the plurality of peptide monomers comprise an aggregation-prone domain; wherein the solution comprises a phosphate buffer or polyphosphates to promote the formation of a structurally ordered cluster of peptide monomers, resulting in a first-generation synthetic amyloid fibril, thereby stabilizing synthetic amyloid fibril formation.

[0016] Aspects disclosed herein include method for stabilizing prion-like mini-tau fibril formation of a plurality of peptide monomers, the method comprising: combining a plurality of peptide monomers, or peptide fragments thereof, in a solution to form a mixture of peptide monomers, wherein at least a portion, optionally all, of the plurality of peptide monomers are full-length tau monomers, or fragments thereof, or comprise a prion-like mini-tau peptide, optionally, wherein at least a portion, optionally all, of the plurality of peptide monomers comprise a post-translational phosphorylation of at least one residue, wherein the solution comprises a phosphate buffer or polyphosphates to promote the formation of a structurally ordered cluster of the peptide monomers, resulting in a first-generation prion-like mini-tau fibril, thereby stabilizing prion-like mini- tau fibril formation.

[0017] Aspects disclosed herein include a method of confirming phosphate or phosphoryl group ordering in the synthetic phosphorylated amyloid fibril or the prion-like mini-tau fibril of any aspect disclosed herein, the method comprising: analyzing the fibril using31P solid-state NMR lineshape analysis, Multiple Quantum Spin Counting (MQSC) with Magic Angle Spinning (MAS), or a combination thereof; indicating the presence of ordered phosphate clusters by evaluating the presence of: a characteristic, narrow,31P solid-state NMR spectral linewidth, a Multiple Quantum Coherence Order (MQCO) of 3 or more, or a combination thereof; thereby confirming phosphate or phosphoryl group ordering in the synthetic phosphorylated amyloid fibril or the prion-like mini-tau fibril.

[0018] Aspects disclosed herein include a monoclonal antibody formulated to selectively bind an epitope of a synthetic phosphorylated amyloid fibril or a prion-likeLVM Ref.72-24WO; 340383 mini-tau fibril, wherein the epitope is characterized by three or more phosphorylated residues arranged in a structurally ordered, fibrillar conformation.

[0019] Without wishing to be bound by any particular theory, there may be discussion herein of beliefs or understandings of underlying principles relating to the compounds and methods disclosed herein. It is recognized that regardless of the ultimate correctness of any mechanistic explanation or hypothesis, an embodiment of the invention can nonetheless be operative and useful. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG.1: Spectroscopy Guided Workflow for Tau Fibril Synthesis.

[0021] FIG.2: Synthetic mini-tau peptide design with the 19-residue peptide sequence of interest highlighted.

[0022] FIG.3: nsTEM of synthetic mini-tau fibrils constructed from synthetic mini-tau peptides: Mini-Tau 1 (SEQ ID NO: 2), Mini-Tau 2 (SEQ ID NO: 11 (DNIKHVLGGGSPhosVQIVYKPV)), and Mini-Tau 3 (SEQ ID NO: 12 (DNIKHVLGGGSVQIVYPhosKPV)).

[0023] FIG.4A: Schematic showing localized hotspot hydrated with more structured water drives entropy driven seeded aggregation of tau. FIG.4B: ODNP-derived experimental diffusivity by kσ (proportional to D). FIG.4C: Computed local water tetrahedrality.

[0024] FIG.5: ThT fluorescence assay for screening aggregation competent mini-tau peptides.

[0025] FIG.6: nsTEM images of mini-tau 1 peptide with corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and (GPT) complement strands. The conformation of the mini-tau 1 is controlled by the complement strand leading to different morphologies of the mini-tau fibrils.

[0026] FIG.7: ThT fluorescence data of 0N4R WT monomer (left) and 0N4R WT P301L monomer (right) aggregation with mini-tau 1 (orange) and mini-tau 2 (pink) mini- tau fibrils as active seeds.LVM Ref.72-24WO; 340383

[0027] FIG.8: Generational seeding strategy for homogenous pathogenic tau fibril synthesis. Seeding tau monomers with previous generations of synthetic fibrils generates more homogenous populations. AD refers to Alzheimer’s disease, CTE refers to chromic traumatic encephalopathy, and IM refers to Intermediate.

[0028] FIG.9: DEER data for the mini-tau fibrils generated from mini-tau 2 (left), mini-tau 1 (middle), mini-tau 3 (right). Distance distributions for each mini-peptide acts as a fingerprint to identify the disease-specific fibril of interest.

[0029] FIG.10: DEER data for full length tau monomers in pathogenic tau fibrils.

[0030] FIG.11A: Experimental DEER signal (top) and P(r) (bottom) of tau 187, labelled at sites 351-373, heparin induced, PSP seeded, and CBD seeded fibrils. FIG. 11B: Spectrograms (left 2) and similarity gradient plot (rightmost) of DEER signal of heparin vs CBD induced, heparin vs PSP induced, and PSP vs CBD induced fibrils.

[0031] FIGs.12A-12H: Fibril formation and stability of jR2R3 peptides. FIG.12A: (upper) The domains of the longest isoform of tau, 2N4R (UniProt accession number (P10636-8)), consisting of an N-terminal domain, the N1 and N2 repeat domains (which are spliced out in the 0N4R isoform), a Proline-rich domain, the 4 repeat domains R1-R4 and a C-terminal domain. (lower left): Sequence of example jR2R3 peptide variants. (lower right): The sequences of tau resolved in tauopathy structures. FIG.12B: Tauopathy fibril structures: CBD type II (PDB#: 6TJX), GPT (PDB #: 7P6a), PSP (PDB #: 7P65), GGT type I (PDB #: 7P66), 0N4R Snake (PDB #: 6QJH), and AD PHF (PDB #: 5O3L). FIG.12C: Maximum ThioflavinT fluorescence after incubation with heparin at 37˚C for 18 hours. Samples contained 50µM protein with 12.5µM heparin (n=3). FIG. 12D: nsTEM of all jR2R3 variant fibrils formed with heparin. FIG.12E: ThioflavinT fluorescence after incubation with guanidinium hydrochloride (GdnHCl) at 37˚C for 18 hours. Samples were aggregated for 18 hours prior to denaturation. (n=3). FIG.12F: AFM images of denatured samples. Fibrils were observed to degrade in a GdnHCl concentration dependent manner. FIG.12G: Seeding of 0N4R tau(P301L) with jR2R3 fibrils (n=3). FIG.12H: Transfection of HEK 293 cells expressing tau187 with jR2R3- (P301L). Timelapse imagery shows accumulation of endogenous tau in aggregates upon transfection with jR2R3-P301L, but not jR2R3. (Right) nsTEM of cellular aggregates from the transfected cells.LVM Ref.72-24WO; 340383

[0032] FIGs.13A-13G: Structure of jR2R3-P301L fibrils. FIG.13A: Example cryoEM image of jR2R3-P301L fibrils. FIG.13B: Representative 2D class average of the singlet class of fibrils. (left) initial class used for initial model building. (right) 2D projection after refinement. FIG.13C: EM map of the fibril viewed from the side and down the axis of the fibril colored by the estimated resolution of the map. FIG.13D: Atomic structure of a single jR2R3-P301L fibril layer (PDB ID #8V1N). FIG.13E: Schematic of the jR2R3- P301L fibril layer. Pink dots are glycine residues, blue dots are positively charged side chains, red dots are negatively charged sidechains, and white are hydrophobic side chains. FIG.13F: (far left) Structure of the jR2R3-P301L inner strand-loop-strand chain alongside other conformations of the segment in tauopathy structures. H299 and Y310 are colored in yellow to highlight differences in the orientation of sidechains across the structures. FIG.13G: Depicts that CBD and PSP each shared 6 residues with jR2R3- P301L, and GGT shared 4 residue orientations.

[0033] FIGs.14A-14E: Electron paramagnetic resonance of jR2R3 and jR2R3- P301L Fibrils. FIG.14A: CW EPR spectra of jR2R3 and jR2R3-P301L before and after fibrilization. FIG.14B: Simulated spectra of the 3 components used to fit FIG.14A. FIG. 14C: Examples of the types of species that contribute to the 3 components used to fit CW EPR spectra. FIG.14D: The proportion of the spectra attributed to mobile (blue), immobile (orange) and immobile, spin-exchanging, species (yellow). FIG.14E: DEER probability distribution (P(r)) of jR2R3(294-305) and jR2R3-P301L(294-305) fibers. The expected P(r) of jR2R3 in the GPT and CBD conformation is shown in dashed black and Red respectively. Time domain signal is shown in inset.

[0034] FIGs.15A-15H: Free energy landscapes of (FIG.15A) jR2R3 and (FIG.15B) jR2R3-P301L from α-carbon distances in REMD simulations show differences in energy well depths and pathways to opening. FIGs.15A and 15B also show six jR2R3-P301L clusters from the various regions of the energy landscape; balls denote K298, and Q307 (black) and V300 and S305 (red), intramolecular hydrogen bonds are depicted as blue dashed lines, and the VQIVYK section is colored gold. FIG.15C: jR2R3-P301L is seen to form more oligomers with 5 or more intermolecular backbone hydrogen bonds in dimer simulations than jR2R3, but these oligomer conformations rarely occur when one of the monomers is clamped & pinched, i.e. one of the monomers is in the bottom left region of the energy landscape. Error bars show 90% confidence intervals. FIG.15A-i:LVM Ref.72-24WO; 340383 A common cluster with many intermolecular backbone hydrogen bonds (depicted as orange lines). FIG.15A-ii: A common cluster in which one monomer is clamped & pinched and only forms two intermolecular backbone hydrogen bonds. FIG.15A-iii: Residues 295-313 of the CBD fold, which has many intermolecular H-bonds but no intramolecular H-bonds. FIG.15D: Backbone dihedral entropy of residues 296-312 in both jR2R3 and jR2R3-P301L. jR2R3-P301L can unpinch then unclamp or unclamp and then unpinch. This flexibility is likely due to the lower rigidity of the amino acid backbone as illustrated by the higher backbone dihedral entropy at residues 300 and 301 in jR2R3-P301L compared to jR2R3. FIG.15E: Local water mapping of residues around jR2R3 and jR2R3-P301L done by characterizing the populations of water with tetrahedral (109.5˚) angles between water oxygens around the peptide surfaces. This mapping shows an increased percentage (~0.20%) of tetrahedral water around residue 301 in jR2R3-P301L compared to jR2R3. FIG.15F: Electron-1H cross-relaxivity parameter, kσ, from ODNP measurements using spin-labels attached at V300C, 295C, and 314C of jR2R3-P301L. kσ is proportional to the translational diffusion dynamics of water. Therefore, these kσ values show that hydration water dynamics are slowed significantly at this site compared to those in jR2R3 (orange and blue). The change in kσ occurs only at residue 300, which indicates that this structuring of water occurs nearby the mutation site. Additionally, to investigate whether change in hydration dynamics was a result of the peptide sequence the same ODNP measurements were made with truncated peptide sequences jR2∆R3 and jR2∆R3-P301L which showed no significant change in kσ. FIG.15G: Dewetting free energy calculations for both jR2R3 and jR2R3- P301L from Nw = 0 (completely dewetted) to Nw = 1.5 (complete saturation). At Nw = 0, there is a difference of 0.44 ± 0.02 kBT between jR2R3 and jR2R3-P301L, suggesting that dewetting is easier with the jR2R3-P301L peptide compared to jR2R3. FIG.15H: Dimerization of jR2R3-P301L releases tetrahedrally enhanced hydration water, which leads to an increase in the entropy of the system.

[0035] FIG.16: The diversity of morphologies observed in jR2R3 and jR2R3-P301L fibrils populations.

[0036] FIG.17A: Demonstration of the consistency of morphologies observed with NS-TEM and AFM. FIG.17B: AFM images of jR2R3 and jR2R3-P301L fibrils (top). The height of the fibrils recorded by AFM were quantified and are plotted as histogramsLVM Ref.72-24WO; 340383 (bottom panel). FIG.17C: Expected P(r) distance distributions between sites 294 and 304 for the 4 strand-loop-strand motifs of jR2R4 that have been reported in the literature. Note that DEER measurements shown in FIGs.14A-14B were measured between sites 294 and 304. Site 305 is not able to be simulated with the published structures as the sidechain is directed inward in the fold and placing a nitroxide radical leads to steric clashes. FIG.17D: The structure of the strand-loop-strand motif in the 4 structures simulated in FIG.17C.

[0037] FIGs.18A-18E: Stabilizing features of the jR2R3-P301L fibrils. FIG.18A: The jR2R3-P301L structure fit within the associated EM map. FIG.18B: The SLS fold is held together at the extremities by an interaction between D295 and K311. FIG.18C: The SLS fold interface with the inner strand is formed by a three residue bridge between S305 of the SLS strand, Q307 of the inner strand, and Q307 of the SLS strand. S305 of the inner strand may also be forming a hydrogen bond network with G303 of the SLS strand. FIG.18D: the GGG loop of the SLS strand may be held together by a hydrogen bond between the carbonyl of P301L and the amine of G304. FIG.18E: Fourier shell correlation curve of the jR2R3-P301L EM map shown in FIG.13C.

[0038] FIGs.19A-19D: EPR spectra (blue) fit to multiple spin components, the resulting fit (reconstruction), and the contribution by each component (immobile, mobile, spin-exchanged) (FIG.19A) jR2R3; (FIG.19B) jR2R3 fibers; (FIG.19C) jR2R3-P301L; (FIG.19D) jR2R3-P301L fibers.

[0039] FIG.20: DEER probability distributions of jR2R3-(P301L) spin-labeled at 294- 314 (Left) and 294-305 (Right). REMD simulations are shown in dashed lines for the 294-314 distance distribution.

[0040] FIGs.21A-21B: Heat map of the end-to-end distance and the radius of gyration of jR2R3 and jR2R3-P301L. FIG.21C: Contact map of jR2R3 and jR2R3- P301L. For each frame of the REMD trajectory two residues were considered to be in contact if the minimum distance between any two atoms in the residue were less than 3Å. The color bar represents the fraction of the trajectory in which the two residues were in contact. The upper right diagonal shows the pairwise distribution of jR2R3-P301L, and the lower left shows jR2R3.LVM Ref.72-24WO; 340383

[0041] FIG.22A: Pairwise map of regions of the free energy landscape of jR2R3 depicted in FIG.15A. FIG.22B: Pairwise map of regions of the free energy landscape of jR2R3-P301L depicted in FIG.15B. Boxed regions indicate area integrated for calculation of FIG.22C and FIG.22D. FIGs.22C-22D: The estimate free-energy of conformational for jR2R3 and jR2R3-P301L in the unclamp (FIG.22C) and unpinch (FIG.22D) modes.

[0042] FIGs.23A-23C: V300 dihedral free energies for (FIG.23A) jR2R3 and (FIG. 23B) jR2R3-P301L. The proceeding proline in jR2R3 constrains V300’s dihedrals. FIG. 23C: jR2R3-P301L’s free energy landscape when only the conformations in the top left of the V300 dihedral free energy landscape are considered. The restricted dihedrals block an unfolding mode (see arrows) where the V300-S305 contact first gets “unpinched” and then the K298-Q307 contact is “unclamped”.

[0043] FIGs.24A-24D: Hydrogen bonding propensity of (FIG.24A) jR2R3, (FIG. 24B) jR2R3 P301L, (FIG.24C) jR2R3 P301S, and (FIG.24D) jR2R3 P301V is measured by β-sheet or α-helix assignment with the DSSP algorithm.90% confidence error bars are shown. Proline lacks a hydrogen bond donor – the amide nitrogen – so it is generally less prone to forming α-helices and β-sheets.

[0044] FIGs.25A-25D: Intermolecular backbone hydrogen bonds for (FIG.25A) jR2R3 and (FIG.25B) jR2R3-P301L dimer simulations. FIGs.25C-25D show jR2R3 (FIG.25C) and jR2R3-P301L (FIG.25D) hydrogen bond probabilities when one of the monomers is closed in a hairpin, i.e. K298-Q307 α-carbon distances < 7Å and V300- S305 α-carbon distances < 8Å.90% confidence error bars are shown.

[0045] FIGs.26A-26D: Differences with other simulations of this section of tau in the literature. We observe that wildtype and P301L conformations do not differ to a large extent, contrary to what other researchers have seen with different force fields(37, 38). We use the force field a99SB-disp(39) with TIP4P-D water while Chen, et. al. used a99SB-ILDN with SPCE water and Stelzl, et. al. used a99SB*-ILDN-q with TIP3P water. FIGs.26A-26B: We observed only minor differences between the wildtype and P301 in their RMSD to various hairpins while previous studies saw significant differences albeit for symmetrical trimers instead of monomers. See, Chen et al., Nat. Comm. (2019) 10:2493. FIG.26A: RMSD distribution with reference to the “best hairpin”, which is theLVM Ref.72-24WO; 340383 conformation with the highest number of β-sheet residues as determined by the DSSP algorithm. FIG.26B: RMSD distribution with reference to the encapsulated hairpin in the PSP disease fold (PDB 7P65, residues 295-313). FIG.26C: Cumulative distribution function (CDF) of the distance between O(V300)-N(G303) which interact via hydrogen bonding; this hydrogen bond is depicted with a dashed line. Stelzl, et. al. saw ~5% and ~20% of P301L and WT conformations respectively with an O(V300)-N(G303) distance below 4Å while we see 22-23% for both P301L and WT. FIG.26D: CDF of the minimum Cα RMSD of SEQ ID NO: 19 (VPGGG) to the closest representative of the NMR ensemble of microtubule-bound tau structures (PDB 2MZ7). Stelzl, et. al. observed ~4% and ~15% of P301L and WT conformations respectively within 1Å of one of the 20 structures in the NMR microtubule-bound ensemble while we see 17-18% for both P301L and WT. Six of the 20 microtubule-bound structures from are depicted with SEQ ID NO: 19 (VPGGG) shown in blue.

[0046] FIG.27: The most populous clusters of jR2R3 and jR2R3-P301L used for INDUS measurements. Clusters were calculated by applying the Daura algorithm on monomer REMD simulations.

[0047] FIG.28A: the percent probability of surrounding waters having hexagonal character surrounding the V300 and P301(L) residues. FIG.28B: Difference in relative tetrahedrality (i.e. tetrahedral water fraction divided by the bulk tetrahedral water fraction) for V300’s backbone and P / L301’s sidechain; 90% confidence error bars are shown. FIG.28C: Difference in the number or hexagonal and pentagonal water rings formed within 4.25 Å of each residue’s heavy atoms; jR2R3 P301L has notably more hexagonal and pentagonal rings formed at V300 and P / L301. FIG.28D: Difference in jR2R3 and jR2R3-P301L’s tetrahedral water fraction around each residue’s backbone and sidechain. V300’s backbone and P / L301’s sidechain show the largest difference in water tetrahedral fraction. FIG.28E: Shannon entropy (Sθ) measurements of water’s 3- body angle distribution for bulk water, around P301 & L301 residues, and around jR2R3 & jR2R3-P301L peptides with varying temperature. This Shannon entropy is correlated with the thermodynamic excess entropy (Monroe & Shell, 2019). P301 shows a larger increase in Shannon entropy with temperature than L301.LVM Ref.72-24WO; 340383

[0048] FIGs.29A-29F: Backbone rigidity, residue hydrophobicity and sidechain length all contribute to the ease of extension for jR2R3. Pairwise energy landscapes as described in FIGs.15A-15B for (FIG.29A) jR2R3, (FIG.29B) jR2R3(P301S), (FIG. 29C) jR2R3(P301V), (FIG.29D) jR2R3-P301L. All the P301 mutants have an energetically accessible unpinch-then-unclamp mode, unlike jR2R3. The free energy wells become less deep with hydrophobicity and hydrophobic length. P301L has a longer hydrophobic side chain than P301V that seems to stabilize intermediates along the unfolding pathway. For each peptide, we conducted REMD simulations of both the HID and HIE protonation states of H299. We show the landscape that most easily opens: HIE for jR2R3 and jR2R3-P301L; HID for P301V; and 50% HID & 50% HIE (Boltzmann-weighted) for P301S. FIGs.29E-29F: ThT fluorescence of jR2R3-P301X peptides plotted against different hydropathy scales. (FIG.29E) Tanford-Nozaki and (FIG.29F) Kyte-Dolittle.

[0049] FIG.30A: Schematic showing overview of the tau construct 2N4R. PDB structure representing 4R tauopathies CBD with PDB ID: 6VH7. Strand-loop-strand designated as jR2R3 (SEQ ID NO: 5) and jR2R3-P301L (SEQ ID NO: 2), are displayed with the PHF6 sequence (SEQ ID NO: 1) underlined, and residues mutated relative to jR2R3 are shown in bold. jR2R3 / jR2R3 P301L sequences represent the R2 / R3 splice junction of tau spanning from D295 to V313. FIGs.30B-30C: Post-translational modifications (PTMs) of jR2R3-P301L (SEQ ID NO: 2) in CBD configuration are presented. Stacking of the monomers is proposed as displayed. Serine 305 (FIG.30B) and Tyrosine 310 (FIG.30C) are the residues at which the phosphoryl group is added.

[0050] FIGs.31A-31B: Monitoring the fibril aggregation of S305p(top) and Y310p(bottom) jR2R3-P301L fibrils (SEQ ID NO: 2) using the Thioflavin T (ThT) fluorescence assays (left) and TEM micrographs (right) for 100 mM NaCl (FIG.31A) and 1M NaCl (FIG.31B) conditions. The scale bar is uniform for all TEM images (500 nm).

[0051] FIG.32A: Fiber denaturation experiment on fibrils with 100mM NaCl condition, normalized ThT fluorescence as a concentration of Guanidinium hydrochloride (GdnHCl) concentration after 24 hours of incubation at a temperature of 37°C. S305pretains the most fluorescence at the highest concentration, indicating a more stable fiber compared to both jR2R3-P301L and Y310p. FIG.32B: Seeding, equalLVM Ref.72-24WO; 340383 amounts of each seed (bottom-most line in each graph) were added to their corresponding monomer type, S305p(FIG.32B, top) shows the highest increase in ThT fluorescence, indicating more fiber formation compared to Y310p(FIG.32B, bottom).

[0052] FIGS.33A-33F:31P NMR spectra of S305pjR2R3-P301L and Y310pjR2R3- P301L samples and the corresponding deconvolution of the spectra using DMfit software. Spectra were obtained at 10 kHz MAS frequency and 100 K temperature under DNP. A signal enhancement of 40-fold was attained with DNP for each spectrum. Each spectra was acquired with 128 scans for signal averaging except for 1M NaCl Y310p(FIG.33F) and a recycle delay of 5 seconds was used. We applied coif3 wavelet denoising using NERD software from Cornell University to obtain a good signal-to-noise ratio for the 1M NaCl Y310pspectra (FIG.33F). A similar line broadening of 50 Hz was applied to each spectrum. FIG.33A: monomer, FIG.33B: 100 mM NaCl, and FIG.33C: 1 M NaCl conditions of S305pjR2R3-P301L samples; FIG.33D: monomer, FIG.33E: 100 mM NaCl, and FIG.33F: 1 M NaCl conditions of Y310pjR2R3-P301L samples.

[0053] FIGs.34A-34D: Even and odd spin counting profiles and MQCO profiles obtained from multi-cosine function curve fitting of S305pjR2R3 P301L fibrils and Y310pjR2R3 P301L fibrils at 10 kHz MAS frequency and with 8 ms excitation time during SR218 DQ recoupling sequence under DNP and a repetition time of 5 s. FIG.34A: 100 mM NaCl S305p, FIG.34B: 1 M NaCl S305p, FIG.34C: 100 mM NaCl Y310p, and FIG. 34D: 1 M NaCl Y310p. The x-axis of the spin counting profiles is represented by the experimental index (j), where each phase is incremented by 360° / experimental index. All spin-counting profiles' integrals were normalized to the integral of the first experiment (j = 0).

[0054] FIGs.35A-35D: Numerical SIMPSON simulations of MQ-SC coherence orders with different geometries of a six-spin system: FIG.35A: Dimer, FIG.35B: Trimer, FIG.35C: six-spin parallel configuration, and FIG.35D: six-spin geometry with structure jR2R3 solved from our lab. Description of the spin system of each configuration is given in Table 4.

[0055] FIG.36: Fibril stacking diagram for S305P. The dotted lines represent a spacing distance of 4.8 Å.LVM Ref.72-24WO; 340383

[0056] FIGs.37A-37B: Overlay of31P NMR spectra of FIG.37A: S305pjR2R3 P301L and FIG.37B: Y310pjR2R3 P301L samples.

[0057] FIGs.38A-38B: The effect of1H-31P heteronuclear decoupling on31P spectra of 100mM NaCl S305pjR2R3-P301L fibrils using the decoupling power on and power off on proton channel. The line shape fitting and parameters are shown in right panel, the narrower peak corresponds to decoupling power on, and the broader peak corresponds to decoupling power off.

[0058] FIGs.39A-39B: Spin counting experiments with different SR218 mixing times of 9.6 ms (FIG.39A) and 16 ms (FIG.39B) on31P spectra of 1M NaCl S305pjR2R3 P301L fibrils.

[0059] FIGs.40A-40D: Even and odd spin counting profiles and subsequent Fourier transformed MQCO profiles of S305pjR2R3 fibrils and Y310pjR2R3 fibrils at 10 kHz MAS frequency and with 8 ms excitation time during SR218 DQ recoupling sequence under DNP. FIG.40A: 100 mM NaCl S305p, FIG.40B: 1 M NaCl S305p, FIG.40C: 100 mM NaCl Y310p, and FIG.40D: 1 M NaCl Y310p.

[0060] FIGs.41A-41B: Even MQ-SC profiles and MQCOs extracted from conventional FT method for monomers of S305pand Y310pjR2R3-P301L at 10 kHz MAS rate and 100 K temperature under DNP. The x-axis of the spin counting profiles is represented by the experimental index (j), where each phase is incremented by 360° / experimental index. All spin-counting profiles' integrals were normalized to the integral of the first experiment (j = 0). FIG.41A: S305pjR2R3-P301L monomers. FIG. 41B: Y310pjR2R3-P301L monomers.

[0061] FIG.42: TEM images for stability studies on jR2R3-P301L (SEQ ID NO: 2), S305pand Y310pfibril samples. Scale bar 500 µM.

[0062] FIGs.43A-43B: Schematic representation of the two strategies discussed in Examples 18 and 19 herein. FIG.43A: Linear arrangement of phosphoryl groups covalently attached to peptides via serine, threonine or tyrosine residues. These assemblies are stabilized by structured water molecules, with phosphoryl groups shown in gold and water-mediated interactions highlighted in blue. FIG.43B: Similar ordered clusters formed by free phosphate groups assembling near positively charged residuesLVM Ref.72-24WO; 340383 (highlighted in red), in a fibril formed by a peptide or a longer protein construct, also stabilized by water binding. The same color scheme is used to depict phosphate / phosphoryl clusters and their associated wire-like interactions.

[0063] FIGs.44A-44D: All spectral data were obtained at 10 kHz MAS frequency and 100 K temperature under DNP. A signal enhancement of 40-fold was attained with DNP for each spectrum. FIG.44A:31P NMR spectra of S305PjR2R3 P301L and Y310PjR2R3 P301L samples and the corresponding deconvolution of the spectra using DMfit soft-ware. FIG.44A: Overlay of monomer spectra of S305Pand Y310P(spectra of S305Pand Y310Pfibrils formed in 100 mM NaCl are provided in FIG.33B and FIG.33C, respectively); FIG.44B: Pulse sequence of1H-31P FSLG-HETCOR. FIG.44C: Overlay plot of1H-31P HETCOR spectra of S305Pmonomers (red) and S305P fibrils (blue) prepared in 100mM NaCl with a CP contact time of 200 ^s, FIG.44D: Overlay plot of1H-31P HETCOR spectra of S305Pfibrils prepared in 100mM NaCl with different CP contact times of 150, 300 and 800 ^s.

[0064] FIGs.45A-45D:31P NMR dipolar spectra (FIG.45D) simulated using SIMPSON for different geometries of FIG.45A: linear geometry, FIG.45B: zigzag geometry, FIG.45C: cluster geometry (shortest distances turned off), respectively at static condition. Powder pattern was performed with a crystal file zcw986. The spin system was generated using the MagResView software for different geometries. Isotropic chemical shifts and scalar couplings were not considered in the simulations.

[0065] FIGs.46A-46F: Numerical SIMPSON simulations of MQCOs for different geometries of a six-spin system and eight-spin system: FIG.46A: a tilted top view of the jR2R3 P301L (PDB ID #8V1N) fibril structure with phosphorylation at site S305 with a distance of 21.3 Å shown between the two adjacent cores, FIG.46B: A distance of 4.8 Å is seen between the two vertical strands S305PjR2R3, FIG.46C: trimer geometry, FIG. 46D: six-spin parallel configuration, FIG.46E: eight-spin parallel configuration, and FIG. 46F: proto fibril geometry in a triple stack from the structure of jR2R3.

[0066] FIGs.47A-47E: Visualization of structured water bridges formed near residues S305Pof an eight-layer fibril (PDB ID #7QL4) and comparative analysis of water dynamics from simulations. FIG.47A: Cryo-EM density map of the in vitro paired helical filament-like structure from Alzheimer’s disease (PDB: 7QL4) in the transparentLVM Ref.72-24WO; 340383 gray superimposed with the atomic structure. Spheres represent the non- phosphorylated serine at residue 305. FIG.47B: Probability density of three-body angle distribution of adjacent phosphorous atoms in the phosphoryl groups, shown alongside bridging waters in a representative structure drawn from the linear end of the distribution. FIG.47C: Rotational anisotropy autocorrelation function ^2(^) of the water bridges (blue), neighboring waters (red), and bulk water (gray) on a semi-log scale with exponential fits (black). Bulk water was fitted with a single exponential function (^=0.53 ps). Neighboring waters were fitted with a triexponential function (^1=0.18 ps, ^2=1.8 ps, ^3=61.5 ps with weights ^1=0.52, ^2=0.42, and ^3=0.06). Water bridges were fitted with a triexponential function (^1=0.24 ps, ^2=2.2 ps, ^3=97.0 ps with weights ^1=0.57, ^2=0.30, and ^3=0.13). FIG.47D: Hydrogen bond lifetime correlation function ^(^) of the various waters on a semi-log scale with exponential fits (black). A single exponential fit was used for bulk water (^=0.29 ps) and a triexponential fit was used for neighboring water (^1=0.29 ps, ^2=5.3 ps, ^3=24.9 ps with weights ^1=0.24, ^2=0.57, and ^3=0.19). Water bridges fit well to a triexponential function (^1=0.51 ps, ^2=16.8 ps, ^3=101.9 ps with weights ^1=0.29, ^2=0.37, and ^3=0.34). FIG.47E: Differential three-body angle distributions, ^3b(^) – ^3bpure(^). The inset plot shows the three-body angle distributions of neighboring waters and water bridges with a peak consistent with icosahedral geometry (^≈64°). The three-body angle distribution of bulk water shows a peak consistent with tetrahedral geometry (^≈109.5°). The black curve represents the three- body angle distribution of pure water at 310 K. A representative water bridge is shown depicting the three-body angle (O-O-O).

[0067] FIGs.48A-48F: Representative TEM for the new conditions of tau peptide formation. FIG.48A depicts results from a plastic vial, with no motion. FIG.48B depicts results from a dark environment with no motion. FIG.48C depicts results from a light environment with no motion. FIG.48D depicts results from a plastic vial, with motion. FIG.48E depicts results from a glass vial with no motion. FIG.48F depicts results from use of Nafion Film with motion. All size bars are 200 nm.

[0068] FIGs.49A-49E: Characterization and structure determination of the fibers and crystals using micro ED and NMR.

[0069] FIG.50: Diagram for MD phosphate structure.LVM Ref.72-24WO; 340383

[0070] FIGs.51A-51B: Numerical SIMPSON simulations of MQ coherence orders of a six-spin system: FIG.51A: triple stack of proto fibril geometry from the structure of jR2R3 with 21 Å distance between the core and the counter strand, FIG.51B: same geometry of FIG.51A with small variations in the chemical shifts. Description of the spin system of each configuration is given in Table 6, below.

[0071] FIG.52: Stack plot of31P NMR spectra of S305pjR2R3 P301L sample acquired at different MAS frequencies of 3 kHz, 5 kHz and 10 kHz MAS frequencies at room temperature and 9.4 T field. *Indicates the side bands due to magic angle spinning.

[0072] FIGs.53A-53F:31P NMR dipolar spectra simulated using SIMPSON for different geometries: linear geometry of 6spin (FIG.53A), 4spins (FIG.53B), and 2spins (FIG.53C), linear geometry cluster geometry with shorter distances turned on / off (D), respectively at static condition. Powder pattern was performed with a crystal file rep986. The spin system was generated using the MagResView software for different geometries. Isotropic chemical shifts and scalar couplings were not considered in the simulations.

[0073] FIG.54: The31P spectra of 100 mM NaCl S305p 31P spectra acquired with different spin-echo time periods of 400, 800, and 2000 µs.

[0074] FIGs.55A-55D: Additional supporting data for even and odd spin counting profiles and MQCO profiles obtained from the same multi-cosine function curve fitting of S305pjR2R3 P301L fibrils and Y310pjR2R3 P301L fibrils described for FIGs.34A-34D. FIG.55A: 100 mM NaCl S305p, FIG.55B: 1 M NaCl S305p, FIG.55C: 100 mM NaCl Y310p, and FIG.55D: 1 M NaCl Y310p.

[0075] FIG.56A: Overlay of31P spectra for comparing heparin-induced fibril samples and non-heparin-induced fibril samples in 50 mM phosphate buffer: Cl-dGAE fibrils (blue); heparin-induced Cl-dGAE fibrils (orange); and control buffer (green). FIG.56B: Zoomed view of the narrow peak in the31P spectrum of Cl-dGAE fibrils in 50 mM phosphate buffer (inset: full spectrum). FIG.56C depicts the even-ordered spin counting profile for the non-heparin-induced Cl-dGAE fibrils, and FIG.56D depicts the corresponding multi-quantum coherence orders (MQCOs) chart.LVM Ref.72-24WO; 340383 STATEMENTS REGARDING CHEMICAL COMPOUNDS AND NOMENCLATURE

[0076] In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. The following definitions are provided to clarify their specific use in the context of the invention.

[0077] The following abbreviations are used herein: AD refers to Alzheimer’s disease; AGD refers to argyrophilic grain disease; CBD refers to corticobasal degeneration; CSA refers to chemical shift anisotropy; CTE refers to chronic traumatic encephalopathy; IDP refers to intrinsically disordered protein; MTBR refers to microtubule-binding domain; MQCO refers to multiple quantum coherence order; MQSC refers to multiple quantum spin count; NFT refers to neurofibrillary tangle; PHF refers to paired helical filaments; PRR refers to polyproline rich region; PSP refers to progressive supranuclear palsy; PTM refers to post-translational modification; SLS refers to strand- loop-strand; and WT refers to wild-type (also referred to herein as naïve).

[0078] As used herein, the term "about" is used to mean approximately, in the region of, roughly, or around. Unless otherwise stated, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10%. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth.

[0079] As used herein, the terms “activation”, “activate”, “activating” and the like in reference to a protein-activator (e.g. agonist)

[0080] As used herein, the terms “administer” and “administering” means to introduce, such as to introduce to a subject a compound or composition. The term is not limited to any specific mode of delivery, and can include, for example, oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini- osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). In embodiments, administration includes direct administration to a tissue.LVM Ref.72-24WO; 340383 Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “co-administer” it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies (e.g. an immunotherapy agent, a small molecule inhibitor). The compounds and compositions of the invention can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compound individually or in combination (more than one compound or agent). The compositions of the present invention can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions. The compositions of the present invention may additionally include components to provide sustained release and / or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos.4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed.7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res.12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol.49:669-674, 1997). In another embodiment, the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cellLVM Ref.72-24WO; 340383 resulting in endocytosis. By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul.13:293-306, 1996; Chonn, Curr. Opin. Biotechnol.6:698-708, 1995; Ostro, Am. J. Hosp. Pharm.46:1576-1587, 1989).

[0081] As used herein, the terms “ameliorate,” “ameliorating,” “ameliorating,” and like terms, when applied to a symptom of a disease or condition of a subject, refers to producing any beneficial change in that symptom relative to an appropriate untreated (or differently treated) control or baseline. Such beneficial change includes, but is not limited to, reducing or attenuating the severity, frequency, duration, or progression of the symptom; delaying the onset of the symptom; lessening the negative impact of the symptom on the subject’s quality of life; or otherwise improving a measurable clinical, biochemical, or patient-reported parameter associated with the symptom. Amelioration may be partial or complete, temporary or sustained, and does not require elimination of the symptom or a cure of the underlying disease or condition.

[0082] Amino acids refers to naturally-occurring amino acids, unnatural (non- naturally occurring) amino acids, and / or combinations of these. Amino acids include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, asparagine, glutamine, serine, threonine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid and glutamic acid. As used herein, reference to “a side chain residue of a natural α-amino acid” specifically includes the side chains of the above-referenced amino acids. Peptides are comprised of two or more amino acids connected via peptide bonds. The term, “amino acid” includes any known amino acids, including, but not limited to, alpha amino acids, beta amino acids, gamma amino acids, delta amino acids, and the like. In some embodiments, the term refers to alpha amino acids. Unnatural (non-naturally occurring) amino acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in either the L- or D-configuration that function in a manner similar to the naturally-occurring amino acids. Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.LVM Ref.72-24WO; 340383

[0083] As used herein, the terms “biological sample”, “sample”, and “test sample” are used interchangeably herein to refer to any material, biological fluid, tissue, or cell obtained or otherwise derived from an individual. This includes blood (including whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, and serum), dried blood spots (e.g., obtained from infants), sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, bronchial brushing, synovial fluid, joint aspirate, organ secretions, cells, a cellular extract, and cerebrospinal fluid. This also includes experimentally separated fractions of all of the preceding. For example, a blood sample can be fractionated into serum, plasma or into fractions containing particular types of blood cells, such as red blood cells or white blood cells (leukocytes). If desired, a sample can be a combination of samples from an individual, such as a combination of a tissue and fluid sample. The term “biological sample” also includes materials containing homogenized solid material, such as from a stool sample, a tissue sample, or a tissue biopsy, for example. The term “biological sample” also includes materials derived from a tissue culture or a cell culture. Any suitable method for obtaining a biological sample can be employed; exemplary methods include, e.g., phlebotomy, swab (e.g., buccal swab), and a fine needle aspirate biopsy procedure. A “biological sample” obtained or derived from an individual subject includes any such sample that has been processed in any suitable manner after being obtained from the individual subject.

[0084] As used herein, the term “complement strand” refers to a peptide, a peptidomimetic, or a peptide-derived oligomer that is designed or selected to associate with at least a portion of a target peptide strand. In aspects, the association between the complement strand and the target peptide strand is a non-covalent association, for example, β-sheet hydrogen bonding, coiled-coil packing, an electrostatic salt-bridge, π- π stacking, or hydrophobic zippering. In aspects, the complement strand associates with the target peptide strand in an orientation that produces a measurable effect relative to the target peptide strand alone, such as increased conformational stability, altered aggregation propensity, or a detectable binding signal. The term “complement strand” includes complementary peptides. In aspects, the complement strand is a complementary peptide that is designed or selected for its sequence complementarity to the target peptide strand. In aspects, the complement strand is supplied to the targetLVM Ref.72-24WO; 340383 peptide strand as a discrete peptide. In aspects, the complement strand is tethered to the target strand via a linking sequence. In aspects, the complement strand is incorporated into a multimeric scaffold.

[0085] As used herein, “effective amount” is an amount sufficient to show a meaningful benefit in a subject, cell, or tissue to be treated (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce transcriptional activity, increase transcriptional activity, ameliorate one or more symptoms of a disease or condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, delayed progression, and / or amelioration of a symptom or symptoms of a disease or condition, which could also be referred to as a “therapeutically effective amount.” The meaningful benefit observed in the subject, cell, or tissue to be treated can be to any suitable degree (10, 20, 30, 40, 50, 60, 70, 80, 90% or more). The exact effective amount may vary depending upon the biological effect desired in the subject, cell and / or tissue to be treated, disease or condition to be treated, and / or the specific characteristics of the active agent. In this respect, any suitable dose of the active agent can be administered to the subject (e.g., human), cell, or tissue. Various general considerations taken into account in determining the “effective amount” are known to those of skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0086] The term “fragment” refers to a portion, but not all, of a composition or material, such as a peptide composition or material. In an embodiment, a fragment of a peptide refers to 50% or more of the sequence of amino acids, optionally 70% or more of the sequence of amino acids and optionally 90% or more of the sequence of amino acids.

[0087] As used herein, the term “group” may refer to a functional group of a chemical compound. Groups of the present compounds refer to an atom or a collection of atoms that are a part of the compound. Groups of the present invention may be attached toLVM Ref.72-24WO; 340383 other atoms of the compound via one or more covalent bonds (e.g., peptide bonds). Groups may also be characterized with respect to their valence state.

[0088] As used herein, the terms “inhibition”, “inhibit”, “inhibiting” and the like in reference to a protein-inhibitor (e.g. antagonist) interaction means negatively affecting (e.g. decreasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the inhibitor. In some embodiments inhibition refers to reduction of a disease or symptoms of disease. In some embodiments, inhibition refers to a reduction in the activity of a signal transduction pathway or signaling pathway. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down- regulating signal transduction or enzymatic activity or the amount of a protein.

[0089] As used herein, the term “isomers” refers to compounds (e.g., peptides) having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms. Isomers include structural isomers and stereoisomers such as enantiomers.

[0090] The terms, “patient”, “subject”, and “subject in need thereof” refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a compound or pharmaceutical composition, as provided herein. Non- limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, a patient is human. In some embodiments, a patient is a mammal. In some embodiments, a patient is a mouse. In some embodiments, a patient is an experimental animal. In some embodiments, a patient is a rat. In some embodiments, a patient is a test animal.

[0091] The term “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” refers to a substance that aids the administration of an active agent to and absorption by a subject. Typically, the pharmaceutically acceptable carrier is one that is chemically inert to the active agents and one that has no detrimental side effects or toxicity under the conditions of use. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), polar or semi-polarLVM Ref.72-24WO; 340383 solvents and co-solvents (e.g., lower alcohols, polyhydric alcohols, glycol ethers, dimethyl sulfoxide (DMSO)), oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0092] The term “pharmaceutically acceptable salt” or “salt” is intended to include nontoxic salts synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. For example, an inorganic acid (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, or hydrobromic acid), an organic acid (e.g., oxalic acid, malonic acid, citric acid, fumaric acid, lactic acid, malic acid, succinic acid, tartaric acid, acetic acid, trifluoroacetic acid, gluconic acid, ascorbic acid, methylsulfonic acid, or benzylsulfonic acid), an inorganic base (e.g., sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or ammonium hydroxide), an organic base(e.g., methylamine, diethylamine, triethylamine, triethanolamine, ethylenediamine, tris(hydroxymethyl)methylamine, guanidine, choline, or cinchonine), or an amino acid (e.g., lysine, arginine, or alanine) can be used. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typical. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445, and Journal of Pharmaceutical Science, 66, 2-19 (1977). For example, they can be a salt of an alkali metal (e.g., sodium or potassium), alkaline earth metal (e.g., calcium), or ammonium.

[0093] Thus, the compounds of the present invention may exist as salts. The present invention includes such salts. These salts may be prepared by methods known to those skilled in the art.LVM Ref.72-24WO; 340383

[0094] Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0095] The terms, “prion-like mini-tau fibril” and “mini-tau fibril” are used interchangeably herein and refer to a synthetic peptide fibril that template full-length tau monomers. In aspects, the mini-tau fibrils enforce the conformation found in pathogenic tau fibrils in vitro and / or in vivo. In aspects, the mini-tau fibril comprises an active fibril surface, i.e. a prion template, and is capable of deweting with a tau monomer substrate. In aspects, the mini-tau fibrils act as templates (i.e., active seeds) that guide and stabilize full-length tau to fold and adopt a disease-specific fibril core conformation. In aspects, mini-tau fibril acts as a template to fold tau fibril monomers and proliferates pathologic disease-specific tau fibrils.

[0096] The terms, “prion-like mini-tau peptide” and “mini-tau peptide” are used interchangeably herein and refer to a synthetic peptide designed to encompass a small section of tau that spans a critical portion of the filament core. In aspects, the mini-tau peptide sequence is identified from postmortem tau fibril samples. In aspects, the mini- tau peptide is designed to aggregate together to form mini-tau fibrils with prion properties. In aspects, the mini-tau peptide is designed to incorporate post-translational modifications (PTMs). In aspects, the mini-tau peptide is designed to form mini-tau fibrils under controlled environmental conditions to allow for conformational tuning. In aspects, the mini-tau peptide has 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 5. In aspects, the mini-tau peptide has 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 2. In aspects, the mini-tau peptide has 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 11. In aspects, the mini-tau peptide has 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 12. In certainLVM Ref.72-24WO; 340383 examples and aspects disclosed herein, the mini-tau peptide is or comprises a jR2R3 peptide. Unless otherwise specified, the term, “jR2R3”, generically refers to any peptide fragment or amino acid sequence that spans the R2 / R3 splice junction of any tau protein isoform, such as the 04NR tau isoform UniProt accession number (P10636-8), (i.e., includes at least the residues defining the R2 / R3 splice junction and may further include one or more N- and / or C-terminal extensions, linkers, analytical tags, conservative substitutions, post-translational modifications, other modifications, or any combination thereof). In certain aspects and examples provided herein, “jR2R3” is used in a specific sense to denote an expressly identified amino acid sequence (e.g., by SEQ ID NO, residue modifications, etc.); in such instances, the reference to “jR2R3” is to that particular sequence as stated. A person of ordinary skill in the art will understand from the surrounding context which usage is intended, for example, “jR2R3 comprising residues 294-313 of tau” or “jR2R3 (SEQ ID NO: 5)” signals the specific usage, whereas “jR2R3” without such qualifiers signals generic usage. The disclosure of specific sequences is illustrative and non-limiting and does not narrow the generic meaning of “jR2R3”.

[0097] to refer to a specific amino acid sequence, such specific uses will be apparent to those of skill in the art based on the surrounding description and context of the aspect or example.

[0098] As used herein, the term “moiety” refers to a chemically distinct or structurally recognizable portion of a larger compound. The term “moiety”, when used in reference to a protein or peptide, includes both contiguous amino acid segments (e.g., a receptor- binding domain, an enzyme active site loop, a signal peptide) and non-contiguous amino acid segments that form a recognizable motif (e.g., the heavy-chain variable region of an antibody fragment).

[0099] Except where otherwise specified, the term “molecular weight” refers to an average molecular weight. Except where otherwise specified, the term “average molecular weight,” refers to number-average molecular weight. Number average molecular weight is defined as the total weight of a sample volume divided by the number of molecules within the sample.LVM Ref.72-24WO; 340383

[0100] As used herein, the term “sequence complementarity” refers to the ability of two peptides (complementary peptides) to non-covalently interact with each other based on the compatibility of their amino acid sequences. In aspects, complementary peptides interact through non-covalent interactions between their amino acid side chains, such as hydrogen bonding, electrostatic interactions, hydrophobic interactions, or a combination thereof. In aspects, the spatial pattern of backbone directionality and side-chain physicochemical properties (e.g., polarity, charge, hydrophobicity, hydrogen-bond donor / acceptor capacity) of the complementary peptides are sufficiently compatible to drive specific, non-covalent association under the stated conditions.

[0101] As used herein, the term "sequence homology" or "sequence identity" means the proportion of amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the fraction of matches over the length of sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; for example, wherein gap lengths of 5 amino acids or less, optionally 3 amino acids or less, are usually used.

[0102] With respect to the terms, “sequence identity” and “sequence complementarity”, sequence identity reports the percentage of positions at which two aligned amino acid sequences contain the same amino acid residue, and sequence complementarity evaluates compatibility (e.g., interactive potential and specific binding affinity) between two amino acid sequences. Therefore, a complement strand may, but does not necessarily, exhibit high (e.g., 75% or more, 85% or more, 95% or more, or 99%) sequence identity to its target peptide strand. For example, an antiparallel β-sheet complement peptide strand designed with an alternating hydrophobic-polar pattern can bind tightly to a target peptide strand presenting a corresponding polar-hydrophobic face, even if the complement peptide strand exhibits low (e.g., 10% or less) sequence identity to the target peptide strand.

[0103] The term “substantially equal”, “substantially equivalent”, or “substantially unchanged”, when used in conjunction with a reference value describing a property or condition, refers to a value that is within 20%, within 10%, optionally within 5%, optionally within 1%, optionally within 0.1%, or optionally is equivalent to the providedLVM Ref.72-24WO; 340383 reference value. For example, a diameter is substantially equal to 100 nm (or, “is substantially 100 nm”) if the value of the diameter is within 20%, optionally within 10%, optionally within 5%, optionally within 1%, within 0.1%, or optionally equal to 100 nm. The term “substantially greater”, when used in conjunction with a reference value describing a property or condition, refers to a value that is at least 1%, optionally at least 5%, optionally at least 10%, or optionally at least 20% greater than the provided reference value. The term “substantially less”, when used in conjunction with a reference value describing a property or condition, refers to a value that is at least 1%, optionally at least 5%, optionally at least 10%, or optionally at least 20% less than the provided reference value.

[0104] As used herein, the terms “treat,” “treating,” “treatment” and like terms refer to any indicia of success in the treatment of an injury, disease, pathology or condition in a subject, including any objective or subjective parameter such as abatement; remission; amelioration of symptoms or making the injury, pathology or condition more tolerable to a subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a subject's physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric exams, and / or a psychiatric evaluation. Treatment may be partial or complete, temporary or sustained, and does not require permanent eradication of the disease or condition or relief of every symptom in every treated subject. For example, demonstration of treatment can be established by one or more objective endpoints, including, but not limited to, a statistically significant improvement in a clinical score or functional test (e.g., memory test, walking distance, oculomotor evaluation) and / or a statistically significant modulation of a condition- or disease-associated biomarker (e.g., tau protein levels). DETAILED DESCRIPTION OF THE INVENTION

[0105] In the following description, numerous specific details of the devices, device components and methods of the present invention are set forth in order to provide a thorough explanation of the precise nature of the invention. It will be apparent, however, to those of skill in the art that the invention can be practiced without these specific details.LVM Ref.72-24WO; 340383

[0106] The microtubule-associated protein tau is a pathological protein that constitutes insoluble neurofibrillary tangles associated with Alzheimer’s Disease (AD), frontotemporal lobar degeneration, and many other neurodegenerative diseases, collectively referred to as tauopathies. Natively, tau is an intrinsically disordered protein (IDP) in monomer form that adopts a large ensemble of disordered conformations. In tauopathies, tau proteins misfold and stack into insoluble amyloid fibrils, with the paired helical filaments (PHFs) of AD being the best-known example. The development of effective diagnostics and therapeutics remains a major obstacle in the generation of experimental tau fibril models that replicate the key structure and property of tauopathy fibrils. Recently, cryogenic electron microscopy (Cryo-EM) allowed the determination of tau fibril structures in multiple tauopathies, including AD, Pick’s Disease (PD), chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain disease (AGD), aging-related tau astrogliopathy (ARTAG), globular glial tauopathy (GGT), familial British dementia (FBD), familial Danish dementia (FDD), and limbic-predominant neuronal inclusion body 4R tauopathy (LNT). These results suggest that the structure of tau fibrils is specific to the disease.

[0107] Post-mortem tissue from tauopathy patients is scarce, and heterogenous with patient-to-patient variability that makes it a difficult material from which to develop therapeutics. Additionally, the slow development of tauopathies over a period of decades, forces researchers to use artificial methods to induce and accelerate pathological tau aggregation. In this context, tools such as mutations or cofactors are critical to achieve prion-like propagation of pathological tau within an accelerated timeframe. The majority of current in vitro and in vivo tauopathy models for developing therapeutic or diagnostic strategies rely on pathogenic MAPT mutations, such as P301L or P301S, often used in tandem with V337M or R406W. The P301 site is mutated in the majority of models of tau aggregation including mice (3xTg, hTau.P301S, JNPL3, PS19, PLB1-triple, TauP301L, among others), cell lines designed to assess or propagate seeding of aggregation, and in vitro studies of tau aggregation. No MAPT mutations are linked to AD, and none of the patient-derived tauopathy structures solved to-date include P301L nor any other exonic mutation to tau. These mutations, especially P301L / S, are nonetheless routinely used in AD and other tauopathy models, out of necessity and / or lack of alternate options.LVM Ref.72-24WO; 340383

[0108] It is critical to gain a molecular-level understanding of the effect of these mutations on the misfolding mechanism of tau and the resulting fibril structures to justify and validate their use to model tauopathies.

[0109] The compositions, methods, and related kits provided herein are useful for producing disease-specific tau fibril models to aid the development of therapeutic molecules or tools, tauopathy-specific antibodies or diagnostic positron emission tomography (PET) imaging agents.

[0110] Native pathological tau fibrils have a “prion-like” property to cross the cell membrane and continuously replicate seeding-competent tau fibrils. A prion is a misfolded protein within ordered, self-assembled, fibrils that incorporate naive monomers into their structure by inducing templated shape changes in in the monomer to match the fibrillar structure. This process can therefore indefinitely extend the prion fibril by in register stacking and templating if naïve tau monomers are available. The self-replicating characteristic of a tau prion is of particular interest if a prion that adopts a known or desired fold can be designed and generated. We propose that the fold leading to a SLS structure is a concrete example of misfolding that imparts this prion property to the propagating fibril.

[0111] For templated aggregation to occur, the interface between the active fibril surface, i.e. the prion template, and the approaching tau monomer substrate must dewet. However, for the approaching tau monomer to adopt the shape of the template, a precisely coordinated process is required. We propose that in-register stacking along a fibril-growing interface must be directed via a hyper-localized tau site that serves as an anchoring pin and dewets to form the initial intermolecular contact. The release of structured water has been predicted computationally to be entropically favored and to drive amyloid aggregation. Structuring of water lining the active sites of enzymes has been shown to facilitate directed binding, but water-directed assembly to template amyloid strain has not been experimentally demonstrated. Solid-state NMR has been used to describe the hydration state of mature tau filaments, but the structure and thermodynamics of solvation water of dynamic tau prior to aggregation are not described.LVM Ref.72-24WO; 340383

[0112] Aspects disclosed herein include compositions, methods, and kits useful for addressing the shortcomings of previously disclosed methods for the synthesis of disease-specific pathogenic fibrils. In aspects, the disease-specific pathogenic fibril is a disease-specific pathogenic tau fibril.

[0113] In aspects, the kits described herein comprise a variety of mini synthetic tauopathy replicas capable of templating pathogenic tau fibrils. Any composition, method, or kit disclosed herein may be useful for accelerating the screening process to find specific binders. In aspects, the seeds of the compositions, methods, and kits disclosed herein are capable of aggregating tau protein in vitro, in bacterial cells, and / or mammalian cells with high efficiency and specificity. For example, in aspects, the mini synthetic tauopathy replica comprises 4R tau isoforms and mixed 3R / 4R fibrils. In aspects, the compositions, methods, and kits described herein are free from biological debris, highly sequence-specific, and customizable with mutations and PTMs at desired residues.

[0114] Aspects disclosed herein include a “Tau Seeding Kit” (also referred to herein as “TauSeeK”). In aspects, TauSeeK is useful for providing researchers with the ability to design or choose a peptide-based active seed (e.g., a prion-like mini-tau fibril) and ensure they proliferate a desired folded shape in full-length tau through real-time spectroscopy. To design these agents that target the pathogenic filaments and bind to specific tauopathy neurofibrillary tangles, well-defined synthetic replicas that adopt critical features of pathogenic tau fibrils are needed. In aspects, in order to access these fibrils synthetically, we have designed prion-like mini-tau peptides that form active- seeding prion-like mini-tau fibrils, and prion-like mini-tau fibrils that template full-length tau monomers and enforce the conformation found in pathogenic tau fibrils both in vitro and in vivo.

[0115] In aspects, the prion-like mini-tau peptides disclosed herein encompass a small section of tau that spans the critical portion of the filament core identified from postmortem tau fibril samples and aggregate together to form mini-tau fibrils with prion properties. In aspects, the prion-like mini-tau peptides are designed to incorporate post- translational modifications (PTMs). In aspects, any suitable PTM may be selected, including, but not limited to, glycosylation, phosphorylation, methylation, ubiquitination,LVM Ref.72-24WO; 340383 acetylation, lipidation, or any combination thereof. Preferably, the PTM supports, enhances, and / or induces the stabilization of fibrils. In aspects, the PTM supports, enhances, and / or induces the stabilization of fibrils via long-range ordering. In aspects, the PTM comprises phosphorylation of at least one residue of the prion-like mini-tau peptide. In aspects, the prion-like mini-tau fibrils are formed under controlled environmental conditions to allow for conformational tuning. The key property of prion- like mini-tau peptides and prion-like mini-tau fibrils is that they adopt the critical fold of pathogenic tau fibrils, but the specific sequence of the prion-like mini-tau peptide is unimportant. This approach ensures that the final mini-tau fibril adopts the critical fold of tauopathy-specific neurofibrillary tangles identified from postmortem tau fibril samples.

[0116] In aspects, upon aggregation of the prion-like mini-tau peptides, the resultant mini-tau fibrils then act as templates that guide and stabilize full-length tau to fold and adopt the disease-specific fibril core conformation. The core hypothesis being that if a mini-tau fibril adopts the critical feature of pathological fibril, it will be able to template full-length tau monomers to form synthetic tau fibrils that contain the critical protein fold. The mechanism of pathogenic tau fibril formation is complex and dependent on factors such as the proteoform of tau monomer substrate (sequence, mutation, PTM), cofactors, and aggregation conditions. Despite this complexity, using these mini-tau fibrils as active seeds for full-length tau aggregation, we have generated synthetic tau fibrils that exhibit similar characteristics to patient-derived samples, such as paired helical fiber (PHF) morphology and prion-like properties. These mini-tau fibrils have been demonstrated to act as active seeds for full-length tau both in vitro and in vivo, allowing for their application in a variety of research settings.

[0117] Structural verification of synthetic tau fibrils is critical to ensuring the reproducibility of any method, with cryoEM providing high-resolution, molecular level structural information. However, cryoEM is time intensive and requires high-quality, homogeneous samples, making it challenging to apply to high-throughput environments. On the other end of the spectrum, ThT assays and nsTEM are fast enough to be used for screening purposes, but don’t allow for molecular level verification and validation of the fibril critical fold. To bridge this gap, we have developed spectroscopic methods to ensure that produced tau fibrils have the desired conformation for a given tauopathy while also being fast and simple enough for widespread use. This spectroscopicLVM Ref.72-24WO; 340383 workflow allows for the rational design and tuning of mini-tau peptides, enabling the synthesis of tau fibrils that adopt the target structures of various human derived, pathogenic disease-specific tau fibrils. In aspects, we can ensure the mini-tau fibrils act as a template by verifying the propagation of the critical fold in full length tau monomers.

[0118] In aspects, the compositions, methods, and kits disclosed herein provide access to prion-like mini-tau fibrils and pathogenic disease-specific fibrils (e.g., to the scientific community) for application towards therapeutic, imaging agent, and antibody development. In aspects, the compositions, methods, and kits described herein provide the scientific foundations for the rational design and tuning of synthetic disease-specific fibrils, such as pathogenic disease-specific tau fibrils. In aspects, the compositions, methods, and kits described herein provide a systematic way to confirm the structure and prion properties of mini-tau peptides and fibrils.

[0119] In aspects, provided herein are methods and compositions comprising a disease-associated structural motif of a protein or peptide fibril characterized by 3 or more phosphoryl and / or phosphate groups associated in a phosphoryl / phosphate cluster within the protein or peptide fibril. In aspects, the phosphoryl / phosphate cluster is a structurally-ordered phosphoryl / phosphate cluster. In aspects, the disease-associated structural motif comprises 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, or 20 or more phosphate / phosphoryl groups, optionally 100 or less, 80 or less, 60 or less, 50 or less, 30 or less, or 25 or less stacked phosphate / phosphoryl groups. In aspects, the phosphoryl / phosphate cluster is characterized by supramolecular stacking of phosphate or phosphoryl groups into linear arrangements within the protein or peptide fibril. In aspects, the compositions and methods provided herein are useful for developing highly-specific binding agents (e.g., antibodies) against any proteinopathy characterized by 3 or more phosphoryl and / or phosphate groups associated in a phosphoryl / phosphate cluster within a protein or peptide fibril.

[0120] In aspects, provided herein are fibril epitopes (e.g., tau fibril epitopes), antibodies, compositions, and methods that enable selective binding to phosphoryl / phosphate group clusters displayed on the surface of amyloid fibrils. In aspects, these clusters may arise from fibrillar assembly of phosphorylated peptides andLVM Ref.72-24WO; 340383 are designed based on sequences from amyloid fibril forming proteins, for example, tau protein. In aspects, such clusters comprise three or more phosphoryl / phosphate “wires” that stabilize fibrils (e.g., tau fibrils or any other amyloid fibril forming protein). In aspects, the phosphates and / or phosphate moieties for the phosphoryl / phosphate wires are provided by phosphoryl groups of TauSeek-derived peptides, fibrils, and / or proteins (e.g., a TauSeeK-derived peptide having a site-specific phosphorylated residue). In aspects, the phosphates and / or phosphate moieties for the phosphoryl / phosphate wires are provided by phosphate ions present in a buffer used during fibril formation. This ordered arrangement can serve as an epitope for developing highly specific binding agents, such as antibodies, against aggregated tau. In aspects, provided herein are methods of producing antibodies (e.g., monoclonal antibodies and recombinant antibodies) using tau protein or peptide fibrils containing 3 or more phosphoryl or phosphate clusters in a row comprising phosphoryl / phosphate wires, arranged linearly, as immunogens. In aspects, the antibodies disclosed herein demonstrate high specificity towards hyperphosphorylated pathogenic tau fibrils or other amyloid fibrils. In aspects, the antibodies disclosed herein only bind when three or more phosphorylated residues are presented in a stacked, spatially aligned configuration. In aspects, the antibodies disclosed herein do not bind to residues that are presented singly, doubly, or in non- fibrillar form because that would be a common, not distinct enough, signature.

[0121] In aspects, the fibril epitopes, antibodies, compositions, and methods provided herein are useful in the generation of structure-controlled pathological fibril models stabilized by long range ordering of phosphoryl or phosphate assemblies. In aspects, the fibril epitopes are tau fibril epitopes. In aspects, the fibril epitopes are tau fibril epitopes designed or generated using a TauSeeK-derived, peptide-based active seed (e.g., a prion-like mini-tau fibril). In aspects, the fibril epitopes (e.g., tau fibril epitopes), antibodies, compositions, and methods provided herein are useful in generating shape-controlled amyloid fibril models stabilized by long range ordering of phosphoryl or phosphate assemblies. In aspects, provided herein are methods for verifying the ordering of phosphate assemblies by31P NMR and MQSC NMR. In aspects, provided herein are diagnostic assays employing antibodies for detecting disease-relevant tau assemblies containing phosphoryl / phosphate clusters as signatures. In aspects, provided herein are therapeutic applications employing antibodies to target or neutralize pathological fibrils.LVM Ref.72-24WO; 340383

[0122] In aspects, the peptides, antibodies, antibody fragments, and epitopes disclosed herein are conjugated to one or more analytical tags. In aspects, the analytical tags are employed to facilitate detection, quantification, capture, sorting, and / or imaging in vitro or in vivo. Suitable tags include, without limitation, optical reporters (e.g., organic fluorophores such as fluorescein, rhodamine, and other dyes; bioluminescent or chemiluminescent enzymes or substrates; lanthanide chelates for time-resolved fluorescence), radiolabels (e.g., 14C labeling), enzymatic labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferases), affinity or hapten tags (e.g., biotin, digoxigenin, His6, HA, Myc), fluorescence / energy-transfer pairs (e.g., FRET / quencher systems), isotopic tags, photoactivatable or photoswitchable dyes, contrast agents (e.g., Gd3 or Mn2+ chelates), or any combination thereof. In aspects, the analytical tag comprises a spin label incorporated into an amino acid residue to enable MQSC by31P solid-state NMR, electron paramagnetic resonance (EPR) spectroscopy (e.g., continuous-wave (CW), double electron-electron resonance (DEER)), or related measurements. In aspects, the analytical tag is attached directly to the peptide, antibody, antibody fragment, or epitope. In aspects, the analytical tag is attached indirectly to the peptide, antibody, antibody fragment, or epitope via a linker (e.g., PEG spacers; cleavable linkers such as disulfide, enzymatic, acid labile, or photocleavable linkers; or other suitable linkers). In aspects, the stoichiometry and / or conjugation site of the analytical tag is selected to preserve binding specificity and / or affinity.

[0123] Aspects disclosed herein include pharmaceutical compositions. The pharmaceutical composition of the present invention can be prepared into various forms according to different administration routes. For example, the pharmaceutical composition can be administered in any of the following ways: oral administration, spray inhalation, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or administration via an explanted reservoir.

[0124] Aspects of the Invention

[0125] Various aspects are contemplated herein, several of which are set forth in the paragraphs below. It is explicitly contemplated that any aspect or portion thereof can beLVM Ref.72-24WO; 340383 combined to form an aspect. In addition, it is explicitly contemplated that any aspect (e.g., Aspect A13) that references an aspect (e.g., Aspect A1) for which there are sub- aspects having the same top level number (e.g., Aspect A1a, A1b, A1c, and so forth) necessarily includes reference to those sub-aspects A1a, A1b, A1c, and so forth. In other words, if Aspect A13 refers to Aspect A1, and there are Aspects A1a and A1b present, then Aspect A13 refers to Aspects A1a or A1b. Furthermore, although the aspects below are subdivided into aspects A, B, C, D, and so forth, it is explicitly contemplated that aspects in each of subdivisions A, B, C, D, etc. can be combined in any manner. Moreover, the term “any preceding aspect” means any aspect that appears prior to the aspect that contains such phrase (in other words, the sentence “Aspect B13: The method of any one of aspects B1-B12, or any preceding aspect, …” means that any aspect prior to aspect B13 is referenced, including aspects B1-B12 and all of the “A” aspects). For example, it is contemplated that, optionally, any method or composition of any of the below aspects may be useful with or combined with any other aspect provided below. Further, for example, it is contemplated that any embodiment described elsewhere herein, including above this paragraph, may optionally be combined with any of the below listed aspects. In some instances in the aspects below, or elsewhere herein, two open ended ranges are disclosed to be combinable into a range. For example, “at least X” is disclosed to be combinable with “less than Y” to form a range, in which X and Y are numeric values. For the purposes of forming ranges herein, it is explicitly contemplated that “at least X” combined with “less than Y” forms a range of X- Y inclusive of value X and value Y.

[0126] Aspect A1. A method for generating a peptide, the method comprising: identifying a region of a disease-specific fibril; selecting a structural motif of the region, wherein the structural motif contributes to a characteristic structure of the disease-specific fibril; synthesizing a peptide comprising an amino acid sequence encoding the structural motif; wherein the peptide mimics the characteristic structure of the disease-specific fibril, thereby generating a peptide.LVM Ref.72-24WO; 340383

[0127] Aspect A1a. A method for generating a peptide, such as a prion-like mini-tau peptide, the method comprising: identifying a region, such as a filament-core region, of a disease-specific fibril, such as a disease-specific tau fibril; selecting a structural motif of the region, wherein the structural motif contributes to a characteristic structure, such as a critical protein fold, of the disease-specific fibril; synthesizing a peptide, such as a prion-like mini-tau peptide, comprising an amino acid sequence encoding the structural motif; wherein the peptide mimics the characteristic structure, such as a critical protein fold, of the disease-specific fibril, thereby generating a peptide, such as a prion-like mini-tau peptide.

[0128] Aspect A1b. A method for generating a prion-like mini-tau peptide, the method comprising: identifying a filament-core region of a disease-specific tau fibril; selecting a structural motif of the region, wherein the structural motif contributes to a characteristic structure, such as a critical protein fold, of the disease-specific fibril; synthesizing a peptide, such as a prion-like mini-tau peptide, comprising an amino acid sequence encoding the structural motif; wherein the peptide mimics the characteristic structure, such as a critical protein fold, of the disease-specific fibril, thereby generating a peptide, such as a prion-like mini-tau peptide.

[0129] Aspect A2. The method of aspect A1, wherein the region comprises a filament-core region, and wherein the disease-specific fibril comprises a disease-specific tau fibril.

[0130] Aspect A2a. The method of aspect A2, wherein the filament core region comprises a microtubule-binding region, a proline-rich region, a N-terminus domain, or a combination thereof.

[0131] Aspect A3. The method of aspect A1 or A2, wherein the filament-core region is or comprises the microtubule-binding domain of the disease-specific tau fibril.LVM Ref.72-24WO; 340383

[0132] Aspect A4. The method of any preceding aspect, wherein the filament-core region is or comprises a R1 region, a R2 region, a R3 region and / or a R4 region of the disease-specific tau fibril.

[0133] Aspect A4a. The method of any preceding aspect, wherein the filament-core region is or comprises a R2 region and / or a R3 region of the disease-specific tau fibril.

[0134] Aspect A4b. The method of any preceding aspect, wherein the filament-core region is a R2 region and a R3 region of the disease-specific tau fibril.

[0135] Aspect A5. The method of any preceding aspect, wherein the peptide, such as a prion-like min-tau peptide, comprises an aggregation-prone domain.

[0136] Aspect A6. The method of any preceding aspect, wherein the structural motif has 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) of sequence identity of SEQ ID NO: 1 (VQIVYK).

[0137] Aspect A7. The method of any preceding aspect, wherein the structural motif has 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 4 (VQIINK).

[0138] Aspect A8. The method of any preceding aspect, wherein the prion-like mini- tau peptide further comprises a linking sequence.

[0139] Aspect A9. The method of aspect A8, or any preceding aspect, wherein the linking sequence is a flexible linker or a cleavable linker.

[0140] Aspect A10. The method of aspect A8 or A9, or any preceding aspect, wherein the linking sequence comprises glycine, serine, or a combination of glycine and serine.

[0141] Aspect A11. The method of any one of aspects A8-A10, or any preceding aspect, wherein the linking sequence comprises between 1 and 20 amino acid residues, optionally between 1 and 10 amino acid residues, optionally between 1 and 5 amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues).LVM Ref.72-24WO; 340383

[0142] Aspect A12. The method of any one of aspects A8-A11, or any preceding aspect, wherein the linking sequence comprises 3 glycine residues, optionally wherein the linking sequence is GGG.

[0143] Aspect A13. The method of any preceding aspect, wherein the disease of the disease-specific fibril is a 4R tauopathy.

[0144] Aspect A14. The method of any preceding aspect, wherein the disease of the disease-specific fibril is Alzheimer’s Disease, Pick’s Disease, chronic traumatic encephalopathy, corticobasal degeneration, progressive supranuclear palsy, argyrophilic grain disease, aging-related tau astrogliopathy, globular glial tauopathy, familial British dementia, familial Danish dementia, and limbic-predominant neuronal inclusion body 4R tauopathy, or any combination thereof.

[0145] Aspect A15. The method of any preceding aspect, wherein the method further comprises: tuning the peptide, such as the prion-like mini-tau peptide, to enforce a desired conformation and / or improve fibril formation of the prion-like mini-tau peptide.

[0146] Aspect A16. The method of aspect A15, or any preceding aspect, wherein the tuning comprises introducing a post-translational modification, a point mutation, a water- structuring additive, or any combination thereof, to the peptide, such as the prion-like mini-tau peptide.

[0147] Aspect A17. The method of aspect A15 or A16, or any preceding aspect, wherein the tuning step comprises introducing a point mutation to at least one residue (e.g., 1, 2, 3, 4, or 5 residues, optionally, less than 30 residues, less than 20 residues, or less than 10 residues) of the peptide, such as the prion-like mini-tau peptide, optionally wherein the point mutation comprises a site-specific proline to leucine substitution mutation.

[0148] Aspect A18. The method of any one of aspects A15-A17, or any preceding aspect, wherein the tuning step comprises a post-translational phosphorylation of at least one residue (e.g., 1, 2, 3, 4, or 5 residues, optionally, less than 30 residues, less than 20 residues, or less than 10 residues) of the peptide, such as the prion-like mini-tauLVM Ref.72-24WO; 340383 peptide, optionally wherein the least one residue comprises a serine residue of the structural motif.

[0149] Aspect A19. The method of any one of aspects A15-A18, or any preceding aspect, wherein the tuning comprises contacting the peptide, such as prion-like mini-tau peptide, with a complement strand.

[0150] Aspect A20. The method of aspect A19, or any preceding aspect, wherein the complement strand has 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of: SEQ ID NO: 14 (GGGQVEVKSEKLDFKDRVQSKIGSLDNITH); SEQ ID NO: 15 (LDFKDRVQSKIGSLDNGGGHKLTFRE); or SEQ ID NO: 16 (FKDRVQSKIGSLDNITHVPG).

[0151] Aspect A21. The method of any one of aspects A15-A20, or any preceding aspect, wherein the desired conformation is a U-shaped conformation.

[0152] Aspect A22. The method of any one of aspects A15-A20, or any preceding aspect, wherein the desired conformation is a strand-loop-strand (SLS) conformation.

[0153] Aspect A23. The method of any preceding aspect, the method further comprising evaluating an aggregation competency of the peptide, such as the prion-like mini-tau peptide.

[0154] Aspect A24. The method of aspect A23, or any preceding aspect, wherein the evaluating step comprises high throughput screening.

[0155] Aspect A25. The method of any preceding aspect, wherein the peptide, such as the prion-like mini-tau peptide, has 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of: SEQ ID NO: 2 (DNIKHVLGGGSVQIVYKPV); SEQ ID NO: 5 (DNIKHVPGGGSVQIVYKPV); SEQ ID NO: 6 (DNIKHVPGG); SEQ ID NO: 7 (DNIKHVLGG); SEQ ID NO: 8 (VPGGGSVQIVYKPV); SEQ ID NO: 9 (VLGGGSVQIVYKPV);LVM Ref.72-24WO; 340383 SEQ ID NO: 10 (DNIKHVPG); SEQ ID NO: 11 (DNIKHVLGGGSPhosVQIVYKPV); SEQ ID NO: 12 (DNIKHVLGGGSVQIVYPhosKPV); SEQ ID NO: 17 (VQIVYKPGGGNHKLTF); or SEQ ID NO: 18 (KVQIINKPGGGKLTFRE).

[0156] Aspect A26. A composition comprising the peptide, such as the prion-like mini-tau peptide, of any preceding aspect and a pharmaceutically acceptable carrier.

[0157] Aspect A27. A method for generating a fibril the method comprising: selecting a peptide generated by the method of any one of aspects A1-A25, or any preceding aspect; and introducing a plurality of the peptide to a solution, thereby generating a fibril.

[0158] Aspect A27a. A method for generating a fibril, such as a prion-like mini-tau fibril, the method comprising: selecting a peptide, such as a prion-like mini-tau peptide, generated by the method of any one of aspects A1-A25, or any preceding aspect; and introducing a plurality of the peptide, such as the prion-like mini-tau peptide, to a solution, thereby generating a fibril, such as a prion-like mini-tau fibril.

[0159] Aspect A27b. A method for generating a prion-like mini-tau fibril, the method comprising: selecting a prion-like mini-tau peptide, generated by the method of any one of aspects A1-A25, or any preceding aspect; and introducing a plurality of the the prion-like mini-tau peptide to a solution, thereby generating a prion-like mini-tau fibril.

[0160] Aspect B1. A method for generating a fibril, the method comprising: generating a peptide, wherein the generating step comprises: identifying a region of a disease-specific fibril; selecting a structural motif of the region, wherein the structural motif contributes to a characteristic structure of the disease-specific fibril;LVM Ref.72-24WO; 340383 synthesizing a peptide comprising an amino acid sequence encoding the structural motif; wherein the peptide mimics the characteristic structure of the disease-specific fibril, introducing a plurality of the peptide to a solution, thereby generating the fibril.

[0161] Aspect B1a. A method for generating a fibril, such as a prion-like mini-tau fibril, the method comprising: generating a peptide, such as a prion-like mini-tau peptide, wherein the generating step comprises: identifying a region, such as a filament core region, of a disease-specific fibril, such as a disease-specific tau fibril; selecting a structural motif of the region, wherein the structural motif contributes to a characteristic structure, such as a critical protein fold, of the disease-specific fibril; synthesizing a peptide, such as a prion-like mini-tau peptide, comprising an amino acid sequence encoding the structural motif; wherein the peptide mimics the characteristic structure, such as the critical protein fold, of the disease-specific fibril, introducing a plurality of the peptide, such as the prion-like mini-tau peptide, to a solution, thereby generating the fibril, such as the prion-like mini-tau fibril.

[0162] Aspect B1b. A method for generating a prion-like mini-tau fibril, the method comprising: generating a prion-like mini-tau peptide, wherein the generating step comprises: identifying a filament-core region of a disease-specific tau fibril; selecting a structural motif of the filament-core region, wherein the structural motif contributes to a critical protein fold of the disease-specific tau fibril; synthesizing a mini-tau peptide comprising an amino acid sequence encoding the structural motif;LVM Ref.72-24WO; 340383 wherein the mini-tau peptide mimics the critical protein fold of the disease- specific tau fibril, introducing a plurality of the prion-like mini-tau peptide to a solution, thereby generating the prion-like mini-tau fibril.

[0163] Aspect B2. The method of aspect A27 or B1, or any preceding aspect, wherein the solution comprises heparin.

[0164] Aspect B3. The method of any one of aspects A27-B2, or any preceding aspect, wherein the solution comprises at least one cofactor and / or a complement strand.

[0165] Aspect B4. The method of aspect B3, or any preceding aspect, wherein the solution comprises a complement strand, and wherein the complement strand has 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of: SEQ ID NO: 14 (GGGQVEVKSEKLDFKDRVQSKIGSLDNITH); SEQ ID NO: 15 (LDFKDRVQSKIGSLDNGGGHKLTFRE); or SEQ ID NO: 16 (FKDRVQSKIGSLDNITHVPG).

[0166] Aspect B5. The method of any one of aspects A27-B4, or any preceding aspect, wherein the method further comprises stabilizing the fibril (e.g., the prion-like mini-tau fibril) to support the characteristic structure, such as the critical protein fold, of the plurality of the peptides, such as the prion-like mini-tau peptides.

[0167] Aspect B6. The method of aspect B5, or any preceding aspect, wherein the solution comprises a complement strand, and the stabilizing step comprises modifying the complement strand to promote selective synthesis of the fibril (e.g., the prion-like mini-tau fibril) that are specific to a tauopathy.

[0168] Aspect B7. The method of aspect B6, or any preceding aspect, wherein the modifying the complement strand step comprises designing the complement strand to mimic the conformation of the disease-specific fibril (e.g., the disease-specific tau fibril).

[0169] Aspect B8. The method of any one of aspects A27-B7, or any preceding aspect, wherein the solution comprises a complement strand and the solution isLVM Ref.72-24WO; 340383 characterized by a molar ratio of about 4:1:10, optionally, 1:1:10, 2:1:10, 3:1:10, 5:1:10, 10:1:10, 20:1:10, 30:1:10, 4:1:1, 4:1:2, 4:1:3, 4:1:4, 4:1:5, 4:1:10, 4:1:20, 4:1:30, or any sub-range thereof, of the peptide:heparin:the complement strand (e.g., prion-like mini- tau peptide:heparin:the complement strand).

[0170] Aspect B9. The method of any one of aspects A27-B8, or any preceding aspect, wherein the disease-specific fibril is a disease-specific tau fibril, and wherein the disease-specific tau fibril is associated with a tauopathy, and the tauopathy is Alzheimer’s Disease, Pick’s Disease, chronic traumatic encephalopathy, corticobasal degeneration, progressive supranuclear palsy, argyrophilic grain disease, aging-related tau astrogliopathy, globular glial tauopathy, familial British dementia, familial Danish dementia, and limbic-predominant neuronal inclusion body 4R tauopathy, or any combination thereof.

[0171] Aspect B10. The method of any one of aspects B3-B9, or any preceding aspect, wherein the at least one cofactor comprises: heparin; RNA; arachidonic acid; polyphosphates; phosphate ions; sulfate ions; a salt, optionally, wherein the salt comprises NaCl or MgCl2; or any combination thereof.

[0172] Aspect B11. The method of any one of aspects A27-B10, or any preceding aspect, wherein the fibril (e.g., the prion-like mini-tau fibril) comprises an active fibril surface, optionally wherein the active fibril surface comprises at least one peptide.

[0173] Aspect B11a. The method of aspect B11, or any preceding aspect, wherein the at least one peptide is a prion-like mini-tau peptide.

[0174] Aspect B11b. The method of aspect B11 or B11a, or any preceding aspect, wherein the at least one peptide comprises an aggregation-prone domain.LVM Ref.72-24WO; 340383

[0175] Aspect B12. The method of any one of aspects A27-B11, or any preceding aspect, the method further comprising evaluating one or more morphological feature of the fibril (e.g., the prion-like mini-tau fibril).

[0176] Aspect B13. The method of aspect B12, or any preceding aspect, wherein the morphological feature comprises fibril formation, average internuclear distance distributions for each peptide of the fibril; homogeneity of the fibril (e.g., the prion-like mini-tau fibril), multiple quantum coherence order (MQCO), or any combination thereof.

[0177] Aspect B14. The method of aspect B12, or any preceding aspect, wherein the morphological feature comprises average internuclear distance distributions for each prion-like mini-peptide, wherein the average internuclear distance distribution is about 4.8 angstroms.

[0178] Aspect B15. The method of aspect B12, or any preceding aspect, wherein the morphological feature comprises MQCO, optionally wherein the fibril (e.g., the prion-like mini-tau fibril) is characterized by an average of at least 331P spins (e.g., 331P spins, 431P spins, 531P spins, 631P spins, 731P spins, 831P spins, 931P spins, 1031P spins) within a spin cluster.

[0179] Aspect B16. The method of any one of aspects B11-B15, or any preceding aspect, wherein the evaluating step comprises electron-electron double resonance spectroscopy (DEER).

[0180] Aspect B17. A composition comprising the fibril (e.g., the prion-like mini-tau fibril) generated by the method of any one of aspects A27-B16, or any preceding aspect, and a pharmaceutically acceptable carrier.

[0181] Aspect B18. A method for generating pathogenic fibrils, the method comprising: selecting one or more fibril generated by the method of any one of aspects A27- B16, or any preceding aspect; providing a first set of monomers; and contacting a plurality of the fibrils with the first set of monomers in a solution, thereby generating pathogenic fibrils.LVM Ref.72-24WO; 340383

[0182] Aspect B18a. A method for generating pathogenic fibrils, such as pathogenic tau fibrils, the method comprising: selecting one or more fibril, such as one or more prion-like mini-tau fibril, generated by the method of any one of aspects A27-B16, or any preceding aspect; providing a first set of monomers, such as a first set of tau monomers; and contacting a plurality of the fibrils with the first set of monomers in a solution, thereby generating pathogenic fibrils, such as pathogenic tau fibrils.

[0183] Aspect B18b. A method for generating full-length pathogenic tau fibrils, the method comprising: selecting one or more prion-like mini-tau fibril generated by the method of any one of aspects A27-B16, or any preceding aspect; providing a first set of tau monomers; and contacting a plurality of the prion-like mini-tau fibrils with the first set of tau monomers in a solution, thereby generating full-length pathogenic tau fibrils.

[0184] Aspect B18c. The method of any one of aspects B18-B18b, wherein the fibrils act as templates that stabilize the first set of monomers to adopt a disease-specific fibril formation, such as a disease-specific fibril core formation.

[0185] Aspect C1. A method for generating pathogenic fibrils, the method comprising: selecting one or more fibril comprising a plurality of peptides wherein at least a portion of the peptides mimic a characteristic structure of a disease-specific fibril; providing a first set of monomers; and contacting a plurality of the fibrils with the first set of monomers in a solution to induce aggregation of the first set of monomers, thereby generating pathogenic fibrils.

[0186] Aspect C1a. A method for generating pathogenic fibrils, such as pathogenic tau fibrils, the method comprising:LVM Ref.72-24WO; 340383 selecting one or more fibril, such as one or more prion-like mini-tau fibril, comprising a plurality peptides, such as a plurality of prion-like mini-tau peptides, wherein at least a portion of the peptides mimic a characteristic structure, such as a critical protein fold, of a disease-specific fibril, such as a disease-specific tau fibril; providing a first set of monomers, such as tau monomers; and contacting a plurality of the fibrils with the first set of monomers in a solution to induce aggregation of the monomers, thereby generating pathogenic fibrils, such as pathogenic tau fibrils.

[0187] Aspect C1b. A method for generating full-length pathogenic tau fibrils, the method comprising: selecting one or more prion-like mini-tau fibril comprising a plurality prion-like mini-tau peptides wherein at least a portion of the prion-like mini-tau peptides mimic a critical protein fold of a disease-specific tau fibril; providing a first set of tau monomers; and contacting a plurality of the prion-like mini-tau fibrils with the first set of tau monomers in a solution to induce aggregation of the first set of tau monomers, thereby generating full-length pathogenic tau fibrils.

[0188] Aspect C1c. The method of any one of aspects C1-C1b, wherein the fibrils act as templates that stabilize the first set of monomers to adopt a disease-specific fibril formation, such as a disease-specific tau fibril core formation.

[0189] Aspect C2. The method of aspect B18 or C1, or any preceding aspect, wherein the solution is characterized by a molar ratio of about 1:1 of the fibrils:the first set of monomers (e.g., 1:1 of the prion-like mini-tau fibrils:the first set of tau monomers).

[0190] Aspect C3. The method of aspect B18 or C2, or any preceding aspect, wherein the solution is characterized by a molar ratio of about 4:1 of the fibrils:the first set of monomers (e.g., 4:1 of the synthetic mini-tau fibrils:the first set of tau monomers).

[0191] Aspect C4. The method of any one of aspects B18-C3, or any preceding aspect, wherein the solution further comprises at least one cofactor.LVM Ref.72-24WO; 340383

[0192] Aspect C5. The method of any one of aspects B18-C4, or any preceding aspect, wherein the method is performed in vitro, optionally wherein the method comprises bacterial cells or mammalian cells.

[0193] Aspect C6. The method of any one of aspects B18-C5, or any preceding aspect, the method further comprising: tuning the structure of the pathogenic fibrils (e.g., the full-length pathogenic tau fibrils) to enforce a desired conformation and / or improve aggregation of the first set of monomers (e.g., the first set of tau monomers).

[0194] Aspect C7. The method of aspect C6, or any preceding aspect, wherein the tuning comprises adjusting the pH, temperature, cofactor, or a combination thereof, of the solution of the contacting step.

[0195] Aspect C8. The method of any one of aspects B18-C7, or any preceding aspect, the method further comprising evaluating one or more morphological feature of the fibrils (e.g., the full-length pathogenic tau fibrils) for the disease-specific fibril core formation (e.g., the disease-specific tau fibril core formation).

[0196] Aspect C9. The method of aspect C8, or any preceding aspect, wherein the morphological feature comprises fibril formation, average internuclear distance distributions for each peptide (e.g., each prion-like mini-peptide) of the fibrils (e.g., the prion-like mini-tau fibrils); homogeneity of the fibrils (e.g., the prion-like mini-tau fibrils), or any combination thereof.

[0197] Aspect C10. The method of aspect C8 or C9, or any preceding aspect, wherein the evaluating step comprises electron-electron double resonance spectroscopy (DEER).

[0198] Aspect C11. The method of aspect C10, or any preceding aspect, further comprising distinguishing distinct signals, wherein the distinct signals comprise a DEER signal, a noise, an artifact, one or more pathogenic fibrils (e.g., one or more full-length pathogenic tau fibrils), or any combination thereof.LVM Ref.72-24WO; 340383

[0199] Aspect C12. The method of aspect C11, or any preceding aspect, wherein the distinguishing step comprises a frequency pattern recognition, wherein the frequency pattern recognition comprises the use of a discretized continuous wavelet transform (CWT) and a structure similarity index measure (SSIM) analysis.

[0200] Aspect C13. A method for generating a subsequent generation of pathogenic fibrils (e.g., a subsequent generation of full-length pathogenic tau fibrils), the method comprising: combining the pathogenic fibrils (e.g., the full-length pathogenic tau fibrils) of any one of aspects B18-C12, or any preceding aspect, with a second set of monomers (e.g., a second set of tau monomers) in a second solution to induce aggregation of the second set of monomers, thereby generating a subsequent generation of pathogenic fibrils (e.g., full- length pathogenic tau fibrils).

[0201] Aspect C13a. A method for generating a subsequent generation of full-length pathogenic tau fibrils, the method comprising: combining the full-length pathogenic tau fibrils of any one of aspects B18-C12, or any preceding aspect, with a second set of tau monomers in a second solution to induce aggregation of the second set of tau monomers, thereby generating a subsequent generation of the full-length pathogenic tau fibrils.

[0202] Aspect C13b. The method of aspect C13 or C13a, wherein the fibrils (e.g., the prion-like mini-tau fibrils) act as templates that stabilize the monomers (e.g., the tau monomers) to adopt a disease-specific fibril formation (e.g., a disease-specific tau fibril core formation).

[0203] Aspect C14. The method of aspect C13, or any preceding aspect, further comprising sonicating the pathogenic fibrils (e.g., the pathogenic tau fibrils) prior to the combining step.

[0204] Aspect C15. A composition comprising the pathogenic fibrils (e.g., the pathogenic tau fibrils) of any one of aspects B18-C14, or any preceding aspect.LVM Ref.72-24WO; 340383

[0205] Aspect D1. Use of the composition, such as diagnostic use, of any one of aspects A26, B17, or C15, or any preceding aspect, for evaluating a tauopathy, optionally wherein the tauopathy is Alzheimer’s Disease, Pick’s Disease, chronic traumatic encephalopathy, corticobasal degeneration, progressive supranuclear palsy, argyrophilic grain disease, aging-related tau astrogliopathy, globular glial tauopathy, familial British dementia, familial Danish dementia, and limbic-predominant neuronal inclusion body 4R tauopathy, or any combination thereof.

[0206] Aspect D2. Use of the method, such as diagnostic use, of any preceding aspect for evaluating a tauopathy, optionally wherein the tauopathy is Alzheimer’s Disease, Pick’s Disease, chronic traumatic encephalopathy, corticobasal degeneration, progressive supranuclear palsy, argyrophilic grain disease, aging-related tau astrogliopathy, globular glial tauopathy, familial British dementia, familial Danish dementia, and limbic-predominant neuronal inclusion body 4R tauopathy, or any combination thereof.

[0207] Aspect E1. A kit for generating fibrils, the kit comprising at least one peptide generated from the method of any one of aspects A1-A25, or any preceding aspect.

[0208] Aspect E1a. A kit for generating fibrils, such as prion-like mini-tau fibrils, the kit comprising at least one peptide, such as at least one prion-like mini-tau peptide, generated from the method of any one of aspects A1-A25, or any preceding aspect.

[0209] Aspect E1b. A kit for generating prion-like mini-tau fibrils, the kit comprising at least one prion-like mini-tau peptide generated from the method of any one of aspects A1-A25, or any preceding aspect.

[0210] Aspect E2. A kit for generating fibrils, the kit comprising at least one peptide wherein each peptide independently comprises a sequence having 75% or greater sequence identity (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100% sequence identity) of: SEQ ID NO: 2 (DNIKHVLGGGSVQIVYKPV); SEQ ID NO: 11 (DNIKHVLGGGSPhosVQIVYKPV); SEQ ID NO: 12 (DNIKHVLGGGSVQIVYPhosKPV); SEQ ID NO: 17 (VQIVYKPGGGNHKLTF);LVM Ref.72-24WO; 340383 SEQ ID NO: 18 (KVQIINKGGGKLTFRE); SEQ ID NO: 21 (SphosVQIVYKPGGGNHKLTF); or SEQ ID NO: 22 (SphosKVQIINKPGGGKLTFRE); or any combination thereof.

[0211] Aspect E2a. A kit for generating fibrils, such as prion-like mini-tau fibrils, the kit comprising at least one peptide, such as prion-like mini-tau peptide, wherein each peptide independently comprises a sequence having 75% or greater sequence identity (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100% sequence identity) of: SEQ ID NO: 2 (DNIKHVLGGGSVQIVYKPV); SEQ ID NO: 11 (DNIKHVLGGGSPhosVQIVYKPV); SEQ ID NO: 12 (DNIKHVLGGGSVQIVYPhosKPV); SEQ ID NO: 17 (VQIVYKPGGGNHKLTF); SEQ ID NO: 18 (KVQIINKGGGKLTFRE); SEQ ID NO: 21 (SphosVQIVYKPGGGNHKLTF); or SEQ ID NO: 22 (SphosKVQIINKPGGGKLTFRE); or any combination thereof.

[0212] Aspect E2b. A kit for generating prion-like mini-tau fibrils, the kit comprising at least one prion-like mini-tau peptide wherein each prion-like mini-tau peptide independently comprises a sequence having 75% or greater sequence identity (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100% sequence identity) of: SEQ ID NO: 2 (DNIKHVLGGGSVQIVYKPV); SEQ ID NO: 11 (DNIKHVLGGGSPhosVQIVYKPV); SEQ ID NO: 12 (DNIKHVLGGGSVQIVYPhosKPV); SEQ ID NO: 17 (VQIVYKPGGGNHKLTF); SEQ ID NO: 18 (KVQIINKGGGKLTFRE); SEQ ID NO: 21 (SphosVQIVYKPGGGNHKLTF); or SEQ ID NO: 22 (SphosKVQIINKPGGGKLTFRE); or any combination thereof.LVM Ref.72-24WO; 340383

[0213] Aspect E3. A kit for the development of tauopathy therapeutics, imaging agents, and / or other technologies for the identification and treatment of tauopathies, the kit comprising a pathogenic fibril (e.g., a pathogenic tau fibril) of any one of aspects B18- C14, or any preceding aspect.

[0214] Aspect E4. A kit for the development of tauopathy therapeutics, imaging agents, and / or other technologies for the identification and / or treatment of tauopathies, the kit comprising: a tau protein; a tau protein fragment, optionally wherein the tau protein fragment comprises an amino acid sequence spanning the R2 / R3 region of a tau protein; a spin labeled full-length pathogenic tau fibril, or a tau fibril fragment thereof, of any preceding aspect; or an isotope labeled full-length pathogenic tau fibril, or a tau fibril fragment thereof, of any preceding aspect; or any combination thereof.

[0215] Aspect E4a. A kit for the development of tauopathy therapeutics, imaging agents, and / or other technologies for the identification and / or treatment of tauopathies, the kit comprising: a tau protein, optionally a tau protein comprising a P301L mutation; a tau protein fragment comprising an amino acid sequence spanning the R2 / R3 region of a tau protein, optionally wherein the tau protein fragment comprises a P301L mutation; a spin labeled full-length pathogenic tau fibril, or a tau fibril fragment thereof, of any preceding aspect; or an isotope labeled full-length pathogenic tau fibril, or a tau fibril fragment thereof, of any preceding aspect; or any combination thereof.

[0216] Aspect E4b. A kit for the development of tauopathy therapeutics, imaging agents, and / or other technologies for the identification and / or treatment of tauopathies, the kit comprising: 0N4R isoform tau protein (UniProt accession number (P10636-8));LVM Ref.72-24WO; 340383 a tau protein fragment having 75% or greater sequence identity (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100% sequence identity) of SEQ ID NO: 13 (NVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQI VYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITH VPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGS IDMVDSPQLATLADEVSASLAKQGL); a tau protein fragment having 75% or greater sequence identity (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100% sequence identity) of SEQ ID NO: 23 (NVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVLGGGSVQI VYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITH VPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGS IDMVDSPQLATLADEVSASLAKQGL); a spin labeled full-length pathogenic tau fibril, or a fragment thereof, of any preceding aspect; or an isotope labeled full-length pathogenic tau fibril, or a fragment thereof, of any preceding aspect; or any combination thereof.

[0217] Aspect F1. A synthetic phosphorylated amyloid fibril comprising a structurally ordered cluster of peptide monomers, wherein at least a portion, optionally all, of the peptide monomers comprise a site-specific phosphorylated residue, wherein the phosphoryl groups of the site-specific phosphorylated residues are aligned in a linear, stacked conformation along the axis of the synthetic phosphorylated amyloid fibril.

[0218] Aspect F2. The synthetic phosphorylated amyloid fibril of aspect F1, or any preceding aspect, wherein the spacing between the phosphoryl groups of the site- specific phosphorylated residues of adjacent strands is between about 4 Å to about 5 Å, optionally about 4.5 Å to about 4.9 Å (e.g., about 4.5 Å, about 4.6 Å, about 4.7 Å, about 4.8 Å, or about 4.9 Å).

[0219] Aspect F3. The synthetic phosphorylated amyloid fibril of aspect F1 or F2, or any preceding aspect, wherein the structurally ordered cluster comprises three or moreLVM Ref.72-24WO; 340383 peptide monomers, wherein each peptide monomer independently comprises a site- specific phosphorylated residue.

[0220] Aspect F4. The synthetic phosphorylated amyloid fibril of aspect F3, or any preceding aspect, wherein the phosphorylated residues of the three or more peptide monomers are aligned in a linear, stacked conformation across adjacent peptide monomers.

[0221] Aspect F5. The synthetic phosphorylated amyloid fibril of any one of aspects F1-F4, or any preceding aspect, wherein at least one of the peptide monomers, optionally all of the peptide monomers, is derived from the microtubule-binding repeat (MTBR) region of tau.

[0222] Aspect F6. The synthetic phosphorylated amyloid fibril of any one of aspects F1-F5, or any preceding aspect, wherein each of the peptide monomers is independently derived from the microtubule-binding repeat (MTBR) region of tau.

[0223] Aspect F7. The synthetic phosphorylated amyloid fibril of any one of aspects F1-F6, or any preceding aspect, wherein the site-specific phosphorylated residue is a phospho-serine, a phospho-threonine, or a phospho-tyrosine.

[0224] Aspect F8. The synthetic phosphorylated amyloid fibril of any one of aspects F1-F7, or any preceding aspect, wherein each of the peptide monomers independently is a full-length tau monomer, a tau protein fragment, or independently comprises a prion-like mini-tau peptide.

[0225] Aspect F9. The synthetic phosphorylated amyloid fibril of any one of aspects F1-F8, or any preceding aspect, wherein at least one, optionally all, of the peptide monomers is a tau peptide fragment.

[0226] Aspect F10. The synthetic phosphorylated amyloid fibril of aspect F9, or any preceding aspect, wherein the tau peptide fragment has 75% or greater sequence identity (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100% sequence identity) of SEQ ID NO: 41 (IKHVPGGGSVQIVYKPVDLSKVTSKSGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKI GSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAE).LVM Ref.72-24WO; 340383

[0227] Aspect F11. The synthetic phosphorylated amyloid fibril of any one of aspects F1-F10, or any preceding aspect, wherein the phosphoryl groups of the site-specific phosphorylated residues form hydrogen-bonded arrays, electrostatically stabilized networks, or any combination thereof.

[0228] Aspect F12. The synthetic phosphorylated amyloid fibril of aspect F11, or any preceding aspect, wherein the hydrogen-bonded arrays and / or electrostatically stabilized networks contribute to fibril stability and enhanced seeding activity.

[0229] Aspect G1. A method for generating a synthetic phosphorylated amyloid fibril comprising: synthesizing a peptide monomer, or a peptide fragment thereof, comprising an aggregation-prone domain; performing a post-translational phosphorylation of at least one residue of the peptide monomer, or the peptide fragment thereof; combining a plurality of the peptide monomer, or the peptide fragment thereof, to form a mixture of peptide monomers and / or peptide fragments thereof; optionally, tuning at least one condition of the combining step to promote the formation of a structurally ordered cluster of peptide monomers; thereby generating a synthetic phosphorylated amyloid fibril.

[0230] Aspect G2. The method of aspect G1, or any preceding aspect, wherein the at least one condition of the tuning step comprises pH adjustment, buffer selection, salt selection, salt concentration, temperature, agitation, cofactor addition, or any combination thereof.

[0231] Aspect H1. A method for stabilizing synthetic amyloid fibril formation of a plurality of peptide monomers, the method comprising: combining a plurality of peptide monomers, or peptide fragments thereof, in a solution to form a mixture of peptide monomers, wherein at least a portion, optionally all, of the plurality of peptide monomers comprise an aggregation-prone domain; wherein the solution comprises a phosphate buffer or polyphosphates to promote the formation of a structurally ordered cluster of peptide monomers, resulting in a first-generation synthetic amyloid fibril,LVM Ref.72-24WO; 340383 thereby stabilizing synthetic amyloid fibril formation.

[0232] Aspect H2. The method of aspect H1, or any preceding aspect, wherein at least a portion, optionally all, of the plurality of peptide monomers, or peptide fragments thereof, comprise a post-translational phosphorylation of at least one residue.

[0233] Aspect H3. The method of aspect H1 or aspect H2, or any preceding aspect, wherein at least a portion, optionally all, of the plurality of peptide monomers, or peptide fragments thereof, do not comprise a post-translational phosphorylation.

[0234] Aspect H4. The method of any one of aspects H1-H3, or any preceding aspect, wherein at least a portion, optionally all, of the plurality of peptide monomers are tau monomers, or fragments thereof, or comprise a prion-like mini-tau peptide.

[0235] Aspect H5. The method of any one of aspects H1-H5, or any preceding aspect, wherein the structurally ordered cluster of peptide monomers is characterized by in-register stacking of the peptide monomers.

[0236] Aspect H6. The method of any one of aspects H1-H5, or any preceding aspect, wherein the structurally ordered cluster of peptide monomers comprises synthetic phosphorylated amyloid fibrils.

[0237] Aspect H7. The method of aspect H6, or any preceding aspect, wherein the synthetic phosphorylated amyloid fibrils comprise long-range phosphate ordering.

[0238] Aspect H8. The method of any one of aspects H1-H1, or any preceding aspect, wherein the solution comprising a phosphate buffer or polyphosphates promotes the formation of the structurally ordered cluster of peptide monomers by providing a phosphate-containing environment, and wherein the phosphate-containing environment induces the formation of synthetic phosphorylated amyloid fibrils, wherein the synthetic phosphorylated amyloid fibrils comprise long-range phosphate ordering.

[0239] Aspect H9. The method of any one of aspects H1-H8, or any preceding aspect, the method further comprising: introducing the first-generation synthetic amyloid fibril to the solution of the combining step; andLVM Ref.72-24WO; 340383 repeating the steps of aspect H1 resulting in a subsequent generation synthetic amyloid fibril.

[0240] Aspect I1. A method of confirming phosphate or phosphoryl group ordering in the synthetic phosphorylated amyloid fibril or the prion-like mini-tau fibril of any preceding aspect, the method comprising: analyzing the fibril using31P solid-state NMR lineshape analysis, Multiple Quantum Spin Counting (MQSC) with Magic Angle Spinning (MAS), or a combination thereof; indicating the presence of ordered phosphate clusters by evaluating the presence of: a characteristic, narrow,31P solid-state NMR spectral linewidth; a Multiple Quantum Coherence Order (MQCO) of 3 or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more); or a combination thereof; thereby confirming phosphate or phosphoryl group ordering in the synthetic phosphorylated amyloid fibril or the prion-like mini-tau fibril.

[0241] Aspect J1. An immunogen comprising the synthetic phosphorylated amyloid fibril, the fibril, or the prion-like mini-tau fibril of any preceding aspect.

[0242] Aspect K1. A method of eliciting an immune response in a subject, the method comprising administering to the subject the immunogen of aspect J1, or any preceding aspect.

[0243] Aspect L1. The synthetic phosphorylated amyloid fibril or the prion-like mini- tau fibril of any preceding aspect, wherein the fibril presents an epitope characterized by three or more phosphorylated residues or phosphate moieties arranged in a structurally ordered, fibrillar conformation.

[0244] Aspect L2. The synthetic phosphorylated amyloid fibril or the prion-like mini- tau fibril of aspect L2, or any preceding aspect, wherein the structurally ordered, fibrillar conformation is characterized by in-register stacking of the three or more phosphorylated residues or phosphate moieties.LVM Ref.72-24WO; 340383

[0245] Aspect M1. A monoclonal antibody formulated to selectively bind the epitope of aspect L1, or any preceding aspect.

[0246] Aspect N1. A monoclonal antibody formulated to selectively bind an epitope of a synthetic phosphorylated amyloid fibril or a prion-like mini-tau fibril, wherein the epitope is characterized by three or more phosphorylated residues or phosphate moieties arranged in a structurally ordered, fibrillar conformation.

[0247] Aspect O1. The monoclonal antibody of aspect M1 or N1, or any preceding aspect, wherein the monoclonal antibody does not bind to non-fibrillar, singly phosphorylated, or non-phosphorylated peptides of the synthetic phosphorylated amyloid fibril or the prion-like mini-tau fibril.

[0248] Aspect O2. The monoclonal antibody of any one of aspects M1-O1, or any preceding aspect, wherein the monoclonal antibody comprises an analytical tag.

[0249] Aspect P1. A method of generating a monoclonal antibody, the method comprising: immunizing a subject with the immunogen of aspect J1 or any preceding aspect; screening sera or hybridoma supernatants; selecting the sera or the hybridoma supernatants characterized by selective binding properties, wherein the selective binding properties comprise binding to multivalent phosphorylated fibrils and do not bind to monomeric or singly phosphorylated peptides; and isolating clones of the sera or the hybridoma supernatants characterized by the selective binding properties; thereby generating the monoclonal antibody.

[0250] Aspect Q1. A pharmaceutical composition comprising the monoclonal antibody of any one of aspects M1-P1, or any preceding aspect, and a pharmaceutically acceptable carrier.

[0251] Aspect R1. Use of the monoclonal antibody of aspect M1, or any preceding aspect, in a diagnostic evaluation of a biological sample, the method comprising:LVM Ref.72-24WO; 340383 contacting a biological sample with the monoclonal antibody of aspect M1, or any preceding aspect; and evaluating a subsample of the biological sample having the analytical tag.

[0252] The invention can be further understood by the following non-limiting examples.

[0253] EXAMPLES

[0254] The following Examples comprise some exemplary but not limiting methods, materials, processes, techniques, compositions, formulations, etc., useful in the practice of the invention as well as exemplary but not limiting data, discussion, and hypotheses, without wishing to be bound by any particular theory. It is recognized that regardless of the ultimate correctness of any mechanistic explanation or hypothesis, an embodiment of the invention can nonetheless be operative and useful.

[0255] Introduction for Examples 1 and 2

[0256] In aspects, we used Overhauser Dynamic Nuclear Polarization (ODNP) to measure the diffusivity of near-surface water around a jR2R3 peptide in its monomer state that reflect on the hydration water structure, and computational tools to quantify the solvation free energy, structure, and dynamics of site-specific hydration water properties and thermodynamics around tau monomers in their IDP state. We hypothesized that localized water directs templated aggregation to faithfully propagate the shape of the mother fibril to the daughter fibril. One of the objectives was to debut a new concept that tau peptide segments that fold and stack to fibrils can serve as “mini- prions” that nucleate the formation of tauopathy fibrils. We presented the design criteria for such a peptide on the example of a 19-residue peptide spanning the R2 / R3 splice junction.

[0257] In aspects, this study presents a first step towards designing a tauopathy specific aggregation pathway by engineering a synthetic minimal tau prion building block (also referred to herein as a “prion-like mini-tau peptide”), jR2R3, that can template and propagate distinct disease folds. In aspects, the prion-like mini-tau peptide was jR2R3. In aspects, we present the discovery that P301L—among the widest used mutations in cell and animal models of Alzheimer’s Disease—destabilizes an aggregation-prohibitingLVM Ref.72-24WO; 340383 internal hairpin and enhances surface water structure at a hyper-localized site that serves as pinning site to promote templated aggregation. In aspects, we suggest that P301L may be a more suitable mutation to include in modeling 4R tauopathies than Alzheimer’s Disease, and that mutations are powerful tools for the purpose of designing of tau prion models as therapeutic tools.

[0258] Because the use of aggregation-accelerating mutations is compulsory at the current state of understanding of tauopathy mimicking fibrils, P301L was used as a model to scrutinize the molecular consequences of this mutation on the energetic and structural properties of tau in its IDP state prior to aggregation and fibril state.

[0259] To understand the effect of P301L on the structural property of the resulting tau fibril, we selected a tau sequence that contains the P301 site to recreate the pathological property of tau fibril, namely seeding competency to achieve shape propagation by templated aggregation. The predominant region of tau observed in the core of tauopathy fibrils consists of four pseudo-repeat domains (R1 through R4) (FIG. 12A), where alternative splicing led to tau isoforms that contained all four repeats (4R tauopathies) or those that lack the R2 domain (3R tauopathies). P301 is a site in the R2 domain, where the P301L mutation has been reported to selectively recruit 4R tau (over 3R) tau. We hence turned to 4R tauopathies, such as CBD, PSP, AGD or GPT in which the building block of the characteristic NFTs is the 4R isoform.

[0260] The tauopathy fibril core contains intricately folded tau segments that are precisely arranged within a well-defined protofibril structure. Such an architecture cannot assemble spontaneously in a single step without error. We posited that there should be a nucleating contact and folding event of a core shape that then induced the rest of the tau protein to form a disease fold. Since our focus for this example was 4R tauopathy, we designed a 4R tauopathy-mimicking peptide candidate for nucleating the formation of a minimal 4R tauopathy prion. By using a short peptide with fewer degrees of freedom we aimed to narrow the aggregation pathway towards a specific fold. The core of every 4R tauopathy fibril structure solved to date, including CBD, PSP, AGD, GGT and GPT (the fibrils found in an LNT patient), contains a strand-loop-strand (SLS) motif made of two opposing β-strands connected by a loop and stabilized by intramolecular side chain contacts between hydrophobic residues and / or salt bridge interactions. In 4RLVM Ref.72-24WO; 340383 tauopathies, the SLS motifs include the P301 residue near the loop portion, and SEQ NO.1: (VQIVYK), the hydrophobic hexapeptide motif referred to as PHF6. In all tau fibrils with solved structures, the PHF6 segment is present, but the SLS motif is only present in 4R tauopathy fibrils. AD fibrils contain a mixed population of 4R and 3R tau while the R2 region is not resolved in the cryo-EM structure. (FIG.12B). Heparin induced tau fibrils also contain the PHF6 segment, but generate a heterogenous population of structures that are all distinct from tauopathy fibrils solved to date. Based on this, we selected a 19-amino-acid peptide spanning residues 295-313 that spans the R2 / R3 splice junction of tau and includes the P301 site as a 4R tauopathy-mimicking peptide candidate, referred to herein as “jR2R3” (SEQ ID NO: 2). While the P301L mutation has been reported to open the protective beta hairpin, we posited that this stand-alone peptide would form fibrils that replicate the SLS fold seen in 4R tauopathies. Moreover, we predicted that the resulting fibrils would have seeding competency if jR2R3 adopted the SLS structure, ensuring that an active fibril end interface was maintained that could recruit a naïve tau monomer.

[0261] Example 1 – TauSeeK Platform

[0262] TauSeeK can provide access to pathologic disease-specific fibril through the following workflow: (1) Identification of the desired disease-specific tau fibril; (2) Selection and aggregation of mini-tau peptide and cofactors for synthesis of mini-tau fiber; (3) Use of mini-tau fibril as template to fold tau fibril monomers and proliferate pathologic disease-specific tau fibrils; (4) Structural verification at the molecular level of synthetic tau fibrils and confirmation of prion properties. These steps constitute a robust and reliable method to assemble tau fibrils. The specific details of the which will be described further herein.

[0263] One goal of the TauSeeK Platform is to provide access to prion-like mini-tau fibrils and pathogenic disease-specific tau fibrils to the scientific community for application towards therapeutic, imaging agent, and antibody development. Another goal is to disclose the scientific foundations for the rational design and tuning of synthetic tau fibrils and provide a systematic way to confirm the structure and prion properties of mini-tau peptides and fibrils.LVM Ref.72-24WO; 340383

[0264] Towards goal one, we have designed two individual kits, TauSeeK1 and TauSeeK2, and an associated suite of spectroscopic tools that will be made available to researchers.

[0265] Towards goal two, we have extensively explored methods to identify, design, and tune synthetic tau fibrils. These new developments and the validation of the experimental results have been enabled by a set of NMR and EPR spectroscopy methods that allow for real-time, dynamic, molecular level structural information on the assembly of intrinsically disordered proteins, discussed in detail in Example 2 below. The spectroscopic methods developed here have effectively opened the door to carefully designed tau fibrils owing to the simplicity and speed of data collection and processing.

[0266] Example 1A – TauSeeK1 Platform

[0267] TauSeeK1 is aimed at researchers interested in generating mini-tau fibrils as active seeds for full-length tau monomers. This kit will allow researchers to seed their own tau fibrils based on their needs by following simple procedures for fibrilization and spectroscopic analysis.

[0268] TauSeeK1 is composed of a mini-tau peptide, tuned for a specific tauopathy, which allows for the fibrilization of full-length tau monomers. These mini-tau peptides have been verified to proliferate the conformation of a disease-specific fibril though spectroscopic methods. These kits may contain one or more, optionally all, of the following mini-tau peptides: a mini-tau peptide having 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 2 (DNIKHVLGGGSVQIVYKPV), also referred to herein as “Mini-Tau-1”; a mini-tau peptide having 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity SEQ ID NO: 11 (DNIKHVLGGGSPhosVQIVYKPV), also referred to herein as “Mini-Tau-2”, “jR2R3- pS305”, or “S305PjR2R3-P301L”;LVM Ref.72-24WO; 340383 a mini-tau peptide having 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 12 (DNIKHVLGGGSVQIVYPhosKPV), also referred to herein as “Mini-Tau-3”, “jR2R3- pY310”; or “Y310PjR2R3-P301L”; a “mini-AD chimera” or “mini-AD seed” (e.g., 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 17 (VQIVYKPGGGNHKLTF)), which, in some cases, comprises SEQ ID NO: 1 (VQIVYK), a linking sequence (e.g., GGG), and SEQ ID NO: 3 (HKLTF). a “mini-CBD chimera” or “mini-CBD seed” (e.g., 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 18 (KVQIINKPGGGKLTFRE), which, in some cases, comprises SEQ ID NO: 4 (VQIINK), a linking sequence (e.g., GGG), and SEQ ID NO: 3 (HKLTF).

[0269] For mini-tau fibril synthesis, a researcher selects a given mini-tau peptide based on the specific tauopathy of interest and follows a simple workflow for the generation of synthetic tau fibrils, as depicted in FIG.1. The overall workflow shown in FIG.1 is as follows: (1) Mini-tau peptide is selected based on the desired disease- specific fibril. (2) Mini-tau fibrils are synthesized according to the Mini-Tau Fibril Synthesis Procedure. Prion property of mini-tau fibrils is confirmed using molecular-level spectroscopy. (3) Purified fibrils are sonicated to reveal more seeding active fibril termini. (4) Pathogenic tau fibrils are synthesized according to the Full-Length Tau Fibril Synthesis Procedure, and uniform shape and prion properties are confirmed using molecular level spectroscopy. (5) Uniform pathogenic tau fibrils are synthesized through a generational seeding strategy according to the Generational Seeding Procedure. Each of these procedures may be performed according to the examples and “Exemplary Experimental Aspects for Examples 1 and 2” below.

[0270] Example 1B – TauSeeK2 Platform

[0271] TauSeeK2 is aimed at researchers interested in obtaining synthetic tau fibrils with the same structure as a disease-specific pathogenic tau fibril. This kit will allow researchers to utilize the tau fibrils for the development of tauopathy therapeutics,LVM Ref.72-24WO; 340383 imaging agents, and other critical technologies for the identification and treatment of tauopathies.

[0272] TauSeeK2 comprises pathogenic tau fibrils synthesized from full-length tau monomers and appropriate active seeds. The full-length tau monomers seeded from the mini-tau fibrils of TauSeeK1 may include one or more, optionally all, of the following: a full-length pathogenic tau monomer (e.g., tau isoform, 0N4R (UniProt accession number (P10636-8)) having the pathogenic P301L substitution); a full-length wild-type tau monomer (i.e., a tau isoform without the pathogenic P301L substitution); a tau protein fragment having the pathogenic P301L substitution (e.g., tau187 (SEQ ID NO: 13 (NVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKP VDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKI ETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLAD EVSASLAKQGL)); a tau protein fragment without the pathogenic P301L substitution (e.g., (SEQ ID NO: 23 (NVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVLGGGSVQIVYKP VDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKI ETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLAD EVSASLAKQGL)) a spin labeled full-length pathogenic tau fibril, or a fragment thereof; and an isotope labeled full-length pathogenic tau fibril, or a fragment thereof.

[0273] The goal of TauSeeK2 is to provide biological debris-free pathogenic tau fibrils to researchers who will develop therapeutics and imaging agents for tauopathies. As a result, the kit may be designed for researchers with no expertise in structural biology and to be as straightforward as possible. In aspects, the only requirement for the utilization of TauSeeK2 is knowledge of the disease-specific fibril so that an appropriate active seed and monomer may be chosen to create a relevant pathogenic tau fibril.LVM Ref.72-24WO; 340383 Outside of this, there is no synthetic overhead for the end user in this aspect. In this example, all the synthetically generated tau fibrils have their structure and prion properties confirmed using molecular-level spectroscopy, such as DEER. This confirmation allows for the reproducible implementation of TauSeeK2 in complex research environments.

[0274] Example 2 – Design Principles and Structural Verification

[0275] To improve the implementation of mini peptide based active seeds in research communities there are two requirements that must be addressed by the TauSeeK platform. Firstly, the mechanistic impact of design choices must be well understood. This mechanistic understanding allows for the rational design of mini-peptides that will ultimately allow for the generation of various tauopathy disease-specific fibrils. Secondly, there must be a method that is real-time and provides molecular level structural information for the characterization of synthetic tau fibrils. This structural characterization will confirm that the mini-tau peptide active seeds allow for structural propagation of full-length tau monomers and the formation of homogenous fibril populations that will be of great value to tauopathy researchers.

[0276] Example 2A – Design of Mini-Tau Seeds

[0277] Identification of Tau Protein Monomer Critical Fold. Identification of the critical protein fold of tau monomers in each disease-specific tau fibril is required for the rational synthesis of mini-tau peptides. FIG.2 demonstrates how mini-tau peptides may be rationally designed to mimic the critical protein fold of tau fibril monomers in corticobasal degeneration (CBD) and progressive supranuclear Palsy (PSP). Identification of a strand-loop-strand (SLS) structure within each tau fibril monomer guides the selection of the peptide sequence for mini-tau fiber synthesis. The highlighted sections of the tau fibril monomers correspond to an 18-residue polypeptide that spans the R2 / R3 section of full-length tau. These mini-tau peptides crucially contain the P301 residue, the hydrophobic, fibrilization prone PHF6 motif, and a counter strand which may stabilize a variety of U-shaped folds that form an SLS structure.

[0278] For mixed 3R4R tauopathies, there is a different pinning site than 4R tauopathies that propagates the disease-specific protein critical fold. This pinning siteLVM Ref.72-24WO; 340383 still retains the SEQ ID NO: 1 (VQIVYK) PHF6 motif, but instead of a U-shaped fold made of the PHF6 and an adjacent counter strand, a more remote peptide sequence acts as the stabilizing counter strand. As a result, a mini-tau peptide that attempts to replicate this pinning site preferably contains the PHF6 region, a linker, and the counter strand. Based on these preferences, we have developed both a mini-AD seed and a mini-CBD seed that contain the PHF6 region, a short linking sequence, and the stabilizing counter strand. This construction effectively recreates the critical fold of tau in AD or CBD disease-specific fibrils.

[0279] Design and Tuning of Mini-Tau Peptide. To further enforce the desired SLS conformation and improve the aggregation of mini-tau peptides to form mini-tau fibrils, site-specific mutations, and post-translational modifications (PTMs) may be incorporated into the mini-tau peptides (FIG.3). Mutation of residue 301 from proline to leucine further enforces the SLS conformation of the mini-tau peptide, corresponding to SEQ ID NO: 2 (FIG.3 (left), mini-tau 1). Additionally, phosphorylation of residues on the mini-tau peptide can influence the conformation of the SLS motif and result in mini-tau fibrils with different morphologies (FIG.3 (middle), mini-tau 2 and FIG.3 (right), mini-tau 3).

[0280] Even when a nucleating peptide core is identified, how these peptides can associate in such a highly concerted fashion, only stacking strictly in register, and forming highly organized fibrils with complex folding architecture is unclear. We have identified a pinning site on any amyloid protein that has prion-like activity so that the tau monomer can preferentially bind to the fibrillar surface along the same site and stack in register and parallel to fibrils. We discovered that in case of tau that site P301L is such a location. Using a combination of experimental and computational techniques, we found that P301L harbors a more structured, slower-diffusing hydration water layer.

[0281] Alterations to the hydration water create an entropic driving force that allows P301L to easily dewet and initiate the aggregation of tau monomers. In the scenario where the mini-tau fibrils serve as prions to recruit full-length tau monomers, the fibril end surface harbors the hyperlocalized hydrophobic site near P301L and facilitates the in-register association with longer tau monomers (FIG.4A). Importantly, we believe that this is a universal characteristic of amyloid fibrils with in-register stacking and seeding competency. Critically, we have shown that PTMs and mutations can dramatically shiftLVM Ref.72-24WO; 340383 the pinning site which position has consequences for fibrillar templating. For example, phosphorylation of site 305 of tau results in the moving of the pinning site from 301 to 305, and results in highly fortified fibril (a portion of the result is shown in FIG.4B). The pinning sites for a given tau isoform can be identified by ODNP (data shown in FIG.4B and quantified in FIG.4C) and solution state NMR through a 2D HSQC.

[0282] To identify aggregation competent mini-tau peptides that will form high-fidelity seeds, high throughput screening is necessary to inform the design and tuning of the peptide sequence. A variety of mini-tau peptide sequences with post-translational modifications and point-mutations can be examined and the extent of fibrilization quantified using ThT staining fluorescence microscopy. Desirable mini-tau peptides preferably demonstrate rapid aggregation under the given conditions that ideally will translate to rapid, in-register aggregation of full-length tau monomers. Once ideal mini- tau peptides have been synthesized, the global morphology of the fibril can be visualized by nsTEM (FIG.5).

[0283] Through the high throughput workflow described above, we have identified distinct mini-tau peptides: SLS1 (jR2R3), mini-tau 1 (jR2R3-P301L), mini-tau 2 (jR2R3- pS305), and mini-tau 3 (jR2R3-pY310). Additionally, it is believed the technology described above constitutes marked improvements to the rational design and utility of these mini-tau peptides through incorporation of one or more of PTMs, the use of complement strands, and the tuning of aggregation conditions guided by DEER spectroscopy. These methods are believed to have allowed for the synthesis of mini-tau fibrils with higher populations of desired disease conformers that can effectively seed full length human tau.

[0284] Development of Mini-Tau Peptide Aggregation Conditions. Once a viable mini-tau peptide with incorporated PTMs has been identified, complementary peptide strands can further enforce the desired tau fibril monomer conformation in the mini-tau peptides. This may be achieved through intramolecular hydrogen bonding between the mini-tau peptide and the complement strand that further enforces the desired critical protein fold of the mini-tau peptide. It is believed that modification of the complement strand can affect the same mini-tau peptide in distinct ways. Specifically, combinations of jR2R3-P301L mini-tau peptide and complement strands result in different folding ofLVM Ref.72-24WO; 340383 the jR2R3-P301L mini-tau peptide. Careful design of complement strands has allowed for the selective synthesis of mini-tau fibrils that are specific to a given tauopathy.

[0285] In this example, the complement strands have been designed based on identification of intramolecular hydrogen bonds in the solved cryoEM structures for different disease-specific tau fibrils. These complementary peptides were designed to mimic the outer β-sheet of tau monomers in disease-specific tau fibrils and therefore reinforce the desired U-shaped conformation of the mini-tau peptide. In this example, complement strands were synthesized for CBD (SEQ ID NO: 14 (GGGQVEVKSEKLDFKDRVQSKIGSLDNITH)), PSP (SEQ ID NO: 15)), and GPT (SEQ ID NO: 16 (FKDRVQSKIGSLDNITHVPG)). FIG.6 shows the tau monomer conformation in CBD, PSP, and GPT (FIG.6, left, middle, and right, respectively). The models of the tau protein shown in FIG.6 highlight both the SLS motif and the outer β-sheet that enforces the U-shaped structure. Based on these monomer conformations, we have designed three complement strands to enforce the same SLS motif in the SLS1 mini-tau peptide. The CBD complement (SEQ ID NO: 14 (GGGQVEVKSEKLDFKDRVQSKIGSLDNITH)), PSP complement (SEQ ID NO: 15 (LDFKDRVQSKIGSLDNGGGHKLTFRE)), and GPT complement, and the computed lowest conformations of SLS1 and the corresponding complement strand are shown in FIG.6. The PSP complement (SEQ ID NO: 15 (LDFKDRVQSKIGSLDNGGGHKLTFRE)) was design to include a linking sequence, thus the resulting sequence corresponds to a single peptide that is a modification from the actual PSP sequence. The design of the PSP complement strand was intended to mimic the disease conformation that wraps around the 19 amino acid SLS core. There is a section in PSP that does not wrap around the SLS core, and therefore was excluded from the PSP complement strand, thus reducing the size the complement strand with a higher specificity to better interact with the SLS and match the alignment.

[0286] Based on these computed conformations, SLS1 adopts different conformations based on the identity of the complement strand, which results in a different mini-tau fibril topology.

[0287] Combinations of mini-tau peptides, complements and cofactors can similarly be screened though ThT fluorescence microscopy and nsTEM in a similar manner to theLVM Ref.72-24WO; 340383 initial peptide sequence screen. This will allow for the rapid development of conditions for forming mini-tau fibrils with homogenous morphologies. We have found that the addition of salts (e.g., NaCl or MgCl2) or the use of different buffers has an impact on the rate of aggregation and the final structure of the mini-tau fibrils. As a result, the high throughput application of these methods is applicable to the development of effective aggregation conditions.

[0288] Example 2B – Synthesis of Pathogenic Tau Fibrils

[0289] Seeding of Tau Monomers with Active Seeds. Mini-tau fibrils may act as an active seed for full-length human tau protein aggregation, and therefore, full-length tau fibrils can be constructed from monomeric tau protein and an active seed. This allows for a fully synthetic pathogenic tau fibril, free of biological debris for use in research applications.

[0290] The mini-tau fibrils in this invention are competent for seeding of full-length tau protein following the Full-Length Tau Fibril Synthesis Procedure, described below. With the addition of different cofactors and salts, different disease-specific tau fibrils may be synthesized from the same mini-tau fibril seed and full-length tau monomers. In this example, we have demonstrated the successful aggregation and fibrilization of tau protein 0N4R (UniProt accession number (P10636-8)) and tau 187 protein fragment (SEQ ID NO: 13) with fibrils constructed from the jR2R3-P301L (SEQ ID NO: 2) and jR2R3 mini-tau peptide (SEQ ID NO: 5 (DNIKHVPGGGSVQIVYKPV)). The jR2R3- P301L (top line, FIG.7) forms active seeds that display a much higher rate of monomer fibrilization than the jR2R3 mini-tau peptide (bottom line, FIG.7), which suggests the importance of mini-peptide design and the effects that the one-dimensional structure of the TauSeeK peptides may have on the formation of mini-tau peptides and pathogenic tau fibrils.

[0291] Generational Homogenization of Pathogenic Tau Fibrils. Due to the prion-like properties of these tau fibrils, they may also act as active seeds for the aggregation of more full-length tau monomer. This allows for generational seeding of tau monomers with synthetic tau fibrils. Once a tau fibril has been synthesized, they may be used as seeds for fibrilization of tau monomers by sonicating the fiber and performing the tau fibril synthesis with these seeds. The advantage of this method is that subsequentLVM Ref.72-24WO; 340383 generations of tau fibrils made in this way demonstrate a more uniform shape throughout the fibril populations (FIG.8). This self-seeding strategy allows for the generation of homogenous samples of disease-specific tau fibrils which will be of value to the scientific community.

[0292] Example 2C – Verification of Fibril Synthesis

[0293] To verify that a given mini-tau peptide is competent for the generation of pathogenic disease-specific tau fibrils, mini-tau peptides may be confirmed to form homogeneous mini-tau fibrils and the ability of the mini-tau fibrils to enforce a given conformation of the monomer may be assessed. Confirmation of these two qualities of a mini-tau peptide is preferable to support the reproducibility of the invention described above. One possible method to assess both the homogeneity of the mini-tau fibrils and the ability to aggregate full-length tau in a conformer specific manner is Double Electron- Electron Resonance (DEER) spectroscopy. We have developed a set of tests that allow for the confirmation of the prion-like properties of mini-tau peptides and mini-tau fibrils for use in different research settings.

[0294] DEER-Guided Aggregation Development. It is preferable that the morphology of the mini-tau fibrils be well defined and uniform to allow for reliable aggregation of full- length tau monomers. To verify the formation of a single population of mini-tau fibrils, we have developed DEER spectroscopy methods that allow for real-time analysis of the homogeneity of our mini-tau fibrils. We have prepared a variety of doubly spin-labelled mini-tau peptides and obtained data for the distance distributions between the spin labels under a variety of different aggregation conditions (FIG.9). With this data, it is possible to reliably verify the morphology of a mini-tau fibril formed under various aggregation conditions. DEER can evaluate whether amorphous or well-defined fibrils are forming, and whether the target intra-molecular distance distribution, P(r), is emerging and being amplified with the aggregation conditions chosen (FIG.9). This allows for molecular level verification of the structures of the mini-tau fibrils, which provides a way to ensure that the synthetic mini-tau fibrils are uniform and are propagating the desired disease-specific critical conformation.

[0295] Spectroscopic Verification of Pathogenic Tau Fibrils. DEER can also be used to identify and tune the conditions for tau monomer aggregation. It is preferable to haveLVM Ref.72-24WO; 340383 tracking tools to probe how changes in pH, temperature, salt concentration or cofactors and seed affect overall fibril structure. In this way, DEER allows for the rational design of aggregation conditions for full-length tau monomers. many peaks and which distance region are present. FIG.10 shows the probability distribution for four distinct tauopathy fibril monomers, which serves as a fingerprint for identifying each disease-specific tau fibril. Tau 187 with spin labels at sites 351 and 373 can allow the aggregation of tau monomers to be monitored in real-time using DEER spectroscopy. In FIG.10 (left), there are three population distributions for three separate aggregation conditions of full- length tau, using CBD seeds, PSP seeds, and heparin as aggregation inducers. For these three aggregation conditions, there are distinct population distributions that can be compared in an automated fashion.

[0296] Real time structure screening does not require perfect signals but more an assessment of how many peaks is present and in which distance region. Even with this limited requirement, it is not easy to objectively access and compare imperfect PDS signal of difficult biological systems, such as tau, because DEER of heterogenous fibrils usually results in convoluted ^(^), encompassing multiple populations of various shapes (FIG.10, left) for cell lysate-seeded fibrils. For such samples, P(r) cannot be reliably extracted often due to insufficient length of the DEER time domain signal or poor SNR. We have developed a novel frequency pattern recognition method using discretized continuous wavelet transform (CWT) with structure similarity index measure (SSIM) analysis that will compare PDS time domain signals through their frequency composition. By comparing the frequency information directly, this method eliminates errors from transforming DEER time domain data to P(r); therefore, allowing the most robust, sensitive, and accurate comparison. By calculating the CWT, we can distinguish between the DEER signal, noise, and artifacts from the experiments by decomposing the DEER signal into different frequency components, thereby allowing a much more detailed comparison of the data than visually comparing the raw ^(^) and the extracted ^(^). By utilizing the SSIM analysis, we can further compare the CWT of the DEER data because SSIM provides errors based on 1) perception as it accounts for the structural and textural information in addition to the magnitude of errors, and 2) saliency by giving more importance to the significant parts of the structures. This will enable theLVM Ref.72-24WO; 340383 comparison of imperfect PDS data of heterogeneous fibril structures, and other difficult biological samples.

[0297] Frequency Pattern Recognition for PDS can differentiate practically identical DEER signals and localize small differences. First, the DEER time traces are decomposed into frequency components by CWT. The calculated CWT will be converted into spectrograms measuring the range of frequencies present along the DEER trace. These spectrograms were compared using SSIM analysis by 1) numerical SSIM index, where SSIM index < 1 denotes a meaningful difference in frequency profile and by 2) similarity gradient plots, where the location with frequency differences can be visually identified. An example is the comparison of the DEER data of disease seeded and heparin induced fibrils. By comparing P(r) and the raw DEER data, we can clearly differentiate heparin induced fibrils from seeded fibrils, while the progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) seeded fibrils look similar (SSIM 0.93). However, the similarity gradient shows differences in the higher frequency region (i.e., short distances region), validating the P(r) difference (FIG.10, right). Using this pattern recognition technique, we can validate similarity between replicates to show reproducibility of each condition and can validate differences between different aggregation conditions. This may effectively allow researchers to confirm the morphology of synthetic tau fibrils.

[0298] Exemplary Experimental Aspects for Examples 1 and 2:

[0299] Mini-Tau Fibril Synthesis Procedure and Materials (without cofactors or complement strands): Peptides obtained from Genescript, with uncapped termini, were dissolved in ddH2O and stored immediately as 100 μM aliquots at −80 °C. The aggregation process was carried out in 50 mL Eppendorf tubes. The stock peptide solution was diluted to a concentration of 50 μM using 20 mM HEPES, pH 7.4. The aggregation took place under continuous shaking at 200 rpm in a 37 °C incubator over 24 hours. The resultant fibrils were isolated using a 50 kDa cutoff concentrator and rinsed three times with MilliQ water to remove excess heparin and unaggregated peptide. The fibrils were then lyophilized overnight using a FreeZone 2.5 L, −84 °C Benchtop Freeze Dryer. Upon quantification of the fibril mass, the seed stock is reconstituted to 1 mM using 20 mM HEPES, pH 7.4, and stored at −80 °C.LVM Ref.72-24WO; 340383

[0300] Mini-Tau Fibril Synthesis Procedure and Materials (with cofactors and complement strands): Peptides obtained from Genescript, with uncapped termini, were dissolved in ddH2O and stored immediately as 100 μM aliquots at −80 °C. The aggregation process was carried out in 50 mL Eppendorf tubes. The stock peptide solution was diluted to a concentration of 50 μM using 20 mM HEPES, pH 6.4. A salt cofactor was added (NaCl or MgCl2) at a concentration of 50 mM or 200 mM.200 μM heparin (average molecular weight 16 kDa) was added to achieve about a 4:1:10 ratio of mini-tau peptide:heparin:complement strand; however, other ratios were tested and shown to be effective, for example, 4:1:16. Other ratios include, but are not limited to, 4:1:12, 4:1:14, 4:1:16, 4:1:18, 4:1:20, 4:1:30, 2:1:10, 2:1:12, 2:1:14, 2:1:16, 2:1:18, 2:1:20, and 2:1:30 (including any sub-ratio therein) of mini-tau- peptide:heparin:complement strand. The aggregation took place under continuous shaking at 200 rpm in a 37 °C incubator over 24 h. The resultant fibrils were isolated using a 50 kDa cutoff concentrator and rinsed three times with MilliQ water to remove excess heparin and unaggregated peptide. The fibrils were then lyophilized overnight using a FreeZone 2.5 L, −84 °C Benchtop Freeze Dryer. Upon quantification of the fibril mass, the seed stock is reconstituted to 1 mM using 20 mM HEPES, pH 7.4, and stored at −80 °C.

[0301] Full-Length Tau Fibril Synthesis Procedure: Mini-tau fibrils, either obtained from TauSeeK1 or prepared previously were sonicated to reveal active seeding termini. The aggregation process was carried out in 50 mL Eppendorf tubes. The stock fibril solution was diluted to a concentration of 50 μM using 20 mM HEPES, pH 6.4 with no additional salt additives. About a 1:1 molar ratio of mini-tau fibril:tau monomer was aggregated at 4˚C and the reaction was allowed to occur over two weeks to ensure full aggregation. Different ratios of mini-tau fibrils to monomer may be used, with varying impacts on the rate of aggregation. For example, a 4:1 molar ratio of mini-tau fibril: tau monomer may be used with little to no change to the observed morphology; however, there may be a lower rate of aggregation. Other molar ratios include, but are not limited to, 2:1, 3:1, 5:1, 10:1, 20:1, 30:11:2, 1:3, 1:4, 1:5, 1:10, 1:20, and 1:30 (including any sub-ratio therein) of mini-tau fibril:tau monomer. The resultant fibrils were isolated and rinsed three times with MilliQ water to remove excess heparin and unaggregated monomer. The fibrils were then lyophilized overnight using a FreeZone 2.5 L, −84 °CLVM Ref.72-24WO; 340383 Benchtop Freeze Dryer. Upon quantification of the fibril mass, the seed stock is reconstituted to 1 mM using 20 mM HEPES, pH 7.4, and stored at −80 °C.

[0302] ThT Assay: All experiments were performed with a BioTek Synergy 2 fluorescent plate reader. In each well the mini-tau peptide (50 μM), ThT (20 μM), and heparin (12.5 μM) in buffer were distributed into a 384-well plate (Corning low volume non-binding surface black with clear flat bottom) to a total volume of 30 μL. The plate reader temperature was set to 37 °C and allowed to equilibrate, the samples were shaken under the programmed high shaking speed in between measurements. ThT fluorescence intensity was measured at (excitation=440 nm, emission=485 nm) every 2 minutes until a plateau was reached. These experiments were done in triplicate at least three times with independent samples.

[0303] TEM Assay: For transmission electron microscopy (TEM) analysis, five μL of fibril samples were applied to a glow-discharged copper grid (Electron Microscopy Science, FCF-200-Cu) for 20 s and blotted dry with filter paper. Samples were stained with 5 μL 1.5 w / v % uranyl acetate and immediately blotted dry. An additional 5 μL of uranyl acetate was added for 60 seconds and blotted dry. Samples were analyzed using a Thermo Scientific Talos G2200X TEM / STEM microscope operated at 200 kV and room temperature. Grids were then imaged with a Ceta II CMOS 4k x 4k camera at the indicated magnifications.

[0304] DEER Spectroscopy: DEER signal was collected for 12-24 hours until an optimal SNR was achieved. All DEER time traces were transformed into distance distributions using DeerLab software package for Python. The time traces were phase corrected and truncated by 300 ns to remove possible "2+1"-artifact. One-step analysis was done using the DeerLab fit function with the following models: ex-4deer model with t1, t2, and pulse length set to experiment parameters, bg_strexp model with the stretch parameter freeze to 3 (for soluble protein) and bg_homfractal model with the fractal dimensionality set to fit between 1 to 3 (for fibril samples), and dipolarmodel using Tikhonov regularization. The uncertainty analysis was done using bootstrapping method with 100 samples. The time domain fitting results are presented both as fitting to the primary data and the distance distribution fits are presented with 95% confidence intervals. samples were corrected using spectra collected of the singly labelled speciesLVM Ref.72-24WO; 340383 (jR2R3-(P301L)-314C) to account for the shift in geometric dimensions. The DEER experiments were performed with a pulsed Q-band Bruker E580 Elexsys spectrometer, equipped with a Bruker QT-II resonator and a 300 W TWT amplifier with an output power of 20 mW for the recorded data (Applied Systems Engineering, Model 177Ka). The temperature of the cavity was maintained at 65 K using a Bruker / ColdEdge FlexLine Cryostat (Model ER 4118HV-CF100). The bridge is equipped with an Arbitrary Wave Generator to create shaped pulses for increased sensitivity. The samples were made in D2O buffers with 30 % (v / v) deuterated glycerol (used as the cryoprotectant). To perform an experiment, approximately 40 μL of sample was added to a 3 mm OD, 2 mm ID quartz capillary and flash frozen in liquid nitrogen to preserve sample conformations.

[0305] Example 3 – N-terminal Segment Preceding the PHF6 motif

[0306] We selected a 19-amino-acid peptide spanning residues 295-313 of 4R tau that we henceforth refer to as jR2R3 (SEQ ID NO.5: (DNIKHVPGGGSVQIVYKPV)) that spans residues 295-313, the R2 / R3 splice junction, of tau which includes the 301 site, the fibrilization prone PHF6 motif and an N-terminal segment that we posit serves as an intramolecular counter-strand. There are many possible variations of this peptide that can adopt a characteristic fold to stack to SLS fibrils in 4R tauopathies, but we start with 295-313 because we believe it is a common region of tau that folds and stacks to a salt bridge-stabilized SLS motifs. Six different variants and fragments of the jR2R3 tau peptide were synthesized. They include the jR2R3 peptide (SEQ ID NO.5), the P301L mutant, jR2R3-P301L (SEQ ID NO.2), the N-terminal half (295-303) of the peptide, jR2∆ (SEQ ID NO.6 (DNIKHVPGG)), the N-terminal half (295-303) of the peptide, jR2∆ P301L mutant, jR2∆-P301L (SEQ ID NO.7 (DNIKHVLGG)), and the C-terminal half (300-313) of the peptide including the 301 site, i.e. j∆R3 (SEQ ID NO.8 (VPGGGSVQIVYKPV)) and j∆R3-P301L (SEQ ID NO.9 (VLGGGSVQIVYKPV)) (FIG. 12A).

[0307] To test aggregation capabilities, each peptide was incubated at a 4:1 peptide:heparin molar ratio (15kDa average MW, Galen Labs, HEP001) in a 50µM peptide solution. With the addition of heparin, all fragments containing the PHF6 segment, i.e. jR2R3, jR2R3-P301L, j∆R3 and j∆R3-P301L) form fibrils according to ThTLVM Ref.72-24WO; 340383 and negative stain TEM (nsTEM) (FIGs.12C and 12D). Neither of the N-terminal half constructs (jR2∆ and jR2∆-P301L), each lacking the PHF6 motif, showed ThT fluorescence intensity, nor were fibrils observed in nsTEM. Because neither jR2∆ nor jR2∆-P301L showed indications of aggregation, we concluded that the N-terminal half of jR2R3 did not have aggregation propensity, and that PHF6 facilitated aggregation.

[0308] Fibrils made of full length jR2R3 and jR2R3-P301L showed significant differences in ThT fluorescent intensity (p=2.3*10-6). In contrast, while there was no significant difference in the ThT fluorescence between the fibril made of the C-terminal half peptides, j∆R3 and j∆R3-P301L. The complete 19-residue peptide with the P301L mutation, jR2R3-P301L, showed significantly greater ThT fluorescence intensity compared to the C-terminal half of this peptide, j∆R3-P301L, suggesting the N-terminal half of jR2R3-P301L contributes significantly to its fibril forming propensity. The chemical nature of leucine that replaces proline in the P301L mutation of jR2R3-P301L allows for one additional backbone hydrogen bond to stabilize the cross-b-sheets in amyloid fibrils, which might be a significant stabilizing contributor to the resulting fibrils. However, no enhancement was observed in the quantity of j∆R3-P301L over j∆R3 fibrils (FIG.12C). We therefore conclude that the effect of P301L is more complex than the addition of an extra H-bond. The pronounced enhancement of aggregation by the P301L mutation only observed in the full jR2R3-P301L, but not in the two C-terminal half peptides, suggests that the P301L mutation alters the relationship between the aggregation-inducing C-terminal segment 300-313 and the N-terminal segment 295- 300. These results support our hypothesis that jR2R3-P301L as a stand-alone peptide folds into aggregation-competent conformations, stabilized in strand-loop-strand fibrils, even without the rest of the tau protein sequence.

[0309] The heparin-induced aggregation of longer 4R tau, including full-length 0N4R tau (UniProt accession number (P10636-8)) and tau187 (residues 255 – 441 of the longest tau isoform, SEQ ID NO: 13), each containing the P301L mutation, produces a heterogeneous tau fibril population that does not include a strand-loop-strand motif (FIG. 12B). Fibrils made of jR2R3 peptides without heparin appeared less uniform, less straight, and did not show the characteristic PHF appearance compared to heparin- induced jR2R3-P301L fibrils according to nsTEM, Because the purpose of this exampleLVM Ref.72-24WO; 340383 is to produce homogeneous tau peptide fibrils with prion properties, we proceeded to assess jR2R3 fibrils induced by heparin for the remainder of the work.

[0310] Example 4 – jR2R3 Fibrils for Seeding Fibrilization of 4R Tau

[0311] Once the fibril-forming capacity and condition was established for jR2R3 and jR2R3-P301L, the competency of the resulting fibrils to recruit naive tau to seed the formation of new fibrils was tested. We first tested whether jR2R3 and jR2R3-P301L fibrils are competent for recruiting peptide monomers of their own kind. The jR2R3- P301L fibrils are more competent seeds than jR2R3 fibrils---the lag time is shorter and the ThT fluorescence amplitude is greater. jR2R3-P301L fibrils are competent seeds, also for recruiting jR2R3 monomers, but not as efficient as for recruiting jR2R3-P301L monomers. In other words, the jR2R3-P301L fibrils have self-seeding competency, implying that heparin is diluted out in fibrils generated by multiple rounds of seeding.

[0312] Next, we tested whether jR2R3 fibrils exert prion-like seeding properties onto longer tau proteins. The competency of jR2R3 fibrils as “mini prions” was evaluated by adding them to tau187 monomers (SEQ ID NO: 13), an N-terminus truncated hTau40 spanning residues 255-441, in a 1:20 seed:monomer molar ratio. Both jR2R3 and jR2R3-P301L fibrils showed seeding competency in recruiting naïve (i.e., WT) tau187 monomers (SEQ ID NO: 13) (FIG.20). Notably, jR2R3-P301L fibril seeds induced aggregation of tau187 with a shorter lag time and reaching more quickly a maximum fluorescence intensity compared to jR2R3 fibril seeds. The ability of the 19-residue jR2R3-P301L peptide fibrils to recruit 4R tau that is 10 times its length implies that the jR2R3-P301L filament ends (either both or one) serve as potent templates to induce misfolding of soluble tau (of either WT or P301L) to adopt aggregation-competent conformations and states. The faster kinetics of jR2R3-P301L fibril-seeded aggregation, in comparison to jR2R3 seeds of the same quantity, corroborates the concept that the conformation of the tau fold adopted in the strand-loop-strand fibril structure lies at the core of the templating competency of jR2R3-P301L fibrils.

[0313] Next, the capability of jR2R3-P301L fibrils to seed aggregation of tau expressed in a cellular environment was tested. Purified fibrils were transfected into H4 neuroglioma cells expressing fluorescently labeled tau187. The formation of puncta in cells was used as a measure of seeding capabilities of tau. Cells were imaged after 24LVM Ref.72-24WO; 340383 hours, and jR2R3 was able to seed cells more than jR2R3. In a companion paper published recently, PNAS 2024 Vol.121 No.15 e2320456121, which is hereby incorporated by reference in its entirety for all purposes, the jR2R3-P301L seeds have been shown to selectively propagate 4R tauopathy, but not 3R tauopathy properties in a 3R-mimicking cell line, establishing isoform selective prion competency of jR2R3-P301L fibrils.

[0314] Example 5 – jR2R3-P301L Fibrils are more Stable than jR2R3 Fibrils

[0315] To compare the stability of the jR2R3 and jR2R3-P301L fibrils, we performed a guanidinium hydrochloride (GdnHCl) denaturation assay. Fibrils were incubated with GdnHCl at concentrations ranging from 100 mM to 1 M, and ThT fluorescence measured after equilibration. A decrease in fluorescence was observed in a GdnHCl concentration-dependent manner for all peptide fibrils. More stable fibrils resist denaturation under more aggressive conditions, so a more rapid decrease in ThT fluorescence with increasing GdnHCl concentration indicates a less stable fibril. All values were normalized to the fluorescence of the corresponding fibrils before denaturation (FIG.12E). At the highest tested GdnHCl concentration (1 M), all peptides exhibited greater than 99% loss of fluorescence. The AFM images of jR2R3 and jR2R3- P301L fibrils subjected to 1 M GdnHCl treatment showed a monomer-like peptide film, indicating a complete breakdown of the filaments. At 500 mM GdnHCl concentration, the peptide fibrils showed clear differences with jR2R3-P301L fibrils experiencing a net loss of fluorescence intensity of 61% ± 13%, and the jR2R3 fibrils of 92% ± 1%. The AFM of jR2R3 peptide fibrils in 500 mM GdnHCl showed a lower total fibril density, a reduction in fibril length and quantity than jR2R3-P301L (FIG.12F).

[0316] The fibrils of j∆R3 and j∆R3-P301L had similar stabilities to each other but were less stable than the fibrils made of the longer jR2R3-P301L peptides; all fluorescence was lost at GdnHCl concentrations greater than 250 mM. If there was a stand-alone local effect originating from the 301 site, one would expect the enhanced aggregation induced by P301L to be observed in the shorter peptides as well, but no difference in the stability between j∆R3 and j∆R3-P301L fibrils was detected. This result again indicates that the P301L mutation allows jR2R3-P301L to form more stable fibrilsLVM Ref.72-24WO; 340383 than jR2R3 by orienting the N-terminus as a stabilizing flanking region within the jR2R3- P301L fibrils.

[0317] Example 6 – jR2R3-P301L Fibrils Display Helical Filament Morphologies

[0318] To qualitatively evaluate the morphology and quantity of the amyloid fibrils formed, negative stain TEM (nsTEM) was used to visualize the fibrils (FIG.12D). Filamentous aggregates were observed with peptides jR2R3, jR2R3-P301L, j∆R3 and j∆R3-P301L after incubation with heparin for 18 hours. The jR2R3 and jR2R3-P301L constructs form fibril populations with distinctly different morphologies compared to that of the half peptides. jR2R3-P301L form longer and more well-defined fibrils than any other tau peptide variant. Multiple morphologies were observed within the same jR2R3- P301L fibril populations (FIG.16), with the most prevalent one involving helical filaments with a width of 40-80 Å and a crossover length of approximately 700 Å (FIG.12D). Other morphologies of jR2R3-P301L fibrils include straight, ribbon-like, or bundled filaments (FIG.16). Fibrils made of jR2R3 were less abundant than jR2R3-P301L, and a straight filament morphology was observed more frequently than PHF filaments (FIG.12D). Notably, the C-terminal-only peptides j∆R3 and j∆R3-P301L formed narrow, highly tortuous fibrils with similar morphology to each other (FIG.12D), corroborating our earlier finding that the P301L mutation does not influence the fibrilization path of these half-peptides in the absence of the N-terminal counter-strand. AFM was also conducted to get better statistical information and found similar results: jR2R3 and jR2R3-P301L were forming distinctly different morphologies and quantities of fibrils (FIG.12F). Specifically, jR2R3-P301L fibrils are more abundant, and appear longer, thicker and better defined.

[0319] Example 7 – jR2R3-P301L Peptides Adopt Fold Consistent with Strand- Loop-Strand Fibril Structure

[0320] We next tested the hypothesis that the jR2R3 fibrils adopt a fold that forms a strand-loop-strand fibril structure. For this, single particle analysis cryogenic Electron Microscopy (cryo EM) was attempted on vitrified samples of jR2R3-P301L and jR2R3 fibrils. However, the jR2R3 fibrils lacked the homogeneity and helical symmetry needed to produce high-quality 2D classes from cryo EM data. In contrast, jR2R3-P301L fibrils shows a homogeneous PHF morphology, suggesting that they adopt well-defined foldsLVM Ref.72-24WO; 340383 that lead to greater stability than the jR2R3 form. The jR2R3-P301L fibrils were subjected to helical reconstruction using RELION. As a result, a 3D EM map was resolved with an estimated resolution of 3.0 Å according to the Fourier shell correlation (FSC) curve (FIG.13C, FIGs.18A-18E). This EM map displays a filament core with a 21-screw symmetry that includes a pair of protofilaments (FIG.13D). Each protofilament is composed of two jR2R3-P301L peptide chains. The outer chains adopt a strand-loop- strand structure, as hypothesized, while the inner chains adopt a more extended conformation wrapping around the exterior of the strand-loop-stranded chain (FIGs.13D and 13E).

[0321] The interface of the protofilaments lies between302GGG304of both inner chains. The two inner chains further stabilize the protofilament interface through intermolecular hydrophobic interactions across sites L301 of one chain, and V306 of the opposite chain. The interface between the inner (extended) and outer (SLS) chains of the protofilament are stabilized by an H-bond between site S305 of the extended chain and G303 of the SLS chain, and by a 3-residue bridge between S305 and Q307 of the SLS chain and Q307 of the extended chain. The SLS-shaped outer chain adopts the closest conformation to the GPT fibrils (SEQ ID NO: 10 (DNIKHVPG)) (RMSD: 1.00 Å), but also has a global RMSD most similar to CBD (RMSD: 2.75 Å) (Table 1). In addition to RMSD, the sidechain orientations of the tauopathy SLS folds were compared to those of jR2R3-P301L fibers. Sidechains were characterized as either internally or externally oriented in relation to the axis of the fold. Proline and glycine were ignored, and the remaining 13 residues in the jR2R3-P301L SLS strand were considered. By this criteria, GPT is the most similar fold to jR2R3-P301L with 11 matching residue orientations. CBD and PSP each shared 6 residues with jR2R3-P301L, and GGT shared 4 residue orientations (FIG.13G). Table 1. RMSD of jR2R3-P301L and Tauopathy Folds (Å) Aligned fold GPT CBD GGT PSPLVM Ref.72-24WO; 340383 301-305 2.59 1.83 2.69 2.93

[0322] The SLS structure of jR2R3-P301L, however, differs from the GPT and CBD folds at site Y310, which breaks the β-sheet and faces outwards from the SLS fold. The break in the β-sheet places K311 in an inward-facing orientation, in proximity to the D295 sidechain. It is likely that this conformation is stabilized by a D295-K311 salt bridge, though the local resolution of the EM map at this location is not high enough to resolve such a bond (FIGs.18A-18D). The apex of the strand-loop-strand might also be stabilized by P301L mutation. A backbone H-bond is formed between L301 and G304 that might help to develop the tight GGG turn that forms the SLS (FIG.18D).

[0323] Within jR2R3-P301L fibrils, the jR2R3-P301L monomer adopts a SLS fold and structure that is close to that found in 4R tauopathies, specifically tau fibrils in GPT. The ability for jR2R3-P301L to adopt a fold and structure similar to that in 4R tauopathy fibrils suggests that P301L will not preclude the exact replication of tauopathy structures but may inherently favor structures like CBD and GPT that have tighter GGG turns in the jR2R3 region. Indeed, a recent publication of P301S Schweighauser et al.68found a similar structure of a strand-loop-strand.

[0324] Example 8 –P301L Mutation of jR2R3 Results in a Greater Population of Strand-Loop-Strand Fibrils

[0325] While the cryo-EM analysis revealed a subpopulation of the jR2R3-P301L fibril that was ordered enough for helical reconstruction, cryo-EM does not offer insight into the complete ensemble distribution of fibril structures because fibrils with greater disorder will not be captured in an electron micrograph. A combination of electron paramagnetic resonance (EPR) techniques was used to describe the composition and structure of the complete jR2R3(-P301L) fibril populations. Continuous wave (CW) EPR was used to describe the extent of incorporation of tau into fibrils, and a combination of CW EPR and DEER was used to describe the proximity of two labeled sites within a single tau chain incorporated in the fibrils. EPR spectroscopy of biomolecules uses site-LVM Ref.72-24WO; 340383 directed spin labeling (SDSL) to attach a nitroxide radical to cysteine residues. Cysteines were added to the termini of jR2R3 and jR2R3-P301L and spin-labeled with MTSL (S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate (e.g., SEQ ID NO: 39 and SEQ ID NO: 40, respectively). SDSL was performed on jR2R3 and jR2R3-P301L with a cysteine added to each terminus (sites 294 and 314) to measure end-to-end distances. To ensure that intramolecular distances are measured within the densely packed fibrils, 10% of the peptides were spin labeled and were mixed with jR2R3(-P301L) peptides without any cysteine mutations (see Exemplary Experimental Aspects for Examples 3-11).

[0326] The CW X-band EPR spectra of fibrils were acquired and lineshape analysis conducted to estimate the fraction of spin labeled tau proteins free or incorporated in fibrils, as well as spin labels within 10 Å of each other that show exchange-coupled spectral features. The acquired CW EPR spectra (FIG.14A) were fit to a 3-component system (FIG.14B) using the MultiComponent software that relies on nonlinear least squares fitting of the spectra, (see Exemplary Experimental Aspects for Examples 3-11). The spectra were assumed to be composed of 3 major sub-populations identified in earlier studies of tau fibrils: (i) a mobile component describing soluble monomers, (ii) an immobile component describing labeled peptides embedded in a fibril that is tumbling slowly, and (iii) a dipolar and / or spin-exchange broadened component describing spin- labels in close proximity (<15 ÅC) of another label (FIGs.14B and 14C). This third component mostly originates from conformations in which the two end labels closely approach each other in a U-shape fold of a SLS arrangement. They also can be due to spin labeled tau stacked in register in b-sheet arrangements, but the probability of direct stacking of spin labeled tau is low given that only 10% of the tau are doubly spin labeled.

[0327] The CW EPR fit of jR2R3 and jR2R3-P301L monomers revealed 86% ± 7% of the tau peptide to be mobile species, i.e. in monomer states. In contrast, the CW EPR spectra of jR2R3 and jR2R3-P301L fibrils contained greater than 95% of the tau peptide population in an aggregated state, in either an immobile or broadened state, indicating a near complete incorporation of labeled protein into aggregates. Of these, 50% of jR2R3- P301L and 22% of jR2R3 corresponded to dipolar or spin-exchange broadened populations. Having solved the high-resolution cryo-EM structure by SPA, we canLVM Ref.72-24WO; 340383 interpret these CW EPR results: 50% of jR2R3-P301L form the SLS structure facing the fibril surface, while 50% of jR2R3-P301L stabilize the U-shape folded peptide as counterstrands in the interior fibril cross section. Since 95% of jR2R3-P301L fibrils are embedded in aggregates, and 50% of the peptides show a broadened state corresponding to populations adopting a close end-to-end spin label distance, the other 50% of the peptides that form extended counterstrand will not show a broadened CW EPR features. It is highly likely, therefore, that jR2R3-P301L peptides form a homogenous fibril population with the jR2R3-P301L structure resolved by cryoEM. In contrast, less than half of the jR2R3 populations are embedded in fibrils with a similar architecture. With only 22% of the jR2R3 fibrils forming conformations with the termini in close proximity, we conclude that jR2R3 peptides are forming different fibrils populations from jR2R3-P301L fibrils, instead forming less ordered, or more extended conformations within the jR2R3 fibrils. This also answers the initial question that we posed: P301L biases the aggregation pathway by promoting the formation of an SLS- like core structure common to 4R tauopathy fibrils.

[0328] Next, DEER was used to extract the probability distribution, P(r), of the intramolecular distance between a select pair of labeled sites. DEER is a pulsed EPR technique that probes the distribution of dipolar coupling oscillations between pairs of spin. DEER can resolve a quantitative P(r) distribution between above 1.5 and 8 nm, as described in Jeschke, et al., Direct conversion of EPR dipolar time evolution data to distance distributions, J. Magn. Reson.155, 72-82 (2002) and Ibáñez, et al., DeerLab: a comprehensive software package for analyzing dipolar electron paramagnetic resonance spectroscopy data. J. Magn. Reson.1, 209-224 (2020), each of which is hereby incorporated by reference in its entirety for all purposes, and specifically for teachings related to DEER spectroscopy methodology. DEER cannot detect distances much below about 1.5 nm due to spectral overlap that leads to mixing of the spin states excited by the pump and probe pulses that hence hinders the generation of quantifiable DEER modulations.

[0329] To capture the differences in the fibril structure and homogeneity of jR2R3 and jR2R3-P301L folds by DEER, an additional set of peptides was prepared by SDSL, with a pair of spin labels placed diagonally across the presumed SLS fold, one at the N- terminal 294 site end and another at serine residue site 305 near the center of theLVM Ref.72-24WO; 340383 peptide (e.g., spin-labeled, jR2R3: SEQ ID NO: 26 (KspinDNIKHVLGGGSspinVQIVYKPV); spin-labeled, jR2R3-P301L: SEQ ID NO: 27 ((KspinDNIKHVPGGGSspinVQIVYKPV)). There were no distinguishable differences in the P(r294-314) of jR2R3 and jR2R3-P301L monomers. The P(r294-314) was broad, with a mean distance of 26.5 Å for both peptides (see, FIG.20, left). The populations of Ree were within two standard deviations of each other at all distances. However, as predicted by CW EPR analysis, the end-to-end distance distributions of the major jR2R3 and jR2R3-P301L populations in the fibril state were too short to be measured by DEER, which is consistent with the jR2R3 peptide adopting a SLS-like fold in which the end spin labels are less than 1.5 nm apart leading to broadened CW EPR features, as discussed in the previous paragraph. Hence, changes in the distance distribution, P(r294-314), upon fibrilization could not be evaluated.

[0330] The P(r294-305) of jR2R3 (e.g., spin-labeled, jR2R3: SEQ ID NO: 24 (KspinDNIKHVLGGGSspin)) and jR2R3-P301L (e.g., spin-labeled, jR2R3: SEQ ID NO: 25 (KspinDNIKHVPGGGSspin)) monomers were also broad and indistinguishable from each other, with a mean distance of 2.4 nm and a span of 2-5 nm (see, FIG.20, right). The expected maximum likelihood distances between 294 and 305 in tauopathy-like SLS structures are 2.8 nm (PSP), 3.1 nm (GGT), 3.6 nm (CBD) and 3.9 nm (GPT) (FIG. 17A), so the mean fibril P(r) was expected to increase compared to the IDP monomer state if SLS structures were formed. Indeed, DEER of jR2R3-P301L(294-305) fibrils (FIG.14E), show a marked extension compared to the mean distance of the monomer ensemble. The 294-305 distance with the highest probability in jR2R3-P301L fibrils was 3.5 nm, indicating that the majority population of jR2R3-P301L extend along the N- terminal half and adopt conformations closer to CBD or GPT folds. The jR2R3 fibrils had a shorter, most probable, 294-305 distance of 2 nm, indicating that the peptides that adopt the characteristic extended N-terminus conformation as part of SLS arrangements do not constitute the dominant populations, unlike in jR2R3-P301L fibrils. The narrowing of P(R) of fibrils made of jR2R3(294-305) compared to the monomer state suggests that distinct folds, and not amorphous aggregates are formed, however, the conformations of the majority jR2R3 population do not meet the criteria of a strand-loop-strand motif.

[0331] We surmised that the majority of the jR2R3-P301L population adopts a SLS- like conformation within the fibrils, consistent with the cryoEM structure of jR2R3-P301L fibrils. Such a distance extension upon fibril formation was not observed with the wildLVM Ref.72-24WO; 340383 type jR2R3(294-305) without P301L. These results show that the P301L mutation of the jR2R3 peptide facilitates the conformational change and assembly of jR2R3-P301L into a strand-loop-strand fold and enhances cross-β stacking along the amyloid fibril axis.

[0332] Example 9 – jR2R3 and jR2R3-P301L have Distinct Free Energies Despite Adopting Conformational Ensembles with Comparable End-to-End Distance Distributions

[0333] Amyloid fibril elongation and seeding is predicated on the recruitment of soluble monomers to the end surface of the fibril. An outstanding question is what interactions are required for the self-assembly to occur in a highly ordered manner that results in structure propagation. The dramatic differences in the quantity, structure, and stability of jR2R3 and jR2R3-P301L fibrils offer the opportunity to scrutinize whether differences in biophysical properties are already engrained in the jR2R3 and jR2R3- P301L peptide monomers prior to their fibrilization. Here, we build upon significant prior work on the effect of P301L on the conformational properties of tau peptide monomers. P301L has been reported to skew tau towards more extended conformations according to SAXS, NMR, and cross-link mass spectroscopy. Though the experimental methods, and length scale of these measurements varied from 100s of amino acid proteins, down to 17 residue peptides, the observation of less compact structures has been consistently observed. A previous study by Chen et. al. suggested that the proline-301 residue helps form a protective β-hairpin in solution of the PHF6 segment that inhibits aggregation of the WT form. The proposed β-hairpin was observed through molecular dynamics (MD) simulations in trimers of peptides spanning residues 295-311, and the occurrence of hairpin conformations according to cross-linking mass spectrometry. We next explore the conformational properties and site-specific hydration water of and around jR2R3 and jR2R3-P301L monomers using MD simulations and experimental techniques.

[0334] To capture the conformational ensembles of jR2R3 and jR2R3-P301L, we used an integrated experimental and computational approach. A computational ensemble of each peptide was computed with replica exchange molecular dynamics (REMD), a method for sampling the conformational space of peptides, including IDPs. The computed P(r) was validated against DEER-derived experimental P(r) to select theLVM Ref.72-24WO; 340383 forcefield that yields the closest agreement. Then, the simulated conformational and energetic landscapes of jR2R3(-P301L) in solution state were analyzed.

[0335] The experimentally measured distance distribution of end-to-end distances, P(Ree), of jR2R3 and jR2R3-P301L monomers were indistinguishable from each other, with a mean Ree of 26.5 Å for both peptides (FIG.20, left). The populations of Ree were within two standard deviations of each other at all distances. The P(R294-305) of diagonal distances across jR2R3 and jR2R3-P301L monomers are similarly indistinguishable (FIG.20, right) within experimentally achievable DEER sensitivities. The P(Ree) of jR2R3 and jR2R3-P301L were simulated by REMD. The force field a99SB-disp, recently optimized for IDPs, provided the closest agreement with experimentally derived P(Ree) by DEER that yield a mean end-to-end distances of 25 Å (FIG.20). Other, widely used, computational forcefields to generate IDP ensembles tend to generate conformational ensembles that are skewed towards more compact conformations.

[0336] While DEER measurements were useful in validating the forcefields used for REMD, the ensemble measurements of distances by DEER only provided a single pairwise distribution, but did not allow for residue-level conformational analysis of other pairs. Hence, MD simulations were needed to gain a more complete, single molecule- level, insight. Next, pairwise contact frequencies of the simulated peptide ensemble were calculated, which showed a tendency for both jR2R3 and jR2R3-P301L to adopt U-shaped conformations. These U-shaped conformations (e.g. FIG.15A at i and iii) tend to be contain intra-molecular contacts between two regions of jR2R3: the N-terminal region I297 to V300 and the C-terminal region S305 to Y310 (FIG.21C).

[0337] We next examined the conformational free energy landscape, which is proportional to the log of the probability distribution of distances (FIG.15A), to uncover the energetic landscape of conformational fluctuations. Infrequent fluctuations can be highly relevant, particularly if they populate an aggregation pathway between aggregation-prohibiting and aggregation-prone conformers. For the jR2R3(-P301L) peptides, we focused on the pathway and free energy of breaking or forming the aggregation prohibiting, internal β-hairpin, seen in the simulated contact maps and discussed above. We monitored the free energy of the jR2R3 series peptides as a function of distances between two pairs of residues, across K298-Q307 (e.g., spin-LVM Ref.72-24WO; 340383 labeled jR2R3: SEQ ID NO: 28 (DNIKspinHVLGGGSVQspinIVYKPV) & spin-labeled, jR2R3-P301L: SEQ ID NO: 30 (DNIKspinHVPGGGSVQspinIVYKPV)) and V300-S305 (e.g., spin-labeled, jR2R3: SEQ ID NO: 29 (DNIKHVspinLGGGSspinVQIVYKPV) & spin- labeled, jR2R3-P301L: SEQ ID NO: 31 (DNIKHVspinPGGGSspinVQIVYKPV)), that form the main stabilizing contacts of the β-hairpin (FIG.15A). The region towards shorter distances near the bottom or left of the free energy landscape represents conformations that are “pinched” across V300-S305 (a shorter peptide region) or “clamped” across K298-Q307 (a larger peptide region) (FIG.15A at i) that is populated with the lowest energy internal β-hairpin conformations. This population was found to hinder dimer formation in simulations (FIG.15B), and hence fibrilization by extension. The top right of the landscape represents open conformations (such as the ones shown in FIG.15A at vi) that are less stable (i.e. higher free energy) and that we hypothesize to be aggregation competent. We found in previous studies suggesting that the region of tau around the PHF6 segment populates extended conformations in the earliest stages of aggregation, and that these extended conformations may be part of aggregation-prone intermediates. Additionally, these extended conformations are reported to be favored by multiple tau disease mutations, suggesting that the effect observed with the P301L mutation may be a universal trait pathological tau mutations.

[0338] We next investigated possible transition pathways from the aggregation prohibiting β-hairpin to open conformations. The computed free energy landscapes shown in FIG.15A reveal two primary modes of unfolding towards aggregation prone conformations: “unpinching” near the ends of the β-hairpin at the V300-S305 contact and “unclamping” at the K298-Q307 contact, near the turn region of the β-hairpin; these modes are illustrated in the supplementary animation. The free energy landscape of jR2R3-P301L has shallower energy barriers compared to WT to break or form aggregation prohibiting β-hairpin conformations and allows for additional pathways to achieve unfolding that are not readily accessible to jR2R3.

[0339] Many, if not all, 4R tauopathy disease folds are held in place by a strong network of intermolecular backbone H-bonds. However, no intramolecular backbone H- bonds are found in the published structures of tau fibrils. We hypothesize that jR2R3- P301L aggregates more readily because of its shallower (around 4 kJ / mol) free energy barriers (FIG.15A and FIGs.22A-22D) to break its aggregation-inhibiting intra-LVM Ref.72-24WO; 340383 molecular contacts and allow inter-molecular H-bonds to form and stabilize the fibrils. Replica exchange simulations of jR2R3 and jR2R3-P301L dimers were performed to test the effect of intramolecular H-bonds on the formation of intermolecular contacts. The most common intramolecular conformation, i.e. the pinched & clamped population prevalent in jR2R3, prevented the dimer from forming intermolecular H-bonds to stabilize the fibrils (FIG.15B and FIGs.25A-25D). In contrast, jR2R3-P301L readily escaped pinched & clamped conformations and populated conformations that allow intermolecular H-bonds to form.

[0340] For jR2R3’s hairpin to open it must first unclamp (i.e. break the K298-Q307 contact) and then unpinch (i.e. break the V300-S305 contact). However, jR2R3-P301L’s hairpin can just as readily unpinch and then unclamp or unclamp then unpinch (FIGs. 22A-22D). jR2R3 lacks this additional unfolding pathway due to its stiffness near residues V300 and P301, as quantified by the lower backbone dihedral entropy found only locally around these sites (FIGs.15A-15B). To test the effect of backbone entropy on unpinching, jR2R3-P301L’s V300 backbone dihedrals were artificially constrained to mimic JR2R3’s V300 backbone. Under the constrained condition, the unpinch-then- unclamp mode was no longer energetically favorable even for jR2R3-P301L (FIGs.23A- 23C). The free energy differences between the different conformational wells of jR2R3 and jR2R3-P301L appear largely due to (a) the increased backbone flexibility around residues 300 and 301 in jR2R3-P301L originating from proline’s absence and (b) increased hydrogen bonding capacity of jR2R3-P301L unlocked by its more exposed conformations (FIGs.24A-24D). The third factor is subject of the next section, the local structuring of water around jR2R3-P301L, but not jR2R3, that direct tau assembly via inter-molecular association around these sites.

[0341] Example 10 – The Local Water Structure is Perturbed Near the P301L Mutation Site of jR2R3-P301L

[0342] While the free energy landscape of jR2R3 and jR2R3-P301L suggests that the P301L mutation lowers the energetic barrier to adopt the aggregation-prone states, a preference for populating an aggregation prone conformation does not provide the driving force for a tightly choreographed, in-register association and templated folding of tau peptides into fibrils with prion competency. We hypothesize that structuring of waterLVM Ref.72-24WO; 340383 is the driving interaction between the monomer and the fibril end surface, and that such features are engrained in differences in the monomer property. We looked to determine parameters that reflect on the effective hydrophobicities—a surface with a positive free energy of hydration, ∆Ghydration—that contribute to the aggregation propensity of jR2R3- P301L.

[0343] Previous work showed that local water hydration dynamics measured by Overhauser dynamic nuclear polarization (ODNP) have a direct correlation with the local water structure, such as the H-O-H three body angle of hydrogen bonded water, and solvation thermodynamic properties. ODNP relies on cross-relaxation of1H nuclei of water molecules induced by the spin flip of the electron spin of the nitroxide spin label, and its efficiency is highly sensitive to the equilibrium dynamics of local water within ~5- 8 Å of the spin label. Hence, if site-specific spin labels are employed then site-specific local water dynamics can be accessed as well. Specifically, we obtained the electron-1H cross-relaxivity parameter, kσ, from ODNP measurements. In the ranges measured here, k^is proportional to the translational diffusion dynamics of water with correlation time in the 10’s ps to 100’s ps range. Diffusion at the kσ timescale corresponds to translationally diffusing hydration water bound to the protein surface with similar or stronger hydrogen bond strength compared to bulk water.

[0344] A single spin-label was attached at three locations, site V300C, the N- terminus (294C) and C-terminus (314C) of the jR2R3(-P301L) peptides (e.g., spin- labeled jR2R3: SEQ ID NO: 32 (CspinDNIKHCspinPGGGSVQIVYKPVCspin) & spin- labeled, jR2R3-P301L: SEQ ID NO: 33 (CspinDNIKHCspinLGGGSVQIVYKPVCspin)), one at a time. At site 300, the measured k^values were 57.9 ± 32 s-1*M-1and 27.3 ± 13 s-1*M-1for jR2R3 and jR2R3-P301L monomers, respectively (FIG.15D). The surrounding hydration water dynamics near site 300 / 301 were significantly slowed down compared to in jR2R3 near sites 300 and 301 (p=0.05). These are in the range of expected k^values for water on protein or peptide surfaces, and are depressed 2-3 fold compared to the value for bulk water of 95.4 s-1*M-1.

[0345] To understand whether the hydration dynamics are slower across the entire jR2R3-P301L surface or only near the 300 site, ODNP measurements were performed at the N- or C- termini. These sites (jR2R3-294C, jR2R3-314C, jR2R3-P301L-294C, andLVM Ref.72-24WO; 340383 jR2R3-P301L-314C) displayed k^of 50 ± 5 s-1*M-1(FIG.15D). No significant difference in hydration dynamics between jR2R3 and jR2R3-P301L were found at either the N- or C-terminal sites. The observation that hydration dynamics were only perturbed around the 300C site in jR2R3-P301L compared to jR2R3, but not near the N- and C-terminal end indicate that the change in water dynamics is a local effect that selectively alters the water structure nearby the mutation site.

[0346] The structural and dynamic properties of hydration water are modulated by the surface geometry and chemical composition of the peptide surface. To determine if the ordering of water near site 300 in jR2R3-P301L was dependent on the local peptide sequence, we performed ODNP measurements of the truncated jR2∆-P301L and jR2∆ peptides spin labeled at the V300C site (FIG.15D). No significant difference in k^was observed between either of the truncated peptides, suggesting that the conformational properties of the peptide contribute to the local structuring of water detected near site 300, in jR2R3-P301L but not in jR2R3.

[0347] We next computed the hydration water diffusivity via the mean-squared displacement (MSD) of water oxygens within the hydration shell near the 301 residue, and around the entire jR2R3 peptide (see Exemplary Experimental Aspects for Examples 3-11 for details). The results show slower computed water diffusivity near the localized 301L site and averaged over the entire peptide surface of jR2R3-P301L compared to jR2R3.

[0348] Example 11 – Disrupted Hydration Shell Around P301L Surface Favors Dehydration

[0349] We hypothesized that slower water near the 300 site of the jR2R3-P301L peptide indicates that structured water is wrapping around the side chain, also referred to as wrap water, and has a lower entropy relative to free bulk water that, upon liberation, adds an entropic driving force for dewetting the jR2R3-P301L peptide assembly interface that can promote protein-protein association. To test this hypothesis, we computed the free energy of dewetting of the protein surface near the 301 residue (see simulation details in Exemplary Experimental Aspects for Examples 3-11) and the local water-protein interaction using indirect umbrella sampling (INDUS). The dewetting free energy for the P301(L) hydration shell, defined as the volume within 0.55 nm of anyLVM Ref.72-24WO; 340383 heavy atom of the residue, capture the properties of the first two hydration layers. Due to the intrinsically disordered nature of the jR2R3-P301L tau peptides, these calculations had to consider multiple conformations to fully describe the ensemble hydration state. Specifically, we obtained the dewetting free energy of site 301 for six most probable conformations of jR2R3 and jR2R3-P301L identified through Daura cluster analysis of the REMD simulations (FIG.27). The average dewetting free energy of residue 301 in jR2R3 and jR2R3-P301L peptides was calculated by a weighted average of this representative conformational subset. The 301L site of jR2R3-P301L exhibited a lower dewetting free energy per water molecule at all levels of hydration levels compared to the 301P site of jR2R3. In particular at Nw=0 (where the residue was totally dewetted) the differential free energy is 0.44 ± 0.02 kBT (Fig.4F). The greater hydrophobicity around site 301L sensitively depends on the environment surrounding this site. Furthermore, the aggregation propensity of a library of jR2R3-P301X mutants was measured with ThT assays (SEQ ID NOs: 2, 5, and SEQ ID NO: 34 (DNIKHVSGGGSVQIVYKPV), SEQ ID NO: 35 (DNIKHVYGGGSVQIVYKPV), SEQ ID NO: 36 (DNIKHVAGGGSVQIVYKPV), SEQ ID NO: 37 (DNIKHVWGGGSVQIVYKPV), SEQ ID NO: 38 (DNIKHVVGGGSVQIVYKPV)). The quantity of aggregated tau showed a moderate correlation with the nominal single-site hydropathy of the 301X residue (see FIGs.29A-29F), suggesting that the local water structuring is still influenced sensitively by the side chain moiety of site 301.

[0350] We next mapped out the local water structure in the hydration layer around jR2R3 and jR2R3-P301L by characterizing the population of water with tetrahedral, i.e. 109.5°, angles between water oxygens all around the peptides. Tetrahedral water structure is a good measure of local hydrophobicity, given that such structuring is characteristic of the hydration shell around small (<1 nm) hydrophobes. Residue 301L of jR2R3-P301L has ~0.20% more tetrahedral hydration waters than jR2R3’s 301P (FIG. 15C); while seemingly small, Jiao, et. al. reported that a similar increase was associated with a ~0.03 Å2 / ps reduction in water diffusivity around a peptoid system. The extent of tetrahedral ordering of water was alternatively evaluated by the number of pentagonal and hexagonal H-bonded water rings in the hydration layer around each residue of jR2R3 and jR2R3-P301L. Residues 300 and 301 in jR2R3-P301L have more pentagonal and hexagonal water rings around them than in jR2R3 (FIGs.28A-28C),LVM Ref.72-24WO; 340383 which we can attribute to the hydrophobic L301 side chain and decreased backbone rigidity in jR2R3-P301L (FIGs.15A-15B). Finally, we investigated the entropy of jR2R3- P301L and jR2R3’s hydration waters across the surface of the entire peptide and in isolation near the 301 residue. The Shannon entropy (Sθ) of water’s 3-body angle was calculated because Sθ was reported by Monroe and Shell (2019) to correlate with the excess thermodynamic entropy. At ambient temperature L301 and jR2R3-P301L had a lower Sθ than P301 & jR2R3 respectively, which persists and becomes more pronounced at higher temperatures (FIG.28D). jR2R3-P301L’s hydration waters therefore had a greater entropy difference compared to bulk water (i.e. a large ΔShydration- bulk), hence facilitating hydrophobic aggregation driven by site-specific, localized, dewetting that increases ΔShydration-bulk.

[0351] Discussion Corresponding to Examples 3-11.

[0352] These studies present the discovery and design of a tau fragment that can form amyloid fibrils with a tauopathy-like fold and prion-like seeding competency. A combined experimental and computational study was conducted to describe the mechanism of action of the P301L mutation that is the single most frequently used disease mutation to model AD. The generation of disease-specific fibril structures must satisfy two criteria. First, fibrils must be composed of layers of tau arranged in a cross-β sheet that are a hallmark of amyloid fibrils and second, the arrangement of tau folds within each layer must match those observed in tauopathy patients. To produce the strand-loop-strand (SLS) folding motif and structure found in 4R tauopathies, we identified a 19-residue peptide (jR2R3 of SEQ ID NO: 5) located at the junction of the R2 and R3 domains of tau, as a model peptide that can fold and stack into strand loop strand (SLS) motifs found in different 4R tauopathies. We found that with the addition of the P301L mutation (e.g., SEQ ID NO: 2), jR2R3-P301L forms an SLS fold with a similar structure to those found in GPT and CBD tau filaments.

[0353] The jR2R3(-P301L) peptide fibrils seed fibrilization of jR2R3(-P301L) monomers and template full-length tau fibril formation in a prion-like manner. While jR2R3 and jR2R3-P301L were both able to seed fibrils formation in vitro, cellular seeding assays were only able to detect seeding competency from jR2R3-P301L. The jR2R3 peptide was designed to mimic 4R tauopathies, and Longhini et. al. report thatLVM Ref.72-24WO; 340383 the seeding by jR2R3-P301L is isoform-selective to 4R tau. We propose that templated seeding of much larger tau by the jR2R3-P301L fibril is initiated by pinning the tau monomer to the fibrillar surface at a site located in the core region of the active fibril end.

[0354] This study suggests that the molecular consequences of the P301L mutation are at least two-fold. P301L appears to lower the energetic barrier for opening the aggregation-prohibiting β-hairpin conformations to make the H-bonding backbone functionalities available to form cross-β sheets. P301L also appears to unlock the aggregation competency of tau by harboring localized structured water near site 300 / 301 that should induce hyper-localized dewetting around the 300 and 301 sites to form intermolecular hydrophobic contacts. Using a combination of ODNP measurements and analysis of REMD and INDUS simulations, we found that P301L creates a more structured, slower-diffusing hydration water layer. These alterations to the hydration water create an entropic driving force that allowed P301L to more easily dewet. If the dewetting of jR2R3-P301L is energetically favorable compared to jR2R3, it follows that the nucleation or elongation of the jR2R3-P301L amyloid fibril may be more favored than the WT form due the dehydrated nature of the fibril folds. In the scenario where jR2R3-P301L peptide fibrils serve as prions to recruit full-length tau monomers, the fibril end surface might harbor the hyper-localized hydrophobic site near P301L and facilitate the in-register association with longer tau monomers.

[0355] The jR2R3-P301L structure may offer some insight into design considerations for tuning such structures to achieve exact replication of 4R tauopathy structures in vitro. We identify three design considerations: i) The charge and identity of the terminal residues, ii) the presence of stabilizing counter-strands, and iii) the presence or incorporation of sites that harbor structured water. i) The charge, and sequence identity of the chain termini have an impact on the jR2R3-P301L fibril structure in the SLS chain. We observe an interaction between D295 and K311 that is likely to be caused by a salt bridge between the sidechains. While K311 is not the C- terminal residue, it is the final resolved residue in the SLS chain, and the final positively charged residue in the chain. Charge interactions are not widely observed at the termini of the resolved tauopathy structures, with hydrophobic interactions dominating, but a similar interaction is observed between K298 and E380 in PSP. We hypothesize that careful selection of the charge sequence may allow the location of such charges in theLVM Ref.72-24WO; 340383 SLS structure to be constrained, and the fibril structure to be biased towards specific conformations. ii) In addition to sharing the strand-loop-strand motif of 4R tauopathies, the jR2R3-P301L also contains multiple layers of folds with the extended chain forming a pseudo-fold around the exterior of the SLS chain. Shi et. al. described 4R tauopathies with either a 3-fold or 4-fold ultrastructural motif. In these folds, the SLS motif is shielded from solvent by an additional layer of protein wrapping around its exterior. jR2R3-P301L partially follows this pattern, with the extended chain wrapping around the exterior of the SLS chain, shielding the PHF6 region from solvent. iii) The structuring of surface water at specific sites on active interface of fibrils through mutation, post-translational modification, or other water-structuring additives should also be explored in the tuning of tauopathy structures. We have observed that at a single P301L site differences in water structuring contribute to enhanced aggregation. It is possible that this represents a more universal phenomena that can be leveraged to structure other regions of tau fibrils. There are a variety of pathogenic tau SNPs that introduce hydrophobic moieties into tau including G272V, G303V, S352L

[0356] We have observed that P301L may be a viable mutation to use in 4R tauopathy models of disease. The cryo-EM structure of jR2R3-P301L successfully produced the hallmark strand loop strand motif of 4R tauopathies, while introducing the P301L mutation. Other mutations, including P301S, may have an effect on fibril structures of tau.

[0357] Exemplary Experimental Aspects for Examples 3-11:

[0358] Peptide Production. Peptides were produced by Genscript to >95% purity with no additional modifications or capping beyond addition of spin labelled cysteines at the sites noted. Peptides were hydrated in 20mM ammonium acetate buffer pH7.4 or in ultra pure H2O to a concentration of 2 mg / mL and were immediately aliquoted and stored at - 80 ˚C until use.

[0359] ThT. Thioflavin T (ThT) assays for β-sheet content were conducted with 50µM protein content unless otherwise noted, and 20µM ThT in a 384-well Corning plate. A BioTek synergy2 plate reader was used with temperature control set at 37˚C. Excitation used a 440 nm filter, and emission was detected at 485 nm.LVM Ref.72-24WO; 340383

[0360] TEM. Negative stain transmission electron microscopy was performed with a 200kV FEI Tecnai G2 Sphera Microscope.200-mesh Formvar copper grids were glow discharged for 45s with an PELCO easiGlow discharge cleaning system.4 µL of sample was applied to grid for 1 minute, blotted with whatman paper, then 4 µL of 1% uranyl acetate was applied to the grids surface and immediately blotted. An additional 4 µL of stain was applied for 1 minute before blotting until dry.

[0361] CryoEM. Multiple sample concentrations were tested for vitrification, and 10 μM protein samples, in a 20 mM ammonium acetate buffer (pH 7.4) with 10 mM NaCl were determined to provide adequate sample coverage and ice thickness for data collection. Samples of jR2R3 and jR2R3-P301L fibrils were incubated at 37˚C on an Eppendorf thermomixer at 600 rpm for 18 hrs. Quantifoil 2 / 2 grids were prepared with either one application of 5 μL, or 2 applications of 3 μL. A single blot of 2 s was applied with a Vitrobot, in the case of 2 applications, a manual blot was applied between the first and second sample application by touching the torn edge of a filter paper to the backside of the grid. Grids were sent to PNCC for further screening and data collection. Grids were screened with a 200kV Arctica microscope. Full data collection was conducted a Titan Krios microscope with a falcon 3 detector, and bioquantum energy filter. SerialEM was used to control image acquisition. Fibrils were observed to partition towards the edge of holes, so 5 micrographs were collected per 2 μm hole, focused around the edge of the holes. Micrographs had an electron exposure of 50 e- / Å2, and a defocus range from -1.0 μm to -2.0 μm. Micrographs were motion corrected using the Motioncor2 implementation in RELION-4.0. CTF correction was performed with CTFFind4. Fibrils were manually picked in RELION.1024 pixel particles were extracted 3 asymmetrical units apart and downscaled to 256 pixels.2D classification was performed and two distinct fibrils populations emerged: a singlet (FIG.13A), and doublet fibril (FIG.14A). The doublet fibril did not contain enough particles to proceed to 3D classification or refinement. Eight 2D classes of the singlet type were used to create an 2D initial model that was used in 3D refinement. Particles were reextracted with a 386 pixel box size for 3D refinement. Particles went through multiple rounds of 3D refinement followed by CTF correction and particle polishing until a final resolution of 3.0 Å was resolved as determined by the Fourier Shell Correlation (FSC=0.143) (FIG.18B). After initial 3D refinement, a C2 symmetry and a 21 screw pseudo symmetry wereLVM Ref.72-24WO; 340383 tested, and the 21 symmetry was determined to provide a better refinement of the structure and was imposed for future refinements.

[0362] Initial model building was conducted in ModelAngelo. The handedness of the map was inverted and provided a better fit to the jR2R3 peptide. Initial models were corrected and refined manually in coot.5 layers of the fibril were refined in Phenix, and the model validation was conducted in Phenix.

[0363] Fibril Seeding. jR2R3 fibers were purified of excess heparin by concentration and washing in 100kDa cutoff Amicon ultra concentrator tubes. Multiple washes with DI water and concentration through the filter were performed, and then the solution was lyophilized. Fiber mass was determined by weighing after lyophilization, and fibers were resuspended to a stock solution of 2 mg / mL (1 mM). jR2R3 or jR2R3-P301L fibrils were added to 0N4R WT tau monomer (UniProt accession number (P10636-8)) and 0N4R(P301L) tau monomer at a 5% molar ratio, and ThT fluorescence intensity was measured.

[0364] HEK Cell Seeding. Cellular seeding was conducted as reported in Longhini, et al., A Small Tau Fragment Specifically Templates Four Repeat Tau Aggregates Through Multiple Generations. bioRxiv, 2023.08.31.555649 (2023), which is hereby incorporated by reference in its entirety, and specifically for teachings and methods related to cellular seedings. H4 cells stably expressing mClover3-Tau187-P301L were plated at a density of 20,000 cells / well of a 96-well plate. Cells were maintained in DMEM supplemented with 10% FBS and 1% Penicillin / Streptomycin. After cells were attached, 2 uM jR2R3 or jR2R3 P301L fibrils in 10 uL of optiMEM were combined with 1.25 uL Lipofectamine 2000 in 8.75 uL optiMEM for 15 minutes, after which they were added to cells. Cells were imaged after 24 hours for visible puncta.

[0365] Peptide Spin-Labeling. Peptides were functionalized with an a nitroxide free- radical, MTSL (S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate). MTSL was incubated at 10x molar concentration with peptides in 4M GdnHCl overnight at 4˚C. Labeled protein was passaged through two GE PD G- 10 desalting columns and exchanged into 20 mM ammonium acetate buffer pH 7.0, with no salt present to prevent preliminary aggregation.LVM Ref.72-24WO; 340383

[0366] Peptides used to measure DEER of fibrils were labelled by mixing the DEER double mutant with 20x concentration of WT protein. MTSL was added in 10x molar excess and the solution was incubated at 4 ˚C for 1hr. Next, heparin was added in a 4:1 molar ratio (tau:heparin) and the solution was incubated at 37 ˚C for 48 hrs; however other ratios are also used, including, but not limited to, 1:1, 2:1, 3:1, 5:1, 10:1, 20:1, 30:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30 (including any sub-ratio therein), of tau:heparin. To remove unbound spin-labels, fibrils were concentrated using a 50 kDa cutoff Amicon ultra concentrator, then diluted with buffer and concentrated again. This was repeated until at least 100x dilution of the original buffer was achieved.

[0367] DEER. DEER signal was collected for 12-24 hours until an optimal SNR was achieved. All DEER time traces were transformed into distance distributions using DeerLab software package for Python. The time traces were phase corrected and truncated by 300 ns to remove possible "2+1"-artifact. One-step analysis was done using the DeerLab fit function with the following models: ex-4deer model with t1, t2, and pulse length set to experiment parameters, bg_strexp model with the stretch parameter freeze to 3 (for soluble protein) and bg_homfractal model with the fractal dimensionality set to fit between 1 to 3 (for fibril samples), and dipolarmodel using Tikhonov regularization. The uncertainty analysis was done using bootstrapping method with 100 samples. The time domain fitting results are presented both as fitting to the primary data and the distance distribution fits are presented with 95% confidence intervals. samples were corrected using spectra collected of the singly labelled species (jR2R3-(P301L)- 314C) to account for the shift in geometric dimensions. The DEER experiments were performed with a pulsed Q-band Bruker E580 Elexsys spectrometer, equipped with a Bruker QT-II resonator and a 300 W TWT amplifier with an output power of 20 mW for the recorded data (Applied Systems Engineering, Model 177Ka). The temperature of the cavity was maintained at 65 K using a Bruker / ColdEdge FlexLine Cryostat (Model ER 4118HV-CF100). The bridge is equipped with an Arbitrary Wave Generator to create shaped pulses for increased sensitivity. The samples were made in D2O buffers with 30 % (v / v) deuterated glycerol (used as the cryoprotectant). To perform an experiment, approximately 40 μL of sample was added to a 3 mm OD, 2 mm ID quartz capillary and flash frozen in liquid nitrogen to preserve sample conformations.LVM Ref.72-24WO; 340383

[0368] CW EPR. CW EPR measurements were carried out at room temperature with a Bruker EMX X-band spectrometer operating at 9.8 GHz (EMX; Bruker Biospin, Billerica, MA) and a dielectric cavity (ER 4123D; Bruker Biospin, Billerica, MA). A sample of 4.0 μL volume was loaded into a quartz capillary tube with 0.6 mm internal diameter (CV6084; VitroCom) and sealed at one end with critoseal, and then placed in the dielectric cavity for measurements. CW EPR spectra were acquired by using 6 mW of microwave power, 0.5 gauss modulation amplitude, 200 gauss sweep width, and 10 scans of signal averaging.

[0369] ODNP. ODNP samples were prepared with 3.5 µL in a quartz capillary tube with 0.6 mm internal diameter (CV6084; VitroCom) and sealed at one end with Critoseal. The other end was sealed with beeswax. All measurements took place under 18.0±0.2 ˚C with constant convective cooling to prevent sample heating. An NMR probe built in-house was used, and the sample was irradiated with microwave at the central electron hyperfine transition. NMR enhancement as a function of mw power was determined with a series of 20 mw power up to 6 W. A T1(p) enhancement curve was performed with a series of 5 inversion recovery experiments as previously described. T1,0 was determined in separate experiments using unlabeled peptides to be ~2.0 s for all peptides used. Data was processed using the hydrationGUI by Tom Casey, a package for processing ODNP data using DNPLab. Most experiments used the ‘automatic process’ function but were manually inspected after processing for aberrations in the integration windowing, or phase correction. If an error was detected (such as a failure to correctly center the NMR peak, windowing and phase correction was performed manually, and a constant 10ppm window was used.

[0370] CWEPR Spin Counting. Spin-labeling efficiency was quantified by CWEPR. A standard ladder of 4-HydroxyTEMPO was measured at 50, 100, 200, 500 µM concentration and cwEPR spectra were recorded. The maximum of the baseline- corrected, integrated spectra was recorded, and fit to a linear regression vs. concentration. All samples were measured under the same conditions, and the max of the baseline-corrected, integral spectra was converted to an effective spin label concentration. The spin label concentration was used in ODNP parameter calculations, and to determine the labeling efficiency. Spin labelling efficiency was calculated by SLeff=[SL] / [Protein].LVM Ref.72-24WO; 340383

[0371] Multi-Component CWEPR Spectra Fitting. Fitting of cwEPR was performed using MultiComponent, a software package from Christian Altenbach. The program is written in LabVIEW (National Instruments) and can be freely downloaded from the following site: http: / / www.chemistry.ucla.edu / directory / hubbell-wayne-l. Fitting was done using similar procedures as described previously. Briefly, the mobile component was determined by fitting the A, G and R tensors to the solution peptides. The A and G tensors were determined first, and then R was allowed to vary. Next a second component was fit to fibrils formed with 10% spin-dilution and a single labelled site at V300C. These fibrils should display minimal exchange-coupling but exist in the slow- motion regime due to the size of the fibril. Fibrils were previously determined have an axially symmetric diffusion tensor (αD = 0˚, βD = 36˚, γD = 0˚) and an ordering parameter of 20. Only the rotational correlation time (R) was fit to this component. Finally, a 3rdcomponent was added to fit doubly-labeled peptides in fibrils. Component 3 used the same rotational correlation time (R) as the second component, as all spin labels in the fibrils should be in similar rotational correlation regimes. A Heisenberg spin-exchange component (ωss=140MHz) previously empirically determined for tau fibrils was added to component 3. Solution state peptides were fit to only the mobile (component 1) and immobile (component 2) components. When fit with a 3rd, spin-exchanged, component the covariance of component 2 and component 3 was too large to obtain trustworthy fits.

[0372] DEER Pulse Program. The following 4-pulsed DEER sequence was applied to all samples: ^^^^ / 2 – ^1 – ^^^^ – (^−^^^^^) – (^2−^) – ^^^^ – ^2 – echo. ^(^) is recorded as the integral of the refocused echo as a function ofdelay, ^, between the Hahn echo and pump pulse. Rectangular observe pulses and chirp pump pulse were used with the following pulse durations: ^^^^ / 2= 12 ns, ^^^^= 24 ns, ^^^^^= 100 ns. The chirp pump pulse was applied with a frequency width of 60 MHz to excite a distinct spin population, referred to as B spins, while the observe spulse was set 33 G up field from the center of the pump frequency range to probe another distinct spin population, A spins. ^1 was set to 180 ns and ^2 was set according to the SNR profile of the dipolar signal. Deuterium ESEEM suppression was done by incrementing ^1 with 16 ns steps, n=8 times. The data was acquired with resolution of 16 ns, 16-step phase cycling, and signal averaged until desirable SNR.LVM Ref.72-24WO; 340383

[0373] DEER P(r) Simulations. The expected P(r) distributions of the jR2R3 in the tauopathy fibril cores (FIG.14B and FIG.17C) were predicted using the DEER-PREdict package. A 298K MTSL library was used to simulate all possible conformations of the labels once tethered to the defined structure extracted from. To form the protein backbone all residues except 294-314 were deleted from each PDB structure. Three chains (A, E, C) were retained, and the labels were attached to the desired label sites of the middle chain (A) to account for clashes with adjacent chains.

[0374] AFM Sample Preparation. jR2R3 and jR2R3-P301L solution mixtures were prepared similarly as TEM samples. Peptide samples were solvated and aliquoted in 20 mM Ammonium Acetate (NH4Ac) buffer with a final concentration of 2 mg / mL. The peptide samples were stored in microcentrifuge tubes at 200 µM concentration in the freezer with -20 / -80 ℃.200 µM peptide aliquots were thawed and added into 20 mM NH4Ac with heparin to a final concentration of 25 / 50 µM of peptide and 4:1 concentration ratio of peptide to heparin. The peptide mixtures were then incubated at 37 ℃ and shaken at 650 rpm for 23 hr. An aliquot of 4-5uL was drop-casted on a freshly cleaved mica surface (TedPella, Redding, CA) after the incubation period. The drop- casted solution was left on mica surface for about 1 min and then the excess liquid was blotted off and dried in a desiccator overnight.

[0375] AFM Imaging. The topography and phase images were acquired by MFP-3D Atomic Force Microscopy (Asylum Research, Goleta, CA) in Tapping Mode Amplitude Modulation using high resolution silicon nitrate tip (NanoAndMore, CA), with a radius <10 nm and a cantilever with spring constant about 2 N / m and a resonant frequency around 70 kHz.

[0376] Fibril Stability Assay. Guanidinium Hydrochloride denaturation assays were conducted under the same conditions as ThT assays. Reported values were detected after signal reached equilibration at least 5 hrs after initial incubation. Fibrils were diluted 2x from initial concentration into the denaturation buffer to reach the reported GdnHCl concentration.

[0377] REMD. All replica-exchange molecular dynamics (REMD) simulations of the jR2R3 and jR2R3-P301L peptides were performed using the Gromacs package (versions 2019.6 and 2020.1). A 7 nm rhombic dodecahedral box was used withLVM Ref.72-24WO; 340383 periodic boundary conditions. The peptides were simulated with ~7900 TIP4P-D waters and neutralizing chlorine ions with the AMBER99SB-Disp force field. Monomer simulations were initialized with the peptide in an extended conformation and with histidine in the HIE protonation state, although the HID protonation state was initialized for simulations in the supplementary figure. Dimer simulations were initialized with the top two clusters (from clustering with the Daura algorithm) placed ~2 nm apart. Monomer and dimer simulations had zwitterionic termini. REMD simulations had 60 temperatures from 300K to ~455K. The particle mesh Ewald (PME) method with a grid- spacing of 0.12 nm was used for calculating the electrostatic interactions.2 fs time- steps were used with a leap-frog integrator.1.2 nm cutoff distance was used for all the nonbonded interactions. The bonds with hydrogen atoms were constrained using the SETTLE algorithm. The REMD systems were simulated for 400-610 ns for monomers, and 500-750 ns for dimers. See Supplement for details on the equilibration and how the total equilibration time was chosen.

[0378] MTSL was attached to a cysteine at residues 294 and 314 (note that jR2R3 and jR2R3-P301L for these experiments was SEQ ID NO: 39 (CspinDNIKHVPGGGSVQIVYKPVCspin) and SEQ ID NO: 40 (CspinDNIKHVLGGGSVQIVYKPVCspin), respectively) and parameterized for a99SB-disp to compare simulation with the DEER end-to-end distance distribution.

[0379] Simulations with the MTSL probe were in an 8 nm rhombic dodecahedral box with ~12,000 TIP4P-D molecules. One sodium ion was inserted to neutralize the system. The same AMBER99SB-disp force field was used, and force field parameters for MTSL were optimized (details below).64 replicas from 292.7 K to 455.6 K were used which resulted in average exchange rates between 20-30% for the attempted exchanges every 3 ps. These temperatures were used for MTSL simulations: 292.7, 294.5, 296.3, 298.1, 300.0, 301.9, 303.8, 305.7, 307.7, 309.7, 311.7, 313.7, 315.8, 317.9, 320.0, 322.1, 324.3, 326.5, 328.7, 330.9, 333.2, 335.5, 337.8, 340.1, 342.5, 344.9, 347.3, 349.7, 352.2, 354.7, 357.2, 359.7, 362.3, 364.9, 367.5, 370.1, 372.8, 375.5, 378.2, 380.9, 383.7, 386.5, 389.3, 392.1, 395.0, 397.9, 400.8, 403.7, 406.7, 409.7, 412.7, 415.8, 418.9, 422.0, 425.2, 428.4, 431.6, 434.9, 438.2, 441.6, 445.0, 448.5, 452.0, 455.6 KLVM Ref.72-24WO; 340383

[0380] The simulations without the probe used the same temperatures, except starting at 300.0 K instead of 292.7 K. Those 60 replicas also resulted in 20-30% average exchange rates. The MTSL simulations also constrained every bond with the LINCS(4) algorithm; the simulations without the MTSL only constrained the bonds with hydrogen atoms with the SETTLE algorithm

[0381] Optimization of the MTSL Force Field. To develop the force field parameters for the spin probe, we performed quantum-chemical calculations of an MTSL molecule, where the RSO2 group was replaced by a terminal SH group to mimic the MTSL- cysteine disulfide bond. The structure of the MTSL molecule in vacuum was optimized in two steps: First at the Hartree-Fock (HF) level with the 6-31G* basis set and next at the DFT level, using the B3LYP functional with the 6-311++G(d,p) basis set. Next, the optimized geometry was used for the Merz-Singh-Kollman (MK) population analyses using the HF level of theory and the 6-31G* basis set. The use of HF / 6-31G* level of theory for deriving the electrostatic potentials (ESP) on the MTSL atoms was consistent with the development of the AMBER94 and the AMBER99SB*-ILDN-Q force fields- based on which models the atomic partial charges for the AMBER99SB-Disp force field were primarily developed. The partial charges of the MTSL atoms were derived by fitting the ESP data using the restrained electrostatic potential (RESP) method. The equilibrium bond-lengths and the bond-angles were determined from the DFT-optimized structure of MTSL. The parameters for the proper and the improper dihedrals were derived from the pyrroline parameters reported by Xue and Skrynnikov (the authors adapted the parameters for the AMBER-based force fields from a CHARMM-based force-field, originally developed by Roux et al.). All other bonded and non-bonded parameters were derived from the AMBER99SB-Disp force field by analogy, considering similar hybridizations of the atoms. The MTSL probe was attached to the β-carbon of a cysteine residue by removing the hydrogen atom attached to the terminal sulphur atom of the probe. The partial charge of the terminal sulphur atom was adjusted accordingly. All the quantum-chemical calculations were performed using the Gaussian 16 suite. The RESP fitting was carried out using the AmberTools suite (version 20).

[0382] Equilibration Procedure. The systems were energy minimized for gradient descent after being solvated. Systems were first simulated for 5 ns in NPT ensemble at 1 bar pressure and 300 K temperature. THE MTSL simulations used a BerendsenLVM Ref.72-24WO; 340383 thermostat (0.1 ps relaxation time) and Berendsen barostat (1.0 ps relaxation time) for the NPT equilibration. The simulations without MTSL used a v-rescale thermostat (0.5 ps time constant for temperature coupling) and the Parrinello-Rahman barostat (3 ps time constant for pressure coupling for NPT equilibration(14). Next, 5 ns long NVT simulations with the Nose-Hoover thermostat (3 ps relaxation time) were performed at 300 K temperature using the average box-size from the final 3 ns of the previous NPT step. Then the system was copied and simulated at the various replica temperatures for 5ns without exchange. Finally, the production REMD simulation started with exchange.

[0383] The monomers with MTSL were simulated for 400 ns per replica, and the last 200 ns were used for data analysis. The monomers without MTSL were simulated for 410-610 ns per replica, of which the first 50 ns were discarded. The dimers (without MTSL) were simulated for 500-750 ns per replica. jR2R3 dimers were equilibrated for 110 ns and jR2R3-P301L were equilibrated for 80 ns of the 750 ns.

[0384] The equilibration time was decided for each measurement by using a heuristic that maximizes the number of effectively uncorrelated measurements. The autocorrelation function was integrated for equilibration times in 10 ns increments starting from 50 ns, and the equilibration time with the most effectively uncorrelated measurements was chosen. Dimer simulation equilibration times were chosen by maximizing the number of effectively uncorrelated intermolecular H-bond counts. Autocorrelation functions were measured using pymbar’s timeseries module.

[0385] INDUS. The top 6 clusters from a Daura clustering analysis of the REMD monomer simulations were further analyzed with INDUS. GROMACS (version 4.5.3) was modified to bias the coarse-grained number of waters, Nv, in the hydration volume of the P301 or L301 residue using the Indirect Umbrella Sampling (INDUS) method. The Gaussian coarse-graining function employed in INDUS is parameterized with a standard deviation of σ = 0.01 nm and a truncation length rc = 0.02 nm. The water dynamics is governed by the Hamiltonian: H = H0 + 1 / 2 κ (Nv - N*)2, where H0 is the unbiased Hamiltonian, and the second term represents the harmonic biasing potential with strength κ = 10 kBT. The overlap in the Nv values that are obtained from sampling over the different N*water molecules (i.e. “for N*in {-6..3..54}”), enable us to estimate the dehydration free energy of the target residue for the Nv values, as characterized by theLVM Ref.72-24WO; 340383 logarithm of the unbiased probability distribution Pv(N), of observing N water molecules in the hydration volume of the target residue that is calculated using the Unbinned Weighted Histogram Analysis Method (UWHAM).

[0386] The rectangular simulation box with size Lx = Ly = Lz = 7.12 nm that contains a peptide and ~11800 water molecules were simulated by using the leapfrog integrator to integrate the equations of motion with a time-step of 2 fs. The oxygen-hydrogen bonds in water were constrained using the SETTLE algorithm, and all the peptide bonds involving hydrogen atoms were constrained using the LINCS algorithm. The short-range van der Waals and Coulombic interactions were truncated using a cut-off of 1.0 nm, and long-range electrostatics were calculated using the particle-mesh Ewald (PME) algorithm. After solvating the 6 most probable conformations obtained from the REMD simulations for each jR2R3 and jR2R3-P301L peptides, the systems were energy minimized through the steepest descent approach (FIG.50). The following MD equilibration simulations were performed prior to the production phase. First, the systems were simulated in an NVT ensemble at temperature T = 300 K for 500 ps, by using the stochastic velocity-rescale thermostat with a time constant of 0.5 ps. Next, a 1.0 ns NPT simulation was performed for every system by using the Berendsen barostat to maintain the temperature and pressure at 1bar. Following this NPT equilibration, we performed a 3.0 ns NPT run for every system by using the stochastic velocity-rescale thermostat with a time constant of 0.5 ps, and with employing the Parrinello-Rahman barostat with a time constant of 1 ps, to maintain the temperature and pressure at 300 K and 1 bar. All the production runs were performed for 10 ns at 300 K and 1 bar, by using the stochastic velocity-rescale thermostat and the Parrinello-Rahman barostat; the first 2 ns were discarded to reach the convergence phase.

[0387] Water Structure Analysis. The “tetrahedral water” fraction for each residue, was calculated by integrating the water triplet angle distribution from 100-120° for each residue. The water triplet angle distribution is the distribution of angles formed by a central water oxygen and the oxygens adjacent waters. The water triplet angle distribution has been shown to reveal signatures of water structuring in response to different types of solutes and interfaces. The distribution of hydration waters 4.25 Å around each residue was computed; the 4.25 Å cutoff corresponds to the second minimum in the RDF between the heavy atoms and water oxygen atoms. NeighboringLVM Ref.72-24WO; 340383 waters are defined as those whose oxygens lie within 3.4 Å of a central water. We computed these water angles for each frame of the converged trajectory, which was saved every 10 ps, and we created a histogram of the angles with 5° bins. Each frame has statistically independent water triplet angles measurements based on measured autocorrelation times. All confidence bars were determined by bootstrapping independent measurements 1000 or more times with replacement. The Shannon entropy of this distribution was calculated by summing the p*log(p) of each of the 5° bins.

[0388] Water rings were counted using code from this repository: https: / / github.com / vitroid / cycless.git. We look at waters 4.25 Å around each residue and determine if two waters are connected based on whether the O-H distance between two waters is closer than 2.45 Å. We count pentagonal or hexagonal water rings when five or six waters in the residue’s hydration layer form a closed, connected graph.

[0389] Residue Backbone Entropy. Residue backbone entropy was measured by analyzing the regions of Ramachandran space that each residue occupied. The Φ and Ψ dihedral space was coarse-grained into four quadrants as follows; A: {ΦΒ : , (−120°<Ψ<50°)}, C : {(−100°<Φ<0°), (Ψ>50° or Ψ<−120°)}, D:the residue backbone entropy (Sres) is measured by using the equation (E1), calculating the Shannon entropy of the Ramachandran regions occupied by each residue, ^^^^^^ = − ∑^ ^^ ∙ ln (^^) (E1)

[0391] where space pi probability that the residue will be in that region.

[0392] Other Analysis of MD Simulations. Energy landscapes were generated by measuring the 2D probability density, taking a logarithm and scaling by Boltzmann’s constant and temperature, and applying gaussian interpolation to smooth the landscape. Residues were assigned secondary structure with the DSSP algorithm. The DauraLVM Ref.72-24WO; 340383 algorithm was used to cluster the most probably conformations; a 0.3 nm root-mean- squared deviations cutoff was used for the backbone Cα-C-N atoms.

[0393] All error bars for MD analyses are 90% confidence intervals, unless otherwise stated. We used pymbar’s timeseries module to calculate the statistical inefficiency. Independent measurements were bootstrapped 1000 times with replacement and sorted to determine the confidence levels. ChimeraX and Pymol were used to visualize conformations. MDAnalysis and mdtraj were used for data analysis. The unzipping and unpinching mode animations were created by visualizing one of the first principal components of the α-carbons coordinates for a clamped and pinched subset of the jR2R3-P301L trajectory, respectively.

[0394] Background for Examples 12-17.

[0395] Since tau proteins stack in register and parallel to elongate into pathological fibrils, phosphoryl groups from adjacent tau strands with 4.8 Å separation must find an energetically favorable spatial arrangement. At first glance, this appears to be an unfavorable configuration due to the proximity of negative charges between phosphate groups from adjacent neighboring tau fibrils. However, as described in the following examples, a counterhypothesis was tested that phosphoryl groups within the fibril core- forming segments favorably assemble into highly ordered, hydrogen-bonded, one- dimensionally extended wires under biologically relevant conditions. We selected two phosphorylation sites associated with neurodegeneration, serine 305 (S305p) and tyrosine 310 (Y310p), on a model tau peptide jR2R3-P301L (tau295-313) spanning the R2 / R3 splice junction of tau, that readily aggregate into a fibril with characteristics of a seed-competent mini prion. Using multiple quantum spin counting (MQ-SC) by31P solid- state NMR of phosphorylated jR2R3-P301L tau peptide fibrils, enhanced by dynamic nuclear polarization, we find that at least six phosphorous spins must neatly arrange in 1D within fibrils or in 2D within a protofibril to yield the experimentally observed MQ- coherence orders of four. We found that S305pstabilizes the tau fibrils and leads to more seeding-competent fibrils compared to jR2R3 P301L or Y310p. This study introduces a new concept that phosphorylation of residues within a core forming tau segment can mechanically facilitate fibril registry and stability due a hithertoLVM Ref.72-24WO; 340383 unrecognized role of phosphoryl groups to form highly ordered, extended, 1D wires that stabilize pathological tau fibrils.

[0396] Research suggests that phosphorylation occurs before aggregation, and phosphorylation at different sites has diverse impacts on biological processes and pathogenic developments. Hyperphosphorylation of soluble tau may contribute to neurodegeneration by weakening association of tau with microtubules, and either causing microtubules to destabilize or increase free intracellular tau available for aggregation. While there are downstream biological consequences of phosphorylation, this studies described in the following examples addressed an important unknown in the field, namely whether there are direct, stand-alone, molecular consequences of phosphorylation on filament formation and stability in vitro, outside the cellular environment. This understanding was key to the synthetic replication of tau NFTs found in tauopathies with molecularly defined protein folds and quaternary structures that are diagnostic hallmarks of different tauopathies including AD, CTE, CBD, PSP, AGD and several other diseases.

[0397] Example 12 – Aggregation Assays of Phosphorylated jR2R3-P301L Tau Fibrils at Two Sites

[0398] Phosphorylated jR2R3-P301L tau fibrils at serine 305 or tyrosine 310 were synthesized using the preparation method described in the Materials and Methods section for Examples 12-16, below. FIG.30A depicts the 19-residue jR2R3 peptide sequence (D295 – V313) derived from the R2 and R3 regions of the 2N4R tau isoform, and the phosphorylation mutations at S305 (FIG.30B) and Y310 (FIG.30C) illustrated in a CBD conformation with the monomer forms stacked in-register and parallel along the fibril axis. The S305pand Y310pfibrils were prepared in a solution of either 100 mM or 1M NaCl during aggregation. High salt concentration such as 1M NaCl screens electrostatic interactions and sequester water away from the protein, thereby amplifying the effect of the hydrophobic association of tau. The high salt concentration is not physiologically relevant, but the effect of such conditions on the protein can nonetheless mimic physiologically relevant conditions mimicking strong electrostatic screening, crowding and / or enhanced hydrophobic effects. Hence, the high salt concentration ofLVM Ref.72-24WO; 340383 1M NaCl was examined in addition to 100 mM NaCl to prepare the phosphorylated fibrils. A ThT assay was used to monitor the formation and stability of fibrils.

[0399] The aggregation of S305pand Y310pfibrils exhibited strong fluorescence owing to binding of ThT (FIGs.31A-31B), indicative of β-sheet formation by fibrillization. In particular, fibrils made of S305pshowed higher fluorescence than Y310pprepared under both the 100 mM (FIG.31A) and the 1 M (FIG.31B) salt concentration conditions (compare top panels, showing results for S305p, to bottom panels, showing results for Y310p, in each of FIGs.31A-31B). The morphology and final fibril formation of S305pand Y310pwere examined using negative stain transmission electron microscopy (TEM). TEM images showed that S305pfibrils exhibited higher quality fibrils displaying paired helical filaments and ribbon-like structures compared to Y310pfibrils under both the 100 mM (FIG.31A) and the 1 M (FIG.31B) salt concentration conditions (compare top panels, showing results for S305p, to bottom panels, showing results for Y310p, in each of FIGs.31A-31B).

[0400] Example 13 – Stability Revealed by Fiber Denaturation Experiments

[0401] The stability of S305pand Y310pfibrils was tested by incubating each of the fibrils to guanidinium hydrochloride (GdnHCl) at varying concentrations: 0 mM, 250 mM, 500 mM, and 1 M. GdnHCl is a denaturant that can disrupt the natural structure of proteins by breaking hydrogen bonds and disturbing β-sheet secondary structures.49The ThT fluorescence was measured after equilibrating the samples for 24 hours. Fibrils that withstand denaturation under more severe conditions are considered more stable. Therefore, a greater decrease in ThT fluorescence with increasing GdnHCl concentration indicates lower fibril stability.

[0402] The stability of the jR2R3-P301L (SEQ ID NO: 2) fibrils prepared under 100 mM NaCl condition are shown in FIG.32A and FIG.42. To standardize the results, all values were compared to the initial fluorescence of the corresponding fibrils before denaturation. Under denaturing conditions with GdnHCl, all peptide fibrils exhibited reduced fluorescence intensity, as the fibers treated with GdnHCl at least partially disassemble into monomers. Notably, at 1M GdnHCl concentration, the peptide fibrils displayed distinct differences in their stability: S305pfibrils showed a net loss of 28% ±LVM Ref.72-24WO; 340383 0.96% and Y310pa net loss of 52% ± 0.57% in fluorescence, whereas jR2R3-P301L fibrils without any phosphorylation showed the greatest net loss of 63% ± 0.97%.

[0403] The TEM images (FIG.42) exhibit the characteristic features of denaturation, with more fragmentation observed in Y310pcompared to S305por jR2R3-P301L (SEQ ID NO: 2) fibrils with increasing GdnHCl concentration. These results show that phosphorylated jR2R3-P301L fibrils are more stable compared to non-phosphorylated jR2R3-P301L fibrils. Of these, S305pfibrils exhibited the highest stability. These results suggest higher orders of associations in phosphorylated fibril.

[0404] Example 14 – Seeding Competency Studies

[0405] Since the S305pfibrils were observed to be more stable compared to the Y310pfibrils (see, Example 13, above), we hypothesized that this would improve the fibers’ ability to be more seeding active. The templating of tau to adopt the same structural folding is an important attribute of a competent seed, and therefore a well- packed fiber was predicted to be properly aligned to allow additional monomers to stack on more readily upon adopting the fold of the fibrillar seed.

[0406] Using absolute ThT fluorescence as a guide to assess seed performance, the comparison of the ThT fluorescence of the seed to the seeded monomer, we demonstrated that the S305pfibrils were more seeding active than the Y310pfibrils (FIG. 32B). This finding is consistent with the discovery described above that S305pfibrils were more stable than the Y310pfibrils.

[0407] Example 15 – SSNMR MAS DNP Studies of Vitrified Fibrils

[0408] After analyzing the fibril morphology, stability, and quality, (see, Examples 12- 14, above) the S305pand Y310pfibril samples were scaled up for solid-state NMR studies.

[0409] Conventional one-dimensional1H-31P CP spectra were recorded under MAS at 10 kHz and DNP at 100 K. The31P NMR line shape and width reflect the spatial distribution of phosphoryl groups. The31P NMR spectra were analyzed using a Gaussian / Lorentzian model with the DMfit software (shown in FIGs.33A-33F).50TheLVM Ref.72-24WO; 34038331P NMR spectra of vitrified monomers of both S305p(FIG.33A) and Y310p(FIG.33D) showed a broad Gaussian lineshape, as expected.

[0410] The31P NMR spectra of the S305pjR2R3-P301L fibrils prepared in 100 mM (FIG.33B) or 1M solution (FIG.33C) showed a significantly narrowed line as compared to the monomer (FIG.33A). Notably, the dominant component with a chemical shift of 0.99 ppm (70%) was a narrowed Lorentzian line, with a full width at half maximum (FWHM) of 220 Hz compared to 800 Hz of the monomer sample with a Gaussian lineshape (compare FIG.33B to FIG.33A). The other two components of the S305pfibril included one Lorentzian component centered at 4.4 ppm with an FWHM of 570 Hz (5 %) and a Gaussian component centered at -1.67 ppm with an FWHM of 1010 Hz (25 %), shown in FIG.33B. We attribute the two broader components to fibril populations with powder orientation of the31P spectral components.

[0411] In contrast, the31P NMR spectra of the Y310pjR2R3-P301L fibrils, prepared in either 100 mM (FIG.33E) or 1M solution (FIG.33F), showed no narrowed31P NMR spectral component.

[0412] The31P NMR spectral narrowing, only seen in S305pfibril, suggests that S305pfibrils are stabilized by closely coupled phosphoryl groups. Random orientation of phosphoryl groups in space will result in a broadened Gaussian lineshape, as observed for Y310pfibrils, while one-dimensionally stacked31P would yield mutually commuting dipolar Hamiltonians that produce dipolarly-narrowed Lorentzian31P NMR lineshapes, as observed in S305pfibrils.

[0413] The line broadening in solid state NMR spectra is mainly caused by anisotropic interactions: chemical shift anisotropy (CSA), homonuclear, and heteronuclear dipolar couplings. CSA fits were performed using the CSA-MAS model in the DMfit50software (fit values and CSA parameters shown in Table 2 and Table 3, respectively, below). The CSA and asymmetry parameter (η) values for S305pmonomers and fibrils were similar, as well as for Y310pfibrils and monomers, indicating CSA did not significantly contribute to the line width. The main sources of heteronuclear dipolar couplings are1H-31P,13C-31P, and15N-31P dipolar couplings. Applying heteronuclear decoupling on protons during acquisition eliminated1H-31P couplings. We confirmed this on the31P NMR of 100 mM NaCl S305p,showing a narrower and moreLVM Ref.72-24WO; 340383 intense peak at 0.99 ppm with decoupling on, while other peaks remained unaffected (FIG.38A). The contribution from13C-31P and15N-31P couplings was negligible due to their smaller magnitudes at isotopic abundance, indicating heteronuclear dipolar couplings are not the primary source of spectral broadening. Therefore, the primary cause of line broadening is the31P-31P homonuclear dipolar couplings. The reduction in linewidth observed in the31P spectra of S305pfibrils at both NaCl conditions, as depicted in FIGs.33B-33C, indicates an averaging effect on the31P-31P homonuclear dipolar couplings. We believe that this averaging of the31P homonuclear dipolar couplings can only be achieved by parallel stacking of the fibrils in a single orientation across several stacking copies of tau under MAS.

[0414] In contrast, the31P NMR spectra of Y310pexhibited broad linewidths, similar to that of the monomer. This observation suggests that the incomplete averaging of homonuclear dipolar couplings in Y310pfibrils is a result of heterogeneity or local disorder present within the sample contributing to the broad linewidths. Therefore, we concluded that S305pfibrils are lined by one-dimensional (1D) phosphoryl wires with better local homogeneity / order compared to Y310pfibrils.

[0415] Decoupling effect on the31P spectra: We verified the effect of heteronuclear decoupling on31P spectra of 100 mM NaCl S305pusing the decoupling power on and power off (shown in FIG.38B) on the proton channel during the acquisition. With decoupling power applied on protons, the peak at 0.99 ppm became much narrower with an increase in intensity when compared to the spectra with the power-off condition. The rest of the peaks were not much affected. We deconvoluted the31P spectra obtained by decoupling off condition and shown in FIG.38A. The peak at 0.99 ppm for S305pexhibited the Lorentzian line shape in both cases when the decoupling was applied on1H and not applied on (FIG.38A). However, the peak at 4.4 ppm showed a Lorentzian nature when the decoupling was applied on1H and showed a Gaussian nature when the decoupling was not applied on the1H channel. But the peak at -1.67 ppm showed Gaussian nature in both cases of decoupling and non-decoupling case. Therefore, we could say that the peak at 0.99 ppm has the effect of1H-31P heteronuclear coupling, others were not affected by the protons. The line shape fitting and the parameters are shown in Table 2 and FIG.38A.LVM Ref.72-24WO; 340383 Table 2. Line Shape Fitting of the31P spectra by Gaussian / Lorentzian Using DMfit. Gaussian depicted as “G”. Lorentzian depicted as “L”. “nodec” refers to decoupling power off. Sample type 0.9 ppm 4.4 ppm -1.6 ppm S305PG L G L G LTable 3. CSA Parameters Obtained from DMfit Using the CSA-MAS Model. Asymmetry Linewidth Sample type δiso (ppm) CSA (ppm)

[0416] Example 15.1 – Additional Data from SSNMR MAS DNP Studies of Vitrified Fibrils

[0417] SSNMR studies were performed under MAS at 10 kHz and with DNP at 100 K on S305Pand Y310Pfibril samples at natural isotopic abundance. First, one-dimensional1H-31P CP spectra were recorded. The 31P NMR lineshape and width directly reflect on the symmetry of the spatial distribution of dipolar coupled phosphoryl groups. The31PLVM Ref.72-24WO; 340383 NMR spectra of vitrified monomers of both S305Pand Y310Pshow a broad Gaussian lineshape (FIG.44A), characteristic of an inhomogeneously broadened line from anisotropy of31P that may originate from NMR chemical shift,31P-31P homonuclear or31P-X heteronuclear dipolar couplings, and structural heterogeneity or disorder, to be discussed below. In contrast, the31P NMR spectra of the S305Pfibrils prepared in 100 mM or 1 M NaCl solution showed a dominant (42 %) spectral component with a significantly narrowed line (FIG.33B) compared to that of the monomer (FIG.44A). This result implies, even without in-depth analysis, that the phosphoryl groups experience higher order spatial arrangements of31P within the S305Pfibrils than in vitrified S305Pmonomers where the31P nuclei’s spatial distribution must be a powder average. However, a spectral decomposition is needed to dissect key details.

[0418] The31P NMR spectra were analyzed using a Gaussian / Lorentzian model with the DMfit software (shown in FIG.44A and FIGs.33A-33F). The monomer spectra of S305Pand Y310Pcould be fit with a Gaussian lineshape, centered at 0.9 and -3.9 ppm, and with a full width at half maximum (FWHM) of 800 Hz and 960 Hz, respectively. The deconvolution of the31P spectra of the S305PjR2R3-P301L fibrils, prepared in either 100 mM (FIG.33B) or 1 M (FIG.33C) salt solution resulted in three distinct spectral components centered around 4.4 ppm, 0.99 ppm, and -1.67 ppm with FWHM of 570 Hz, 220 Hz and 1010 Hz, respectively. These components represent three physically distinct phosphoryl group populations.

[0419] The narrowest spectral component centered at a chemical shift of 0.99 ppm is the dominant component (42%) adopting a Lorentzian line with a FWHM of 220 Hz—this is characteristic of a homogeneously broadened line originating from higher order spatial31P arrangements. The spectral component centered at 4.4 ppm is a Lorentzian with an FWHM of 570 Hz (10 %) and the one centered at -1.67 ppm a Gaussian with an FWHM of 1010 Hz (48 %). These two broader components were attributed to fibril populations with imperfect alignments or amorphous aggregates with powder orientation of the31P nuclei. The31P NMR spectra of the Y310Pfibrils, prepared in either 100 mM or 1M salt solution, showed no spectral component with a narrow and Lorentzian31P NMR linewidth.LVM Ref.72-24WO; 340383

[0420] Line broadening in solid state NMR spectra is mainly caused by anisotropic interactions from chemical shift anisotropy (CSA), homonuclear and heteronuclear dipolar couplings or structural heterogeneity and disorder. First, we considered the effect of CSA. CSA fits were performed with the CSA-MAS model using the DMfit 45 software (fit values shown in Table 3). The CSA and asymmetry parameter (η) values between S305Pmonomers and fibrils were similar, as well as between Y310Pfibrils and monomers, indicating CSA does not significantly contribute to the31P NMR line width. We next considered the anisotropy of heteronuclear dipolar couplings between1H-31P,13C-31P, and15N-31P. Applying heteronuclear decoupling on protons during acquisition would eliminate or reduce the effects of1H-31P couplings. Notably, line-narrowing effects upon proton decoupling was only measurable for the narrowest Lorentzian component of the31P NMR spectra of S305Pfibrils centered at 0.99 ppm with decoupling narrowing the FWHM from 400 to 220 Hz, while no effect was seen for the broader spectral components for the S305Pfibrils prepared in 100 mM NaCl solution (FIG.38A). These results show that1H-31P couplings are not the major contributors to the broad Gaussian31P NMR spectral population (FIGs.44A-44D and FIGs.38A-38B). The contribution from13C-31P and15N-31P couplings is negligible due to their smaller magnitudes at natural isotope abundance for13C and15N. Hence, heteronuclear dipolar couplings are not considered to be the primary source of spectral broadening for the broad components. Next, we compare homogeneous line broadening by31P T2 relaxation and inhomogeneous line broadening by31P-31P dipolar couplings in the31P spectra of S305P. We acquired CP-echo spectra with varying echo times (400 µs, 800 µs, and 2000 µs) that would yield different linewidths if they were homogeneously broadened by T2. The results showed that the31P spectral line shape and width of S305Pfibrils did not change visibly with different echo times (FIG.54), indicating that the observed line broadening is due to inhomogeneous broadening.

[0421] It was hypothesized that the primary line broadening mechanism was31P-31P homonuclear dipolar couplings or structural heterogeneity in which31P in different structural arrangements adopt slightly different chemical shifts. Random orientation of closely associated phosphoryl groups in space will result in a broadened Gaussian lineshape from a powder distribution of31P-31P dipolar vectors, as observed for Y310Pfibrils. The reduction in linewidth observed in the31P spectra of S305Pfibrils, observed at both NaCl conditions (FIGs.33B (100 mM NaCl) and FIG.33C (1 M NaCl)), cannotLVM Ref.72-24WO; 340383 be due to dynamic averaging of the31P-31P homonuclear dipolar couplings given that the sample is a vitrified solid. Instead, the narrowing may originate from enhanced structural homogeneity in neatly and stably packed fibrils that display minimal variation in the isotropic31P chemical shifts (different from CSA).

[0422] Another possibility is that an extended 1D arrangement of the phosphoryl groups results in the31P-31P dipolar vectors aligning unidirectionally along the given fibril axis. Even though θ, the angle between the dipolar vector and B0, will be different between different fibrils, the θ of adjacent phosphoryl groups within a given fibrils experiencing the strongest dipolar coupling remains unchanged. This arrangement should lower the effective dipolar coupling between multiple 1D-arranged31P spins, and hence resulting in dramatic31P line narrowing in the S305Pfibrils. This is in contrast to the31P NMR lineshape of Y310Pfibrils that remained as broad as that of vitrified monomers.

[0423] We further verified the effect of the geometrical arrangement of multiple31P spins by numerical simulations. To understand the orientational dependence of phosphoryl groups on the line shape of the31P spectra, we considered linear, zigzag and randomly clustered spatial arrangements of spins (see, FIGs.45A-45C), and performed numerical simulations to generate dipolar spectra under static conditions. The linear arrangement resulted in the narrowest linewidth, the zigzag and cluster arrangement showed a slightly greater broadening compared to the linear arrangement (FIG.45D). The dipolar couplings from the31P spins adopting closer distances than 4.8 A in the cluster configuration were turned off. This narrowed the31P NMR linewidth, but it remained still broader than that of the linear geometry. This underscored the finding that even if no chemical shift variation is present from structural heterogeneity and the closest31P-31P distance kept at 4.8 A, the geometrical arrangement of multiple31P spins still tune the31P NMR linewidth. We could further underscore this by comparing the31P NMR linewidth of 6 contiguously coupled31P spins as shown in FIG.45A, to that of 4 contiguously coupled31P spins and 2 contiguously coupled31P spins in the same linear arrangements (FIGs.40A-40D).

[0424] At a MAS frequency of 3 kHz, the orientation dependence of the dipolar vectors is averaged out and produces a single narrow peak in all simulated cases. ThisLVM Ref.72-24WO; 340383 is distinct from the experimental spectra of S305Pat 3 kHz MAS frequency (shown in FIG.37C), suggesting that the experimental dipolar coupling strength experienced by the31P spins is much greater than modeled here, potentially due to closer or higher order packing of the phosphoryl groups in their extended arrangements. This exercise demonstrates that a linear arrangement of dipolar coupled31P spins in an extended arrangement has special consequences on the resulting31P NMR spectrum that is distinct from a random distribution of dipolar coupled31P spins.

[0425] The experimental results together with the simulated31P NMR lineshape data suggested that the phosphoryl groups in S305Pfibrils are neatly and unidirectionally packed but not in Y310Pfibrils. In contrast, the phosphoryl group of Y310Pfibrils may experience significant structural heterogeneity and packing disorder within the fibrils.

[0426] Example 16 – MQ-SC Experiments

[0427] Multiple quantum spin count (also referred to herein as “MQ-SC”) experiments were performed to obtain direct evidence for phosphoryl group clustering and 1D ordering. The goal was to determine the minimal cluster size by analyzing the highest possible extracted MQ coherence order (also referred to herein as “MQCO”), which is equal to the number of coupled spins present within a spin cluster. In the structure of the phosphorylated jR2R3-301L fibrils, the positions of31P spins in each of S305pand Y310presidue of peptide molecule will dictate the spatial arrangement of31P spins in S305Por Y310Presidues within β-sheets. The different spin network geometries that MQ-SC experiments are sensitive to include parallel and anti-parallel arrangements, or trimer and protofibril arrangements with an internuclear distance of 4-5 Å. In an in- register, parallel β-sheet structure, the31P spins on adjacent tau fibrils should form a nearly linear arrangement with internuclear distances of ~4.8 Å if the phosphoryl groups associate with each other. However, if the phosphoryl groups repel or do not associate with each other, the protein region harboring the phosphoryl sites would be disordered and no significant MQ-CO would be observed.

[0428] MQ-SC experiments were performed on both fibril and monomer samples of S305pand Y310pat both 100 mM and 1M NaCl concentrations. We recently demonstrated the utility of the MQ-SC method on vitrified samples of adenosine triphosphate (ATP), amorphous calcium phosphate (ACP), crystalline hydroxyapatiteLVM Ref.72-24WO; 340383 (HAp) (Ca5(PO4)3OH, and simulated body fluid (mSBF) under DNP using SR218 pulse sequence to determine the number of31P spins present in the clusters. See, Nowotarski et al., J. Phys. Chem. Lett.2024, 7084-7094, which is hereby incorporated by reference in its entirety, and specifically for said demonstration of the utility of the MQ-SC method and multi-cosine fitting methods for extracting MQCOs. The SR218 sequence is an R- symmetry-based pulse sequence used for double quantum excitation and reconversion in the MQ-SC experiments, and is a super cycled version of R218, described as a π / 2- pulse sandwiched R218R2−18 pulse sequence that is minimally sensitive to CSA. It requires the radio frequency power to be set to equal to half the MAS frequency. The excitation time of 8 ms was found to be optimal when the MQ-SC experiment was performed on 1M NaCl S305pfibrils with different excitation times of SR218 sequence (shown in FIG.39A (9.6 ms), FIG.39B (16 ms), and FIG.40B (8 ms)). The excitation time of 8 ms was used during MQ-SC experiments on all fibrils / monomers in this study. The phase of the excitation block was incremented in a phase step 2π / j (j=number of experiments) to extract the multi-quantum coherences, and all the remaining phases in the experiment were kept constant.

[0429] FIGs.34A-34D illustrate the MQ-SC profiles of even and odd spin-counting and the corresponding coherence orders extracted for S305pat 100 mM NaCl (FIG. 34A) and 1M NaCl (FIG.34B) conditions as well as Y310pat 100 mM NaCl (FIG.34C) and 1M NaCl (FIG.34D) conditions. Here, we utilized multi-cosine fitting to extract the MQCOs, as demonstrated in a recent study of our group (Nowotarski et al., 2024), that relies on the knowledge that the spin counting profile takes the form of multi-cosine waveforms as elucidated by the Average Hamiltonian Theory described by Olyer and Tycko. Oyler et al., J. Phys. Chem. B 2002, 106(33), 8382–8389, which is hereby incorporated by reference in its entirety. The spectra of S305pfibrils prepared at 100 mM (FIG.34A) and 1M NaCl (FIG.34B) concentration showed the highest (MQCOs) up to four. In contrast, the maximum MQCO observed for Y310pfibrils was two (FIGs.34C- 34D), which can originate from a pair of dipolar coupled phosphoryl groups, which was initially believed to suggest an absence of in-register stacking of phosphates in Y310pfibrils.

[0430] Additional experimentation further demonstrated the narrow component in the S305Pspectra is the dominant spectral component, and was found to have MQCOs upLVM Ref.72-24WO; 340383 to 4, while the two broader components of S305p fibrils did not exhibit MQCO beyond 2. The same multi-cosine fitting of FIGs.34A-34D were used for FIGs.55A-55D, respectively, to extract the MQCOs. The S305Pfibrils prepared at 100 mM (FIG.55A) and 1M NaCl (FIG.55B) concentration both yielded MQCOs of 4 above error. In contrast, the maximum MQCO observed for Y310Pfibrils was 3 (FIGs.55C-55D). This suggests a weaker in-register stacking propensity of phosphoryl groups in Y310Pfibrils as compared to S305Pfibrils. Control MQ-SC experiments were performed on monomeric S305Pand Y310Ppeptides not subjected to aggregation reactions. The MQ- SC profiles of the monomers show random fluctuations (displayed in FIGs.51A (S305P) and 51B (Y310P)) as a function of phase increment of the excitation block, and no MQCO could be identified. Even though an MQCO of 3 in Y310Pfibrils is less than that of 4 in S305P fibrils, the phosphoryl group stacking in Y310Pfibrils still has greater order than in vitrified peptide monomers that show powder orientation.

[0431] The extracted MQCO intensities and the error values corresponding to each sample of FIGs.55A-55D are listed in Table 7, below. Because MQ-SC profiles will show fluctuations even in the absence of any MQCO, it is the intensity relative to the error bar that determines whether the MQCO count is real.

[0432] Table 7. MQCO intensities and the error values shown in FIGs.55A-55D. 100mM S305P1 M S305P100mM Y310P1 M Y310PMQCO or 2 29 28 29 28 28 28

[0433] To investigate the potential structure of S305pfibrils that give rise to an MQCO of four, we performed MQ-SC simulations with six31P geometry networks using the SIMPSON software. The SIMPSON simulations of MQ-SC were conducted usingLVM Ref.72-24WO; 340383 parameters identical to those employed in the experimental setup described above, with the spin systems of different geometries described in Table 4, below. Even and odd MQ- SC simulations were carried out for different geometries, such as a dimer, trimer, linear (also referred to herein as parallel) chain of six spins, and prototypical paired helical filament (PHF) geometry. This allowed for an exploration of the spin arrangements and their corresponding multi-quantum coherences in each of these geometries. A distance of 4.8 Å was used between two successive31P spins, corresponding to the inter-strand β-sheet distance.

[0434] FIGs.35A-35D present the results of the MQ-SC simulations that show the extracted MQCOs for different geometries. Additional depictions of the results are also provided in FIGs.46C-46E and FIGs.51A-51B and Table 4, below; additional discussion of these results is provided below in Example 16.1. Dimer geometry simulations (FIG.35A) produced MQCOs of 1 and 2 with odd and even spin counting simulations, respectively, as expected from the spin system. The MQSC simulations of the trimer spin system showed the MQCOs of 1, 3, and 2 for odd and even spin counting simulations, respectively, according to the expected MQCOs of 1, 2, and 3 in the trimer spin system. Six spin network simulations of the linear arrangement of31P spins showed the MQCOs of 1 and 3 with odd spin counting and MQCOs of 2 and 4 with even spin counting, respectively, displayed in FIG.35C, not exceeding the MQCO of 4 above the noise level in the MQCO charts. Spin counting simulations for a paired helical geometry displayed MQCOs of 2 and 4 with even spin counting simulations and 1 and 3 with odd spin counting simulations, respectively. Based on the comparison of simulation results of the linear chain comprising six spins and experimental results of S305p, it indicates that31P spins in each peptide molecule in the fibril are adopting a parallel in-register packing arrangement of the beta sheets. In addition, the simulations of the prototypical PHF geometry are also plausible structures, even though these structures lead to higher MQCOs than measured. With the decreasing probability of observing higher-order quantum coherence and the stringent requirement of T1 and T2 relaxation of31P NMR, weak dipolar couplings between31P spins as well as isotropy of31P chemical shifts to successfully carry out MQ-SC experiments, it is exceedingly difficult to observe MQCOs exceeding four, regardless of the defect-free order number of phosphoryl groups. Therefore, a measured MQCO of four in fibrils made of phosphorylated S305 tau peptides implies that there is in-register parallel β-sheetLVM Ref.72-24WO; 340383 stacking with neat phosphoryl group ordering, while the data is also consistent with prototypical PHF geometries. In contrast, Y310Pfibrils did not show MQCOs of more than two, indicating a lack of parallel in-register stacking. The MQ-SC experiments show that the phosphoryl groups form tightly ordered stacks that reinforce the supramolecular fibril assembly despite the net negative charge of the phosphoryl groups that were assumed to experience repulsive forces. Our observation suggests that water-mediated, H-bonded, clustering of phosphoryl groups is favorable, that in turn impacts the folding and aggregation pathway and resulting fibril property.

[0435] The MQ-SC results are consistent with a 1D array of the phosphoryl groups in S305pfibrils that lead to line narrowing of the31P spectra, and random distribution of phosphoryl groups in Y310pfibrils causing broad line widths. Control MQ-SC experiments were performed on monomeric S305pand Y310ppeptides not subject to aggregation. The MQ-SC profiles of the monomers show random fluctuations (displayed in FIG.41A (S305pmonomers) and FIG.41B (Y310pmonomers)) as a function of phase increment of the excitation block, and accordingly no MQCO could be counted upon Fourier Transformation. This is expected since there is no local ordering in the monomer samples in the absence of fibrillization. We emphasize that MQ-SC is the only method to offer direct insight into the number of ordered phosphates in extended structures, sort of directly visualizing the spatial ordering of phosphoryl groups. Table 4. Description of the Spin Systems used for SIMPSON Simulations of FIGs.35A- 35D. Dipolar bij / (2π) αPC βPC γPCLVM Ref.72-24WO; 340383 dipolar 46 -22.3 -180.0 90.0 0.0 dipolar 56 -178.2 -180.0 90.0 0.0LVM Ref.72-24WO; 340383 dipolar 46 -20.7 90.3 90.4 0.0 dipolar 56 -167.1 89.9 90.2 0.0LVM Ref.72-24WO; 340383 dipolar 12 -178.2 90.0 90.0 0.0 dipolar 13 -22.3 90.0 90.0 0.0FIGs.45A-45D. Dipolar bij / (2π) αPC βPC γPCLVM Ref.72-24WO; 340383 dipolar 45 -178.2 179.6 90.0 0.0 dipolar 46 -22.3 179.6 90.0 0.0LVM Ref.72-24WO; 340383 dipolar 45 -178.2 103.0 90.0 0.0 dipolar 46 -22.3 41.7 90.0 0.0

[0438] To investigate the assembly structure of phosphoryl groups in S305p fibrils that give rise to an MQCO of 4, we performed MQ-SC simulations of31P arrangements of 6 to 8 spins using the SIMPSON software. The SIMPSON simulations of MQSC were conducted using parameters identical to those employed in the experiment for31P spin networks of different geometries (Tables 4 and 5, above). Even and odd MQ-SC simulations were carried out for a trimer, linear chain of 6 and 8 spins, and a triple-stack of a prototypical protofibril geometry. This allowed for the exploration of31P spin arrangements and their corresponding MQCOs in each of these geometries. A distance of 4.8 Å was used between stacked31P spins corresponding to the inter-strand β-sheet spacing.

[0439] The resolved cryo-EM structure of the jR2R3-P301L tau peptide fibril studied here, without phosphorylation of sites 305 or 310, show the closest inter-tau distance at site 305 within the protofibril to be at least 21 Å spaced apart by counter strands between them, while ongoing cryo-EM structure studies of the phosphorylated P301L jR2R3 structure suggests a similarly long31P -31P distance within protofibrils contained within the same cross section. The results for the 21 Å case are shown in FIG.46F and FIGs.51A-51B. The trimer geometry simulations (FIG.46C) produced MQCOs of 1, 2 and 3, a linear 6 spin arrangement showed MQCOs of up to 4, and a linear 8 spin arrangement of31P spins showed MQCOs of up to 5. Spin counting simulations for the protofibril geometry in a triple stack, and the31P -31P distance of 21 Å between tau strands within the protofibrils of the same cross section (FIG.46F and FIG.51B) displayed MQCOs of up to 3, suggesting that the MQCOs were not affected by distant31P -31P spacings between protofibrils (shown in Table 6, below). We also verified that small changes in the chemical shifts did not affect the MQCOs in the simulation of the triple stack of protofibrils (FIG.51B) However, the MQCOs would be reduced with larger variations in the chemical shifts. Close31P -31P spacings in protofibrils rivaling the b- sheet spacing of 4.8 Å would render it difficult to discern between a linear stack of 6LVM Ref.72-24WO; 340383 peptides vs a triple-stack of protofibril pairs packed within 5 Å between tau strands. Still, the numerical simulation of MQCOs showed that31P spins were adopting a parallel in- register packing arrangement of at least six tau molecules neatly stacked in a β-sheet if the inter protofibril31P -31P distance was larger than 5 Å. Even if the inter protofibril31P -31P distance was < 5 Å, an MQCO of 4 implies parallel in-register and neat packing of at least three phosphoryl groups. In other words, it is difficult to observe MQCOs beyond 4 in biological samples, unless the31P spin arrangement adopts higher order packing. This was especially true for vitrified samples that have shorter T2 relaxation times and for dipolar couplings smaller than 180 Hz between31P spins at 4.8 Å separation. Hence, an MQCO of 4 in S305Pfibrils implies that the in-register parallel β-sheet stacked fibrils are stabilized by neat phosphoryl group stacking with high ordering. Furthermore, phosphoryl groups may be reinforcing tau association even in the absence of strict spatial ordering, given that the Y310P fibrils are more stable than jR2R3-P301L fibrils according to the GdnHCl assay, despite the lack of long-range spatial ordering of the phosphoryl groups in Y310p fibrils.

[0440] All experimental evidence taken together showed that the phosphoryl groups associate and reinforce the supramolecular fibril assembly despite the net negative charge of the phosphoryl groups. We suggest that water-mediated, H-bonded, phosphoryl groups form extended phosphoryl group “wires” that reinforce the tau fibrils. To obtain experimental evidence, we examined the correlation of31P spectral components with water.2D1H-31P HETCOR experiments were performed on S305p monomers and fibrils prepared in 100 mM NaCl to establish the1H-31P correlations between water and the phosphoryl groups (FIGs.44C-44D). The 2D1H-31P HETCOR spectra with 200 ^s CP contact time (τCP) shows a narrow signal along the31P dimension for the S305p fibril samples and broader signal for the monomer samples, consistent with earlier discussed31P 1D NMR lineshapes. The broad31P component correlates with1H at 4.0-4.5 ppm corresponding to bulk water, while only the narrow31P component observed in the S305Pfibril sample shows correlations with an additional1H species centered at ~5.5 ppm. Given that bulk water’s1H resonance is expected at 4.0- 4.5 ppm, the correlation with this1H signal corresponds to interaction between the P- OH- of the phosphoryl group and surrounding bulk water. The downfield shifted1H signal at 5.5 ppm indicates water protons in exchange with acidic water protons. It isLVM Ref.72-24WO; 340383 described in the literature that water protons directly associated with phosphoric acids and phosphoryl groups show a downfield chemical shift from bulk water chemical shift due to chemical exchange between water and the exchangeable OH protons in H3PO4, H2O, and H3O+ at 8.6 ppm. We suggest that the1H signal at 5.5 ppm correspond to hydrogen-bonded water protons bridged between two phosphoryl group oxygens, and hence more acidic. It is also possible that water sandwiched between phosphoryl groups are in exchange with phosphoryl group protons that are expected to yield a1H chemical shifts of around 5-6 ppm. The1H-31P peak centered at 4 ppm along the1H dimension is observed at all contact times tested for cross-polarization (CP) down to τCP = 150 μs, while the 5.5 ppm peak requires τCP = 200 μs or longer, suggesting that31P components corresponding to the narrow spectral linewidth is further away from or interacts with fewer protons compared to the31P belonging to the broader spectral component.

[0441] The1H peak at 4 ppm broadened along the31P spectral dimension, suggesting that both the narrow and broad31P components of the S305p jR2R3 P301L fibril were in proximity of bulk water. However, the 5.5 ppm1H peak did not broaden along the31P dimension, even for long CP contact time up to τCP = 800 μs. This is a surprising result suggesting that this water proton sandwiched between the phosphoryl group does not interact with disordered phosphoryl groups that are not arranged in an extended 1D wire, and do not readily exchange with bulk water protons. The chemical shift value together with the features of the HETCOR data establishes that this resonance can be assigned to a discrete population of water molecules bridging adjacent phosphoryl groups.

[0442] Table 6. Description of the Spin Systems used for SIMPSON Simulations of FIGs.51A-51B. Six-spin jR2R3-21ÅLVM Ref.72-24WO; 340383 Dipolar bij / (2π) αPC βPC γPC

[0443] Example 16.2 – Molecular Dynamics Simulations

[0444] Still, we did not know the geometrical arrangement and energetics of such a water-mediated extended phosphoryl “wire” and hence turned to molecular dynamics (MD) simulations performed in explicit water. We considered an in vitro paired helical filament-like structure from Alzheimer’s disease (PDB ID #7QL4) as a representative and suitable model structure for the beta-sheet packing and spacing (displayed in FIG. 47A). The exact fibril fold structure is not as important for the purpose of studying the role of water molecules that stabilize phosphoryl groups along the fibril axis. Serine at residue 305 was converted to dianionic phosphoserine in the serine phosphorylated fibrils. A previous study investigating the effect of phosphorylation on fibril aggregation propensity reported the dianionic phosphoryl group to be the dominant species at pH = 7.5, monoanionic at pH = 3.6, and neutral at pH = 1.1. See, Valette, et al., Phosphorylation as a Tool to Modulate Aggregation Propensity and to Predict FibrilLVM Ref.72-24WO; 340383 Architecture, (2012), ChemBioChem 13(2), 271-281, which is hereby incorporated by reference in its entirety. Given the pKa of 5.6 for the monoanionic-dianionic equilibrium of phosphoserine reported in the literature and considering that the samples were studied at pH = 7.4, the dianionic form of the phosphoryl group was expected to predominate. Energy minimization of the structure, followed by NVT and NPT equilibration simulations, was carried out. A 1 ns trajectory was run to capture the water structure, dynamics, bonding of waters with and surrounding the phosphoryl groups. The details of the MD simulation are provided in the “Exemplary Experimental Aspects for Examples 12-16” section, below. When screening through the frames in the trajectory of energetically favorable structures, water molecules have been found to form bridges between the phosphoryl groups by establishing hydrogen bonds (the hydrogen bond criterion is provided in SI 2.0). The three-body P-P-P angle between the phosphorous atoms in neighboring layers is important to assess the linearity of the phosphoryl groups in the fibril (FIG.47B). The distribution highlights that the linear arrangements are stabilized and enforced by water mediated hydrogen bonds between dianionic phosphoryl groups.

[0445] For comparative analysis of the hydration water populations interacting with the phosphoryl groups, three distinct water groups were defined: bulk waters located at least 10 Å from the protein; neighboring waters within 5 Å of residue 305; and bridging waters which were hydrogen bonded to the oxygens connecting two adjacent phosphoryl groups. Various parameters were calculated for all three types of water: the rotational anisotropy autocorrelation function (ACF) ^((^), hydrogen bond lifetime correlation function ^(^), and the differential three-body angle distributions ^3b(^) – ^3bpure(^). The definitions of the rotational anisotropy autocorrelation function (ACF) ^((^), and hydrogen bond lifetime correlation function ^(^) are provided in SI. The rotational anisotropy ACF provides insights on restrictions in structural dynamics of water in confined regions. Rotational anisotropy decay times and hydrogen bond lifetimes for different waters were extracted by fitting the corresponding ACFs and hydrogen bond lifetime correlation functions to exponential functions. Bulk water data was fitted with a single exponential function while bridging and neighboring waters required a triexponential fit, yielding three distinct lifetimes corresponding to three different populations. The fitting plots are shown in FIGs.47C–47D. Bridging watersLVM Ref.72-24WO; 340383 showed an order of magnitude longer decay times of ^2 = 2.2 ps with 30% and ^3 = 97.0 ps with 13% of its populations compared to neighboring waters (^1 = 0.18 ps, ^2 = 1.8 ps with 52% and 42% populations) and bulk waters with a single ^ = 0.53 ps. Similarly, bridging waters showed significantly longer hydrogen bond lifetimes of ^1=0.51 ps, ^2=16.8 ps, ^3=101.9 ps compared to both bulk and neighboring waters. Water structuring also plays a crucial role in driving the interactions between waters and the fibril surface. The three-body (O-O-O) angle distribution, ^3b(^) has been shown to capture changes in water structure near the fibril surface and was calculated for all waters within a cutoff distance of 3.5 Å from a phosphoryl group. The differential three- body angle distribution, Δ^3b(^) revealed that neighboring waters exhibit higher population in the tetrahedral range (100° < ^ < 120°), slightly exceeding that of bridging waters.

[0446] Bulk waters exhibited characteristic icosahedral (^ = 64°) populations along with a positive feature near ^ = 50°, which corresponds to the relative population of over coordinated water molecules. The population of tetrahedral angles for each water group was computed by integrating over the tetrahedral region of P3b(^) (100° < ^ < 120°). Pure water at 310 K was observed to be 21.40% tetrahedral, bulk water was observed to be 20.63% tetrahedral, neighboring waters are observed to be 18.25% tetrahedral, and water bridges were observed to be 16.51% tetrahedral. The higher tetrahedrality observed in neighboring waters relative to bridging waters based on ΔP3b(^) suggest structured water formation around the phosphoryl groups, likely driven by local hydrophobic effects commonly referred to as "wrap waters." These two kinds of waters, bridging and neighboring waters, could be attributed to the two proton chemical shifts of 4 and 5.5 ppm observed in 2D1H-31P HETCOR spectra, with the down field chemical shift of 5.5 ppm corresponding to the bridging waters and the 4 ppm population to neighboring waters. However, rapid exchange between neighboring and bulk water renders them spectroscopically indistinguishable. The MD-derived observations are also consistent with the conclusions drawn from experiments, namely that the bridging water has a long lifetime and does not readily exchange with bulk water. Most importantly, the computational study demonstrated that water molecules mediate the formation of extended phosphoryl "wires" by forming hydrogen bonds between phosphoryl groups.LVM Ref.72-24WO; 340383

[0447] These MQ-SC results reinforce the earlier stated hypothesis that the phosphoryl groups in S305p fibrils form a stabilizing 1D wire that is responsible for, or at least contributes to, generating31P NMR features with dramatic line narrowing. The phosphoryl groups in Y310p fibrils still associated favorably and increased fibril stability, but did not form an extended 1D arrangement. We emphasize that MQ-SC is the only known method that can offer direct insight into the number of ordered phosphates in extended structures and the spatial ordering of phosphoryl groups that are spectroscopically indistinguishable.

[0448] Example 17 – Water Structure Directing the Assembly of Tau Peptides

[0449] This example relates to processing and methods for their use in the diagnosis, research, and treatment of neurodegenerative diseases. Specifically, by controlling the structure of ordered water for the formation of tau protein into conformationally directed morphologies. More specifically, this example provides a process for generating fibers with specific disease relevant shape through the ordering of water that facilitates the elongation of tau into filaments. This property ensures high specificity towards aggregated pathogenic tau fibrils. Thus, a rational design of disease relevant folding can occur when the conditions of ordered water structure are met.

[0450] Tau peptides have been used to study the effect of water structure which has been a key component of the stacking process. By developing the conditions necessary to generate ordered water through temperature, motion, container material properties light irradiation, and electro-magnetic fields, we have tuned the properties and behavior of tau aggregation. In aspects, the conditions necessary to generate ordered water structure include: selection of a vial and its material (glass nafion film), selection of temperature (37°C, RT, 4°C), rate of motion (shaking, rotating, flow), selection of the light source (IR, microwave), or a combination thereof.

[0451] A critical tau fragment spanning D295 to V313 of 4R tau was used, this fragment forms the junction between the R2 and R3 repeat domains and contains the P301L mutation (jR2R3-P301L) (e.g., SEG ID NO: 5). This peptide has been shown to form fibrils adopting pathological folds and displaying prion properties. We select from the multiple conditions stated above in this Example 17, and test each of their effects on a tau peptide jR2R3-P301L (tau295-313) that readily aggregates into fibrils with prion-LVM Ref.72-24WO; 340383 like seeding competency. Influencing the water and therefore the solvent-protein interactions, a critical step in developing 4R tauopathies including Corticobasal Degeneration (CBD), Progressive Supranuclear Palsy (PSP), and Argyrophilic Grain Disease (AGD) can be achieved.

[0452] We confirmed the ordering of monomers within the jR2R3-P301L fiber (tau295-313) using negative stain TEM to show how each condition causes a distinct morphology, specifically inducing crystallinity (see, FIG.48). Micro Electron Diffraction of tau peptide crystals, show...

Claims

LVM Ref.72-24WO; 340383 WE CLAIM:

1. A method for generating a prion-like mini-tau peptide, the method comprising: identifying a filament-core region of a disease-specific tau fibril; selecting a structural motif of the filament-core region, wherein the structural motif contributes to a critical protein fold of the disease-specific fibril; synthesizing a mini-tau peptide comprising an amino acid sequence encoding the structural motif; wherein the mini-tau peptide mimics the critical protein fold of the disease- specific tau fibril, thereby generating a prion-like mini-tau peptide.

2. The method of claim 1, wherein the filament-core region of the disease- specific tau fibril comprises a microtubule-binding region, a proline-rich region, a N-terminus domain, or a combination thereof.

3. The method of claim 1 or claim 2, wherein the filament-core region is or comprises the microtubule-binding domain of the disease-specific tau fibril.

4. The method of any preceding claim, wherein the filament-core region is or comprises a R2 region and / or a R3 region of the disease-specific tau fibril.

5. The method of any preceding claim, wherein the prion-like min-tau peptide comprises an aggregation-prone domain.

6. The method of any preceding claim, wherein the structural motif has 75% or greater sequence identity of SEQ ID NO: 1 (VQIVYK).

7. The method of any preceding claim, wherein the structural motif has 75% or greater sequence identity of SEQ ID NO: 4 (VQIINK).LVM Ref.72-24WO; 340383 8. The method of any preceding claim, wherein the prion-like mini-tau peptide further comprises a linking sequence.

9. The method of claim 8, wherein the linking sequence is a flexible linker or a cleavable linker.

10. The method of claim 8 or 9, wherein the linking sequence comprises glycine, serine, or a combination of glycine and serine.

11. The method of any one of claims 8-10, wherein the linking sequence comprises between 1 and 20 amino acid residues, optionally between 1 and 10 amino acid residues, optionally between 1 and 5 amino acid residues.

12. The method of any one of claims 8-11, wherein the linking sequence comprises 3 glycine residues, optionally wherein the linking sequence is GGG.

13. The method of any preceding claim, wherein the disease of the disease- specific fibril is a 4R tauopathy.

14. The method of any preceding claim, wherein the disease of the disease- specific fibril is Alzheimer’s Disease, Pick’s Disease, chronic traumatic encephalopathy, corticobasal degeneration, progressive supranuclear palsy, argyrophilic grain disease, aging-related tau astrogliopathy, globular glial tauopathy, familial British dementia, familial Danish dementia, and limbic- predominant neuronal inclusion body 4R tauopathy, or any combination thereof.

15. The method of any preceding claim, wherein the method further comprises: tuning the prion-like mini-tau peptide to enforce a desired conformation and / or improve fibril formation of the prion-like mini-tau peptide.LVM Ref.72-24WO; 340383 16. The method of claim 15, wherein the tuning comprises introducing a post- translational modification, a point mutation, a water-structuring additive, or any combination thereof, to the prion-like mini-tau peptide.

17. The method of claim 15 or 16, wherein the tuning step comprises introducing a point mutation to at least one residue of the prion-like mini-tau peptide, optionally wherein the point mutation comprises a site-specific proline to leucine substitution mutation.

18. The method of any one of claims 15-17, wherein the tuning step comprises a post-translational phosphorylation of at least one residue of the prion-like mini-tau peptide, optionally wherein the least one residue comprises a serine residue of the structural motif.

19. The method of any one of claims 15-18, wherein the tuning comprises contacting the prion-like mini-tau peptide with a complement strand.

20. The method of claim 19, wherein the complement strand has 75% or greater sequence identity of: SEQ ID NO: 14 (GGGQVEVKSEKLDFKDRVQSKIGSLDNITH); SEQ ID NO: 15 (LDFKDRVQSKIGSLDNGGGHKLTFRE); or SEQ ID NO: 16 (FKDRVQSKIGSLDNITHVPG).

21. The method of any one of claims 15-20, wherein the desired conformation is a U-shaped conformation.

22. The method of any one of claims 15-20, wherein the desired conformation is a strand-loop-strand (SLS) conformation.

23. The method of any preceding claim, the method further comprising evaluating an aggregation competency of the prion-like mini-tau peptide.LVM Ref.72-24WO; 340383 24. The method of claim 23, wherein the evaluating step comprises high throughput screening.

25. The method of any preceding claim, wherein the prion-like mini-tau peptide has 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of: SEQ ID NO: 2 (DNIKHVLGGGSVQIVYKPV); SEQ ID NO: 5 (DNIKHVPGGGSVQIVYKPV); SEQ ID NO: 6 (DNIKHVPGG); SEQ ID NO: 7 (DNIKHVLGG); SEQ ID NO: 8 (VPGGGSVQIVYKPV); SEQ ID NO: 9 (VLGGGSVQIVYKPV); SEQ ID NO: 10 (DNIKHVPG); SEQ ID NO: 11 (DNIKHVLGGGSPhosVQIVYKPV); SEQ ID NO: 12 (DNIKHVLGGGSVQIVYPhosKPV); SEQ ID NO: 17 (VQIVYKPGGGNHKLTF); or SEQ ID NO: 18 (KVQIINKPGGGKLTFRE).

26. A composition comprising the prion-like mini-tau peptide of any preceding claim and a pharmaceutically acceptable carrier.

27. A method for generating a prion-like mini-tau fibril, the method comprising: selecting a prion-like mini-tau peptide generated by the method of any one of claims 1-25; and introducing a plurality of the prion-like mini-tau peptide to a solution, thereby generating a prion-like mini-tau fibril.

28. A method for generating a prion-like mini-tau fibril, the method comprising: generating a prion-like mini-tau peptide, wherein the generating step comprises:LVM Ref.72-24WO; 340383 identifying a filament-core region of a disease-specific tau fibril; selecting a structural motif of the filament-core region, wherein the structural motif contributes to a critical protein fold of the disease-specific fibril; synthesizing a mini-tau peptide comprising an amino acid sequence encoding the structural motif; wherein the mini-tau peptide mimics the critical protein fold of the disease-specific tau fibril, introducing a plurality of the prion-like mini-tau peptide to a solution, thereby generating the prion-like mini-tau fibril.

29. The method of claim 27 or 28, wherein the solution comprises heparin.

30. The method of any one of claims 27-29, wherein the solution comprises at least one cofactor and / or a complement strand.

31. The method of claim 30, wherein the solution comprises a complement strand, and wherein the complement strand has 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of: SEQ ID NO: 14 (GGGQVEVKSEKLDFKDRVQSKIGSLDNITH); SEQ ID NO: 15 (LDFKDRVQSKIGSLDNGGGHKLTFRE); or SEQ ID NO: 16 (FKDRVQSKIGSLDNITHVPG).

32. The method of any one of claims 27-31, wherein the method further comprises stabilizing the mini-tau fibrils to support the critical protein folds of the plurality of the synthetic mini-tau peptides.

33. The method of claim 32, wherein the solution comprises a complement strand, and the stabilizing step comprises modifying the complement strand toLVM Ref.72-24WO; 340383 promote selective synthesis of the synthetic mini-tau fibrils that are specific to a tauopathy.

34. The method of claim 33, wherein the modifying the complement strand step comprises designing the complement strand to mimic the conformation of the disease-specific tau fibril.

35. The method of any one of claims 27-34, wherein the solution comprises a complement strand and the solution is characterized by a molar ratio of about 4:1:10, optionally, 1:1:10, 2:1:10, 3:1:10, 5:1:10, 10:1:10, 20:1:10, 30:1:10, 4:1:1, 4:1:2, 4:1:3, 4:1:4, 4:1:5, 4:1:10, 4:1:20, 4:1:30, or any sub-range thereof, of the prion-like mini-tau peptide:heparin:the complement strand.

36. The method of any one of claims 27-35, wherein the disease-specific tau fibril is associated with a tauopathy, and the tauopathy is Alzheimer’s Disease, Pick’s Disease, chronic traumatic encephalopathy, corticobasal degeneration, progressive supranuclear palsy, argyrophilic grain disease, aging-related tau astrogliopathy, globular glial tauopathy, familial British dementia, familial Danish dementia, and limbic-predominant neuronal inclusion body 4R tauopathy, or any combination thereof.

37. The method of any one of claims 30-36, wherein the at least one cofactor comprises: heparin; RNA; arachidonic acid; polyphosphates; phosphate ions; sulfate ions; a salt, optionally, wherein the salt comprises NaCl or MgCl2; or any combination thereof.LVM Ref.72-24WO; 340383 38. The method of any one of claims 27-37, wherein the prion-like mini-tau fibril comprises an active fibril surface, optionally wherein the active fibril surface comprises at least one prion-like mini-tau peptide having an aggregation- prone domain.

39. The method of any one of claims 27-38, the method further comprising evaluating one or more morphological feature of the prion-like mini-tau fibril.

40. The method of claim 39, wherein the morphological feature comprises fibril formation, average internuclear distance distributions for each prion-like mini- peptide of the prion-like mini-tau fibrils; homogeneity of the prion-like mini-tau fibrils, multiple quantum coherence order (MQCO), or any combination thereof.

41. The method of claim 39, wherein the morphological feature comprises average internuclear distance distributions for each prion-like mini-peptide, wherein the average internuclear distance distribution is about 4.8 angstroms.

42. The method of claim 39 wherein the morphological feature comprises MQCO, optionally wherein the prion-like mini-tau fibril is characterized by an average of at least 431P spins within a spin cluster.

43. The method of any one of claims 38-42, wherein the evaluating step comprises electron-electron double resonance spectroscopy (DEER).

44. A composition comprising the prion-like mini-tau fibril generated by the method of any one of claims 27-43 and a pharmaceutically acceptable carrier.

45. A method for generating full-length pathogenic tau fibrils, the method comprising: selecting one or more prion-like mini-tau fibril generated by the method of any one of claims 27-43;LVM Ref.72-24WO; 340383 providing a first set of tau monomers; and contacting a plurality of the prion-like mini-tau fibrils with the first set of tau monomers in a solution, wherein the prion-like mini-tau fibrils act as templates that stabilize the tau monomers to adopt a disease-specific tau fibril core formation, thereby generating full-length pathogenic tau fibrils.

46. A method for generating full-length pathogenic tau fibrils, the method comprising: selecting one or more prion-like mini-tau fibril comprising a plurality prion- like mini-tau peptides wherein at least a portion of the prion-like mini-tau peptides mimic a critical protein fold of a disease-specific tau fibril; providing a first set of tau monomers; and contacting a plurality of the prion-like mini-tau fibrils with the first set of tau monomers in a solution to induce aggregation of the first set of tau monomers, wherein the prion-like mini-tau fibrils act as templates that stabilize the tau monomers to adopt a disease-specific tau fibril core formation, thereby generating full-length pathogenic tau fibrils.

47. The method of claim 45 or 46, wherein the solution is characterized by a molar ratio of about 1:1 of the prion-like mini-tau fibrils:the first set of tau monomers.

48. The method of claim 45 or 46, wherein the solution is characterized by a molar ratio of about 4:1 of the synthetic mini-tau fibrils:the first set of tau monomers.

49. The method of any one of claims 45-48, wherein the solution further comprises at least one cofactor.LVM Ref.72-24WO; 340383 50. The method of any one of claims 45-49, wherein the method is performed in vitro, optionally wherein the method comprises bacterial or mammalian cells.

51. The method of any one of claims 45-50, the method further comprising: tuning the structure of the full-length pathogenic tau fibrils to enforce a desired conformation and / or improve aggregation of the first set of tau monomers.

52. The method of claim 51, wherein the tuning comprises adjusting the pH, temperature, cofactor, or a combination thereof, of the solution of the contacting step.

53. The method of any one of claims 45-52, the method further comprising evaluating one or more morphological feature of the full-length pathogenic tau fibrils for the disease-specific tau fibril core formation.

54. The method of claim 53, wherein the morphological feature comprises fibril formation, average internuclear distance distributions for each prion-like mini- peptide of the prion-like mini-tau fibrils; homogeneity of the prion-like mini-tau fibrils, or any combination thereof.

55. The method of claim 53 or 54, wherein the evaluating step comprises electron-electron double resonance spectroscopy (DEER).

56. The method of claim 55, further comprising distinguishing distinct signals, wherein the distinct signals comprise a DEER signal, a noise, an artifact, one or more full-length pathogenic tau fibrils, or any combination thereof.

57. The method of claim 55, wherein the distinguishing step comprises a frequency pattern recognition, wherein the frequency pattern recognitionLVM Ref.72-24WO; 340383 comprises the use of a discretized continuous wavelet transform (CWT) and a structure similarity index measure (SSIM) analysis.

58. A method for generating a subsequent generation of full-length pathogenic tau fibrils, the method comprising: combining the full-length pathogenic tau fibrils of any one of claims 45-57 with a second set of tau monomers in a second solution to induce aggregation of the second set of tau monomers, wherein the prion-like mini-tau fibrils act as templates that stabilize the tau monomers to adopt a disease-specific tau fibril core formation, thereby generating a subsequent generation of the full-length pathogenic tau fibrils.

59. The method of claim 58, further comprising sonicating the full-length pathogenic tau fibrils prior to the combining step.

60. A composition comprising the full-length pathogenic tau fibrils of any one of claims 45-58.

61. Use of the composition of any one of claims 26, 44, or 60 for evaluating a tauopathy, optionally wherein the tauopathy is Alzheimer’s Disease, Pick’s Disease, chronic traumatic encephalopathy, corticobasal degeneration, progressive supranuclear palsy, argyrophilic grain disease, aging-related tau astrogliopathy, globular glial tauopathy, familial British dementia, familial Danish dementia, and limbic-predominant neuronal inclusion body 4R tauopathy, or any combination thereof.

62. Use of the method of any preceding claim for evaluating a tauopathy, optionally wherein the tauopathy is Alzheimer’s Disease, Pick’s Disease, chronic traumatic encephalopathy, corticobasal degeneration, progressive supranuclear palsy, argyrophilic grain disease, aging-related tau astrogliopathy, globular glial tauopathy, familial British dementia, familialLVM Ref.72-24WO; 340383 Danish dementia, and limbic-predominant neuronal inclusion body 4R tauopathy, or any combination thereof.

63. A kit for generating prion-like mini-tau fibrils, the kit comprising at least one prion-like mini-tau peptide generated from the method of any one of claims 1- 25.

64. A kit for generating prion-like mini-tau fibrils, the kit comprising at least one prion-like mini-tau peptide wherein each prion-like mini-tau peptide comprises a sequence having 75% or greater sequence identity (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100% sequence identity) of: SEQ ID NO: 2 (DNIKHVLGGGSVQIVYKPV); SEQ ID NO: 11 (DNIKHVLGGGSPhosVQIVYKPV); SEQ ID NO: 12 (DNIKHVLGGGSVQIVYPhosKPV); SEQ ID NO: 17 (VQIVYKPGGGNHKLTF); or SEQ ID NO: 18 (KVQIINKGGGKLTFRE); or any combination thereof.

65. A kit for the development of tauopathy therapeutics, imaging agents, and / or other technologies for the identification and treatment of tauopathies, the kit comprising a full-length pathogenic tau fibril of any one of claims 45-59.

66. A kit for the development of tauopathy therapeutics, imaging agents, and / or other technologies for the identification and treatment of tauopathies, the kit comprising: 0N4R isoform tau protein (UniProt accession number (P10636-8)); a tau protein fragment having 75% or greater sequence identity (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100% sequence identity) of SEQ ID NO: 13 (NVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGS VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLLVM Ref.72-24WO; 340383 DNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLS NVSSTGSIDMVDSPQLATLADEVSASLAKQGL); a tau protein fragment having 75% or greater sequence identity (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100% sequence identity) of SEQ ID NO: 23 (NVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVLGGGS VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSL DNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLS NVSSTGSIDMVDSPQLATLADEVSASLAKQGL); a spin labeled full-length pathogenic tau fibril, or a fragment thereof, of any preceding claim; or an isotope labeled full-length pathogenic tau fibril, or a fragment thereof, of any preceding claim; or any combination thereof.

67. A synthetic phosphorylated amyloid fibril comprising a structurally ordered cluster of peptide monomers, wherein at least a portion, optionally all, of the peptide monomers comprise a site-specific phosphorylated residue, wherein the phosphoryl groups of the site-specific phosphorylated residues are aligned in a linear, stacked conformation along the axis of the synthetic phosphorylated amyloid fibril.

68. The synthetic phosphorylated amyloid fibril of claim 67, wherein the spacing between the phosphoryl groups of the site-specific phosphorylated residues of adjacent strands is between about 4.5 Å to about 4.9 Å (e.g., about 4.5 Å, about 4.6 Å, about 4.7 Å, about 4.8 Å, or about 4.9 Å).

69. The synthetic phosphorylated amyloid fibril of claim 67 or 68, wherein the structurally ordered cluster comprises three or more peptide monomers, wherein each peptide monomer independently comprises a site-specific phosphorylated residue.LVM Ref.72-24WO; 340383 70. The synthetic phosphorylated amyloid fibril of claim 69, wherein the phosphorylated residues of the three or more peptide monomers are aligned in a linear, stacked conformation across adjacent peptide monomers.

71. The synthetic phosphorylated amyloid fibril of any one of claims 67-70, wherein at least one of the peptide monomers is derived from the microtubule- binding repeat (MTBR) region of tau.

72. The synthetic phosphorylated amyloid fibril of any one of claims 67-71, wherein each of the peptide monomers is independently derived from the microtubule-binding repeat (MTBR) region of tau.

73. The synthetic phosphorylated amyloid fibril of any one of claims 67-72, wherein the site-specific phosphorylated residue is a phospho-serine, a phospho-threonine, or a phospho-tyrosine.

74. The synthetic phosphorylated amyloid fibril of any one of claims 67-73, wherein each of the peptide monomers independently is a full-length tau monomer, a tau fragment, or independently comprises a prion-like mini-tau peptide.

75. The synthetic phosphorylated amyloid fibril of any one of claims 67-74, wherein at least one, optionally all, of the peptide monomers is a tau fragment.

76. The synthetic phosphorylated amyloid fibril of claim 75, wherein the tau fragment has 75% or greater sequence identity (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100% sequence identity) of SEQ ID NO: 41 (IKHVPGGGSVQIVYKPVDLSKVTSKSGSLGNIHHKPGGGQVEVKSEKLDFK DRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAE).LVM Ref.72-24WO; 340383 77. The synthetic phosphorylated amyloid fibril of any one of claims 67-76, wherein the phosphoryl groups of the site-specific phosphorylated residues form hydrogen-bonded arrays, electrostatically stabilized networks, or any combination thereof.

78. The synthetic phosphorylated amyloid fibril of claim 77, wherein the hydrogen- bonded arrays and / or electrostatically stabilized networks contribute to fibril stability and enhanced seeding activity.

79. A method for generating a synthetic phosphorylated amyloid fibril comprising: synthesizing a peptide monomer, or a peptide fragment thereof, comprising an aggregation-prone domain; performing a post-translational phosphorylation of at least one residue of the peptide monomer, or the peptide fragment thereof; combining a plurality of the peptide monomer, or the peptide fragment thereof, to form a mixture of peptide monomers and / or peptide fragments thereof; optionally, tuning at least one condition of the combining step to promote the formation of a structurally ordered cluster of peptide monomers; thereby generating a synthetic phosphorylated amyloid fibril.

80. The method of claim 79, wherein the at least one condition of the tuning step comprises pH adjustment, buffer selection, salt selection, salt concentration, temperature, agitation, cofactor addition, or any combination thereof.

81. A method for stabilizing synthetic amyloid fibril formation of a plurality of peptide monomers, the method comprising:LVM Ref.72-24WO; 340383 combining a plurality of peptide monomers, or peptide fragments thereof, in a solution to form a mixture of peptide monomers, wherein at least a portion, optionally all, of the plurality of peptide monomers comprise an aggregation-prone domain; wherein the solution comprises a phosphate buffer or polyphosphates to promote the formation of a structurally ordered cluster of peptide monomers, resulting in a first-generation synthetic amyloid fibril, thereby stabilizing synthetic amyloid fibril formation.

82. The method of claim 81, wherein at least a portion, optionally all, of the plurality of peptide monomers, or peptide fragments thereof, comprise a post- translational phosphorylation of at least one residue.

83. The method of claim 81 or claim 82, wherein at least a portion, optionally all, of the plurality of peptide monomers, or peptide fragments thereof, do not comprise a post-translational phosphorylation.

84. The method of any one of claims 81-83, wherein at least a portion, optionally all, of the plurality of peptide monomers are full-length tau monomers, or fragments thereof, or comprise a prion-like mini-tau peptide.

85. The method of any one of claims 81-84, wherein the structurally ordered cluster of peptide monomers is characterized by in-register stacking of the peptide monomers.

86. The method of any one of claims 81-85, wherein the structurally ordered cluster of peptide monomers comprises synthetic phosphorylated amyloid fibrils.

87. The method of claim 85, wherein the synthetic phosphorylated amyloid fibrils comprise long-range phosphate ordering.LVM Ref.72-24WO; 340383 88. The method of any one of claims 81-87, wherein the solution comprising a phosphate buffer or polyphosphates promotes the formation of the structurally ordered cluster of peptide monomers by providing a phosphate-containing environment, and wherein the phosphate-containing environment induces the formation of synthetic phosphorylated amyloid fibrils, wherein the synthetic phosphorylated amyloid fibrils comprise long-range phosphate ordering.

89. The method of any one of claims 81-88, the method further comprising: introducing the first-generation synthetic amyloid fibril to the solution of the combining step; and repeating the steps of claim 79, resulting in a subsequent generation synthetic amyloid fibril.

90. A method of confirming phosphate or phosphoryl group ordering in the synthetic phosphorylated amyloid fibril or the prion-like mini-tau fibril of any preceding claim, the method comprising: analyzing the fibril using31P solid-state NMR lineshape analysis, Multiple Quantum Spin Counting (MQSC) with Magic Angle Spinning (MAS), or a combination thereof; indicating the presence of ordered phosphate clusters by evaluating the presence of: a characteristic, narrow,31P solid-state NMR spectral linewidth; a Multiple Quantum Coherence Order (MQCO) of 3 or more; or a combination thereof; thereby confirming phosphate or phosphoryl group ordering in the synthetic phosphorylated amyloid fibril or the prion-like mini-tau fibril.

91. An immunogen comprising the synthetic phosphorylated amyloid fibril or the prion-like mini-tau fibril of any preceding claim.LVM Ref.72-24WO; 340383 92. A method of eliciting an immune response in a subject, the method comprising administering to the subject the immunogen of claim 91.

93. The synthetic phosphorylated amyloid fibril or the prion-like mini-tau fibril of any preceding claim, wherein the fibril presents an epitope characterized by three or more phosphorylated residues or phosphate moieties arranged in a structurally ordered, fibrillar conformation.

94. The synthetic phosphorylated amyloid fibril or the prion-like mini-tau fibril of claim 93, wherein the structurally ordered, fibrillar conformation is characterized by in-register stacking of the three or more phosphorylated residues or phosphate moieties.

95. A monoclonal antibody formulated to selectively bind the epitope of claim 94.

96. A monoclonal antibody formulated to selectively bind an epitope of a synthetic phosphorylated amyloid fibril or a prion-like mini-tau fibril, wherein the epitope is characterized by three or more phosphorylated residues or phosphate moieties arranged in a structurally ordered, fibrillar conformation.

97. The monoclonal antibody of claim 95 or 96, wherein the monoclonal antibody does not bind to non-fibrillar, singly phosphorylated, or non-phosphorylated peptides of the synthetic phosphorylated amyloid fibril or the prion-like mini- tau fibril.

98. The monoclonal antibody of any one of claims 95-97, wherein the monoclonal antibody comprises an analytical tag.

99. A method of generating a monoclonal antibody, the method comprising: immunizing a subject with the immunogen of claim 91; screening sera or hybridoma supernatants;LVM Ref.72-24WO; 340383 selecting the sera or the hybridoma supernatants characterized by selective binding properties, wherein the selective binding properties comprise binding to multivalent phosphorylated fibrils and do not bind to monomeric or singly phosphorylated peptides; and isolating clones of the sera or the hybridoma supernatants characterized by the selective binding properties; thereby generating the monoclonal antibody.

100. A pharmaceutical composition comprising the monoclonal antibody of any one of claims 95-99 and a pharmaceutically acceptable carrier.

101. Use of the monoclonal antibody of claim 98 in a diagnostic evaluation of a biological sample, the method comprising: contacting a biological sample with the monoclonal antibody of claim 98; and evaluating a subsample of the biological sample having the analytical tag.

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