TAU protein modification pattern in biofluids for the diagnostic of tauopathies

Abstract

Methods of differentiating and quantifying modifications in the tau protein in bio-fluids to detect, classify, and treat a plurality of tauopathies are disclosed. The disclosed methods include liquid chromatographic mass spectrometric analyses on isolated tau protein fragments to identify and quantify deamidated or isomerized asparagines or aspartic acids indicative of a neuropathology associated with a tauopathy.

Description

TITLE OF THE INVENTIONTAU PROTEIN MODIFICATION PATTERN IN BIOFLUIDS FOR THE DIAGNOSTIC OF TAUOPATHIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No. 63 / 571 ,862 filed on March 29, 2024, which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.MATERIAL INCORPORATED-BY-REFERENCE

[0003] The Sequence Listing, which is a part of the present disclosure, includes a computer readable form comprising nucleotide and / or amino acid sequences of the present invention. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0004] The present disclosure generally relates to systems and associated methods of diagnosing tauopathies.BACKGROUND OF THE INVENTION

[0005] Many neurological disorders are characterized by protein aggregation in the brain. The brain aggregation of Tau protein characterized neuropathology in patients with Alzheimer's Disease (AD), Progressive Supranuclear Palsy, Corticobasal Degeneration (CBD), Pick disease (PiD), Chronic Traumatic Encephalopathy (CTE), Argyrophilic grain disease (AGD), AGD and ageing-related tau astrogliopathy (ARTAG), Frontotemporal Lobar degeneration (FTLD) associated to MAPT (Microtubule- Associated Protein Tau) mutation or behavioral variant frontotemporal dementia (bvFTD) and others defined as tauopathies. Tauopathies are divided into primary andsecondary tauopathies. Primary tauopathies are neurological disorders primarily due to tau aggregation and include most of the tauopathies. AD is a secondary tauopathy characterized by tau accumulation consecutive to the initial aggregation of Abeta42 deposition in the brain. AD can be diagnosed using the relative decrease of Abeta42 level in biofluids in response to the corresponding enrichment of this peptide as aggregate in the brain. Cerebrospinal fluid (CSF) Abeta42 biomarkers can be used to differentiate AD from other neurological disorders including primary tauopathies. In addition to Abeta42-derived biomarkers, tau hyperphosphorylation at several residues (T217, T231 , T181 , T205, S208, T153, T111 ) in the CSF and blood plasma is elevated in AD. AD tau hyperphosphorylation is detected simultaneously with brain Abeta aggregation even at an asymptomatic stage when tau aggregation is not detectable by Tau PET (Positron Emission Tomography) imaging.

[0006] Recently, the measures of tau peptides MTBR-tau275 and MTBR-tau282 from tau microtubule binding region (MTBR) relative to the level of total tau from the Mid-domain have been proposed for the differential diagnosis of CBD versus PSP. However, these measures are restricted to the identification of CBD participants confirmed by neuropathology and are typically enabled to identify tau abnormality in PSP participants and participants diagnosed with corticobasal syndrome (CBS, presumable with CBD tau pathology).

[0007] CryoEM of tau aggregates extracted from the brain has revolutionized the understanding of tau aggregation in many tauopathies by resolving tau domain structures forming the core of the aggregates. CryoEM data confirmed tau in aggregates is divided into the domain included in the aggregates defined as "core” and the soluble domains surrounding the core called "fuzzy coat”. The tau sequences included in the core differ depending on the considered tauopathy which implies the tau soluble domains in the fuzzy coat also vary depending on the tauopathy. To date, CryoEM has been used to resolve tau aggregate structures from AD, CTE, PSP, CBD, PiD, AGD, or MAPT 10+16 in human brain extracts.

[0008] According to tau half-life measurement by SILK in the brain and CSF, soluble tau remains between 20 to 40 days in the central nervous system. Conversely,tau in the aggregates should remain in the brain for an infinite amount of time unless aggregated tau partially solubilizes over time. During this period, the tau fuzzy coat may be exposed to various modifications including phosphorylation, ubiquitination, acetylation, methylation, or deamidation. Extensive tau deamidation has been reported in brain AD tau aggregates on residues localized on the fuzzy coat.

[0009] Protein deamidation spontaneously occurs through the hydrolysis of the amide group on the side chain of Asparagine or Glutamine. There are no known repair mechanisms for this modification. Newly synthesized proteins are free of this post- translational modification which progressively appears on the protein in a timedependent manner. Because the deamidation process requires the formation of a succinimide intermediate before hydrolysis, deamidation rates depend on the residues surrounding the asparagine (mostly on Cter), as well as the secondary and tertiary structure of proteins. Used as a molecular clock, the measure of deamidation rates can provide an estimation of protein half-life and turnover. Asparagine deamidation half-live ranges from a few days to more than 1 year depending on protein sequence and structure. Several months-old proteins with protein domains having a conformational- free structure would have deamidated asparagines to a rate theoretically ranging from 10% to 100%. Similar rates would be expected on glutamine for several years to decades-old proteins, considering the longer deamidation half-life of glutamine residues. When the protein domain contains the asparagine residue as a fixed conformation, the formation of the succinimide intermediate is highly restrained due to the steric restrictions, and the asparagine deamidation process is reduced.SUMMARY OF THE INVENTION

[0010] Among the various aspects of the present disclosure is the provision of methods for differentiating and quantifying tau protein modifications as sampled from bio-fluids for the detection, classification, and treatment of a plurality of tauopathies.One aspect of the present disclosure provides for a method for recognizing and classifying a tauopathy comprising: sampling at least one of a full-length tau protein or a truncated tau protein; digesting the at least one full-length tau protein or the at least one truncated tau protein into a plurality of fragments by employing one of LysC, LysN, orTrypsin; precipitating the plurality of tau protein fragments; performing liquid chromatographic mass spectrometric analyses on one or more of the tau protein fragments to differentiate and quantify the deamidated or isomerized asparagines or aspartic acids contained within the tau protein fragment; comparing the deamidation of the asparagines or isomerization of aspartic acids within the tau protein fragment amongst one another to determine if a tauopathy is present, which type of tauopathy is present, or the stage of progression of the tauopathy.

[0011] In some embodiments, the tauopathy is a neurological disorder.

[0012] In some embodiments, the tauopathy is Alzheimer’s Disease.

[0013] In some embodiments, the sample used for sampling is taken from cerebrospinal fluid.

[0014] In some embodiments, the sample used for sampling is taken from biofluids or plasma.

[0015] In some embodiments, the precipitation is immunoprecipitation.

[0016] In some embodiments, the deamidation of asparagines and / or isomerization of aspartic acids within the tau protein fragment are compared to determine if a tauopathy is present, which type of tauopathy is present, or the stage of progression of the tauopathy. The resulting characterization of a tauopathy is communicated to a healthcare provider for use in diagnosis and / or for the selection of one or more appropriate treatment plans to treat, slow the progression of, or prevent one or more tauopathies.

[0017] Another aspect of the present disclosure provides for a method for recognizing and classifying a tauopathy comprising: sampling, from cerebrospinal fluid or plasma, at least one of a full length tau protein or a truncated tau protein; digesting the at least one full length tau protein or the at least one truncated tau protein into a plurality of fragments by employing Trypsin; precipitating the plurality of tau protein fragments; performing liquid chromatographic mass spectrometric analyses on one or more of the tau protein fragments in order to differentiate and quantify the phosphorylation of one or more of T111 , T153, T181 , T205, S208, T217, and T231 taupeptides; classifying a patient’s tauopathy or lack thereof based on the differentiation and quantification of the phosphorylation of the at least one or more tau peptides; selecting a treatment or prevention plan for one or more tauopathies given the classification based on phosphorylation results used by a healthcare provider or used to inform a healthcare provider.

[0018] Other objects and features will be in part apparent and in part pointed out hereinafter.DESCRIPTION OF THE DRAWINGS

[0019] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0020] FIG. 1 is a schematic of various tauopathies and their relationships to one another related to particular peptide sequences.

[0021] FIG. 2 is a table of the prediction of tau asparagine residues deamidation half-life according to recombinant peptides deamidation studies. These are the peptide sequences to be considered for the measure of tau deamidation.

[0022] FIG. 3 is a graph and associated table that shows the deN167 iso3 / iso4 ratio differentiates controls from participants clinically identified as PSP or CBS with an Area Under ROC curve of 0.925.

[0023] FIG. 4A is a graph of % deN167 / N167isoaii from CSF samples of stratified participant groups with distinct pathologies.

[0024] FIG. 4B is a graph of % deN167 iso3 from CSF samples of stratified participant groups with distinct pathologies.

[0025] FIG. 4C is a graph of deN167 iso3 / (iso1 + iso2) ratio from CSF samples of stratified participant groups with distinct pathologies.

[0026] FIG. 4D is a graph of deN167 iso3 / iso4 ratio from CSF samples of stratified participant groups with distinct pathologies.

[0027] FIG. 4E is a graph of CSF 243-254 peptide levels obtained by HJ32.11 tauimmunoprecipitation from CSF samples of stratified participant groups with distinct pathologies.

[0028] FIG. 4F is a graph of CSF 260-267 peptide levels obtained by HJ32.11 tau immunoprecipitation from CSF samples of stratified participant groups with distinct pathologies.

[0029] FIG. 4G is a graph of % isoD252 obtained by HJ32.11 tau immunoprecipitation from CSF samples of stratified participant groups with distinct pathologies.

[0030] FIG. 4H is a graph of deN255243-256 peptide level amounts from CSF samples of stratified participant groups with distinct pathologies.

[0031] FIG. 4I is a graph of % deN255 243-256 peptide levels obtained by HJ32.11 tau immunoprecipitation from CSF samples of stratified participant groups with distinct pathologies.

[0032] FIG. 4J is a graph of % deN255 243-256 iso2 peptide levels obtained by HJ32.11 tau immunoprecipitation from CSF samples of stratified participant groups with distinct pathologies.

[0033] FIG. 4K is a graph of % deN255 243-257 peptide levels obtained by HJ32.11 tau immunoprecipitation from CSF samples of stratified participant groups with distinct pathologies.

[0034] FIG. 4L is a graph of % deN265 260-267 peptide levels obtained by HJ32.11 tau immunoprecipitation from CSF samples of stratified participant groups with distinct pathologies.

[0035] FIG. 4M is a graph of % deN265 iso2 260-267 peptide levels obtained by HJ32.11 tau immunoprecipitation from CSF samples of stratified participant groups with distinct pathologies.

[0036] FIG. 4N is a graph of % deN265 260-267 peptide levels obtained by HJ32.11 tau immunoprecipitation using HJ34.8 antibodies from CSF samples of stratified participant groups with distinct pathologies.

[0037] FIG. 40 is a graph of % deN279 peptide levels obtained by HJ32.11 tau immunoprecipitation using HJ34.8 antibodies from CSF samples of stratified participant groups with distinct pathologies.

[0038] FIG. 4P is a graph of % deN286aii peptide levels obtained by HJ32.11 tau immunoprecipitation using HJ34.8 antibodies from CSF samples of stratified participant groups with distinct pathologies.

[0039] FIG. 4Q is a graph of % deN286 / N286 iso4 peptide levels obtained by HJ32.11 tau immunoprecipitation using HJ34.8 antibodies from CSF samples of stratified participant groups with distinct pathologies.

[0040] FIG. 4R is a graph of 3R / 4R 275 peptide ratio levels obtained by HJ32.11 tau immunoprecipitation using HJ34.8 antibodies from CSF samples of stratified participant groups with distinct pathologies.

[0041] FIG. 4S is a graph of 3R / 4R 282 peptide ratio levels obtained by HJ32.11 tau immunoprecipitation using HJ34.8 antibodies from CSF samples of stratified participant groups with distinct pathologies.

[0042] FIG. 5 is a graph and associated table of T217 hyperphosphorylation ratio that differentiates controls from participants clinically identified as PSP or CBS with an AUC of 0.887.

[0043] FIG. 6A is a graph of CSF pT111 / T111 ratio from CSF samples of stratified participant groups with distinct pathologies.

[0044] FIG. 6B is a graph of CSF pT 153 / T 153 ratio from CSF samples of stratified participant groups with distinct pathologies.

[0045] FIG. 6C is a graph of CSF pT181 / T181 ratio from CSF samples of stratified participant groups with distinct pathologies.

[0046] FIG. 6D is a graph of CSF pS199 / S199 ratio from CSF samples of stratified participant groups with distinct pathologies.

[0047] FIG. 6E is a graph of CSF pS202 / S202 ratio from CSF samples of stratified participant groups with distinct pathologies.

[0048] FIG. 6F is a graph of CSF pT205 / T205 ratio from CSF samples of stratified participant groups with distinct pathologies.

[0049] FIG. 6G is a graph of CSF pS208 / S208 ratio from CSF samples of stratified participant groups with distinct pathologies.

[0050] FIG. 6H is a graph of CSF pT217 / T217 ratio from CSF samples of stratified participant groups with distinct pathologies.

[0051] FIG. 6I is a graph of CSF pT231 / T231 ratio from CSF samples of stratified participant groups with distinct pathologies.

[0052] FIG. 7 is a schematic representing the hypothesis that depending on the type of tau aggregates, protein domains staying in the "fuzzy coat” are different and that asparagines in the fuzzy coat can be deamidated with time but asparagines in the cores cannot.

[0053] FIG. 8 is a schematic showing the strategy to monitor CSF tau deamidation rate on 5 tau asparagines by extracting appropriate pools of tau fragments using immunoprecipitation.

[0054] FIG. 9A is a graph of the amounts of CSF tau fragments recovered by HJ32.11 immunoprecipitation in samples from stratified participants with distinct pathologies.

[0055] FIG. 9B is a graph of the amounts of CSF tau fragments recovered by HJ34.8 antibody treatment in samples from stratified participants with distinct pathologies.

[0056] FIG. 9C is a graph of the amounts of CSF tau fragments recovered by HJ32.11 immunoprecipitation and HJ34.8 antibody treatment in samples from stratified participants with distinct pathologies.

[0057] FIG. 9D is a schematic of the relevant tau peptide sequences and how HJ32.11 and HJ34.8 interacts with the sequences.

[0058] FIG. 10A is a graph of the ratio of CSF pT217 / T217 from participants by group analysis of Tau TANGLES and Tau SILK groups and related subsets with distinctpathologies.

[0059] FIG. 10B is a graph of the ratio of CSF pT205 / T205 from participants by group analysis of Tau TANGLES and Tau SILK groups and related subsets with distinct pathologies.

[0060] FIG. 11A is a graph of % deN167 isoS / total from HJ8.5 / HJ8.7 / Tau1 extract from CFS samples from participants with distinct pathologies.

[0061] FIG. 11 B is a graph of the deN167 iso3 / iso4 ratio from H J8.5 / H J8.7 / Tau1 extract from CFS samples from participants with distinct pathologies.

[0062] FIG. 11 C is a graph of % deN265 from HJ34.8 extract from CFS samples from participants with distinct pathologies.

[0063] FIG. 11 D is a graph of % deN279 from HJ34.8 extract from CFS samples from participants with distinct pathologies.

[0064] FIG. 11 E is a graph of % deN286 from HJ34.8 extract from CFS samples from participants with distinct pathologies.

[0065] FIG. 11 F is a schematic representing the distinct isomer patterns differentiating distinct SILK and TANGLES pathologies.

[0066] FIG. 12 is a schematic representing a deamidation hypothesis adjusted with results from the study on proteolytic resistant fragments by Mass Spectrometry (MALDI-TOF).

[0067] FIG. 13 is an LC-MS spectrum of LysC CSF digest of N 167 (K.GQANATRIPAK.T [164, 174]) focusing on quantifying the N167 deamidation rate.

[0068] FIG. 14A is a plot of avg. deN167 / N167 iso3 / iso4 vs. avg. deN167 / N167 (%) iso3 from CSF samples from CFS samples identifying the TANGLES cohort.

[0069] FIG. 14B is a plot of avg. deN167 / N167 iso3 / iso4 vs. avg. deN167 / N167 (%) iso3 from CSF samples from CFS samples identifying the QC-M, QC-H, and QC-L cases.

[0070] FIG. 14C is a plot of avg. deN167 / N167 iso3 / iso4 vs. avg. deN167 / N167 (%) iso3 from CSF samples from CFS samples identifying the Tau SILK cohort.

[0071] FIG. 15A is a pair of LC-MS spectra plots of D252 peptide (LQTAPVPMPDLK).

[0072] FIG. 15B is a magnified section of the D252 peptide spectra plots in FIG. 15A showing peaks representative of deamidated (isoD) isomers.

[0073] FIG. 16A is a graph of the measure of N255 (LQTAPVPMPDLKNVK) modification in tryptic digest from CSF tau species extracted with HJ32.11 .

[0074] FIG. 16B is the raw LC-MS spectra data used to quantify N255 modification as seen in FIG. 16A.

[0075] FIG. 16C is another graph of the measure of N255 (LQTAPVPMPDLKNVK) modification in tryptic digest from CSF tau species extracted with HJ32.11.

[0076] FIG. 16D is the raw LC-MS spectra data used to quantify N255 modification as seen in FIG. 16C.

[0077] FIG. 16E is a graph of the measure of 14N from N255 in tryptic digest from CSF tau species extracted with HJ32.11 .

[0078] FIG. 16F is a graph of the measure of 13C15N from N255 (LQTAPVPMPDLK) in tryptic digest from CSF tau species extracted with HJ32.11 .

[0079] FIG. 17A is an LC-MS spectra plot of N265 (K.IGSTENLK.H [260, 267], SEQ ID NO: 18) that identifies deaminated isomers (iso2, iso3).

[0080] FIG. 17B is a plot representing relative abundance of N265 isomers in a set of replicates as quantified by the LC-MS plot seen in FIG. 17A.

[0081] FIG. 17C is a plot of avg. de / N265 / N265 vs. avg. 14N / 13C15N 260-267 from N265 CSF tau species extracted with HJ32.11 .

[0082] FIG. 17D is another representation of the plot of avg. de / N265 / N265 vs. avg. 14N / 13C15N 260-267 from N265 CSF tau species extracted with HJ32.11 seen in FIG. 17C. showing a cluster of negative controls in the bottom left, and the best fit curve line for iso2 samples.

[0083] FIG. 18A is an LC-MS spectra plot of N279 (K.VQIINK.K [275, 280]) thatidentifies deaminated isomers.

[0084] FIG. 18B is a plot representing relative abundance of N279 isomers in a set of replicates as quantified by the LC-MS plot seen in FIG. 18A.

[0085] FIG. 18C is a plot of avg. de / N279 / N279 vs. avg. 14N / 13C15N 275-280 from N279 CSF tau species extracted with HJ32.11 .

[0086] FIG. 18D is another representation of the plot of avg. de / N279 / N279 vs. avg. 14N / 13C15N 275-280 from N279 CSF tau species extracted with HJ32.11 seen in FIG. 18C. showing a cluster of negative controls in the bottom left, and the best fit curve line for pathological samples.

[0087] FIG. 19A is an LC-MS spectra plot of N286 (K.LDLSNVQSK.C [282, 290], SEQ ID NO:20) that identifies deaminated isomers.

[0088] FIG. 19B is a plot representing relative abundance of N286 isomers in a set of replicates as quantified by the LC-MS plot seen in FIG. 19A.

[0089] FIG. 19C is a plot of avg. de / N286 / N286 vs. avg. 14N / 13C15N 282-290 from N286 CSF tau species extracted with HJ32.11 .

[0090] FIG. 19D is another representation of the plot of avg. de / N286 / N286 vs. avg. 14N / 13C15N 282-290 from N286 CSF tau species extracted with HJ32.11 seen in FIG. 19C. showing a cluster of negative controls in the bottom left, and the best fit curve line for pathological samples.

[0091] FIG. 20A is a plot of deN265 / N265 vs. deN279 / N279 from the overall cohort of CSF patient samples as described in Example 9.

[0092] FIG. 20B is a plot of deN286 / N286 vs. deN279 / N279 from the overall cohort of CSF patient samples as described in Example 9.

[0093] FIG. 20C is a plot of deN265 / N265 vs. deN286 / N286 from the overall cohort of CSF patient samples as described in Example 9.

[0094] FIG. 21 is an LC-MS spectra of K ESPLQTPTE GSEEPGSETSDAK.S [45, 67] peptide showing the normal aspartic acid (D) peak (right peak) and the D isomer peak (isoD, left peak).

[0095] FIG. 22A is a graph of the dN167 / N167 ratio in CSF samples from participants in healthy controls (HC), corticobasal syndrome (CBS) + progressive supranuclear palsy Richardson Syndrome (PSP-RS), and Parkinson Disease (PD) groups.

[0096] FIG. 22B is a graph of the dN167 / N167 ratio in CSF samples from participants in HC, CBS, PSP-RS, and PD groups.

[0097] FIG. 22C is a graph of the isoD / D 45-67 ratio in CSF samples from participants in HC, CBS + PSP-RS, and PD groups.

[0098] FIG. 22D is a graph of the isoD / D 45-67 ratio in CSF samples from participants in HC, CBS, PSP-RS, and PD groups.

[0099] FIG. 23A is a graph of the % isoD252 243-254 in CSF of stratified groups with distinct pathologies using the HJ32.11 protocol.

[0100] FIG. 23B is a graph of the % deN255 243-256 in CSF of stratified groups with distinct pathologies using the HJ32.11 protocol.

[0101] FIG. 23C is a graph of the % deD255 243-257 in CSF of stratified groups with distinct pathologies using the HJ32.11 protocol.

[0102] FIG. 23D is a graph of the % deN265 260-267 in CSF of stratified groups with distinct pathologies using the HJ32.11 protocol.

[0103] FIG. 23E is a graph of the % deN265 260-267 in CSF of stratified groups with distinct pathologies using the HJ34.8 protocol.

[0104] FIG. 23F is a graph of the % deN279 in CSF of stratified groups with distinct pathologies using the HJ34.8 protocol.

[0105] FIG. 23G is a graph of the % deN286 in CSF of stratified groups with distinct pathologies using the HJ34.8 protocol.

[0106] FIG. 23H is a graph of the 14N / 13C15N 243-254 ratio in CSF of stratified groups with distinct pathologies using the HJ32.11 protocol.

[0107] FIG. 23I is a graph of the 14N / 13C15N 260-267 ratio in CSF of stratified groups with distinct pathologies using the HJ32.11 protocol.

[0108] FIG. 23J is a graph of the 3R / 4R 275 ratio in CSF of stratified groups with distinct pathologies using the HJ34.8 protocol.

[0109] FIG. 23K is a graph of the 3R / 4R 282 ratio in CSF of stratified groups with distinct pathologies using the HJ34.8 protocol.

[0110] FIG. 24 is a schematic showing the four isomers of asparagine following deamidation.DETAILED DESCRIPTION OF THE INVENTION

[0111] The present disclosure is based, at least in part on the discovery that differential tau asparagine deamidation patterns may be used to identify distinct categories of tauopathies and their relative progressions. This is in part because asparagine-containing peptides located in the fuzzy coat of tau aggregates are extensively modified by deamidation, and asparagine-containing peptides located in the core of tau aggregates are less likely deamidated due to their involvement in the quaternary structure of tau filaments.

[0112] The measure of tau deamidation status is performed by measuring the ratio between signals obtained for the deamidated species at a given asparagine residue normalized to the signal of an unmodified peptide with intact asparagine and the corresponding non deamidated species. FIG, 24 shows the 4 different isomers due to the asparagine modification, including L-isoAsp, L-Asp, D-isoAsp and D-Asp isomers. Non-deamidated and deamidated species can be simultaneously differentiated and quantified by LC-MS analysis thanks to a mass shift increase of about 0.984 Da after deamidation and a change of hydrophobicity leading to different retention time on reverse-phase chromatography for deamidated peptides.

[0113] Tau asparagine deamidation pattern can be measured using asparagines deamidation status from residues located in the tau mid-domain region (N167), tau microtubule binding region (N255, N265, N279, N286, N327, N359, N368) or tau C- terminus (N381 , N410). The prediction of asparagine site turnover based on the study of synthetic peptides is used to predict tau asparagines prone to rapid deamidation (N167, N381 , N368, N279) and those with longer turnover (N265, N286, N410, N255, N296,N327). The abundance of deamidated tau species will be a function of the deamidation turnover of each respective site as well as the protein domain turnover and the protein domain conformation.

[0114] Most of the tau deamidation can be measured on full-length tau. Full- length tau is found primarily in brain and cell cultures using tau purification by chemical extraction of immunopurification preferentially with antibodies targeting epitopes not covering tau asparagines.

[0115] In biofluids or cell media, tau is truncated. The measure of tau asparagines deamidation can be performed on different tau fragments extracted using antibodies covering multiple domains of the tau sequence.

[0116] What follows are some non-limiting examples of measuring deamidation and or isomerization of tau asparagines and aspartic acids.

[0117] In one non-limiting example, deamidation on N167 can be measured on tau extracts obtained by immunoprecipitation (for example using HJ8.5, HJ8.7, Tau1 , or other antibodies targeting epitopes located in the Nter and mid domain of tau) or chemical extraction. Chemical extraction refers to a method that precipitates proteins but leaves soluble tau fragments which can be enriched by solid phase extraction. N167 and deamidated N167 isomers can be measured on peptides from the digestion of tau extract obtained by using LysC (to generate the peptide GQANATRIPAK (SEQ ID NO:1 ) and corresponding deamidated peptides isomers) or by LysN (to generate the peptide KGQANATRIPA (SEQ ID NO:2) and corresponding deamidated peptides isomers). Trypsin generates the peptide GQANATR (SEQ ID NO:3) which can be used for the measure, but this peptide is experimentally too hydrophilic to be accurately measured by reversed-phase liquid chromatography.

[0118] By way of another non-limiting example, the isomerization of aspartic acid D252 through succinimide formation and hydrolysis can be measured (see Example 9) within an LQTAPVPMPDL (SEQ ID NO:4) tau sequence.

[0119] By way of another non-limiting example, N255 and deamidated N255 isomers can be measured on tau extracts obtained by immunoprecipitation (for exampleusing HJ8.5, HJ8.7, Tau1 , or other antibodies targeting epitopes located in the Nter and mid domain of tau or using antibodies targeting the R1 MTBR domain as HJ32.11 ) or chemical extraction. Trypsin generates the peptide LQTAPVPMPDLK (SEQ ID NO:5) that does not carry an asparagine but instead carries an aspartic acid (D) residue that is used as a surrogate for the N255 non-modified sequence. The deamidation on N255 promotes a missed cleavage leading to the peptide LQTAPVPMPDLKdeNVK (SEQ ID NO:6), in which deN is a deamidated asparagine. The endogenous cleavage after V256 and the deamidation at N255 lead to the deamidated peptide LQTAPVPMPDLKdeNV (SEQ ID NO:7).

[0120] By way of another non-limiting example, N265 and deamidated N265 isomers can be measured on tau extracts obtained by immunoprecipitation (for example using HJ8.5, HJ8.7, Tau1 , or other antibodies targeting epitopes located in the Nter and mid domain of tau or using antibodies targeting the R1 MTBR domain as using HJ32.11 or HJ34.8) or chemical extraction. The trypsin and LysC digestions will generate the peptides IGSTENLK (SEQ ID NO:8) and IGSTEdeNLK (SEQ ID NO:8) and the LysN digestion KIGSTENLK (SEQ ID NO:9) and KIGSTEdeNLK (SEQ ID NO:9) which can be used to measure the N265 deamidation rate.

[0121] By way of another non-limiting example, N279 and deamidated N279 isomers can be measured on tau extracts obtained by immunoprecipitation (for example using HJ8.5, HJ8.7, Tau1 , or other antibodies targeting epitopes located in the Nter and mid domain of tau or using antibodies targeting the R1 MTBR domain as using HJ32.11 or HJ34.8) or chemical extraction. The trypsin and LysC digestions will generate the peptides VQIINK (SEQ ID NO: 10) and VQIIdeK (SEQ ID NO: 10) and the LysN digestion KVQIIN (SEQ ID NO:11 ) and KVQIIdeNL (SEQ ID NO:12) which can be used to measure the N279 deamidation rate.

[0122] Another non-limiting example includes that N286 and deamidated N286 isomers can be measured on tau extracts obtained by immunoprecipitation (for example using HJ8.5, HJ8.7, Tau1 or other antibodies targeting epitopes located in the Nter and mid domain of tau or using antibodies targeting the R1 MTBR domain as using HJ32.11 or HJ34.8) or chemical extraction. The trypsin and LysC digestions will generate thepeptides LDLSNVQSK (SEQ ID N0:13) and LDLSdeNVQSK (SEQ ID N0:13) and the LysN digestion KLDLSNVQS (SEQ ID NO:14) and KLDLSdeNVQS (SEQ ID NO:14) which can be used to measure the N279 deamidation rate.MOLECULAR ENGINEERING

[0123] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0124] The terms "heterologous DNA sequence", "exogenous DNA segment" or "heterologous nucleic acid," as used herein, each refers to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling or cloning. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

[0125] Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

[0126] A “promoter” is generally understood as a nucleic acid control sequence that directs the transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates the transcription of an operably linked gene inresponse to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0127] A "transcribable nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit the translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).

[0128] The “transcription start site” or "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1 . With respect to this site, all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e. , further protein encoding sequences in the 3' direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

[0129] "Operably-linked" or "functionally linked" refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for anRNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects the expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0130] A "construct" is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.

[0131] A construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3'-untranslated region (3' UTR). Constructs can include but are not limited to the 5' untranslated regions (5' LITR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.

[0132] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".

[0133] "Transformed," "transgenic," and "recombinant" refer to a host cell or organism such as a bacterium, cyanobacterium, animal, or plant into which aheterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term "untransformed" refers to normal cells that have not been through the transformation process.

[0134] "Wild-type" refers to a virus or organism found in nature without any known mutation.

[0135] Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above-required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680- 688; Sanger et al. (1991 ) Gene 97(1 ), 119-123; Ghadessy et al. (2001 ) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and / or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.

[0136] Nucleotide and / or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned,the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity = X / Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0137] Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example, the exchange of Glu by Asp, Gin by Asn, Vai by He, Leu by He, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur / selenium- containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. The amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of these artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.

[0138] “Highly stringent hybridization conditions” are defined as hybridization at 65 DC in a 6 X SSC buffer (i.e. , 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (Tm) of a DNA duplexbetween the two sequences. If a particular duplex has a melting temperature lower than 65 DC in the salt conditions of a 6 X SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 DC in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence can be determined using the following formula: Tm = 81 .5 DC + 16.6(log10[Na+]) + 0.41 (fraction G / C content) - 0.63(% formamide) - (600 / I). Furthermore, the Tm of a DNA:DNA hybrid is decreased by 1 -1.5DC for every 1 % decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).

[0139] Host cells can be transformed using a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN- 10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated into the host cell genome.

[0140] Exemplary nucleic acids which may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA that is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

[0141] Host strains developed according to the approaches described herein can be evaluated by any number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN- 10: 0954523253).

[0142] Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNAinterference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing). Traits influential in defining optimal siRNA sequences include G / C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.Genome Editing

[0143] As described herein, Zbtb46 signals can be modulated (e.g., reduced, eliminated, or enhanced) using genome editing. Processes for genome editing are well known; see e.g. Aldi 2018 Nature Communications 9(1911 ). Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

[0144] For example, genome editing can comprise CRISPR / Cas9, CRISPR-Cpf1 , TALEN, or ZNFs. Adequate expression of Zbtb46 by genome editing can result in protection from autoimmune or inflammatory diseases, and promote a hot immune microenvironment in tumors.

[0145] As an example, clustered regularly interspaced short palindromic repeats(CRISPR) / CRISPR-associated (Cas) systems are a new class of genome-editing tools that target desired genomic sites in mammalian cells. Recently published type II CRISPR / Cas systems use Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes to a 20-nucleotide DNA sequence and immediately preceding an NGG motif recognized by Cas9 (thus, a (N)20NGG target DNA sequence). This results in a double-strand break three nucleotides upstream of the NGG motif. The double-strand break instigates either non- homologous end-joining, which is error-prone and conducive to frameshift mutations that knock out gene alleles, or homology-directed repair, which can be exploited with the use of an exogenously introduced double-strand or single-strand DNA repair template to knock in or correct a mutation in the genome. Thus, genomic editing, for example, using CRISPR / Cas systems could be useful tools for therapeutic applications for cancer therapy, particularly immunotherapy-resistant cancer, to target cells by the incorporation of Zbtb46 signals.

[0146] For example, the methods as described herein can comprise a method for altering a target polynucleotide sequence in a cell comprising contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein.

[0147] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0148] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to beobtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0149] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0150] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0151] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0152] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0153] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0154] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as nonlimiting examples.EXAMPLES

[0155] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure and thus can be considered to constitute examples of modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0156] The measurement of tau asparagine deamidation / tau aspartic acid isomerization as performed on both full-length tau and different tau fragments extracted using antibodies covering multiple domains of the tau sequence has multiple applications / embodiments including but not limited to the applications / embodiments discussed hereinafter.(1 ) Identification of AD-like deamidation profile linked to R3-R4 domains aggregation (3R+4R tauopathies).

[0157] One non-limiting embodiment includes the identification of an AD-like deamidation profile linked to R3-R4 domain aggregation (3R+4R tauopathies). That is, in AD, the fuzzy coat includes a large part of the tau sequence with N167, N255, N265, N279, and N286 expected to be part of the asparagines with less conformational restriction, able to form succinimide intermediate and be deamidated after succinimide hydrolysis. Consistently, N255, N265, N279, and N286 are abnormally deamidated in CSF samples from participants identified as AD and with high CSF tau hyperphosphorylation at T217 (>7.5%), abnormal CSF MTBR-tau243 and abnormal CSF 42 / 40 ratio. The abnormality status is defined in comparison to results from participants with low CSF tau phosphorylation at T217 (<2.5%) and a normal betaamyloid 42 / 40 ratio. The presence of tau peptides with higher deamidation rate suggests part of the tau peptides found in CSF from participants with tau aggregates, as measured by CSF tau MTBR-tau243 level, partially originated from tau domains with abnormally long half-life which are part of the fuzzy coat in AD tau aggregates. This demonstrates AD tau aggregates get their fuzzy coat digested and released extracellularly as tau fragments in the CSF. Of note, the abnormal deamidation on N265 seems higher in the HJ32.11 extract than in the HJ34.8 extract. N 167 is slightly more deamidated in participants identified as AD. This asparagine residue is expected to have a short half-life (22,5 days) leading to an abundant deamidation rate in normal tau (deN167 / N167 >50%). The contribution of tau species with a much longer half-life may not drastically change the N167 deamidation rate due to the high abundance ofdeamidation in normal tau. Alternatively, it was found that one of the less abundant isomers from N167 deamidation (isomer 3 according to the LC elution order) can be used to identify abnormal deN167 isomerization status linked to AD. This abnormality can be measured by different normalizations of the deN167 isomer3 (iso3) using signals from non-deamidated N167 and deamidated N167 isomers. For example, deN167 iso3 is normalized to the sum of signals for all measured N167 and deN167 species (deN167 iso3 %) or normalized to the signal of deN167 isomer4 (iso4) (deN167 iso3 / iso4 ratio). These two variables are abnormal in participants identified as AD.

[0158] Abnormal D252 isomerization is observed in all participants identified as AD and those with intermediate pT217 hyperphosphorylation.

[0159] Overall, CSF tau abnormality on deN167 iso3 abundance, and abnormally high deamidation rate on N255, N265, N279, N286, and D252 isomerization may indicate the presence in the brain of AD-like tau aggregates. This pattern should be expected in 3R+4R tauopathies where the core includes R3 and R4 domains.Combined with CSF AB42 / 40 expected to be low only in AD, this pattern can be used to differentiate AD from other 3R+4R tauopathies such as CTE and PART.(2) Identification of CBD / PSP-like deamidation profile linked to R2-R3-R4 domains aggregation (4R tauopathies)

[0160] Another non-limiting embodiment includes the identification of CBD / PSP- like deamidation profile linked to R2-R3-R4 domain aggregation (4R tauopathies). That is, In CBD and PSP, the 4R-specific domain including asparagine residues N279 and N286 are part of the core of tau aggregates and are expected to be less prone to deamidation than residues expected to be part of the fuzzy coat as N167 and N255. N265 is not part of the aggregation core domains resolved by CryoEM but is close to the interface between the core and the fuzzy coat.

[0161] We identified abnormal deN167 isomer3 relative abundances in most participants with CBS and PSP. Participants with PSP may have lower deN167 isomer4 relative abundance compared to CBS participants. Accordingly, the deN167 iso3 / iso4 ratio may be higher in PSP participants than in CBS participants. Outstandingly, this ratio differentiates controls from participants clinically identified as PSP or CBS with anArea Under ROC curve of 0.925.

[0162] Abnormal D252 isomerization is observed in part of the CBS and PSP participants with a degree of abnormality like what is observed in participants with intermediate pT217 hyperphosphorylation. Though we observed higher N255 deamidation in many PSP / CBS participants, the separation from participants with low pT217 phosphorylation is not significant. It is hypothesized this comes from the limited LC-MS assay specificity which is expected to be improvable notably by using a corresponding internal standard. No significant N265, N279, and N286 abnormal deamidation statuses are observed in CSF tau from PSP and CBS participants. The normal deamidation status on N279 and N286 is predicted by the model where these asparagine residues are immobilized in both PSP and CBD tau aggregates. The lake of high deamidation rate on residue N265 could be related to its location close to the interface between the fuzzy coat and the aggregation core. This location at the edge may prevent the release of tau fragments including IGSTENLK (SEQ ID NO:8) and IGSTEdeNLK (SEQ ID NO:8) sequences, the corresponding domains being more likely truncated before the release or remaining attached to the core. Alternatively, N265 may have a restricted conformation in the PSP / CBD aggregates due to its proximity to the respective cores.

[0163] Overall, CSF tau abnormalities on deN167 iso3 and iso4 abundance, D252 isomerization, abnormal deamidation on N255, and normal modification status on N265, N279, N286, and D252 may indicate the presence in the brain of CBD-like or PSP-like tau aggregates. This pattern should be expected in 4R tauopathies where the core includes R2, R3, and R4 domains. This CSF measure can be used in combination with CSF AB42 / 40 expected to be normal in CBS and PSP to confidently identify a primary tauopathy.(3) Prediction of PiD deamidation profile linked to R1 -R3-R4 domains aggregation (3R tauopathy)

[0164] Another non-limiting embodiment includes the prediction of PiD deamidation profile linked to R1-R3-R4 domains aggregation (3R tauopathy). In PiD, only 3R tau isoforms form aggregates. It is not expected that an abnormally highdeamidation rate on the 4R-specific residues N279 and N286 will occur since the corresponding domains remain soluble and have a normal turnover. The asparagines N255 and N265 are part of the PiD aggregation core resolved by CryoEM and should not be able to form succinimide intermediates leading to an abnormal deamidation rate over time. D252 is unlikely to isomerize due to its proximity to the core. It is predicted that only deN167 iso3 and iso4 abnormal abundance can be detected in PiD aggregates and CSF tau.

[0165] Overall, CSF tau abnormalities on deN167 iso3 and iso4 abundance and normal modification status on N255, N265, N279, N286, and D252 may indicate the presence in the brain of PiD-like tau aggregates. This pattern should be expected in PiD 3R tauopathy where the core includes R1 , R3, and R4 domains. This CSF measure can be used in combination with CSF AB42 / 40 expected to be normal in PiD to confidently identify a primary tauopathy.(4) Characterization of the tau deamidation profiles in CSF from MAPT mutation carriers

[0166] Yet another non-limiting embodiment includes the characterization of the tau deamidation profiles in CSF from MAPT mutation carriers. In MAPT R406W, the modification pattern observed for participants with cognitive symptoms is like the modification pattern observed in the CSF of AD participants. This is consistent with the detection pattern of a 3R+4R tauopathy as predicted for this MAPT mutation-related tauopathy. Further, in MAPT 10+16, the modification pattern observed for participants with cognitive symptoms is like the modification pattern observed in PSP / CBS participants CSF except for N265 deamidation recovered by HJ32.11 which appears to be more deamidated than PSP / CBS participants. This is consistent with the detection pattern of a 4R tauopathy as predicted for this MAPT mutation-related tauopathy with a specificity on N265 deamidation recovered by HJ32.11.(5) Differentiation of various tauopathies

[0167] In one non-limiting embodiment, the extracts obtained by HJ32.11 tau immunoprecipitation of CSF extracts already immunoprecipitated using anti N-ter / Mid- domain and HJ34.8 antibodies can be used to measure the abundance of 243-254 and260-267 peptides. In AD and R406W participants, the CSF level of 243-254 is significantly elevated in response to AD pathology measured by Tau PET as recently reported (Horie et al. Nature Medicine 2023). The tryptic peptide deN255243-256 CSF concentration provides an even better separation of AD participants as previously disclosed with the ArgC peptides 241 -256 and deN255241 -256. Part of the PSP / CBS has elevated 243-254 CSF level compared to controls (ptau217 neg group) and the deN255 243-256 is not significantly elevated. Unlike AD, part of the CBS participants with high 243-254 CSF levels have also elevated 260-267 levels. This relationship can be used to separate CBS from AD participants.

[0168] In another non-limiting embodiment, the extracts obtained by HJ34.8 tau immunoprecipitation of CSF extracts already immunoprecipitated using anti N-ter / Mid- domain antibodies can be used to measure the relative abundance of 3R (VQIVYKPVDLSK, SEQ ID NO:15) to 4R specific peptides (VQIINK, 275 [SEQ ID NO: 10] and LDLSNVQSK 282 [SEQ ID NO: 13], FIGS. 4R and 4S). The 3R / 4R-282 ratio is elevated in ptau-217 positive participants with intermediate to high values. Higher 3R / 4R-282 ratios are also observed in some of the CBS / PSP participants. This ratio may be lower in 10+16 and R406W participants. Similar trends are observed using the 3R / 4R 275 ratio.(6) Tau phosphorylation profile for characterization of primary tauopathies

[0169] In addition to the tau deamidation / isomerization patterns discussed in the above four embodiments, in another non-limiting embodiment, tauopathies can be detected using abnormal hyperphosphorylation detection combined with CSF42 / 40 ratio (negative in primary tauopathies, positive in AD). Hyperphosphorylation on T111 , T153, T181 , T205, S208, T217, and T231 is typical for AD participants with cognitive decline. In preclinical AD, these p-tau sites are also abnormally phosphorylated to a lesser extent except for phosphorylation at T205 which becomes significantly hyperphosphorylated closely to the emergence of symptoms.

[0170] In CBS / PSP CSF, significant hyperphosphorylation is detected on T217 and T231 in most of the participants. Outstandingly, the T217 hyperphosphorylation ratio differentiates controls from participants clinically identified as PSP or CBS with anAUC of 0.887.

[0171] Other sites found modified in AD are less likely to be modified in PSP / CBS. S208 might be slightly abnormal in CBS cases. T205 may be sporadically abnormal in participants with CBS.

[0172] In the CSF of FTD 10+16 participants, abnormal hyperphosphorylation is observed on T217, T205, and slightly on T231. Hypophosphorylation (abnormally low phosphorylation) is observed on T111 , T153, S199 and S202.

[0173] In the CSF of MAPT R406W participants, abnormal hyperphosphorylation is observed on T217 and T205 and may be slightly higher on T231 and T111. Other phosphorylation sites are normal.(7) Immunoprecipitation with Tau1 / HJ8.5 / HJ8.7 on CSF samples

[0174] Immunoprecipitation with Tau1 / HJ8.5 / HJ8.7 on a cohort of 107 CSF samples. This included 34 individuals with clinical diagnosis of Parkinson Disease.(PD) expected to have low risk of tau pathology, 20 individuals with clinical diagnosis of progressive supranuclear palsy Richardson Syndrome (PSP-RS) expected to have a high risk of tau pathology associated to 4R tau aggregation, 45 individuals with clinical diagnosis of corticobasal syndrome (CBS) expected to have a high risk of tau pathology associated to 4R tau aggregation, and 8 individuals cognitively unimpaired healthy controls (HC)

[0175] It is noted that the differential diagnosis of PSP-RS versus PD is challenging particularly at early stage of clinical syndrome, the individuals selected should be at a more advanced stage when the diagnosis confidence is better, and most PD individuals has been confirmed positive to a CSF alpha synuclein seeding assay.

[0176] In addition to isomers described above, the measure of the aspartic acid isomerization on a tau peptide containing two aspartic acid residues prone to isomerization (D54 and D65) is added. This peptide, located within the N-terminus domain of tau isoforms 1 N and 2N, is recovered by the HJ8.5, HJ8.7 or Tau 1 antibodies (FIG. 21 ). The sequence associated with this isomerization is KESPLQTPTEDGSEEPGSETSDAKS (SEQ ID NO:16q) [45, 67], In summary the firsttau immuno precipitation step can measure D isomerization at D54 / D65 in addition to previously reported N267 deamidation.

[0177] It is shown that both N167 deamidation and D isomerization on peptide 45-67 increase in the groups at risk of tauopathy (FIGS. 22A, 22B, 22C, and 22D).(8) Modified amyloid positivity stratification

[0178] In this example, amyloid negative CDRO participants are divided between Young Normal Control (YNC Average Age 42.1 [19.5-60.7] and Aged Matched Controls (>60 yrs Average 72.0 [63.0-82.2]). Amyloid positive participants are divided according to clinical status. 2 participants have neuropathic confirmation of cortico basal degeneration (CBD). Others are stratified as described in previous Examples.

[0179] This stratification confirms abnormal deamidation / isomerization of most of the asparagine residue recovered by HJ32.11 and HJ34.8 are abnormal in symptomatic amyloid positive participants corresponding to a clinical diagnosis of AD. This deamidation profile is consistent with 3R+4R aggregates profile (FIGS. 23A, 23B, 23C, 23D, 23E, 23F, 23G, 23H, 23I, 23J, and 23K). Notably, the % deN255 on 243-256 peptide recovered by HJ32.11 seems to be significantly impacted by age (AUC 0.8629, FIG. 23B) and the 14N / 13C15N 260-267 measure may be higher in CBD and participants with CBS symptoms (FIG. 23I).(9) Deamidation and isomerization of tau asparagines and aspartic acids

[0180] Succinimide formation from Asn is typically faster than from Asp. Weak acid to alkaline pH environment destabilize Asn and Asp by promoting the succinimide formation and its subsequent hydrolysis. Protein conformation and microenvironment affect Asn deamidation and Asp isomerization as do pH, temperature, buffer. LC-MS can measure: 1 ) deamidation rate Asp / Asn 2) Asp enantiomers relative abundance (when separated by LC).

[0181] 5 Asn in the MTBR having relatively long half time compared to other Asn are of note. This range of half-lives is significantly superior to known tau half-life. Corresponding deamidation on these residues would be detectable in CSF for a tau pool staying sequestered in aggregates and then released as soluble based on thesoluble / insoluble exchange hypothesis.

[0182] The hypothesis of this example is: (1 ) depending on the type of tau aggregates, protein domains staying in the "fuzzy coat” are different and (2) asparagines in the fuzzy coat can be deamidated with time but asparagines in the cores cannot (FIG. 7). To investigate this hypothesis, a strategy to monitor CSF tau deamidation rate on 5 tau asparagines by extracting appropriate pools of tau fragments using immunoprecipitation is developed (FIG. 8).

[0183] CSF tau fragments were characterized as recovered by HJ32.11 and HJ34.8 (FIGS. 9A, 9B, 9C, and 9D). Group analysis using CSF from Tau TANGLES and TAU SILK was performed (FIGS. 10A and 10B). Tau SILK samples are categorized according to CSF %ptau217, Neg: 7.5%. Most of the CBS / PSP are in the intermediate range of value for %ptau217. All FTD 10+16 (n=2) have higher %ptau217. The R406W TANGLES participants previously identified as ptau217 positive were not part of the study. Of note, based on current knowledge R406W and P301 L cryoEM structure is unknown and the 10 +16 group should have a CBD-like aggregate structure. Therefore, group analysis using Tau TANGLES and TAU SILK asparagine modification patterns was performed (FIGS. 11 A, 11 B, 11 C, 11 D, 11 E, and 11 F).

[0184] Next, another hypothesis was adjusted with results from the study on proteolytic resistant fragments by Mass Spectrometry (MALDI-TOF) (FIG. 12), and specific peptides were interrogated. The first peptide interrogated was N167 (FIGS. 13, 14A, 14B, and 14C). The low steric hindrance of A168 leads to a fast N167 deamidation. More than 50% of N167 deamidation was observed by measuring the ratio between Asn167 and Asp167 LC-MS signals. Further, N167 deamidation leads to 4 Asp isomers observable in CSF using LysC digestion. Based on the literature the most abundant isomer should be [3-L-Asp 4, and co-elution between Isol and Iso2 was observed.

[0185] Therefore, deamidation rate, and isomerization and enantiomerization process can be measured. It is hypothesized that these measures depend on: i) the average age of tau species measured in CSF since their synthesis in cells, and ii) adopted tau conformations during tau species life. deN167 isomer relative abundance inTauSILK / TANGLES cohort was quantified. The relative abundance of deN167 isomer3 increases in most of the TANGLES CSF samples, QC-M, QC-H (according to ptau217) and some of the Tau SILK participants. This separation is not observed using the overall deamidation rate using all isomers, neither using iso1 +2 or iso4, but the ratio iso3 / iso4 appears to provide such separation. It is hypothesized that abnormal tau conformation promotes the formation of a conformer less likely to be found when tau has its normal conformation.

[0186] D252 and D255 isomerization was measured in tryptic digest from CSF tau species extracted with HJ32.11 (FIGS. 15A, 15B, 16A, 16B, 16C, 16D, and 16F). N265 deamidation was interrogated using fragment KIGSTENLKH (SEQ ID NO: 18) (FIGS. 17A, 17B, 17C, and 17D). Based on the spectra, 1 is a large isomer surrounded by at least 2 others. LC resolution might be critical to study isomers in more detail. Higher abundance in general was observed, which is consistent with the location of the peptide around HJ34.8 epitope. It is noted that some of the Tau SILK samples have abnormally high deamidation and part of the TANGLES may be on the lower range for both normal and deamidated peptides.

[0187] N279 deamidation was also interrogated using fragment KVQIINKK ([275, 280], SEQ ID NO: 19) as illustrated in FIGS. 18A, 18B, 18C, and 18D). 1 peak is a large isomer and 3 minor isomers on the right are observed. The high modification abundance in general is consistent with the shorter half-life expected for this site (NK). It is noted that some of the Tau SILK samples have abnormally high deamidation, and part of the TANGLES may be on the lower range for both normal and deamidated peptides.

[0188] N286 deamidation was interrogated (FIGS. 19A, 19B, 19C, and 19D) using fragment KLDLSNVQSKC ([282, 290], SEQ ID NQ:20). 3 isomers were detected. It is interesting to observe that LDL peptide is typically low and its deamidated version is low or undetected in the TANGLES cohort compared to Tau SILK. Abnormal deamidation rate and higher deamidation level are observed for TAU SILK samples. This may be consistent with a release of the AD fuzzy coat (able to be modified on this domain). For the 4R tauopathies from TANGLES, lower 4R and less deamidation wouldbe expected assuming this domain is part of the core of aggregation.

[0189] Finally, deamidation association was interrogated (FIGS. 20A, 20B, and 20C). It is shown that when one site is deamidated, the 2 others are deamidated as well, and most of them are Tau SILK. It is proposed that Tau PET may provide positivity detection. No obvious deamidation for TANGLES on N265 vs N279 was observed.

Claims

1. CLAIMSWhat is claimed is:

1. A method for detecting and classifying a tauopathy, the method comprising: providing a biological sample comprising at least one of a full-length tau protein or a truncated tau protein; b. digesting the at least one full-length tau protein or the at least one truncated tau protein into a plurality of fragments with an enzyme selected from LysC, LysN, Trypsi, and any combination thereof; c. selectively precipitating at least one subset of the plurality of tau protein fragments comprising at least one corresponding amino acid sequence using at least one antibody, each antibody targeting one of the corresponding amino acid sequences; d. performing liquid chromatographic mass spectrometric analyses on the precipitated at least one subset of the plurality of tau protein fragments to differentiate and quantify the deamidated or isomerized asparagines or aspartic acids contained within the at least one subset of the plurality of tau protein fragments; e. comparing the deamidation of asparagines or isomerization of aspartic acids within the tau protein fragment at least one subset of the plurality of tau protein fragments to determine at least one of: a presence of a tauopathy, a type of tauopathy present, and a stage of progression of the tauopathy.

2. The method of claim 1 wherein the tauopathy is a neurological disorder.

3. The method of claim 1 wherein the tauopathy is Alzheimer’s Disease.The method of claim 1 wherein the sample used for sampling is taken from cerebrospinal fluid.The method of claim 1 wherein the sample used for sampling is taken from bio-fluids or plasma.

6. The method of claim 1 wherein the precipitation is immunoprecipitation.

7. The method of claim 1 , wherein at least one of the presence of the tauopathy, the type of tauopathy present, and the stage of progression of the tauopathy is used to select one or more of an appropriate treatment or prevention of the tauopathy.

8. A method for detecting and / or classifying Alzheimer disease comprising: a. sampling, from cerebrospinal fluid or plasma, at least one of a full- length tau protein or a truncated tau protein; b. digesting the at least one full-length tau protein or the at least one truncated tau protein into a plurality of fragments; c. precipitating the plurality of tau protein fragments; d. performing liquid chromatographic mass spectrometric analyses on plurality of the tau protein fragments to differentiate and quantify a normalized level of deamidated N167 selected from: i. a ratio of deamidated N167 isomer 3 to all measured N167 and deN167; or e. a ratio of deamidated N167 isomer 3 to deamidated N167 isomer 3; and f. classifying Alzheimer disease based on an abnormal normalized level of deamidated N167.

9. The method of claim 8, further comprising selecting a treatment or prevention plan for the Alzheimer disease based on the abnormal normalized level of deamidated N167.

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