TRANSACTIVE RESPONSE DNA-BINDING PROTEIN 43KDa (TDP-43) MUTANTS AND USES THEREOF

Abstract

The invention relates to TDP-43 protein mutants and uses thereof. The invention further relates to methods for the preparation of TDP-43 protein mutants aggregates, including fibrils and uses thereof. The invention further relates to use the TDP-43 protein mutants for the identification and screening of modulators of TDP-43 protein aggregation and related methods.

Description

[0001] TRANSACTIVE RESPONSE DNA-BINDING PROTEIN 43KDa (TDP-43) MUTANTS

[0002] AND USES THEREOF

[0003] Field of the Invention

[0004] The present invention pertains generally to the field of prevention of transactive response DNA- binding protein 43kda (TDP-43) misfolding / aggregation.

[0005] Background of the Invention

[0006] Amyotrophic Lateral Sclerosis (ALS), is a neurodegenerative disease characterized by the loss of motor neurons leading to death of progressive weakness and respiratory failure less than three years from symptom onset (Coupe et al., 2012, Eur. Neurol. Rev., 8, 38). The mechanisms underpinning the pathogenesis of ALS are not fully understood, but it is believed to be linked to a combination of genetic and environmental factors Yu et al., 2017, Transl. Neurodegener., 6, 15). Over 90% cases of ALS are sporadic and don’t have a family history. One of the main molecular hallmarks of ALS is the presence of cytoplasmic inclusions in motor neurons (Blokhuis et al. 2013, Acta Neuropathol. (Berl.) 125, 777- -794) composed of misfolded and aggregated forms of the protein Transactive response DNA binding protein of 43 kDa (TDP-43). These inclusions are found in 97% of ALS patients, including sporadic and about 50% of familial cases (Prasad et al., 2019, Front. Mol. Neurosci., 12, 25). TDP-43 cytoplasmic inclusions, composed of a mixture of hyperphosphorylated, truncated, ubiquited TDP-43 accumulate in affected neurons in the brain and spinal cord of ALS and FTD patients (Neumann et al., 2006, Science 314, 130- 133; Arai et al., 2006, Biochem. Biophys. Res. Commun., 351, 602-611). TDP-43 inclusions are also found in the brain of individuals affected by several other neurodegenerative diseases, including progressive muscular atrophy, Alzheimer disease, Parkinson disease (NDDs), and newly defined Limbic- predominant age-related TDP-43 encephalopathy (LATE), collectively referred to as TDP-43 proteinopathies Josephs et al., 2014, Acta Neuropathol. (Berl), 127, 811-824; Dugger et al., 2017, Cold Spring Harb. Perspect. Biol. 9, a028035; Nelson et al., 2019, Brain 142, 1503-1527; de Boer etal., 2021, J. Neurol. Neurosurg. Psychiatry 92, 86-95).

[0007] Recent studies suggest that the common co-occurrence of TDP-43 pathology with other pathological hallmarks of NDDs, such as Lewy bodies, amyloid plaques, and neurofibrillary tangles, influence the clinical heterogeneity of NDDs and rate of disease progression (de Boer et al., 2021, supra; Kawakami et al, 2019, Acta Neuropathol. (Berl.), 138, 751-770). Therefore, understanding the mechanisms of TDP-43 aggregation and pathology formation and developing effective strategies to prevent these processes could have wide ranging implications for several neurodegenerative diseases. TDP-43 consists of 414 amino acids (Uniprot ID: Q13148), comprising of a Ubiquitin-like N- terminal domain (NTD) with a nuclear localization signal (NLS), two folded RNA recognition motifs: RRM1 and RRM2, a nuclear export signal (NES), and a C-terminal domain (CTD) which is highly aggregation-prone (Figure 1) (Franqois-Moutal et al., 2019, Front. Mol. Neurosci., 12, 301). The structure of full-length protein is yet to be determined and the C-terminal domain responsible for the initiation of TDP-43 misfolding and aggregation is highly disordered, thus making structure-aided drug design challenging. However, several structures of isolated domains such as the NTD, RRMs have been solved using nuclear magnetic resonance and / or X-ray crystallography (Loughlin et al., 2019, Curr. Opin. Struct. Biol., 59, 134- -142; Wright et al., 2020, iScience, 23, 101159).

[0008] S333 phosphorylation was detected from Sarkosyl-insoluble abnormal TDP-43 species of a female ALS patient with abundant and widespread TDP-43 pathologies (Kametani etal., 2016, Sci. Rep. 6, 23281). Furthermore, previous studies showed that co-expressing TDP-43 and kinase CKl l- 317 enhances in SH-SY5Y cells, enhances TDP-43 phosphorylation at S333 and leads to increase aggregation and cytotoxicity (Nonaka et al, 2016, J. Biol. Chem. 291, 5473 5483). In vitro, coincubation of casein kinase- 1 with recombinant TDP-43 resulted in increased phosphorylation primarily at S342 (Kametani et al., 2009, Biochem. Biophys. Res. Commun., 382, 405- -409).

[0009] Although these findings may suggest that these residues could undergo phosphorylation in vivo, no other studies have been carried out to investigate their effect on TDP-43 functions, aggregation or pathology formation (Figure 1) (Eck et al., 2021, GeroScience, 43, 1605- -1614).

[0010] Gruijs da Silva et al., 2022, The Embo Journal, 41, 1-23 reports the observation that disease-linked TDP-43 hyperphosphorylation at C-terminal Serines (S403 / S404; S409 / S410) suppressed TDP-43 condensation and aggregation and concluded that it was the number of phosphorylation sites, but not their exact position which would be critical for the suppression of TDP-43 condensation.

[0011] Other references such as XXX [please complete] show that phosphorylatons at serines sites such as S369, S379, S403, S404, S409 and S410 have the opposite effect on TDP-43 aggregation. Nonaka et al., 2016, Journal of Biological Chemistry, 29(11), 5473, 5483 reports phosphorylation sites of aggregated TDP-43 by CKdl-317 as being S92, S292, S305, S317, S333, S389, S393, S395, S403, S404, S409 and S410.

[0012] Furthermore, several Cryo-EM structures of the aggregation-prone full CTD (276-414) or fragments derived from this domain (Guenther et al., 2018, Nat. Struct. Mol. Biol., 25, 463- -471; Jiang et al., 2016, Rep. 6, 23928; Cao et al., 2019, Nat. Struct. Mol. Biol., 26, 619 -627; Li et al., 2021, Nat. Commun., 12, 1620) have been solved and reveal different folds. Interestingly, all these structures differ dramatically from the core structure of TDP-43 filaments isolated from FTD-ALS brains (Arseni et al., 2022, Nature, 601, 139 143). These findings suggest that accurate understanding and modeling the mechanisms of TDP-43 aggregation and pathology formation requires working with the full-length TDP-43. However, TDP-43 exhibits a high propensity to aggregate during or immediately after purification. This has precluded systematic studies to determine the biophysical, biochemical and structural determinants of TDP-43 physiological functions, or early aggregation events on the pathway to TDP-43 filaments and inclusion formation. Furthermore, it has precluded the development of high-throughput assays to screen for inhibitors of TDP-43 misfolding and aggregation.

[0013] Since TDP-43 misfolds, aggregates, and accumulates in the form of neuronal cytoplasmic inclusions are one of the primary diagnostic pathological biomarkers of NDDs, and key determinants of disease symptomology and progression, TDP-43 misfolding and aggregation has emerged as the most actively pursued therapeutic strategy to treat ALS and other NDDs, with an emphasis on targeting TDP-43 aggregates, neutralizing their activity, or promoting their clearance.

[0014] However, most existing full-length recombinant TDP-43 purification strategies have failed to produce a stable monomeric form of the protein in acceptable purity. Briefly, TDP-43 protein samples used in published studies are either 1) not pure, i.e., contain truncated fragments (Carlomagno et al, 2014, PLoSONE9, e90452; McGurk et al., 2018, Biochemistry 57, 6923 6926: Staderini, et al., 2022, Protein Sci. n / a, e4509) generated as fusion proteins, and when the tags were removed, no additional steps were implemented to isolate the cleaved native protein in pure form; or 3) purified after refolding from inclusion bodies without reassessment of their folding and functional properties (Furukawa et al, 2011, J. Biol. Chem., 286, 18664- -18672; Shenoy et al., 2020, FEBSJ. 287, 2449-2467 ; Capitini et al., 2021, Amyloid Int. J. Exp. Clin. Investig. Off. J. Int. Soc. Amyloidosis, 28, 56-65). Therefore, in most cases, it is not clear to what extent the refolding and resolubilization conditions alter the native confirmation and / or oligomerization states of the protein.

[0015] Currently, the great majority of anti -TDP-43 aggregation approaches focus on targeting the aggregated forms of the protein, TDP-43 oligomers, fibrils, or inclusions to neutralize their toxicity or block their seeding activity and spreading to different brain regions (de Boer et al., 2021, supra; Tamaki et al., 2018, Sci. Rep. 8, 6030; Swarup et al., 2011, Brain 134, 2610-2626). None of the drugs emerging from drug discovery programs based on these approaches have entered the clinic. Therefore, there is a need for the development of methods that enable in vitro handling of TDP-43 protein and the identification of agents capable of preventing and / or reverting the misfolding / aggregation of this protein. Summary of the Invention

[0016] A general object of this invention is to provide a method for the identification of modulators of TDP-43 protein aggregation.

[0017] One of the specific objects of this invention is to provide analogues of TDP-43 protein which would behave in a similar manner as the wild-type but which would be stable in monomeric form and which the aggregation could be triggered in a controlled manner.

[0018] It is advantageous to provide a TDP-43 mutant protein which can be purified as a stable monomeric form, while retaining the ability to bind to the natural ligands of TDP-43 and its physiological cellular properties.

[0019] It is advantageous to provide a TDP-43 mutant protein which is stable in a monomeric form to enable biophysical and functional characterization of the protein.

[0020] It is advantageous to provide a TDP-43 mutant protein which is stable in a monomeric form but which aggregation can be triggered in a controlled manner into oligomers and fibrils similar to those formed by the WT protein as this would enable the identification of proteins, small molecules and nucleic acid-based molecules that bind to TDP-43 and modulate its structure and pathogenic or functional properties.

[0021] Objects of this invention have been achieved by providing an isolated TDP-43 mutant protein according to claim 1.

[0022] Another of the specific objects of this invention is to provide a method for the preparation of oligomers and fibrils of TDP-43 mutant protein similar to those formed by the WT protein.

[0023] Objects of this invention have been achieved by providing a method according to claim 11.

[0024] Another of the specific objects of this invention is to provide a method for the identification of modulators of TDP-43 protein structure and aggregation.

[0025] It is advantageous to provide a method for the identification of modulators of TDP-43 protein aggregation wherein the modulators could interfere with the monomeric form of TDP-43 protein.

[0026] Objects of this invention have been achieved by providing a method according to claim 12.

[0027] Another of the specific objects of this invention is to provide a method / kit useful in an assay for screening TDP-43 anti-aggregation compounds. Disclosed herein is an isolated TDP-43 mutant protein, said mutant comprising at least one phosphomimetic mutation of at least one Serine from the C-terminal sequence located between positions 279 and 360 of the TDP-3 wild type sequence.

[0028] Also disclosed herein is a use of an isolated TDP-43 mutant protein of the invention for the preparation of oligomers and fibrils of TDP-43 mutant protein.

[0029] Also disclosed herein is a use of an isolated TDP-43 mutant protein of the invention for raising monomer-specific antibodies.

[0030] Also disclosed herein is a method for the in vitro preparation of oligomers and fibrils of TDP-43 mutant protein.

[0031] Also disclosed herein is a method for the identification of modulators of TDP-43 aggregation, wherein said method comprises: a) Providing a TDP-43 mutant protein according to the invention in aqueous solution at a temperature from about 4°C to 25°C; b) Adding to said mutant in aqueous solution, at least one UG / TG rich ligand and incubating the mixture at a temperature from about 4°C to 25°C; c) Subjecting the co-incubated mutant to a temperature increase sufficient to trigger TDP-43 mutant protein aggregation, typically at or above 25°C but lower than 40°C (e.g. to about 37 °C); d) Determining the protein mutant aggregation extent in presence of a candidate modulator compared to the protein mutant aggregation extent in absence of a candidate modulator.

[0032] Also disclosed herein is a kit for assaying TDP-43 aggregation, said kit comprising at least one reaction vessel comprising at least one TDP-43 mutant protein of the invention and instructions for use.

[0033] Also disclosed herein is the use of a kit according to the invention in a method according to the invention.

[0034] The present invention is based on the unexpected finding that phosphorylation or phosphorylationlike mutation(s) at a small number (e.g. as low as 1 or 2) of specific Serine positions allow the stabilizing of the TDP-43 protein in monomeric form at a temperature from about 4°C to room temperature. A mutant according to the invention was obtained in highly pure form (> 98%) and in stable monomeric form at room temperature for up to 100 h and exhibited similar subcellular distribution and binding affinity to the UG / TG-rich natural nucleotides as the WT protein. Further, the present invention is based on the unexpected finding that the monomeric TDP-43 protein mutant could be induced to aggregate in a controlled manner on the simple action of a temperature increase, typically at or above 25°C but lower than 40°C (e.g. to about 37 °C) which undergoes rapid aggregation and forms fibrils similar to those formed by the WT protein.

[0035] Other features and advantages of the invention will be apparent from the claims, detailed description, and figures.

[0036] Brief Description of the drawings

[0037] Figure 1 materializes the ALS / FTD-relevant phosphorylation sites found in the sequence of TDP- 43.

[0038] Figure 2 describes the production and the characterization of aggregation propensity of WT and S333D / S342D mutant TDP-43 core peptide 279-360 as described in Example 1. A: Schematic depiction of production protocol of the TDP-43 core peptide 279-360 (WT and S333D / S342D mutant). B: Comparison of ThT-based aggregation kinetics of TDP-43 peptide 279-360 (WT and S333D / S342D). n=5. C: TEM analysis of the aggregation process of samples from (B) over time. Scale bar: 200 nm.

[0039] Figure 3 shows the expression, purification and characterization of the full-length WT TDP-43 and TDP-43 S333D / S342D as described in Example 2. A: Schematic depiction of the protocol of TDP-43 production and purification. SDS-PAGE analysis of the Elis-SUMO fusion TDP-43 after IMAC purification, and native TDP-43 purification from SEC for WT; B: and S333D / S342D (C). SEC purification chromatography and subsequent SDS-PAGE analysis of oligomeric and monomeric fractions of WT (D) and S333D / S342D (E) TDP-43. (F) and (G) Size distribution analysis of fractions C2 from (D) and (E) by DLS, respectively. (H) and (I) TEM analysis of fractions C2 from (D) and (E), respectively. EM grids were made within 10 min after the elution of selected fractions. Scale bar: 200 nm.

[0040] Figure 4 reports the long-term stability studies of WT TDP-43 and TDP-43s333D / s342D s described in Example 2. A: Protein sedimentation-based aggregation assay of WT TDP-43 and TDP- 43 S333D / S342D, analysis of remaining soluble protein in the supernatants by UPLC. n=3. SDS-PAGE analysis of supernatants and pellets from the sedimentation assay for WT TDP-43 (B) and TDP- 43 S333D / S342D (C). (D) CD analysis of the WT TDP-43 sample at Time 0 (starting point). (E) CD analysis of TDP-43 S333D S342D sample at Time 0 and 72 h incubation at R.T. (F) TEM analysis of the samples during aggregation assay (0-44 h) at R.T. (G) TEM analysis of the TDP-43 S333D / S342D sample during aggregation assay after 72 h and 1-week incubation at R.T. Figure 5 provides prediction of fibril-disrupting phosphomimetic mutations of TDP-43 amyloids as described in Example 3. A: Surface representation of the patient-derived TDP-43 fibril (PDB: 7py2) based on residue properties. Serine residues were highlighted. (B) AAG prediction (kcal / mol) of mutations from Serine (S) to Aspartate (D), Glutamate (E) C: Comparison of AAG values (kcal / mol) for serine residues when mutated to Aspartate for cyro-EM structures formed by TDP- 43 C-terminal core peptides.

[0041] Figure 6 shows the binding affinity of TDP-43 with its nucleotide ligand TG12, TDP-43 S333D / S342D aggregation and its monomer stabilization by oligonucleotide ligands as described in Example 4. Direct binding studies of TG12 ligand with WT TDP-43 (A) and TDP-43 S333D / S342D (B). (C) Stability studies of TDP-43 S333D / S342D at R.T. and 37 °C. Supernatants were analyzed by UPLC. n=3. (D) Time-dependent inhibition of TDP-43 S333D / S342D aggregation at 37 °C in the presence and absence of 20 pM UG12, analyzed by plate-based DLS. n=3. (E) Time-dependent inhibition of TDP-43 S333D / S342D aggregation at 37 °C in the presence of 20 pM UG12, TG12 or TG8, analyzed by plate-based DLS. n=3. (F) Dose-dependent inhibition of TDP-43 S333D / S342D aggregation in the presence of UG12, TG12 or TG8 at 37 °C, analyzed by plate-based DLS, after 3 h aggregation induction. n=3.

[0042] Figure 7 shows the results of a screening assay according to the invention as described in Example 5 for identifying compound targeting native TDP-43 stabilization using a mutant of the invention m TDP-43. A: Workflow of the compound screening. B: Results of SPR binding assay between selected hit compounds (Bromoctriptine and Caspofungin) and the native mTDP-43. C: Representative TEM images showing the absence of fibrils in the presence of selected hit compounds in the aggregation assay of native mTDP-43. Scale bar: 200 nm.

[0043] Figure 8 shows the liquid-liquid phase separation studies of WT TDP-43 and TDP-43 S333D / S342D as described in Example 6. A: Representative bright field microscopic images of TDP-43 condensates of different concentrations of WT TDP-43 and TDP-43s333D / s342o in the absence and presence of Ulp-1 cleavage for 1 h. Number (B) and size (C) quantification of droplets formed during TDP-43 LLPS. D: Representative bright field microscopic images of TDP-43 ageing condensates after 3 h incubation in LLPS buffer. E: SDS-PAGE analysis of supernatants from solubility assay of His- SUMO TDP-43 samples cleaved by Ulp-1 in LLPS buffer. F: Representative bright field microscopic images of TDP-43 phase separation co-incubated with TG12 for 3 h. Scale bar: 25 pm.

[0044] Figure 9 represents the subcellular localization studies of WT and S333D / S342D TDP-43 as described in Example 7. A: Immunocytochemistry analysis of WT and S333D / S342D TDP-43 expression and subcellular localization in cells. B: Western blot analysis of WT and TDP- 43s333D / s342oin cells following subcellular fractionation.

[0045] Figure 10 shows the protein dynamic studies of TG12 and TDP-43S333D / S342D complex by HDX- MS described in Example 8. A: Schematic depiction of HDX-MS experimental setup to study TDP- 43 S333D / S342D. B: Global differences in FDD exchange level difference for TDP-43s333D / s342o in the presence and absence of TG12. Each point represents a peptide plotted on the x-axis by the residue at the center of the peptide (peptide centroid). Regions showing peptides with significant difference in exchange are highlighted in cyan squares. C: Surface representation of TGI 2 binding and deuteration exchange rate plots of selected peptides in the RRMs (PDB: 4bs2). Star indicates that difference in exchange is > 14 %, > IDa and p < 0.05 (unpaired student t-test). D: Uptake plots for a representative selection of peptides (mean + / - SD, n=3, * P < 0.05).

[0046] Detailed description of embodiments of the invention

[0047] The expression “phosphomimetic mutation” refers to a modification of an amino acid chemical structure resulting in group that is phosphorylated or which mimics a phosphorylated group, in particular which is negatively charged at physiologic pH of 7.4. Typically, a phosphomimetic mutation is selected from an Asp or Glu mutation (including L or D amino acids and non- hydrolyzable synthetic derivative of these mutants such as phosphonates.

[0048] Phosphomimetic mutations could be induced by natural enzymes that regulate phosphorylation at these residues.

[0049] The expression “TDP-43 mutant protein” according to the invention refers to a protein comprising the sequence of wild-type human TDP-43 but having at least one phosphomimetic mutation at least one Serine from the C-terminal sequence located between positions 279 and 360. Said mutant may have further conservative mutations outside those positions.

[0050] The expression “candidate modulator” refers to an agent of interest for its possible impact on TDP- 43 protein aggregation (e.g. small molecules, antibodies, nucleic acid-based molecule, etc.), that stabilize the TDP-43 monomer, modulate the formation of TDP-43 aggregates or induce disaggregation of already formed TDP-43 aggregates. Candidate modulators include native state stabilizers, aggregation inhibitor, and disaggregating candidate compounds.

[0051] The expression “UG / TG rich ligand” refers to DNA or RNA sequence rich in TG or UG repeats respectively, including native TDP-43 ligands such as UG12, TG8 or TG12 such as described in Rengifo-Gonzalez et al., 2021, Biochemistry and Chemical Biology Structural Biology and Molecular Biophysics, https: / / doi.org / 10. 7554 / eLife.676O5. The expression “aggregate” includes the formation of a multimeric assembly of the protein which includes fibrils, fibrillar species, soluble and insoluble oligomeric species such as described in Lansbury et al., 2006, Nature, 443(7113):774-9. dot 10.1038 / nature05290.

[0052] According to a particular aspect, is provided an isolated TDP-43 mutant protein, said mutant comprising at least one phosphomimetic mutation of at least one Serine from the C-terminal sequence located between positions 279 and 360 of the TDP-3 wild type sequence.

[0053] According to a further particular aspect, is provided an isolated TDP-43 mutant protein, said mutant comprising at least one phosphomimetic mutation of at least one Serine from the C-terminal sequence located between positions 279 and 360 of the TDP-3 wild type sequence.

[0054] According to a particular aspect, is provided an isolated TDP-43 mutant protein according to the invention wherein said mutant comprises at least one phosphomimetic mutation of at least one Serine located at positions selected from 292, 305, 317, 332, 333, 342, 347 and 350 or a combination thereof.

[0055] According to a particular aspect, is provided an isolated TDP-43 mutant protein according to the invention wherein said mutant comprises at least two phosphomimetic mutations of at least one Serine from the C-terminal sequence located between positions 279 and 360.

[0056] According to a particular aspect, the phosphomimetic mutation is a Serine to Aspartic acid mutation.

[0057] According to a particular aspect, the phosphomimetic mutation is a Serine to Glutamic acid mutation.

[0058] According to another further particular aspect, is provided an isolated TDP-43 mutant protein comprising a sequence of SEQ ID NO: 6.

[0059] According to a particular aspect, is provided an isolated TDP-43 mutant protein according to the invention wherein said mutant comprising at least the S333D and / or the S342D mutation(s).

[0060] According to a particular aspect, the isolated TDP-43 mutant protein is a mutant comprising a sequence selected from SEQ ID NO: 3, 4 or 5.

[0061] According to a particular aspect, the isolated TDP-43 mutant protein comprises a sequence of SEQ ID NO: 3

[0062] According to another particular aspect, is provided a method for in vitro preparation of oligomers and fibrils of TDP-43 mutant protein. According to a further particular aspect, is provided a method for in vitro preparation of oligomers and fibrils of TDP-43 mutant protein, said method comprising the following steps:

[0063] - Providing a TDP-43 mutant protein according to the invention in aqueous solution at a temperature from about 4°C to 25°C;

[0064] - Subjecting the mutant to a temperature increase sufficient to trigger TDP-43 mutant protein aggregation at or above 25°C but lower than 40°C, typically from about 30°C to about 38°C (typically, about 70% of the protein is aggregated in about 3h in this latter range of temperature);

[0065] - Isolating the formed oligomers and fibrils of TDP-43 mutant protein.

[0066] According to a further particular aspect, is provided a method for in vitro preparation of oligomers and fibrils of TDP-43 mutant protein of the invention, wherein the formed oligomers and fibrils are isolated by ultracentrifugation, for example at 20,000g for about 15 min at a temperature comprised between 4°C and 25°C.

[0067] According to another particular aspect, is provided a method for the identification of modulators of TDP-43 aggregation, wherein said method comprises: a) Providing a TDP-43 mutant protein according to the invention in aqueous solution at a temperature from about 4°C to 25°C; b) Adding to said mutant in aqueous solution, at least one UG / TG rich ligand and incubating the mixture at a temperature from about 4°C to 25°C; c) Subjecting the co-incubated mutant to a temperature increase sufficient to trigger TDP-43 mutant protein aggregation; at or above 25°C but lower than 40°C (e.g. to about 37 °C); d) Determining the extent of protein mutant aggregation in presence of a candidate modulator compared to the protein mutant aggregation extent in absence of a candidate modulator.

[0068] According to a particular aspect, a TDP-43 mutant protein according to the invention is provided in aqueous solution at a temperature from about 20°C to 25°C.

[0069] According to a particular aspect, a TDP-43 mutant protein according to the invention is provided in aqueous solution at concentration from about 2 to about 5 pM.

[0070] According to a more particular aspect, a TDP-43 mutant protein according to the invention is provided in aqueous solution comprising phosphate-buffered saline (PBS) at a pH around 7.5 (e.g. 7.4) or Tris-Buffered Saline (TBS) buffer at a pH around 8 (e g. 8.0).

[0071] According to a particular aspect, a candidate modulator is provided in aqueous solution at a concentration from about 5 to about 20 pM.

[0072] According to a particular aspect, a candidate modulator is provided in aqueous solution as a mixture with the TDP-43 mutant protein according to the invention. According to another particular aspect, a candidate modulator is added to the mixture of the TDP- 43 mutant protein according to the invention containing at least one UG / TG rich nucleotide ligand just before triggering the aggregation by a temperature increase.

[0073] According to a particular aspect, the mutant is subjected to a temperature increase up to about 37°C and incubated at this temperature for about 1 to about 4 h (e.g. 3 h).

[0074] According to a particular aspect, the extent of TDP-43 mutant protein aggregation, is assessed by dynamic light scattering.

[0075] According to a particular aspect the extent of TDP-43 mutant protein aggregation, in particular fibril formation extent is assessed by electron microscopy or light scattering.

[0076] According to another particular embodiment, the TDP-43 mutant protein aggregate formation extent can be assessed by atomic force microscopy.

[0077] Referring to the figures, in particular first to Figure 7A, is provided an illustration of a method for the identification of modulators of TDP-43 aggregation according to an embodiment of the invention. The illustrated method generally comprises the steps of: a) Providing a TDP-43 mutant protein according to the invention in aqueous solution at a concentration from about to about 2 to about 5 pM (e.g. 2.5 pM) to a reaction well containing a pre-loaded candidate compound at a concentration from about 5 to about 20 mM; b) Adding at least one UG / TG-rich nucleotide ligand (e g. TG12 at 10 pM) in aqueous solution at a concentration from 10 to 20 pM to the mixture and thoroughly mixing the mixture at room temperature for about 15 min; c) Subjecting the co-incubated mutant to a temperature increase sufficient to trigger TDP-43 mutant protein aggregation, typically at about 37°C for about 3 h; d) Determining the extent of protein mutant aggregation by DLS at 20°C in presence of a candidate modulator compared to the protein mutant aggregation extent in absence of a candidate modulator.

[0078] Wells with TG12 (e.g. 10 pM in 14 wells) are used as a positive control in a method of the invention for the identification of modulators of TDP-43 aggregation, as TG12 showed the best aggregation inhibition of mTDP-43 and DMSO was used as negative control (e.g. 60 nL in each well, 14 replicates). In total, when six plates were screened, and the Z’ score was calculated as 0.56 based on the controls. According to another particular aspect, is provided a kit for assaying TDP-43 aggregation, said kit comprising at least one reaction vessel comprising at least one TDP-43 mutant protein of the invention and instructions for use.

[0079] According to another particular aspect is provided a method for the identification of modulators of TDP-43 protein aggregation according to the invention wherein said method comprises the steps of:

[0080] - Providing a kit for assaying early stage of TDP-43 aggregation according to the invention; Mixing together the reaction components of the kit.

[0081] A kit according to the invention can be further used in combination with downstream biophysical or biochemical assays such as for example electron and atomic force microscopy and circular dichroism for the further characterization of the candidate modulator or aggregates.

[0082] Therefore, the method, the aggregates and the uses thereof represent useful tools for use in the diagnosis and identification of agents useful for the prevention or treatment of TDP-43 proteinopathies such as Amyotrophic Lateral Sclerosis (ALS).

[0083] Further, the stability of the mutant monomers of the invention provides unique opportunities to identify or develop monomer-specific antibodies as potential diagnostics or therapeutics.

[0084] The invention having been described, the following examples are presented by way of illustration, and not limitation.

[0085] EXAMPLES

[0086] The following abbreviations refer respectively to the definitions below:

[0087] OD (optical density)

[0088] Materials

[0089] TDP-43 constructs (S333D / S342D mutant for core peptide 279-360 and full length) used in the following examples were purchased from Genscript, and amplified using NucleoBond Xtra Midi kit from MACHEREY-NAGEL. The Luria Broth (Miller's LB Broth) was from CondaLab. ER2566 E. coli (E6901S) and One Shot® BL21 (DE3) competent cells were purchased respectively from New England BioLabs and Invitrogen. Phenyl methane sulfonyl fluoride (PMSF) was purchased from Axonlab (A0999.0005). Complete protease inhibitor tablets (11697498001) were from Roche. Isopropyl-B-D-thiogalactopyranoside (IPTG) was ordered from Applichem (A1008,0025). 1,4-Dithiothreitol (DTT) was from AppliChem, Thioflavin T (ThT) was from Sigma, and Trifluoroacetic acid (TFA) was from Acros Organic, p-18 well uncoated chamber coverslips (81817) were form Ibidi. The rabbit primary anti-TDP-43 antibody (full length, 18280- 1-AP) was purchased from Proteintech. The secondary goat anti -rabbit antibody with Alexa680 was purchased from Invitrogen (A-21109). The PageRuler prestained protein ladder (26617), and SeeBlue Plus2 pre-stained protein standard (LC5925) were purchased from Thermo Scientific. The filter-trap plates were from Merck. Uranyl formate (22450) and TEM grids were purchased from Electron Microscopy Sciences. UG12, TG12 and TG8 oligonucleotides were ordered from MicroSynth.

[0090] Methods

[0091] Recombinant Protein expression and purification

[0092] His-SUMO-TDP-43

[0093] The fusion proteins were produced following as described in Kumar et al., 2023, Neuroscience, in press. Briefly, the plasmid was transformed into ER2566 or BL21 cells using a standard chemical competent method. A well-grown single colony was then added to 200 mL LB medium containing lx Kanamycin, and the small culture was incubated at 30 °C overnight with shaking at 180 rpm. The overexpressed culture was diluted with pre-culture LB medium into 3 L with an ODeoo of 0.05. The resulting bacterial culture was incubated at 30 °C until the ODeoo reached 0.45-0.55. The overexpression was induced with 0.4 mM IPTG, and cells were grown afterwards at 14 °C overnight.

[0094] Cells from 3 L culture were harvested by centrifugation at 4000 rpm for 12 min at 4 °C. The pellets were then resuspended in 120 mL lysis buffer on ice (30 mM Tris, 15 mM Imidazole, 0.5 M NaCl, 10% v / v Glycerol, 1 mM DTT, pEI 8.0). 2 mM PMSF and 4 tablets of complete protease inhibitors were quickly added and dissolved under stirring. Cells were lyzed by sonication (5 min, pulse on 10 s, pulse off 10 s, 70% amplitude) using a Vibra cell VCX130 from Sonics. Supernatants were separated by centrifugation (45 min, 14,000 rpm, 4 °C), followed by the addition of 24 pL Benzonase Nuclease HC (Novagen), and mild stirring at R.T. for 30 min. Then the crude supernatants were filtered (0.45 pm syringe filter membrane) and loaded quickly into a 20 mL preequilibrated His-trap affinity column (GE healthcare). After that, weakly bound and tag-free proteins were washed-off by 5 column volumes of Buffer A (30 mM Tris, 15 mM Imidazole, 0.5 M NaCl, 10% v / v Glycerol, 1 mM DTT, pH 8.0) at a flow rate of 2.0 mL / min. Imidazole gradient elution with Buffer B (30 mM Tris, 500 mM Imidazole, 0.5 M NaCl, 10% v / v Glycerol, 1 mM DTT, pH 8.0) from 0 to 40% and then to 100% was carried out to collect fusion proteins. Fractions (5 mL per tube) were analyzed to identify the quality of fusion proteins using SDS-PAGE, LTQ- MS, and analytical C4 reversed-phase ultra-high performance liquid chromatography (UPLC). Theoretical molecular weights of WT and S333D / S342D His-SUMO-TDP-43 are 57775.2 Da and 57758 Da, respectively.

[0095] Native TDP-43

[0096] The WT native protein was purified by two different methods. The reverse IMAC strategy was as described in Kumar et al., 2023, Neuroscience, in press. The second strategy using SEC was to get monomeric native protein as the TDP-43 S333D / S342D. In brief, the His-SUMO tag of the fusion protein (WT or S333D / S342D) was cleaved overnight at 4 °C using Ulp-1 (1: 10 ratio), and checked by UPLC and SDS-PAGE. Subsequently, the cleaved sample was centrifuged (20,000 g, 5 min, 4 °C) to remove preformed aggregates. 1 mL supernatant was injected into the SEC column (Superdex 200, 100 / 300 increase, Cytia) and washed with buffer (30 mM Tris, 300 mM NaCl, 1 mM DTT, pH 8.0) in 0.4 mL / min to separate native monomers from oligomers. Fractions (0.5 mL per tube) were analyzed by SDS-PAGE, LTQ-MS and UPLC to check the quality of the monomeric proteins. Theoretical molecular weights of native WT and TDP-43 S333D / S342D are 44608 Da and 44664 Da, respectively.

[0097] TDP-43 core peptides 279-360

[0098] Both the WT and S333D / S342D core peptides 279-360 and their fusion proteins were prepared following the same protocol as described in Kumar et al., 2023, Neuroscience, in press. Samples were analyzed by SDS-PAGE, LTQ-MS, and UPLC. Theoretical molecular weights of WT and S333D / S342D core peptides 279-360 are 8041.08 Da and 8421.97 Da, respectively.

[0099] ThT kinetics-based aggregation assay

[0100] TDP-43 core peptides 279-360

[0101] Lyophilized TDP-43 core peptide 279-360 (WT or S333D / S342D) was treated with HFIP / TFA (1 :1) to disaggregate for 30min at 30 °C. The buffer was then evaporated under mild nitrogen stream. The remaining protein film was dissolved in fibril forming buffer (30 mM Tris, 100 mM NaCl at pH 7.4) to give a final peptide concentration of 200 pM (2.5% DMSO, v / v). A stock solution of ThT at 1 mM was also prepared in advance. For ThT-based aggregation assay, 600 pL of each sample was prepared, with the following composition: 10 pM core peptide (WT or S333D / S342D), and 20 pM ThT. For each sample, 5 replicates of 110 pL samples were pipetted into polybase black 96-well plate with optical bottom (Corning, 3615). 5 replicates of 20 pM ThT were used as negative controls. The plate was sealed, and ThT kinetics were carried out in a FLUOstar Omega plate reader (BMG Labtech) by recording the ThT fluorescence online every 300 s, using a 440 nm excitation filter and a 480 nm emission filter set at 25 °C with shaking at 100 rpm for every 5 s before each measurement. The aggregation curve of every sample shown were the average of 5 independent recordings, and error bars show the standard deviation of the measurements. During the aggregation assay, 5 pL samples were taken at different timepoints (0, 6, 12, 20, 30, 48, 72 h) for TEM analysis.

[0102] WT TDP-43 core peptide aggregation in the presence of nucleotide ligands

[0103] The final peptide concentration was 5 pM, and the concentration of TG12 and UG12 ligands were 10 pM. Peptide sample without ligand addition as the positive control. For each sample, 3 replicates of 95 pL / well were performed. Other settings were the same as above.

[0104] Sedimentation assay

[0105] Aggregation assay

[0106] Monomeric protein samples (WT and S333D / S342D, 4 pM) purified from SEC were aliquoted into 50 pL in Eppendorf tubes. Samples were incubated at a fixed temperature (4, 25, or 37 °C) in the plate incubator. At each timepoint (0, 1, 3, 5, 20, 28, 44, 72, 96 h), samples were centrifuged (15 min, 20,000 g, 4 °C) to precipitate insoluble aggregates, and the supernatants were used for UPLC, SDS-PAGE, and CD analysis. Before each centrifugation, 5 pL of each sample was also taken for TEM analysis. The solubility curves were plotted based on the peak intensities from UPLC, with 3 independent replicates.

[0107] Aggregation inhibition assay

[0108] Monomeric TDP-43 S333D / S342D (4 pM) were aliquoted into 50 pL in Eppendorf tubes. A gradient concentrations of nucleotide ligands were added to paralleled tubes and mixed well, making final concentrations from 0 to 50 pM (2-time dilutions). The tubes were incubated on ice for 10 min before the aggregation induction at 37 °C in a plate incubator. After 3 h incubation, samples were carefully loaded into a 96-well filter plate (Merck, MSGVS2210) with pore size of 0.22 pm. The filtrates were then analyzed by UPLC, SDS-PAGE and CD. The solubility curves were plotted based on the peak intensities from UPLC, with 3 independent replicates.

[0109] Far-TJV circular dichroism (CD) spectroscopy

[0110] CD spectra of TDP-43 samples, loaded in a quartz cuvette with 1 mm path length, were collected using Jasco J-815 CD spectrophotometer operated at 20 °C operated within the range of 200-250 nm. The sample volumes were 150 pL each. Data acquirements used the following parameters: data pitch, 0.2 nm, bandwidth, 1 nm; scanning speed, 50 nm / min, digital integration time, 2 s. A spectra of each sample was the average of 10 repeats followed by a binomial approximation. The processed spectra were obtained by subtracting the baseline signal (quartz cuvette) from the protein spectra with no further smoothening. The raw data were converted to mean residue ellipticity (0MRW). Cellular thermal shift assay

[0111] SH-SY5Y cells were cultured using DMEM medium (LifeTech) with 10% FBS (LifeTech) in a T75 flask. Cells were counted and 20 million cells were used for each experiment.

[0112] Thermal melting assay

[0113] Cell pellets from 20 million cells were resuspended in lx PBS buffer (1.5 mL) with a protease and phosphotase inhibitor cocktail (1%, v / v, prepared in-house). The sample was then lyzed from 3 freeze-thaw cycles using liquid nitrogen, followed by centrifugation (20,000 g, 20 min, 4 °C) to get clear cell lysates. After that, the lysate was aliquoted into 70 pL in 20 PCR tubes (Thermo Scientific, AB0620). TG12 was added to the tubes with a final concentration of 10 pM, and the same volumes of ligand buffer were added to the rest tubes as negative controls. Samples were then incubated at R.T. for 30 min, before individually heated to different temperatures ranging from 44 to 71 °C (interval: 3 °C) for 3 min using thermocycler (Biometra TRIO Touch) Samples were immediately treated with a controlled cooling to 20°C for 1 min. Resulted samples were transferred into 1.5 mL eppendorf tubes and centrifuged at 20,000 g for 20 min at 4 °C. Lammeli buffer was added into the supernatants, and heated at 95 °C for 5 min. The assay was done in 3 independent replicates.

[0114] Dynamic light scattering (DLS)

[0115] Cuvette format

[0116] DLS measurements of the hydrodynamic radius (R were performed at 25 °C with a Nanostar instrument (Wyatt technology) using a 100 pL disposable cuvette (Wyatt technology). 10 pL of samples were loaded and measured using an averaging time of 30 s (10 acquisitions each). Particle translational diffusion coefficients were calculated from decay curves of autocorrelation of light scattering data and converted to hydrodynamic radius (Rh) with the Stokes-Einstein equation. Histograms of mass versus R were calculated using Dynamics software (v7.10.1.21).

[0117] Plate format

[0118] DLS measurements of the hydrodynamic radius (Rh) were performed at R.T. or 37 °C using a DynaPro plate reader II (Wyatt technology) using a 384-well black microplate with optical transparent bottom (Corning, 3540). 30 pL of samples were loaded and measured in 30 s (10 acquisitions each) with auto-attenuation. Each sample was repeated 3 times in parallel. Results were analyzed by Dynamic software (v7.10.1.21).

[0119] Transmission electron microscopy (TEM)

[0120] Prior to the sample loading, Formvar and carbon-coated 200 mesh containing copper EM grids (Electron Microscopy Sciences) were glow-discharged for 30 s at 20 mA using a PELCO easiGlow™ Glow Discharge Cleaning System (TED PELLA, Inc). Subsequently, 5 gL of samples were placed onto the EM grids and waited for 5 min. Samples on the grids were then carefully blotted out using the edge of filter papers and air-dried for 30 s. After that, grids were washed 3 times with ultrapure water and 0.7 % (w / v) uranyl formate solution (prepared in-house), respectively. Grids were then examined using a Tecnai Spirit BioTWIN™ electron microscope. The microscope was equipped with a LaB6 gun, operated at an acceleration voltage of 80kV and images were captured using a 4K x 4K charge-coupled device camera (FEI Eagle).

[0121] In vitro phase separation studies and bright field microscopy

[0122] His-SUMO fusion proteins (WT and S333D / S3342D) were centrifuged at 20,000 g, 20 min, 4 °C to remove any preformed aggregates. Samples were then buffer exchanged into LLPS inducing buffer (20 mM HEPES, 150 mM NaCl, 1 mM DTT, pH 7.4) using the PD-10 desalting column. Concentrations of the resulting protein samples were adjusted to 5, 10 and 15 gM. Samples were then aliquoted in 100 gL into eppendorf tubes, followed by the addition of Ulp-1 (5% v / v). In the case of co-incubation with the ligand, TG12 was also added (Protein: TG12 = 1 :2) together with Ulp-1. After that, samples were mixed thoroughly and quickly transferred into wells of Pluronics- 68-coated chambered coverslips (g-slide 18 well glass bottom, ibidi, Cat No: 81817). Images were acquired after 1 h incubation with Ulp-1 and / or TG12 using bright-field microscopy on a Zeiss LSM 700 Inverted microscope (Apochromat 40x / 1.30 oil objective, and Axiocam MRm (B / W) camera). For the analysis of droplets, images were processed by Image J / Fiji software. Droplets were counted and measured by size, using the command Analyze Particles, excluding particles with a circularity below 0.2 and / or an area smaller than 2 pixels. To have time-dependent observation of condensates aging effect, images were also acquired after 3 h incubation at each concentration, with / without Ulp-1 cleavage and TG12 treatment.

[0123] Biacore™ binding assay

[0124] Fusion proteins (WT and S333D / 342D) purified from IMAC were used for binding studies. Interactions between TDP-43 and TG12 were analyzed by Biacore 8K system (GE Healthcare, Uppsala, Sweden) using amine coupling. Briefly, 30 gg / mL proteins (diluted in NaAc, pH 4.0) were respectively immobilized on sensor chip CM5 (Series S) after surface activation by EDC and NHS (1 :1). A gradient concentration of TG12 (7.8-4000 nM) in the running buffer (lx PBS + 0.02% v / v Tween-20) were injected as analytes. The association and dissociation time for each sample was 120 s. Results were analyzed by Biacore™ Evaluation software (8K version). HDX-MS studies

[0125] HDX-MS experiments were performed following a well-established protocol with minimal modifications (Lentini et al., 2021, Nat. Commun. 12, 3788). HDX reactions were done in 50 pL volumes with a final protein concentration of 1.6 pM. Briefly, 80 pM of TDP-43 S333 / 342D in 8 pL, in absence or in presence of 50 M TG12, were pre-incubated on ice for 5 min before the initiation of deuteration with D2O.

[0126] Deuterium exchange reaction was initiated by adding 42 pL of D2O exchange buffer (10 mM Tris, 300 mM NaCl in D2O, pH 8.0) to the protein sample. For reactions with the TDP-43-TG12 complex, D2O buffer also contained 50 pM TGI 2. Reactions were carried-out on ice for 3 incubation times (3, 30, 300 s) and quenched by the sequential addition of 20 pL of ice-cold quench buffer 1 (4 M Gdn-HCl / I M NaCl / 100 mM NaH2PO4 pH 2.4 / 1% formic acid). Samples were immediately frozen in liquid nitrogen and stored at -80 °C for up to two weeks. All experiments were repeated in triplicate.

[0127] To quantify deuterium uptake into the protein, samples were thawed and injected in a UPLC system immersed in ice with 0.1% formic acid as the liquid phase. The protein was digested via two immobilized pepsin columns (Thermo #23131), and peptides were collected onto a VanGuard precolumn trap (Waters) The trap was subsequently eluted, and peptides separated with a C18, 300 A, 1.7 pm particle size Fortis Bio 100 x 2.1 mm column over a gradient of 8 - 30% buffer C for 20 min at 150 pL / min (Buffer B: 0.1% formic acid; buffer C: 100% acetonitrile). Mass spectra were acquired on an Orbitrap Velos Pro (Thermo), for ions from 400 to 2200 m / z using an electrospray ionization source operated at 300 °C, 5 kV of ion spray voltage. Peptides were identified by data- dependent acquisition of a non-deuterated sample after MS / MS and data were analysed by Mascot. Deuterium incorporation levels were quantified using HD examiner software (Sierra Analytics), and quality of every peptide was checked manually. Results are presented (Fig. 10) as percentage of maximal deuteration compared to theoretical maximal deuteration. Changes in deuteration level between two states were considered significant if >10% and >0.8 Da and p< 0.05 (unpaired / -test).

[0128] Cellular sub-localization of WT and S333D / S342D TDP-43 in cells

[0129] WT and S333D / S342D TDP-43 were synthesized and subcloned into the SinPGK-WHV vector (Thermo Fisher Scientific) using the Gateway pDONR Vector kit (Life Technologies, Cat#12536- 017) and LR Clonase II enzyme mix (Life Technocogies, Cat#l 1791-020). The resulting plasmids were then transformed into One ShotTM Stbl3TM E. coli competent cells and subjected to MaxiPrep preparation. HeLa-ATCC cells were plated in 6-well plate containing coverslips at a density of 150,000 cells per well. Cells were transfected with 2.5 pg of DNA plasmid per well using Lypofectaming 2000 reagent during 6 h followed by changing the culture media. Gene expression was allowed to occur during 48 h prior to fixation with 4% formaldehyde solution for 30 min. Nuclei was counterstained with DAPI, mutant TDP-43 proteins were detected using a mouse monoclonal Anti-HA tag antibody (Abeam, #abl8181). Endogenous full-length TDP-43 was detected with a TDP-43 antibody (Proteintech, #18280-l-AP).

[0130] Transfected cells were fixed with 4% formaldehyde solution for 30 min at room temperature, followed by three washes with lx PBS. To permeabilize the cells, a solution containing 0.5% Triton X-100 and 3% BSA was applied for 1 h at room temperature, and the cells were then washed three times with lx PBS. To block non-specific binding, cells were treated with 3% BSA in PBS for 30 min at room temperature. Primary antibodies, including mouse monoclonal anti-HA tag antibody (abl8181) and TDP-43 antibody (10782-2-AP), were diluted to 1 / 100 in PBS containing 0.5% Triton X-100 and incubated with cells for 2 h at room temperature. After three washes with PBS, secondary antibodies conjugated with fluorophores were added and incubated for 1 h at room temperature. Cells were then washed three times with PBS and counterstained with DAPI solution. Finally, coverslips were mounted on slides with mounting medium. The HA-tag was visualized in red, while full-length TDP-43 was visualized in green.

[0131] Total, cytosolic, and nuclear proteins were separated on a 16% tri cine gel and transferred onto a 0.22 pm nitrocellulose membrane using a semi-dry system from Life Technologies. The membrane was then blocked for 30 min using Blocking Buffer from LiCore, followed by incubation with primary antibodies against either Ha-tag (from Abeam) or TDP-43 (from Proteintech). The primary antibodies were diluted in lx PBS, and the membranes were incubated for 2 h at room temperature while shaking. The secondary antibodies used were Anti-Mouse 680 and Anti-Rabbit 8000, which were used for counterstaining. The LiCore system was used for protein detection and visualization.

[0132] Example 1: Comparison of the aggregation ability of WT TDP-43 C-terminal portion 279- 360 and mutants in this portion

[0133] In order to assess whether introducing phosphorylation or at least one phosphomimetic mutation at some of the Ser residues C-terminal portion 279-360 could stabilize TDP-43 and prevent its aggregation, the double mutant S333D / S342D (SEQ ID NO: 2) was generated.

[0134] The TDP-43 C-terminus core peptide 279-360 (SEQ ID NO: 1) corresponds to the proteaseresistant core of TDP-43 filaments isolated from the brain of an FTD-ALS patient and to the sequence (282-360) that formed aggregates found in ALS patient’s brain (Arseni et al., 2022, supra) and to correspond to the protease resistant amyloid core of TDP-43 filaments derived from recombinant full-length TDP-43 (Kumar et al., 2023, Neurosciene, In press)' . Finally, amyloid fibrils of C-terminal fragments but not the intact full-length protein bind to the amyloid dyes, Thioflavin T and Congo red, thus enabling real-time kinetic studies of TDP-43 fibril formation (Kumar etal., 2023, supra), therefore, this core peptide was used to study the behavior of mutants.

[0135] The TDP-43 C-terminus core peptide 279-360 (SEQ ID NO: 1) without (WT) or with S333D / S342D mutation (SEQ ID NO: 2) was produced using the same expression system recently developed for the full-length TDP-43 protein (Kumar et al., 2023, supra). Briefly, the peptide was produced by fusion to the His-SUMO protein. After cleavage from SUMO, WT and mutant 279- 360-TDP-43 were purified by reverse phase HPLC (Figure 2A).

[0136] The kinetics and extent of aggregation of both peptides using the ThT (Thioflavin T) kinetics-based aggregation assay and electron microscopy as detailed above.

[0137] As shown in Figure 2B, mutant 279-360-TDP-43S333D / S342D showed a much longer lag-phase time and reduced ThT fluorescence compared to the WT peptide. Time-dependent TEM analysis (Figure 2C) showed that the WT peptide formed predominantly short fibrils which were already present as early at 12 h. In contrast, no aggregates were found in the 279-360-TDP -43 S333D / S342D sample at this timepoint. Even after 48 h incubation, only a few fibrils were found in the 279-360- TDP-43S333D / S342D samples, whereas extensive large fibrils and clumps of aggregates were seen in the WT sample at this timepoint. These results demonstrated the first proof of concept that mimicking phosphorylation at S333 and S342 could significantly disrupt the interaction of the core peptides, and inhibit its aggregation and fibrillization.

[0138] Example 2: Comparison of the aggregation ability of WT TDP-43 and mutants in this portion To validate the findings using the TDP-43 amyloid core peptide 270-360, the effect of these mutations on the stability and aggregation of the monomeric full-length (FL) TDP-43 protein was investigated as follows:

[0139] The FL-TDP-43 was prepared by a method based on expressing the protein initially as a His- SUMO-fusion protein as described above.

[0140] The protein was purified by affinity chromatography (IMAC) (Figure 3A-B), followed by a secondary reverse IMAC purification after removal of the His-SUMO tag using Ulp-1 to generate the full-length protein under native conditions. While this method led to the production of highly pure TDP-43 protein, size exclusion chromatography (SEC) analysis of the protein after Ulp-1 cleavage revealed that it had a high propensity to undergo rapid oligomerization. Therefore, the reverse IMAC step was replaced with a SEC. As shown in Figure 3D, WT TDP-43 eluted as two main peaks with elution volumes of 8-10 mL and 14-16 mL representing TDP-43 oligomers and monomers, respectively. SDS-PAGE analysis showed that most of the protein elutes in the monomer peak. However, analysis of the fractions corresponding to the monomeric peak by dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed the presence of oligomers of different sizes ranging from 4 nm to 70 nm (Figure 3F, H), despite the fact that samples were analyzed within only 10 min after their elution from the column. These results suggested that monomeric full-length TDP-43 is unstable and aggregates quickly even at low concentrations (1 pM) at 4 °C, thus precluding studies to investigate its function, structure, and interactome. In contrast, FL TDP-43 S333D / S342D (SEQ ID NO: 3) was expressed and purified using the same method, and it eluted mainly as a single peak with an elution volume (14-16 mL) corresponding to TDP-43 monomers (Figure 3E). The eluted monomeric TDP-43 S333D / S342D fractions were then analyzed by DLS, showing an average hydrodynamic size of 4 nm corresponding to TDP-43 monomers, which confirmed the homogeneity of the sample, and absence of oligomers (Figure 3G). In addition, the size distribution of oligomeric samples was also analyzed. TEM analysis of the monomeric samples C2 revealed the absence of TDP-43 aggregates was only seen in the mutant sample (Figure 3H, 31). Overall, these results demonstrate that the introduction of phosphomimetic mutations at S333 and S342 stabilize monomeric TDP-43 and inhibits its oligomerization and aggregation.

[0141] To further characterize the stability of TDP-43 S333D / S342D monomers, the levels of remaining soluble WT and mutant TDP-43 at room temperature were determined by analysis of the amount of soluble protein in supernatant fractions, obtained after centrifuging the samples at 20,000 g, using UPLC. As shown in Figure 4A, approximately 30% of WT TDP-43 was lost within the first 20 min after purification by SEC. After 44 h, < 5% of protein remained soluble. In contrast, approximately 90- 95% of TDP-43 S333D / S342D remained soluble after 96 h of incubation under the same conditions. These findings were confirmed by SDS-PAGE analysis and quantification of the remaining protein in the supernatants and pellets at different timepoints (Figure 4B-C). Consistent with these findings, circular dichroism (CD) analysis of the supernatants showed dramatic reduction in the signal for WT TDP-43 after 24 h (Figure 4D), whereas the CD spectrum of TDP-43 S333D / S342D remained unchanged even after 72 h incubation at room temperature (Figure 4E).

[0142] Finally, the extent of aggregation by WT and TDP-43 S333D S342D was also monitored and compared by TEM. As shown in Figure 4F, the WT protein formed small oligomers (average size: 25 nm) after the SEC purification, which grew rapidly to large fibrils and amorphous aggregates within the first 5 h. In contrast, no fibrils or large aggregates were observed in the TDP-43 S333D / S342D samples during the first 72 h incubation, and only rare small oligomers were observed. After 1 week, TDP- 43 S333D / S342D formed fibrils and oligomers resembling those formed by the WT protein (Figure 4F- G). Altogether, these findings demonstrate that the TDP-43S333D / S342D is stable and resistant to aggregation at room temperature for up to 90 h, but retains the ability to form fibrils and oligomers after extended period of aggregation.

[0143] Example 3: In Silico mapping of the Serine in cryo EM structure from WT

[0144] All serine residues in the cryo-EM structure of brain-derived TDP-43 filaments (Arseni et al., supra) were mapped and residue properties of the protein surface were analyzed (Figure 5A). Several serine residues located in relatively hydrophobic regions with high potential of preventing or disrupting TDP-43 aggregation when mutated to glutamate or aspartate or upon phosphorylation of the protein were identified. These residues are S317, S332, S333, S347 (Figure 5A). Towards assessing the secondary structure disrupting potential of these mutations, we adopted a neural network-based computational method which is called DeepDDG (http: / / protein.org.cn / ddg.html) (Cao etal., 2019, J. Chem. Inf. Model., 59, 1508-1514). Delta delta G (AAG) is the change in the change in Gibbs free energy (double changes intended), and is a measure of the change in energy between the folded and unfolded states (AGfoiding) of a given protein, and the change in AGfoiding when a point mutation is present. It has been considered as an excellent predictor of whether a point mutation will change the protein stability or not.

[0145] As summarized in Figure 5B, the AAG values dropped upon the introduction of aspartate / glutamate mutation all serine residues, which indicated decreased structure stability of the fibril. Among the residues, mutations at S317, S332, S333 and S347 demonstrated the maximal disrupting effect, followed by mutations at S350, S305, and S292. These findings suggest that introducing single or double S to E / D mutations at these residues could stabilize monomeric TDP-43 in a similar manner as the S333D / S342D mutations. Furthermore, those findings also suggest phosphorylation of monomeric TDP-43 at these residues has high potential of stabilizing monomeric TDP-43 and could serve as a mechanism for regulating its native structure and functions.

[0146] Taken into consideration of the structure polymorphism of the protein, the serine residues were also mapped in the fibrillar structures obtained in vitro. It was found that the localization of these residues was different in some structures, i.e, the S333D / S342D pair. Therefore, AAG values were calculated for all these residues when mutated to aspartate in different structures, and the detailed comparison was shown in Figure 5C. Similar to the patient brain derived fibril (7py2), AAG values for S332, S333 and S342 are very low for most structures, indicating similar destabilizing effect on TDP-43 fibrillization. In addition, there are also other mutation sites showing promising fibril destabilization, i.e, S305 in the entire C-terminal aggregates formed in vitro. Therefore, these residues are also important to stabilize native TDP-43 structure when mutated to aspartates. The list of mutations that we have identified as top candidates for stabilizing the native state of TDP- 43 and / or inhibits its aggregation were highlighted in Figure 5B.

[0147] Example 4: Comparison of the binding properties of the WT and TDP-43S333D / S342D mutants with oligonucleotide ligands, and their aggregation inhibition on TDP-43S333D / S342D.

[0148] The effect of the Serine mutations on the properties of the full-length (FL) TDP-43 protein was investigated as follows:

[0149] To determine whether the S333D / S342D mutations alter the ability of TDP-43 to bind to its natural nucleotide ligands, the binding of WT TDP-43 and TDP-43 S333D / S342D to TG12, which was previously shown to bind to WT TDP-43 with high affinity we assessed and compared using surface plasmon resonance (SPR). The binding of TG12 to both WT and TDP-43 S333D / S342D proteins were very similar, ranging from 122 nM to 158 nM, indicating that the mutations do not compromise the ability of TDP-43 to bind its ligands (Figure 6A, B). As shown in Figure 6C at R.T, TDP- 43 s333D / s342D remains monomeric for days. However, at 37 °C, itundergoes rapid aggregation within 5 h, resulting in the conversion of - 70% of the protein to insoluble aggregates. Therefore, in order to determine whether the strong binding of TDP-43 S333D / S342D to TG12 would translate into stabilization of the monomeric TDP-43 S333D / S342D and inhibit its aggregation, aggregation of TDP- 43S333D / S342D was compared in the absence or the presence of the nucleotide ligands.

[0150] To validate our findings, the size-distribution changes of TDP-43 S333D / S342D in the absence or the presence of the oligonucleotide ligands was monitored using a microwell plate based DLS assay. In the absence of ligands, the average hydrodynamic radius of TDP-43 S333D / S342D increased in a time-dependent manner from 4 nm to 2000 nm upon aggregation induction at 37 °C, and reached a plateau in 3 h (Figure 6E). In the presence of UG12, TG8 or TG12 ligands, we observed a significant delay in the increase of TDP-43 hydrodynamic radius, and the resulting maximum radius of TDP-43 proteins was approximately 140 nm after 5 h of incubation, suggesting that these ligands strongly inhibited TDP-43 oligomerization. The order of inhibitory effect was as follows: TGI 2 > TG8 > UG12 (Figure 6E, F), and correlated well with their reported binding affinity to TDP-43 (Cassel et a , 2010, SLAS Discov. 15, 1099-1106). The TG12 ligand totally abolished the oligomerization of TDP-43 S333D / S342D, as judged by the fact that the hydrodynamics radius (4 nm) of TG12-treated protein samples remained unchanged for up to 5 h. As predicted, all ligands showed dose-dependent aggregation-inhibitory activity (Figure 6G). Example 5: Example of a screening assay using mutants of the invention

[0151] The mutant form of the TDP-43 according to the invention remains monomeric for days at 4 °C or room temperature and can be induced to aggregate and form TDP-43 fibrils by increasing the temperature to 37 °C. Therefore, those properties are ideal for the development of HTS assay as those provide necessary conditions to incubate TDP-43 monomers with compounds prior to the aggregation of the protein.

[0152] The following assay was designed to screen for agents able to modulate TDP-43 aggregation. The overall workflow of the screening was shown in Figure 7A. a) Providing a mutant (mTDP-43) according to the invention mTDP-43 (S333D / S342D) was purified as described in Kumar et al., 2023, supra and 2.5 pM mTDP-43 as determined by nanodrop™ was added at room temperature in Tris buffer (30 mM Tris, 300 mM NaCl, ImM DTT, pH8.0) to reaction wells which (384-well black microplate with optical transparent bottom (Corning, 3540) were then sealed. b) Incubating the mutant with UG / TG rich ligands

[0153] The samples were co-incubated with different TDP-43 natural ligands UG12, TG8 or TG12 (10 pM) at R.T. for 30 min. c) Subjecting the co-incubated mutant to a temperature from about 35 to about 38°C Then, the plate comprising the reaction vessels was loaded into the well-calibrated 37 °C chamber of a dynamic light scattering (DLS) plate reader. d) Measuring the protein mutant aggregation extent

[0154] DLS measurements of the hydrodynamic radius (Rh) were performed automatically using a DynaPro plate reader II (Wyatt technology) every 20 min for 3 h using the method described above (10 acquisitions, 3 s each). The average Rh values were plotted versus the incubation time.

[0155] The results showed that DLS can nicely monitor the aggregation process of TDP-43 aggregation and that the UG / TG-rich ligands can effectively inhibit the aggregation of mTDP-43.

[0156] The same experiment was conducted with the 2.5 pM mutant protein samples pre-incubated with different ligands at R.T. with a final concentration ranging from 0.625 to 80 pM. The non-treated protein sample was used as a control. Then all samples were added to a DLS plate in triplicate, followed by aggregation induction at 37 °C in the plate reader. After 3 h, DLS measurements were done as described above. The average Rh values were plotted versus the ligand concentrations. The results showed that the UG / TG-rich ligands dose-dependently inhibit the aggregation of mTDP-43. Test compound screening using the library of FDA-approved drugs

[0157] In order to assay the role of candidate compounds on the aggregation of mTDP-43, a candidate compound was preloaded into each of the reaction vessels before the addition of the mutant protein under step a). A library of FDA-approved drugs (1350 compounds) was used as candidate compounds to validate the assay.

[0158] Briefly, 60 nL of candidate compounds at 10 mM in DMSO were preloaded to the 384-well DLS plate using the acoustic liquid handling system. mTDP-43 protein samples were freshly prepared and the concentration was adjusted to 2.5 p\1. Then 30 pL protein was added to wells using a multichannel pipette. The plate was sealed and centrifuged briefly for 1 min at 1,000 g to remove any bubbles, followed by thorough mixing for 15 min at R.T. on a plate shaker. TG12 was used as positive control (10 pM in 14 wells), as it showed the best aggregation inhibition of mTDP-43 and DMSO was used as negative control (60 nL in 14 wells).

[0159] Under step c), the plate was then incubated at 37 °C for 3 h to induce protein aggregation, previous to the DLS measurements at 20 °C using the same settings above (step d). In total six plates were screened, and the Z’ score was calculated as 0.56 based on the controls (Zhang et al., 1999, SLAS Discovery, 4(2), 67-73, https: / / doi.org / 10.1177 / 108705719900400206). An excellent Z’ -score of approximately 0.56-0.6x was obtained. Out of the mono dose-based screening, 50 hit compounds with reduced average hydrodynamic radius on TDP-43 aggregation were picked out. SPR results in Figure 7B showed that some compounds can bind to the mTDP-43 with moderate low- micromolar affinity. In addition, TEM analysis on the protein samples that co-incubated with different compounds at 37 °C demonstrated the presence of predominantly TDP-43 oligomers of different sizes (Figure 7C) and the absence of fibrils or large aggregates that are commonly observed in the absence of compounds. Collectively, the results indicated that these compounds may inhibit the fibrillization of TDP-43 through specific binding to form oligomers.

[0160] These data support that an assay using mutants according to the invention can take advantage of the increased stability of the TDP-43 mutants of the invention can lead to a robust high-throughput screening assay to identify several compounds that stabilize monomeric TDP-43 or block its fibrillization.

[0161] Therefore, those data support that an assay using mutants according to the invention is very promising for conducting HTS of large chemical libraries against different TDP-43 targets, including the monomeric TDP-43 for developing effective anti -aggregation drugs for TDP-43 proteinopathies. Such assays could identify small molecules that would bind directly to the C- terminal amyloidogenic domain and prevent its self-assembly or molecules that bind to the functional domain and inhibit TDP-43 aggregation through allosteric effects.

[0162] Since the heterogeneity of the aggregation pathway and structural diversity of TDP-43 aggregates renders targeting TDP-43 pathologies with a single agent challenging or ineffective, an effective approach would be to prevent TDP-43 misfolding in the first place. The above assay approaches are independent of the structure of the different types of TDP-43 aggregates or the nature of the toxic aggregate species since they are designed to prevent aggregation in the first place and could also lead to the identification of molecules that alter TDP-43 aggregation and prevent the formation of toxic or seeding-competent species.

[0163] Example 6: Effects of the mutants on the LLPS of TDP-43.

[0164] Recent studies have shown that TDP-43 and other RNA-binding ALS-related aggregation-prone proteins (e.g., FUS, SOD1) undergo liquid-liquid phase separation (LLPS) and suggested that this process could play a role in both regulating the normal function of the protein and / or the initiation of TDP-43 aggregation and fibrillization (Pakravan et al., 2020, J. Mol. Cell Biol., 13, 15 -28). It is reported that the CTD of TDP-43, especially the short-conserved region (316-346) promotes its condensation by forming weak homomeric contacts (Hallegger et al., 2021, Cell 184, 4680- 4696. e22). To compare the LLPS behavior of the WT and TDP-43 S333D / S342D, His-SUMO fusion TDP-43 proteins were purified by IMAC, followed by buffer exchange into the HEPES buffer. Subsequently, phase separation was induced by SUMO protease Ulpl-mediated cleavage of the His-SUMO tag, and release of the TDP-43 proteins. The extent of in situ phase separation was visualized and followed by bright field microscopy. Both proteins did not show phase separation when fused to the His-SUMO tag, but showed rapid phase separation and formation of distinct types of condensates upon addition of Ulp-1 (Figure 8A). Compared to the WT TDP-43, the TDP- 43 S333D / S342D samples formed much less droplets in the studied concentrations (5-15 pM), and the number of droplets decreased with the decreasing concentrations (Figure 8B). In addition, size analysis revealed the droplets formed by TDP-43 S333D / S342D samples were much bigger (10-35 pm2) than the WT (1-3 pm2) at all tested concentrations, and the size increased with increasing protein concentrations. Similar concentration-dependent decrease / increase in droplet size were observed for the WT samples, although it was not as significant as observed for TDP-43 S333D / S342D (Figure 8C). After 3 h incubation, some condensates from WT samples started aging at 15 pM, where more rigid structure formed. For the mutant samples, droplets were still expanding gradually in size and were more liquid-like, but showed no indication of condensate-aging effect (Figure 8D). In addition, supernatants of the phase separated protein samples at each timepoint were also taken for SDS-PAGE analysis after imaging. The gels in Figure 8E showed that the solubility of the WT protein was not significantly decreased compared to TDP-43S333D / S342D during LLPS, although it seemed to undergo liquid-to-solid phase transition after 3 h of incubation.

[0165] Therefore, this suggests that the mutations in the mutant of the invention does not seem to abolish the phase separation function of TDP-43 and rendered the condensates more dynamic and liquidlike, which may not affect TDP-43 physiological functions related to its LLPS ability.

[0166] Example 7: Effect of the mutations on the subcellular localization

[0167] In order to determine if the monomer stabilizing mutations alter the subcellular localization of TDP- 43, WT or TDP-43 S333D / S342D were expressed in HeLa cells and assessed their subcellular localization by immunohistochemistry. Both WT and TDP-43 S333D / S342D were fused to an HA tag to allow selective monitoring of their subcellular localization and comparison to the endogenous TDP-43. As shown in Figure 9A, WT and S333D / S342D TDP-43 exhibited a predominantly nuclear localization, similar to that of endogenous TDP-43. The western blot analysis of TDP-43 followed by subcellular fractionation also confirmed the observation that TDP-43 S333D / S342D was mainly localized in the nucleus as the WT TDP-43 (Figure 9B). Therefore, these results demonstrate that these mutations do not alter the normal subcellular localization of TDP-43.

[0168] Example 8: The use of mutant TDP-43 to study TDP-43 dynamics and mechanism of stabilization upon ligand binding

[0169] To investigate TDP-43 dynamics, the dynamics of monomeric TDP-43 S333D S342D (10 pM) in the absence and presence of 50 pM TG12 ligand (Figure 10A) was compared using Hydrogen / Deuterium Exchange coupled to Mass Spectrometry (HDX-MS) which allows for the investigation of protein dynamics in solution, as well as for the identification of ligand binding sites and allosteric conformational changes that may occur in other regions of the protein upon ligand binding by monitoring exchange of protein amide hydrogens with the solvent. The hydrogendeuteration exchange reactions were performed by diluting concentrated TDP-43 S333D / S342D with deuterated buffer at 4°C for three reaction times (3, 30 and 300 sec) followed by quenching and analysis of deuteration levels.

[0170] The overall peptide coverage of protein-TG12 complex analyzed by mass spectrometry was of 74% and 95 % in presence and absence of TG12 respectively. Peptides from each domain of TDP- 43 s333D / s342D could be analyzed. As shown in Figure 10B, comparison of H / D exchange levels in the presence and absence of TG12 demonstrated significant differences in two regions. Significant exchange rate difference occurred in the RRMs (108-119 and 221-231) where TG12 had previously been shown to bind (Figure 10C). Interestingly, there were other regions outside the RRMs that also showed protection from exchange upon TG12 binding found in the NTD (43-50), the RRMs (133-149), and CTD (288-303, 332-340, 361-374, 361-374 and 406-415), however not to a significant level (Figure 10D). No increase in H / D rate were observed, indicating that TG12 binding does not destabilize any structural element. Overall, the mutations in the mutant of the invention enable to generate homogenous and stable TDP-43 monomers to study the mechanism of nucleotide ligand induced protein stabilization by HDX-MS, which revealed that the main binding sites of TG12 lies in the RRMs, and that binding leads to a global stabilization of TDP-43 structure in an allosteric manner.

[0171] LIST OF SEQUENCES

[0172] Human (Homo sapiens) Transactive response DNA-binding protein 43 kDa ( TDP-43) core peptide 279-360 (synthetic) SEQ ID NO : 1

[0173] NP GGFGNQGGFG NSRGGGAGLG NNQGSNMGGG MNFGAFS INP AMMAAAQAAL QSSWGMMGML ASQQNQSGPS GNNQNQGNMQ

[0174] Human (Homo sapiens) Transactive response DNA-binding protein 43 kDa ( TDP-43) core peptide 279-360 mutant S333D / S342D (synthetic) SEQ ID NO : 2

[0175] NP GGFGNQGGFG NSRGGGAGLG NNQGSNMGGG MNFGAFS INP AMMAAAQAAL QSDWGMMGML ADQQNQSGPS GNNQNQGNMQ

[0176] Human (Homo sapiens) full length Transactive response DNA-binding protein 43 kDa (TDP-43) mutant S333D / S342D (synthetic) SEQ ID NO : 3

[0177] MSEYIRVTED ENDEPIE I PS EDDGTVLLST VTAQFPGACG LRYRNPVSQC MRGVRLVEGI LHAPDAGWGN LVYWNYPKD NKRKMDETDA SSAVKVKRAV QKTSDLIVLG LPWKTTEQDL KEYFSTFGEV LMVQVKKDLK TGHSKGFGFV RFTEYETQVK VMSQRHMIDG RWCDCKLPNS KQSQDEPLRS RKVFVGRCTE DMTEDELREF FSQYGDVMDV FI PKPFRAFA FVTFADDQIA QSLCGEDLI I KGISVHISNA EPKHNSNRQL ERSGRFGGNP GGFGNQGGFG NSRGGGAGLG NNQGSNMGGG MNFGAFS INP AMMAAAQAAL QSDWGMMGML ADQQNQSGPS GNNQNQGNMQ REPNQAFGSG NNSYSGSNSG AAIGWGSASN AGSGSGFNGG FGSSMDSKSS GWGM

[0178] Human (Homo sapiens) full length Transactive response DNA-binding protein 43 kDa (TDP-43) mutant S333D (synthetic) SEQ ID NO : 4

[0179] MSEYIRVTED ENDEPIE I PS EDDGTVLLST VTAQFPGACG LRYRNPVSQC MRGVRLVEGI LHAPDAGWGN LVYWNYPKD NKRKMDETDA SSAVKVKRAV QKTSDLIVLG LPWKTTEQDL KEYFSTFGEV LMVQVKKDLK TGHSKGFGFV RFTEYETQVK VMSQRHMIDG RWCDCKLPNS KQSQDEPLRS RKVFVGRCTE DMTEDELREF FSQYGDVMDV FI PKPFRAFA FVTFADDQIA QSLCGEDLI I KGISVHISNA EPKHNSNRQL ERSGRFGGNP GGFGNQGGFG NSRGGGAGLG NNQGSNMGGG MNFGAFS INP AMMAAAQAAL QSDWGMMGML ASQQNQSGPS GNNQNQGNMQ REPNQAFGSG NNSYSGSNSG AAIGWGSASN AGSGSGFNGG FGSSMDSKSS GWGM

[0180] Human (Homo sapiens ) full length Transactive response DNA-binding protein 43 kDa (TDP-43) mutantS342D (synthetic) SEQ ID NO : 5 MSEYIRVTED ENDEPIE I PS EDDGTVLLST VTAQFPGACG LRYRNPVSQC MRGVRLVEGI LHAPDAGWGN LVYWNYPKD NKRKMDETDA SSAVKVKRAV QKTSDLIVLG LPWKTTEQDL KEYFSTFGEV LMVQVKKDLK TGHSKGFGFV RFTEYETQVK VMSQRHMIDG RWCDCKLPNS KQSQDEPLRS RKVFVGRCTE DMTEDELREF FSQYGDVMDV FI PKPFRAFA FVTFADDQIA QSLCGEDLI I KGISVHISNA EPKHNSNRQL ERSGRFGGNP GGFGNQGGFG NSRGGGAGLG NNQGSNMGGG MNFGAFS INP AMMAAAQAAL QSSWGMMGML ADQQNQSGPS GNNQNQGNMQ REPNQAFGSG NNSYSGSNSG AAIGWGSASN AGSGSGFNGG FGSSMDSKSS GWGM

[0181] Consensus sequence of a mutated region of a mutant of the invention of Human (Homo sapiens) full length Transactive response DNA- binding protein 43 kDa (TDP-43) ( synthetic) SEQ ID NO : 6

[0182] NP GGFGNQGGFG NXaaRGGGAGLG NNQGXaaNMGGG MNFGAFXaalNP AMMAAAQAAL QXaaXaaWGMMGML AXaaQQNQXaaGPXaa GNNQNQGNMQ

[0183] Wherein each Xaa i s independentl y selected from Ser ( L or D-Ser ) , Asp ( L- or D Asp ) , Glu ( L- or D- Glu ) and Pro ( L or D-Pro ) and wherein at least one o f the Xaa is not Ser .

[0184] Human (Homo sapiens) full length Transactive response DNA-binding protein 43 kDa (TDP-43) (wild-type) SEQ ID NO : 7

[0185] MSEYIRVTED ENDEPIE I PS EDDGTVLLST VTAQFPGACG LRYRNPVSQC MRGVRLVEGI LHAPDAGWGN LVYWNYPKD NKRKMDETDA SSAVKVKRAV QKTSDLIVLG LPWKTTEQDL KEYFSTFGEV LMVQVKKDLK TGHSKGFGFV RFTEYETQVK VMSQRHMIDG RWCDCKLPNS KQSQDEPLRS RKVFVGRCTE DMTEDELREF FSQYGDVMDV FI PKPFRAFA FVTFADDQIA QSLCGEDLI I KGISVHISNA EPKHNSNRQL ERSGRFGGNP GGFGNQGGFG NSRGGGAGLG NNQGSNMGGG MNFGAFS INP AMMAAAQAAL QSSWGMMGML ASQQNQSGPS GNNQNQGNMQ REPNQAFGSG NNSYSGSNSG AAIGWGSASN AGSGSGFNGG FGSSMDSKSS GWGM

Claims

Claims1. An isolated TDP-43 mutant protein, said mutant comprising at least one phosphomimetic mutation of at least one Serine from the C-terminal sequence located between positions 279 and 360 of the TDP-3 wild type sequence.

2. A mutant protein according to claim 1, said mutant comprises at least one phosphomimetic mutation of at least one Serine located at positions selected from 292, 305, 317, 332, 333, 342, 347 and 350 or a combination thereof.

3. A mutant protein according to any one of the preceding claims, wherein said mutant comprises a sequence of SEQ ID NO: 6.

4. A mutant protein according to any one of the preceding claims, said mutant comprising at least two phosphomimetic mutations of at least one Serine from the C-terminal sequence located between positions 279 and 360.

5. A mutant protein according to any one of the preceding claims, wherein said phosphomimetic mutation is a Serine to Aspartic acid mutation.

6. A mutant protein according to any one of the preceding claims, wherein said mutant comprising at least the S333D and / or the S342D mutation(s).

7. A mutant protein according to any one of the preceding claims, wherein said mutant comprises or consists in SEQ ID NO: 3, 4 or 5.

8. A mutant protein according to any one of the preceding claims, wherein said mutant comprises or consists in SEQ ID NO: 3.

9. A mutant protein according to any one of claims 1 to 4, wherein phosphomimetic mutation is a Serine to Glutamic acid mutation.

10. Use of a mutant protein according to any one of claims 1 to 9, for the preparation of oligomers and fibrils of TDP-43 mutant protein.

11. A method for the in vitro preparation of oligomers and fibrils of TDP-43 mutant protein, said method comprising the following steps:- Providing a TDP-43 mutant protein according to anyone of claims 1 to 9 in aqueous solution at a temperature from about 4°C to 25°;- Subjecting the mutant to a temperature increase sufficient to trigger TDP-43 mutant protein aggregation, at or above 25°C but lower than 40°C;- Isolating the formed oligomers and fibrils of TDP-43 mutant protein.

12. A method for the identification of modulators of TDP-43 aggregation, wherein said method comprises:- Providing a TDP-43 mutant protein according to the invention in aqueous solution at a temperature from about 4°C to 25°C;Adding to said mutant in aqueous solution, at least one UG / TG rich ligand and incubating the mixture at a temperature from about 4°C to 25°C;Subjecting the mutant to a temperature increase sufficient to trigger TDP-43 mutant protein aggregation at or above 25°C but lower than 40°C;- Determining the protein mutant aggregation extent in presence of a candidate modulator compared to the protein mutant aggregation extent in absence of a candidate modulator.

13. A method according to claim 12, wherein said TDP-43 mutant is provided in aqueous solution at a temperature from about 20°C to 25°C.

14. A method according to claim 12 or 13, wherein said TDP-43 mutant is provided in aqueous solution at a concentration from about 2 to about 5 pM.

15. A method according to any one of claims 12 to 14, wherein the mutant said is subjected to a temperature increase up to about 37°C and incubated at this temperature for about 1 to about 4 h (e.g. 3 h).