Êtes-vous un étudiant de l'EPFL à la recherche d'un projet de semestre?
Travaillez avec nous sur des projets en science des données et en visualisation, et déployez votre projet sous forme d'application sur Graph Search.
Preventing the misfolding or aggregation of TDP-43 is the most actively pursued disease-modifying strategy to treat amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. In our work, we provide proof of concept that native state stabilization of TDP-43 is a viable and effective strategy for treating TDP-43 proteinopathies. In Chapter 2 of this thesis, we provide proof of concept that native state stabilization of monomeric TDP-43 is a viable and more effective therapeutic strategy for treating TDP-43 proteinopathies. Firstly, we used published Cryo-EM structures of TDP-43 fibrils to design C-terminal amino acid substitutions, which mimic native posttranslational modifications, to disrupt TDP-43 aggregation. Secondly, we showed that these mutations (S333D/S342D) stabilize monomeric TDP-43 for days without altering its physiological and cellular properties (subcellular localization). Thirdly, we demonstrated that binding native oligonucleotide ligands stabilizes monomeric TDP-43 and prevents its fibrillization and phase separation in the absence of direct binding with the aggregation-prone C-terminal domain. Fourthly, we showed that the monomeric TDP-43 variant (TDP-43S333D/S342D) could be induced to misfold and aggregate in a controlled manner, thus enabling the design and implementation of the first high-throughput screening assay to identify native state stabilizers of TDP-43. In Chapter 3 of this thesis, taking advantage of the previously reported aggregation-inducible and monomeric TDP-43 variant, we scaled up our screen to identify chemical stabilizers of TDP-43 that capture its functional species. Another 8 hit compounds that showed an apparent inhibitory effect on the size increase of TDP-43 during its aggregation were identified in the DLS-based assay. Our data showed that these compounds can bind to both the variant and wild-type TDP-43 with affinities from low to moderate micromolar range. In addition, all compounds were shown to inhibit the aggregation of full-length TDP-43 in a dose-dependent manner, with some of these compounds also inhibiting the aggregation of its amyloid core peptide 279-360. Electron microscopy analysis indicated that these compounds actually stabilized TDP-43 in its various oligomeric states. Subsequent mass photometry analysis of the protein-ligand complexes revealed that some compounds may maintain TDP-43 as stable monomers. This study serves as a strong support for targeting TDP-43 aggregation using native-state stabilizers to rescue its proteinopathies.In Chapter 4 of this thesis, we developed a new protocol to generate different stable forms of unmodified and small-molecule-induced TDP-43 oligomers and investigated their ability to 1) retain some of the functional properties of TDP-43; 2) seed the aggregation of TDP-43; and 3) exert toxic effects upon addition to neurons. We showed that coincubation of TDP-43 with small molecules, such as epigallocatechin gallate (EGCG), dopamine, and 4-hydroxynonenal (4-HNE), increased the production yield of TDP-43 stable oligomers, which could be further purified by size-exclusion chromatography. Interestingly, despite significant differences in the morphology and size distribution of the different TDP-43 oligomer preparations, they all retained the ability to bind to nucleotide RNA. Surprisingly, none of these oligomer preparations could seed the aggregation of TDP-43 core peptide 279-360. Finally, we showed that all 4 types of TDP-43 oligomers exert very mild cytotoxicity to primary neurons. Our results suggest that functional TDP-43 oligomers, other than the native states, could also be stabilized by chemical modulators and might serve as a new approach to halt TDP-43 aggregation in different NDDs.
Hilal Lashuel, Yllza Jasiqi, Lixin Yang
, ,