Seeding the aggregation of TDP-43 requires post-fibrillization proteolytic cleavage

Despite the strong evidence linking the transactive response DNA-binding protein 43 (TDP-43) aggregation to the pathogenesis of frontotemporal lobar degeneration with TDP-43, amyotrophic lateral sclerosis and several neurodegenerative diseases, our knowledge of the sequence and structural determinants of its aggregation and neurotoxicity remains incomplete. Herein, we present a new method for producing recombinant full-length TDP-43 filaments that exhibit sequence and morphological features similar to those of brain-derived TDP-43 filaments. We show that TDP-43 filaments contain a β-sheet-rich helical amyloid core that is fully buried by the flanking structured domains of the protein. We demonstrate that the proteolytic cleavage of TDP-43 filaments and exposure of this amyloid core are necessary for propagating TDP-43 pathology and enhancing the seeding of brain-derived TDP-43 aggregates. Only TDP-43 filaments with exposed amyloid core efficiently seeded the aggregation of endogenous TDP-43 in cells. These findings suggest that inhibiting the enzymes mediating cleavage of TDP-43 aggregates represents a viable disease-modifying strategy to slow the progression of amyotrophic lateral sclerosis and other TDP-43 proteinopathies.

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Software and code
Policy information about availability of computer code Data collection Cryo-EM images were collected using the EPU 2.6.1 (ThermoFisher Scientific).
Mass spectrometry was performed using LTQ Orbitrap XL (Thermo Fisher, San Jose CA), equipped with an Accela Pump HPLC and a CTC™ThermoPAL™ autosampler (Thermo).
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Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Raw cryo-EM images were deposited to the Electron Microscopy Public Image Archive (EMPIAR) with accession code EMPIAR-10840. Both cryo-EM maps of TDP-43 CP filaments were deposited to the Electron Microscopy Data Bank (EMDB) with accession code EMD-13795. Built atomic model was deposited to the Protein Data Bank (PDB) with accession code PDB ID 7Q3U. Most data from this study are available as Source data within this paper. All other data that are provided in the article and Supplementary information are available from the corresponding author on reasonable request.

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A list of human brain tissues and demographic details associated with the ALS/FTLD-TDP patients and neurologically normal individuals is provided in Supplementary Table 3.

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Samples were chosen based on the neuropathological confirmation and on age of death for both ALS/FTLD-TDP patients and neurologically normal individuals.

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Human postmortem brains were obtained through the guidelines from the University of Pennsylvania, CNDR Brain Bank (doi:10.1016/j.jalz.2013.06.003). All necessary written informed consent forms were obtained from the patients or their next of kin in accordance with University of Pennsylvania Institutional Review Board guidelines and confirmed at the time of death.
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