Gasset-Rosa F, Lu S, Yu H, Chen C, Melamed Z, Guo L, Shorter J, Da Cruz S, Cleveland DW. Cytoplasmic TDP-43 De-mixing Independent of Stress Granules Drives Inhibition of Nuclear Import, Loss of Nuclear TDP-43, and Cell Death. Neuron. 2019 Apr 17;102(2):339-357.e7. Epub 2019 Mar 7 PubMed.
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This work is important because it provides evidence for TDP-43 liquid-liquid phase separation (LLPS), stress granule interactions, and fibrilization/solidification of TDP-43 in cells.
The authors use stable cell lines for exogenous protein expression and not transient transfection, which often leads to protein overexpression. TDP-43 overexpression alone alters protein function and solubility, preventing an accurate interpretation of the phenotypes observed while studying protein localization and solubility.
Briefly, the findings are of paradigm-shifting potential, challenging the idea that stress granules are the crucibles for cytoplasmic TDP-43 pathology in cultured cell models and in disease. The authors elegantly show that solid-like cytoplasmic accumulation of TDP-43, recognized by markers of pathology, occurs independently of stress granule recruitment. These structures are observed upon treatment with preformed fibrils, oxidative stress (arsenite treatment), and increasing cytoplasmic TDP-43 levels.
The authors find that internalization of aggregates derived from recombinant TDP-43 or FUS induces liquid-liquid phase separation of endogenous TDP-43 in the cytoplasm, but not stress granule formation. The cytoplasmic complexes increase with time and some treated cells show depletion of nuclear TDP-43 at later time points, which is a feature that accompanies TDP-43 pathology in ALS and FTD. Even after exposing these cells to oxidative stress, TDP-43 structures fail to significantly co-localize with stress granule markers. These observations contrast numerous previous findings postulating that it is the co-localization of TDP-43 with stress granules that initiates cytoplasmic aggregation.
The experiments studying the ΔNLS TDP-43 mutant add another layer of complexity in understanding TDP-43-stress granule interactions. The authors find only initial co-localization of this mutant with stress granules. At later time points, TDP-43 separates and redistributes into separate cytoplasmic foci in U2OS cells (see Fig4B).
The separation of ΔNLS TDP-43 from stress granules is in agreement with McGurk et al., 2018, which showed that TDP-43 accumulation in stress granules is mediated by poly(ADP-ribose) (PAR) binding through the nuclear localization sequence (NLS). This interaction, however, is not the only required factor for TDP-43-stress granule accumulation, as the Cleveland group also shows that ΔNLS TDP-43 is initially recruited to stress granules during early arsenite treatment.
In future studies, it would be important to increase TDP-43 cytoplasmic localization without disrupting PAR binding to identify the specific mechanisms that induce cytoplasmic TDP-43 granules independent of stress granule accumulation. This is important because, as Gasset-Rosa et al. and McGurk et al. suggest, these stress-granule-independent TDP-43 foci are particularly prone to conversion into aggregates. Therefore, stress-granule recruitment may actually be a protective mechanism to reduce toxic aggregates. This has also been suggested by Alberti and Hyman (Mateju et al., 2017), who found that chaperones are recruited to stress granules and may help in maintaining proper folding of proteins targeted to these complexes. In addition, aberrant stress granules are targeted for degradation by the aggresome to prevent accumulation of misfolded proteins.
Another interesting observation reported here is the conversion of TDP-43 complexes with liquid properties to structures with gel or solid-like properties in cells. This process may be central to understanding the emergence of TDP-43 pathology. We are particularly interested in this process based on our own evidence of a TDP-43 aggregate intermediate, which we suggest acts as a scaffold for large aggregates. Using purified protein, we show that TDP-43 aggregation is initiated by soluble, detergent-resistant oligomers and is followed by fibril or large aggregate formation (French et al., 2019).
References:
McGurk L, Gomes E, Guo L, Mojsilovic-Petrovic J, Tran V, Kalb RG, Shorter J, Bonini NM. Poly(ADP-Ribose) Prevents Pathological Phase Separation of TDP-43 by Promoting Liquid Demixing and Stress Granule Localization. Mol Cell. 2018 Sep 6;71(5):703-717.e9. Epub 2018 Aug 9 PubMed.
Mateju D, Franzmann TM, Patel A, Kopach A, Boczek EE, Maharana S, Lee HO, Carra S, Hyman AA, Alberti S. An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function. EMBO J. 2017 Jun 14;36(12):1669-1687. Epub 2017 Apr 4 PubMed.
French RL, Grese ZR, Aligireddy H, Dhavale DD, Reeb AN, Kedia N, Kotzbauer PT, Bieschke J, Ayala YM. Detection of TAR DNA-binding protein 43 (TDP-43) oligomers as initial intermediate species during aggregate formation. J Biol Chem. 2019 Apr 26;294(17):6696-6709. Epub 2019 Mar 1 PubMed.
View all comments by Yuna AyalaStanford University
The authors show that upon arsenic stress TDP-43 goes initially to stress granules, but when stress persists, TDP-43 demixes from the stress granule and forms a separate type of condensate. While TDP-43 initially remained dynamic in these new condensates, further prolonging arsenic exposure induced a hardening transition. These data are in line with recent reports by McGurk et al. and Mann et al. (McGurk et al., 2018; Mann et al., 2019).
What really sets this paper apart from the two previous reports is that treatment of cells with fibrils from TDP-43 results in the spontaneous phase separation of TDP-43 in the cytoplasm. Compellingly, these condensates persisted for up to a month without maturing into solid-like assemblies, contrary to what happens upon arsenic stress conditions. Moreover, these assemblies were positive for phospho-TDP-43 (an established pathological hallmark) and recruited nuclear transport factors, leading to nuclear transport defects, further depletion of TDP-43 from the nucleus, and eventual cell death. This suggests that cytoplasmic liquid TDP-43 droplets can nonetheless be toxic when they persist, without the need to mature into solid aggregates, as is commonly believed. Additionally it shows that the details of TDP-43 phase separation may be very dependent on the specific stress (arsenic stress vs fibrils in this case). The latter observation also emphasizes that we always need to be careful with extrapolating results made in conditions of arsenic stress to the situation in patients, where likely other stress pathways will be at play.
The finding that TDP-43 can remain in dynamic liquid droplets for extended periods of time that are nonetheless toxic also suggests a provocative possibility: The TDP-43 pathology we have observed for more than a decade in fixed patient material may not necessarily represent aggregates, but could constitute an aberrant liquid/gel phase. This would be in line with the apparent absence of classic amyloid fibrillary structure in TDP-43 pathology. Whether true or not, there is a lot of exciting work ahead to get to the bottom of this!
References:
McGurk L, Gomes E, Guo L, Shorter J, Bonini NM. Poly(ADP-ribose) Engages the TDP-43 Nuclear-Localization Sequence to Regulate Granulo-Filamentous Aggregation. Biochemistry. 2018 Dec 26;57(51):6923-6926. Epub 2018 Dec 17 PubMed.
Mann JR, Gleixner AM, Mauna JC, Gomes E, DeChellis-Marks MR, Needham PG, Copley KE, Hurtle B, Portz B, Pyles NJ, Guo L, Calder CB, Wills ZP, Pandey UB, Kofler JK, Brodsky JL, Thathiah A, Shorter J, Donnelly CJ. RNA Binding Antagonizes Neurotoxic Phase Transitions of TDP-43. Neuron. 2019 Apr 17;102(2):321-338.e8. Epub 2019 Feb 27 PubMed.
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