Recombinant proteins linking the terminal 152 amino acid residues of TDP43 to maltose binding protein (MBP) and/or a 6×-histidine tag were expressed in Escherichia coli, purified and incubated under physiologic conditions of monovalent salt and neutral pH (Materials and Methods). Both fusion proteins were observed to transition into a gel-like state in a manner temporally concordant with the formation of homogeneous polymers as observed by transmission electron microscopy (EM) (SI Appendix, Fig. S1A). Lyophilized gel samples were evaluated by X-ray diffraction, yielding prominent cross-β diffraction rings at 4.7 and 10 Å (SI Appendix, Fig. S1E). When analyzed by semidenaturing agarose gel electrophoresis, the amyloid-like polymers formed from the TDP43 LC domain dissolved such that the protein migrated in the monomeric state (SI Appendix, Fig. S1B). As such, we conclude that the TDP43 LC domain is capable of forming labile, cross-β polymers similar to polymers formed by the LC domains of the FUS protein (14), various hnRNP proteins (15), and the head domains of various intermediate filament proteins (18).

Prior to gelation, solutions containing the 6×His-tagged LC domain of TDP43 became cloudy (SI Appendix, Fig. S1C). Light microscopic examination of the solutions revealed uniform droplets 2–10 µm in diameter. Recognizing that the TDP43 LC domain contains 10 evolutionarily conserved methionine residues, we exposed the observed droplets to varying concentrations of hydrogen peroxide (H₂O₂). Droplet melting was initially observed at 0.06% (19.6 mM) H₂O₂, and droplets fully disappeared at 0.3% (98 mM) H₂O₂ (Fig. 1A). By contrast, no evidence of melting was observed for liquid-like droplets formed from the LC domain of FUS even upon exposure to 1% H₂O₂, presumably because only one methionine is present in this sequence (17). Upon resolving the protein samples variously exposed to H₂O₂ by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the TDP43 protein was observed to migrate more slowly as a consequence of methionine oxidation (Fig. 1B). Direct evidence of H₂O₂-mediated methionine oxidation of the TDP43 LC domain was confirmed by mass spectrometry as will be shown subsequently in this work.

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Fig. 1.

H₂O₂-mediated oxidation of the TDP43 low complexity domain. (A) Liquid-like droplets of TDP-43 LC domain were exposed to indicated concentrations of H₂O₂ for 1 h. (Scale bar, 25 μm.) (B) Coomassie-stained SDS-PAGE analysis of a purified sample of the TDP43 LC domain after exposure to varied concentrations of H₂O₂ ranging from 0% (left) to 1% (right).

In addition to melting liquid-like droplets formed upon self-association of the TDP43 LC domain, H₂O₂ also disassembled hydrogel polymers formed by the TDP43 LC domain. As shown in SI Appendix, Fig. S2, overnight incubation of cross-β polymers formed from a fusion protein linking MBP to the TDP43 LC domain led to polymer disassembly. The same conditions of incubation with H₂O₂ failed to disassemble cross-β polymers made from either a GFP fusion protein linked to the LC domain of the FUS RNA-binding protein, or an mCherry fusion protein linked to the LC domain of the hnRNPA2 RNA-binding protein.

Assuming that liquid-like droplets formed from the TDP43 LC domain might melt as a consequence of methionine oxidation, we tested for droplet reformation upon addition of the MsrA and MsrB methionine sulfoxide reductase enzymes that are known to reduce the two stereoisomeric forms of methionine sulfoxide (19). H₂O₂-mediated oxidation was first quenched by addition of sodium sulfite (Na₂SO₃). We then added the MsrA and MsrB enzymes and supplemented the reaction with thioredoxin, thioredoxin reductase, and NADPH. These agents allow for sequential steps of reduction of the otherwise oxidized TDP43 methionine residues; reduction of the oxidized MsrA and MsrB enzymes; reduction of oxidized thioredoxin; reduction of oxidized thioredoxin reductase; and terminal conversion of NADPH to NADP⁺. The combination of the five reagents allowed reformation of liquid-like droplets (Fig. 2A) and returned the migration pattern of the TDP43 protein to that of the starting protein as deduced by SDS gel electrophoresis (Fig. 2B). Removal of either of the two methionine sulfoxide reductase enzymes partially impeded droplet reformation and partially restored the more rapid migration of the TDP43 substrate protein on SDS gels. By contrast, droplet reformation was fully eliminated upon removable of: 1) both Msr enzymes; 2) thioredoxin; 3) thioredoxin reductase; or 4) NADPH.

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Fig. 2.

Reduction of the oxidized TDP43 LC domain by two methionine sulfoxide reductase enzymes revives formation of liquid-like droplets. (A) Photographs of liquid-like droplets formed by untreated, recombinant TDP43 LC domain (Upper Left), H₂O₂-oxidized protein (Upper Center), protein exposed to both MsrA and MsrB methionine sulfoxide reductase enzymes, thioredoxin, thioredoxin reductase, and NADPH (Upper Right). Lower three images show droplets formed from enzymatic reduction reactions missing only MsrA (Lower Left), only MsrB (Lower Center), or both methionine sulfoxide reductase enzymes (Lower Right). (B) An SDS-PAGE used to resolve electrophoretic migration patterns of the TDP43 LC domain. H₂O₂-mediated oxidation retarded the migration of the TDP43 LC domain (lane 2). Incubation of the oxidized protein with both MsrA and MsrB methionine sulfoxide reductase enzymes, thioredoxin, thioredoxin reductase, and NADPH restored the migration pattern to that of the reduced protein (lane 3). Removal of either Msr enzyme led to partial restoration of the electrophoretic mobility of the protein (lanes 4 and 5). Removal of both Msr enzymes failed to alter the electrophoretic pattern of the oxidized protein (lane 6). Lanes 7 and 8 were loaded with fully reduced and fully oxidized proteins, respectively.

A method of H₂O₂-mediated footprinting was used to characterize cross-β polymers formed from the TDP43 LC domain (SI Appendix, Fig. S3). Following concepts articulated in a recent study of proteome-wide susceptibility of cellular proteins to H₂O₂-mediated oxidation (20), it was reasoned that regions of the TDP43 LC domain directly involved in the formation of structural order might be partially immune to oxidation relative to regions remaining in a state of molecular disorder. Hydrogel preparations of the TDP43 LC domain were exposed for 5 min to a buffer supplemented with varying concentrations of H₂O₂. The reaction was quenched by the addition of 300 mM Na₂SO₃, desalted to remove the excess sodium sulfite, denatured in 6 M guanidine HCl, and fully oxidized by exposure to 1% ¹⁸O-labeled H₂O₂. The terminal step of saturation oxidation with ¹⁸O-labeled H₂O₂ served two purposes. First, it ensured that no spurious ¹⁶O oxidation could take place during mass spectrometry sample preparation and analysis. Second, it allowed for accurate quantitation of the fractional oxidation from mass spectrometry data by measuring the ¹⁸O/¹⁶O ratio for individual methionine residues (20). The protein was then fragmented to completion with chymotrypsin and subjected to mass spectrometry as a means of determining the ratio of ¹⁸O to ¹⁶O for each of the 10 methionine residues resident within the TDP43 LC domain (Materials and Methods).

We interpret low ¹⁸O/¹⁶O ratios equivalent to fully denatured TDP43 to be reflective of unstructured methionine residues, and high ¹⁸O/¹⁶O ratios to be reflective of methionine residues that might be involved in formation of cross-β structure. Fig. 3 shows a varied pattern of protection for the 10 methionine residues localized within the TDP43 LC domain. This pattern corresponds to an H₂O₂ “footprint” of the TDP43 LC domain observed in highly polymeric, hydrogel samples of the protein (Fig. 3 A, Left). A footprint of protein present in liquid-like droplets is shown in Fig. 3A, Center. These similar footprints predict methionine residues M322 and M323 to be substantially protected from H₂O₂-mediated oxidation. More modest levels of protection were observed for methionine residues M336, M337, and M339. The two methionine residues located closest to the carboxyl terminus of the protein (M405 and M414), and the two methionine residues located on the amino terminal side of the LC domain (M307 and M311), were poorly protected from H₂O₂-mediated oxidation in both hydrogel and liquid-like droplet samples of the TDP43 LC domain.

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Fig. 3.

H₂O₂-mediated footprints of TDP43 protein present in hydrogel samples, liquid-like droplets, and HEK293 cells. (A) For recombinant protein samples used to form hydrogels (Left) and liquid-like droplets (Center), samples were exposed to varying concentrations of H₂O₂ for 30 min. After quenching reactions with sodium sulfite, samples were denatured in 6 M guanidine hydrochloride and exposed for 30 min to a 0.5% solution of ¹⁸O-labeled H₂O₂. The ¹⁸O/¹⁶O ratio for each methionine residue within the TDP43 LC domain was then determined from chymotrypsin digested samples by mass spectrometry and normalized relative to monomeric protein to determine protection factors (Materials and Methods). For cellular TDP43 (Right), HEK293 cells were exposed for 5 min to 0.1% H₂O₂. Cells were cryo-mill disrupted and solubilized in 4 M urea. Flag-tagged TDP43 was recovered by immunoprecipitation, exposed to 5% SDS to fully denature the protein, and exposed for 30 min to a 0.5% solution of ¹⁸O-labeled H₂O₂. Following chymotrypsin fragmentation, the ¹⁸O/¹⁶O ratio for each methionine residue was measured by mass spectrometry (Materials and Methods). For all three samples, the region exhibiting most substantial protection from initial ¹⁶O-H₂O₂–mediated oxidation included methionine residues 322, 323, 336, 337, and 339. (B) The location of the footprinted region of 18 amino acids is highlighted in yellow on the sequences of the TDP43 LC domains of 11 vertebrate species ranging from humans (Homo) to fish (Danio). The methionine residue numbers of human TDP43 are indicated on the top of the sequence.

Moving from test tube studies of recombinant protein to living cells, we used CRISPR methods to insert both a 3×Flag epitope tag and green fluorescent protein (GFP) onto the amino terminus of the endogenous TDP43 protein of HEK293 cells (Materials and Methods). Live-cell imaging of the GFP-tagged protein revealed punctate nuclear staining (SI Appendix, Fig. S4) consistent with numerous published studies of TDP43 (21, 22). In order to probe for the presence or absence of structural order within the TDP43 LC domain in living cells, we exposed the CRISPR-modified HEK293 cells for 5 min to varying levels of H₂O₂. The cells were then frozen in liquid nitrogen and powdered using a cryo-mill instrument (Materials and Methods). Cryo-mill powder was resuspended in lysis buffer and separated into soluble and insoluble fractions by centrifugation. As shown in SI Appendix, Fig. S5, graded increases in H₂O₂ led to graded changes in the ratio of soluble/insoluble TDP43. In the absence of added H₂O₂, western blot analysis revealed that ~50% of the cellular TDP43 remained in an insoluble state. This amount of insoluble protein was reduced in a graded manner upon exposure of cells to 0.01%, 0.03%, or 0.1% H₂O₂ and fully eliminated from cells exposed to either 0.3% or 1% H₂O₂. These H₂O₂-mediated decreases in the insoluble fraction of TDP43 were reciprocally balanced by increases in the amount of soluble protein.

Recognizing that H₂O₂-mediated oxidation of TDP43 might influence its solubility, we resuspended cryo-mill powder in lysis buffer supplemented with 4 M urea. Addition of the chaotropic agent allowed for complete solubilization of TDP43 under conditions compatible with immunoprecipitation using anti-Flag antibodies. We thus treated growing cells for 5 min with 0.1% ¹⁶O-labeled H₂O₂, prepared cryo-mill lysate, solubilized the powder with lysis buffer containing 4 M urea, and immunoprecipitated the Flag-tagged TDP43 protein. Postimmunoprecipitation, the protein was denatured in 5% SDS and exposed to 1% ¹⁸O-labeled H₂O₂ in order to saturate oxidation of all methionine residues within the TDP43 LC domain. The sample was quenched with sodium sulfite, desalted, chymotrypsin digested to completion and evaluated by mass spectrometry to measure the ¹⁸O/¹⁶O ratio of each of the 10 methionine residues within the TDP43 LC domain. As shown in Fig. 3A, Right, the oxidation footprint observed for cellular TDP43 was similar to the footprints observed in both hydrogel and liquid-like droplet preparations of the recombinant protein.

To more carefully investigate the methionine oxidation footprint observed in cells, we performed H₂O₂ dose–response analyses using two methodologies. In the first of the two cases, cells were exposed to 0.03%, 0.1%, or 0.3% H₂O₂ and analyzed by the same methods of shotgun mass spectrometry as described above. As shown in SI Appendix, Fig. S6, graded increases in the level of ¹⁶O methionine oxidation were observed as a function of graded increases in H₂O₂ dose. Cellular material was subsequently processed identically for use in an independent series of experiments wherein targeted methods of mass spectrometry were employed instead of shotgun mass spectrometry (Materials and Methods). Additionally, as analyzed by the targeted mass spectrometry approach, we performed each experiment in triplicate. The combination of considerably more robust data availed by the use of the targeted mass spectrometry approach, coupled with three biological replicates, offers enhanced confidence that the various methionine residues localized within the TDP43 LC domain indeed vary in a reproducible manner with respect to sensitivity to H₂O₂-mediated oxidation. In all experiments, methionine residues 322 and 323 were demonstrably more resistant to oxidation relative to the eight other methionines within the TDP43 LC domain.

The location of the methionine oxidation footprint of TDP43, as defined by the boundaries of methionine residues 322 and 339, is highlighted in yellow upon the sequences of the TDP43 LC domains of 11 vertebrate species, ranging from fish to humans (Fig. 3B). The footprinted region colocalizes with an ultraconserved segment of 22 amino acids. In the 500 million years separating fish from humans, not a single amino acid has been changed within this ultraconserved region in any of the 11 species included in this analysis.

The overlapping footprinted and ultraconserved regions of the TDP43 LC domain also colocalize with a cross-β structure described recently from cryo-EM and solid-state NMR studies of TDP43 LC domain polymers (23, 24). Highly related, dagger-like structures were independently resolved by cryo-EM methods for three different cross-β polymers formed from the TDP43 LC domain (Fig. 4). Structural overlap among the three independent fibrils was observed to initiate at proline residue 320 of the TDP43 LC domain and persist for 15 residues to tryptophan residue 334. In Fig. 4, we compare the methionine oxidation footprint described in this study with the cross-β structures resolved by the Eisenberg laboratory. The two methionine residues most protected from H₂O₂-mediated oxidation, M322 and M323, are disposed wholly within the cross-β structure. The moderately protected methionine residues M336, M337, and M339 are located on the immediate C-terminal side of the cross-β structure. Finally, all five of the oxidation-exposed residues of M307, M311, M359, M405, and M414 were located outside of the Eisenberg cross-β structure.

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Fig. 4.

Correspondence of TDP43 LC domain oxidation footprint and molecular structure of cross-β polymers as deduced by cryo-EM. Arrows connect methionine residues protected from H₂O₂-mediated oxidation (TDP43 LC domain hydrogel footprint) with their position either within the cross-β polymer core (M322 and M323), or on the immediate, C-terminal side of the polymer core (M336, M337, and M339). Molecular model of TDP43 LC domain polymer core is derived from cryo-EM structures resolved for three independent TDP43 LC domain fibrils (23).

The potential roles of different methionine residues in helping establish the biological utility of the TDP43 LC domain were investigated by introducing methionine-to-valine (M-to-V) substitutions into the protein sequence. Each of the 10 M-to-V variants was expressed, purified, and tested in assays for liquid-like droplet formation and cross-β polymerization as deduced by thioflavin-T staining. Eight of the 10 M-to-V variants generated liquid-like droplets indistinguishable from droplets formed from the native LC domain of TDP43 (SI Appendix, Fig. S7A). The M337V variant produced smaller droplets that were noticeably less translucent than those formed by the native LC domain, and the M323V variant yielded amorphous aggregates instead of liquid-like droplets.

The cross-β polymerization capacity of the native LC domain of TDP43 was compared with that of the nine droplet-competent M-to-V variants under three conditions: 1) in the absence of H₂O₂-mediated oxidation; 2) following mild oxidation; and 3) following extensive oxidation. Partial and full oxidation reactions were carried out in the presence of 6 M guanidine such that oxidation would not be influenced by any form of protein structure (Materials and Methods). After quenching with sodium sulfite, samples were analyzed by SDS-PAGE to ensure equivalent levels of protein integrity and degree of oxidation (Fig. 5A). Polymerization was initiated via dilution out of 6 M guanidine and monitored by spectrophotometry using thioflavin-T fluorescence (Fig. 5B). In the absence of oxidation, all nine variants were observed to polymerize as rapidly as the native LC domain, with mildly enhanced polymerization observed for the M336V, M337V, and M339V variants. Under saturating conditions of H₂O₂-mediated oxidation, none of the 10 proteins displayed any evidence of polymerization (SI Appendix, Fig. S7B). Finally, under conditions of partial oxidation, no polymerization was observed for the native TDP43 LC domain. Mild oxidation similarly prevented polymerization for six of the M-to-V variants. Surprisingly, the M337V variant and, to lesser extents, the M336V and M339V variants, polymerized despite partial oxidation.

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Fig. 5.

Polymerization assays for nine M-to-V variants within the TDP43 LC domain. (A) Recombinant protein samples were prepared from the native (WT) TDP43 LC domain and nine M-to-V variants. Samples were prepared in the reduced state (−) as well as after exposure for 25 min to either 0.03% (partially oxidized) or 1% (heavily oxidized) H₂O₂ in the presence of 6 M guanidine HCl. The reduced, partially oxidized, and heavily oxidized samples of each protein were resolved by SDS-PAGE. (B) Oxidation reactions were quenched with sodium sulfite, and polymerization was monitored by thioflavin-T (ThT) fluorescence immediately following 20× dilution into polymerization buffer (Materials and Methods). All 10 of the reduced samples exhibited robust evidence of polymerization, with the M337V variant reproducibly exhibiting an enhanced rate of polymerization (Left). Upon partial oxidation resulting from exposure to 0.03% H₂O₂, polymerization was substantially inhibited for all samples save for the M337 variant (Right). No evidence of polymerization was observed for the native protein or any of the nine M-to-V variants subsequent to exposure to 1% H₂O₂ (SI Appendix, Fig. S7B).

Here, we describe studies of the low complexity domain of the TDP43 protein. We offer the following observations pertinent to the study of this protein. First, the LC domain of TDP43 assembles into a cross-β structure that accounts for localized protection from H₂O₂-mediated oxidation. The morphological properties of the observed pattern of oxidation protection yield a footprint that is similar whether taken from highly polymerized, hydrogel samples of the protein, liquid-like droplet samples of the protein, or the TDP43 protein endogenous to living cells (Fig. 3).

Second, the location of this footprinted region of the TDP43 LC domain corresponds precisely to a region of extreme evolutionary conservation (Fig. 3). The footprinted region is also coincident in location to that of a cross-β polymer core of the TDP43 LC domain characterized at a molecular level by cryo-EM (Fig. 4) (23). Indeed, the idiosyncratic properties of the TDP43 LC domain footprint correlate with the molecular structure of the cross-β polymers resolved by Eisenberg and coworkers. Some degree of transient confusion may, however, accompany publication of the present work because Eisenberg’s dagger-like polymers were reported to be assembled irreversibly (23), whereas our polymers are readily disassembled upon exposure to hydrogen peroxide or assayed by semi-denaturating detergent agarose gel electrophoresis (SI Appendix, Figs. S1 and S2). Nonetheless, it is our belief that our TDP43 LC domain polymers are likely to be assembled in a manner analogous to Eisenberg’s dagger-like structures.

Our observations are not easily reconciled with the reported presence of localized α-helical structure within the TDP43 LC domain (9, 10, 25–27). Solution NMR spectroscopic measurements have given evidence of α-helical secondary structure between residues 322 and 344 of the LC domain in a limited population of TDP-43 molecules. Intriguingly, it is this same region that has been shown to: 1) form structurally precise cross-β interactions at physiological pH (7.4–7.5) as deduced by both cryo-EM and solid-state NMR approaches (23, 24, 28); 2) be protected in a structure-dependent manner from H₂O₂-mediated oxidation (Fig. 3A); and 3) be strikingly conserved through evolution (Fig. 3B). All of the five reports describing this α-helical structure made note that the helix was partially observed only under conditions of acidic pH (lower than 6.1). In two of the five studies, under near-neutral pH (6.5 and above), the TDP43 LC domain was observed to quickly adopt secondary structure heavily dominated by β-conformation (9, 27).

Given that the locations of the α-helical and β-enriched structures of the TDP43 LC domain precisely colocalized, they cannot coexist. Knowing that intracellular pH is neutral, not acidic, we predict that the TDP43 LC domain prefers to adopt a β-conformation in living cells. To this end, we make note that our methionine oxidation footprints observed at neutral pH were indistinguishable as visualized in hydrogel polymers of the TDP43 LC domain (which undoubtedly exist in a cross-β structural conformation), liquid-like droplets, and living cells (Fig. 3).

The third observation of note presented in this study is that M-to-V mutation of residues 336, 337, and 339 of the TDP43 LC domain yielded proteins that polymerize more rapidly than normal and are partially resistant to H₂O₂-mediated inhibition of polymerization (Fig. 5). We make note that the M337V mutation has been observed in independent kindreds by human genetic studies of ALS (29, 30) and that CRISPR-mediated introduction of this single amino acid change into the endogenous TDP43 gene of mice leads to profound neuropathology (31, 32).

We hypothesize that methionine residues 336, 337, and 339 may be of particular importance to a “redox switch” evolutionarily crafted into the LC domain of TDP43. Whereas methionine residues located outside of the cross-β core of the LC domain are far easier to oxidize than those located proximal to the polymer core, we imagine that oxidation of “easy-to-hit” methionine residues may trigger a cascade that loosens the structure—eventually allowing for oxidation of methionine residues 336, 337, and/or 339. Once oxidized within the four amino acid region specifying these “cardinal” methionine residues, we offer that polymerization of the TDP43 LC domain is effectively blocked until the MsrA and MsrB methionine sulfoxide reductase enzymes are able to reduce the protein and revive capacity for LC domain self-association.

Extending from these thoughts, it is possible to appreciate the potential hazard of the M337V mutation, as it might aberrantly allow sustained cytoplasmic aggregation of TDP43 under conditions of partial oxidation. Oxidation-resistant self-assembly of the TDP43 LC domain by the M337V variant might result from a more tightly assembled cross-β structure as hinted by the polymerization assays shown in Fig. 5. It is likewise possible that replacement of methionine 337 with an amino acid side chain that is not susceptible to oxidation also contributes to oxidation resistance of the cross-β assembly. Either or both of these alterations in function of the TDP43 LC domain might account for the disease-causing penetrance of the M337V mutation (29, 30).

In many regards, the observations articulated herein parallel similar studies of the low complexity domain of the yeast ataxin-2 protein (16, 17). Those studies provided evidence that the 24 methionine residues within the yeast ataxin-2 LC domain engender redox sensitivity via the same mechanisms described herein for TDP43. The biologic utility of the yeast ataxin-2 redox sensor can be understood from comprehensive studies of that system. Yeast ataxin-2 is required for the metabolic state of mitochondria to be properly coupled to the TOR pathway and autophagy (17). Since the yeast ataxin-2 protein forms a cloud-like structure surrounding mitochondria, it is sensible to imagine that its methionine-rich LC domain is proximally poised to sense the presence or absence of mitochondria-generated reactive oxygen species. Indeed, detailed experiments have been undertaken to validate the importance of oxidation-mediated changes in the polymeric state of the yeast ataxin-2 LC domain as a key event in coupling the metabolic state of mitochondria to the activity state of TOR and its influence on autophagy (17).

Unlike yeast ataxin-2, TDP43 is primarily a nuclear protein. Why, then, would the LC domain of TDP43 be endowed with the apparent capacity to sense reactive oxygen species? We speculate that when TDP43 shuttles out of the nucleus as a part of ribonucleoprotein complexes housed within RNA granules, oxidation-sensitive self-association of its LC domain might allow for the “unfurling” of RNA granules in the proximity of mitochondria. This might facilitate localized translation of certain mRNAs in the proximity of mitochondria, perhaps useful for optimizing synthesis of mitochondria-destined proteins in the immediate vicinity of mitochondria.

How might this redox regulation be supported by physiological levels of oxidants? The half-maximal concentration of H₂O₂ required for melting droplets of the TDP43 LC domain in vitro is roughly 0.2% (~60 mM) (estimated from Fig. 1B) vs. 0.33% (~100 mM) for the yeast ataxin-2 LC domain (16). These are indeed very high H₂O₂ concentrations. For yeast ataxin-2, we have observed that the addition of as little as 0.0025% H₂O₂ to yeast cells can result in inhibition of the protein’s function (i.e., inhibition of autophagy) (16). This is the equivalent of ~0.7 mM H₂O₂. We speculate that local production of H₂O₂ within particular microcompartments in the cell (e.g., proximal to mitochondria-rich domains) could result in a much higher effective local H₂O₂ concentrations that are comparable to the concentration used in our studies. Lastly, we suspect that even partial oxidation of key methionine residues may be sufficient to tip the balance between monomeric and polymeric states (Fig. 5B). Based on these considerations, we predict that TDP43 LC domain may also be responsive to physiological levels of oxidants in cells.

The ability of MsrA and MsrB to reduce methionine sulfoxides in TDP43 and reverse its aggregation state may be pertinent to age-related alterations that occur in these enzymes. In this regard, it is notable that the accumulation of methionine sulfoxides and reductions in the activity of the Msr enzymes are well-described hallmarks of aging and age-related neurodegenerative disorders (33). It is, thus, possible that aging may disrupt regulation of the TDP43 “redox switch” and that this dysregulation may play a role in exacerbating the pathological features of aging and some neurodegenerative disorders.

We close with consideration of the prominent role of TDP43 in neuronal granules (4, 6). Neuronal granules are found in dendritic extensions of neurons where they are understood to facilitate localized translation proximal to active synapses (34). Knowing that active synapses are mitochondria-rich (35), we offer the simplistic idea that the LC domain of TDP43 might suffer enhanced oxidation upon encountering mitochondria-enriched synapses. If so, we further hypothesize that oxidation-induced dissolution of cross-β polymers otherwise adhering TDP43 to neuronal granules and their resident mRNAs might assist in facilitating synapse-proximal translation.