Mass spectrometric analysis of accumulated TDP-43 in amyotrophic lateral sclerosis brains

Results

Immunoblot analysis revealed that abundant abnormal TDP-43 was recovered in the Sarkosyl-insoluble fractions of brains from both ALS cases as shown in Fig. 1A. A phosphorylation-independent anti-TDP-43 detected normal TDP-43 of 43 kD in the fractions of both ALS and control brains, but also detected abnormal TDP-43 bands, including full-length phosphorylated TDP-43 of 45 kD, ~25 kD fragments and smears in ALS brains (Fig. 1A). The pS409/410 antibody, which specifically recognizes the abnormal phosphorylation of Ser409 and 410 strongly reacted with these pathological TDP-43 bands and additional fragments of 18 ~ 24 kD (Fig. 1A). No such bands were detected in the same fraction of control brain. Intensities of pS409/410 immunoreactive bands in both ALS cases were 5 ~ 10 times higher compared with those from the other cases previously analyzed in our lab (data not shown). Therefore, we thought these two cases are good for the protein chemical analysis of pathological TDP-43.

After SDS-PAGE, the gel portions corresponding to these bands were cut out as shown in Fig. 1B and digested with trypsin and chymotrypsin. Digests were analyzed with nano-flow LC-MS/MS. As shown in Tables 1 and 2, we identified 229 peptides from brain of case 1 and 92 peptides from brain of case 2; this difference may reflect the differences in total amounts of pathological TDP-43 in these brains and the numbers of fractions. These peptides covered about 90% of the intact molecule (Tables 1 and 2). Identified peptides (representative), modifications and cleavage sites are shown and summarized in Figs 2 and 3. Further cleavage site peptides are shown in supplemental Fig. S1. Other proteins contaminating the Sarkosyl-insoluble fractions are listed in supplemental Table S1.

Discussion

Accumulation of filamentous inclusions composed of abnormally phosphorylated full-length TDP-43 (45 kDa) and its fragments (35 and 17–27 kDa) is a defining feature of TDP-43 proteinopathies²⁶. It has been reported that overexpression of full-length TDP-43 with or without mutations in cultured cells and animals leads to fragmentation of the protein, generating 35-kDa (CTF-35) and 25-kDa (CTF-25) C-terminal fragments via caspase-mediated TDP-43 cleavage³⁰,³⁴,⁴⁰. Furthermore, activation of calpain, a Ca²⁺-dependent cysteine protease, by upregulation of Ca²⁺-permeable AMPA receptors generates C-terminally cleaved TDP-43 fragments (~35 kDa) and causes mislocalization of TDP-43 in motor neurons³⁶. Those reports noted that the ~35 kDa fragments were localized in the cytoplasm and formed insoluble aggregates.

However, no such TDP-43 peptides cleaved by caspase or calpain reported were not detected in the pathological TDP-43 in the brains of patients in this study. On the other hand, uncleaved peptides by caspase or calpain were detected as the major molecules in the digests. Furthermore, several novel intrinsically cleaved TDP-43 peptides were identified in the pathological TDP-43, i.e., three and five N-terminal peptides (Cys164-Lys165, Tyr214-Gly215 and Asn279-Pro280 in case 1, and Arg55-Leu56, Val75-Asn76, Val108-Leu109, Val130-Leu131 and Asn279-Pro280 in case 2) (Figs 3A,B and 4 and Table 3) and two C-terminal peptide (Asn291-Ser292 in case 1 and Gly400-Phe401 in case 2) (Figs 3A,B and 4), strongly suggested that these cleaved fragments under pathological conditions was different from that under TDP-43-overexpressing conditions.

Cellular expression of TDP-43 is tightly regulated at the transcriptional and post-translational levels. Under pathological conditions, however, formation of TDP-43 aggregates within the cell nucleus or cytoplasm results in reduced free nuclear TDP-43, and therefore the TDP-43 binding-receptor sensor detects a fall in protein levels and responds with increased TDP-43 production; the produced TDP-43 is then sequestered by TDP-43 aggregates². Hence, TDP-43 production under pathological conditions does not necessarily represent overexpression. It is possible that TDP-43 aggregate formation under pathological conditions is different from that under TDP-43-overexpressing conditions.

It has been recently reported that C9ORF72 repeat expansions in mice cause TDP-43 pathology. In these mice, insoluble phosphorylated TDP-43 was predominantly present in monomeric form and high-molecular-weight or truncated pTDP-43 species were not detected⁴¹. Further, recombinant full-length human TDP-43 forms structurally stable, spherical oligomers that are neurotoxic in vitro and in vivo⁴². Indeed, cellular aggregate formation or accumulation of TDP-43 C-terminal fragments (CTFs) is not primarily responsible for development of the observed disease phenotype in mutant or wild-type TDP-43 mice⁴³,⁴⁴,⁴⁵. These findings suggest that full-length TDP-43, not the cleavage fragments, participates at an early stage in TDP-43 pathology. Fragmentation may occur after the accumulation of TDP-43.

We identified some intrinsically cleaved TDP-43 peptides at the N-terminus and C-terminus of Sarkosyl-insoluble TDP-43 in this study. These cleavage sites were neither the caspase cleavage sites nor the calpain cleavage sites of TDP-43 as reported previously⁴⁶,⁴⁷. Several cleavage sites deduced from these N-terminal peptides exist in the region of residues 55–280 as shown in Table 3 and Fig. 4. Therefore, full-length TDP-43 may be gradually processed at N-terminal sites after the formation of insoluble aggregates as shown in Fig. 4. Our findings that cleavage site is correlated with the distribution of molecular weight size are consistent with this idea. Further studies using antibodies for these cleavage sites will clarify the gradual processing mechanism.

In our previous studies, we identified phosphorylation sites by immunochemical methods²⁷ and also identified casein kinase-1 phosphorylation sites on recombinant TDP-43 ³⁸. Almost all Ser and Thr residues in C-terminal Gly-rich half were able to be phosphorylated in vitro experiment³⁸. In the present work, however, a part of accumulated TDP-43 was phosphorylated, not all. Non-phosphorylated peptides were major products from aggregated TDP-43 digests from these ALS brains. We protein-chemically identified several novel phosphorylation sites by directly analyzing the accumulated TDP-43 from two ALS patients. Phosphorylation sites identified in case 1 and case 2 corresponded to the sites identified in casein kinase-1 phosphorylated TDP-43. Although 9 phosphorylation sites were common to both cases, more phosphorylation sites were identified in case 2, as shown in Fig. 3A,B.

It has known that non-phosphorylated intact TDP-43 forms fibrous aggregate⁴², suggesting abnormal phosphorylation is not first event in TDP-43 pathology. The level of phosphorylation may change according to the time when TDP-43 aggregates exist in cytoplasm. Phosphorylation-triggered degradation is a common strategy for elimination of regulatory proteins in many important signaling processes. Phosphorylation of TDP-43 may occur in elimination process. The impairment of this process due to TDP-43 aggregation will induce abnormal phosphorylation of TDP-43 aggregate. As the time has passed, the level of phosphorylation seems to become more. Therefore, the individual difference may occur in the level of phosphorylation.

Furthermore, it seems reasonable to speculate that heavily phosphorylated peptides may not be detected because of low recovery from the column and low ionization efficiency in mass analysis. S409/S410-phosphorylated TDP-43 was immunochemically included in almost all fractions as shown in Fig. 1. However, we found small amount of peptide including S409 with phosphorylation in case 2, while we found no peptide S409/S410 with phosphorylation in case1. A peptide including S409/S410, which derived from chymotrypsin digestion, has at least 5 phosphorylation sites. We have already reported that TDP-43 C-terminal region including these phosphorylation sites was heavily phosphorylated²⁶,²⁷,³². Therefore, we might not detect C-terminal region peptide including S409/S410 residues. It is possible that there are some differences in phosphorylation states of accumulated TDP-43 and in the detection efficiency in the mass analysis between the cases.

Indeed, there were individual differences in other modifications, as shown in Fig. 3A,B. However, we found that common modifications in case 1 and case 2 were focused in the 180–330 region (Fig. 3A,B, and Table 4). This suggests that the 180–330 region of TDP-43 had the same structure in both case 1 and case 2.

Interestingly, inclusions within the brain of ALS and FTLD-TDP patients are readily labelled with antibodies that recognize the C-terminus of TDP-43, but not with N-terminal TDP-43 antibodies. In contrast, spinal cord inclusions are labelled with both N- and C-terminal TDP-43 antibodies, suggesting that they are composed of full-length TDP-43 ⁴⁸. This regional heterogeneity in terms of C-terminal fragment formation suggests that these fragments may not be necessary for TDP43-mediated neurodegeneration⁴⁹. Furthermore, TDP-43 fragments generated during neurodegeneration were not C-terminal fragments, but rather were derived from a central portion of human TDP-43 ⁴⁵. These results indicate that a common structure region, residues 180–330 of TDP-43, is significant for neurodegeneration

It has already reported that a Gly-rich region (287–322), which contains multiple glycine repeats, may contribute significantly to fiber formation as well as aggregation propensity⁵⁰,⁵¹. Recently, it has also been reported that the 274–414 GQN-rich region (C-terminal region) has prion-like properties³³,³⁵, and that RRM2 domain of TDP-43 plays a key role in forming proteinaceous aggregates⁵². The common structure region, residues 180–330 of TDP-43, includes RRM2 and a part of the GQN-rich region. Therefore, this common structure region may be the core region of TDP-43 aggregates. The identification of N-terminal and C-terminal peptides in this study suggests that most of the cleavage sites exist in the N-terminal and C-terminal non-core regions. Proteolytic enzymes may readily access these regions and cleavage may occur gradually from distal regions. Our data provide important insight into the mechanism of TDP-43 accumulation, though further studies will be needed to clarify the molecular pathology of TDP-43 in detail.

Methods

Additional Information

How to cite this article: Kametani, F. et al. Mass spectrometric analysis of accumulated TDP-43 in amyotrophic lateral sclerosis brains. Sci. Rep. 6, 23281; doi: 10.1038/srep23281 (2016).

Supplementary Material