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

Abstract

TDP-43 is the major disease-associated protein involved in the pathogenesis and progression of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions linked to TDP-43 pathology (FTLD-TDP). Abnormal phosphorylation, truncation and cytoplasmic mis-localization are known to be the characteristics for the aggregated forms of TDP-43, and gain of toxic abnormal TDP-43 or loss of function of physiological TDP-43 have been suggested as the cause of neurodegeneration. However, most of the post-translational modifications or truncation sites in the abnormal TDP-43 in brains of patients remain to be identified by protein chemical analysis. In this study, we carried out a highly sensitive liquid chromatography-mass spectrometry analysis of Sarkosyl-insoluble pathological TDP-43 from brains of ALS patients and identified several novel phosphorylation sites, deamidation sites, and cleavage sites. Almost all modifications were localized in the Gly-rich C-terminal half. Most of the cleavage sites identified in this study are novel and are located in N-terminal half, suggesting that these sites may be more accessible to proteolytic enzymes. The data obtained in this study provide a foundation for the molecular mechanisms of TDP-43 aggregation and ALS pathogenesis.

An Amyloid-Like Pathological Conformation of TDP-43 Is Stabilized by Hypercooperative Hydrogen Bonds

“…Indeed, based on the similarities of the characteristic bands in SDS-PAGE chromatograms previously described (Nonaka et al, 2013; Kametani et al, 2016), our conformer may represent the structural core of Type A TDP-43 proteinopathy, the most common class of TDP-43 maladies (Mackenzie et al, 2011). As TDP-43 Type A aggregates are remarkably more heat-stable as seeds than Type B or Type C aggregates (Nonaka et al, 2013), what remains to be addressed is the basis of this exceptional stability.…”

“…Based on the apparent protection of residues 341–367 against Ser phosphorylation, Gln/Asn deamidation and Met oxidation in TDP-43 ex vivo aggregates, we therefore propose as a working hypothesis that our conformational model for this segment (Figure 2) may correspond to the pathological conformer present in the ex vivo aggregates of patient 2 described by Kametani et al (2016). Indeed, based on the similarities of the characteristic bands in SDS-PAGE chromatograms previously described (Nonaka et al, 2013; Kametani et al, 2016), our conformer may represent the structural core of Type A TDP-43 proteinopathy, the most common class of TDP-43 maladies (Mackenzie et al, 2011).…”

“…Most phosphorylated residues (18 of 29) are in the C-terminal region, including S342, S347 and S350 in the Q/N-rich subregion. More recently, this laboratory has also analyzed TDP-43 aggregates from two ex vivo ALS brains (Kametani et al, 2016). Remarkably, they found 13 phosphorylation sites in one brain and 15 sites in the second.…”

“…Moreover, the recent mass-spectrometry/proteolysis study of TDP-43 from ex vivo brains of two ALS patients provides insight into the relative importance of the hydrophobic and Q/N rich regions (Kametani et al, 2016). In this report, Kametani and co-workers found that residues 341–346 or 341–360 of the Q/N-rich segment were protected against proteolytic cleavage, deamidation and phosphorylation (in patient 1 and patient 2, respectively).…”

Profilin 1 mutants form aggregates that induce accumulation of prion-like TDP-43

“…Interestingly, the C-terminal fragment-banding patterns of converted host proteins resemble those of insoluble TDP-43 used as seeds, suggesting that the conversion is template-dependent. Moreover, mass spectrometric analysis of insoluble TDP-43 from brains of ALS patients suggested that RRM2, the glycine-rich domain, and a part of the Q/N-rich domain form the core region of TDP-43 aggregates 23 . These results suggest that seed-dependent accumulation of prion-like, conformationally changed TDP-43 via interaction at the C-terminal region has a pivotal role in the pathogenesis of ALS and FTLD-TDP.…”