Antibody against TDP-43 phosphorylated at serine 375 suggests conformational differences of TDP-43 aggregates among FTLD–TDP subtypes

Abstract

Aggregation of hyperphosphorylated TDP-43 is the hallmark pathological feature of the most common molecular form of frontotemporal lobar degeneration (FTLD–TDP) and in the vast majority of cases with amyotrophic lateral sclerosis (ALS–TDP). However, most of the specific phosphorylation sites remain to be determined, and their relevance regarding pathogenicity and clinical and pathological phenotypic diversity in FTLD–TDP and ALS–TDP remains to be identified. Here, we generated a novel antibody raised against TDP-43 phosphorylated at serine 375 (pTDP-43S375) located in the low-complexity domain, and used it to investigate the presence of S375 phosphorylation in a series (n = 44) of FTLD–TDP and ALS–TDP cases. Immunoblot analysis demonstrated phosphorylation of S375 to be a consistent feature of pathological TDP-43 species, including full-length and C-terminal fragments, in all FTLD–TDP subtypes examined (A–C) and in ALS–TDP. Of particular interest, however, detailed immunohistochemical analysis showed striking differences in the immunoreactivity profile of inclusions with the pTDP-43S375 antiserum among pathological subtypes. TDP-43 pathology of ALS–TDP, FTLD–TDP type B (including cases with the C9orf72 mutation), and FTLD–TDP type C all showed strong pTDP-43S375 immunoreactivity that was similar in amount and morphology to that seen with an antibody against TDP-43 phosphorylated at S409/410 used as the gold standard. In stark contrast, TDP-43 pathology in sporadic and genetic forms of FTLD–TDP type A (including cases with GRN and C9orf72 mutations) was found to be almost completely negative by pTDP-43S375 immunohistochemistry. These data suggest a subtype-specific, conformation-dependent binding of pTDP-43S375 antiserum to TDP-43 aggregates, consistent with the idea of distinct structural TDP-43 conformers (i.e., TDP-43 strains) as the molecular basis for the phenotypic diversity in TDP-43 proteinopathies.

Cryo-EM structure of amyloid fibrils formed by the entire low complexity domain of TDP-43

“…One of the features of ALS and FTLD pathology is phosphorylation of Ser residues within the C-terminal part of the TDP-43 LCD, with the consensus pathological phosphorylation sites at Ser403, Ser404, and Ser409/410, and additional disease-specific phosphorylation sites at Ser379 and Ser369 3 , 38 . The full phosphorylation landscape and population of molecules phosphorylated at specific sites are, however, unknown.…”

Post-Translational Modifications Modulate Proteinopathies of TDP-43, FUS and hnRNP-A/B in Amyotrophic Lateral Sclerosis

“…In line with this, different un-phosphorylatable mutations of Ser to Ala weakly induce phase transition. On the other hand, it is also known that phosphorylated TDP-43 represents one of the predominant components of protein aggregates in ALS and FTD ( Hasegawa et al, 2008 ; Guedes et al, 2017 ), in which TDP-43 is found phosphorylated in its LCD, mainly on Ser 403/404 and 409/410 ( Neumann et al, 2020 ). Nevertheless, whether this phosphorylation occurs before or after aggregation and its possible causative role in stabilizing protein aggregation need to be yet clarified.…”

Cryo-EM structure of amyloid fibrils formed by the entire low complexity domain of TDP-43

“…One of the features of ALS and FTLD pathology is phosphorylation of Ser residues within the C-terminal part of TDP-43 LCD 2,19 . Most of these phosphorylation sites are buried in the interior of the fibril structure that we determined for the non-phosphorylated protein (Extended Data Fig.…”