Fig. 2.

Nitrosative stress triggers formation of SNO-TDP-43 and HMW TDP-43, stimulating TDP-43 aggregation/insolubility in cell-based assays. (A and B) S-nitrosylation of recombinant TDP-43 (amino acids 1 to 265, representing the N-terminal fragment of TDP-43). Recombinant TDP-43(1-265) was incubated with or without SNOC (200 μM), subjected to biotin-switch assay (to assess SNO-TDP-43), and analyzed by Coomassie staining for input TDP-43 (A). Top-down mass spectrometry analysis identified a single NO modification on TDP-43(1-265). Left: Control (without “−SNOC”). Right: SNOC exposed (B). (C and D) Exposure to SNOC leads to formation of disulfide-linked, HMW TDP-43. Recombinant TDP-43(1-265) was exposed to SNOC, separated on SDS-PAGE under reducing or nonreducing conditions, and assessed by Coomassie staining. Monomeric (monomer) and dimeric (dimer) species of recombinant TDP-43(aa 1 to 265) are indicated (C). Primary rat cortical neurons were exposed to SNOC, and after 10 or 40 min, cell lysates were prepared and subjected to biotin-switch assay for detection of SNO-TDP-43. Total cell lysates were electrophoresed on 4 to 12% gradient SDS-PAGE gels under nonreducing or reducing conditions, and immunoblotted with anti-TDP-43 antibody (D). (E and G) In response to NO exposure, TDP-43 increased in the insoluble fraction of neuroblastoma SH-SY5Y cells (E) and primary rat cortical neurons (G). SH-SY5Y cells were exposed to SNOC (200 μM), and after 60 min, RIPA soluble (S) and insoluble (I) fractions were prepared. Immunoblots were conducted with anti-TDP-43 antibodies. Primary cultures of rat cortical neurons were exposed to NMDA (50 μM) in the presence or absence of the NOS inhibitor, l-NAME; the insoluble fraction was analyzed by immunoblot. (F) Recruitment of TDP-43 into SGs after nitrosative stress. SH-SY5Y cells were exposed to the relatively long-lived NO donor GSNO (200 μM), and after 1 h, immunofluorescence images of G3BP (SG marker, red) and TDP-43 (green) were visualized by confocal microscopy. Boxed area is shown at higher magnification. (Scale bar, 10 µm.)