Fig. 4.

TDP-43 aggregation induces generation of endogenous NO-related species, and nonnitrosylatable mutant TDP-43 rescues neurotoxicity. (A) SH-SY5Y cells were transfected with empty vector (control) or WT TDP-43. To block NOS activity, cells were incubated with l-NAME. Seventy-two hours after transfection, culture medium was collected, and nitrite/nitrate levels were determined using Griess reagent (n = 4). (B and C) Primary rat cortical neurons were exposed to 50 nM recombinant TDP-43(1-265) or control protein (BSA) for 24 h, and the fluorescence response of DAF-FM, an indicator of NO-related species, was measured. Representative images of DAF-FM-labeled cells (B). (Scale bar, 50 µm.) Quantification of changes in DAF-FM fluorescence intensity after exposure to recombinant TDP-43(1-265) (n = 5) (C). (D) Primary rat cortical neurons were exposed to 50 nM recombinant TDP-43(1-265) or control protein (BSA) for 72 h, and cell death was measured by TUNEL assay. Representative images of TUNEL-positive cells (D, Left), and quantification of cell death after exposure to recombinant TDP-43(1-265) (n = 3) (D, Right). (Scale bar, 50 µm.) (E) Effects of SNO-TDP-43 on neuronal cell death. SH-SY5Y cells were transfected with WT TDP-43 or TDP-43(C173A/C175A) mutant constructs, exposed to 100 µM SNOC, and analyzed 24 h later for cell death by TUNEL staining (n = 4 separate experiments). Graphs indicate mean + SEM; *P = 0.0033 (A, control vs. TDP-43), 0.0181 (A, TDP-43 vs. TDP-43 + l-NAME), 0.0426 (D), and 0.0032 (E) by two-tailed Student’s t test. **P = 0.0336 (C) by one-tailed Student’s t test.