Fig. 5.

TDP-43 regulates STMN2 expression and CREB phosphorylation in a NO-dependent manner. (A and B) Analysis of STMN2 cryptic exon expression in SH-SY5Y cells transfected with TDP-43 siRNA (siTDP-43) (n = 3). (C and D) Exposure of cells to recombinant TDP-43(1-265) increased STMN2 cryptic exon expression; NOS inhibitor l-NAME significantly reversed this effect (D, n = 3). Expression of STMN2 cryptic exon was monitored by standard RT-PCR (A and C) and qRT-PCR (B and D). GAPDH levels were measured by qRT-PCR as an internal standard. (E) Intracerebral injection of recombinant TDP-43(1-265) results in appearance of phospho-TDP43-positive cytosolic SGs. Four weeks after injection, confocal immunohistochemical images of phospho-TDP-43 (green) and G3BP (SG marker, red) were obtained in cortical sections adjacent to the injection site. (Scale bar, 10 µm.) (F and G) Intracerebral injection of recombinant TDP-43(1-265) induced down-regulation of pCREB expression in vivo. Coinjection of l-NAME to block NOS activity abrogated this result. Representative double-labeled immunofluorescence images of pCREB and total CREB (F), and quantification of pCREB expression (n = 27 brain sections analyzed in nine mice) (G). (Scale bar, 20 µm.) (H and I) Intracerebral injection of recombinant TDP-43(1 to 265) decreases MAP2 expression in vivo. Coinjection of l-NAME abrogated this effect. Representative immunofluorescence images for MAP2-positive neuronal dendrites (H), and quantification of MAP2 expression (n = 27 brain sections analyzed in nine mice) (I). (Scale bar, 40 µm.) Histograms represent mean + SEM; *P = 0.0378 (B), *P = 0.0222 (D), **P = 0.0456 (D), *P = 0.0258 (G), and *P = 0.0042 (I) by Student’s t test; n.s., not significant.