Fig. 4.

S-Nitrosylation of Drp1 regulates dimerization, GTPase activity, mitochondrial fragmentation, and neuronal damage. (A) HEK cells expressing enhanced GFP (EGFP)–tagged WT-Drp1 or Drp1(C644A) were exposed to 200 μM SNOC or control decayed SNOC. Cell extracts were subjected to SDS–polyacrylamide gel electrophoresis, with or without dithiothreitol (DTT) to reduce disulfide bonds and thus inhibit dimer formation, and immunoblotted. (B) GST-fused WT-Drp1, Drp1(C644A), and DN-Drp1(K38A) were expressed and purified from bacteria, exposed to SNOC, and assayed for GTPase activity 30 min later (*P < 0.01). (C) Atomic-resolution model of Drp1 superimposed onto electron-density map of homologous domains of dynamin dimer: GTPase (yellow), forming the head; middle (green) and GED (magenta) domains, forming the stalk. (D) S-Nitrosylation of Drp1 enhances mitochondrial fission. Cortical neurons transfected with mito-DsRed2 (Ct), mito-DsRed2 plus WT-Drp1, mito-DsRed2 plus DN-Drp1, mito-DsRed2 plus Drp1(C644A), or mito-DsRed2 plus Drp1(C505A) were exposed to SNOC and scored for fragmented mitochondria 1 hour later (*P < 0.05). (E) SNO-Drp1 increases apoptotic cell death. Cortical neurons transfected with plasmid (p)EGFP, pEGFP plus WT-Drp1, pEGFP plus Drp1(C644A), or pEGFP plus DN-Drp1(K38A) were exposed to SNOC, and 18 hours later stained for NeuN (to identify neurons) and Hoechst 33342 (to assess nuclear morphology). Values are means + SEM (n ≥ 3, *P < 0.02). (F) Naturally secreted Aβ decreases dendritic spine density in cortical neurons via NO. Cultured cortical neurons were cotransfected with EGFP and pcDNA3 (Ct), WT-Drp1, or Drp1(C644A) and exposed for 5 days to 7PA2-conditioned medium containing oligomerized Aβ (+) or control (−). At the right are representative images of dendritic spines of neurons transfected with EGFP-Drp1 (n ≥ 7, *P < 0.002).