NO induces mitochondrial fission and S-nitrosylation of Drp1. (A) Cortical neurons transfected with mito-DsRed2 were exposed to 200 μM SNOC. Fluorescent images show mitochondrial morphology before and 1 hour after SNOC. (B) SNOC induced mitochondrial fragmentation in a dose-dependent manner. Values are means + SEM (n = 3, *P < 0.05). (C) Cortical neurons were exposed to SNOC or decayed (old) SNOC for <15 min and subjected to biotin-switch assay.

S-Nitrosylation of Cys⁶⁴⁴ in Drp1. (A) HEK293 cells transiently transfected with green fluorescent protein (GFP)–fused constructs for wild-type or representative cysteine mutant Drp1 were exposed to SNOC, lysed, and subjected to immunoprecipitation with antibody to GFP. Precipitated Drp1 was analyzed for S-nitrosylation with the DAN assay by monitoring NO release spectrofluoro-metrically in relative fluorescence units (RFU). Values are means + SEM (n = 3, *P < 0.01). (B) nNOS-HEK293 cells transfected with GFP-fused constructs for wild-type (WT) or representative cysteine mutant Drp1 were exposed to A23187 and subjected to biotin-switch assay. (C) Homology modeling of Drp1 atomic structure: Predicted ribbon structure of human Drp1 showing putative acid/base S-nitrosylation motif around Cys⁶⁴⁴. Yellow, GTPase domain; green, middle (M) domain; magenta, GED domain; C, Cys; E, Glu; K, Lys.

SNO-Drp1 formation in vitro and in vivo. (A and B) Biotin-switch assay of nNOS-HEK293 cells activated with A23187 for 1 to 3 hours. (C) SNO-Drp1 formation by biotin-switch assay in neuronal cells. Left: N2a/APP695 cells were harvested after 30 hours in conditioned medium containing endogenously secreted Aβ soluble oligomers or 30 min after SNOC exposure. Right: Cortical neurons were exposed to 10 μM Aβ25–35 or control Aβ35-25 for 6 hours. (D) SNO-Drp1 detection in vivo by biotin-switch assay of representative human postmortem control (Ct), AD, or Parkinson’s disease (PD) brains. (E) Relative amounts of SNO-Drp1 formation. Biotin-switch assays and immunoblot analyses were quantified by densitometry, and the relative ratio of SNO-Drp1 to total Drp1 was calculated for all samples. Values are means + SEM (n ≥ 4 for each group, *P < 0.02).

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).