S-Nitrosylation of DRP1 does not affect enzymatic activity and is not specific to Alzheimer's disease

Abstract

Mitochondrial dysfunction and synaptic loss are among the earliest events linked to Alzheimer's disease (AD) and might play a causative role in disease onset and progression. The underlying mechanisms of mitochondrial and synaptic dysfunction in AD remain unclear. We previously reported that nitric oxide (NO) triggers persistent mitochondrial fission and causes neuronal cell death. A recent article claimed that S-nitrosylation of dynamin related protein 1 (DRP1) at cysteine 644 causes protein dimerization and increased GTPase activity and is the mechanism responsible for NO-induced mitochondrial fission and neuronal injury in AD, but not in Parkinson's disease (PD). However, this report remains controversial. To resolve the controversy, we investigated the effects of S-nitrosylation on DRP1 structure and function. Contrary to the previous report, S-nitrosylation of DRP1 does not increase GTPase activity or cause dimerization. In fact, DRP1 does not exist as a dimer under native conditions, but rather as a tetramer capable of self-assembly into higher order spiral- and ring-like oligomeric structures after nucleotide binding. S-nitrosylation, as confirmed by the biotin-switch assay, has no impact on DRP1 oligomerization. Importantly, we found no significant difference in S-nitrosylated DRP1 (SNO-DRP1) levels in brains of age-matched normal, AD, or PD patients. We also found that S-nitrosylation is not specific to DRP1 because S-nitrosylated optic atrophy 1 (SNO-OPA1) is present at comparable levels in all human brain samples. Finally, we show that NO triggers DRP1 phosphorylation at serine 616, which results in its activation and recruitment to mitochondria. Our data indicate the mechanism underlying nitrosative stress-induced mitochondrial fragmentation in AD is not DRP1 S-nitrosylation.

Figure 1

S-Nitrosylation of DRP1 does not alter GTPase activity. A) Kinetics of DRP1 GTPase activity. Recombinant wild-type DRP1 or mutant DRP1^K38A were pre-treated with either 200 μM of fresh SNOC, aged (decayed) SNOC, or left untreated and then subjected to the GTPase assay. Data indicate mean steady state values (n=3). B) Detection of SNO-DRP1. Biotin-switch assay of the DRP1 protein after the GTPase assay of the experiment shown in (A). Proteins were separated by SDS-PAGE and immunoblots were probed with either avidin-horseradish peroxidase for detection of SNO-DRP1 (top) or with anti-DRP1 antibodies (bottom). C) Initial rate of GTP hydrolysis at 500 μM GTP in the presence of DRP1 pretreated with either 200 μM fresh or aged SNOC.

Figure 2

Recombinant and brain DRP1 form tetramers under native conditions irrespective of S-nitrosylation. A) DRP1 runs as tetramer or higher order oligomer in native gels. Recombinant DRP1 isoforms were left untreated or pre-treated with aged or fresh SNOC (200 μM), separated on native gels and stained with Coomassie blue. B) Reducing conditions do not break the tetramers in native gels. Recombinant DRP1 isoforms were separated under non-reducing (-DTT) or reducing conditions (+DTT) on native gels and Coomassie blue stained. C) Human brain DRP1 is mostly a tetramer. Brain extracts from normal controls (lanes 1, 2, 3), AD (lanes 4, 5, 6) or PD patients (lanes 7, 8, 9) were separated on native gels and immunoblots were probed with anti-DRP1 antibodies. D) S-nitrosylation of DRP1 does not trigger DRP1 dimerization. Recombinant DRP1 (699 amino acid isoform) was left untreated or treated with aged or fresh SNOC (200 μM), heat denatured under non-reducing (-DTT) or reducing conditions (+DTT) and separated by SDS-PAGE, immunoblotted, and probed with anti-DRP1 antibodies.

Figure 3

S-Nitrosylation has no effect on DRP1 assembly. Electron micrographs of negatively stained recombinant DRP1 (699aa isoform) in the presence of non-hydrolyzable GTP analog GMP-PNP and corresponding 4× zoom of regions of interest (A) − SNOC and (B) + SNOC. C) Control at 100 mM NaCl with neither nucleotide nor SNOC. D) Control at 100 mM NaCl with 200 μM SNOC without nucleotide. Scale bar, 200 nm.

Figure 4

SNO-DRP1 is not specific to AD. A) DRP1 is endogenously biotinylated. Immunoblot of NeutrAvidin agarose from normal (N), AD or PD human brain lysates probed with anti-DRP1 antibodies. B) Immunoblots of pre-cleared input brain DRP1 protein used in the biotin-switch assay, with actin serving as loading control. C) SNO-DRP1 (arrows) in human normal, AD, or PD brain including the biotin and ascorbate controls using the biotin-switch assay and DRP1 immunoblotting. ID number of postmortem samples is indicated above each blot. D) Ratios of SNO-DRP1 to DRP1 in normal (n=4), AD (n=4), and PD (n=4) brain samples. Ratios were calculated from densitometric values using the ImageJ Gel Analysis software. The SNO-DRP1 signals were corrected for the signals obtained in ascorbate controls. Data are ± S.E.M..

Figure 5

Presence of SNO-OPA1 in normal, AD, and PD brains. Immunoblot of input proteins used in the biotin-switch assay probed with anti-OPA1 or anti-Actin controls (left panels). SNO-OPA1 (arrows) in human normal (N), AD, or PD brain, including the biotin and ascorbate controls, was detected by the biotin-switch assay and OPA1 immunoblotting (right panel).

Figure 6

NO triggers DRP1 Ser616 phosphorylation and recruitment to mitochondria. A) Immunoblots of Ser616 phosphorylated DRP1 and total DRP1 in the cytosolic or mitochondrial fractions. Actin and COX IV were used as loading controls and markers for the cytosolic and mitochondrial fractions, respectively. HEK293 cells were exposed either to aged or fresh SNOC (300 μM) and the cytosolic and mitochondrial fractions were isolated after various time points. B) Immunoblots of phospho-DRP1 (upper panel), total DRP1 (middle panel) and actin (lower panel) in human normal, AD, or PD brain samples. C) Ratios of p-DRP1 Ser616 to DRP1 of normal (n=5), AD (n=5), or PD (n=5) brains. Ratios were obtained by densitometric measurements of immunoblots using the ImageJ Gel Analysis software. Samples were normalized with the actin loading control.