Mitochondrial Dynamics Associated with Oxygen-Glucose Deprivation in Rat Primary Neuronal Cultures

Abstract

Our objective was to investigate the mitochondrial dynamics following oxygen-glucose deprivation (OGD) in cultured rat cortical neurons. We documented changes in morphology, protein expression, and DNA levels in mitochondria following OGD and examined the roles of mitochondrial fission [dynamin-related protein 1 (Drp1), fission protein-1 (Fis1)] and fusion [mitofusin-1 (Mfn1), mitofusin-2 (Mfn2), and optic atrophy-1 protein (OPA1)] proteins on mitochondrial biogenesis and morphogenesis. We tested the effects of two Drp1 blockers [15-deoxy-Δ12,14-Prostaglandin J2 (PGJ2) and Mitochondrial Division Inhibitor (Mdivi-1)] on mitochondrial dynamics and cell survival. One hour of OGD had minimal effects on neuronal viability but mitochondria appeared condensed. Three hours of OGD caused a 60% decrease in neuronal viability accompanied by a transition from primarily normal/tubular and lesser number of rounded mitochondria during normoxia to either poorly labeled or small and large rounded mitochondria. The percentage of rounded mitochondria remained the same. The mitochondrial voltage-dependent anion channel, Complex V, and mitoDNA levels increased after OGD associated with a dramatic reduction in Drp1 expression, less reduction in Mfn2 expression, an increase in Mfn1 expression, with no changes in either OPA1 or Fis1. Although PGJ2 increased polymerization of Drp1, it did not reduce cell death or alter mitochondrial morphology following OGD and Mdivi-1 did not protect neurons against OGD. In summary, mitochondrial biogenesis and maintained fusion occurred in neurons along with mitochondrial fission following OGD; thus Mfn1 but not Drp1 may be a major regulator of these processes.

Sustained mitochondrial functioning in cerebral arteries after transient ischemic stress in the rat: a potential target for therapies

“…Complex V produces ATP from ADP via phosphorylation in the presence of the respiratory chain's electron transport-generated proton gradient across the inner mitochondrial membrane (35). These findings concerning VDAC and complex V subunit-␣ are in agreement with our previous findings on rat primary cortical neurons after OGD (40). However, there were no differences between protein levels in the contralateral and control groups.…”

“…We performed Western blot analysis on isolated MCAs (23,40). Proteins were extracted by homogenizing MCAs using a Radnoti glass tissue grinder in 4°C Nonidet P-40 lysis buffer (Invitrogen, Frederick, MD) containing a proteinase and phosphatase inhibitor (both at 5 l/ml, Sigma-Aldrich).…”

“…DNA was harvested by homogenizing isolated MCAs in 200 l Nuclei Lysis solution using a DNA extraction kit (Promega, Madison, WI). Levels of mitochondrial (mt)DNA were measured using the mitochondrial cytochrome b gene (MT-CYB, Rn03296746_s1), which was normalized to the nuclear heat shock protein 70 gene (Hspa 1a, Rn00583013_s1, Applied Biosystems) (40). Triplicates of the samples were run using the following protocol: 1 cycle at 95°C for 15.05 min and 45 cycles at 95°C for 15 s and 60°C for 1 min.…”

Local Mitochondrial-Endolysosomal Microfusion Cleaves Voltage-Dependent Anion Channel 1 To Promote Survival in Hypoxia

“…They are dynamic organelles that continuously undergo fission and fusion during cell growth (3,4). Under stress conditions, such as nutrient depletion or hypoxia, mitochondria either fragment or are degraded by HIF-dependent mitophagy (mitochondrial removal by autophagy) (5) or hyperfuse together to form elongated or rounded structures that optimize ATP production and promote cell survival (6)(7)(8)(9)(10)(11).…”

Mitochondrial Quality Control as a Therapeutic Target

“…Several groups have screened chemical libraries and uncovered three novel molecules: Mdivi-1 (Cassidy-Stone et al, 2008), Dynasore (Macia et al, 2006), and P110 (Qi et al, 2013). Among these, Mdivi-1 has been extensively investigated in experimental rodent models of epilepsy Xie et al, 2013), ischemia (Park et al, 2011;Zhang et al, 2013), oxygen-glucose deprivation (Wappler et al, 2013), rhabdomyolysis (Tang et al, 2013), and conditions involving endosome aggregation and vesicle fusion during exocytosis (Chlystun et al, 2013). In all of these states, Mdivi-1 exhibited beneficial effects on cells and tissues by restricting mitochondrial fission and maintaining normal fission-fusion balance and cell function.…”