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.
Highlights
Mitochondria undergo fission and fusion under physiologic conditions to maintain optimal morphological characteristics necessary to match ATP production to cellular needs
We investigated the effect of 3 h oxygen-glucose deprivation (OGD) on mitochondrial biogenesis from 0 h to 24 h following reoxygenation in neurons to determine: (1) mitochondrial fission (Drp1 and fission protein 1 (Fis1)) and fusion (Mfn1, Mfn2, and optic atrophy-1 protein (OPA1)) protein changes with western blot (WB); (2) changes in mitochondrial protein expression measuring respiratory chain complex proteins
Western blots revealed the presence of dynamin-related protein 1 (Drp1) (Fig. 3A), Mfn1 (Fig. 4A), Mfn2 (Fig. 4C), OPA1 (Fig. 4E), and Fis1 (Fig. 4G) in the cell lysates
Summary
Mitochondria undergo fission and fusion under physiologic conditions to maintain optimal morphological characteristics necessary to match ATP production to cellular needs. Maintaining a balance between fission and fusion is important in neurons because of high neuronal energy demand and long mitochondrial transport distances especially in motor neurons [1,2]. In neural cells the balance shifts toward fission compared with nonneural cells in order to maintain small, highly motile mitochondria consistent with need [2]. We postulated that unique neuronal requirements necessitate a different mode of mitochondrial dynamics regulation compared with other cell types especially under stress conditions. The major proteins involved in fission/ fusion are dynamin-related protein 1 (Drp1), mitofusin-1(Mfn1), mitofusin-2 (Mfn2), and optic atrophy-1 protein (OPA1). Dynamin-related protein 1 induces mitochondrial fission after translocating to the mitochondrial outer membrane and polymerizing and binding with fission protein 1 (Fis1) [3,4] with Drp activity regulated by post translational modificationssuch as phosphorylation [3,5,6]
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