Abstract 3903: Dynamin-related Protein 1 Independent Apoptosis In Neurons Following Oxygen-glucose Deprivation

Background and purpose: Previous studies showed that mitochondrial (mito) fission occurs prior to apoptosis and that a key initiator is the mito fission protein dynamin-related protein 1 (Drp1). However, little information is available concerning mito dynamics in neurons due to OGD. Therefore, we investigated mito fission and mito fusion responses in cultured neurons in an in vitro model of brain ischemia. Methods: Primary rat cortical neurons were isolated from E18 Sprague Dawley fetuses. Neurons were exposed to OGD 9 days after culturing for 3 hr followed by up to 24 hr of reoxygenation. Mito dynamics were investigated by measuring the expression of: 1) mito fission [Drp1; mito fission 1 (Fis1)] and mito fusion [mitofusin-2 (Mfn2); optic atrophy-1 (OPA1)] proteins; 2) electron transport chain proteins (complex II 70 kDa subunit, complex IV subunit I, complex V alpha subunit); 3) mito DNA; and 4) apoptosis protein Bax. We also examined neuronal and mito morphology using transmission electron microscopy. The effects of the Drp1 inhibitors 15d-Prostaglandin J 2 (2.5-20 µM) (15d-PGJ 2 ) and Mdivi-1 (2.5-250 µM) were also investigated. Results: 3hr OGD followed by 3-24 hr recovery resulted in an increase in the level of mito DNA and enhanced expression of complex IV and V proteins. During this time, abnormal morphology, including enlarged and/or swollen mitochondria, was often observed. However, many mitochondria still retained normal morphology. Drp1 levels decreased before virtually disappearing by 6 hr of reoxygenation following OGD while Fis1 expression did not change. Bax expression increased with an increasing number of apoptotic cells during this time. Mfn2 decreased after OGD while OPA1 did not change significantly. 15d-PGJ 2 treatment dose-dependently increased cell death both in control and OGD-exposed neurons. Furthermore, 15d-PGJ 2 treatment surprisingly increased Drp1 both in controls and in OGD-exposed cells, and also increased the mito DNA. Additionally, 15d-PGJ 2 treatment itself increased the number of larger mitochondria in the neurons. However, Mdivi-1 did not have any effects. Conclusions: Despite a lower fission/fusion protein ratio, the increase in mitochondrial size indicates a shift to enhanced fusion in neurons following OGD. In addition, apoptosis induction seems to be Drp1 independent in this model. Additionally, 15d-PGJ 2 treatment appears to decrease survival in neurons following OGD. Thus, mito dynamics are affected by OGD and 15d-PGJ 2 treatment and play a role in cell survival/death. </jats:p

Mitochondrial dynamics associated with oxygen-glucose deprivation in rat primary neuronal cultures.

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

Antibodies used in this study.

<p>Antibodies used in this study.</p

Changes in Drp1 protein expression in neurons following OGD.

<p>Representative Drp1 blots are shown with the corresponding β-actin blot using standard western blot protocol and monoclonal Drp1 antibody in control and OGD samples (A) in non-treated and proteinase inhibitor (PI)-treated control and OGD samples (B). Drp1 is highly expressed in neurons under normoxic condition and decreases following 3 h OGD. Proteinase inhibitor treatment results in higher Drp1 expression in OGD cells but is still greatly reduced compared to control. (C) A representative immunoblot with its corresponding β-actin blot using non-denaturing conditions and monoclonal Drp1 antibody. Arrow indicates monomer sized Drp1. Note the two very faint bands of approximately 26 kDa and 32 kDa. Control neurons express high amount of dimer/trimer sized Drp1 that decrease following 3 h OGD. Relative immunoband intensity of Drp1 bands and Drp1 dimer/monomer band ratios are presented as means ± s.e.m. of <i>n</i> ≥3 independent experiments (D-E). *p<0.05 **p<0.005 ***p<0.001 vs. control. Drp1 = Dynamin-related protein 1, PI = proteinase inhibitor, OGD = oxygen-glucose deprivation, R/O = reoxygenation.</p

Transmission electron microscopic (TEM) images of mitochondrial changes in neurons after OGD.

<p>This figure shows representative TEM images of control and OGD cells. In control cells the mitochondria are mainly small, tubular, with normal density (A, top picture), however, there are some mitochondria that are larger and have normal, or increased intra-mitochondrial density (A, bottom picture). Following 1 h OGD there were several larger and elongated mitochondria in the cortical neurons (B). Several cells also showed the nuclear and cytoplasmic signs of apoptosis (condensed/peripheralized nuclear chromatin and/or cytoplasm, cytoplasmic vacuolization) or necrosis (decreased cytoplasmic density, no detectable plasma membrane) even after 1 h OGD. Following 3 h OGD several mitochondria are large, swollen, show a loss in their internal cristae structure, and their matrix is less dense compared with control (arrowheads). There are, however, several larger, but morphologically intact mitochondria with normal or high density, and some cells have both kinds of mitochondria (Fig. C). Furthermore, most of the cells following 3 h of OGD showed apoptotic or necrotic signs in the TEM pictures. With increasing time after 3 h OGD there were increased numbers of large, swollen mitochondria with rupture/loss of internal cristae (D, arrowheads), whereas some cells still had morphologically intact mitochondria (D, stars). Bars represent 500 nm, stars = mitochondria with intact internal membrane structure, arrowheads = damaged mitochondria, ch = nuclear chromatin, m = mitochondrion, N = nucleus. OGD = oxygen-glucose deprivation.</p

Mfn1, Mfn2, OPA1, and Fis1 protein expression in neurons following OGD.

<p>Representative western blots are shown for Mfn1 (A), Mfn2 (C), OPA1 (E), and Fis1 (G) with their corresponding β-actin blots. Panels B, D, F, and H show relative intensity bar graphs for the same proteins as means ± s.e.m. of <i>n</i>≥3 independent experiments. Mfn1 increased after OGD, whereas Mfn2 decreased after three, but did not change after one h OGD. OPA1 did not change significantly following OGD. Fis1 protein expression does not change following OGD in neurons. *p<0.05 ***p<0.001 vs. control, <i>n</i>≥3 independent experiments. mitofusin1 = Mfn1, mitofusin2 = Mfn2, optic atrophy-1 protein = OPA1, OGD = oxygen-glucose deprivation.</p

Changes in VDAC, complex II and V proteins, and mtDNA expression following 3 h OGD.

<p>Representative western blots for VDAC, Complex II, and Complex V with their corresponding β-actin blots (A, B, C), together with relative intensity values for these proteins (D). VDAC and Complex V proteins showed increased expression, whereas Complex II protein expression did not change significantly following OGD. (E) Shows the changes in mtDNA expression following 3 h of OGD. MtDNA/nuclear DNA ratio significantly increased 12 h following 3 h OGD. *p<0.05 **p<0.005 ***p<0.001 vs. control, n≥3 independent experiments. mtDNA = mitochondrial DNA, OGD = oxygen-glucose deprivation, VDAC = voltage-dependent anion channel.</p

The effect of PGJ2 or Mdivi-1 treatment on cortical neurons under normal and OGD conditions.

<p>(A) Shows a representative Drp1 western blot of control, and OGD cells with and without PGJ₂ treatment. With the non-denaturing western blot method an additional band (above the 227 kDa molecular weight marker) appeared on the western blot due to PGJ₂ treatment both in control, and in OGD samples, but it was not detectable after 24 h reoxygenation. (B) Shows the changes in viability of the cells due to PGJ₂ treatment and 3 h OGD. PGJ₂ treatment decreased cell viability in 15 and 20 µmol/L concentrations and further decreased cell viability after 3 h OGD in 10, 15, and 20 µmol/L concentrations. (C) Shows a representative Drp1 western blot of control, and OGD cells with and without Mdivi-1 treatment. The treatment did not change Drp1 expression in our experiments. (D) Shows the changes in viability of the cells due to Mdivi-1 treatment and 3 h OGD. Mdivi-1 was not protective in 3 h OGD, and at higher doses (75, 100 µmol/L) it decreased cell viability in control neurons. Each panel represents <i>n</i>≥3 independent experiments. *p<0.05 vs. control, † p<0.05 vs. untreated OGD, Co = Control, Drp1 = Dynamin-related protein 1, Mdivi-1 = Mitochondrial division inhibitor-1, PGJ₂ = 15-deoxy-D12,14-prostaglandin J₂, R/O = reoxygenation, OGD = oxygen-glucose deprivation.</p