Elevated hydrostatic pressure triggers release of OPA1 and cytochrome C, and induces apoptotic cell death in differentiated RGC-5 cells.

PURPOSE: This study was conducted to determine whether elevated hydrostatic pressure alters mitochondrial structure, triggers release of the dynamin-related guanosine triphosphatase (GTPase) optic atrophy type 1 (OPA1) or cytochrome C from mitochondria, alters OPA1 gene expression, and can directly induce apoptotic cell death in cultured retinal ganglion cell (RGC)-5 cells. METHODS: Differentiated RGC-5 cells were exposed to 30 mmHg for three days in a pressurized incubator. As a control, differentiated RGC-5 cell cultures were incubated simultaneously in a conventional incubator. Live RGC-5 cells were then labeled with MitoTracker Red and mitochondrial morphology was assessed by fluorescence microscopy. Mitochondrial structural changes were also assessed by electron microscopy and three-dimensional (3D) electron microscope tomography. OPA1 mRNA was measured by Taqman quantitative PCR. The cellular distribution of OPA1 protein and cytochrome C was assessed by immunocytochemistry and western blot. Caspase-3 activation was examined by western blot. Apoptotic cell death was evaluated by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. RESULTS: Mitochondrial fission, characterized by the conversion of tubular fused mitochondria into isolated small organelles, was triggered after three days exposure to elevated hydrostatic pressure. Electron microscopy confirmed the fission and noted no changes to mitochondrial architecture, nor outer membrane rupture. Electron microscope tomography showed that elevated pressure depleted mitochondrial cristae content by fourfold. Elevated hydrostatic pressure increased OPA1 gene expression by 35+/-14% on day 2, but reduced expression by 36+/-4% on day 3. Total OPA1 protein content was not changed on day 2 or 3. However, pressure treatment induced release of OPA1 and cytochrome C from mitochondria to the cytoplasm. Elevated pressure also activated caspase-3 and induced apoptotic cell death. CONCLUSIONS: Elevated hydrostatic pressure triggered mitochondrial changes including mitochondrial fission and abnormal cristae depletion, alteration of OPA1 gene expression, and release of OPA1 and cytochrome C into the cytoplasm before the onset of apoptotic cell death in differentiated RGC-5 cells. These results suggest that sustained moderate pressure elevation may directly damage RGC integrity by injuring mitochondria.

Opa1-mediated cristae opening is Bax/Bak and BH3 dependent, required for apoptosis, and independent of Bak oligomerization.

Controversy surrounds the role and mechanism of mitochondrial cristae remodeling in apoptosis. Here we show that the proapoptotic BH3-only proteins Bid and Bim induced full cytochrome c release but only a subtle alteration of crista junctions, which involved the disassembly of Opa1 complexes. Both mitochondrial outer membrane permeabilization (MOMP) and crista junction opening (CJO) were caspase independent and required a functional BH3 domain and Bax/Bak. However, MOMP and CJO were experimentally separable. Pharmacological blockade of MOMP did not prevent Opa1 disassembly and CJO; moreover, expression of a disassembly-resistant mutant Opa1 (Q297V) blocked cytochrome c release and apoptosis but not Bax activation. Thus, apoptosis requires a subtle form of Opa1-dependent crista remodeling that is induced by BH3-only proteins and Bax/Bak but independent of MOMP.

Intraocular pressure elevation induces mitochondrial fission and triggers OPA1 release in glaucomatous optic nerve.

PURPOSE: To determine whether elevation of intraocular pressure (IOP) triggers mitochondrial fission and ultrastructural changes and alters optic atrophy type 1 (OPA1) expression and distribution in the optic nerve (ON) of glaucomatous DBA/2J mice. METHODS: IOP in the eyes of DBA/2J mice was measured, and mitochondrial structural changes were assessed by conventional electron microscopy (EM) and EM tomography. Cytochrome c oxidase IV subunit 1 (COX), OPA1, and Dnm1, a rat homologue of dynamin-related protein-1, mRNA were measured by quantitative (q)PCR. COX and OPA1 protein distribution was assessed by immunocytochemistry and Western blot. RESULTS: Excavation of the optic nerve head (ONH), axon loss, and COX reduction were evident in 10-month-old glaucomatous ONHs of eyes with >20 mm Hg IOP elevation. EM analysis showed mitochondrial fission, matrix swelling, substantially reduced cristae volume, and abnormal cristae depletion in 10-month-old glaucomatous ONH axons. The mean length of mitochondrial cross section in these axons decreased from 858.2 +/- 515.3 nm in 3-month-old mice to 583.3 +/- 298.6 nm in 10-month-old glaucomatous mice (P < 0.001). Moderate reductions of COX mRNA were observed in the 10-month-old DBA/2J mice's ONHs. Larger reductions of OPA1 immunoreactivity and gene expression were coupled with larger increases of Dnm1 gene expression in 10-month-old glaucomatous ONH. Subcellular fractionation analysis indicates increased release of both OPA1 and cytochrome c from mitochondria in 10-month-old glaucomatous ONs. CONCLUSIONS: IOP elevation may directly damage mitochondria in the ONH axons by promoting reduction of COX, mitochondrial fission and cristae depletion, alterations of OPA1 and Dnm1 expression, and induction of OPA1 release. Thus, interventions to preserve mitochondria may be useful for protecting against ON degeneration in glaucoma.