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Abstract #23528 Published in IGR 11-2
Elevated hydrostatic pressure triggers release of OPA1 and cytochrome C, and induces apoptotic cell death in differentiated RGC-5 cells
Ju W-K; Kim K-Y; Lindsey JD; Angert M; Patel A; Scott RT; Liu Q; Crowston JG; Ellisman MH; Perkins GAMolecular Vision 2009; 15:- 120-134
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-dimenstional (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(plus or minus)14% on day 2, but reduced expression by 36(plus or minus)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 cell integrity by injuring mitochondria.
W.-K. Ju. Hamilton Glaucoma Center, University California San Diego, 9415 Campus Point Dr., La Jolla, CA 92037-0946, United States. danielju@glaucoma.ucsd.edu
Classification:
2.13 Retina and retinal nerve fibre layer (Part of: 2 Anatomical structures in glaucoma)
3.6 Cellular biology (Part of: 3 Laboratory methods)
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
3.3 Immunohistochemistry (Part of: 3 Laboratory methods)
