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

Ju WK, Kim KY, Lindsey JD, Angert M, Patel A, Scott RT, Liu Q, Crowston JG, Ellisman MH, Perkins GA, Weinreb RN - Mol. Vis. (2009)

Bottom Line: Electron microscopy confirmed the fission and noted no changes to mitochondrial architecture, nor outer membrane rupture.Elevated pressure also activated caspase-3 and induced apoptotic cell death.These results suggest that sustained moderate pressure elevation may directly damage RGC integrity by injuring mitochondria.

View Article: PubMed Central - PubMed

Affiliation: The Sophie and Arthur Brody Optic Nerve Laboratory, Hamilton Glaucoma Center, University of California San Diego, La Jolla, CA 92037-0946, USA. danielju@glaucoma.ucsd.edu

ABSTRACT

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.

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Thy1.1 and BDNF immunocytochemistry in differentiated RGC-5 cells. Thy1.1 (A) and BDNF (C) immunoreactivity are present in differentiated RGC-5 cells. Higher magnification showed that Thy1.1 (B) and BDNF (D)-positive RGC-5 cells extend neurites (arrowheads). Size bar represents 20 µm (B and D).
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f6: Thy1.1 and BDNF immunocytochemistry in differentiated RGC-5 cells. Thy1.1 (A) and BDNF (C) immunoreactivity are present in differentiated RGC-5 cells. Higher magnification showed that Thy1.1 (B) and BDNF (D)-positive RGC-5 cells extend neurites (arrowheads). Size bar represents 20 µm (B and D).

Mentions: Apoptosis was induced in differentiated RGC-5 cells exposed to elevated hydrostatic pressure for 3 days. Specific morphological changes of apoptotic cell death included cell body shrinkage and compaction of the nucleus with Hoechst, compared to non-pressurized control cells (Figure 5). The TUNEL-positive apoptotic cells showed bright green labeling of fragmented nuclear DNA (Figure 5B), compared to non-pressurized control cells. To assess apoptotic cell death after elevated hydrostatic pressure, we counted apoptotic-condensed nuclei with Hoechst. The percentage of cells with condensed nuclei appearance was approximately 28.4±6.2% in the cultures exposed to elevated hydrostatic pressure at 3 days, compared to 4.9±3.1% in the non-pressurized control cells (n=3, Figure 5C). Using western blot, RGC-5 cells exposed to elevated hydrostatic pressure showed cleavage of caspade-3 at 3 days. The presence of caspase-3 activation was assessed by the observation of 17 kDa subunit that derived from the cleavage of the 32 kDa proenzyme caspase-3. In contrast, no cleavage of caspase-3 was detected in non-pressurized control cells (Figure 5D). Additionally, we confirmed that differentiated RGC-5 cells expressed BDNF and Thyl.1 as well as extended neurites (Figure 6).


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

Ju WK, Kim KY, Lindsey JD, Angert M, Patel A, Scott RT, Liu Q, Crowston JG, Ellisman MH, Perkins GA, Weinreb RN - Mol. Vis. (2009)

Thy1.1 and BDNF immunocytochemistry in differentiated RGC-5 cells. Thy1.1 (A) and BDNF (C) immunoreactivity are present in differentiated RGC-5 cells. Higher magnification showed that Thy1.1 (B) and BDNF (D)-positive RGC-5 cells extend neurites (arrowheads). Size bar represents 20 µm (B and D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2629709&req=5

f6: Thy1.1 and BDNF immunocytochemistry in differentiated RGC-5 cells. Thy1.1 (A) and BDNF (C) immunoreactivity are present in differentiated RGC-5 cells. Higher magnification showed that Thy1.1 (B) and BDNF (D)-positive RGC-5 cells extend neurites (arrowheads). Size bar represents 20 µm (B and D).
Mentions: Apoptosis was induced in differentiated RGC-5 cells exposed to elevated hydrostatic pressure for 3 days. Specific morphological changes of apoptotic cell death included cell body shrinkage and compaction of the nucleus with Hoechst, compared to non-pressurized control cells (Figure 5). The TUNEL-positive apoptotic cells showed bright green labeling of fragmented nuclear DNA (Figure 5B), compared to non-pressurized control cells. To assess apoptotic cell death after elevated hydrostatic pressure, we counted apoptotic-condensed nuclei with Hoechst. The percentage of cells with condensed nuclei appearance was approximately 28.4±6.2% in the cultures exposed to elevated hydrostatic pressure at 3 days, compared to 4.9±3.1% in the non-pressurized control cells (n=3, Figure 5C). Using western blot, RGC-5 cells exposed to elevated hydrostatic pressure showed cleavage of caspade-3 at 3 days. The presence of caspase-3 activation was assessed by the observation of 17 kDa subunit that derived from the cleavage of the 32 kDa proenzyme caspase-3. In contrast, no cleavage of caspase-3 was detected in non-pressurized control cells (Figure 5D). Additionally, we confirmed that differentiated RGC-5 cells expressed BDNF and Thyl.1 as well as extended neurites (Figure 6).

Bottom Line: Electron microscopy confirmed the fission and noted no changes to mitochondrial architecture, nor outer membrane rupture.Elevated pressure also activated caspase-3 and induced apoptotic cell death.These results suggest that sustained moderate pressure elevation may directly damage RGC integrity by injuring mitochondria.

View Article: PubMed Central - PubMed

Affiliation: The Sophie and Arthur Brody Optic Nerve Laboratory, Hamilton Glaucoma Center, University of California San Diego, La Jolla, CA 92037-0946, USA. danielju@glaucoma.ucsd.edu

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus