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Anthony P. Leonard, Nathan Perron, Cécile Nasarre, Craig Beeson, Bärbel Rohrer; Mitochondrial Fusion is Linked to Bioenergetic Capacity and Cell Survival In a Cell-based Model of PDE6ß-dependent RP. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5471.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis Pigmentosa (RP) is a chronic, progressive retinal degenerative disease with limited therapies. Our data and those of others have suggested that metabolism is reduced with the onset of cell death in the rd1 mouse model of RP (i.e., lack of function of PDE6ß resulting in calcium cytotoxicity; Acosta et al., 2005; Lohr et al., 2006). The present study was undertaken to establish a molecular mechanism for a novel class of neuroprotective compounds identified in a high throughput screen designed to identify agents effective in slowing degeneration in a cell-culture based model of PDE6ß-dependent RP.
The mouse retina-derived 661w cone photoreceptor cell line was grown under standard conditions. Calcium cytotoxicity was induced by either treating the cells directly with Ca2+ ionophore A23187 or by opening the cGMP-gated cation channels using phosphodiesterase inhibitor IBMX. Cell viability was measured using tetrazolium dye reduction; mitochondrial respiratory capacity was analyzed via Seahorse extracellular flux assays; levels of relevant proteins were analyzed via Western blots of whole cell lysates; and mitochondrial morphology was visualized by tetramethylrhodamine methyl ester (TMRM) and epifluorescence microscopy.
A class of compounds sharing a common pharmacophore was identified that rescues 661w cells from Ca2+-mediated damage by increasing mitochondrial respiratory capacity. While basal and maximal respiratory capacity are decreased in IBMX-treated cells exposed to vehicle, addition of either lead compound CB11 or CB12 significantly restored both energy parameters. Impaired mitochondrial metabolism was characterized by increased mitochondrial fission, which was also reversed by CB11 or CB12 pretreatment. Interestingly, CB12 treatment was correlated with increased protein levels of the mitochondrial fusion protein mitofusin 2 (Mfn2) and decreased levels of the pro-fission protein dynamin-related protein 1 (Drp1).
Taken together, these data suggest that the induction of a pro-fusion phenotype is associated with mitochondrial protection and cell survival. This study is the first known linkage between mitochondrial fission and bioenergetic capacity in RP and represents a novel mechanism and potential treatment for disease-mediated mitochondrial damage in the retina.
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