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Mausumi Bandyopadhyay, Nathan Perron, Cecile Nasarre, Craig Beeson, Baerbel Rohrer; Increasing mitochondrial respiratory capacity is protective in models of photoreceptor cell degeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1951.
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© ARVO (1962-2015); The Authors (2016-present)
Alterations in energy metabolism are associated with rod degeneration. An increase in glycolysis over mitochondrial respiration has been identified prior to the onset of rod loss in the rd1 mouse model of retinitis pigmentosa (RP). We hypothesize that preserving mitochondrial respiratory capacity (MRC) might lead to prolonged cell survival. Hence, we sought to identify compounds, which target MRC, for neuroprotection in the rd1 retina.
The DIVERSet library (ChemBridge), a unique, non-proprietary collection of 50,000 synthetic small molecules that covers the maximum pharmacophore diversity with the minimum number of compounds, was used. Three rounds of screening, increasing in complexity, were performed. As a Primary Screen, cell survival assays (MTS) were performed, identifying compounds that protect against Ca2+ cytotoxicity induced by Ca2+ ionophore A23187. As a Secondary Screen, compounds were analyzed using Seahorse extracellular flux (XF) assays, measuring lactate secretion (glycolysis) and oxygen consumption (mitochondrial ATP production). As a Tertiary Screen, compounds were tested in rd1 retina/RPE explants to determine their effect on rod degeneration. Postnatal day 10 explants were treated every 48 hrs with compounds for a total of 10 days. Cultures were sectioned and counterstained to count rows of photoreceptors in ten regions.
A23187 was titrated to result in 50% cell death after 24 hrs. Twelve compounds were identified to protect against Ca2+ cytotoxicity. For XF assays, IBMX was titrated to cause a 50% decrease of the maximal (FCCP-uncoupled) oxygen consumption rate after 24 hrs. Pretreatment with six compounds significantly preserved uncoupled mitochondrial function. In rd1 explants, four compounds slowed rod cell loss, resulting in 2-4-fold increase in thickness of the outer nuclear layer after 10 days in culture.
These results suggest that impaired energy metabolism in photoreceptors is a major contributor to RP; and our High-Content Screening protocol identified novel compounds for the treatment of RP and other forms of neurodegeneration.
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