Abstract
Purpose: :
Oxidative stress and calcium overload contribute to the pathology of retinal degeneration. We hypothesize that these stressors cause decreases in mitochondrial ATP production and respiratory capacity that contribute to retinal cell dysfunction and degeneration. We used the 661W photoreceptor cell line to study the effects of calcium and oxidative stress on mitochondrial energy metabolism and cell viability. We have also identified neuroprotective agents based on their ability to improve defects in cellular metabolism due to oxidative and calcium stress.
Methods: :
To induce calcium or oxidative stress, 661W cells were treated with IBMX (PDE inhibitor), A23187 (calcium ionophore), paraquat or menadione (redox cyclers). Metabolic responses to the stresses were assessed from changes in extracellular acid release (ECAR) and oxygen consumption (OCR) as measured with a Seahorse Biosciences extracellular flux analyzer. Cell viability was assessed from metabolic and dye exclusion assays. A 50,000 member library of agents from the DIVERSet collection (ChemBridge) were screened for their ability to protect cells from calcium-induced cell death.
Results: :
Basal OCR and ECAR were measured during and after oxidant or calcium stress. Maximal mitochondrial capacity was measured as the increase in OCR following uncoupling with FCCP protonophore in the presence of oligomycin, an ATPase inhibitor. Although the stressors had little or no effect on baseline OCR/ECAR rates, maximal mitochondrial capacity was reduced by stress and the decreases measured at 45 min post-treatment were found to be predictive of cell death measured at 24 hrs.
Conclusions: :
Defects in mitochondrial pathways that produce ATP underlie a number of retinal pathologies, a phenotype that was reproduced by calcium and oxidative stress in 661W cells. These stressors lead to a diminished mitochondrial capacity which can be seen very early in pathogenesis. Thus, perturbation in energy metabolism is likely to serve as an early biomarker in retinal degeneration; and agents that ameliorate the dysregulation of energy metabolism could be developed into therapeutic strategies for treatment of retinal damage.Funding Source Name/Number: This work was supported by NIH grant 1S10RR024582-01, Foundation Fighting Blindness Translational Research Acceleration Program, and an unrestricted grant to MUSC from Research to Prevent Blindness, Inc., New York, NY. BR is a Research to Prevent Blindness Olga Keith Weiss Scholar.
Keywords: metabolism • photoreceptors • calcium