Abstract
Purpose :
Impaired mitochondrial electron transport chain (ETC) function resulting in a decreased mitochondrial membrane potential (MMP) is a primary mechanism of disease in numerous neurovascular and neurodegenerative diseases involving the eye. There is a critical need to develop alternate mechanisms to maintain MMP in order to improve mitochondrial function. We propose the expression of type 1 opsins into the inner mitochondrial membrane (IMM), and when stimulated with light, pump hydrogen ions (H+), across the inner IMM to produce a gradient independent of the oxidative phosphorylation mechanism of the ETC. We hypothesize that Opsins have the potential to Restore Cellular Aerobics (ORCA).
Methods :
Multiple plasmids were designed and tested in Cos-7 cells. Plasmid design includes an IMM localization sequence and protein tags flanking the opsin sequence with a fluorescent marker. Type 1 opsins utilized are Bacteriorhodopsin (bR), Deltarhodopsin (dR), and Xanthorhodopsin (xR). Rotenone and antimycin-a, toxins inhibiting complex I & III of the ETC, were used to establish a depolarized mitochondrial model in order to characterize opsin function. Characterization of opsins were established by measuring cellular viability, mitochondrial morphology, and quantification of mitochondrial cationic dye (JC1). Live cell imaging using confocal microscopy to stimulate and subsequently image mitochondria were conducted to optimize opsin function.
Results :
Western blots of mitochondrial isolates from Cos-7 cells and immunofloucrescent microscopry confirmered successful localization of opsins to mitochondria following transfection. Preliminary light stimulaiton experiments (Figure:1) demonstrates a decrease in mitochondrial potential in the presence of toxins, subsequent increase in light stimulated cells, but no change in non-light stimulated cells. Experiments currently underway include further characterization of light-stimulation protocol, its effects and rescue of in-vitro diseased cell states with ORCA.
Conclusions :
Optogenetics remains a remarkable neuroscientific tool with incredible promise for the field of retina. Our group aims to deepen the clinical translation of optogenetic tools and principles beyond that of initiating phototransduction by modulating sub-cellular retinal physiology. To our knowledge, the work presented herein potentially represents a novel mechanism for establishing an electrochemical gradient within the mitochondria.
This is a 2020 ARVO Annual Meeting abstract.