Abstract
Purpose :
Impaired mitochondrial function such as decreased adenosine triphosphate (ATP) production and increased reactive oxygen species (ROS) generation are early abnormalities in age-related neurodegeneration and glaucoma. Although traditional enzymatic cycling assays have been used to detect whole-cell ATP and ROS levels, they lack spatiotemporal resolution and are endpoint rather than real-time measurements. This study aimed to monitor ATP and H2O2 dynamics in live retinal ganglion cells (RGCs) using genetically encoded fluorescent biosensors to provide insights on the mechanistic roles of mitochondrial dysfunction in aging and glaucoma.
Methods :
Primary RGCs were purified from Sprague Dawley rats (P6) via an immunomagnetic bead-based method. Genetically encoded fluorescent biosensors, PercevalHR and mito-Hyper4 (Hyper4 supplemented with a mitochondrial localization sequence), were used for the detection of changes in ATP:ADP ratio and mitochondrial H2O2, respectively. Oligomycin, an ATP synthase inhibitor, was used to induce mitochondrial dysfunction in RGCs. Live-cell confocal imaging was used to capture real-time intracellular ATP:ADP ratio and mitochondrial H2O2 variations in RGCs. Fluorescence intensity and ratios were calculated from a region of interest drawn around each analysed RGC and was compared to the corresponding baseline.
Results :
A total of 50 RGCs were imaged for this study. Only single RGCs with neurite extensions and biosensor fluorescence were selected for imaging. The average fluorescent intensity ratio (F500/F420) of RGCs transduced with PercevalHR and mito-Hyper4 was 16.5 and 7.9, respectively. After addition of oligomycin, PercevalHR showed a 7.1% drop at 20 min, 4.7% drop at 80 min, and a 22.6% drop at 12 h. In contrast, RGCs with mito-Hyper4 showed no change at 20 min, but increased by 16.2% at 60 min, 24.3% at 2 h, and returned to baseline at 24 h (Fig 1).
Conclusions :
PercevalHR and mito-Hyper4 biosensors provided the sensitivity required for spatiotemporal ATP and H2O2 dynamic measurements in subcellular localizations of live primary RGCs. This study suggested that ATP and H2O2 biosensors are promising tools for elucidating the underlying mechanisms of RGC mitochondrial dysfunction in aging and glaucoma.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.