Analysis of mitochondrial dynamics and function in the retinal pigment epithelium by high-speed high-resolution live imaging

Abstract

Mitochondrial dysfunction is strongly implicated in neurodegenerative diseases including age-related macular degeneration (AMD), which causes irreversible blindness in over 50 million older adults worldwide. A key site of insult in AMD is the retinal pigment epithelium (RPE), a monolayer of postmitotic polarized cells that performs essential functions for photoreceptor health and vision. Recent studies from our group and others have identified several features of mitochondrial dysfunction in AMD including mitochondrial fragmentation and bioenergetic defects. While these studies provide valuable insight at fixed points in time, high-resolution, high-speed live imaging is essential for following mitochondrial injury in real time and identifying disease mechanisms. Here, we demonstrate the advantages of live imaging to investigate RPE mitochondrial dynamics in cell-based and mouse models. We show that mitochondria in the RPE form extensive networks that are destroyed by fixation and discuss important live imaging considerations that can interfere with accurate evaluation of mitochondrial integrity such as RPE differentiation status and acquisition parameters. Our data demonstrate that RPE mitochondria show localized heterogeneities in membrane potential and ATP production that could reflect focal changes in metabolism and oxidative stress. Contacts between the mitochondria and organelles such as the ER and lysosomes mediate calcium flux and mitochondrial fission. Live imaging of mouse RPE flatmounts revealed a striking loss of mitochondrial integrity in albino mouse RPE compared to pigmented mice that could have significant functional consequences for cellular metabolism. Our studies lay a framework to guide experimental design and selection of model systems for evaluating mitochondrial health and function in the RPE.

Targeting complement C3a receptor resolves mitochondrial hyperfusion and subretinal microglial activation in progranulin-deficient frontotemporal dementia

“…Given the early transcriptional changes in mitochondrial ROS metabolism, we decided to investigate mitochondrial dynamics and function by high-speed super-resolution live imaging of RPE explants from wildtype and Grn −/− mice 30 . Live imaging of MitoTracker-labeled mitochondria and volumetric reconstructions of mitochondrial networks showed increased connectivity or hyperfusion of mitochondria in 4-mo Grn −/− RPE that was even more pronounced in 17-mo mice (Figures 5A-5C).…”

Daily Light Onset and Plasma Membrane Tethers Regulate Mitochondria Redistribution within the Retinal Pigment Epithelium

“…Throughout the day after light onset, we found mitochondria redistributed away from the basal region of the cell towards the apical surface. Mitochondrial movement within the RPE has been studied in cell culture models by fluorescence microscopy but has not been shown in tissues at the resolution of electron microscopy that allows precise determination of apical vs. basal distribution [14,34,35]. The fixative used in electron microscopy can cause some sample shrinkage, but generally good preservation is maintained, as has been shown in correlative live cell imaging and electron microscopy studies [36,37].…”

“…Therefore, mitochondrial movement and regions of close apposition between mitochondrial and other organelle membranes, known as membrane contact sites (MCSs), can likely aid in adapting and responding to changes in the local environment [12]. Previous studies including our work have shown that mammalian RPE is heavily populated with a heterogeneous population of mitochondria based on membrane potential and ATP production, and these mitochondria predominantly localize to the basal region of the cell [13][14][15].…”

Recent Advances in Our Understanding of Age-Related Macular Degeneration: Mitochondrial Dysfunction, Redox Signaling, and the Complement System

“…Dysfunction in mitochondrial homeostasis, through abnormal dynamics and metabolic activities, represent a critical sign of advanced cellular age and may evolve into AMD [239]. Triggers related to AMD, such as tobacco smoking and complement activation, may induce mitochondrial fragmentation and dysfunction [240][241][242]. In addition, a state of hyperfusion, characterized by an abnormal increase of mitochondrial fusion, along with decreased mitochondrial turnover, has been identified as a driver of RPE senescence [162].…”