Abstract
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in developed countries, characterized by the death of retinal pigment epithelial (RPE) cells and photoreceptors. Previous studies report an accumulation of damaged and dysfunctional mitochondria in RPE of human donors with AMD. Understanding how damaged mitochondria accumulate in AMD is an important step in discovering disease mechanisms and identifying therapeutic targets. In this report, we assessed mitochondrial fission and fusion by quantifying proteins and measured mitochondrial autophagy (mitophagy) via protein analysis and advanced imaging techniques using mitochondrial targeted mKeima in primary human RPE from donors with or without AMD. We report disease-specific differences in mitochondrial proteins that regulate fission, fusion, and mitophagy that were present at baseline and with treatments to stimulate these pathways. Data suggest AMD RPE utilize receptor-mediated mitophagy as a compensatory mechanism for deficits in the ubiquitin-mediated mitophagy pathway. These changes in mitochondrial homeostasis could lead to the buildup of damaged and dysfunctional mitochondria observed in the RPE of AMD donors.
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Introduction
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in developed countries. This multifactorial disease involves complex genetic and environmental factors whose effects accelerate with age1,2. AMD affects 30% of individuals 75–85 years of age, with a global estimate of 128 million cases in 2020 and 288 million cases by 2040, a consequence of the world’s increasing elderly population3,4. The two forms of AMD include “wet” and “dry”, with wet AMD caused by the abnormal growth of blood vessels into the retina. While wet AMD is less common, it has a number of effective therapies to halt or prevent vision loss5. Dry AMD is the most common form of the disease, accounting for approximately 85% of all AMD cases6. Vision loss associated with dry AMD is caused by the death of retinal pigment epithelium (RPE) and photoreceptors. RPE are essential for maintaining a healthy retina as they are responsible for key functions, such as the transport of nutrients to photoreceptors and the directed secretion of growth factors7. Currently, there are no effective treatments for dry AMD, due to our incomplete understanding of the cellular events causing disease pathology. Thus, there remains an urgent need to identify the underlying mechanisms causing AMD in order to successfully develop therapeutic interventions.
One of the prevailing hypotheses is that RPE mitochondrial defects drive AMD pathology8. This hypothesis is supported by numerous studies in human retina from eye bank donors. Analysis of electron microscopy images found donors with AMD had significantly fewer mitochondrial number, reduced surface area, and an altered cristae morphology9. Additionally, proteomic studies of human RPE tissue found an altered mitochondrial proteome, with multiple proteins in the electron transport chain decreasing in AMD RPE10,11. There are also reports of increased mitochondrial DNA damage with progression of AMD severity12,13. Consistent with the mitochondrial defects observed in tissue, cultures of human primary RPE showed significantly decreased mitochondrial function in RPE from donors with AMD14,46. Additional compounds designed to protect mitochondria from oxidative damage (N-acetyl-L-Cysteine; NAC), remove damaged mitochondria via increased autophagy (rapamycin), upregulate mitochondrial biogenesis (pyrroloquinoline; PQQ), or improve oxidative phosphorylation (nicotinamide mononucleotide, NMN) have been investigated in our lab47. We found that RPE from AMD donors responded to the drugs as detected by the increase in mitochondrial function. In contrast, RPE from donors without AMD did not respond to the drugs. These results are consistent with the idea that mitochondrial dysfunction in the diseased cells can be ameliorated by treatments that target mitochondrial defects. Our study supports the idea that mitochondrial defects drive AMD, and targeting pathways of mitochondrial homeostasis may be a viable treatment option.
This study uncovers potential mechanisms leading to mitochondrial damage and dysfunction associated with AMD that may initiate the metabolic crisis in the retina. We observed disease-specific differences under basal conditions and in response to two mitochondrial stressors, FCCP and CoCl2. The AMD-associated changes in mitochondrial proteins reported in this study could lead to the buildup of damaged and dysfunctional mitochondria that begins to disrupt the delicate retinal ecosystem and lead to the eventual death of RPE and photoreceptors in AMD. Experiments using different stressors and analytical methods may further identify mechanisms that explain these AMD-associated changes in mitochondrial homeostasis, providing new therapeutic targets to treat AMD.
Data availability
All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).
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Acknowledgements
The authors wish to acknowledge the contribution of personnel from the Lions Gift of Sight (St. Paul, MN) for their assistance in procuring human donor eyes and processing eye tissue. The authors also thank the donors and their families for their essential contributions to the research. This work was supported in part by the National Institutes of Health (NIH) National Eye Institute (NEI) F31-EY031558 (to CRF), T32-EY025187 (to CRF), R01EY026012 (to DAF), and R01EY028554 (to DAF), NIH National Institute of Aging (NIA) T32-AG029796 (to CRF), Diana Jacobs Kalman/AFAR Scholarships for Research in the Biology of Aging (to CRF), VitreoRetinal Surgery Foundation Fellowship (to CRF), University of Minnesota Undergraduate Research Opportunities Program (to AAS), the Elaine and Robert Larson Endowed Vision Chair, the Lindsay Family Foundation, and an anonymous benefactor for AMD research.
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Conceptualization: C.R.F., A.A.S., D.A.F.; Methodology: C.R.F., A.A.S., M.C.E., D.A.F.; Investigation: C.R.F., A.A.S., M.C.E., S.R.M.; Writing—Original Draft: C.R.F.; Writing—Review and Editing: C.R.F., A.A.S., M.C.E., S.R.M., D.A.F.; Supervision: C.R.F. and D.A.F.; Funding Acquisition: C.R.F. and D.A.F.
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Fisher, C.R., Shaaeli, A.A., Ebeling, M.C. et al. Investigating mitochondrial fission, fusion, and autophagy in retinal pigment epithelium from donors with age-related macular degeneration. Sci Rep 12, 21725 (2022). https://doi.org/10.1038/s41598-022-26012-5
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DOI: https://doi.org/10.1038/s41598-022-26012-5
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