| Author: | Abokyi, Samuel |
| Title: | Antioxidative role of autophagy in the protection against in vitro and in vivo oxidative stress-induced models of age-related retinal degeneration |
| Advisors: | Tse, Dennis (SO) To, Chi-ho (SO) Chan, Ho-lung Henery (SO) |
| Degree: | Ph.D. |
| Year: | 2021 |
| Subject: | Retinal degeneration Retina -- Diseases Retinal degeneration -- Age factors Hong Kong Polytechnic University -- Dissertations |
| Department: | School of Optometry |
| Pages: | xviii, 229 pages : color illustrations |
| Language: | English |
| Abstract: | Age-related macular degeneration (AMD) is a common cause of visual impairment in the elderly population. There are very limited therapeutic options for AMD with the predominant therapies targeting VEGF-A in the retina of patients afflicted with the"wet form" of AMD. Recently, it is becoming clear that oxidative damage of the retinal pigment epithelium (RPE) is an earlier event in the pathophysiology of AMD. This thesis, therefore, sought to investigate potential molecular mechanisms and targets for protection against oxidative damage in the RPE and retinal degeneration. With the aim of finding effective cellular defense mechanisms that modulate oxidative stress in RPE cells, this study investigated changes in autophagy and Nuclear factor erythroid 2-related factor 2 (NRF2) in the presence of the hydroquinone (HQ, an oxidant found in cigarette smoke). In vitro, our results showed that human RPE cells were more susceptible to oxidative damage from HQ compared to H2O2. The dysregulation of cellular antioxidative pathways such as autophagy and NRF2 were found to be involved. The autophagosome marker LC3-II was increased by both HQ and H2O2, suggesting autophagy activation by the oxidants. However, HQ exposure induced NRF2 and p62 inhibition at both transcription and protein levels, unlike H2O2. Based on these results, we hypothesized that autophagy upregulation could be an effective approach for inhibiting oxidative damage in human RPE cells. Next, we assessed the role of autophagy in antioxidants' protection of human RPE cells against oxidative stress. Antioxidant vitamin supplements and the thiols are recommended for managing AMD. We demonstrated in this study that vitamin supplements and thiols upregulated autophagy flux via the transcription factor EB (TFEB), resulting in their protection against HQ-or H2O2 -induced oxidative damage in human RPE cells. Trehalose is a natural dietary molecule reported to induce autophagy and antiaging and neuroprotective effects in several animal models of neurodegenerative diseases. Hence, we investigated the protective effect of trehalose-induced autophagy against oxidative stress in human RPE cells. Incubation of the cells with trehalose upregulated autophagy-related genes and protein markers of macroautophagy and chaperonemediated autophagy (CMA). Additionally, we found that trehalose rescued human RPE cells from HQ-induced oxidative damage in an autophagy dependent manner. These findings warranted investigation into the neuroprotection of trehalose in oxidative stress-mediated retinal degenerative diseases. Lastly, we studied the contribution of autophagy to oxidative stress and the development of retinal degeneration in normal ageing wild-type BALB/c mice. Mice monitored from age 2-18 months, using electroretinography (ERG) and spectral-domain optical coherence tomography (SD-OCT), demonstrated retinal degenerative changes in older mice which were accompanied with autophagy decline and increased oxidative stress. Immunoblot and quantitative real-time PCR results showed a downregulation of autophagy-related genes and/or proteins including ATG5 and ATG7, LC3-II, LAMP-2A, and TFEB in the neural retina of older mice with degenerative changes. Oral administration of trehalose improved TFEB-mediated autophagy and cone-mediated response decline associated with aging in BALB/c mice. Collectively, our data reveal the significance of TFEB-mediated autophagy in the protection of human RPE cells and retina against oxidative damage. More importantly, it supports the targeting of TFEB-induced autophagy as a novel therapeutic approach for preventing age-related decline in cone-mediated function. |
| Rights: | All rights reserved |
| Access: | open access |
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