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Title: Molecular mechanisms of retinal pigment epithelial cell homeostasis in the aging eye
Author: Rajapakse, Dinusha Prashadini
ISNI:       0000 0004 6494 932X
Awarding Body: Queen's University Belfast
Current Institution: Queen's University Belfast
Date of Award: 2017
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Retinal pigment epithelial (RPE) cells have been in the forefront of ocular cell research due to their central roles in maintaining retinal health and homoeostasis. Damages or dysfunctions to RPE cells are recognised as a cause of many age-related retinal diseases including age-related macular degeneration (AMD). During ageing, RPE cells undergo numerous structural, functional and morphological changes that have been implicated in disease progression, therefore much research is now invested in maintaining RPE cell homeostasis with age. This thesis focuses on using in vitro models to determine how RPE cells respond to oxidative insults caused by continuous exposure to photoreceptor outer segments (POS). RPE cells decline in number with age and are also considered to be post-mitotic therefore how they repair damage during ageing remains poorly defined. Compelling evidence suggests that RPE cells undergo multinucleation during ageing. In Chapter 3, POS and oxidised photoreceptor outer segments (oxPOS) treatments were found to dose-dependently suppress RPE cell proliferation. After 48 h treatment with oxPOS 33% of RPE cells were multinucleated compared to 5% in untreated controls (P<0.05). Furthermore, POS treatment downregulated IL-6, IL-8 and MCP-1 expression in RPE cells whereas oxPOS had no change or upregulated inflammatory gene expression. Suppression of inflammatory genes by POS may contribute to the immune suppressive microenvironment of the sub-retinal space. The immune suppressive effect may be lost/ reduced in the ageing eyes where oxPOS accumulate. To understand the underlying mechanism leading to POS and oxPOS-induced RPE cell multinucleation experiments were carried out, in Chapter 4, to determine if multinucleation was as a result of cell fusion or cell cycle incompletion. The findings demonstrated that RPE multinucleation was not due to cell-cell fusion, but to the failure of cytokinesis. Interestingly, the phagocytic activity of multinucleated RPE cells was not different from that of mononuclear RPE cells. Thus the mechanism as to how POS and oxPOS induce RPE multinucleation was elucidated. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. The regulation of oxidative stress in activation of the PKC pathway and stages of cell-cycle has been previously implicated in cell multinucleation. In Chapter 5, experiments were conducted to determine involvement of the PKC pathway in oxPOS-induced RPE multinucleation. Active PKC levels significantly increased following oxPOS treatment (P<0.001) of RPE cells as well as upregulated the mRNA expression of PKC a, 5, <, i and p compared to untreated controls. Pharmacological inhibition of PKC< down-regulated p27 kip1 expression and suppressed oxPOS-induced RPE cell multinucleation. This multinucleation was partially reversed by suppressing p27 kip1 by using a small molecular inhibitor, A2CE which further confirmed the association of p27 kip 1 in oxPOS-induced RPE multinucleation process. POS and oxPOS treatment increased ROS but RPE cells remained healthy and functional. One potential factor that has been linked to improved AMD patient symptoms in clinical trials is zinc supplementation through its purported anti-oxidative effect in various cell types, including RPE. As such, the protective role of zinc on RPE when exposed to oxPOS was explored using a low zinc and zinc supplementation in vitro model in Chapter 6. Under low zinc conditions, exposure to oxPOS adversely impacted RPE cell phagocytosis, mitochondrial health, decreased the expression of autophagy marker Beclin-1, increased oxidative stress and apoptosis of cells whereas this was not the case under zinc supplemented conditions. Moreover, ZnT2 and ZnT3 were highlighted as transporters implicated in maintenance of RPE homeostasis. The overall research findings within this thesis highlight that, in the presence of POS and oxPOS, RPE cells remain non-proliferative however the cells undergo DNA synthesis and even stages of cell-cycle and nuclear replication in order to repair damage with the aid of PKC pathway activation. Furthermore, zinc and zinc transporters play a critical role in maintaining the integrity of RPE cells under prolonged oxPOS exposure. Further understanding of multinuclear RPE functions would provide valuable information as to whether these cells are beneficial or detrimental to the ageing eye. Better understanding of zinc and, in particular the role zinc transporters, in RPE could lead to potential therapeutic targets for age-related disease conditions such as AMD.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available