Kalargyrou, Aikaterini; (2019) Determining the molecular mechanisms mediating cytoplasmic material transfer between photoreceptors in the transplantation paradigm. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Retinal degenerations are a complex group of disorders that all culminate in the same final common path, the loss of the light sensing cells of the eye, the photoreceptors. Photoreceptor replacement strategies aim to reverse the loss of vision by transplanting healthy cells to replace those lost through degeneration. Over the past decade, research has shown that transplanting photoreceptor precursors into models of retinal dysfunction results in restoration of visual function. Until recently, this was thought to be attributed solely to donor photoreceptor cells integrating into the retina. However, we have recently demonstrated that the observed rescue was instead largely due to exchange of RNA and/or protein between donor and remaining host photoreceptor cells, a mechanism we named material transfer. Since this process appears to render host cells functional, the mechanisms by which this occurs are of significant interest. In this PhD thesis, I sought to determine the molecular mechanism underlying material transfer. I hypothesized that this may involve direct physical contacts or indirect shedding and uptake of information packaged in extracellular vesicles (EVs). I first developed a robust protocol to maintain primary rod precursors in an isolated culture system to enable the study of both molecular mechanisms. I established that cultured photoreceptors release vesicles bearing the phenotypical and molecular characteristics of EVs, accompanied with the molecular signature of the cell of origin. By employing the Cre-loxP system I confirmed that photoreceptor-derived EVs can alter gene expression in glia cells, both in vitro and in vivo, but not in other photoreceptors, strongly indicating that EVs are not the primary mediators of material transfer in the transplantation paradigm. However, a combination of imaging methods, alongside pharmacological inhibition of the actin cytoskeleton of photoreceptor cultures, revealed transient tubulovesicular processes between photoreceptors, that are capable of transferring fluorescent reporters, organelles, and lipids. These fine structures are typically destroyed during fixation, impeding comprehensive assessment in vivo. Finally, I demonstrated and characterized a few examples of donor-host contacts in vivo, when fluorescent reporters were tagged to the membrane of donor cells. Taken together the above findings support that physical connections are most likely the mechanism underlying photoreceptor communication during material transfer.

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