Determining the molecular mechanisms mediating cytoplasmic material transfer between photoreceptors in the transplantation paradigm

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

A protocol for isolation and culturing of mouse primary postmitotic photoreceptors and isolation of extracellular vesicles

Here, we present a protocol for isolating and culturing mouse photoreceptors in a minimal, chemically-defined medium free from serum. We describe steps for retina dissection, enzymatic dissociation, photoreceptor enrichment, cell culture, extracellular vesicles (EVs) enrichment, and EV ultrastructural analysis. This protocol, which has been verified for cultured cells derived from multiple murine strains, allows for the study of several aspects of photoreceptor biology, including EV isolation, and cell-cell interactions such as nanotubes (NTs)

Extracellular vesicles in the retina - putative roles in physiology and disease

The retina encompasses a network of neurons, glia and epithelial and vascular endothelia cells, all coordinating visual function. Traditionally, molecular information exchange in this tissue was thought to be orchestrated by synapses and gap junctions. Recent findings have revealed that many cell types are able to package and share molecular information via extracellular vesicles (EVs) and the technological advancements in visualisation and tracking of these delicate nanostructures has shown that the role of EVs in cell communication is pleiotropic. EVs are released under physiological conditions by many cells but they are also released during various disease stages, potentially reflecting the health status of the cells in their cargo. Little is known about the physiological role of EV release in the retina. However, administration of exogenous EVs in vivo after injury suggest a neurotrophic role, whilst photoreceptor transplantation in early stages of retina degeneration, EVs may facilitate interactions between photoreceptors and Müller glia cells. In this review, we consider some of the proposed roles for EVs in retinal physiology and discuss current evidence regarding their potential impact on ocular therapies via gene or cell replacement strategies and direct intraocular administration in the diseased eye.</p

Nanotube-like processes facilitate material transfer between photoreceptors

Neuronal communication is typically mediated via synapses and gap junctions. New forms of intercellular communication, including nanotubes (NTs) and extracellular vesicles (EVs), have been described for non-neuronal cells, but their role in neuronal communication is not known. Recently, transfer of cytoplasmic material between donor and host neurons (“material transfer”) was shown to occur after photoreceptor transplantation. The cellular mechanism(s) underlying this surprising finding are unknown. Here, using transplantation, primary neuronal cultures and the generation of chimeric retinae, we show for the first time that mammalian photoreceptor neurons can form open-end NT-like processes. These processes permit the transfer of cytoplasmic and membrane-bound molecules in culture and after transplantation and can mediate gain-of-function in the acceptor cells. Rarely, organelles were also observed to transfer. Strikingly, use of chimeric retinae revealed that material transfer can occur between photoreceptors in the intact adult retina. Conversely, while photoreceptors are capable of releasing EVs, at least in culture, these are taken up by glia and not by retinal neurons. Our findings provide the first evidence of functional NT-like processes forming between sensory neurons in culture and in vivo

Branquiocráneo y dentición del pargo Lutjanus griselus (Pisces: Lutjanidae)

Summary: Human vision relies heavily upon cone photoreceptors, and their loss results in permanent visual impairment. Transplantation of healthy photoreceptors can restore visual function in models of inherited blindness, a process previously understood to arise by donor cell integration within the host retina. However, we and others recently demonstrated that donor rod photoreceptors engage in material transfer with host photoreceptors, leading to the host cells acquiring proteins otherwise expressed only by donor cells. We sought to determine whether stem cell- and donor-derived cones undergo integration and/or material transfer. We find that material transfer accounts for a significant proportion of rescued cells following cone transplantation into non-degenerative hosts. Strikingly, however, substantial numbers of cones integrated into the Nrl−/− and Prph2rd2/rd2, but not Nrl−/−;RPE65R91W/R91W, murine models of retinal degeneration. This confirms the occurrence of photoreceptor integration in certain models of retinal degeneration and demonstrates the importance of the host environment in determining transplantation outcome. : Pearson and colleagues demonstrate that transplanted cone photoreceptors can both undergo incorporation into the host neural retina and engage in cytoplasmic material transfer with host rod and cone photoreceptors. They show that the host environment plays a crucial role in determining the relative contributions of these two mechanisms to transplantation outcome. Keywords: photoreceptor, retina, blindness, retinal dystrophy, Nrl−/−, function, transplantation, material transfer, fusion, integratio