Lamb, William David Benjamin; (2021) Potential of Muller glia-derived extracellular vesicles for retinal neuroprotection. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Müller glia cells retain progenitor-like characteristics in the adult human eye and can partially restore visual function upon intravitreal transplantation into animal models of glaucoma. Based on observations that this effect does not depend on cell replacement of the degenerated retina, efficacy is thought to depend upon the cell transfer of neuroprotective agents to retinal ganglion cells (RGC). In an attempt to identify candidate molecules, this work first characterised the ability of Müller glia to synthesise and secrete a range of neurotrophic growth factors. Since certain pro-inflammatory cytokines are known to be upregulated in the glaucomatous eye, this study also investigated the modulatory effects of exogenous cytokines on the expression of neurotrophin genes and proteins. Recently it has been demonstrated that cells can communicate through the release of nano-sized membrane-bound organelles called extracellular vesicles (EV). These contain bioactive molecules that induce functional changes when internalised by recipient cells. Small and large sub-populations of EV (sEV and lEV) were purified from Müller glia cultures and their contents were characterised, revealing the enrichment of characteristic proteins and several RNA species. Of particular interest are the findings of small non-coding microRNAs (miRNA) present in the sEV, that directly regulate gene expression through silencing of mRNA. Sequencing of miRNAs recovered from small EV subsets indicated preferential enrichment with transcripts that target genes involved in cell growth and survival, including PTEN, the master inhibitor of the AKT/mTOR pathway. On this basis, a putative mechanism for the neural pro-survival effects induced by neurotrophic factors previously ascribed to Müller glia was suggested. Labelling of EV with a lipophilic dye confirmed the direct internalisation of these vesicles into primary RGC in culture. Since vesicle uptake has been shown to be cell type specific, it seems likely that Müller glia derived EV express membrane molecules that facilitate this internalisation, making them appealing for use as vehicles for the delivery of neuroprotective molecules to retinal neurons. Finally, Müller EV function was tested in vivo using a NMDA-model of RGC depletion. Adult rats receiving intravitreal delivery of 3x109 small EV showed significantly improvement in RGC function when compared to vehicle control, as determined by the negative scotopic threshold response (nSTR) of the dark-adapted electroretinogram (ERG). Taken together, the data presented in this thesis suggests that EV represent a significant part of the neuroprotective Müller cell signalling activity, likely in addition to the release of neurotrophic growth factors. EV enriched in neuroprotective molecules may represent a more stable, and less immunogenic alternative to whole cell transplantation, particularly given their minute size and low toxicity profiles. This work provides new insight into the neuroprotective secretome of Müller glia, adding further support to previous studies demonstrating their potential utility in a cell-based glaucoma therapy.

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