Abstract
PurposeRetinal bipolar cells survive even in the later stages of inherited retinal degenerations (IRDs) and so are attractive targets for optogenetic approaches to vision restoration. However, it is not known to what extent the remodelling that these cells undergo during degeneration affects their function. Specifically, it is unclear if they are free from metabolic stress, receptive to adeno-associated viral vectors, suitable for opsin-based optogenetic tools and able to propagate signals by releasing neurotransmitter. MethodsFluorescence activated cell sorting (FACS) was performed to isolate labelled bipolar cells from dissociated retinae of litter-mates with or without the IRD mutation Pde6brd1/rd1 selectively expressing an enhanced yellow fluorescent protein (EYFP) as a marker in ON-bipolar cells. Subsequent mRNA extraction allowed Illumina® microarray comparison of gene expression in bipolar cells from degenerate to those of wild type retinae. Changes in four candidate genes were further investigated at the protein level using retinal immunohistochemistry over the course of degeneration. ResultsA total of sixty differentially expressed transcripts reached statistical significance: these did not include any genes directly associated with native primary bipolar cell signalling, nor changes consistent with metabolic stress. Four significantly altered genes (Srm2, Slf2, Anxa7 & Cntn1), implicated in synaptic remodelling, neurotransmitter release and viral vector entry had immunohistochemical staining colocalising with ON-bipolar cell markers and varying over the course of degeneration. ConclusionOur findings suggest relatively few gene expression changes in the context of degeneration: that despite remodelling, bipolar cells are likely to remain viable targets for optogenetic vision restoration. In addition, several genes where changes were seen could provide a basis for investigations to enhance the efficacy of optogenetic therapies.
Highlights
3 have allowed retinal gene replacement to become a reality for patients suffering from 4 certain inherited retinal degenerations (IRDs)(Russell et al, 2017)
2 Our findings suggest relatively few gene expression changes in the context of degeneration: 3 that despite remodelling, bipolar cells are likely to remain viable targets for optogenetic
2 Advances in retinal gene therapy delivery methods, such as adeno-associated virus (AAV), 3 have allowed retinal gene replacement to become a reality for patients suffering from 4 certain inherited retinal degenerations (IRDs)(Russell et al, 2017)
Summary
3 have allowed retinal gene replacement to become a reality for patients suffering from 4 certain inherited retinal degenerations (IRDs)(Russell et al, 2017). AAV delivered optogenetic tools have been shown to restore electrophysiological and behavioural responses to light in animal models of IRDs(Cehajic-Kapetanovic et al, 2015; De Silva et al, 2017; Doroudchi et al, 2011) by rendering surviving cells in the degenerate retina sensitive to light. This general principle of survivor cell stimulation has been demonstrated clinically with electronic retinal prostheses already in clinical use for vision restoration(Edwards et al, 2018; Luo and da Cruz, 2016).
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