Genetic Analysis of Photoreceptor Degeneration-induced Retinal Remodeling

Abstract

Purpose: : Photoreceptor cell degeneration elicits significant remodeling of the retinal circuitry which may impede efforts to restore vision with pharmaceutical, stem cell or gene therapies. Our goal was to protect the second order neurons from degeneration-induced remodeling by genetically manipulate the number and type of surviving photoreceptors in a zebrafish model of photoreceptor degeneration.

Methods: : Zebrafish homozygous for the pde6c^w59 allele were used as a model of cone degeneration (Stearns et al. 2007). Bipolar cell morphology was visualized with the nyx::MYFPQ16 transgenic line, in which ON-bipolar cells express YFP (Shroeter et al. 2006). Increased rod numbers were obtained by placing the pde6c^w59 allele on the lots-of-rods (lor/tbx2b^p25bbtl) background (Alvarez-Delfin et al 2009) or by antisense morpholino knockdown of a second photoreceptor-specific transcription factor. Photoreceptor subtypes, synaptic terminals, and glutamate receptor expression by OFF-bipolar cells and horizontal cells were accessed on immunolabeled retinal sections. Images were captured with a Zeiss 510 confocal microscope outfitted with high NA objective lens.

Results: : In the larval zebrafish retina, cones account for ~90-95% of all photoreceptors. Larvae homozygous for the pde6c^w59 allele demonstrated rapid cone degeneration and secondary rod loss. Cone degeneration induced changes of ON-bipolar cell morphology, disrupted the continuity of the outer plexiform layer, increased neurite outgrowth and decreased labeling for glutamate receptor expression on bipolar cell and horizontal cell dendritic processes. In contrast, rod and cone numbers in retinas of juvenile and adult zebrafish, are approximately equal. In homozygous pde6c^w59 adults, immunolabeling for ribeye demonstrated that rod synaptic structure was maintained, and the cone degeneration was not associated with alterations of glutamate receptor expression on bipolar cells or horizontal cells. These data suggest that the surviving rods protected the inner retina from degeneration-induced remodeling. To test this hypothesis in larvae, we used two different genetic manipulations to increase the number of rods in wildtype and pde6c^w59 mutant embryos. We observed that increasing the number of rods to approximately twenty-five percent of the original cone number was sufficient to offer partial protection of the inner retina from cone degeneration-induced remodeling.

Conclusions: : These results suggest that the survival of a small percentage of the total number of photoreceptors can offer significant protection from degeneration-induced retinal remodeling. Further studies are underway to investigate the molecular mechanisms influencing this relationship.

Keywords: retina: distal (photoreceptors, horizontal cells, bipolar cells) • genetics • protective mechanisms