Abstract
Purpose :
Inherited retinal disorders like retinitis pigmentosa often lead to blindness due to the death of photoreceptors. Photoreceptor death induces remodeling of bipolar cells (BCs), their synaptic partners. Remodeling is likely mediated by changes in gene expression, but the gene networks involved in BC rewiring are poorly understood. This is a significant hurdle to developing effective retinal therapies as visual information can only be transmitted if retinal synapses remain intact. Thus, vision restoration for retinitis pigmentosa is dependent not only on halting photoreceptor death, but also on preventing or reversing BC remodeling. The purpose of this study was to examine how progressive photoreceptor degeneration impacts the transcriptomes of BCs.
Methods :
Mice with a neomycin cassette inserted into the Cngb1 locus (Cngb1neo/neo) were crossed with Grm6;GFP mice to create mice that exhibit a slow photoreceptor degeneration and express GFP in ON BCs. Littermates heterozygous for neomycin were used as controls. At designated timepoints (P30: 30% rod loss and 2% cone loss; P90: 70% rod loss and 5% cone loss; and P210: 96% rod loss and 35% cone loss), mice were sacrificed, retinas dissociated using papain, and FACS sorted into GFP+ and GFP- cell populations, which yielded populations of sorted BCs for RNA extraction and bulk RNA sequencing using Illumina MiSeq.
Results :
We found over 2000 genes differentially expressed in BCs throughout photoreceptor degeneration in the Cngb1neo/neo ON BC transcriptome. Developmental genes were largely upregulated over time in Cngb1neo/neo ON BCs, but downregulated in heterozygous cells. Additionally, genes related to process outgrowth were upregulated in Cngb1neo/neo, but stationary in heterozygotes. This is in line with anatomy which suggests ON BCs extend dendrites during degeneration to find new synaptic partners. However, genes associated with synaptogenesis were largely stationary for both groups, indicating new synapses are not being formed.
Conclusions :
Transcriptomic assessment of remodeling ON BCs show large changes in gene expression as degeneration progresses, independent of post-development age-related changes. These results provide new avenues for therapeutic targets related to preventing or reversing remodeling, as well as inducing neuroplasticity for cell replacement and regenerative technologies for photoreceptor degenerations.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.