Abstract
Purpose :
Photoreceptor death induces remodeling of bipolar cell (BC) dendrites. This remodeling is accompanied by changes in gene expression, but the gene networks that are involved in this rewiring are poorly understood. Furthermore, gene therapies are being developed to halt and rescue photoreceptors from degeneration. The halting of photoreceptor death at different timepoints in the degeneration process is likely to have distinct consequences on the morphologies and gene expression patterns of postsynaptic BCs. The purpose of this study was to examine how progressive photoreceptor degeneration impacts the transcriptomes of BCs and how these transcriptomes are altered by the genetic rescue of rods from degeneration.
Methods :
Mice with a floxed neomycin cassette inserted into the Cngb1 locus (Cngb1neo/neo) were crossed with UBC-cre and Grm6-GFP mice to create a triple transgenic line. These mice express green fluorescent protein (GFP) in BCs and exhibit a slow form of rod degeneration (all rods are lost ~6 months). Also, this line allows for genetic rescue of rods by tamoxifen-induced cre-recombination that triggers the expression of Cngb1 from the endogenous locus. Littermates heterozygous for the neomycin cassette were used as controls. At designated ages, mice were sacrificed, retinas dissociated using papain, and FACS sorted into GFP+ and GFP- cell populations, which yielded relatively pure populations of sorted BCs for RNA extraction and transcriptomic analysis.
Results :
We found significant BC gene expression differences between groups that depended on the amount of rod degeneration and the time-point of rod rescue: WT vs degenerating (P30, P90, P210), untreated vs treated, and early treatment (Tx at P30) vs late treatment (Tx at P90). There were fewer differentially expressed genes between late-stage degenerating bipolar cells (P90 untreated) and late treated bipolar cells (Tx at P90, sac at P150), indicating that late therapy is not fully reversing pathology.
Conclusions :
These results identify gene networks in postsynaptic BCs that respond to rod degeneration and death. These results also point toward the development of novel therapies to ameliorate blinding conditions and increase the effectiveness of vision restoration.
This is a 2021 ARVO Annual Meeting abstract.