Abstract
Purpose :
Patients with degenerative retinal dystrophies suffer from various degrees of blindness as a result of acute or gradual photoreceptor cell death. While gene supplementation therapies attempt to treat affected individuals with monogenic disorders at an early disease stage, there is a clinical demand for universal treatment that can halt or even reverse the effects of degeneration at later stages. We have shown that NMDA-induced ablation of retinal ganglion cells and amacrine cells, together with Ascl1 overexpression in murine Müller glia (MG) induces the MG to generate neurons. This work investigates the potential for glia-to-neuron reprogramming in retinas that suffered photoreceptor ablation, which better mimics the pathology of patients with late-stage retinal degeneration.
Methods :
To induce photoreceptor degeneration, adult mice homozygous or heterozygous for the Rpe65 Leu450 were subjected to light-damage by exposing them to ~20,000 lux of white light for 6 hours. To test glia-to-neuron conversion, transgenic mice were used with inducible MG-specific expression of the proneural transcription factor Ascl1 (Glast-CreER/LNL-tTA/TetO-Ascl1-GFP) or Ascl1 in combination with other transcription factors. One week after light-damage, when most photoreceptors in the central retina were ablated, the transgenic mice were injected with tamoxifen to induce the expression of neurogenic factors in MG followed by intravitreal injection of Trichostatin-A (TSA) alone or with γ-secretase inhibitor DAPT. Four weeks later, the number and types of glial-derived neurons was assessed.
Results :
Light-damaged retinas showed uniform recombination, confirming that GLAST-controlled gene expression in MG was effective during and after photoreceptor cell death After 4 weeks of Ascl1 expression in MG, approximately 30% of the recombined MG expressed the bipolar/photoreceptor marker Otx2+, similar to the effects previously reported with NMDA-induced retinal injury. The proportions of GFP+Chx10+ (~40%), GFP+Otx2+ (~30%) and GFP+Sox2+ (~65%) populations were similar between eyes that received TSA or TSA+DAPT, suggesting that Notch pathway inhibition with DAPT in this context did not improve the reprogramming outcome.
Conclusions :
This work sets the stage for understanding how reprogramming paradigms could translate in models of photoreceptor degeneration, with the aim of regenerating the retina at late disease stages.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.