Cell death in neurodegenerative diseases is often thought to be governed by apoptosis; however, an increasing body of evidence suggests the involvement of alternative cell death mechanisms in neuronal degeneration. We studied retinal neurodegeneration using 10 different animal models, covering all major groups of hereditary human blindness (rd1, rd2, rd10, Cngb1 KO, Rho KO, S334ter, P23H, Cnga3 KO, cpfl1, Rpe65 KO), by investigating metabolic processes relevant for different forms of cell death. We show that apoptosis plays only a minor role in the inherited forms of retinal neurodegeneration studied, where instead, a non-apoptotic degenerative mechanism common to all mutants is of major importance. Hallmark features of this pathway are activation of histone deacetylase, poly-ADP-ribose-polymerase, and calpain, as well as accumulation of cyclic guanosine monophosphate and poly-ADP-ribose. Our work thus demonstrates the prevalence of alternative cell death mechanisms in inherited retinal degeneration and provides a rational basis for the design of mutation-independent treatments.

Retinitis pigmentosa (RP) is a group of genetically heterogeneous, severe retinal diseases commonly leading to legal blindness. Mutations in the CNGB1a subunit of the rod cyclic nucleotide-gated (CNG) channel have been found to cause RP in patients. Here, we demonstrate the efficacy of gene therapy as a potential treatment for RP by means of recombinant adeno-associated viral (AAV) vectors in the CNGB1 knockout (CNGB1(-/-)) mouse model. To enable efficient packaging and rod-specific expression of the relatively large CNGB1a cDNA (~4 kb), we used an AAV expression cassette with a short rod-specific promoter and short regulatory elements. After injection of therapeutic AAVs into the subretinal space of 2-week-old CNGB1(-/-) mice, we assessed the restoration of the visual system by analyzing (i) CNG channel expression and localization, (ii) retinal function and morphology and (iii) vision-guided behavior. We found that the treatment not only led to expression of full-length CNGB1a, but also restored normal levels of the previously degraded CNGA1 subunit of the rod CNG channel. Both proteins co-localized in rod outer segments and formed regular CNG channel complexes within the treated area of the CNGB1(-/-) retina, leading to significant morphological preservation and a delay of retinal degeneration. In the electroretinographic analysis, we also observed restoration of rod-driven light responses. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this work provides a proof-of-concept for the treatment of rod channelopathy-associated RP by AAV-mediated gene replacement.

In the mammalian retina, light signals generated in photoreceptors are passed to bipolar and horizontal cells via synaptic contacts. In various pathological conditions, these second-order neurons extend neurites into the outer nuclear layer (ONL). However, the molecular events associated with this neurite outgrowth are not known. Here, we characterized the morphological synaptic changes in the CNGA3/CNGB1 double-knockout (A3B1) mouse, a model of retinitis pigmentosa. In these mice, horizontal cells looked normal until postnatal day (p) 11, but started growing neurites into the ONL 1 day later. At p28, the number of sprouting processes decreased, but the remaining sprouts developed synapse-like contacts at rod cell bodies, with an ultrastructural appearance reminiscent of ribbon synapses. Hence, neurite outgrowth and ectopic synaptogenesis in the A3B1 retina were precisely timed events starting at p12 and p28, respectively. We therefore performed microarray analysis of retinal gene expression in A3B1 and wild-type mice at those ages to evaluate the genomic response underlying these two events. This analysis identified 163 differentially regulated genes in the A3B1 retina related to neurite outgrowth or plasticity of synapses. The global changes in gene expression in the A3B1 retina were consistent with activation of signaling pathways related to Tp53, Smad, and Stat3. Moreover, key molecules of these signaling pathways could be localized at or in close proximity to outgrowing neurites. We therefore propose that Tp53, Smad, and Stat3 signaling pathways contribute to the synaptic plasticity in the A3B1 retina.