Autophagy disruption and mitochondrial stress precede photoreceptor necroptosis in multiple mouse models of retinitis pigmentosa

Abstract

Purpose : One of the greatest barriers to developing treatments for retinitis pigmentosa (RP) is the heterogeneity of this disorder, with causative mutations identified in over 280 genes. Therefore, despite recent advances in gene therapies for RP, developing treatments that are applicable to a broad range of causative alleles is a priority for eye health. Our work characterises the contribution of different cell death mechanisms to photoreceptor loss throughout disease progression using humanised mouse models of RP. We hypothesise that mutations in multiple genes all result in a final common cell death pathway that could be a target for therapy.

Methods : We performed single cell RNA sequencing (scRNA-seq) to identify transcriptional changes in degenerating photoreceptors in Rpgr^Ex3d8 mutants compared to wild type littermates at 18 months old. We validated these findings using electron microscopy and immunofluorescent imaging at several disease stages (6, 12 and 18 months). Further, we repeated the validation experiments in a Pde6b mutant mouse model of RP (Pde6b^atrd2) at equivalent disease stages (postnatal days P13, P16 and P18).

Results : Rpgr^Ex3d8 mutant photoreceptors have increased expression of genes associated with necroptosis, lysosomal and mitochondrial function. Rpgr^Ex3d8 mutants exhibit accumulation of autophagosomes in photoreceptor inner segments from 6 months and a significant increase in autophagosome markers at 12 months (n=3, p=0.04). This was also observed in Pde6b^atrd2 mutants at P16 (n=3, p=0.016), suggesting autophagy dysregulation in both models. Both Rpgr^Ex3d8 and Pde6b^atrd2 photoreceptors show progressive disruption of mitochondrial morphology, indicative of mitochondrial stress. We also observed a progressive increase in photoreceptor necroptosis in both Rpgr^Ex3d8 (n=3, p<0.0001 at 18 months) and Pde6b^atrd2 mutants (n=3, p=0.027 at P18) compared to age-matched wild type controls.

Conclusions : We show that mitochondrial damage and autophagy dysregulation contribute significantly to photoreceptor degeneration in two mouse models of RP with different genetic causes. Necroptosis may be the primary mechanism of cell death in both models. These insights provide potential therapeutic targets for novel RP treatments that could be applicable to patients with a wide range of causative alleles.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.