The molecular signaling leading to cell death in hereditary neurological diseases such as retinal degeneration is incompletely understood. Previous neuroprotective studies have focused on apoptotic pathways; however, incomplete suppression of cell death with apoptosis inhibitors suggests that other mechanisms are at play. Here, we report that different signaling pathways are activated in rod and cone photoreceptors in the P23H rhodopsin mutant rat, a model representing one of the commonest forms of retinal degeneration. Up-regulation of the RIP1/RIP3/DRP1 axis and markedly improved survival with necrostatin-1 treatment highlighted necroptosis as a major cell-death pathway in degenerating rod photoreceptors. Conversely, up-regulation of NLRP3 and caspase-1, expression of mature IL-1β and IL-18 and improved cell survival with N-acetylcysteine treatment suggested that inflammasome activation and pyroptosis was the major cause of cone cell death. This was confirmed by generation of the P23H mutation on an Nlrp3-deficient background, which preserved cone viability. Furthermore, Brilliant Blue G treatment inhibited inflammasome activation, indicating that the 'bystander cell death' phenomenon was mediated through the P2RX7 cell-surface receptor. Here, we identify a new pathway in cones for bystander cell death, a phenomenon important in development and disease in many biological systems. In other retinal degeneration models different cell-death pathways are activated, which suggests that the particular pathways that are triggered are to some extent genotype-specific. This also implies that neuroprotective strategies to limit retinal degeneration need to be customized; thus, different combinations of inhibitors will be needed to target the specific pathways in any given disease.

Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c w59 mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c w59 embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed.Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c w59 mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.

Tissue fusion is an essential morphogenetic mechanism in development, playing a fundamental role in developing neural tube, palate and the optic fissure. Disruption of genes associated with the tissue fusion can lead to congenital malformations, such as spina bifida, cleft lip/palate and ocular coloboma. For instance, the Pax2 transcription factor is required for optic fissure closure, although the mechanism of Pax2 action leading to tissue fusion remains elusive. This lack of information defining how transcription factors drive tissue morphogenesis at the cellular level is hampering new treatments options. Through loss- and gain-of-function analysis, we now establish that pax2 in combination with vax2 directly regulate the fas-associated death domain (fadd) gene. In the presence of fadd, cell proliferation is restricted in the developing eye through a caspase-dependent pathway. However, the loss of fadd results in a proliferation defect and concomitant activation of the necroptosis pathway through RIP1/RIP3 activity, leading to an abnormal open fissure. Inhibition of RIP1 with the small molecule drug necrostatin-1 rescues the pax2 eye fusion defect, thereby overcoming the underlying genetic defect. Thus, fadd has an essential physiological function in protecting the developing optic fissure neuroepithelium from RIP3-dependent necroptosis. This study demonstrates the molecular hierarchies that regulate a cellular switch between proliferation and the apoptotic and necroptotic cell death pathways, which in combination drive tissue morphogenesis. Furthermore, our data suggest that future therapeutic strategies may be based on small molecule drugs that can bypass the gene defects causing common congenital tissue fusion defects.

Retinitis pigmentosa (RP) is a group of inherited neurological disorders characterized by rod photoreceptor cell death, followed by secondary cone cell death leading to progressive blindness. Currently, there are no viable treatment options for RP. Due to incomplete knowledge of the molecular signaling pathways associated with RP pathogenesis, designing therapeutic strategies remains a challenge. In particular, preventing secondary cone photoreceptor cell loss is a key goal in designing potential therapies. In this study, we identified the main drivers of rod cell death and secondary cone loss in the transgenic S334ter rhodopsin rat model, tested the efficacy of specific cell death inhibitors on retinal function, and compared the effect of combining drugs to target multiple pathways in the S334ter and P23H rhodopsin rat models. The primary driver of early rod cell death in the S334ter model was a caspase-dependent process, whereas cone cell death occurred though RIP3-dependent necroptosis. In comparison, rod cell death in the P23H model was via necroptotic signaling, whereas cone cell loss occurred through inflammasome activation. Combination therapy of four drugs worked better than the individual drugs in the P23H model but not in the S334ter model. These differences imply that treatment modalities need to be tailored for each genotype. Taken together, our data demonstrate that rationally designed genotype-specific drug combinations will be an important requisite to effectively target primary rod cell loss and more importantly secondary cone survival.