T-type voltage-gated channels, Na+/Ca2+-exchanger, and calpain-2 promote photoreceptor cell death in inherited retinal degeneration
Jie Yan, Lan Wang, Qianlu Yang, Qianxi Yang, Xinyi He, Yujie Dong, Zhulin Hu, Mathias W. Seeliger, Kangwei Jiao, François Paquet‐Durand
Abstract
Abstract Inherited retinal degenerations (IRDs) are a group of untreatable and commonly blinding diseases characterized by progressive photoreceptor loss. IRD pathology has been linked to an excessive activation of cyclic nucleotide-gated channels (CNGC) leading to Na + - and Ca 2+ -influx, subsequent activation of voltage-gated Ca 2+ -channels (VGCC), and further Ca 2+ influx. However, a connection between excessive Ca 2+ influx and photoreceptor loss has yet to be proven. Here, we used whole-retina and single-cell RNA-sequencing to compare gene expression between the rd1 mouse model for IRD and wild-type ( wt ) mice. Differentially expressed genes indicated links to several Ca 2+ -signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca 2+ -chelator BAPTA-AM or inhibitors of different Ca 2+ -permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca 2+ -release-activated channel (CRAC), and Na + /Ca 2+ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca 2+ -dependent protease calpain-2. Effects on the retinal activity of poly(ADP-ribose) polymerase (PARP), sirtuin-type histone-deacetylase, calpains, as well as on activation of calpain-1, and − 2 were monitored, cell death was assessed via the TUNEL assay. While rd1 photoreceptor cell death was reduced by BAPTA-AM, Ca 2+ -channel blockers had divergent effects: While inhibition of T-type VGCC and NCX promoted survival, blocking CNGCs and CRACs did not. The treatment-related activity patterns of calpains and PARPs corresponded to the extent of cell death. Remarkably, sirtuin activity and calpain-1 activation were linked to photoreceptor protection, while calpain-2 activity was related to degeneration. In support of this finding, the calpain-2 inhibitor NA-184 protected rd1 photoreceptors. These results suggest that Ca 2+ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca 2+ -levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca 2+ -signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs.