T-type voltage-gated channels, Na+/Ca2+-exchanger, and calpain-2 promote photoreceptor cell death in inherited retinal degeneration

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 Ca²⁺-influx, subsequent activation of voltage-gated Ca²⁺-channels (VGCC), and further Ca²⁺ influx. However, a connection between excessive Ca²⁺ 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 Ca²⁺-signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca²⁺-chelator BAPTA-AM or inhibitors of different Ca²⁺-permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca²⁺-release-activated channel (CRAC), and Na⁺/Ca²⁺ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca²⁺-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, Ca²⁺-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 Ca²⁺ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca²⁺-levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca²⁺-signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs. Video Abstract.

Calcium channels; HDAC; PAR; Retinitis pigmentosa; SOCE; cGMP.