Targeting Caspase-12 to Preserve Vision in Mice With Inherited Retinal Degeneration.

Mice expressing a mutant form of human rhodopsin with a threonine to methionine substitution at position 17 (T17M) develop severe retinal degeneration and thus mimic the human autosomal dominant retinitis pigmentosa (ADRP).1,2 The study of molecular mechanisms underlying the retinopathy in these mice has demonstrated that the unfolded protein response (UPR), along with inflammatory signaling, significantly contributes to the progression of retinal degeneration.3 Thus, the activation of all three UPR branches, including protein kinase R–like endoplasmic reticulum kinase (PERK), activating transcriptional factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1), has been detected in T17M retinas as early as postnatal (P) day 15. Moreover, we have recently revealed that the rate of retinal degeneration could be significantly altered in these mice by modifying the expression of UPR hallmark proteins. For example, we found that full knockout of the UPR proapoptotic CHOP protein in degenerating retinas dramatically accelerated the rate of retinal degeneration4,5 while full ablation of the UPR downstream marker, the endoplasmic reticulum (ER) stress–associated caspase (Csp)-7, significantly slowed the rate of retinal degeneration.6 In addition to Csp-7, Csp-12 localizes to the Golgi complex and is also activated during ER stress.7 Several studies have suggested that Csp-12 acts as an initiator Csp during ER stress–induced apoptosis, activating an effector Csp-3 or -7, which then cleaves key substrates required for normal cellular functions, thus leading to apoptosis.8 Recently, it has also been reported that Csp-12–deficient mice are resistant to ER stress–induced apoptosis, but their cells do undergo apoptosis in response to other stimuli. Thus, in experiments with primary cortical neurons, the link between ER stress and Csp-12 activation has been proposed through demonstration of the reduced susceptibility of Csp-12−/− neurons to Aβ-induced UPR activation and subsequent cell death.9 These experiments have clearly demonstrated that activation of Csp-12 during ER stress plays a proapoptotic role and contributes to neuronal death in Alzheimer's disease.9 Recent studies have proposed that there are two mechanisms for ER stress–mediated Csp-12 activation that act in different compartments of the cell. The first involves the interaction of proCsp-12 with the IRE1–TRAF2 complex (IRE1 UPR arm) on the cytosolic side of the ER membrane.10 This interaction occurs as a result of altered complex formation between IRE1α and TRAF2 (tumor necrosis factor receptor-associated factor 2) in response to ER stress and modified activity of Jun N-terminal inhibitory kinase (JIK) against its binding partner IRE1a.10 This suggests that Csp-12 activated by TRAF2 plays a crucial role in signal transduction from IRE1 to downstream signaling molecules and thus links the stress sensor molecule IRE1 to the activation of Csp-12. The second mechanism for Csp-12 activation occurs in the cytosol, where an increase in Ca2+ concentration results in calpain activation. The μ-calpain activation in turn precedes expression/activation of Csp-12 and is required for activation/cleavage of Csp-12.11 Given that a cytosolic Ca2+ increase could also occur in response to ER stress, this pathway along with the above-mentioned TRAF2-pathway points to Csp-12 as the ER stress–induced Csp promoting apoptosis. Involvement of Csp-12 in the mechanisms of several different retinopathies has been recently described in a number of studies.12–16 Although a combination of inhibiting Csp-12, upregulating the ER-resident chaperone BiP, and inhibiting CAP-dependent translation, has been proposed as the most efficient way to maintain BBS-12 photoreceptors,13 the direct impact of inhibiting Csp-12 on loss of photoreceptor function and cell death has yet to be shown, since Csp-12 has not been previously assigned a direct role in retinal degeneration. The Csp-12 inhibitor INH (the synthetic peptide Z-Ala-Thr-Ala-Asp(O-methyl)-fluoromethyl-ketone) in this study has been used in combination with other drugs such as guanabenz and valproic acid, pointing to the necessity of conducting a study demonstrating a direct link between Csp-12 activation and retinal degeneration. Therefore, in the current study we explored the role of Csp-12 in the mechanism of retinal pathogenesis in T17M mice. Our goal was to determine if ER stress–induced apoptosis played a role in ADRP photoreceptor demise and whether the therapeutic approach of targeting ER stress–promoted apoptosis was sufficient to rescue ADRP photoreceptors. Building on a previous study that looked exclusively at Csp-7 in T17M mice, the current work genetically modulated Csp-12 activity in T17M mice and compared the results to our previous findings. We also attempted to answer the question whether blocking apoptosis overall proved sufficient to rescue ADRP retinas and prevent further loss of retinal integrity. Our data point to inhibition of Csp-12 as a potential therapeutic approach that significantly slows retinal degeneration and together with other nonapoptotic targets could dramatically delay photoreceptor cell death.