Molecular mechanisms underlying apoptosis in retinitis pigmentosa, as in other neurodegenerative diseases, are still elusive, and this fact hampers the development of a cure for this blinding disease. We show that two apoptotic pathways, one from the mitochondrion and one from the endoplasmic reticulum, are coactivated during the degenerative process in an animal model of retinitis pigmentosa, the rd1 mouse. We found that both AIF and caspase-12 translocate to the nucleus of dying photoreceptors in vivo and in an in vitro cellular model. Translocation of both apoptotic factors depends on changes in intracellular calcium homeostasis and on calpain activity. Knockdown experiments defined that AIF plays the major role in this apoptotic event, whereas caspase-12 has a reinforcing effect. This study provides a link between two executor caspase-independent apoptotic pathways involving mitochondrion and endoplasmic reticulum in a degenerating neuron.rd1 mouse ͉ photoreceptor ͉ retinal stem cells R etinitis pigmentosa (RP) is a form of retinal degeneration resulting from rod photoreceptor cell death and leading to blindness. Despite the remarkable genetic heterogeneity of this disease, photoreceptors undergo a common mode of cell death: apoptosis. An autosomal recessive form of RP is caused by mutations in the rod-specific ␤-catalytic subunit of the phosphodiesterase gene PDE6B (1). The naturally occurring retinal degeneration (rd1) mouse is the animal model for this type of RP (2). The rd1 mouse has elevated levels of cGMP (3, 4), and this elevation results in elevated intracellular calcium (5). Ca 2ϩ concentration within the cytosol as well as Ca 2ϩ tides and ebbs within various organelles, such as mitochondria, nucleus, and endoplasmic reticulum (ER), are important in regulating many cellular functions such as neuronal survival or cell death.There are instances, most notably after Ca 2ϩ overload, in which the cell-death pathway elicited differs from classical caspasemediated apoptosis. Calpains are cysteine proteases activated by calcium during apoptotic processes (6). Calpain I and II (-and m-calpain) are expressed in the retina (7), and recent reports showed activation of calpain and cathepsin D in rd1 mice (5, 8). Several proteins are known targets of calpain protease activity, such as caspase-12 and apoptosis-inducing factor (AIF). Caspase-12, localized to the ER (9), can be activated by m-calpain in the presence of the pancaspase inhibitor zVAD.fmk (10). Interestingly, caspase-12 has been linked to neuronal degeneration in neurotoxicity caused by amyloid-␤ protein (9), by prion protein (11), and in animal models of ALS (12). The cleaved active form of caspase-12 participates to the apoptotic event by translocation to the nucleus (13); however, it is unclear whether caspase-12 can induce chromatin fragmentation. AIF also directly translocates to the nucleus to execute DNA fragmentation that culminates in cell death (14). The translocation of AIF from mitochondria to the nucleus has been implicated in neuronal d...

The bacterial CRISPR/Cas system has proven to be an efficient tool for genetic manipulation in various organisms. Here we show the application of CRISPR-Cas9 technology to edit the human Rhodopsin (RHO) gene in a mouse model for autosomal dominant Retinitis Pigmentosa. We designed single or double sgRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. By delivering Cas9 and sgRNAs in a single plasmid we induced an efficient gene editing in vitro, in HeLa cells engineered to constitutively express the P23H mutant RHO allele. Similarly, after subretinal electroporation of the CRISPR/Cas9 plasmid expressing two sgRNAs into P23H RHO transgenic mice, we scored specific gene editing as well as significant reduction of the mutant RHO protein. Successful in vivo application of the CRISPR/Cas9 system confirms its efficacy as a genetic engineering tool in photoreceptor cells.

SummaryThe autosomal dominant form of retinitis pigmentosa (adRP) is a blindness-causing conformational disease largely linked to mutations of rhodopsin. Molecular simulations coupled to the graph-based protein structure network (PSN) analysis and in vitro experiments were conducted to determine the effects of 33 adRP rhodopsin mutations on the structure and routing of the opsin protein. The integration of atomic and subcellular levels of analysis was accomplished by the linear correlation between indices of mutational impairment in structure network and in routing. The graph-based index of structural perturbation served also to divide the mutants in four clusters, consistent with their differences in subcellular localization and responses to 9-cis retinal. The stability core of opsin inferred from PSN analysis was targeted by virtual screening of over 300,000 anionic compounds leading to the discovery of a reversible orthosteric inhibitor of retinal binding more effective than retinal in improving routing of three adRP mutants.

Activation of Bax was observed in all three models of retinitis pigmentosa and leads to neurodamage by localization at the mitochondrion. Our data suggest that Bax can be envisaged as one of the promising target molecules for restraining photoreceptor degeneration.

Background: Pigment epithelium-derived factor (PEDF) interacts with its receptor PEDF-R to exert cytoprotection. Results: Alanine scanning of a small fragment (17-mer) of PEDF reveals key interacting residues for binding PEDF-R and alternative retinoprotective peptide versions with higher efficacy. Conclusion: The 17-mer contains a novel PEDF-R binding region important for retinoprotection. Significance: Altered PEDF peptides could be exploited pharmacologically to improve protection of photoreceptors from degeneration.