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Cellular and molecular mechanisms of the light response in photoreceptor cells of the mammalian retina

Final Report Summary - RODCELL (Cellular and molecular mechanisms of the light response in photoreceptor cells of the mammalian retina)

Project context and objectives

Genetic defects in proteins involved in the transduction of light in photoreceptor cells of the retina lead to severe retinal degeneration and blindness. Mutations that primarily affect rod function lead to retinitis pigmentosas. These diseases affect 1 in 4000 individuals, initially causing night blindness. However, as the gradual loss of rods eventually compromises cone cell viability, they lead to a complete loss of visual function. Mutations that primarily affect cone function lead to cone dystrophies (CDs) or macular degenerations, diseases characterised by the loss of central vision. There is no current cure for these diseases. The first aim of this proposal was to establish mouse models of the autosomal dominant cone dystrophies (adCDs) caused by mutations in the genes encoding guanylate cyclase activating protein 1 (GCAP1) and 2 (GCAP2), two Ca2+-binding proteins that regulate cGMP synthesis in rods and cones, in order to study the pathways leading to cell death in vivo. The second aim was to study the novel role of the guanylate cyclase activating proteins in the synaptic terminal of rod cells, where they are likely playing an important role in the dynamic assembly / disassembly of synaptic ribbons with changes in illumination. Ribbons are hallmark specialisations of sensory neurons required to sustain their characteristic high tonic neurotransmitter release. Characterising the mechanisms that govern their dynamic turnover will contribute to understand how they are assembled and how they work. We perform our studies in animal models because photoreceptors are such compartmentalised and specialised neurons that there is no cell culture system that reproduces their complexity. That is why we were also interested in introducing to the lab more efficient and affordable strategies to do mouse genetics as a basis to support these studies and future gene function studies in photoreceptor cells.

For the first aim, we have characterised a mouse model of retinal degeneration caused by transgenic expression in rods of a mutant form of GCAP2 impaired to bind Ca2+, the mutant EF-GCAP2 in which the three functional EF-hands have been disrupted. To date, ten different mutations in guanylate cyclase activating protein 1 (GCAP1) have been linked to autosomal dominant cone dystrophies, a disease characterised by the loss of colour vision and central visual acuity, and to macular dystrophies. Generally, the mutations leading to cone dystrophy affect one of the Ca2+ binding sites or alter protein structure in a way that impair Ca2+-binding. GCAPs stimulate retGC catalytic activity when intracellular Ca2+ decreases in response to light. It is the 'Ca2+-empty' form of GCAPs that activate cGMP synthesis. In vitro, mutants impaired to bind Ca2+ remain active with increasing [Ca2+], leading to constitutive activation of the cyclase independently of the lighting conditions. Therefore, it has been proposed that GCAP1 mutations would lead to photoreceptor cell death in vivo by causing constitutive cGMP synthesis, leading to abnormally high levels of cGMP. As a consequence, a higher fraction of the cGMP-channels would be kept open, increasing the inward current of calcium. Elevated intracellular calcium would induce apoptosis. There are two recent studies characterising transgenic mice expressing Y99C-GCAP1 in rod photoreceptors that would support this hypothesis.

However, by doing morphological and biochemical analysis of mice expressing an EF-GCAP2 transgene in rods, in the course of this project we have observed that GCAP proteins that are severely impaired at binding Ca2+ are deleterious for the cell even when they are not active and they do not traffick to the light-sensitive compartment of the cell. The mutant form EF-GCAP2, with the three functional EF-hands disrupted is inactive and accumulates at proximal compartments of the cell when expressed in vivo in rod photoreceptors, failing to be transported to the outer segment where phototransduction takes place. Therefore, we conclude that certain human-disease causing mutations that affect Ca2+ binding affinity in GCAP proteins will likely affect their activity and ability to traffick, probably by decreasing their thermal stability. Accumulation of mutant unstable proteins at the inner segment of the cell in vivo leads to toxicity by a mechanism that is independent of cGMP metabolism and possibly involves pathways related to conformational disorders, such as the heat response. In order to characterise the basis of the pathology in this mouse model, experiments are under way to characterise the protein-protein interactions that the mutant GCAP2 establishes at the inner segment. Pull-down experiments with recombinant GCAP2, as well as immunoprecipitation experiments with anti-GCAP2 polyclonal and monoclonal antibodies have been performed, and a number of GCAP2 putative interacting partners have been identified, that are currently being validated. These results point to GCAP2 having new molecular targets, such as proteins involved in the heat response, but also in vesicular trafficking, protein synthesis, synaptic vesicle recycling and lipid metabolism. That is, results indicate that GCAPs have new Ca2+-sensor functions at the inner segment and synaptic terminal of photoreceptor cells, where they might be involved in different processes. We will continue the characterisation of new GCAP functions, as this knowledge will be very relevant to unveil the pathology underlying retinal dystrophies caused by GCAP mutations. This study focused on expressing a mutant protein in rods because a rod phenotype is easier to analyse in the mouse retina, where rods constitute 95 % of photoreceptor cells. We have here set the ground to perform a subsequent study in cones. Characterising the cGMP-independent basis of toxicity of GCAP mutants in rods and cones will help in guiding combined therapies for patients in the future.

In the second aim, we have addressed the role of GCAP2 in the synaptic terminal of photoreceptor cells. In the course of this study we have analysed the rod and cone synaptic phenotype of GCAP2 mouse models of loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic mice overexpressing GCAP2 in rods), both by ultrastructural analysis of their retinas and assessment of visual function by electroretinogram. Transmission electron microscopy analysis of synaptic terminals in the different mouse models revealed that overexpression of GCAP2 in rods leads to a substantial shortening of the ribbon synapse. The ribbon synapse is a specialised organelle in the synaptic terminals of sensory neurons in the retina and inner ear that serves to anchor synaptic vesicles and the neurosecretion exocytic machinery to the immediacy of the active zone. From our results, we infer that GCAP2 plays a role at mediating the dynamic morphological changes that assemble / disassemble ribbons during dark/light adaptation, known to be Ca2+-dependent. We have also demonstrated at the ultrastructural level that GCAP2 colocalises with Ribeye, a unique and major protein component of the synaptic ribbon. An intriguing observation was that the effect of overexpressing GCAP2 at shortening ribbon length was substantially magnified in the absence of GCAP1, which points to GCAP1 being involved in this process, somehow opposing GCAP2 function. These results are relevant to the extent that they contribute to understand how synaptic ribbons are rearranged in photoreceptor cells depending on illumination, and the extent to which these remodelling changes affect synaptic strength.

In the course of this study, we have introduced innovative, more practical and affordable strategies to do mouse genetics in the lab, by developing protocols of in vivo DNA electroporation to transiently or stably express heterologous DNA in murine photoreceptors. We can now get transient transgenesis by electroporation of living mice (neonates) after plasmid DNA subretinal injection. This technology, originally developed by the laboratory of Connie Cepko at Harvard, United States of America (USA) (Matsuda and Cepko, 2004. PNAS 101, pp 16 - 22), is fast and affordable. However, it has the limitation that the levels of transfection are low and typically restricted to the area of injection. Its use is therefore restricted to studies in which conclusions can be extracted from a few sparse cells (e.g. subcellular localisation). For this reason, we have adapted for transgene expression in photoreceptors the method for stable transgenesis by DNA electroporation into the male germ line at p30 and subsequent breeding originally developed by Subeer Majumdar (Suveera Dhup and Subeer Majumdar, 2008. Nature Methods 5, pp. 601 - 603). This method is fast and affordable, and it does not require the specialised equipment and skilled personnel of a transgenic core facility. We are obtaining approximately 25 - 30 % of transgene integration with this technique, and we are working now towards using the methodology to target gene expression, by using siRNAs.

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