Calcium dynamics in degenerating photoreceptors

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Photoreceptor degeneration is a pathological process where an inherited mutation leads to loss of photoreceptors and blindness. Even though models of photoreceptor degeneration have been studied extensively, the exact pathway(s) along which photoreceptor cell death proceeds are still unclear. This knowledge, however, is crucial for developing neuroprotective strategies to prevent or delay degeneration.Previous studies indicate that dysregulation of Ca2+ homeostasis may play a role in photoreceptor cell death. For instance, it was suggested that accumulation of the second messenger cGMP in degeneratingphotoreceptors leads to elevated Ca2+ levels via activation of cyclic nucleotide-gated channels. This results in a rise in Ca2+-dependent, calpain-type proteolytic activity, a marker for looming photoreceptor cell death.Here we aim at a better understanding of the pathophysiology underlying degeneration of mammalian cone photoreceptors, focusing on the role of Ca2+. We will employ 2P imaging to record Ca2+ levels and light-evoked response from cones in acute retinal slices of mutants (cpfl1, rd1, rd10) crossbred with transgenic mice that express a Ca2+ biosensor in cones. Cpfl1 carries a phosphodiesterase 6 (Pde6) gene mutation in cones, leading to primary cone degeneration. In contrast, rd1 and rd10 carry Pde6 gene mutations in rods; here cones display secondary degeneration: they die after rods have vanished. Other than in rd1, degeneration in rd10 starts after retinal development is concluded, thus rd10 is useful for differentiating between development- and degeneration-related alterations in cone Ca2+. In the first part, we will follow the disease progression by recording cones at different time-points to find out if alterations in Ca2+ dynamics take place before or after degeneration onset. In parallel histological and biochemical experiments, we will monitor the presence and activity of markers related to Ca2+ homeostasis and cell death. This way, we will be able to compile a degeneration-stage resolved framework of Ca2+-related processes during cone degeneration. We are particularly interested in comparing primary and secondary cone degeneration, because it is still unclear if they are based on the same or on different mechanisms.In the second part, we will investigate if aberrant cone Ca2+ dynamics in mutant mice can be influenced by pharmacological treatment and if such measures may be exploited for neuroprotective approaches. We willstudy the effects of compounds known to regulate Ca2+ homeostasis. After verifying effects in acute slices, we will employ long-term organotypic retinal explant cultures exposed to the selected compounds and evaluate their effect on cone survival using histological and biochemical methods.We expect this project not only to improve our understanding of cone degeneration, but also to contribute to a more comprehensive picture of cell death and neuronal degeneration