Calcium dynamics in degenerating photoreceptors

光感受器退化是一种病理过程，其中遗传突变导致光感受器丧失和失明。尽管感光细胞退化的模型已被广泛研究，但感光细胞死亡进行的确切途径沿着仍不清楚。然而，这些知识对于开发神经保护策略以预防或延迟变性是至关重要的。先前的研究表明，Ca 2+稳态失调可能在感光细胞死亡中起作用。例如，有人认为，第二信使cGMP在退化的光感受器中的积累通过激活环核苷酸门控通道导致Ca 2+水平升高。这会导致Ca 2+依赖性钙蛋白酶型蛋白水解活性的上升，这是一个迫在眉睫的感光细胞死亡的标志物。在这里，我们旨在更好地了解哺乳动物视锥细胞退化的病理生理学基础，专注于Ca 2+的作用。我们将采用2 P成像记录的Ca 2+水平和光诱发的反应，从急性视网膜切片的突变体（cpfl 1，rd 1，rd 10）与转基因小鼠杂交表达的Ca 2+生物传感器在锥细胞。Cpfl 1携带视锥细胞中的磷酸二酯酶6（Pde 6）基因突变，导致原发性视锥细胞变性。相比之下，rd 1和rd 10在视杆细胞中携带Pde 6基因突变;在这里，视锥细胞显示继发性变性：它们在视杆细胞消失后死亡。与rd 1不同，rd 10中的变性在视网膜发育结束后开始，因此rd 10可用于区分视锥Ca 2+的发育相关改变和变性相关改变。在第一部分中，我们将通过记录不同时间点的视锥细胞来跟踪疾病进展，以了解Ca 2+动力学的改变是否发生在变性发作之前或之后。在平行的组织学和生物化学实验中，我们将监测与Ca 2+稳态和细胞死亡相关的标志物的存在和活性。这样，我们将能够编译一个退化阶段的解决框架的钙相关过程中锥退化。我们特别感兴趣的是比较原发性和继发性视锥细胞变性，因为目前还不清楚，如果他们是基于相同或不同的mechanism.In第二部分，我们将调查，如果异常的视锥细胞Ca 2+动力学突变小鼠可以通过药物治疗的影响，如果这些措施可以利用神经保护的方法。我们将研究已知调节Ca 2+稳态的化合物的作用。在急性切片中验证效果后，我们将使用长期暴露于所选化合物的器官型视网膜外植体培养物，并使用组织学和生物化学方法评估其对视锥存活的影响。我们期望该项目不仅提高我们对视锥变性的理解，而且有助于更全面地了解细胞死亡和神经元变性

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