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Calcium dynamics in degenerating photoreceptors

退化光感受器中的钙动力学

基本信息

批准号：
266705556
负责人：
Professor Dr. Thomas Euler
金额：
--
依托单位：
Werner Reichardt-Centrum für integrative Neurowissenschaften (CIN)
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2014
资助国家：
德国
起止时间：
2013-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/266705556?language=en
关键词：
Calcium dynamics degenerating photoreceptors

项目摘要

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

光感受器变性是一种病理过程，遗传突变导致光感受器丧失和失明。尽管光感受器变性模型已被广泛研究，但光感受器细胞死亡的确切途径仍不清楚。然而，这一知识对于制定预防或延缓退化的神经保护策略至关重要。先前的研究表明，Ca2+稳态失调可能在感光细胞死亡中发挥作用。例如，有人认为，退化光感受器中第二信使 cGMP 的积累会通过环核苷酸门控通道的激活导致 Ca2+ 水平升高。这导致 Ca2+ 依赖性钙蛋白酶型蛋白水解活性的增加，这是迫在眉睫的感光细胞死亡的标志。在这里，我们的目标是更好地了解哺乳动物视锥光感受器退化的病理生理学，重点关注 Ca2+ 的作用。我们将采用 2P 成像来记录突变体 (cpfl1、rd1、rd10) 与在视锥细胞中表达 Ca2+ 生物传感器的转基因小鼠杂交的急性视网膜切片中的 Ca2+ 水平和视锥细胞的光诱发反应。 Cpfl1 在视锥细胞中携带磷酸二酯酶 6 (Pde6) 基因突变，导致原发性视锥细胞变性。相比之下，rd1和rd10在视杆细胞中携带Pde6基因突变；此处，视锥细胞表现出继发性退化：视杆细胞消失后它们就会死亡。与 rd1 不同，rd10 的变性在视网膜发育结束后开始，因此 rd10 可用于区分视锥细胞 Ca2+ 中与发育相关和变性相关的改变。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的进展，以确定 Ca2+ 动力学的变化是否发生在变性发生之前或之后。在平行的组织学和生化实验中，我们将监测与 Ca2+ 稳态和细胞死亡相关的标记物的存在和活性。这样，我们将能够编译锥体变性过程中 Ca2+ 相关过程的变性阶段解析框架。我们对比较原发性和继发性视锥细胞变性特别感兴趣，因为目前尚不清楚它们是否基于相同或不同的机制。在第二部分中，我们将研究突变小鼠中异常的视锥细胞Ca2+动力学是否会受到药物治疗的影响，以及这些措施是否可以用于神经保护方法。我们将研究已知调节 Ca2+ 稳态的化合物的作用。在急性切片中验证效果后，我们将采用长期暴露于所选化合物的器官型视网膜外植体培养物，并使用组织学和生化方法评估它们对视锥细胞存活的影响。我们期望该项目不仅能提高我们对视锥细胞变性的理解，而且有助于更全面地了解细胞死亡和神经元变性