Gap-Junction-Medicated Cell Death in Rod-Cone Dystrophie

描述(申请人提供)：视网膜色素变性(RP)是一组遗传介导的退行性视网膜疾病，表现出广泛的表型。在各种形式的RP的基础上，病理上存在明显的异质性，但在已知的病例中，相当大比例的病例是由于视紫红质或其他视杆特异蛋白的突变引起的。然而，尽管遗传缺陷仅在视杆感光细胞中表达，否则健康的视锥感光细胞总是会死亡，导致严重的视力障碍。这项提议的主要目的是检验这样一种假设，即疾病从死亡的视杆细胞传播到遗传上正常的视锥细胞是一种“旁观者”效应，由这些光感受器亚型之间存在的缝隙连接介导。根据这一观点，触发细胞凋亡过程的试剂渗透到细胞间缝隙连接通道，将促凋亡信号从患病的视杆带到邻近的视锥细胞。如果缝隙连接是光感受器退化非细胞自主传播的一个重要因素，那么阻断通过这些通道的传输可能会提供一种新的治疗干预形式，使视锥细胞免于遭受其邻居的命运。 我们计划进行的实验将有助于确定，当连接视杆和视锥的缝隙连接蛋白(连接蛋白36)被破坏时，表达杆状特定基因缺陷的转基因小鼠的视锥感光细胞是否表现出显著更高的存活率。Cx36“基因敲除”小鼠是可用的，我们建议将它们与表达各种导致视杆锥体营养不良的杆状特异突变的小鼠杂交。锥体结构和功能将通过一组组织学、免疫细胞化学和电生理学方法进行评估。此外，我们计划进行一系列分子生物学和生物化学研究，以进一步检测哺乳动物光感受器中连接蛋白的表达，并确定能够渗透到细胞间通道的促凋亡剂。

DESCRIPTION (provided by applicant): Retinitis pigmentosa (RP) constitutes a group of genetically-mediated, degenerative retinal diseases that display a broad range of phenotypes. There is appreciable heterogeneity in the pathologies that underlie the various forms of RP, but of the known cases, a substantial percentage arise as a consequence of mutations in rhodopsin or other rod-specific proteins. However, despite the fact that the genetic defect is expressed solely in the rod photoreceptors, otherwise healthy cone photoreceptors invariably die, resulting in severe visual impairment. The principal goal of this proposal is to test the hypothesis that the spread of the disease from dying rods to genetically normal cones is a form of "bystander" effect, mediated by the gap junctions that exist between these photoreceptor subtypes. On this view, agents that trigger the apoptotic process permeate the intercellular gap-junctional channels to carry proapoptotic signals from diseased rods to neighboring cones. If gap junctions are a significant factor in the non-cell-autonomous spread of photoreceptor degeneration, blocking transmission through these channels may provide a novel form of therapeutic intervention that would enable cones to be spared the fate of their neighbors. The experiments we plan to conduct will help to determine whether the cone photoreceptors of transgenic mice that express rod-specific gene defects exhibit a significantly higher survival when the gap-junctional protein (connexin36) that couples their rods and cones is disrupted. Cx36 "knockout" mice are available, and we propose to cross them with mice expressing various rod-specific mutations that result in rod-cone dystrophy. Cone structure and function will be assessed by a panel of histological, immunocytochemical, and electrophysiological methods. In addition, we plan to conduct a series of molecular biological and biochemical studies to examine further connexin expression in mammalian photoreceptors, and to identify proapoptotic agents that are able to permeate the intercellular channels.