PHENOTYPIC EXPRESSION IN INHERITED RETINAL DEGENERATIONS

rd小鼠是遗传性视网膜变性的模型， 它的光传导级联中的缺陷。 cGMP-PDE的β亚基 是这只老鼠视网膜退化的候选基因。 研究 退化的潜在机制和基因修复的潜力， 我们已经表达了正常牛cGMP-PDE β亚基（β-PDE）， rd小鼠。 在这些转基因动物中，我们能够恢复β- PDE酶活性和“拯救”光感受器。 当“拯救” 这些动物的视网膜证实了β-PDE是rd基因， 由这种缺陷基因导致的过程， 所有的视杆细胞和视锥细胞的问题还有待解决。 显然，正常的 特定于杆的PDE的功能对于维持 视网膜的完整性，从而了解其在视网膜的作用， 退化是关键。 该提案的长期目标是 揭示了在视杆细胞光转导中基因缺陷的相关机制 级联损失所有杆以及锥，因为发生在许多人类 遗传性视网膜变性 另一个目标是发展 药物干预来抑制这些机制。 具体来说，我们将测试的想法，有一个'窗口'的 表达的光转导机制可以容忍， 高于或低于该水平的β-PDE表达导致视杆变性。 我们将通过检查适当的转基因、rd/rd、rd/+来检验这一概念 和正常+/+小鼠进行原位杂交和免疫细胞化学 方法. 我们还将检验杂合子rd/+是 适于通过调节β-PDE水平来补偿β-PDE水平的降低， cGMP门控Na+/Ca++通道数量。 视蛋白的不正确分类是所有啮齿动物模型的标志， 视网膜变性 我们将测试视蛋白的正常分类是否 在转基因小鼠中，PDE活性的恢复伴随着PDE活性的恢复而恢复。 rd/rd小鼠。 我们将确定退化梯度的原因 在一些转基因小鼠中观察到， “部门”RP。 最后，我们将制定治疗策略， 干预RD。 我们将测试我们的假设，rd缺陷打开 cGMP门控离子通道数量过多，导致 灾难性的离子流入 几种特异性阻断Na+/Ca++的药物 将使用通道来控制这种流入。 其次，我们将 试图阻止可能造成损失的破坏性级联， 锥体，特别是降解酶的激活和 产生自由基。 我们将使用特定的酶抑制剂， 自由基清除剂干预这些过程。

The rd mouse is a model of an inherited retinal degeneration which has a defect in its phototransduction cascade. The beta-subunit of cGMP-PDE is the candidate gene for retina degeneration in this mouse. To study the mechanisms underlying degeneration and the potential for gene repair, we have expressed a normal bovine cGMP-PDE beta-subunit (beta-PDE) in the rd mouse. In these transgenic animals, we were able to restore the beta- PDE enzyme activity and 'rescue' the photoreceptors. While the 'rescue' of the retina in these animals confirms that the beta-PDE is the rd gene, the processes resulting from this defective gene which lead to the loss of all rods and cones remain to be resolved. Clearly, the normal functioning of rod-specific PDE is fundamental to maintaining the integrity of the retina, thus understanding its role in retinal degeneration is critical. The long-term goal of this proposal is to reveal mechanisms linking a gene defect in the rod phototransduction cascade to the loss of all rods as well as cones, as occurs in many human inherited retinal degenerations. A further goal is to develop pharmacologic interventions to inhibit these mechanisms. Specifically, we will test the idea that there is a 'window' of expression which the phototransduction mechanism can tolerate and that beta-PDE expression above or below that level leads to rod degeneration. We will test this concept by examining appropriate transgenic, rd/rd,rd/+ and normal +/+ mice using in situ hybridization and immunocytochemical methods. We will also test the hypothesis that the heterozygote rd/+ is adapted to compensate for reduced beta-PDE levels by regulating the number of cGMP-gated Na+/Ca++ channels. The improper sorting of opsin is a hallmark of all rodent models of retinal degenerations. We will test if the normal sorting of opsin is restored concomitant with the restoration of PDE activity in transgenic rd/rd mice. We will determine the cause of the gradient of degeneration observed in some transgenic mice which has a pattern similar to 'sectoral' RP. Finally, we will develop strategies for therapeutic intervention in rd. We will test our hypothesis that the rd defect opens an excessive number of cGMP-gated ion channels, resulting in a catastrophic ion influx. Several drugs which specifically block Na+/Ca++ channels will be used to control this influx. Secondarily, we will attempt to block destructive cascades which may cause the loss of the cones, specifically the activation of degradative enzymes and the generation of free radicals. We will use specific enzyme inhibitors and free-radical scavengers to intervene in these processes.