PHENOTYPIC EXPRESSION IN INHERITED RETINAL DEGENERATIONS

RD小鼠是遗传性视网膜变性的模型，这种视网膜变性具有 其光传导级联中的一个缺陷。CGMP-PDE的β亚基 是这只小鼠视网膜退化的候选基因。学习 退化的潜在机制和基因修复的可能性， 我们表达了一个正常的牛cGMP-PDEβ亚基(β-PDE) 第三只老鼠。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。而‘营救’ 证实了β-PDE是RD基因， 这种缺陷基因所导致的导致丢失的过程 所有的杆状和圆锥体的问题仍有待解决。显然，正常的 杆状特异的PDE的功能是维持 视网膜的完整性，从而了解其在视网膜中的作用 退化是至关重要的。这项提议的长期目标是 揭示杆状光导中与基因缺陷相关的机制 级联到所有杆和锥体的丢失，就像在许多人类中发生的那样 遗传性视网膜变性。另一个目标是发展 药物干预以抑制这些机制。 具体地说，我们将测试这样一种想法，即存在一个 光传导机制可以耐受的表达，以及 高于或低于该水平的β-PDE表达会导致视杆细胞退变。 我们将通过检查适当的转基因、RD/RD、RD/+来测试这一概念 和正常+/+小鼠的原位杂交和免疫细胞化学 方法：研究方法。我们还将检验杂合子RD/+是 经调整以补偿降低的β-PDE水平 CGMP门控Na+/Ca++通道数。 视蛋白的不正确分类是所有啮齿动物模型的一个标志 视网膜变性。我们将测试视蛋白的正常分选是否 转基因植株中PDE活性恢复伴随的恢复 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了类似于 ‘行业’RP。最后，我们将制定治疗策略 对研发的干预。我们将测试我们的假设，即RD缺陷打开 过多的cGMP门控离子通道，导致 灾难性的离子流入。几种特异性阻断Na+/Ca++的药物 将使用渠道来控制这种涌入。其次，我们会 试图阻止可能导致损失的破坏性级联 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和 自由基清除剂来干预这些过程。

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.