Genetic Mechanisms of Retinal Degenerative Disease

描述（由申请人提供）：触发光感受器细胞死亡的机制仍然知之甚少，阻碍了对影响150多万人的视网膜变性的有效治疗的发展。这项研究的长期目标是阐明触发光感受器细胞死亡的分子信号。最近，我们发现了一类变性，其中光传导信号的持续激活导致杆细胞死亡。我们假设由持续的转导蛋白信号导致的cGMP水解导致视网膜变性。cGMP的下降增加了封闭构型中环状核苷酸门控（CNG）通道的比例，减少了钙进入光感受器的量。缺钙是变性的一个可能原因。然而，细胞内低钙也可以刺激鸟苷酸环化酶活性合成cGMP。根据cGMP水解和合成的相对速率，这可能导致一个无效的循环，导致cGMP水平降低或升高，通道关闭或打开分别导致钙不足或过量。为了验证这一假设，我们将创造具有影响杆状细胞中钙流入和流出的基因突变的转基因小鼠，即杆状细胞CNG a亚基和杆状细胞Na+， Ca2+/K+交换器。我们的目标是检查cGMP水平的扰动是否会改变细胞内钙，是视网膜变性的机制原因。具体目标为了验证一种假设，即一个突变主要将杆cGMP核苷酸门控通道置于封闭构型，将诱导变性，这与光转导级联的组成激活非常相似。我们将在转基因小鼠中引入R553Q CNGA1敲入突变，该突变将cGMP结合亲和力降低30倍，关闭更大比例的CNG通道。评估对视网膜形态、cGMP和钙水平的影响。具体目标2。为了验证“rd”小鼠视网膜中与cGMP升高相关的视网膜变性是由杆外段过量的Ca2+引起的，并且可以通过减少细胞内Ca2+来改善。我们将通过过度表达杆状视蛋白启动子表达的Na+，Ca2+/K+交换器（NCKX1），从而增加钙的外排，从而从基因上减少“rd”小鼠的细胞内钙。如果这个假设是正确的，那么与rd小鼠相比，rd、NCKX1双突变小鼠的退化速度会减慢。具体目标3。为了验证NCKX1基因突变的假设，该基因调节杆状细胞外段Ca2外排，通过增加细胞内钙导致视网膜变性。将创建I906T棒Na+，Ca2+/K+交换器（NCKX1）敲入突变小鼠。体外研究表明该突变体的转运活性降低。预计钙外排减少会导致细胞内钙升高。如果钙升高是视网膜变性的一个原因，我们预测变性将会发生。这些研究将阐明cGMP和Ca2+在视网膜疾病和正常光感受器功能中的生理关系和作用。

DESCRIPTION (provided by applicant): The mechanisms triggering photoreceptor cell death remain poorly understood, hindering the development of effective therapies for retinal degenerations affecting more than 1.5 million people. The long-term goal of this study is to elucidate molecular signals that trigger photoreceptor cell death. Recently, we identified a subclass of degenerations in which persistent activation of phototransduction signaling caused rod cell death. We hypothesize that cGMP hydrolysis resulting from persistent transducin signaling causes retinal degeneration. The drop in cGMP increases the fraction of rod cyclic nucleotide gated (CNG) channels in the closed configuration, reducing calcium entry into photoreceptors. Calcium depletion is one possible cause of degeneration. However, low intracellular calcium can also stimulate guanylate cyclase activity to synthesize cGMP. Depending on the relative rates of cGMP hydrolysis and synthesis, this may result in a futile cycle that leads to either low or elevated cGMP levels, closing or opening channels to cause insufficient or excess calcium, respectively. To test this hypothesis, we will create transgenic mice with mutations affecting genes that modulate calcium influx and efflux in rod cells, namely the rod CNG a-subunit and the rod Na+, Ca2+/K+ exchanger. Our goal is to examine whether perturbations in cGMP levels, which alter intracellular calcium, is a mechanistic cause of retinal degenerations. Specific Aim 1. To test the hypothesis that a mutation that predominantly sets the rod cGMP nucleotide-gated channel in the closed configuration will induce degeneration much the same as constitutive activation of the phototransduction cascade will. We will introduce an R553Q CNGA1 knock-in mutation in transgenic mice that reduces cGMP binding affinity 30-fold, closing a larger fraction of CNG channels. The effect on retinal morphology, cGMP and calcium levels will be assessed. Specific Aim 2. To test the hypothesis that retinal degeneration associated with elevated cGMP in the "rd" mouse retina is caused by excessive Ca2+ in rod outer segments and can be ameliorated by reducing intracellular Ca2+. We will genetically reduce intracellular calcium in the "rd" mouse by over expressing the Na+,Ca2+/K+ exchanger (NCKX1) expressed from the rod opsin promoter, thus increasing efflux of calcium. If the hypothesis is correct, degeneration in rd, NCKX1 double mutant mice will be slowed compared to the rate of degeneration in rd mice. Specific Aim 3. To test the hypothesis that mutations in the NCKX1 gene, which regulates Ca2 efflux from rod outer segments, cause retinal degeneration by increasing intracellular calcium. An I906T rod Na+,Ca2+/K+ exchanger (NCKX1) knock-in mutant mouse will be created. In vitro studies show reduced transport activity by this mutant. The reduced calcium efflux is predicted to cause a rise in intracellular calcium. If elevated calcium is a cause of retinal degeneration, we predict that degeneration will occur. The proposed studies will elucidate the physiological relationship and role of cGMP and Ca2+ in retinal disease and normal photoreceptor function.