Genetic Mechanisms of Retinal Degenerative Disease

描述(申请人提供)：触发感光细胞死亡的机制仍然知之甚少，阻碍了影响超过150万人的视网膜退化有效治疗方法的开发。这项研究的长期目标是阐明触发感光细胞死亡的分子信号。最近，我们发现了一类持续激活光传导信号导致视杆细胞死亡的退行性变。我们假设持续的转导信号导致的cGMP水解会导致视网膜变性。CGMP的下降增加了闭合构型中的杆状环核苷酸门控(CNG)通道的比例，减少了钙离子进入光感受器。钙缺乏是退化的一个可能原因。然而，细胞内低钙也能刺激鸟苷环化酶合成cGMP。取决于cGMP水解和合成的相对速率，这可能导致一个无用的循环，导致cGMP水平降低或升高，关闭或打开通道，分别导致钙不足或过剩。为了验证这一假设，我们将创建转基因小鼠，其突变影响调控杆状细胞钙内流和外流的基因，即杆状CNGα亚单位和杆状Na+，Ca+/K+交换器。我们的目标是研究改变细胞内钙离子的cGMP水平的扰动是否是视网膜退化的机械性原因。具体目的1.检验假设，即主要将杆状cGMP核苷酸门控通道设置为闭合构型的突变将导致与光转导级联的结构性激活大致相同的退化。我们将在转基因小鼠中引入R553Q CNGA1敲入突变，使cGMP结合亲和力降低30倍，关闭更大比例的CNG通道。将评估对视网膜形态、cGMP和钙水平的影响。具体目的2.验证视网膜变性与cGMP升高相关的假说，即视网膜退行性变是由视杆细胞外节中过多的钙离子引起的，通过降低细胞内钙离子可以改善这一假说。我们将通过过度表达由视杆蛋白启动子表达的Na+，Ca2+/K+交换器(NCKX1)，从而增加钙的外流，从而遗传地减少“RD”小鼠的细胞内钙离子。如果假设是正确的，RD，NCKX1双突变小鼠的退变速度将比RD小鼠的退变速度慢。具体目的3.验证NCKX1基因突变通过增加细胞内钙而引起视网膜变性的假说。NCKX1基因调节视杆细胞外段的钙外流。建立I906T杆状Na+，Ca2+/K+交换器(NCKX1)敲入突变小鼠。体外研究表明，该突变体降低了转运活性。据预测，钙外流减少会导致细胞内钙的增加。如果升高的钙是视网膜退化的原因，我们预测这种退化将会发生。这些研究将阐明cGMP和钙离子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.