Regulation of Calcium Signaling in Retinal Ganglion Cells after Nerve Injury

描述(由申请人提供)： 颅脑钝挫伤或冲击伤是战场上常见的眼外伤和创伤性脑损伤，常伴有多发性视力障碍、单眼或双眼失明。视神经病变的特征是视神经的原发损伤，神经节细胞及其轴突的丢失。神经节细胞的死亡在一定程度上是由损伤后细胞内过多的钙([钙]i)负荷所介导的。与此发现一致的是，给予钙通道拮抗剂可增强视神经挤压后视网膜神经节细胞的存活，抑制L和T型钙电流，并减少继发性神经节细胞死亡。这项研究的基本原理是，通过调节钙通道活性降低神经节细胞内钙水平可能是治疗视网膜损伤的保护策略的重要组成部分。抑制神经节细胞内过高的[Ca~(2+)]i将为神经节细胞存活和损伤后轴突的恢复提供时间窗口。拟议的研究将检验这一假设，即抑制神经损伤后升高的[钙]i可提高神经节细胞的存活率。具体目的1将确定L和T型钙通道及其辅助蛋白(23和124)在小鼠视网膜神经节细胞中的信号作用。实验将测定神经节细胞的L和T型钙通道及其附属蛋白的表达，以及神经节细胞L和T型钙电流的生理和生物物理特性。特定目标2将验证神经节细胞钙信号在视神经挤压或横断后立即和几天后失调的假设。研究将确定视神经损伤后神经节细胞是否存在短期(12和24小时)和长期(10天和20天)的改变，a)L和T型以及23和124Ca~(2+)通道亚单位的表达，以及b)介导神经节细胞钙电流和信号转导的膜机制。具体目标3将验证钙通道拮抗剂和小干扰RNA(SiRNA)反义钙通道亚单位载体调控钙信号转导，提高视神经损伤后神经节细胞存活的假说。研究将确定a)L和T型钙通道拮抗剂洛美利嗪和b)反义T型钙通道(CaV3.1和CaV3.2)，以及23和124钙通道亚单位siRNA载体是否调节钙信号并提高视神经损伤后神经节细胞的存活。拟议的研究将阐明正常和损伤的神经节细胞中的钙信号转导，并开发控制神经损伤后细胞内钙升高的新方法，这将提高神经节细胞的存活，这是挽救视力的关键步骤。这些研究与退伍军人管理局的健康相关目标是一致的，即开发高效和新颖的眼睛损伤和疾病治疗方法。

DESCRIPTION (provided by applicant): Ocular and traumatic brain injuries (TBI) from blunt trauma or blast injury to the head occur with high frequency on the battlefield, and they are often accompanied by multiple visual dysfunctions, acuity loss and blindness in one or both eyes. Optic neuropathies are characterized by primary injury to the optic nerve, and loss of ganglion cells and their axons. Ganglion cell death is mediated in part by excessive intracellular Ca2+ ([Ca2+]i) loads following injury. Consistent with this finding is the enhancement of retinal ganglion cell survival after optic nerve crush with the administration of Ca2+ channel antagonists, which inhibit both L- and T-type Ca2+ currents, and reduce secondary ganglion cell death. The rationale underlying the proposed studies is that reduction of ganglion cell intracellular Ca2+ levels by regulation of Ca2+ channel activity may be an important component of protective strategies for the treatment of retinal injury. Suppression of excessively elevated [Ca2+]i in ganglion cells would provide a temporal window for ganglion cell survival and axonal recovery following injury. Proposed studies will test the hypothesis that suppression of elevated [Ca2+]i following nerve injury enhances ganglion cell survival. Specific aim 1 will define the signaling role of L- and T-type Ca2+ channels, and their accessory proteins (23 and 124) in mouse retinal ganglion cells. Experiments will determine a) the expression of L- and T-type Ca2+ channels and their accessory proteins by ganglion cells, and b) characterize the physiological and biophysical properties of L- and T-type Ca2+ currents of ganglion cells. Specific aim 2 will test the hypothesis that ganglion cell Ca2+ signaling is dysregulated immediately following and several days after optic nerve crush or transection. Investigations will determine if there are short (12 and 24 hours)- and long (10 and 20 days)-term alterations of a) the expression of L- and T-type, and 23 and 124 Ca2+ channel subunits by ganglion cells, and b) membrane mechanisms that mediate Ca2+ currents and signaling in ganglion cells following optic nerve injury. Specific aim 3 will test the hypothesis that Ca2+ channel antagonists and small interfering RNA (siRNA) antisense Ca2+ channel subunit vectors regulate Ca2+ signaling, and enhance ganglion cell survival after optic nerve injury. Investigations will determine if a) the L- and T-type Ca2+ channel antagonist, lomerizine, and b) antisense T-type (CaV3.1 and CaV3.2), and 23 and 124 Ca2+ channel subunit siRNA vectors, modulate Ca2+ signaling and enhance ganglion cell survival following optic nerve injury. Proposed studies will elucidate Ca2+ signaling in normal and injured ganglion cells, and develop novel approaches for controlling elevated intracellular Ca2+ following nerve injury, which will enhance ganglion cell survival, a key step in saving vision. These studies are consistent with the health-related goals of the Veterans Adminstration to develop highly effective and novel treatments for eye injury and disease.