Regulation of Calcium Signaling in Retinal Ganglion Cells after Nerve Injury

描述（由申请人提供）： 在战场上，由头部钝伤或爆炸伤引起的眼外伤和创伤性脑损伤（TBI）发生率很高，并且它们通常伴有多个视觉功能障碍、视力丧失和单眼或双眼失明。视神经病变的特征在于视神经的原发性损伤以及神经节细胞及其轴突的损失。神经节细胞死亡部分由损伤后过量的细胞内Ca 2+（[Ca 2 +]i）负荷介导。与这一发现相一致的是，在视神经挤压后，施用Ca 2+通道拮抗剂可增强视网膜神经节细胞的存活，其抑制L型和T型Ca 2+电流，并减少继发性神经节细胞死亡。提出的研究的基本原理是，通过调节Ca 2+通道活性来降低神经节细胞内Ca 2+水平可能是治疗视网膜损伤的保护策略的重要组成部分。抑制神经节细胞中过度升高的[Ca 2 +]i将为损伤后神经节细胞存活和轴突恢复提供时间窗。拟开展的研究将检验神经损伤后抑制[Ca 2 +]i升高可增强神经节细胞存活的假设。具体目标1将定义L型和T型Ca 2+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传导作用。实验将确定a）L-和T-型Ca 2+通道及其辅助蛋白在神经节细胞中的表达，和B）表征神经节细胞的L-和T-型Ca 2+电流的生理和生物物理特性。具体目标2将检验神经节细胞Ca 2+信号传导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，a）L型和T型以及23和124 Ca 2+通道亚单位的表达，和B）介导Ca 2+电流和神经节细胞信号传导的膜机制是否存在短期（12和24小时）和长期（10和20天）变化。具体目标3将检验Ca 2+通道拮抗剂和小干扰RNA（siRNA）反义Ca 2+通道亚基载体调节Ca 2+信号传导并增强视神经损伤后神经节细胞存活的假设。研究将确定a）L型和T型Ca 2+通道拮抗剂洛美利嗪和B）反义T型（CaV 3.1和CaV 3.2）以及23和124 Ca 2+通道亚基siRNA载体是否调节视神经损伤后的Ca 2+信号传导并增强神经节细胞存活。拟议的研究将阐明正常和受伤的神经节细胞中的Ca 2+信号传导，并开发新的方法来控制神经损伤后细胞内Ca 2+的升高，这将提高神经节细胞的存活率，这是挽救视力的关键一步。这些研究与退伍军人管理局的健康相关目标是一致的，即为眼损伤和疾病开发高效和新颖的治疗方法。

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.