The Role of Gap Junctions in the Progressive Loss of Retinal Neurons in Glaucoma

 描述(申请人提供)：青光眼是全球第二大致盲原因，是一种神经退行性疾病，通常与眼内压(IOP)升高有关，特征是视网膜神经节细胞(RGC)进行性丧失导致视力丧失。在与神经病理相关的各种类型的细胞死亡中，细胞凋亡和坏死的主要途径已经被很好地描述。然而，除了初级细胞死亡的这些内在机制外，通过缝隙连接(GJS)的细胞间通讯似乎在继发性或旁观者细胞死亡中发挥了重要的作用，但人们对此知之甚少。GJS是在相邻细胞之间形成细胞质桥梁的蛋白质，是有毒物质从濒临死亡的细胞传递到邻近细胞导致死亡的管道。CNS的研究表明，事实上，GJS介导的进行性继发性细胞死亡可能是与许多损伤相关的细胞死亡的主要原因，包括缺血、兴奋毒性和创伤。与这些发现一致的是，我们在视网膜上的初步数据显示，阻断或切除GJS可以显著减少实验性青光眼中RGCs和无长突细胞(ACS)的丢失。因此，我们认为GJ介导的继发性细胞死亡是青光眼损伤的重要机制因素，导致大部分RGC和无长突细胞(AC)丢失，从而为神经保护治疗提供了新的靶点。为了验证这一假设，我们建议使用实验性青光眼的小鼠模型来直接证明，药物阻断GJS或基因消融其组成的连接蛋白亚基导致RGCs及其偶联的ACS的丢失显著减少。根据连接蛋白的组成，不同的GJ可以具有选择性的通透性，这表明GJ的组成可能决定了它是否促进了损伤后的细胞死亡。因此，我们将使用选择性连接蛋白被去除的基因敲除小鼠来确定哪些GJ队列支持青光眼细胞丢失，因此应该作为促进神经保护的靶点。最后，我们将进行电生理和行为学实验，以评估通过阻断GJS来增加青光眼患者RGC和AC的生存能力是否会导致视觉功能的保护。在这些实验中，我们将记录视网膜电信号(ERG)，并使用膜片钳和微电极阵列记录技术来评估视网膜功能。我们还将记录视觉诱发电位(VEP)和测量视动反应(OKR)来评估中枢视觉功能。这项研究的结果将阐明与青光眼相关的RGC变性的一个新的重要机制，并由此揭示神经保护治疗的新靶点。虽然拟议的工作将针对青光眼，但出现的治疗策略应该适用于中枢神经系统其他部分出现的神经退行性疾病的治疗。

 DESCRIPTION (provided by applicant): Glaucoma, the second leading cause of blindness worldwide, is a neurodegenerative disease often associated with elevated intraocular pressure (IOP) and characterized by the progressive loss of retinal ganglion cells (RGCs) leading to visual loss. Among the various types of cell death associated with neuropathologies, the major cellular pathways underlying apoptosis and necrosis have been well characterized. However, in addition to these intrinsic mechanisms underlying primary cell death, intercellular communication via gap junctions (GJs) appear to play a major, yet poorly understand, role in secondary or `bystander' cell death. GJs, proteins that form cytoplasmic bridges between neighboring cells, act as conduits by which toxic materials are passed from dying cells to their neighbors leading to their death. Studies in CNS suggest that progressive secondary cell death mediated by GJs may, in fact, account for the majority of cell death associated with a number of insults, including ischemia, excitotoxicity, and trauma. Consistent with these findings, our preliminary data in retina show that blockade or ablation of GJs can significantly reduce the loss of RGCs and amacrine cells (ACs) in experimental glaucoma. We therefore posit that GJ-mediated secondary cell death is a crucial mechanistic element of glaucomatous injury, resulting in the majority of RGC and amacrine cell (AC) loss, and thereby offers a novel target for neuroprotective treatment. To test this hypothesis, we propose to use mouse models of experimental glaucoma to show directly that pharmacological blockade of GJs or genetic ablation of their constituent connexin subunits results in a significant reduction in in the loss o RGCs and the ACs to which they are coupled. Different GJs, based on the composition of their connexins, can have selective permeabilities, suggesting that the makeup of a GJ may determine whether it promotes cell death following injury. We will therefore use knockout mice in which selective connexins are ablated to determine which GJ cohorts support cell loss in glaucoma and should thus be targeted to promote neuroprotection. Finally, we will carry out electrophysiological and behavioral experiments to assess whether increasing RGC and AC survivability in glaucoma by blocking GJs results in preservation of visual function. In these experiments, we will record the electroretinogram (ERG), and use patch-clamp and microelectrode array recording techniques to assess retinal function. We will also record visual evoked potentials (VEP) and measure the optokinetic response (OKR) to assess central visual function. The results of this study should elucidate a new and important mechanism of RGC degeneration associated with glaucoma and, in so doing, reveal a novel target for neuroprotective treatment. While the proposed work will be directed at glaucoma, the therapeutic strategies that emerge should be applicable to the treatment of neurodegenerative diseases seen in other parts of the CNS.