Regulation of Retinal Cell Death in Diabetes

 描述（由申请人提供）：在理解糖尿病如何影响提供视力的视网膜神经元方面存在根本性的差距。正因为如此，目前的治疗方法干预疾病的后期阶段，当血管受到影响时。抗VEGF治疗限制了晚期糖尿病视网膜病变（DR）的损害，但不能解决对神经感觉视网膜的损害。因此，该项目的长期目标是确定导致DR患者视网膜神经节细胞（RGC）功能障碍的机制。总体目标是确定雷帕霉素复合物2（mTORC 2）信号传导的机制靶标在高血糖和蛋白质合成之间的相互作用中的作用，导致DR患者RGC功能障碍和随后的视力丧失。该项目将利用糖尿病大鼠和小鼠，其是DR的标准临床前模型，并且表现出人DR的早期神经退行性变化。中心假设是糖尿病诱导的mTORC 2信号传导缺陷损害视网膜神经节细胞的蛋白质合成、轴突功能和存活。这项工作的基本原理是，确定导致RGC功能障碍和死亡的途径将最终导致糖尿病患者的预防性治疗和更好的视力。该假设得到了强有力的初步数据的支持，这些数据显示：1）RGC中显著的mTOR和Rictor表达; 2）糖尿病人供体眼中mTOR表达降低;和3）成年小鼠中Rictor表达的条件性敲除能力。该假设将在两个特定目标中进行测试：1）确定糖尿病损害视网膜mTORC 2活性的机制以及糖尿病对蛋白质合成的细胞特异性影响，以及2）确定mTORC 2活性受损对视网膜神经节细胞蛋白质合成、存活和形态的影响。第一个目标将检查糖尿病对人类视网膜中mTORC 2复合物成员蛋白表达的影响，使用糖尿病啮齿动物模型来确定糖尿病降低视网膜mTORC 2活性和蛋白合成的分子机制，并检查这些影响的细胞特异性。第二个目标将采用体内空间和细胞特异性靶点功能丧失研究，通过敲除视网膜内层神经元中的mTOR相关蛋白Rictor和Raptor来破坏mTORC复合物，并确定对蛋白质周转、轴突功能和细胞存活的影响。该提议是创新性的，因为它：1）扩展了DR中mTORC 2活性降低的新观察，并研究了神经视网膜中mTOR功能的未探索区域; 2）将是第一个研究导致DR中神经元完整性丧失的视网膜mTORC复合物和蛋白质合成中的细胞特异性改变的人;和3）将使用创新技术，包括翻译组学来定义视网膜mRNA翻译中的细胞特异性变化，以及靶向AAV-cre重组酶载体来删除RGC中的Rictor、Raptor和蛋白磷酸酶PP 2A基因。这项工作意义重大，因为它将阐明临床相关的方法，以恢复负责视网膜疾病中RGC功能丧失的缺陷信号通路，这些疾病对视力具有普遍重要性，并对NEI大胆目标倡议具有影响。

 DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how diabetes impacts the retinal neurons that provide vision. Because of this, current therapies intervene in latter stages of the disease, when blood vessels are affected. Anti-VEGF therapy limits damage in advanced diabetic retinopathy (DR), but does not address damage to the neurosensory retina. Hence, the long-term goal of this project is to define the mechanisms that cause retinal ganglion cell (RGC) dysfunction in patients with DR. The overall objective is to define the roles of mechanistic target of rapamycin complex 2 (mTORC2) signaling in the interactions between hyperglycemia and protein synthesis that lead to RGC dysfunction and subsequent vision loss in DR. The project will utilize diabetic rats and mice, which are the standard pre-clinical models of DR, and which exhibit the early neurodegenerative changes of human DR. The central hypothesis is that diabetes-induced defects in mTORC2 signaling impair protein synthesis, axonal function and survival of retinal ganglion cells. The rationale for this work is that identifying pathways causing RGC dysfunction and death will ultimately lead to preventive treatments and better vision for persons with diabetes. The hypothesis is supported by strong preliminary data showing: 1) prominent mTOR and Rictor expression in RGCs; 2) reduced mTOR expression in diabetic human donor eyes; and 3) the ability to conditionally knockout of Rictor expression in adult mice. The hypothesis will be tested in two specific aims: 1) to define the mechanisms by which diabetes impairs retinal mTORC2 activity and the cell-specific effects of diabetes on protein synthesis, and 2) to define the consequences of impaired mTORC2 activity on retinal ganglion cell protein synthesis, survival and morphology. The first aim will examine the effect of diabetes on mTORC2 complex member protein expression in human retinas, use diabetic rodent models to determine the molecular mechanisms by which diabetes reduces retinal mTORC2 activity and protein synthesis, and examine the cell-specificity of these effects. The second aim will employ in vivo spatial and cell- specific targete loss-of-function studies to disrupt mTORC complexes by knockout of the mTOR-associated proteins Rictor and Raptor in inner retinal neurons and determine the effects on protein turnover, axonal function and cell survival. The proposal is innovative because it: 1) expands the novel observation of diminished mTORC2 activity in DR and investigates the unexplored area of mTOR function in the neural retina; 2) will be the first to examine the cell-specific alterations i retinal mTORC complexes and protein synthesis leading to loss of neuronal integrity in DR; and 3) will use innovative techniques, including translatomics to define cell-specific changes in retinal mRNA translation, and targeted AAV-cre recombinase vectors to delete Rictor, Raptor and protein phosphatase PP2A genes in RGC. The work is significant because it will elucidate clinically relevant means to restore defective signaling pathways responsible for loss of RGC function in retinal diseases that have universal importance to vision and implications for the NEI Audacious Goals Initiatives.