Gene Expression Regulatory Pathways and Retinal Ganglion Cell Neuroprotection

青光眼、外伤性和其他视神经疾病中视网膜神经节细胞(RGC)的丢失导致 永久性的部分或完全失明。可能反对RGC死亡的分子机制仍然是 积极的研究领域和潜在的高影响，作为慢性视神经疾病中RGC生存的桥梁 有很高的保存或恢复视力的潜力。涉及多条信号转导途径 在RGC中的神经保护作用，包括cAMP和神经营养因子诱导的丝裂原活化蛋白激酶 (MAPK)信号通路。这些途径如何协同促进RGC的生存并引发其 下游影响尚不清楚。我们实验室的最新数据支持一个模型，在这个模型中，信号体 由核周支架蛋白肌肉A-激酶锚定蛋白α(mAKAPα/AKAP6α)组成 介导cAMP依赖的信号转导和增强神经保护性MAPK信号转导导致ETS Like-1蛋白(Elk-1)转录因子激活与RGC存活。这种胞内病毒的鉴定 与神经保护相关的cAMP信号间隔提供了一种空间上不同的机制 CAMP作用，并应为提供比全球更强的治疗特异性的策略的设计提供信息 用腺苷环化酶激活剂或cAMP类似物升高cAMP。在本申请中，我们提出了三个具体的 目的是验证这一模型，并阐明cAMP和神经营养之间协同作用的机制。 神经保护中的信号因素。具体目标1：定义神经保护性基因的表达。使用单一的- 细胞RNA转录组测序(scRNA-seq)，我们将研究相似基因转录到什么程度 程序是由不同的神经保护干预措施引起的，包括全身性与 划分营地高度，确定个别RGC亚型是否优先受 CAMP和神经营养因子信号转导，并确定其表达改变可能是关键的候选基因 作为对治疗干预的回应的神经保护。具体目标2：核周的作用 区域cAMP信号在RGC神经保护中的作用。使用新工具促进或抑制cAMP和 在特殊的细胞内隔室，我们将测试在RGC mAKAPα处的cAMP信号是否 信号小体对于视神经挤压后RGC的神经保护是唯一充分和/或必要的。特定的 目的3：cAMP和神经营养因子依赖的RGC神经保护之间的串扰。为了测试 CAMP和神经营养因子是否通过共同调节依赖的ERK1/2促进神经保护 ELK-1激活，视网膜神经节细胞中ELK-1功能增强和丧失的小鼠将受到视神经挤压和 比较它们对外源性神经营养因子和AAV介导的额外治疗的反应 MAKAPα信号室增强。总而言之，这些具体目标将提供分子洞察力 进入信号通路和改变的基因表达，可以在体内提供RGC神经保护，而 为预防RGC病视力丧失的新策略提供概念验证。

Loss of retinal ganglion cells (RGCs) in glaucoma and traumatic and other optic neuropathies results in permanent partial or complete blindness. Molecular mechanisms that may oppose this RGC death remain an area of active investigation and potential high impact, as bridging RGC survival in chronic optic neuropathies has high potential to preserve or restore vision. Multiple signal transduction pathways have been implicated in RGC neuroprotection, including cAMP and neurotrophic factor-induced mitogen-activated protein kinase (MAPK) signaling pathways. How these pathways synergistically promote RGC survival and elicit their downstream effects remains unknown. Recent data from our labs support a model in which signalosomes organized by the perinuclear scaffold protein muscle A-Kinase Anchoring Protein α (mAKAPα/AKAP6α) mediate cAMP-dependent signaling and potentiate neuroprotective MAPK signaling, resulting in Ets Like-1 protein (Elk-1) transcription factor activation and RGC survival. The identification of this intracellular cAMP signaling compartment specifically relevant to neuroprotection provides a mechanism for spatially distinct cAMP action and should inform the design of strategies providing therapeutic specificity greater than global cAMP elevation with adenylyl cyclase activators or cAMP analogs. In this application, we propose three Specific Aims to test this model and to elucidate the mechanism conferring the synergy between cAMP and neurotrophic factor signaling in neuroprotection. Specific Aim 1: Defining Neuroprotective Gene Expression. Using single- cell RNA transcriptome sequencing (scRNA-seq), we will study to what degree similar gene transcription programs are induced by different neuroprotective interventions, including generalized versus compartmentalized cAMP elevation, determine whether individual RGC subtypes are preferentially regulated by cAMP and neurotrophic factor signaling, and identify gene candidates whose altered expression may be critical for neuroprotection in response to therapeutic intervention. Specific Aim 2: Role of Perinuclear Compartmented cAMP Signaling in RGC Neuroprotection. Using new tools to promote or inhibit cAMP and Ca2+ in special intracellular compartments, we will test whether Ca2+-cAMP signaling at RGC mAKAPα signalosomes is uniquely sufficient and/or necessary for RGC neuroprotection after optic nerve crush. Specific Aim 3: Crosstalk Between cAMP- and Neurotrophic Factor-Dependent RGC Neuroprotection. To test whether cAMP and neurotrophic factors promote neuroprotection through co-regulation of ERK1/2-dependent Elk-1 activation, mice with gain- and loss-of-function for Elk-1 in RGCs will be subjected to optic nerve crush and compared for their response to additional treatment with exogenous neurotrophic factors and AAV-mediated mAKAPα signaling compartment enhancement. Together, these Specific Aims will provide molecular insights into the signaling pathways and the altered gene expression that can confer RGC neuroprotection in vivo, while providing proof-of-concept for new strategies to prevent loss of vision in RGC disease.