Gene Expression Regulatory Pathways and Retinal Ganglion Cell Neuroprotection

青光眼和外伤性及其他视神经病变中视网膜神经节细胞（RGC）的丧失导致 永久性部分或完全失明。可能对抗RGC死亡的分子机制仍然存在， 作为慢性视神经病变中RGC存活率的桥接研究和潜在高影响领域 具有很高的保留或恢复视力的潜力。多种信号转导途径已被牵连， RGC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存活并引发其下游 影响仍然未知。来自我们实验室的最新数据支持一种模型，其中信号体是由 核周支架蛋白肌肉A激酶激活蛋白α（mAKAPα/AKAP 6 α）介导的cAMP- 依赖性信号传导和增强神经保护性MAPK信号传导，导致Ets Like-1蛋白（Elk-1） 转录因子激活和RGC存活。识别这种细胞内cAMP信号室 与神经保护特异性相关提供了空间上不同的cAMP作用机制， 告知策略的设计，其提供比腺苷酰的总体cAMP升高更大的治疗特异性 环化酶激活剂或cAMP类似物。在这个应用程序中，我们提出了三个具体的目标来测试这个模型， 阐明cAMP和神经营养因子信号之间协同作用的机制， 神经保护具体目标1：定义神经保护基因表达。使用单细胞RNA 转录组测序（scRNA-seq），我们将研究在何种程度上相似的基因转录程序是 由不同的神经保护干预诱导，包括全身性与区室化cAMP 升高，确定单个RGC亚型是否优先受cAMP和神经营养因子调节， 因子信号传导，并确定其表达改变可能对神经保护至关重要的候选基因。 对治疗干预的反应。特定目的2：核周区室cAMP信号传导在 RGC神经保护。使用新的工具来促进或抑制特定细胞内的cAMP和Ca 2 + 我们将测试RGC mAKAPα信号体上的Ca 2 +-cAMP信号是否是唯一足够的 和/或视神经挤压后RGC神经保护所必需的。具体目标3：cAMP-之间的串扰 和神经营养因子依赖性RGC神经保护作用。为了检测cAMP和神经营养因子 通过共调节ERK 1/2依赖性Elk-1激活促进神经保护， 将对RGC中Elk-1的功能性进行视神经挤压，并比较它们对额外刺激的反应。 用外源性神经营养因子和AAV介导的mAKAPα信号传导隔室处理 增强总之，这些特定目的将提供分子的见解信号通路和 改变的基因表达可以在体内赋予RGC神经保护作用，同时为新的治疗提供概念验证。 预防RGC疾病视力丧失的策略。

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. Identifying 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 therapeutics 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 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.