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。多条信号转导通路被认为与 RGC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。这些通路如何协同促进RGC存活并引发其下游 其影响尚不清楚。我们实验室的最新数据支持这样一种模型，即信号小体由 核周支架蛋白肌肉A-激酶锚定蛋白α(mAKAPα/AKAP6α)介导cAMP 依赖的信号和增强神经保护性MAPK信号，导致Ets like-1蛋白(ELK-1) 转录因子激活与RGC存活。识别这个细胞内cAMP信号隔间 特别是与神经保护相关的为空间上不同的cAMP作用提供了一种机制，应该 告知提供比腺苷全球cAMP升高更强的治疗特异性的策略的设计 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目标来测试该模型和 阐明cAMP和神经营养因子信号转导协同作用的机制 神经保护。具体目标1：定义神经保护性基因的表达。使用单细胞RNA 转录组测序(scRNA-seq)，我们将研究相似基因转录程序的程度 由不同的神经保护干预措施诱导，包括全身性cAMP和分区cAMP 升高，确定个别RGC亚型是否优先受cAMP和神经营养调节 因子信号转导，并确定其表达变化可能对神经保护至关重要的候选基因 对治疗干预的反应。特异性目标2：核周间隔化cAMP信号转导通路 RGC神经保护。利用新工具促进或抑制特殊细胞内cAMP和钙离子 ，我们将测试RGC mAKAPα信号体上的钙-cAMP信号是否唯一足够 和/或视神经挤压后RGC神经保护所必需的。具体目标3：阵营之间的相声- 和神经营养因子依赖的RGC神经保护。检测cAMP和神经营养因子 通过共同调节ERK1/2依赖的ELK-1激活促进神经保护，小鼠有得和失- 视网膜神经节细胞中ELK-1的功能缺失将受到视神经挤压的影响，并比较它们对其他 外源性神经营养因子和腺相关病毒介导的mAKAPα信号室治疗 增强功能。总之，这些特定的目标将提供对信号通路和 基因表达的改变可以在体内提供RGC神经保护，同时为新的 预防RGC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.