Gene Expression Regulatory Pathways and Retinal Ganglion Cell Neuroprotection

基因表达调控途径与视网膜神经节细胞神经保护

• 批准号: 10530683
• 负责人: Jeffrey L Goldberg
• 金额: $ 50.14万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10530683
• 关键词: A kinase anchoring protein; Adenylate Cyclase; Area; Biological Assay; Blindness; Brain-Derived Neurotrophic Factor; Candidate Disease Gene; Catalytic Domain; Cell Death; Cell Survival; Cell membrane; Cells; Cessation of life; Chimeric Proteins; Chronic; Complete Blindness; Crush Injury; Cyclic AMP; Cytosol; Data; Dependovirus; Disease; Eye diseases; Gene Expression; Gene Transduction Agent; Generations; Genes; Genetic Transcription; Glaucoma; Individual; Injury; Intervention; Investigation; Ischemic Optic Neuropathy; MAPK3 gene; Mediating; Mediator; Mitogen-Activated Protein Kinases; Modeling; Molecular; Morbidity - disease rate; Mus; Muscle Proteins; Natural regeneration; Nerve Crush; Nuclear; Nuclear Envelope; Nuclear Matrix-Associated Proteins; Optic Nerve; Optic Nerve Injuries; PDE4D3; Pathway interactions; Predisposition; Proteins; RNA; Regimen; Regulation; Regulatory Pathway; Retinal Ganglion Cells; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Specificity; Testing; Therapeutic; Therapeutic Intervention; analog; axon regeneration; cell regeneration; cellular resilience; design; experimental study; gain of function; in vivo; insight; loss of function; nerve damage; neuroprotection; neurotrophic factor; optic nerve disorder; phosphoric diester hydrolase; preservation; prevent; programs; protein activation; protein expression; regenerative; response; sight restoration; single-cell RNA sequencing; synergism; tool; transcription factor; transcriptome sequencing

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.

青光眼、外伤及其他视神经病变导致视网膜神经节细胞 (RGC) 损失 永久性部分或完全失明。可能阻止 RGC 死亡的分子机制仍然是一个未知数 积极研究和潜在高影响的领域，作为慢性视神经病变中 RGC 生存的桥梁 具有保留或恢复视力的巨大潜力。涉及多种信号转导途径 RGC 神经保护，包括 cAMP 和神经营养因子诱导的丝裂原激活蛋白激酶 (MAPK) 信号通路。这些途径如何协同促进 RGC 存活并引发其 下游影响仍未知。我们实验室的最新数据支持一个模型，其中信号体 由核周支架蛋白肌肉 A 激酶锚定蛋白 α (mAKAPα/AKAP6α) 组织 介导 cAMP 依赖性信号传导并增强神经保护性 MAPK 信号传导，从而导致 Ets Like-1 蛋白 (Elk-1) 转录因子激活和 RGC 存活。该细胞内的鉴定 与神经保护特别相关的 cAMP 信号室提供了一种空间上不同的机制 cAMP 的作用，并应为策略的设计提供信息，提供比全球更大的治疗特异性 使用腺苷酸环化酶激活剂或 cAMP 类似物可升高 cAMP。在此应用中，我们提出了三个具体的 旨在测试该模型并阐明 cAMP 与神经营养细胞之间协同作用的机制 神经保护中的因子信号传导。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诱导的，包括广义与 区室化 cAMP 升高，确定单个 RGC 亚型是否优先受 cAMP 和神经营养因子信号传导，并识别其表达改变可能至关重要的候选基因 用于响应治疗干预的神经保护。具体目标 2：核周的作用 RGC 神经保护中的区室 cAMP 信号传导。使用新工具促进或抑制 cAMP 和 Ca2+ 在特殊的细胞内区室中，我们将测试 RGC mAKAPα 上的 Ca2+-cAMP 信号是否存在 信号小体对于视神经挤压后 RGC 神经保护来说是充分和/或必需的。具体的 目标 3：cAMP 和神经营养因子依赖性 RGC 神经保护之间的串扰。测试 cAMP 和神经营养因子是否通过 ERK1/2 依赖性共同调节来促进神经保护 Elk-1 激活，RGC 中 Elk-1 功能获得和丧失的小鼠将遭受视神经挤压和 比较他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。这些具体目标共同将提供分子见解 进入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 为预防 RGC 疾病导致视力丧失的新策略提供概念验证。