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: 10723138
• 负责人: Jeffrey L Goldberg
• 金额: $ 11.79万
• 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 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10723138
• 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。多个信号转导通路已被牵连 在RGC神经保护中，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存活并引发它们的 下游影响仍不清楚。我们实验室的最新数据支持一种模型， 由核周支架蛋白肌肉A激酶激活蛋白α（mAKAPα/AKAP 6 α）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，导致Ets Like-1蛋白（Elk-1）转录因子激活和RGC存活。这种细胞内的鉴定 与神经保护特异性相关的cAMP信号传导区室提供了空间上不同的 cAMP的作用，并应告知策略的设计，提供治疗特异性大于全球 腺苷酸环化酶激活剂或cAMP类似物升高cAMP。在本申请中，我们提出了三个具体的 目的是验证该模型，并阐明cAMP和神经营养素之间协同作用的机制， 神经保护中的信号传导因子。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，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，并确定其表达改变可能至关重要的候选基因 对治疗干预的神经保护作用。具体目标2：核周蛋白的作用 RGC神经保护中的区室化cAMP信号传导。使用新的工具来促进或抑制cAMP， 我们将检测RGC mAKAPα中的Ca 2 +-cAMP信号传导是否与特定细胞内区室中的Ca 2+有关， 信号体是唯一足够的和/或必要的RGC神经保护后视神经挤压。具体 目的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共同调节ERK 1/2依赖的神经保护作用 对于Elk-1激活，将对RGC中Elk-1功能获得和丧失的小鼠进行视神经挤压， 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信号区室增强。这些具体目标将共同提供分子见解 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， 为预防RGC疾病中视力丧失的新策略提供概念验证。