VRC: The Role of Perinuclear cAMP in Retinal Ganglion Cell Neuroprotection and Optic Nerve Regeneration

VRC：核周 cAMP 在视网膜神经节细胞神经保护和视神经再生中的作用

• 批准号: 10220042
• 负责人: Michael Seth Kapiloff
• 金额: $ 38.52万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10220042
• 关键词: Axon; Behavior; Binding; Biological; Biological Assay; Brain-Derived Neurotrophic Factor; Cell Survival; Cholera Toxin; Chromatin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoprotection; Data; Disease; Electrophysiology (science); Electroretinography; Evoked Potentials; Eye diseases; Failure; Fluorescence Resonance Energy Transfer; Glaucoma; HDAC4 gene; HDAC5 gene; Histology; Histone Deacetylase; Image; In Vitro; Injections; Injury; Ischemic Optic Neuropathy; MAPK7 gene; Morbidity - disease rate; Mus; Natural regeneration; Nerve Crush; Nerve Regeneration; Neurites; Neurons; Nuclear; Nuclear Envelope; Operative Surgical Procedures; Optic Nerve; Optic Nerve Injuries; PDE4D3; PDE4D3 phosphodiesterase; Pathway interactions; Pattern; Peptides; Phosphorylation; Regimen; Reporter; Repressor Proteins; Retina; Retinal Ganglion Cells; Role; Scaffolding Protein; Second Messenger Systems; Signal Transduction; Stains; Testing; Therapeutic; Trauma; Vision; Visual; adeno-associated viral vector; analog; axon growth; axon regeneration; cell type; disability; gene therapy; genetic corepressor; in vivo; insight; live cell imaging; mutant; nerve damage; neuroprotection; neurotrophic factor; novel; novel therapeutic intervention; optic nerve regeneration; preservation; regenerative; response; restoration; sight restoration; targeted treatment; transcription factor

Failure of retinal ganglion cells (RGCs) to survive or regenerate their optic nerve axons underlies permanent visual disability in glaucoma, trauma, and other eye diseases. In this application we propose to study the role of a newly defined perinuclear cAMP second messenger compartment in neuroprotection and axon nerve regeneration. The phosphodiesterase PDE4D3 is specifically associated with the nuclear envelope-associated scaffold protein mAKAPα. Preliminary data show that cAMP signaling at mAKAPα in neurons is tightly regulated by PDE4D3 such that displacement of the PDE using a specific anchoring disruptor peptide promotes perinuclear protein kinase A activity and neurite extension in vitro and RGC survival in vivo following optic nerve crush, mimicking the application of exogenous cAMP analog. The mechanisms how cAMP at mAKAPα contributes to neuroprotection and neurite outgrowth are the focus of this project. For example, we propose that PDE4D3 serves as the fulcrum for crosstalk between cAMP and ERK5 pathways at mAKAPα signalosomes. In addition, potential effectors for mAKAPα-dependent cAMP-PKA neuroprotective signaling are class IIa HDACs (HDACs 4 and 5) that organize co-repressor complexes on chromatin via binding to transcription factors such as MEF2. PKA signaling promotes HDAC4/5 nuclear localization in other cell types, and we propose that mAKAPα and PKA-dependent HDAC phosphorylation results in HDAC nuclear localization in neurons promoting RGC survival following optic nerve injury. The central hypothesis of this project is as follows: Perinuclear cAMP signaling at PDE4D3-regulated mAKAPα signalosomes in neurons promotes class IIa HDAC nuclear localization, enhancing RGC survival and axon regeneration following optic nerve injury. Aim 1) Mechanisms underlying cAMP- dependent neuroprotective and axon regenerative signaling at mAKAPα signalosomes. Using a nuclear- envelope localized PKA activity reporter, we will determine by FRET imaging whether mAKAPα-bound PDE4D3 is regulated by neurotrophin-dependent ERK5 signaling and other upstream signals. The role of class IIa HDACs in neuron survival and axon growth will be studied using primary neuronal cultures, and whether HDAC intracellular localization is regulated by cAMP at mAKAPα signalosomes will be studied by live cell imaging. Aim 2) Targeting of perinuclear cAMP signaling as a therapeutic approach for RGC protection and optic nerve regeneration. To test whether intravitreal gene therapy targeting the mAKAPα compartment is synergistic with neurotrophin therapy that induces ERK5 signaling, adeno-associated virus vectors expressing a PDE4D3 anchoring disruptor peptide will be injected intravitreally before or after optic nerve crush surgery with or without simultaneous injection of BDNF. Histology and vision functional assays will be used to assess preservation and/or restoration of RGC/optic nerve function. To test whether enhanced class IIa HDAC nuclear localization confers neuroprotection, additional mice will be treated with adeno-associated virus vectors expressing mutant HDAC proteins and similarly studied for their role in optic nerve injury.

视网膜神经节细胞（RGC）无法存活或再生其视神经轴突是永久性的基础 青光眼、外伤和其他眼部疾病导致的视力障碍。在此应用中，我们建议研究 新定义的核周 cAMP 第二信使室在神经保护和轴突神经中的作用 再生。磷酸二酯酶 PDE4D3 与核膜相关 支架蛋白mAKAPα。初步数据表明神经元中 mAKAPα 的 cAMP 信号受到严格调控 通过 PDE4D3，使用特定的锚定破坏肽置换 PDE 会促进核周 视神经挤压后体外蛋白激酶 A 活性和神经突延伸以及体内 RGC 存活， 模仿外源cAMP类似物的应用。 mAKAPα 上的 cAMP 的作用机制 神经保护和神经突生长是该项目的重点。例如，我们建议 PDE4D3 作为 mAKAPα 信号体上 cAMP 和 ERK5 通路之间串扰的支点。此外， mAKAPα 依赖性 cAMP-PKA 神经保护信号传导的潜在效应器是 IIa 类 HDAC（HDAC） 4 和 5）通过与 MEF2 等转录因子结合在染色质上组织共阻遏物复合物。 PKA 信号传导促进其他细胞类型中的 HDAC4/5 核定位，我们提出 mAKAPα 和 PKA 依赖性 HDAC 磷酸化导致神经元中 HDAC 核定位，促进 RGC 存活 视神经损伤后。该项目的中心假设如下：核周 cAMP 信号传导 神经元中 PDE4D3 调节的 mAKAPα 信号体促进 IIa 类 HDAC 核定位，增强 视神经损伤后 RGC 存活和轴突再生。目标 1) cAMP 的潜在机制- mAKAPα 信号体上依赖的神经保护和轴突再生信号传导。使用核- 包膜局部 PKA 活性报告基因，我们将通过 FRET 成像确定 mAKAPα 是否结合 PDE4D3 受神经营养蛋白依赖性 ERK5 信号和其他上游信号的调节。 IIa 类 HDAC 的作用 将使用原代神经元培养物来研究对神经元存活和轴突生长的影响，以及 HDAC 是否 细胞内定位由 mAKAPα 信号体上的 cAMP 调节，将通过活细胞成像进行研究。目的 2) 靶向核周 cAMP 信号作为 RGC 保护和视神经的治疗方法 再生。测试针对 mAKAPα 区室的玻璃体内基因治疗是否与 诱导 ERK5 信号转导的神经营养素治疗、表达 PDE4D3 的腺相关病毒载体 锚定破坏肽将在视神经挤压手术之前或之后注射到玻璃体内，无论是否有 同时注射BDNF。组织学和视觉功能测定将用于评估保存情况 和/或 RGC/视神经功能的恢复。测试是否增强IIa类HDAC核定位 赋予神经保护作用，额外的小鼠将接受表达突变体的腺相关病毒载体治疗 HDAC 蛋白并对其在视神经损伤中的作用进行了类似的研究。