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: 9913728
• 负责人: Michael Seth Kapiloff
• 金额: $ 25.52万
• 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 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9913728
• 关键词: Axon; Behavior; Binding; Biological; Biological Assay; Brain-Derived Neurotrophic Factor; Cell Survival; Cholera Toxin; Chromatin; Complex; 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; 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; in vivo; insight; live cell imaging; mutant; neuroprotection; neurotrophic factor; novel; novel therapeutic intervention; optic nerve regeneration; preservation; regenerative; response; 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）不能存活或再生其视神经轴突是永久性视网膜病变的基础。 青光眼、外伤和其他眼病造成的视力残疾。在本申请中，我们建议研究 一个新定义的核周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PDE 4D 3特异性地与核分裂相关的 支架蛋白mAKAPα。初步数据显示，在神经元中，mAKAPα的cAMP信号受到严格调节， 通过PDE 4D 3，使得使用特异性锚定破坏肽置换PDE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， 模拟外源性cAMP类似物的应用。mAKAPα的cAMP作用机制 神经保护和神经突生长是该项目的重点。例如，我们提出PDE 4D 3 作为mAKAPα信号体上cAMP和ERK 5通路之间串扰的支点。此外，本发明还提供了一种方法， mAKAPα依赖性cAMP-PKA神经保护信号的潜在效应物是IIa类HDAC（HDAC 4和5），其通过结合转录因子如MEF 2在染色质上组织共阻遏物复合物。 PKA信号促进HDAC 4/5在其他细胞类型中的核定位，我们认为mAKAPα和 PKA依赖的HDAC磷酸化导致HDAC在神经元中的核定位促进RGC存活 视神经损伤后。该项目的中心假设如下： 神经元中PDE 4D 3调节的mAKAPα信号体促进IIa类HDAC核定位， 视神经损伤后RGC存活和轴突再生。目的1）cAMP的潜在机制 依赖性神经保护和轴突再生信号在mAKAPα信号体。使用核武器- 包膜定位PKA活性报告基因，我们将通过FRET成像确定mAKAPα结合的PDE 4D 3 受神经营养因子依赖性ERK 5信号传导和其他上游信号调节。IIa类HDAC的作用 将使用原代神经元培养物研究HDAC对神经元存活和轴突生长的影响， 将通过活细胞成像研究mAKAPα信号体上cAMP对细胞内定位的调节。目的 2)靶向核周cAMP信号传导作为RGC保护和视神经的治疗方法 再生为了测试靶向mAKAPα区室的玻璃体内基因治疗是否与 诱导ERK 5信号传导的神经营养因子治疗，表达PDE 4D 3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玻璃体内注射， 同时注射BDNF。组织学和视觉功能测定将用于评估保存 和/或RGC/视神经功能的恢复。为了测试增强的IIa类HDAC核定位是否 赋予神经保护作用，另外的小鼠将用表达突变体的腺相关病毒载体治疗。 HDAC蛋白和类似地研究了它们在视神经损伤中的作用。