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）无法生存或再生其视神经轴突永久性 青光眼，创伤和其他眼部疾病的视觉障碍。在此应用中，我们建议研究 一个新定义的神经保护和轴突神经的核周营第二信使室 再生。磷酸二酯酶PDE4D3特别与核包膜相关 支架蛋白质makapα。初步数据表明，神经元中MAKAPα的CAMP信号受到严格调节 通过PDE4D3，可以使用特定的锚固破坏者肽促进PDE的位移会促进核周 蛋白激酶A活性和神经蛋白神经蛋白在体外和RGC生存中的体内生存后，视神经挤压后， 模仿外源营地类似物的应用。 Makapα营地如何贡献的机制 神经保护和神经侵蚀是该项目的重点。例如，我们建议PDE4D3 作为Makapα信号体的CAMP和ERK5途径之间的串扰的支点。此外， MAKAPα依赖性CAMP-PKA神经保护信号的潜在影响是IIA类HDAC（HDACS） 4和5）通过结合转录因子（例如MEF2）在染色质上组织共抑制复合物。 PKA信号传导促进了其他细胞类型中的HDAC4/5核定位，我们建议Makapα和 依赖PKA的HDAC磷酸化导致促进RGC存活的神经元中的HDAC核定位 遵循视神经损伤。该项目的中心假设如下： 神经元中PDE4D3调节的MAKAPα信号体促进IIA类HDAC核定位，增强 视神经损伤后的RGC存活和轴突再生。目标1）营地的机制 - MAKAPα信号体的依赖性神经保护和轴突再生信号传导。使用核 信封局部PKA活性报道，我们将通过fret成像确定makapα结合的PDE4D3是否 受神经营养蛋白依赖性ERK5信号传导和其他上游信号的调节。 IIA级HDACS的角色 在神经元生存和轴突生长中，将使用原发性神经元培养物进行研究，以及HDAC是否是否 细胞内定位由MAKAPα信号体的CAMP调节，将通过活细胞成像研究。目的 2）将核周营信号作为RGC保护和视神经的治疗方法的靶向 再生。测试玻璃室内基因治疗是否针对MAKAPα室是否与 神经营养疗法影响ERK5信号传导，表达PDE4D3的腺相关病毒载体 在视神经挤压手术之前或之后，锚定干扰肽将在有或没有的情况下注射玻璃体 同时注射BDNF。组织学和视力功能测定将用于评估保存 和/或恢复RGC/神经神经功能。测试是否增强了IIA类HDAC核定位 赋予神经保护作用，其他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。