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)无法存活或再生其视神经轴突是永久性的基础 青光眼、创伤和其他眼病的视力残疾。在本应用程序中，我们建议研究 神经保护和轴突神经中新定义的核周cAMP第二信使隔室 再生。磷酸二酯酶PDE4D3与核膜相关的 支架蛋白mAKAPα。初步数据显示，神经元中mAKAPα处的cAMP信号受到严格调控 通过PDE4D3，使使用特定锚定干扰肽的PDE置换促进核周 视神经损伤后蛋白激酶A活性、体外突起伸展及体内RGC存活 模仿外源cAMP类似物的应用。MAKAPα上cAMP的作用机制 神经保护和轴突生长是这个项目的重点。例如，我们建议PDE4D3 作为mAKAPα信号小体上cAMP和ERK5通路之间串扰的支点。此外, α依赖的cAMP-PKA神经保护信号的潜在效应物是IIa类HDAC(HDAC) 4和5)通过与MEF2等转录因子结合，在染色质上组织共抑制物复合体。 PKA信号促进HDAC4/5在其他类型细胞中的核定位，我们认为mAKAPα和 依赖PKA的HDAC磷酸化导致促进RGC存活的神经元中HDAC核的定位 在视神经损伤之后。该项目的中心假设如下：核周cAMP信号在 PDE4D3调节的神经元α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背后的机制- MAKAPα信号体的依赖神经保护和轴突再生信号。使用核弹- 我们将通过FRET成像确定mAKAPα是否与PDE4D3结合 受神经营养素依赖的ERK5信号和其他上游信号的调控。IIa类人类发展援助中心的作用 将使用原代神经元培养来研究神经元的存活和轴突生长，以及HDAC 细胞内定位受cAMP调控，α信号小体将通过活细胞成像进行研究。目标 2)靶向核周cAMP信号作为RGC保护和视神经的治疗途径 再生。为了测试针对mAKAPα区的玻璃体内基因治疗是否与 神经营养因子治疗诱导ERK5信号转导，表达PDE4D3的腺相关病毒载体 无论有无视神经挤压手术前或术后玻璃体内注射锚定干扰素 同时注射脑源性神经营养因子。组织学和视觉功能分析将用于评估保存情况 和/或恢复RGC/视神经功能。测试增强的IIa类HDAC核本地化 给予神经保护，更多的小鼠将被表达突变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类似的研究。