Ubiquitin-system manipulations to probe and mitigate ethanol damage in neurons

泛素系统操作可探测和减轻神经元中的乙醇损伤

• 批准号: 10357893
• 负责人: MICHAEL D. VARNUM
• 金额: $ 21.69万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-25 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10357893
• 关键词: Address; Affect; Alcohol abuse; Alcohols; Autophagocytosis; Biochemical; Biochemistry; Biological; Biological Assay; Blindness; Brain; CRISPR/Cas technology; Candidate Disease Gene; Cells; Child; Data; Defect; Degradation Pathway; Development; Disease; Disease model; Ethanol; Ethanol dependence; Fetal Alcohol Exposure; Fetal Alcohol Spectrum Disorder; Foundations; Functional disorder; Genes; Goals; Health; Health Hazards; Homeostasis; Human; Impaired cognition; Impairment; Knowledge; Link; Measurement; Mediating; Metabolic; Modeling; Molecular; Monitor; Morphology; Nerve Degeneration; Neuraxis; Neurologic; Neurons; Normal Cell; Outcome; Outcome Study; Output; Pathogenicity; Pathway interactions; Pattern; Pharmacology; Photoreceptors; Population; Predisposition; Prevalence; Proteins; Proteomics; Public Health; Reporter; Reporting; Retina; Retinal Cone; Retinal Photoreceptors; Signal Transduction; Societies; System; Testing; Therapeutic Intervention; Tissues; Transgenic Organisms; Ubiquitin; Ubiquitination; Vision; Visual system structure; Work; Zebrafish; alcohol effect; alcohol exposure; base; cellular targeting; gene product; his6 tag; innovation; insight; molecular targeted therapies; multicatalytic endopeptidase complex; overexpression; pressure; protein degradation; protein folding; proteostasis; response; retinal damage; retinal neuron; therapeutic development; visual performance

PROJECT SUMMARY/ABSTRACT The consequences of prenatal alcohol exposure for the immature central nervous system represents a devastating but likely underestimated public health hazard, producing a range of ethanol-induced developmental defects including cognitive impairments having long-term impacts on society. Damage to neurons also includes cells within the retina, producing visual system facets of these fetal alcohol spectrum disorders (FASD). Critical gaps in knowledge remain regarding the specific mechanisms underlying ethanol- mediated damage to neurons, and their intrinsic vulnerabilities and neuroprotective features, which might be monitored, enhanced and/or exploited in some way to rescue neurons from damage. One proposed cellular target for ethanol damage is disruption in neurons of critical protein homeostasis pathways including the ubiquitin system. Our long-term goal is develop an understanding of the molecules and mechanisms producing photoreceptor neuron dysfunction and degeneration, and to elucidate potential rescue strategies. The overall objective of the current proposal is to determine the contribution of ubiquitin-system impairment to tissue damage by ethanol in these specific neurons, and the feasibility of ubiquitin-system manipulations to rescue neuronal function. The central hypothesis of our proposal is that binge-like ethanol exposure disrupts fundamental protein turnover mechanisms in photoreceptor neurons, undermining normal cell proteostasis. Our hypothesis is based on preliminary data from our lab demonstrating that binge-like exposure to ethanol produces loss of visual function in larval zebrafish, an advantageous model for FASD, with partial rescue of function via pharmacological enhancement of autophagy and the ubiquitin-proteasome system; ubiquitin-system impairment in photoreceptor-derived cells and photoreceptor neurons after ethanol exposure; and previously reported evidence for ubiquitin-system changes in the brain with ethanol. We plan to test our central hypothesis and accomplish the main objective of this proposal by completing the following specific aims: (1) determine the effects on ubiquitin-system manipulations on cone photoreceptor neuron function and proteostasis in the context of damaging exposure to alcohol; and (2) interrogate the changing landscape of ubiquitin-modified proteins in cone photoreceptors following ethanol exposure. These studies will involve molecular manipulations, including cell-specific expression of reporter proteins for ubiquitin- system impairment and CRISPR/Cas9 gene disruption specifically in cone photoreceptors; biochemistry; visual performance assays; ERG recordings; and photoreceptor-specific ubiquitin proteomics. The expected outcomes of these studies are an enhanced understanding of the relationship between early alcohol exposure, neuronal damage, and the ubiquitin system, and a mechanistic foundation for the possible development of therapeutic approaches to mitigate neuronal damage caused by alcohol abuse.

项目摘要/摘要 未成熟中枢神经系统的产前酒精暴露的后果代表 毁灭性但可能低估的公共卫生危害，产生了一系列乙醇引起的 发展缺陷，包括认知障碍对社会产生长期影响。损坏 神经元还包括视网膜内的细胞，产生这些胎儿酒精谱的视觉系统方面 疾病（FASD）。关于乙醇的基本机制的知识差距仍然存在 对神经元的介导损害及其内在脆弱性和神经保护特征，这可能是 以某种方式受到监测，增强和/或开发，以挽救神经元免受损害。一个提出的细胞 乙醇损伤的靶标是关键蛋白稳态途径神经元的中断 泛素系统。我们的长期目标是对分子和机制发展了解 产生光感受器神经元功能障碍和变性，并阐明潜在的救援策略。 当前建议的总体目标是确定泛素系统损害的贡献 在这些特定神经元中乙醇的组织损伤，以及泛素系统操纵的可行性 挽救神经元功能。我们提案的中心假设是暴饮暴食的乙醇暴露 破坏光感受器神经元中的基本蛋白质周转机制，破坏正常细胞 蛋白毒酸。我们的假设基于实验室的初步数据，证明了暴饮暴食样 暴露于乙醇会导致幼虫斑马鱼的视觉功能丧失，斑马鱼是FASD的有利模型， 通过促进自噬和泛素 - 蛋白酶体的药理学增强功能的部分营救 系统;乙醇后，光感受器衍生的细胞和光感受器神经元中的泛素系统损伤 接触;并先前报道了用乙醇在大脑中变化的泛素系统变化的证据。我们计划 测试我们的中心假设并完成以下内容的主要目标 具体目的：（1）确定对泛素系统操纵对锥形感光器神经元的影响 在破坏酒精暴露的情况下的功能和蛋白质症； （2）审问变化 乙醇暴露后锥形感受器中泛素改性蛋白的景观。这些研究 将涉及分子操作，包括报告蛋白的细胞特异性表达 - 泛素 - 系统障碍和CRISPR/CAS9基因破坏专门在锥形感受器中；生物化学； 视觉性能测定； ERG记录；和感光特异性泛素蛋白质组学。预期 这些研究的结果是对早期酒精之间关系的增强的理解 暴露，神经元损伤和泛素系统，以及可能的机械基础 开发治疗方法来减轻酗酒造成的神经元损害。