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 产前酒精暴露对未成熟的中枢神经系统的影响代表着一种 毁灭性但可能被低估的公共健康危害，产生一系列乙醇诱导的 发育缺陷，包括对社会有长期影响的认知障碍。损坏 神经元还包括视网膜内的细胞，产生这些胎儿酒精光谱的视觉系统方面 精神障碍(FASD)。关于乙醇的具体机制，人们的认识仍然存在严重差距- 对神经元的介导性损伤，以及它们的内在脆弱性和神经保护功能，这可能是 以某种方式被监测、增强和/或利用，以拯救神经元免受损害。一项建议的蜂窝 酒精损伤的靶点是破坏关键蛋白质稳态通路的神经元，包括 泛素系统。我们的长期目标是发展对分子和机制的理解 造成光感受器神经元功能障碍和变性，并阐明潜在的救援策略。 目前提案的总体目标是确定泛素系统损害的贡献 乙醇对这些特定神经元的组织损伤，以及泛素系统操作的可行性 来挽救神经功能。我们提议的中心假设是，暴饮暴食的酒精暴露 破坏光感受器神经元的基本蛋白质周转机制，破坏正常细胞 蛋白质平衡。我们的假设是基于我们实验室的初步数据表明，类似狂欢的 接触乙醇会导致斑马鱼幼体视觉功能丧失，这是FASD的一个有利模型， 通过药物增强自噬和泛素蛋白酶体部分挽救功能 乙醇对感光细胞和感光神经元泛素系统的损伤 以及之前报道的乙醇对大脑泛素系统变化的证据。我们计划 要检验我们的中心假设，并通过完成以下各项来实现本提案的主要目标 具体目的：(1)确定泛素系统对视锥感光神经元的影响 在酒精损害暴露的情况下的功能和蛋白质平衡；以及(2)询问变化 酒精暴露后视锥感光细胞中泛素修饰蛋白的景观。这些研究 将涉及分子操作，包括泛素报告蛋白的细胞特异性表达- 视锥感光细胞特异性的系统损伤和CRISPR/Cas9基因破坏；生物化学； 视觉表现分析；ERG记录；以及光感受器特定泛素蛋白质组学。预期中的 这些研究的结果是加深了对早期饮酒之间关系的理解 暴露、神经元损伤和泛素系统，以及可能的 开发治疗方法以减轻酒精滥用造成的神经元损伤。