Metabolic Mechanisms of Copper-Dependent Neurodegeneration and Excitability in Menkes Disease

门克斯病铜依赖性神经变性和兴奋性的代谢机制

• 批准号: 10462355
• 负责人: Alicia R Lane
• 金额: $ 4.68万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-06 至 2025-04-05
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10462355
• 关键词: Acute; Affect; Animal Model; Binding; Bioenergetics; Biological; Birth; Brain; Cell Death; Cells; Copper; Data; Disease; Engineering; Epilepsy; Equilibrium; Exhibits; Gene Expression; Genes; Genetic Diseases; Genetic Models; Genetic Transcription; Glycolysis; Goals; Homeostasis; Human; Hypoxia; Infant; Intake; Intellectual functioning disability; Knock-out; Knowledge; Lead; Link; Menkes Kinky Hair Syndrome; Metabolic; Metabolic Pathway; Metabolism; Metals; Mitochondria; Modeling; Molecular; Molecular Target; Mus; Mutant Strains Mice; Mutation; National Research Service Awards; Nerve Degeneration; Nervous system structure; Neurologic; Neurologic Symptoms; Neurons; Neurotransmitters; Nutrient; Oxidative Phosphorylation; Oxygen; Oxygen saturation measurement; Parkinson Disease; Pathology; Pathway interactions; Phenotype; Play; Production; Reactive Oxygen Species; Regulation; Research; Respiration; Role; Testing; Therapeutic; base; calcium indicator; cell injury; cellular engineering; chromatin immunoprecipitation; cytochrome c oxidase; enzyme activity; epileptic encephalopathies; experimental study; extracellular; inorganic phosphate; metabolic phenotype; mitochondrial metabolism; molecular phenotype; mouse model; nano-string; neuroblastoma cell; neuron loss; neuronal excitability; neuropathology; neurotransmission; protein expression; rare genetic disorder; response; transcription factor; transcriptome sequencing; transcriptomics

PROJECT SUMMARY Menkes disease is a rare genetic condition in which the disruption of copper homeostasis induces neurodegeneration and other neurological symptoms soon after birth. The underlying mechanisms of Menkes neuropathology remain unclear, but the metabolic changes observed in Menkes disease and the crucial role of mitochondria in neurons point to dysregulation of cellular bioenergetics as a possible factor. Preliminary data in human cells indicates that copper depletion decreases expression of genes regulated by hypoxia induced factor 1 alpha (HIF-1α). HIF-1α is a transcription factor sensitive to metals and oxygen that regulates cellular bioenergetics by switching metabolism from mitochondrial oxidative phosphorylation to glycolysis. Further, these copper depleted cells exhibit increased mitochondrial respiration. Resolving the newly identified role of HIF-1α in regulating mitochondrial function is central to understanding how copper dyshomeostasis elicits neurodegeneration in Menkes disease. Thus, the overall objective of this F31 NRSA application is to test how copper depletion influences the HIF-1α pathway in neurons to regulate cellular metabolism and influence cell excitability and survival. The central hypothesis that will be tested in this proposal is that neuronal copper depletion selectively downregulates transcriptional activity of the HIF-1α pathway to redirect nutrients through mitochondrial respiration rather than glycolysis, rendering cells hyperexcitable due to production of reactive oxygen species by mitochondria and thus susceptible to cell death. In Aim 1, the HIF-1α pathway will be stimulated in copper depleted and control neuroblastoma cells or primary cultured neurons in order to comprehensively assess gene expression, determine binding of HIF-1α to target genes, and quantify mitochondrial respiration and glycolysis in the context of HIF-1α activity. In Aim 2, genetically encoded calcium indicators will be used in primary neuronal cultures from wildtype or neuronal-specific copper depleted mice while stimulating the HIF-1α pathway to assess how copper depletion affects cell excitability and determine the effect of HIF-1α on these phenotypes. Completion of these aims will clarify the metabolic pathways responsive to copper and their effects on neuronal function. The application of this knowledge will inform our understanding, research, and treatment of neuropathology of diseases known to be associated with dysregulated metals and/or metabolism for which there are currently limited therapeutics.

项目概要 门克斯病是一种罕见的遗传性疾病，铜稳态的破坏会导致 出生后不久的神经退行性变和其他神经系统症状。门克斯的潜在机制。 神经病理学仍不清楚，但在门克斯病中观察到的代谢变化以及 神经元中的线粒体表明细胞生物能量失调是一个可能的因素。 人类细胞表明铜耗竭会降低缺氧诱导因子调节的基因表达 1 α (HIF-1α) 是一种对金属和氧敏感的转录因子，可调节细胞。 通过将代谢从线粒体氧化磷酸化转变为糖酵解来实现生物能量学。 铜耗尽的细胞表现出线粒体呼吸增加，解决了新发现的 HIF-1α 的作用。 调节线粒体功能对于理解铜稳态如何引起 因此，F31 NRSA 应用的总体目标是测试如何发生门克斯病的神经变性。 铜耗竭影响神经元中的 HIF-1α 通路，从而调节细胞代谢并影响细胞 本提案将测试的中心假设是神经元铜。 耗竭选择性下调 HIF-1α 途径的转录活性，从而通过 线粒体呼吸而不是糖酵解，使细胞由于反应性物质的产生而过度兴奋 在目标 1 中，HIF-1α 途径将受到线粒体的影响，从而容易发生细胞死亡。 在铜耗尽并控制神经母细胞瘤细胞或原代培养的神经元中进行刺激，以便 全面评估基因表达，确定HIF-1α与靶基因的结合，并定量 HIF-1α 活性背景下的线粒体呼吸和糖酵解 在目标 2 中，基因编码的钙。 指示剂将用于野生型或神经元特异性铜耗尽小鼠的原代神经元培养物，同时 刺激 HIF-1α 通路以评估铜消耗如何影响细胞兴奋性并确定效果 HIF-1α 对这些表型的影响的完成将阐明响应的代谢途径。 铜及其对神经功能的影响将有助于我们理解， 已知与失调金属和/或相关的疾病的神经病理学研究和治疗 目前治疗方法有限。