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

项目总结 门克斯病是一种罕见的遗传疾病，在这种情况下铜稳态的破坏会导致 出生后不久就会出现神经变性和其他神经症状。门克斯的潜在机制 神经病理学尚不清楚，但在孟克斯病中观察到的代谢变化及其关键作用 神经元中的线粒体表明细胞生物能量学的失调是一个可能的因素。第一季度初步数据 人类细胞提示铜缺乏降低了低氧诱导因子调控基因的表达 1α(HIF-1α)。缺氧诱导因子-1α是一种对金属和氧气敏感的转录因子，调节细胞 通过将代谢从线粒体氧化磷酸化转换到糖酵解的生物能量学。此外，这些 铜缺乏的细胞表现出线粒体呼吸增加。解析新发现的缺氧诱导因子-1α的作用 对线粒体功能的调节是理解铜代谢紊乱如何引起的核心 门克斯病的神经退行性变。因此，此F31 NRSA应用程序的总体目标是测试 铜缺乏影响神经元内缺氧诱导因子-1α途径调节细胞代谢并影响细胞 兴奋性和生存能力。将在这一提议中检验的中心假设是神经元铜 耗竭选择性地下调HIF-1α途径的转录活性，通过 线粒体呼吸而不是糖酵解，使细胞由于产生反应性而过度兴奋 线粒体中的氧物种，因此容易引起细胞死亡。在目标1中，缺氧诱导因子-1α途径将是 在铜的耗竭和对照神经母细胞瘤细胞或原代培养的神经元中刺激，以便 综合评估基因表达，测定缺氧诱导因子-1α与靶基因的结合，并定量 线粒体呼吸和糖酵解在缺氧诱导因子-1α活性的背景下。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缺乏小鼠的原代神经元培养，而 刺激缺氧诱导因子-1α通路以评估铜缺乏如何影响细胞兴奋性并确定其影响 HIF-1α在这些表型上的表达。这些目标的完成将澄清响应于 铜及其对神经功能的影响。这些知识的应用将有助于我们理解， 研究和治疗已知与金属和/或调节失调有关的疾病的神经病理学 新陈代谢，目前治疗方法有限。

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.