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

项目摘要 门克斯氏病是一种罕见的遗传性疾病，其中铜稳态的破坏诱导 神经变性和其他神经系统症状。门克斯的潜在机制 神经病理学尚不清楚，但在Menkes病中观察到的代谢变化和 神经元中的线粒体指出细胞生物能量学的失调是一个可能的因素。的初步数据 人类细胞表明，铜耗竭降低了缺氧诱导因子调控的基因表达， 1 α（HIF-1α）。HIF-1α是一种对金属和氧敏感的转录因子， 通过将代谢从线粒体氧化磷酸化转换为糖酵解来实现生物能量学。此外，这些 铜耗尽的细胞表现出增加的线粒体呼吸。解决新发现的HIF-1α的作用 在调节线粒体功能中的作用是理解铜稳态异常如何 Menkes病的神经变性因此，本F31 NRSA应用程序的总体目标是测试如何 铜缺乏影响神经元中HIF-1α通路调节细胞代谢，影响细胞凋亡， 兴奋性和生存。在这个提议中将要检验的中心假设是， 耗尽选择性下调HIF-1α途径的转录活性，通过 线粒体呼吸，而不是糖酵解，使细胞过度兴奋，由于生产反应性 氧物种的线粒体，从而易于细胞死亡。在目标1中，HIF-1α通路将被 在铜耗尽和对照神经母细胞瘤细胞或原代培养的神经元中刺激， 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 指示剂将用于来自野生型或神经元特异性铜耗尽小鼠的原代神经元培养物， 刺激HIF-1α通路，以评估铜耗竭如何影响细胞兴奋性， HIF-1α对这些表型的影响。这些目标的完成将阐明代谢途径， 铜及其对神经功能的影响。这些知识的应用将有助于我们的理解， 研究和治疗已知与失调金属和/或 目前对其存在有限的治疗方法。

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.