Endoplasmic Reticulum NAD(P)H Dynamics in Dopamine Neurons

多巴胺神经元内质网 NAD(P)H 动力学

• 批准号: 9750034
• 负责人: EDWIN S LEVITAN
• 金额: $ 19.56万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750034
• 关键词: Amphetamine Abuse; Amphetamines; Apoptosis; Autophagocytosis; Binding Proteins; Biochemical; Bioenergetics; Biology; Brain; Buffers; Catecholamines; Cell Nucleus; Cell physiology; Cells; Cellular biology; Chronic; Coenzymes; Coupled; Coupling; Cytoplasm; Cytosol; DNA Damage; Dopamine; Endoplasmic Reticulum; Financial compensation; Fluorescence; Foundations; Future; Glucose-6-Phosphate; Hexoses; Image; Individual; Location; Measures; Metabolism; Microscopy; Mitochondria; Modernization; Modification; NADH; NADP; Neurons; Neurosecretory Systems; Optics; Organelles; Oxidation-Reduction; Oxidative Stress; Oxides; Oxidoreductase; PC12 Cells; Parkinson Disease; Pentosephosphate Pathway; Pharmacology; Prevalence; Proteins; Reaction; Regulation; Research; Role; Signal Transduction; Site; Slice; Steroids; Stress; Substantia nigra structure; Testing; Tissues; Vesicle; autooxidation; biological adaptation to stress; cell type; dopaminergic neuron; drug of abuse; experimental study; inorganic phosphate; insight; neuron loss; neurotoxicity; novel; pyridine nucleotide; response; sensor; therapy development; tool; two-photon

Summary/Abstract for R21 Pyridine nucleotides are redox coenzymes that are essential for bioenergetics, metabolism and DNA damage responses. For decades, autofluorescence from reduced pyridine nucleotides (i.e., NADH and NADPH or NAD(P)H) was used to study redox in the nucleus, cytoplasm and mitochondria, while the endoplasmic reticulum (ER) NAD(P)H pool was assumed to be insignificant. This assumption has now been overturned by our recent brain slice two-photon NAD(P)H imaging experiments that demonstrated that much of somatic NAD(P)H in dopamine neurons is in the ER and coupled to mitochondrial function. We also discovered that an amphetamine acts via dopamine vesicles to rapidly reduce NAD(P)H, thus potentially reducing compensation for redox stress. Therefore, our studies have revealed new NAD(P)H organelle distribution and redox coupling, as well as NAD(P)H regulation by an important abused drug. Here we use fluorescence lifetime microscopy (FLIM) and newly generated ER- targeted indicators for individual pyridine nucleotides and their redox ratios (e.g. NAD+/NADH) to determine the identity and location of pyridine nucleotides involved in the amphetamine response and ER-mitochondria NAD(P)H coupling. Then the roles of shuttles and ER-mitochondria proximity in interorganelle redox coupling will be determined. Finally, the ER NAD(P)H pool will be perturbed to test the novel hypothesis that the ER buffers pyridine nucleotide redox in other organelles. This study will yield fundamental insights into the native and pharmacological control of pyridine nucleotides in organelles and guide future development of therapies for reducing dopamine neuron redox stress associated with Parkinson’s disease and amphetamine abuse.

R21摘要/摘要 吡啶核苷酸是生物能量学所必需的氧化还原辅酶， 代谢和DNA损伤反应。几十年来，减少的自体荧光 吡啶核苷酸（即，用还原型辅酶Ⅱ（NADH和NADPH或NAD（P）H）研究了 细胞核、细胞质和线粒体，而内质网（ER） 假设NAD（P）H池不显著。这个假设现在已经 被我们最近的脑切片双光子NAD（P）H成像实验推翻， 表明多巴胺神经元中的大部分体细胞NAD（P）H位于ER中， 与线粒体功能相关联。我们还发现安非他明的作用是通过 多巴胺囊泡迅速减少NAD（P）H，从而可能减少代偿 氧化还原应激因此，我们的研究揭示了新的NAD（P）H细胞器 分布和氧化还原偶联，以及NAD（P）H调节的一个重要滥用 药在这里，我们使用荧光寿命显微镜（FLIM）和新产生的ER- 单个吡啶核苷酸及其氧化还原比的靶向指示剂（例如， NAD+/NADH）以确定参与的吡啶核苷酸的身份和位置。 安非他明反应和ER-线粒体NAD（P）H偶联。那么， 穿梭和ER-线粒体接近细胞器间的氧化还原偶联将是 测定最后，ER NAD（P）H库将被扰动以测试新的假设 ER缓冲其他细胞器中的吡啶核苷酸氧化还原。这项研究将产生 对吡啶核苷酸的天然和药理学控制的基本见解 并指导未来开发用于减少多巴胺神经元 氧化还原应激与帕金森病和安非他明滥用有关。