Endoplasmic Reticulum NAD(P)H Dynamics in Dopamine Neurons

多巴胺神经元内质网 NAD(P)H 动力学

基本信息

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
项目类别：
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

项目摘要

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 的总结/摘要吡啶核苷酸是生物能学所必需的氧化还原辅酶，几十年来，新陈代谢和 DNA 损伤反应减少。吡啶核苷酸（即 NADH 和 NADPH 或 NAD(P)H）用于研究氧化还原细胞核、细胞质和线粒体，而内质网（ER） NAD(P)H 池被认为是微不足道的。我们最近的脑切片双光子 NAD(P)H 成像实验推翻了这一点证明多巴胺神经元中的大部分体细胞 NAD(P)H 位于 ER 和我们还发现安非他明通过线粒体功能发挥作用。多巴胺囊泡可快速减少 NAD(P)H，从而可能减少补偿因此，我们的研究揭示了新的 NAD(P)H 细胞器。分布和氧化还原耦合，以及 NAD(P)H 通过重要的滥用调节在这里，我们使用荧光寿命显微镜（FLIM）和新生成的 ER-。单个吡啶核苷酸及其氧化还原比的目标指标（例如 NAD+/NADH) 来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 偶联的作用。细胞器间氧化还原耦合中的穿梭和 ER-线粒体接近度将最后，ER NAD(P)H 池将受到扰动以检验新假设。 ER 缓冲其他细胞器中的吡啶核苷酸氧化还原作用。对吡啶核苷酸的天然和药理学控制的基本见解细胞器中并指导未来减少多巴胺神经元的疗法的开发与帕金森病和安非他明滥用相关的氧化还原应激。