Endoplasmic Reticulum NAD(P)H Dynamics in Dopamine Neurons

R21摘要/摘要 吡啶核苷酸是生物能量学中必不可少的氧化还原辅酶， 代谢和DNA损伤反应。几十年来，自体荧光从减少 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，内质网(ER) 假设NAD(P)H池微不足道。这一假设现在已经成为 被我们最近的脑切片双光子NAD(P)H成像实验推翻了 证明多巴胺神经元中的体细胞NAD(P)H大部分在内质网和内质网 再加上线粒体的功能。我们还发现安非他明通过 多巴胺囊泡迅速减少NAD(P)H，从而潜在地减少代偿 治疗氧化还原压力。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的调节作用 毒品。在这里，我们使用荧光寿命显微镜(FLIM)和新生成的ER- 个别吡啶核苷酸及其氧化还原比率的目标指标(例如 NAD+/NADH)，以确定参与的吡啶核苷酸的身份和位置 苯丙胺反应和内质网线粒体NAD(P)H偶联。那么这些角色 细胞器间氧化还原偶联中的穿梭和内质网-线粒体的接近 下定决心。最后，ER NAD(P)H池将被扰动以检验新的假设 内质网缓冲了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控制的基本认识 并指导减少多巴胺神经元的治疗方法的未来发展 氧化还原压力与帕金森氏症和苯丙胺滥用有关。

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.