The effects of alpha-synuclein pathology on noradrenergic neurons

α-突触核蛋白病理学对去甲肾上腺素能神经元的影响

基本信息

批准号：
9321608
负责人：
Laura MacQueen Butkovich
金额：
$ 4.4万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9321608
关键词：
Address Affect Aldehydes Alzheimer&apos s Disease Amino Acid Sequence Animal Model Animals Antioxidants Autonomic Dysfunction Autopsy Axon Biochemical Brain Brain region Catecholamines Cell Count Cell Survival Cells Characteristics Detection Dimerization Disease Progression Dopamine Dopamine-beta-monooxygenase Dopaminergic Cell Experimental Models Goals Health Heritability Histologic Human In Vitro Length Lesion Measurement Mediating Mental Depression Methods Molecular Conformation Motor Mus Mutation Nerve Degeneration Neuraxis Neurites Neurons Norepinephrine Oxidative Stress Parkinson Disease Pathology Pharmacology Physiological Polymers Post Translational Modification Analysis Post-Translational Protein Processing Predisposition Presynaptic Terminals Probability Property Proteins Reactive Oxygen Species Reporting Research Role SNCA gene Sleep disturbances Solubility Stress Substantia nigra structure Synapses System Tertiary Protein Structure Testing Time Toxic effect Transgenic Mice Transgenic Model Transgenic Organisms age related aged alpha synuclein beta pleated sheet brain tissue cytotoxicity dimer dopaminergic neuron early onset generalized anxiety in vivo locus ceruleus structure mRNA Expression monomer mouse model neuron loss neuronal cell body non-motor symptom noradrenergic novel overexpression pars compacta polymerization promoter resilience selective expression uptake vesicular monoamine transporter 2

项目摘要

Project Summary/Abstract A major hallmark of Parkinson’s disease (PD) is presence of α-synuclein (αsyn) positive aggregates and cell loss substantia nigra pars compacta (SNpc), yet studies of PD post-mortem brain tissue report that αsyn aggregation and cell loss in the locus coeruleus (LC) is more severe than, and may precede that in the SNpc. The LC is the major noradrenergic center of the brain, and loss of LC neurons is associated with non-motor symptoms of PD, including sleep disturbances, depression, generalized anxiety, and autonomic dysfunction. In experimental models LC lesion potentiates nigrostriatal degeneration, yet little is known of the mechanisms underlying LC cell loss in PD. To date, we have lacked an appropriate animal model to understand how αsyn pathology specifically affects noradrenergic systems in PD, and whether noradrenergic neurons are readily vulnerable to αsyn pathology. This study will test the hypothesis that αsyn accumulation in LC noradrenergic neurons affects αsyn solubility and shifts its conformation towards toxic oligomeric species, which will potentiate the detrimental effects of oxidative stress, reduce noradrenergic cell viability, and induce cell loss in a time-dependent manner. The Specific aims of the proposed research are 1) to determine how αsyn overexpression in LC neurons affects the biochemical properties and conformation of αsyn and 2) evaluate how αsyn accumulation in LC neurons affects neuronal function, health, and susceptibility to oxidative stress. These aims will be addressed using a novel BAC transgenic mouse (DBH-hSNCA) overexpressing human wild type αsyn under the dopamine-β-hydroxlase (DBH) promoter. Inducing selective expression of human wild type αsyn in noradrenergic neurons will reveal how αsyn pathology affects the LC. Analysis of post- translational modifications, αsyn solubility, and conformation will be used to evaluate characteristics of αsyn accumulation in LC neurons in young and aged transgenic and non-transgenic DBH-hSNCA mice. Vulnerability of αsyn overexpressing LC neurons to conditions of oxidative stress will be examined in vitro and in vivo by inhibiting the vesicular monoamine transporter 2 (VMAT2). VMAT2 inhibition increases cytosolic catecholamines, where they are rapidly digested into reactive aldehyde intermediates, and result in the formation of reactive oxygen species. Following VMAT2 inhibition, in vitro measurements of neuronal heath will include LC neurite length, number of LC neurons, and detection of reactive oxygen species in primary culture. In vivo, mRNA expression of antioxidant molecules will be assessed, and neuronal loss will be determined using unbiased stereological cell counting of LC neurons in young and aged transgenic and non-transgenic DBH-hSNCA mice. Completion of these studies will reveal the structural and functional consequences of an increased αsyn burden in noradrenergic systems and will advance our understanding of how αsyn accumulation in the LC contributes to disease progression in PD.

项目概要/摘要帕金森病 (PD) 的一个主要标志是存在 α-突触核蛋白 (αsyn) 阳性聚集体和细胞黑质致密部 (SNpc) 缺失，但对 PD 死后脑组织的研究报告称 αsyn 蓝斑 (LC) 中的聚集和细胞损失比 SNpc 更严重，并且可能先于 SNpc。 LC 是大脑主要的去甲肾上腺素能中心，LC 神经元的丧失与非运动障碍有关。 PD 症状，包括睡眠障碍、抑郁、广泛性焦虑和自主神经功能障碍。实验模型 LC 损伤会加剧黑质纹状体变性，但其机制知之甚少迄今为止，我们还缺乏合适的动物模型来了解 αsyn 的作用机制。病理学特别影响 PD 中的去甲肾上腺素能系统，以及去甲肾上腺素能神经元是否容易易受 αsyn 病理影响本研究将检验 LC 去甲肾上腺素能中 αsyn 积累的假设。神经元影响 αsyn 的溶解度，并将其构象转变为有毒的寡聚体，这将增强氧化应激的有害影响，降低去甲肾上腺素能细胞活力，并诱导细胞损失所提议研究的具体目标是 1) 确定 αsyn 如何进行。 LC 神经元中的过度表达会影响 αsyn 的生化特性和构象，2) 评估 LC 神经元中 αsyn 的积累如何影响神经功能、健康和对氧化应激的易感性。这些目标将通过使用过度表达人类野生动物的新型 BAC 转基因小鼠 (DBH-hSNCA) 来实现多巴胺-β-羟化酶 (DBH) 启动子下的 αsyn 型诱导人类野生的选择性表达。去甲肾上腺素能神经元中的 αsyn 型将揭示 αsyn 病理学如何影响 LC 后分析。翻译修饰、αsyn 溶解度和构象将用于评估 αsyn 的特性年轻和老年转基因和非转基因 DBH-hSNCA 易感性小鼠中 LC 神经元的积累。 αsyn 过度表达 LC 神经元对氧化应激条件的影响将通过体外和体内检查抑制囊泡单胺转运蛋白 2 (VMAT2) 会增加胞质。儿茶酚胺，它们被快速消化成活性醛中间体，并导致 VMAT2 抑制后，神经元健康状况的体外测量将发生。包括 LC 神经突长度、LC 神经元数量以及原代培养物中活性氧的检测。在体内，将评估抗氧化分子的 mRNA 表达，并确定神经元损失使用年轻和老年转基因和非转基因 LC 神经元的无偏立体细胞计数 DBH-hSNCA 小鼠的完成将揭示结构和功能的后果。增加去甲肾上腺素能系统中的 αsyn 负担，并将增进我们对 αsyn 如何发挥作用的理解 LC 中的积累有助于 PD 的疾病进展。