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

• 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
• 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' 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

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.

项目摘要/摘要 帕金森氏病（PD）的主要标志是存在α-突触核蛋白（α-Syn）阳性聚集体和细胞 损失黑质核酸杆菌（SNPC），但对验尸后脑组织的研究报告了αSyn 基因座（LC）中的聚集和细胞损失比SNPC中的聚集和细胞损失更为严重。 LC是大脑的主要去肾上腺素能中心，LC神经元的丧失与非运动有关 PD的症状，包括睡眠障碍，抑郁症，广义动画和自主神经功能障碍。 实验模型LC病变增强了黑质性变性，但对这些机制知之甚少 PD中的基础LC细胞损失。迄今为止，我们缺乏适当的动物模型来了解αSyn 病理特异性影响PD中的去甲肾上腺素能系统，以及非甲肾上腺素能神经元是否容易 容易受到α-syn病理的影响。这项研究将检验以下假设：α-syn在LC去甲肾上腺素能中积累 神经元影响α-Syn溶解度，并将其构象转移到有毒的寡聚物种上，这将 增强氧化应激的有害作用，降低去甲肾上腺素能细胞活力，并诱导细胞损失 时间依赖的方式。拟议的研究的具体目的是1）确定αSyn的方式 LC神经元中的过表达影响αSyn的生化特性和构象，2）评估 LC神经元中的α-Syn积累如何影响神经元功能，健康和对氧化物胁迫的敏感性。 这些目标将使用过表达人类野生的新型BAC转基因小鼠（DBH-HSNCA）来解决 在多巴胺-β-羟基（DBH）启动子下型αSyn。诱导人类野生的选择性表达 甲肾上腺素能神经元中的αSyn型将揭示αSyn病理如何影响LC。后分析 翻译修饰，αSyn溶解度和构象将用于评估αSyn的特征 在年轻和老年转基因和非转基因DBH-HSNCA小鼠的LC神经元中积累。脆弱性 将过表达LC神经元到氧化应激条件的αSyn的体外和体内将检查氧化应激条件 抑制囊泡单胺转运蛋白2（VMAT2）。 VMAT2抑制增加了胞质 儿茶酚胺迅速消化成反应性醛中间体，并导致 活性氧的形成。遵循VMAT2抑制后，神经heath的体外测量将 包括LC神经蛋白的长度，LC神经元的数量以及原发性培养中活性氧的检测。 在体内，将评估抗氧化剂分子的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的LC神经元的公正立体细胞计数 DBH-HSNCA小鼠。这些研究的完成将揭示一个结构和功能后果 在去甲肾上腺素能系统中增加了αSynburnen，并将促进我们对αSyn的理解 LC中的积累有助于PD的疾病进展。