The effects of alpha-synuclein pathology on noradrenergic neurons

项目摘要/摘要 帕金森病(PD)的一个主要特征是存在α-突触核蛋白(α)阳性集合体和细胞 黑质致密部丢失，但对帕金森病死后脑组织的研究报告α同步 蓝斑(LC)的聚集和细胞丢失比SNPC更严重，而且可能先于SNPC。 LC是大脑的主要去甲肾上腺素能中枢，LC神经元的丢失与非运动性相关 帕金森病的症状，包括睡眠障碍、抑郁、广泛性焦虑和自主神经功能障碍。在……里面 实验性模型LC损毁加重黑质纹状体变性，但其机制知之甚少 帕金森病潜在的LC细胞丢失。到目前为止，我们还缺乏合适的动物模型来理解α同步是如何 病理特异地影响帕金森病患者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。本研究将验证αSYN在LC去甲肾上腺素能体内蓄积的假说 神经元影响αSYN的溶解性，并将其构象向有毒的低聚物种转移，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 依赖时间的方式。拟议研究的具体目标是1)确定α同步 α在LC神经元中的过表达对其生化性质和构象的影响 αSYN在LC神经元中的积累如何影响神经元的功能、健康和对氧化应激的敏感性。 这些目标将通过一种新型的BAC转基因小鼠(DBH-hSNCA)来实现 在多巴胺-α-羟解酶(DBH)启动子下键入βSYN。诱导人野生型基因的选择性表达 去甲肾上腺素能神经元中的αSYN类型将揭示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的特性 年轻和老年转基因和非转基因DBH-hSNCA小鼠LC神经元的蓄积。漏洞 αSYN过表达的LC神经元对氧化应激条件的体外和体内实验将通过 抑制囊泡单胺转运体2(VMAT2)。VMAT2抑制增加胞浆 儿茶酚胺，在那里它们被迅速消化成活性醛中间体，并导致 活性氧物种的形成。在VMAT2抑制后，神经元健康的体外测量将 包括LC突起的长度、LC神经元的数量以及原代培养中的活性氧检测。 在体内，将评估抗氧化剂分子的mrna表达，并确定神经元的丢失。 青年和老年转基因与非转基因LC神经元的无偏立体细胞计数 DBH-hSNCA小鼠。这些研究的完成将揭示一个 增加去甲肾上腺素能系统的α同步负荷，将促进我们对α同步是如何 LC中的蓄积有助于帕金森病的进展。

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.