A Zebrafish Model of Multiple System Atrophy

描述(申请人提供)：多系统萎缩(MSA)是一种进行性神经退行性疾病，可导致帕金森氏症、共济失调和自主神经功能衰竭。MSA对治疗无反应，并在确诊后十年内死亡。MSA的病理包括神经元丢失、星形胶质细胞增生和胶质细胞质内含物(GCI)的形成。GCI主要存在于少突胶质细胞中，含有神经元突触前蛋白--突触核蛋白的纤维沉积。来自体外实验、尸检研究和转基因小鼠模型的证据表明，GCI的形成在机制上参与了MSA的神经元退化。这项工作的长期目标是了解神经胶质细胞相互作用如何参与MSA的发病机制，并开发利用潜在分子生理学的神经保护策略。 作为一种模式生物，斑马鱼具有许多特性，这些特性可能有助于识别由于少突胶质细胞突触核蛋白的表达和沉积而导致神经退化的因素。斑马鱼的大脑含有丰富的少突胶质细胞，保留了与其他脊椎动物大脑相同的基本组织结构，并具有与MSA研究直接相关的专门神经元群体。在斑马鱼中已经开发出强大的遗传和体内药物筛选技术，这些工具可以部署在斑马鱼MSA模型中，以识别调节病理表达的基因和药物化合物。 在这一探索性拨款申请中，我们提出了一种假设，即在斑马鱼脑的少突胶质细胞中表达人？-突触核蛋白将导致神经胶质胞质内含物的形成和进行性神经元的丢失。为了验证这一假设，我们将从我们产生的生殖系嵌合体中建立转基因斑马鱼品系，该嵌合体在斑马鱼髓磷脂P0启动子(目标1)的控制下表达？-突触核蛋白。我们将分析这些转基因FISH与野生型FISH的比较，以寻找形成组织学和生化特性类似于胶质细胞质包涵体的不溶性？-突触核蛋白沉积的证据(目标2)，以及多巴胺能神经元或小脑浦肯野细胞的退化，或髓鞘形成缺陷(目标3)。 这些实验数据将支持异位少突胶质细胞表达β-突触核蛋白和GCI形成在MSA发病机制中的中枢参与。此外，由此产生的斑马鱼模型将是我们未来旨在确定MSA中少突胶质细胞定向神经变性的分子基础的核心研究，并可能进一步发展为高通量体内试验，从而能够分离出治疗MSA的新的神经保护剂。

DESCRIPTION (provided by applicant): Multiple system atrophy (MSA) is a progressive neurodegenerative condition that causes Parkinsonism, ataxia and autonomic failure. MSA is unresponsive to treatment and fatal within a decade of diagnosis. The pathology of MSA includes neuronal loss, astrogliosis and formation of glial cytoplasmic inclusions (GCI). GCIs are found predominantly within oligodendrocytes and contain fibrillar deposits of the neuronal presynaptic protein ?-synuclein. Evidence from in vitro experiments, autopsy studies and transgenic mouse models suggests that GCI formation is mechanistically involved in the neuronal degeneration of MSA. The long-term aim of this work is to understand how glial-neural interactions are involved in the pathogenesis of MSA, and to develop neuroprotective strategies that exploit the underlying molecular physiology. As a model organism, the zebrafish has many properties that may prove favorable for identifying factors that cause neurodegeneration as a consequence of oligodendroglial ?-synuclein expression and deposition. The zebrafish brain is rich in oligodendrocytes, retains the same basic organization of other vertebrate brains and has specialized neuronal populations of direct relevance to the study of MSA. Powerful genetic and in vivo drug screening techniques have been developed in the zebrafish, and these tools could be deployed in a zebrafish MSA model to identify genes and pharmaceutical compounds that modulate the expression of pathology. In this exploratory grant application, we propose to test the hypothesis that expression of human ?-synuclein in oligodendrocytes of the zebrafish brain will lead to the formation of glial cytoplasmic inclusions and progressive neuronal loss. In order to test this hypothesis, we will establish transgenic zebrafish lines from germline chimeras we have generated that express ?-synuclein under control of the zebrafish myelin P0 promoter (aim 1). We will analyse these transgenic fish in comparison with wild-type fish for evidence of formation of insoluble ? -synuclein deposits with histological and biochemical properties similar to glial cytoplasmic inclusions (aim 2), and degeneration of dopaminergic neurons or cerebellar Purkinje cells, or defects in myelination (aim 3). The data generated by these experiments will lend support to the central involvement of ectopic oligodendroglial expression of ?-synuclein and GCI formation in the pathogenesis of MSA. Furthermore, the resulting zebrafish model will be central to our future studies aimed at defining the molecular basis for oligodendrocyte-directed neurodegeneration in MSA, and may be further developed as a high-throughput in vivo assay to enable isolation of novel neuroprotective agents for treatment of MSA.