Variations in lipid homeostasis and signaling affect alpha-synuclein pathobiology

脂质稳态和信号传导的变化影响α-突触核蛋白病理学

• 批准号: 8003757
• 负责人: Daniel J Termine
• 金额: $ 4.76万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8003757
• 关键词: Affect; Animal Model; Binding; Binding Proteins; Cells; Cholesterol; Collaborations; Culture Media; Defect; Disease; Disease Progression; Ergosterol; Eukaryota; Event; Filipin; Genetic; Homeostasis; Human; Institutes; Investigation; Label; Laboratories; Lead; Lesion; Lipid Binding; Lipids; Metabolic; Microscopy; Modeling; Monitor; Neurodegenerative Disorders; Neurons; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Population; Production; Proteins; RNA Interference; Regulation; Signal Transduction; Sphingolipids; Staining method; Stains; Sterol Biosynthesis Pathway; Sterols; Therapeutic; Toxic effect; Variant; Yeast Model System; Yeasts; alpha synuclein; dopaminergic neuron; human disease; macromolecule; overexpression; public health relevance; response; therapeutic target; trafficking

DESCRIPTION (provided by applicant): Synucleinopathies are a class of neurodegenerative disease characterized by pathological lesions composed of aggregates of a-synuclein (a-syn) in select populations of neurons. Alpha-synuclein is a small, abundant lipid-binding protein of unknown function that is prone to misfolding when not bound to lipids. The accumulation of a-syn aggregates in synucleinopathies suggests that aberrations in protein homeostasis might contribute to pathogenesis in these diseases. Because protein homeostasis mechanisms are highly conserved throughout eukaryotes, yeast cells can be used as a tractable model organism to investigate factors that contribute to the pathogenesis of synucleinopathies. The Lindquist laboratory has developed a yeast model over-expressing human a-syn that recapitulates many aspects of a-syn toxicity, and has observed that sterol biosynthesis and trafficking are also perturbed. Disrupting sterol biosynthesis can potentially alter a variety of cellular pathways. In fact, sphingolipid production is responsive to flux through the sterol pathway. Interestingly, both pathways have been implicated independently in a-syn toxicity and disease progression. The contribution of lipids to a-syn toxicity remains unresolved, and understanding how lipids affect synucleinopathy pathogenesis is essential for identifying potential therapeutic targets. The first aim of my proposal will investigate how the yeast lipidome is altered in response to a-syn expression. Lipidomic profiling has performed on yeast expressing nontoxic and toxic levels of a-syn in collaboration with Dr. Clary Clish at the Broad Institute. The lipid species identified up to this point correlate with those predicted to change upon a-syn expression. These results will then be compared to the lipid profiles of yeast co-expressing a-syn and genetic modifiers we discovered using overexpression and deletion screens. This will identify the lipids that change when toxicity is suppressed or enhanced. These targets lipids will then be manipulated in our yeast a-syn model to determine their effect on toxicity. My second aim is to evaluate whether the defects in cellular unesterified ergosterol distribution caused by a-syn expression lead to the mis-trafficking and accumulation of sphingolipids. Fluorescent microscopy will be used to monitor sphingolipid trafficking and localization in yeast cells. In addition, sphingolipids will be quantified using metabolic labeling. The cellular localization of unesterified ergosterol will be determined using filipin stain. Finally, my last aim will be evaluate sterol-sphingolipid regulation defects in cultured DA neurons expressing a-syn. Similar to aim 2, fluorescent microscopy will be used to monitor the trafficking and localization of unesterified cholesterol and sphingolipids, respectively. The amount of sphingolipids and sterols in a-syn expressing DA neurons will be quantified as well. PUBLIC HEALTH RELEVANCE: Lipids are important macromolecules that are involved in many cellular events and have been implicated in several human diseases. Investigation of their contribution to neurodegenerative disease specifically PD, will promote further understanding of lipid homeostasis and a-syn pathobiology. The identification of lipids that might affect a-syn toxicity will be extremely useful in developing lipid-specific therapeutics reducing the pleiotropic effects associated with the general lipid drugs currently employed.

描述（由申请人提供）：突触核蛋白病是一类神经退行性疾病，其特征在于在选定的神经元群体中由α-突触核蛋白（α-syn）聚集体组成的病理性病变。α-突触核蛋白是一种小而丰富的脂质结合蛋白，其功能未知，当不与脂质结合时易于错误折叠。突触核蛋白病中α-syn聚集体的积累表明蛋白质稳态的畸变可能有助于这些疾病的发病机制。由于蛋白质稳态机制在整个真核生物中是高度保守的，因此酵母细胞可以用作易处理的模式生物来研究有助于突触核蛋白病发病机制的因素。Lindquist实验室已经开发了过表达人a-syn的酵母模型，其概括了a-syn毒性的许多方面，并且已经观察到固醇生物合成和运输也受到干扰。破坏甾醇生物合成可以潜在地改变多种细胞途径。事实上，鞘脂的产生对通过固醇途径的流量有反应。有趣的是，这两种途径都独立地涉及a-syn毒性和疾病进展。脂质对a-syn毒性的贡献仍未得到解决，并且了解脂质如何影响突触核蛋白病发病机制对于确定潜在的治疗靶点至关重要。我的建议的第一个目标将调查酵母脂质体是如何改变响应a-syn表达。与布罗德研究所的Clary Clish博士合作，对表达无毒和毒性水平的α-syn的酵母进行了脂质组学分析。到目前为止鉴定的脂质种类与预测在α-syn表达后改变的那些相关。然后将这些结果与我们使用过表达和缺失筛选发现的共表达a-syn和遗传修饰剂的酵母的脂质谱进行比较。这将确定当毒性被抑制或增强时发生变化的脂质。然后将在我们的酵母a-syn模型中操纵这些靶脂质以确定它们对毒性的影响。我的第二个目的是评估由a-syn表达引起的细胞未酯化麦角固醇分布的缺陷是否导致鞘脂的错误运输和积累。荧光显微镜将用于监测酵母细胞中的鞘脂运输和定位。此外，将使用代谢标记对鞘脂进行定量。将使用菲律宾染色确定未酯化麦角甾醇的细胞定位。最后，我的最后一个目标将是评估在培养的DA神经元表达α-syn的甾醇鞘脂调节缺陷。与目标2相似，将使用荧光显微镜分别监测未酯化胆固醇和鞘脂的运输和定位。还将定量表达α-syn的DA神经元中鞘脂和留醇的量。 公共卫生相关性：脂质是重要的大分子，参与许多细胞事件，并与多种人类疾病有关。研究它们对神经退行性疾病特别是PD的贡献，将促进对脂质稳态和a-syn病理生物学的进一步理解。可能影响α-syn毒性的脂质的鉴定将在开发降低与目前使用的一般脂质药物相关的多效性效应的脂质特异性治疗剂中极其有用。