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: 8330269
• 负责人: Daniel J Termine
• 金额: $ 1.05万
• 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 2012-10-21
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8330269
• 关键词: Affect; Anabolism; Animal Model; BODIPY; Binding; Binding Proteins; Cell Culture Techniques; Cells; Cholesterol; Collaborations; Complex; Culture Media; Defect; Development; Disease; Disease Progression; Endosomes; Enzymes; Ergosterol; Eukaryota; Event; Filipin; Gaucher Disease; Gene Deletion; Genes; Genetic; Global Change; Goals; Homeostasis; Human; Individual; Institutes; Intracellular Accumulation of Lipids; Investigation; Label; Lead; Lesion; Lipid Binding; Lipids; Metabolic; Microscopy; Modeling; Monitor; Mutation; Neurodegenerative Disorders; Neuroglia; Neurons; Orthologous Gene; Parkinson Disease; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Population; Predisposition; Production; Proteins; RNA Interference; Regulation; Research Proposals; Risk Factors; Signal Transduction; Sphingolipids; Staining method; Stains; Sterol Biosynthesis Pathway; Sterols; Supplementation; Therapeutic; Toxic effect; Vacuole; Variant; Yeast Model System; Yeasts; abstracting; alpha synuclein; dopaminergic neuron; embryonic stem cell; glucosylceramidase; human disease; insight; macromolecule; mouse model; overexpression; peroxisome; research study; response; synucleinopathy; therapeutic target; trafficking; yeast genetics

Project Abstract Synucleinopathies are a class of neurodegenerative disease characterized by pathological lesions composed of aggregates of alpha-synuclein (alpha-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 alpha-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 lab has developed a yeast model over-expressing human alpha- syn that recapitulates many aspects of alpha-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 alpha-syn toxicity and disease progression. The contribution of lipids to alpha-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 alpha-syn expression. Lipidomic profiling has performed on yeast expressing nontoxic and toxic levels of alpha-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 alpha-syn expression. These results will then be compared to the lipid profiles of yeast co-expressing alpha-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 alpha-syn model to determine their effect on toxicity. My second aim is to evaluate whether the defects in cellular unesterified ergosterol distribution caused by alpha-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 alpha-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 alpha-syn expressing DA neurons will be quantified as well.

项目摘要