Amyloid Beta-Protein: ApoE and Cholesterol Homeostasis

β-淀粉样蛋白：ApoE 和胆固醇稳态

基本信息

批准号：
7569483
负责人：
WELLINGTON GIBSON WOOD
金额：
$ 23.72万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-01-15 至 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7569483
关键词：
Adrenergic Receptor Alleles Alzheimer&apos s Disease Amyloid beta-Protein Amyloid beta-Protein Precursor Apolipoprotein E Astrocytes Blood Vessels C57BL/6 Mouse Caveolae Caveolins Cell membrane Cell physiology Cells Cerebrum Cholesterol Cholesterol Homeostasis Coculture Techniques Complex Conditioned Culture Media Coupled Cyclic AMP DNA Binding Data Epidemiologic Studies Gene Expression Genetic Transcription Golgi Apparatus Hippocampus (Brain)Homeostasis Human Incubated Individual Knockout Mice Laboratories Lead Lipids Medial Mediating Membrane Membrane Microdomains Messenger RNA Mus Nerve Degeneration Neurons Organelles Pathway interactions Patients Pharmaceutical Preparations Phenotype Play Primary Cell Cultures Production Protein Isoforms Proteins Proteomics Rattus Reporting Risk Role Source Structure Synaptophysin Testing Time Transcription Factor AP-2 Alpha Transcriptional Regulation Tretinoin Wood material Work apolipoprotein E-3 apolipoprotein E-4 beta-adrenergic receptor brain tissue caveolin 1 cholesterol trafficking lipid transport neurite growth protein expression research study synaptogenesis trafficking transcription factor vesicle-associated membrane protein

项目摘要

This application focuses on mechanisms of amyloid beta-protein perturbation of cholesterol and apoE homeostasis within astrocytes and resulting consequences on neuronal function. There is a dynamic interaction between cholesterol and amyloid beta-protein (ABeta), a protein that is thought to be an important contributor to neurodegeneration that occurs with Alzheimer's disease. Cholesterol levels modulate amyloid precursor protein expression and ABeta1-42 production. Conversely, ABeta1-42 alters cellular cholesterol dynamics particularly cholesterol trafficking in astrocytes and neurons. The Golgi complex play an important role in protein and lipid trafficking and recent work from our laboratory has shown that ABeta1-42 modified cholesterol distribution within the Golgi complex in astrocytes, reduced cholesterol levels in the plasma membrane and increased apoE levels. These results lead us to hypothesize that: ABeta1-42 disrupts cholesterol and apoE homeostasis in astrocytes and effects are apoE isoform dependent. Mechanisms of ABeta1-42 effects involve a caveolin associated pathway between the Golgi complex and the plasma membrane, and transcriptional regulation of apoE expression that is dependent on stimulation of Beta-adrenergic receptors, cAMP formation and the transcription factor AP-2. Consequences of ABeta1-42 perturbation of astrocyte cholesterol and apoE homeostasis are alterations in neuronal cholesterol domains and the synaptophysin/synaptobrevin complex. To test this we will: 1: Evaluate effects of ABeta1-42 on caveolin levels in the cis-medial and trans- regions of the Golgi complex of astrocytes. Examine proteomics of the Golgi complex regions of astrocytes treated with ABeta1-42 Evaluate caveolae and lipid raft structure and function in astrocytes treated with ABeta1-42. Primary astrocytes from mice expressing human apoE2, apoE3, apoE4, apoE-null mice, caveolin-l-null mice and human astrocytes will be used. 2: Examine the effects of ABeta1-42 on distribution of apoE levels in astrocyte organelles and conditioned media. Evaluate apoE mRNA abundance in astrocytes incubated with ABeta1-42. Evaluate effects of ABeta1-42 on Beta-adrenergic receptors, cAMP formation, transcription factor AP-2 levels and DNA binding of AP-2. Astrocytes of mice expressing human apoE2, apoE3, and apoE4 will be used. 3: Evaluate neuronal lipid raft proteins, lipids and transbilayer cholesterol distribution using neurons of C57BL/6 mice co-cultured with astrocytes from mice expressing human apoE2, apoE3, apoE4, and apoE-null mice. Examine synaptophysin/synaptobrevin complex in neurons of C57BL/6 mice co-cultured with astrocytes from mice expressing human apoE2, apoE3, apoE4, and apoE-null mice. Determine if the neuronal apoE4 and apoE-null phenotype can be "rescued" by incubation with astrocytes of apoE2, apoE3, or wildtype mice. In some experiments astrocytes will be treated with ABeta1-42.

该应用的重点是星形胶质细胞中胆固醇和APOE稳态的淀粉样蛋白β-蛋白扰动的机制，以及对神经元功能的影响。胆固醇和淀粉样蛋白β-蛋白质（ABETA）之间存在动态相互作用，该蛋白被认为是阿尔茨海默氏病发生的神经变性的重要因素。胆固醇水平调节淀粉样蛋白前体蛋白表达和ABETA1-42产生。相反，Abeta1-42改变了细胞胆固醇动力学，尤其是在星形胶质细胞和神经元中的胆固醇运输。高尔基体络合物在蛋白质和脂质运输中起着重要作用，以及我们实验室的最新工作表明，ABETA1-42在星形胶质细胞中高尔基体复合物中修饰的胆固醇分布，质膜膜中胆固醇水平降低并降低了APOE水平。这些结果导致我们假设：Abeta1-42在星形胶质细胞中破坏胆固醇和APOE稳态，效果是APOE同工型的依赖性。 ABETA1-42作用的机制涉及高尔基体配合物与质膜之间的小窝蛋白相关途径，以及APOE表达的转录调控，取决于β-肾上腺素能受体，cAMP形成和转录因子AP-2的刺激。 ABETA1-42星形胶质细胞胆固醇和APOE稳态的扰动是神经元胆固醇结构域的改变以及突触素/突触蛋白突触蛋白络合物的改变。为了测试这一点，我们将：1：评估Abeta1-42对星形胶质细胞高尔基体络合物的顺式中心和转移区域中小窝蛋白水平的影响。检查用Abeta1-42处理的星形胶质细胞的高尔基体复合区域的蛋白质组学评估了用Abeta1-42处理的星形胶质细胞中的小窝和脂质筏结构和功能。将使用来自表达人APOE2，APOE3，APOE4，APOE-NULL小鼠，小窝L-NULL小鼠和人类星形胶质细胞的小鼠的主要星形胶质细胞。 2：检查Abeta1-42对星形胶质细胞器和条件培养基中APOE水平分布的影响。评估与Abeta1-42孵育的星形胶质细胞中的ApoE mRNA丰度。评估Abeta1-42对β-肾上腺素能受体，cAMP形成，转录因子AP-2水平和DNA结合的影响。将使用表达人APOE2，APOE3和APOE4的小鼠的星形胶质细胞。 3：使用与表达人APOE2，APOE3，APOE4和APOE-NULL小鼠的星形胶质细胞共培养的C57BL/6小鼠的神经元的神经元胆固醇分布评估神经元脂质筏蛋白，脂质和跨贝贝胆固醇分布。检查与表达人APOE2，APOE3，APOE4和APOE-NULL小鼠的星形胶质细胞共培养的C57BL/6小鼠神经元中神经元中的突触素/突触纤维复合物。确定是否可以通过与APOE2，APOE3或WildType小鼠的星形胶质细胞孵育来“营救”神经元APOE4和APOE-NULL表型。在某些实验中，星形胶质细胞将用Abeta1-42处理。