Amyloid Beta-Protein: ApoE and Cholesterol Homeostasis

这项应用侧重于淀粉样β蛋白对星形胶质细胞内胆固醇和载脂蛋白E稳态的影响及其对神经元功能的影响的机制。胆固醇和淀粉样β蛋白(ABeta)之间存在着动态的相互作用，这种蛋白被认为是导致阿尔茨海默病神经退化的重要因素。胆固醇水平调节淀粉样前体蛋白的表达和ABeta1-42的产生。相反，ABeta1-42改变了细胞的胆固醇动态，特别是胆固醇在星形胶质细胞和神经元中的运输。高尔基复合体在蛋白质和脂肪运输中起着重要作用，我们实验室最近的工作表明，ABeta1-42改变了星形胶质细胞高尔基复合体中胆固醇的分布，降低了质膜中的胆固醇水平，增加了载脂蛋白E的水平。这些结果使我们假设：ABeta1-42破坏了星形胶质细胞中胆固醇和载脂蛋白E的动态平衡，并且这种作用是载脂蛋白E亚型依赖的。ABeta1-42的作用机制涉及高尔基体和质膜之间的小窝相关途径，以及依赖于β-肾上腺素能受体、cAMP形成和转录因子AP-2刺激的载脂蛋白E表达的转录调节。ABeta1-42对星形胶质细胞胆固醇和apoE稳态的影响是神经元胆固醇结构域和突触素/突触素复合体的改变。为了验证这一点，我们将：1：评估ABeta1-42对星形胶质细胞高尔基复合体顺式、内侧和跨区小窝蛋白水平的影响。检测ABeta1-42处理的星形胶质细胞高尔基复合体区域的蛋白质组学，评估ABeta1-42处理的星形胶质细胞的小凹和脂筏的结构和功能。将使用表达人载脂蛋白2、载脂蛋白E3、载脂蛋白E4的小鼠的原代星形胶质细胞、载脂蛋白E缺失的小鼠、小窝蛋白L缺失的小鼠和人类星形胶质细胞。2：检测ABeta1-42对星形胶质细胞细胞器和条件培养液中载脂蛋白E水平分布的影响。评估ABeta1-42孵育的星形胶质细胞apoE mRNA丰度。评价ABeta1-42对β-肾上腺素能受体、cAMP形成、转录因子AP-2水平和AP-2 DNA结合的影响。表达人载脂蛋白2、载脂蛋白E3和载脂蛋白E4的小鼠星形胶质细胞将被使用。3：通过与表达人apo2、apoE3、apoE4和apoE缺失小鼠的星形胶质细胞共培养的C57BL/6小鼠的神经元，评估神经元脂质移植蛋白、脂质和跨双层胆固醇的分布。检测与表达人apo2、apoE3、apoE4和apoE缺失小鼠的星形胶质细胞共培养的C57BL/6小鼠神经元中突触素/突触素复合体。确定神经元apoE4和apoE缺失表型是否可以通过与apoE2、apoE3或野生型小鼠的星形胶质细胞孵育来“拯救”。在一些实验中，星形胶质细胞将被ABeta1-42处理。

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.