Amyloid Beta-Protein: ApoE and Cholesterol Homeostasis

本申请重点研究淀粉样β蛋白扰动星形胶质细胞内胆固醇和apoE稳态的机制以及对神经元功能产生的影响。胆固醇和β-淀粉样蛋白（ABeta）之间存在动态相互作用，这种蛋白质被认为是阿尔茨海默病引起的神经退行性变的重要促成因素。胆固醇水平调节淀粉样蛋白前体蛋白的表达和 ABeta1-42 的产生。相反，ABeta1-42 改变细胞胆固醇动态，特别是星形胶质细胞和神经元中的胆固醇运输。高尔基复合体在蛋白质和脂质运输中发挥着重要作用，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内的胆固醇分布，降低了质膜中的胆固醇水平并增加了 apoE 水平。这些结果使我们做出假设：ABeta1-42 会破坏星形胶质细胞中的胆固醇和 apoE 稳态，并且其影响是 apoE 同工型依赖性的。 ABeta1-42 作用的机制涉及高尔基复合体和质膜之间的小窝蛋白相关通路，以及依赖于 β 肾上腺素受体刺激、cAMP 形成和转录因子 AP-2 的 apoE 表达的转录调节。 ABeta1-42 扰动星形胶质细胞胆固醇和 apoE 稳态的后果是神经元胆固醇结构域和突触素/突触短蛋白复合物的改变。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复合体顺式内侧和反式区域中caveolin水平的影响。检查经 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 评估经 ABeta1-42 处理的星形胶质细胞的小凹和脂筏结构和功能。将使用来自表达人apoE2、apoE3、apoE4的小鼠、apoE缺失小鼠、caveolin-l缺失小鼠和人星形胶质细胞的原代星形胶质细胞。图 2：检查 ABeta1-42 对星形胶质细胞细胞器和条件培养基中 apoE 水平分布的影响。评估与 Abeta1-42 一起孵育的星形胶质细胞中的 apoE mRNA 丰度。评估 ABeta1-42 对 β 肾上腺素受体、cAMP 形成、转录因子 AP-2 水平和 AP-2 DNA 结合的影响。将使用表达人apoE2、apoE3和apoE4的小鼠星形胶质细胞。图3：使用与表达人apoE2、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筏蛋白、脂质和跨双层胆固醇分布。检查与表达人 apoE2、apoE3、apoE4 和 apoE 缺失小鼠的星形胶质细胞共培养的 C57BL/6 小鼠神经元中的突触素/突触短蛋白复合物。确定神经元 apoE4 和 apoE-null 表型是否可以通过与 apoE2、apoE3 或野生型小鼠的星形胶质细胞一起孵育来“拯救”。在一些实验中，星形胶质细胞将用 Aβ1-42 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.