CHOLESTEROL AND NEURODEGENERATION

描述：(逐字摘自申请者的摘要)我们的中心目标是 格兰特将研究功能失调的胆固醇处理作为 几种神经退行性疾病的细胞发病机制。 越来越多的证据表明，胆固醇运输和 新陈代谢可能是几种神经退行性疾病的重要特征。 我们的实验室专注于两种蛋白质，载脂蛋白D(APOD)和 最近克隆的Niemann-Pick C蛋白1(NCP1)在 胆固醇在神经胶质细胞和外周细胞中的运输。在尼曼-皮克 C病(NP-C)，NPC1突变导致细胞胆固醇积聚 和进行性神经退行性变。我们发现，在这种紊乱中， 细胞内胆固醇的积累与高水平的APOD有关 在胶质细胞中的表达。我们还发现大脑中APOD水平升高 阿尔茨海默氏症和亨廷顿氏症。APOD在卵巢癌中的表达增强 这些疾病主要发生在关系密切的少突胶质细胞中。 与退化的神经元有关。使用共聚焦免疫荧光法 显微镜和一组针对功能域的抗肽抗体 NPC1，我们在细胞质小泡中发现了NPC1，这些小泡由 溶酶体相关膜糖蛋白2(LAMP2)。这些NPC1阳性 囊泡明显不同于充满胆固醇的溶酶体，后者是一种 是NP-C细胞的标志。另一方面，我们发现APOD特别是 与充满胆固醇的溶酶体共定位。此外， 通过可逆地捕获胆固醇在正常细胞中模拟NP-C表型 溶酶体导致胆固醇、NPC1和APOD在相同的囊泡中积累。 这些对NPC1和APOD细胞内定位的研究表明 未被识别的囊泡转运途径控制着后内吞 细胞胆固醇的分布。通过荧光共振能量转移 研究发现，APOD能与胆固醇结合并相互作用 特别是apoE4，而不是apoE2和apoE3。此外，还可以通过 APOD与apoE4结合可破坏APOD与配体的结合。最后，展示了APOD 在缺乏神经营养物质的情况下对培养的神经元产生深刻的神经营养效应 脂类或其他化合物。这些观察结果为研究提供了基础 在本申请中提出的，旨在进一步定义 胆固醇代谢、NPC1、APOD和神经退行性变。我们的具体目标 主要内容有：(1)研究囊泡转运的细胞内途径 NPC1和APOD的表达及其与细胞胆固醇的关系 新陈代谢(2)研究人与人之间物理作用的本质 APOD和apoE，并确定APOD之间是否也存在相互作用 和NPC1(3)调查APOD发挥作用的机制(S) 神经营养因子及其与胆固醇转运的关系(4) 研究神经胶质细胞是否调节神经元胆固醇代谢，以及 若有，机制为何；及(5)探讨发病机制及 NPC1和APOD调控紊乱的功能后果 神经退行性疾病。

DESCRIPTION: (Verbatim from the Applicant's Abstract) The central aim of our grant is to study dysfunctional cholesterol processing as a key feature in the cellular pathogenesis of several neurodegenerative disorders. Increasing evidence suggests that disturbances in cholesterol trafficking and metabolism may be an important feature of several neurodegenerative disorders. Our laboratory has focussed on two proteins, apolipoprotein D (apoD) and the recently cloned Niemann-Pick C protein 1 (NCP1) that are important in cholesterol transport in both neuroglial and peripheral cells. In Niemann-Pick C disease (NP-C), mutations in NPC1 lead to cellular cholesterol accumulation and progressive neurodegeneration. We have found that in this disorder, intracellular accumulation of cholesterol is linked to high levels of apoD expression in glia. We have also found elevated levels of apoD in the brain in Alzheimer's and Huntington's diseases. The increased expression of apoD in these disorders was predominantly in oligodendroglia that were closely associated with degenerating neurons. Using confocal immunofluorescence microscopy and a panel of anti-peptide antibodies to functional domains of NPCI, we have found NPC1 in cytoplasmic vesicles that are delineated by the lysosome associated membrane glycoprotein 2 (LAMP2). These NPC1 positive vesicles are clearly distinct from the cholesterol filled lysosomes that are a hallmark of NP-C cells. On the other hand, we found that apoD specifically colocalizes with the cholesterol filled lysosomes. Furthermore, drugs which mimic the NP-C phenotype in normal cells by reversibly trapping cholesterol in lysosomes cause cholesterol, NPC1 and apoD to accumulate in the same vesicles. These studies of the intracellular localization of NPC1 and apoD suggest an unrecognized vesicular trafficking pathway governing retroendocytic distribution of cellular cholesterol. By fluorescence resonance energy transfer studies, we have found that apoD binds cholesterol and that it interacts specifically with apoE4, but not apoE2 and apoE3. Furthermore, interaction of apoD with apoE4 disrupts ligand binding by apoD. Finally, apoD has been shown to induce profound neurotrophic effects on cultured neurons in the absence of lipids or other cofracts. These observations provide the basis for the studies proposed in this application that aim to further define the link between cholesterol metabolism, NPC1, apoD and neurodegeneration. Our specific aims are: (1) To investigate the intracellular pathway for the vesicular trafficking of NPC1 and apoD and to determine how it relates to cellular cholesterol metabolism (2) To investigate the nature of the physical interaction between apoD and apoE, and determine whether there is also an interaction between apoD and NPC1 (3) To investigate the mechanism(s) through which apoD acts as a neurotrophic factor and how it relates to cholesterol trafficking (4) To investigate whether glial cells regulate neuronal cholesterol metabolism, and if so, by what mechanism, and (5) To investigate the pathogenesis and functional consequence of disordered NPC1 and apoD regulation in neurodegenerative disorders.