CHOLESTEROL AND NEURODEGENERATION

描述：（来自申请人的摘要的逐字）我们的中心目标是 格兰特是研究功能失调的胆固醇加工作为一个关键特征， 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 代谢可能是几种神经变性疾病的重要特征。 我们的实验室专注于两种蛋白质，载脂蛋白D（apoD）和 最近克隆的尼曼-皮克C蛋白1（NCP 1）， 神经胶质细胞和外周细胞中的胆固醇转运。尼曼匹克 C疾病（NP-C），NPC 1突变导致细胞胆固醇蓄积 和进行性神经退化我们发现在这种紊乱中， 胆固醇的细胞内积累与高水平的apoD有关 在胶质细胞中的表达。我们还发现， 老年痴呆症和亨廷顿舞蹈症。apoD表达增加， 这些疾病主要发生在少突胶质细胞中， 与神经元退化有关使用共聚焦免疫荧光 显微镜和一组抗肽抗体的功能结构域， NPCI，我们已经在细胞质囊泡中发现了NPC 1， 溶酶体相关膜糖蛋白2（LAMP 2）。NPC 1阳性 囊泡与充满胆固醇的溶酶体明显不同， NP-C细胞的标志。另一方面，我们发现apoD特异性地 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，模拟正常细胞中的NP-C表型。 溶酶体导致胆固醇、NPC 1和apoD在相同的囊泡中积累。 NPC 1和apoD的细胞内定位的这些研究表明， 未识别的囊泡运输途径控制后内吞 细胞胆固醇的分布。通过荧光共振能量转移 研究中，我们发现apoD与胆固醇结合， 特别是apoE 4，而不是apoE 2和apoE 3。此外， apoD与apoE 4破坏apoD与配体的结合。最后，apoD已经被证明 在缺乏的情况下诱导对培养的神经元的深刻的神经营养作用， 脂质或其它组分。这些观察结果为研究提供了基础 在本申请中提出了旨在进一步定义 胆固醇代谢、NPC 1、apoD和神经变性。我们的具体目标 主要内容有：（1）研究囊泡运输的细胞内途径 NPC 1和apoD之间的关系，并确定其与细胞胆固醇的关系 （2）研究代谢之间的物理相互作用的性质 apoD和apoE，并确定apoD之间是否也存在相互作用 （3）探讨apoD作为一种蛋白质受体的作用机制。 神经营养因子及其与胆固醇运输的关系（4） 研究神经胶质细胞是否调节神经元胆固醇代谢， 如果是，通过什么机制;（5）研究发病机制， NPC 1和apoD调节紊乱的功能后果 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.