CHOLESTEROL AND NEURODEGENERATION

描述：（摘自申请人摘要）本研究的中心目标

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.