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Brain lipid metabolism/alpha-synuclein gene-abated mice

脑脂质代谢/α-突触核蛋白基因减弱的小鼠

基本信息

批准号：
6791847
负责人：
ERIC James MURPHY
金额：
$ 4.89万
依托单位：
UNIVERSITY OF NORTH DAKOTA
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-06-01 至 2005-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6791847
关键词：
Lewy body biological transport biomarker brain cell line fatty acid binding protein fatty acid metabolism gene expression high performance liquid chromatography intracellular transport laboratory mouse lipid metabolism neurons protein structure function synapses thin layer chromatography transfection

项目摘要

Although alpha-synuclein is highly expressed in neuronal synapses, the functional role of this protein remains unclear. Elucidating the physiological function of alpha-synuclein is important as this protein is implicated as a causative factor in familial Parkinson disease (PD), primarily because of its association with Lewy bodies, a pathological marker of PD. In cell lines transfected with either mutant alpha- synuclein or wildtype alpha-synuclein, wildtype but not mutant alpha- synuclein associate with lipid droplets, suggesting that the native protein may be involved in lipid trafficking or metabolism. In a parallel set of studies using alpha-synuclein gene-abated mice, there was a large decrease in the steady-state mass of a number of different phospholipids in both whole brain and isolated synaptosomes compared to control mice. Further, there was a significant decrease in the amount of docosahexaenoic acid in a number of different phospholipid classes, suggesting a decrease in uptake and trafficking of docosahexaenoic acid. These data suggest a down-regulation of neuronal lipid metabolism. Because fatty acid binding proteins can alter phospholipid metabolism and alter phospholipid acyl chain composition, I submit the following hypothesis: alpha-Synuclein directly affects fatty acid uptake and targeting in neurons, essentially functioning as a neuronal fatty acid binding protein. However, this hypothesis that alpha-synuclein has a role in neuronal lipid uptake, trafficking, and metabolism needs to be further elucidated using a combination of whole animal and cell culture studies. The following specific aims are designed to address the potential role of alpha-synuclein in brain lipid metabolism using alpha- synuclein gene abated mice, neuronal cultures derived from these mice, and stably transfected H293 cells expressing mutant and wildtype alpha-synuclein. These aims are: 1. Determine the effect of alpha-synuclein on the uptake and subsequent metabolism of [3H]-palmitic, [3H]-arachidonic, and [3H]- docosahexaenoic acid in brains of control and alpha-synuclein gene- abated mice in vivo using steady-state tracer kinetic analysis. 2. Using cells derived from control and gene-abated mice as well as stably transfected cell lines, determine the effect of alpha-synuclein on cellular fatty acid uptake, metabolism, and trafficking.

虽然α-突触核蛋白在神经元突触中高度表达，但该蛋白的功能作用仍不清楚。阐明α-突触核蛋白的生理功能是重要的，因为这种蛋白质被认为是家族性帕金森病（PD）的致病因素，主要是因为它与路易体（PD的病理标志物）相关。在用突变体α-突触核蛋白或野生型α-突触核蛋白转染的细胞系中，野生型而非突变体α-突触核蛋白与脂滴缔合，表明天然蛋白质可能参与脂质运输或代谢。在使用α-突触核蛋白基因消减小鼠的一组平行研究中，与对照小鼠相比，全脑和分离的突触体中许多不同磷脂的稳态质量大幅降低。此外，在许多不同的磷脂类别中，二十二碳六烯酸的量显著减少，表明二十二碳六烯酸的摄取和运输减少。这些数据表明神经元脂质代谢的下调。由于脂肪酸结合蛋白可以改变磷脂代谢和改变磷脂酰基链的组成，我提出以下假设：α-突触核蛋白直接影响脂肪酸的摄取和神经元的靶向，本质上是作为一个神经元的脂肪酸结合蛋白。然而，这一假设，α-突触核蛋白在神经元脂质摄取，运输和代谢的作用，需要进一步阐明使用整个动物和细胞培养研究的组合。以下具体目的旨在使用α-突触核蛋白基因缺失的小鼠、源自这些小鼠的神经元培养物和表达突变型和野生型α-突触核蛋白的稳定转染的H293细胞来阐明α-突触核蛋白在脑脂质代谢中的潜在作用。这些目标是：1.使用稳态示踪剂动力学分析确定α-突触核蛋白对对照小鼠和α-突触核蛋白基因缺失小鼠脑中[3 H]-棕榈酸、[3 H]-花生四烯酸和[3 H]-二十二碳六烯酸的摄取和随后代谢的影响。2.使用来自对照和基因消减小鼠以及稳定转染细胞系的细胞，确定α-突触核蛋白对细胞脂肪酸摄取、代谢和运输的影响。