Intra- and extra-cellular cholesterol transport systems during brain repair and synaptic remodelling in the adult rodent brain

我们的团队已经记录了一种优雅的机制，通过这种机制，受伤的大脑使用死亡/垂死细胞释放的胆固醇和磷脂来为幸存的神经元提供适当重建新突触网络所需的脂质。我们称这个过程为胆固醇循环/重塑级联。我们的团队已经证明，胆固醇和磷脂的细胞外转运需要载脂蛋白E（apoE），一种高功能的脂质转运蛋白， 在啮齿类动物中，对于脑损伤或神经毒性损伤的突触网络的替换是必要的。作为响应，发现识别apoE-脂蛋白复合物（即HDL）的细胞表面受体介导形成新芽和新突触所需的胆固醇的内化和再分布。在敲除小鼠中，脑apoE或其主要受体的完全缺失显示出损害i）随年龄的突触完整性，ii）损伤诱导的突触恢复和iii）认知性能。不幸的是，我们对这个级联反应的启动知之甚少;即胆固醇和磷脂从细胞内星形胶质细胞隔室向apoE-脂蛋白（HDL）装配线的运输和包装。 细胞内产生的胆固醇通过一系列称为ATP结合盒（ABC）复合物的转运蛋白在外周和CNS中转运。ABC转运蛋白（ABCA和ABCG亚类）参与磷脂和/或胆固醇跨质膜的转运。ABCG 1的产生与apoE的关系 在外周巨噬细胞中也发现了分泌，但在重塑脑中没有发现。 假设：作为对神经元损失的响应，脑胆固醇主要由星形胶质细胞动员，其中ABCA 1、A7或G1的协调表达促进其向细胞表面移动 其中载脂蛋白E等待催化脂质转移到富含载脂蛋白E的HDL复合物。 这些脂蛋白随后被用于喂养需要大量脂质的表达apoE受体的邻近神经元，以重建突触网络并扩展现有网络。 目的：我们建议通过检查i）体内和体外胶质细胞中ABC转运系统对脂质的细胞内转运，ii）在存在/不存在ABC转运蛋白的情况下含apoE的脂蛋白复合物的组装，iii）apoE和ABC与细胞表面脂质的相互作用，iv）apoE-HDL复合物通过脂蛋白受体被神经元结合和内化。 预期结果：我们相信，这些研究将为我们提供至关重要的见解，如导致突触修复激活的分子事件级联和参与脑修复的关键脂质物质的递送 机制等

Our team has documented an elegant mechanism by which the injured brain uses the cholesterol and phospholipids released from dead/dying cells to feed surviving neurons with the lipids which are required for the proper rebuilding new synaptic networks. We call this process the cholesterol recycling/remodeling cascade. Our team has already demonstrated that the extracellular transport of cholesterol and phospholipids by apolipoprotein E (apoE), a highly functional lipid transporter, is required and necessary for the replacement of synaptic networks in response to brain injury or, neurotoxic insult in rodents. In response, cell-surface receptors that recognize apoE-lipoprotein complexes (i.e. HDL) were found to mediate the internalization and redistribution of the cholesterol required for the formation of new sprouts and new synapses. The complete absence of brain apoE or, its main receptor, in knockout mice was shown to compromise i) synaptic integrity with age, ii) lesion-induced synaptic recovery and iii) cognitive performance. Unfortunately, we know very little about the initiation of this cascade; i.e. the transport and packaging of cholesterol and phospholipids from intracellular astroglial compartments towards the apoE-lipoprotein (HDL) assembly line. Cholesterol produced intracellularly is transported via a series of transporters called ATP-Binding cassette (ABC) complexes in both the periphery and in the CNS. The ABC transporters (ABCA and ABCG subclasses) are involved in the transport of phospholipids and/or cholesterol across the plasma membrane. A relationship between ABCG1 production and apoE secretion has also been found in peripheral macrophages but not in the remodelling brain. Hypothesis: In response to neuronal loss, brain cholesterol is mobilized primarily by astrocytes where the coordinated expression of ABCA1, A7 or G1 facilitates its movement toward the cell surface where apolipoprotein E awaits to catalyze the transfer of lipids to apoE-rich HDL complexes.  These lipoproteins are subsequently used to feed neighboring neurons expressing apoE-receptors in need of large amount of lipids to rebuild synaptic networks and to expand existing ones. Objectives: We propose to dissect the molecular cascade regulating cholesterol recycling by examining i) the intracellular transport of the lipids by the ABC transport system in glial cells both in vivo and in vitro, ii) the assembly of apoE-containing lipoprotein complexes in presence/absence of ABC transporters, iii) the interaction of apoE and ABCs with cell surface lipids and, iv) the binding and internalization of the apoE-HDL complex via lipoprotein receptors by neurons. Expected Outcome: We believe that these studies will provide us with crucial insights as to the cascade of molecular events leading to the activation of synaptic repair and the delivery of key lipid species involved in brain repair mechanisms.