Role of apolipoprotein E in age and amyloid beta related neuronal dysfunction

 描述（由申请人提供）：载脂蛋白E是脑内脂质代谢的主要调节剂，并被认为是形成突触生物学的重要因素。此外，二十多年前，APOE 4等位基因被确定为散发性阿尔茨海默病（AD）的最强遗传风险因素。该K99/00申请提出检查APOE在神经退行性背景下的复杂影响，试图消除其作为AD中的APOE神经毒性作用的必要催化剂的作用，同时作为胆固醇和 磷脂是突触的载体，并且在生理环境中作为NMDAR依赖性钙内流的调节剂。利用最近开发的尖端技术，如清醒在体内多光子钙成像，阵列断层扫描，和突触神经小体纯化与lipidomics检测突触脂质含量，我将询问APOE对ApoE介导的神经毒性在突触的功能相关性。在这个奖项的指导阶段，我将解决如何APOE基因破坏是对阿托伐他汀毒性保护。我们的初步数据表明，载脂蛋白E空减少寡聚腺苷酸对钙稳态异常的影响，无论是在设置的转基因小鼠进行清醒的体内钙成像和野生型小鼠暴露于生物产生的寡聚腺苷酸。为了直接检验APOE无效的有益作用是由于APOE-A β相互作用的破坏这一假设，我们将通过腹膜内注射特异性竞争性抑制剂A β 12 - 28 P来解离AD转基因小鼠中的APOE/A β复合物。通过静脉注射AAV 9使用新的基因转移方法，独立阶段将恢复不同APOE亚型的表达，并评估每种变体如何调节神经元活性和突触完整性。我们已经成功地在单一外周输注AAV 9后转导全脑星形胶质细胞。然后，我们将在APOE缺失或APP/APOE缺失动物的脑中表达APOE 2、APOE 3和APOE 4。我们假设，每个变体将不同的形状APOE的生理功能和APOE神经毒性。特别是，我们预计APOE 2将作为神经系统的神经保护因子并限制AAPO 4突触毒性，而APOE 4将对突触有害。这些实验将为我的独立事业奠定基础。通过与海曼博士一起学习结构和功能性突触测定（我的导师），结合新获得的生化组学方法与博士格什顿（脂质组学和蛋白质组学分析专家）和现代基因转移方法与布雷克菲尔德博士，我将在一个很好的位置，以利用新发现的神经退行性疾病，并启动一个独立的职业生涯，既进行仔细的结构，功能和生物化学表型，以及创建和操纵神经系统疾病的遗传模型。

 DESCRIPTION (provided by applicant): Apolipoprotein E is a major regulator of intracerebral lipid metabolism and has been implicated as an important actor shaping synaptic biology. In addition, more than two decades ago, the APOE4 allele was identified as the strongest genetic risk factor for the sporadic form of Alzheimer's disease (AD). This K99/00 application proposes to examine the complex impact of APOE in a neurodegenerative context, trying to disambiguate its role as a necessary catalyst of Aß neurotoxic effects in AD, while acting as a cholesterol and phospholipids carrier to the synapse and as a regulator of NMDAR-dependent calcium influx in a physiological context. Taking advantage of recently developed cutting-edge techniques such as awake in vivo multiphoton calcium imaging, array tomography, and synaptoneurosome purification with lipidomics assays to examine synaptic lipid content, I will interrogate the functional relevance of APOE towards Aß mediated neurotoxicity at the synapse. During the mentored phase of this award, I will address how APOE gene disruption is protective against Aß toxicity. Our preliminary data suggest that APOE null diminishes oligomeric Aß's effects on calcium dyshomeostasis, both in the setting of transgenic mice undergoing awake in vivo calcium imaging and in wild type mice exposed to biologically produce oligomeric Aß. To directly test the hypothesis that the beneficial effects of APOE null are due to disruption of APOE-Aß interactions, we will dissociate the APOE/Aß complexes in AD transgenic mice by intraperitoneal injection of a specific competitive inhibitor, Aß12-28P. Using a novel gene transfer approach by intravenous injection of AAV9, the independent phase will then restore the expression of the different APOE isoforms and evaluate how each variant modulate neuronal activity and synaptic integrity. We have already succeeded in transducing astrocytes brain wide after a single peripheral infusion of AAV9. We will then express APOE2, APOE3 and APOE4 in the brains of APOE-null or APP/APOE-null animals. We postulate that each variant will differentially shape APOE physiological functions and Aß neurotoxicity. In particular, we expect that APOE2 will act as a neuroprotective factor on the neural system and limit Aß synaptotoxicity, while APOE4 will be detrimental at the synapse. These experiments will lay the groundwork for my independent career. By learning structural and functional synaptic assays with Dr. Hyman (my mentor), combined with newly acquired biochemical -omics approaches with Dr. Gerszten (an expert in lipidomics and proteomics analyses) and modern gene transfer approaches with Dr. Breakefield, I will be in an excellent position to take advantage of emerging discoveries in neurodegenerative diseases and to launch an independent career to both carry out careful structural, functional, and biochemical phenotyping and to create and manipulate genetic models of neurological diseases.