Effects of APOE on neuronal network dynamics using multi-electrode arrays

载脂蛋白E （apoE）是一种在中枢神经系统中由神经胶质细胞合成的脂蛋白相关糖蛋白，负责脑内脂质转运。它还与多种中枢神经系统功能有关，包括：神经发育、炎症和突触可塑性。重要的是，APOE是阿尔茨海默病（AD）发展的最强遗传风险因素；它影响疾病发展的早期过程，并且已知在没有AD病理的情况下影响正常的脑功能。人类共有三种等位基因：APOE-ε2、APOE-ε3和APOE-ε4。与非ε4携带者相比，单个ε4等位基因的风险增加2- 3倍，而两个ε4等位基因的风险显著增加12倍。目前，apoe4相关AD风险的机制尚不清楚。我的首要假设是，APOE基因型在AD发病前会影响正常的脑功能，特别是通过影响大脑中有组织神经元群的发育和网络活动。本研究拟探讨：1)APOE基因型是否影响体外神经网络的功能发育；2) apoE如何影响活动诱导的网络动力学和兴奋性神经传递。为了测试这些目标，我将首先使用多电极阵列（MEA）评估培养中神经元网络的形成和功能特性（目的1）。MEA允许同时测量神经元培养系统中59个位点的电活动。皮层组织将从APOE靶向替代（TR）小鼠（一种表达人类APOE代替小鼠APOE的敲入模型）中收获，并在MEAs上培养，随着神经元网络的发育进行重复电生理记录。这个体外模型允许我测试每个apoE亚型对神经网络的贡献，有效地减少存在于其他基于细胞的系统中的混淆。我推测，与apoE2或apoE3相比，apoE4可能会对神经网络的发育产生负面影响，导致整体网络活动下降。接下来，我将利用化学长时程增强（cLTP）来评估APOE基因型对活性诱导的兴奋和神经网络动力学的影响（目的2）。MEAs允许在cLTP过程中对apoE水平和信号传导进行简单的药理学操作，从而从机制上确定apoE亚型及其受体在活性诱导的网络动力学中所起的作用。我假设apoE4对cltp诱导的网络动力学有负面影响。这个跨学科的建议代表了一种理解APOE基因型如何促进大规模网络形成和神经元群体活动的新方法。如果我的假设是正确的，那么从这些研究中获得的信息将对补偿apoe4相关的大脑变化的预防性治疗的发展至关重要。

DESCRIPTION (provided by applicant): Apolipoprotein E (apoE) is a lipoprotein-associated glycoprotein synthesized in the CNS by glial cells, responsible for lipid transport within the brai. It has also been linked to a variety of CNS functions, including: neurodevelopment, inflammation, and synaptic plasticity. Importantly, APOE is the strongest genetic risk factor for the development of Alzheimer's disease (AD); it affects processes early in disease development and is known to influence normal brain function in the absence of AD pathology. There are three common human alleles: APOE-ε2, APOE-ε3, and APOE-ε4. Compared to non-ε4 carriers, a single copy of the ε4 allele confers an increased risk of 2- to 3-fold, while two ε4 alleles dramatically increase AD risk by 12-fold. At present, the mechanisms underlying apoE4-associated AD risk are unknown. My overarching hypothesis is that APOE genotype affects normal brain function before AD pathogenesis, specifically by affecting the development and network activity of organized neuronal populations in the brain. The objective of this research proposal is to investigate: 1) whether APOE genotype affects the functional development of neuronal networks in vitro; and 2) how apoE affects activity-induced network dynamics and excitatory neurotransmission. To test these objectives, I will first assess the formation and functional properties of neuronal networks in culture using multi-electrode arrays (MEA) (Aim 1). The MEA allows simultaneous measurement of electrical activity at 59 sites in a neuronal culture system. Cortical tissue will be harvested from APOE Targeted Replacement (TR) mice, a knock-in model expressing human APOE in place of murine APOE, and cultured onto MEAs for repeated electrophysiological recording as neuronal networks develop. This in vitro model allows me to test the contribution of each apoE isoform on neuronal networks, effectively reducing confounds present in other cell-based systems. I hypothesize that apoE4 may negatively impact the development of neuronal networks and cause overall decreased network activity compared to apoE2 or apoE3. Next, I will assess the influence of APOE genotype on activity-induced excitation and neuronal network dynamics using chemical Long-Term Potentiation (cLTP) (Aim 2). MEAs allow simple pharmacologic manipulation of apoE levels and signaling during cLTP to mechanistically determine the role apoE isoforms and its receptors play in activity-induced network dynamics. I hypothesize that apoE4 negatively affects cLTP-induced network dynamics. This interdisciplinary proposal represents a novel approach to understanding how APOE genotype contributes to large scale network formation and neuronal population activity. If my hypotheses are correct, then the information gained by these studies will be critical for the development of preventative therapeutics that compensate for apoE4-related brain changes.