Project 3 - Animal Models Examining Neurovasculature

项目3 -项目概要/摘要 血脑屏障（BBB）和神经血管单位（NVU）的每种细胞成分功能障碍 - 血管细胞、神经胶质细胞和神经元-在实验中与阿尔茨海默病（AD）的演变有关， 影像学、病理学和流行病学研究。这些发现导致了一种新兴的“神经血管” 该假说认为脑血管功能障碍导致认知能力下降和痴呆 在AD中。项目3支持P01的总体主题，重点是载脂蛋白E（APOE）基因，主要遗传学基因， AD的危险因素，通过实验在细胞、分子水平上询问BBB/神经血管假说， 和系统水平，并以同种型特异性和性别特异性的方式使用新的APOE 3和APOE 4knock， 在（KI）flox/flox小鼠（E3 F; E4 F）中，有和没有来自星形胶质细胞和周细胞的apoE缺失（ BBB相关apoE），以及有和无Aβ和tau病理。根据我们的试点和公布的数据， 假设1）破坏血脑屏障（BBB）蛋白质-蛋白质相互作用（PPI）信号网络 潜在的内皮屏障破坏和血脑屏障功能障碍将先于并预测突触和神经元 在新的APOE KIF细胞系中，APOE 4相对于APOE 3驱动的功能障碍和行为变化， 和2）3 K3 A激活蛋白C（APC），APC的细胞信号类似物，将纠正 BBB和PSD 95突触PPI信号传导网络失调，将保护BBB和神经元功能， 减少AD病理学，从而在APOE/AD模型中延迟发作并减缓疾病进展。到 为了验证我们的假设，我们将单独使用新的E3 F和E4 F小鼠，并与i）特异性 在星形胶质细胞和周细胞中表达Cre重组酶，以确定细胞特异性缺失关键蛋白的影响。 BBB相关的apoE源和ii）与APP/PS1-21小鼠和P301 S tau小鼠研究相互作用 Aβ和tau蛋白通路。为了评估BBB/血管功能障碍，我们将使用：用途：1）先进的分子生物学技术， 通过同时测量CSF中的BBB/NVU细胞特异性和Aβ和tau生物标志物评估BBB 和血浆; 2）新的BBB蛋白质组学分析; 3）BBB和CBF的先进MRI评估;和4） 通过组织分析验证生物流体生物标志物。为了评估突触和神经元功能障碍，我们将 用途：5）PSD 95相互作用分析;和6）Papez回路/DMN中海马通路的病毒带追踪; 6）行为学和7）神经病理学分析。我们将评估apoE等位基因特异性对BBB的影响， 通过生物流体、组织和成像生物标志物、BBB和PSD 95 PPI分析、海马 在不同年龄有和没有apoE缺失的APOE KIF小鼠（雄性，雌性）中的病毒带追踪和行为 从星形胶质细胞和周细胞（AIM 1），与APP/PS1-21和P301 S tau小鼠交叉与AD病理学相关 (AIM 2），并将在不同年龄的APOE KIF小鼠（雄性，雌性）中单独评估3 K3 A-APC治疗， 与APP/PS1-21和P301 S tau小鼠（AIM 3）杂交。项目3将促进我们对 在细胞和分子水平上以apoE亚型特异性和性别特异性方式研究AD的发病机制。

PROJECT 3 – PROJECT SUMMARY/ ABSTRACT Dysfunctionin the blood-brain barrier (BBB) and each of the cellular components of the neurovascular unit (NVU) – vascular cells, glial cells, and neurons – has been linked to Alzheimer’s disease (AD) evolution in experimental, imaging, pathological, and epidemiological studies. These findings have led to an emerging ‘neurovascular hypothesis’ of AD which holds that cerebrovascular dysfunction contributes to cognitive decline and dementia in AD. Project 3 supports the overall theme of P01 focused on apolipoprotein E (APOE) gene, the major genetic risk factor for AD, by interrogating the BBB/neurovascular hypothesis experimentally at the cellular, molecular and systems levels and in an isoform-specific and gender-specific fashion using novel APOE3 and APOE4knock in (KI)flox/flox mice (E3F; E4F) with and without apoE deletion from astrocytes and pericytes (the key sources of BBB-associated apoE), and with and without Aβ and tau pathology. Based on our pilot and published data we hypothesize that 1) Disrupted blood-brain barrier (BBB) protein-protein interaction (PPI) signaling networks underlying endothelial barrier disruption and BBB dysfunction will precede and predict synaptic and neuronal dysfunction and behavioral changes driven by APOE4 relative to APOE3 in new APOE KIF lines both without and with AD pathology; and 2) 3K3A-activated protein C (APC), a cell signaling analog of APC, will correct dysregulated BBB and PSD95 synaptic PPI signaling networks, will protect BBB and neuronal function, and diminish AD pathology, thereby delaying onset and slowing down disease progression in APOE/AD models. To test our hypothesis, we will use novel E3F and E4F mice alone and crossed i) with Cre lines that specifically express Cre recombinase in astrocytes and pericytes to determine the effects of cell-specific deletion of the key BBB-associated apoE source(s) and ii) with APP/PS1-21 mice and P301S tau mice to investigate interactions with Aβ and tau pathways. To evaluate BBB/vascular dysfunction we will use: 1) advanced molecular assessment of the BBB by simultaneously measured BBB/NVU cell-specific and Aβ and tau biomarkers in CSF and plasma; 2) a novel BBB proteomics analysis; 3) advanced MRI assessment of BBB and CBF; and 4) validation of biofluid biomarkers by tissue analysis. To evaluate synaptic and neuronal dysfunction we will use: 5) PSD95 interactomeanalysis; and 6) viral tract-tracing of hippocampal pathways in the Papez circuit/DMN; and 6) behavior and 7) neuropathology analysis. We will evaluate apoE-allele-specific effects on BBB and neuronal dysfunction by biofluid, tissue and imaging biomarkers, BBB and PSD95 PPI analysis, hippocampal viral tract-tracing, and behavior in APOE KIF mice(male, female) at different ages with and without apoE deletion from astrocytes and pericytes (AIM 1), crossed with APP/PS1-21 and P301S tau micein relation to AD pathology (AIM 2), and will evaluate 3K3A-APC therapy in APOE KIF mice (male, female) at different ages, alone and crossed with APP/PS1-21 and P301S tau mice (AIM 3). Project 3 will advance our understanding of the pathogenesis of AD at the cellular and molecular level in an apoE isoform-specific and gender-specific fashion.