Mechanisms of Apolipoprotein E Isoform Conferred Susceptibility and Resistance to Alzheimer's Disease

散发性阿尔茨海默病（AD）的易感性主要受载脂蛋白E基因型的调节。APOE ε4等位基因的单个拷贝使AD风险增加约3倍，2个ε4拷贝使AD风险增加约15倍，而与ε3纯合子相比，ε2等位基因使AD风险减半。ε4有害作用的病因学和ε2赋予的保护作用均未得到充分解释。以往的研究主要集中在apoE在Aβ沉积中的作用，发现apoE以异构体依赖的方式结合Aβ肽，促进Aβ组装成淀粉样纤维，并促进实质斑块和血管沉积的形成，其顺序为E4>>E3>E2，而APP转基因小鼠中ApoE基因敲除可阻止纤维状Aβ沉积的形成。除了对Aβ的纤维化和沉积的催化作用外，有证据表明apoE亚型对Aβ从脑细胞外（或间质）空间的清除、小胶质细胞炎症反应的调节以及突触可塑性和神经元网络功能的调节具有特异性作用，这些都可能有助于APOE基因型对AD易感性的差异作用。虽然APOE基因型与Aβ从脑间质间隙清除的可变速率之间的关系已被公认，但apoE亚型如何差异地参与这一过程以及它是否依赖于apoE/Aβ的直接结合仍存在争议。我们初步的微透析实验表明，在Tg 2576和PDAPP小鼠的脑间质液（ISF）中，apoE与Aβ之间有很大程度的结合，而应用特异性apoE/Aβ拮抗剂，可显著增加未结合的Aβ水平。基于这些数据，我们假设apoE亚型在ISF中与Aβ的结合存在差异，并且apoE/Aβ相互作用的药理学靶向可能增强Aβ清除并防止Aβ寡聚化。将在Specific Aim I中使用APPSW/PS1 dE 9/apoE-TR小鼠（APP/E-TR）对各种人APOE等位基因进行小鼠Apoe基因的靶向置换（TR），并对APP/E-/-小鼠进行各种体内微透析实验，探索该假设。Specific Aim II将研究APOE基因型如何影响炎症小胶质细胞反应。我们的初步研究表明，APP/E4小鼠中的小胶质细胞活化对Aβ沉积和抗A β被动免疫的反应比APP/E2和APP/E3小鼠更大。因此，我们推测，无效的Aβ吞噬作用和有害的小胶质细胞活化可能是ε4等位基因赋予AD易感性的独立机制。将通过从不同年龄的apoE-TR和apoE-/-小鼠以及APP/E-TR和APP/E-/-小鼠中分离的原代CNS小胶质细胞的功能性吞噬作用和转录组学研究来探索这一假设。转录组评估将包括促炎细胞因子和抗炎细胞因子的RT-qPCR以及无偏倚的RNA-Seq，以鉴定小胶质细胞中apoE亚型差异激活的信号通路。Specific Aim III将使用老年apoE-TR小鼠和APP/E-TR小鼠研究apoE亚型如何以Aβ非依赖性和Aβ依赖性方式调节Reelin-Apoer 2/Vldlr信号传导，从而调节突触可塑性和神经元网络完整性。

Susceptibility to sporadic Alzheimer's disease (AD) is foremost modulated by APOE genotype. A single copy of the APOE ε4 allele endows a ~3 fold increase in AD risk, and 2 ε4 copies effect a ~15 fold increase, while an ε2 allele halves AD risk compared to ε3 homozygotes. Neither etiology of ε4 deleterious effect nor ε2 conferred protection is fully explained. Past work primarily focused on the role of apoE in Aβ deposition showed thatapoE in isoform-dependent fashion binds Aβ peptides, facilitates assembly of Aβ into amyloid fibrils and promotes formation of parenchymal plaques and vascular deposits in the rank order of E4>>E3>E2, while the Apoe gene knockout in APP transgenic mice precludes formation of fibrillar Aβ deposits. In addition to the well-studied catalytic effect on fibrillization and deposition of Aβ, there is evidence of apoE isoform-specific effect on the clearance of Aβ from the brain extracellular (or interstitial) space, modulation of microglia inflammatory response, and regulation of synaptic plasticity and neuronal network function, which all may contribute to the differential effect of APOE genotype on AD susceptibility. Though relationship between APOE genotype and variable rate of Aβ clearance from the brain interstitial space is well recognized, how apoE isoforms differentially engage this process and whether it depends on direct apoE/Aβ binding remains disputed. Our preliminary microdialysis experiments indicate substantial degree of binding between apoE and Aβ in the brain interstitial fluid (ISF) of Tg2576 and PDAPP mice while application of specific apoE/Aβ antagonist, dramatically increases unbound Aβ level. Based on these data we hypothesize that apoE isoforms differentially bind Aβ in the ISF and that pharmacological targeting of the apoE/Aβ interaction may enhance Aβ clearance and prevent Aβ oligomerization. This hypothesis will be explored in Specific Aim I using APPSW/PS1dE9/apoE-TR mice (APP/E-TR) with targeted replacement (TR) of the mouse Apoe gene for various human APOE alleles and APP/E-/- mice subjected to various in vivo microdialysis experiments. Specific Aim II will investigate how APOE genotype influences inflammatory microglia response. Our preliminary studies show greater microglia activation in APP/E4 mice in response to Aβ deposition and anti-Aβ passive immunization than in APP/E2 and APP/E3 mice. We thus hypothesize that ineffective Aβ phagocytosis and deleterious microglia activation can be an independent mechanism of ε4 allele conferred susceptibility to AD. This hypothesis will be explored by functional phagocytosis and transcriptomics studies of primary CNS microglia isolated from apoE-TR and apoE-/- mice of various ages and from APP/E-TR and APP/E-/- mice. Transcriptome assessment will include RT-qPCR of pro- and anti-inflammatory cytokines and unbiased RNA-Seq to identify signaling pathways differentially activated by apoE isoforms in microglia. Specific Aim III, using aged apoE-TR mice and APP/E-TR mice, will investigate how apoE isoforms in Aβ-independent and Aβ-dependent way modulate Reelin-Apoer2/Vldlr signaling, which regulates synaptic plasticity and neuronal network integrity.