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

散发性阿尔茨海默病(AD)的易感性首先受APOE基因的调节。与ε3纯合子相比，单拷贝ε4等位基因可使AD风险增加约3倍，2个ε4拷贝可使AD风险增加约15倍，而ε2等位基因可使AD风险减半。ε4有害效应的病因和ε2赋予的保护作用都没有得到充分的解释。过去的工作主要集中在载脂蛋白E在Aβ沉积中的作用，结果表明，载脂蛋白E以异构体依赖的方式结合Aβ多肽，促进Aβ组装成淀粉样纤维，并促进E4&gt；&gt；E3&gt；E2级实质斑块和血管沉积的形成，而APP转基因小鼠的apoE基因敲除阻止了纤维状Aβ沉积的形成。除了对Aβ的纤化和沉积的催化作用已有研究外，还有证据表明载脂蛋白E对Aβ从脑细胞外(或间质)间隙的清除、小胶质细胞炎症反应的调节、突触可塑性和神经元网络功能的调节等方面具有特异性作用，这些都可能是载脂蛋白E基因对AD易感性的差异性效应的原因。虽然载脂蛋白E基因与Aβ从脑间质清除的可变率之间的关系已被很好地认识，但载脂蛋白E亚型如何不同地参与这一过程以及它是否依赖于载脂蛋白E/Aβ的直接结合仍存在争议。我们的初步微透析实验表明，Tg2576和PDAPP小鼠脑间质液中的apoE和Aβ之间有相当程度的结合，而应用特定的apoE/Aβ拮抗剂，显著增加了未结合的Aβ水平。基于这些数据，我们假设apoE亚型在ISF中以不同的方式结合Aβ，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。这一假说将在特定的目标I使用APPSW/PS1dE9/apoE-tr小鼠(APP/E-tr)和接受各种体内微透析实验的APP/E-/-小鼠(APP/E-/-小鼠)进行研究，其中APP/E-tr小鼠具有各种人类APOE等位基因的定向替换(Tr)。《特定目的II》将研究APOE基因如何影响炎性小胶质细胞反应。我们的初步研究表明，APP/E4小鼠对Aβ沉积和抗Aβ被动免疫的反应比APP/E2和APP/E3小鼠更大的小胶质细胞激活。因此，我们推测无效的Aβ吞噬作用和有害的小胶质细胞激活可能是ε4等位基因导致AD易感性的独立机制。这一假说将通过从不同年龄的apoE-tr和apoE-/-小鼠以及APP/E-tr和APP/E-/-小鼠分离的原代CNS小胶质细胞的功能吞噬和转录学研究来探索。转录组评估将包括促炎和抗炎细胞因子的RT-qPCR和无偏见的RNA-Seq，以确定小胶质细胞中apoE亚型差异激活的信号通路。具体目的III，使用老年apoE-tr小鼠和APP/E-tr小鼠，将研究apoE亚型在β非依赖和β依赖的方式中如何调节Reelin-ApoER2/Vldlr信号，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.