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

散发性阿尔茨海默病（AD）的易感性主要由APOE基因型调节。APOE ε4等位基因的1个拷贝使AD风险增加3倍，2个拷贝使AD风险增加15倍，而ε2等位基因与ε3纯合子相比，AD风险减少一半。无论是ε4有害作用的病因还是ε2的保护作用都没有得到充分的解释。过去的研究主要集中在apoE在Aβ沉积中的作用，结果表明apoE以亚型依赖的方式结合Aβ肽，促进Aβ组装成淀粉样原纤维，并促进实质斑块和血管沉积的形成，其顺序为E4 b> >E3>E2，而在APP转基因小鼠中敲除apoE基因可阻止纤维Aβ沉积的形成。除了对Aβ纤维化和沉积的催化作用已得到充分研究外，有证据表明apoE亚型特异性影响Aβ从脑细胞外（或间质）空间的清除，调节小胶质细胞炎症反应，调节突触可塑性和神经网络功能，这些都可能导致apoE基因型对AD易感性的差异影响。虽然APOE基因型与脑间隙Aβ清除率之间的关系已得到广泛认可，但APOE亚型如何以不同的方式参与这一过程，以及它是否依赖于APOE /Aβ的直接结合仍存在争议。我们的初步微透析实验表明，Tg2576和PDAPP小鼠脑间质液（ISF）中apoE和Aβ的结合程度较高，而应用特异性apoE/Aβ拮抗剂可显著提高未结合的Aβ水平。基于这些数据，我们假设apoE亚型在ISF中与Aβ结合的差异，并且apoE/Aβ相互作用的药理学靶向可能增强Aβ的清除并防止Aβ寡聚化。这一假设将在Specific Aim I中通过APPSW/PS1dE9/ Apoe -TR小鼠（APP/E-TR）和APP/E-/-小鼠（APP/E- /-小鼠进行各种体内微透析实验）对小鼠Apoe基因进行靶向替代（TR）。特异性目的II将研究APOE基因型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。因此，我们假设无效的Aβ吞噬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。这一假设将通过从不同年龄的apoE- tr和apoE-/-小鼠以及APP/E- tr和APP/E-/-小鼠中分离的初级中枢神经小胶质细胞的功能吞噬和转录组学研究来探索。转录组评估将包括促炎性和抗炎细胞因子的RT-qPCR和无偏倚RNA-Seq，以确定小胶质细胞中apoE亚型差异激活的信号通路。特异性目的III将利用老年apoE- tr小鼠和APP/E-TR小鼠，研究apoE亚型如何以a β独立和a β依赖的方式调节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.