mRNA sequencing of brain and liver in multiple Alzheimer's Disease mouse models after treatment with either a Non-Targeting Control siRNA or an siRNA targeting mouse Apoe mRNA

The most common genetic risk factor for late onset Alzheimer Disease (AD) is the APOE4 allele, with evidence for gain- and loss-of-function as a primary mechanism. ApoE knockout in mice abrogates AD phenotypes but causes severe atherosclerosis due to the role of liver ApoE in cholesterol homeostasis. Previous attempts to therapeutically block brain-specific ApoE in adult models of AD only modestly reduced ApoE expression and no significant impact on amyloid burden. Here, we optimized a divalent siRNA (di-siRNA) to selectively silence ApoE in brain (>2 months). By measuring transcriptomic changes upon knockdown of ApoE in the CNS in multiple mouse models of AD, we find that ApoE knockdown results in an upregulation of the innate immune response, likely through activation of microglia. Additionally, we find that the changes in the transcriptome were similar, but not identical, two weeks and two months post-treatment. We find that mild reduction of ApoE in the liver does not cause widespread transcriptomic changes, but near complete knockdown of ApoE in the liver results in the dysregulation of many lipid metabolism-related pathways. Overall design: For the brain samples in each mouse model and each treatment (NTC or ApoE siRNA) timepoint (2 Weeks or 2 Months post-treatment) the sample size was 10-20 mice. For the liver samples in each treatment (NTC or ApoE siRNA, bilateral intracerebroventricular or subcutaneous injections) the sample size was 3-4 mice.

晚发性阿尔茨海默病（Alzheimer Disease, AD）最常见的遗传风险因子为APOE4等位基因，现有证据表明其核心致病机制为功能获得与功能丧失。小鼠体内的载脂蛋白E (Apolipoprotein E, ApoE)基因敲除可消除AD相关表型，但由于肝脏ApoE在胆固醇稳态中的关键作用，该操作会引发严重动脉粥样硬化。此前尝试在成年AD模型中通过治疗手段靶向阻断脑源性ApoE，仅能轻度降低ApoE的表达水平，且对淀粉样蛋白负荷无显著影响。

本研究优化了一种二价小干扰RNA (divalent siRNA, di-siRNA)，可在小鼠脑部实现选择性的ApoE基因沉默（持续时长超2个月）。通过在多种AD小鼠模型的中枢神经系统 (central nervous system, CNS)中检测ApoE基因敲低后的转录组变化，本研究发现ApoE敲低会引发先天免疫应答上调，该过程大概率通过小胶质细胞激活介导。此外，治疗后2周与2个月时的转录组变化模式相似但并不完全一致。

本研究还发现，肝脏ApoE的轻度表达下调不会引发广泛的转录组变化，但肝脏ApoE近乎完全敲低则会导致大量脂质代谢相关通路的失调。

实验整体设计：针对每种AD小鼠模型、每种处理方式（非靶向对照siRNA (non-targeting control siRNA, NTC)或ApoE siRNA）以及每个时间点（治疗后2周或2个月）的脑组织样本，每组样本量为10~20只小鼠；针对每种处理方式（NTC或ApoE siRNA，给药方式为双侧脑室内注射或皮下注射）的肝脏样本，每组样本量为3~4只小鼠。