Comparative profiling of hippocampal gene expression in AppNL-G-F knock-in mouse model of Alzheimer's disease.

To identify molecular pathological alterations in Alzheimer’s disease (AD) brains, we had previously performed comparative gene expression profiling with human gene array using RNAs prepared from postmortem human brains donated for the Hisayama study. The hippocampi from AD brains showed the most significant alteration in gene expression profile (PMID: 23595620). In the present study, in order to obtain a comprehensive view of gene expression profiles in AD hippocampus, we applied human transcriptome array and deep RNA-sequencing to the same samples and reanalyzed the gene array data, and performed integrative analysis of all 3 methods. We also applied gene array to hippocampus of 6-month-old App[NL-G-F/NL-G-F] knock-in AD model mice, and at last we performed the interspecies comparison. IPA analysis of the top significantly altered genes derived from integrative analysis of all 3 methods in human AD hippocampus revealed that neurotransmission, synaptic transmission and development of neurons are the top significantly decreased functions. In hippocampi of App[NL-G-F/NL-G-F] mice which exhibit significant amyloidosis but not neuronal degeneration, we found that expression of Gfap (astrocyte marker) and Cd68 (microglia marker) genes are significantly upregulated together with genes categorized in the immune response of cells. Comparing the human and mouse data, we found major common genes are related to recruitment of inflammatory cells. Amyloid-beta accumulation is the major hallmark of AD at 6 month-old App[NL-G-F/NL-G-F] mice, therefore our data suggests that amyloid-beta accumulation increases the recruitment of inflammatory cells in early stage of AD brain, prior to neuronal degeneration. Six month-old female wild-type and App[NL-G-F/NL-G-F] mice harboring three pathogenic App-knock-in mutations, that promote aggressive amyloidosis by increasing total Aβ production (Swedish mutation KM670/671NL) and increasing the Aβ42 aggregation (Arctic mutation E693G) and increasing the Aβ42/Aβ40 ratio (Iberian mutation I716F) under the control of the endogenous APP promoter were used for hippocampal gene expression profiling with the Affymetrix Genechip Mouse Gene 2.0 ST Array. Array data was processed by Affymetrix Transcriptome Analysis Console (TAC) software 4.0.

为明确阿尔茨海默病（Alzheimer’s Disease, AD）患者大脑的分子病理学改变，本团队此前依托桧山研究（Hisayama Study）捐赠的尸检人脑组织提取RNA，采用人类基因芯片开展了对比基因表达谱分析。其中，AD患者大脑海马体的基因表达谱改变最为显著（PMID: 23595620）。
本研究中，为全面解析AD患者海马体的基因表达谱，我们对上述相同样本分别开展人类转录组芯片、深度RNA测序检测，并对原有基因芯片数据进行重新分析，最终整合三种方法的结果开展联合分析。此外，我们还对6月龄App[NL-G-F/NL-G-F]敲入AD模型小鼠的海马体进行基因芯片检测，最终完成跨物种比较分析。
对人类AD海马体三种方法整合分析得到的显著差异基因进行IPA（Ingenuity Pathway Analysis）分析，结果显示神经传递、突触传递及神经元发育是最显著下调的生物学功能。
在仅出现显著淀粉样蛋白沉积但未发生神经元变性的App[NL-G-F/NL-G-F]小鼠海马体中，我们发现胶质纤维酸性蛋白（Gfap, 星形胶质细胞标志物）、CD68（小胶质细胞标志物）的基因表达显著上调，同时伴随细胞免疫应答相关基因的表达升高。
对比人类与小鼠的测序数据，我们发现二者共有的核心差异基因主要与炎症细胞招募相关。6月龄App[NL-G-F/NL-G-F]小鼠的主要AD病理特征为β淀粉样蛋白（Amyloid-beta, Aβ）沉积，因此本研究数据表明，在AD脑内发生神经元变性之前，β淀粉样蛋白沉积即可在疾病早期促进炎症细胞的招募。
本研究采用的实验动物为6月龄雌性野生型小鼠与携带三种致病性App敲入突变的App[NL-G-F/NL-G-F]小鼠：这些突变可通过增加总Aβ生成量（瑞典突变KM670/671NL）、促进Aβ42聚集（北极突变E693G）以及升高Aβ42/Aβ40比值（伊比利亚突变I716F），在内源性APP启动子调控下加速严重淀粉样蛋白沉积。我们采用Affymetrix Genechip Mouse Gene 2.0 ST芯片对上述小鼠的海马体开展基因表达谱分析。
芯片数据采用Affymetrix Transcriptome Analysis Console（TAC）软件4.0进行处理。