Relationships of APOE Genotypes With Small RNA and Protein Cargo of Brain Tissue Extracellular Vesicles From Patients With Late-Stage AD.
Relationships of APOE Genotypes With Small RNA and Protein Cargo of Brain Tissue Extracellular Vesicles From Patients With Late-Stage AD.
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DOI：
10.1212/nxg.0000000000200026
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发表时间：
2022-12
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载脂蛋白E（APOE）基因的变异是散发性阿尔茨海默病（AD）的最大已知危险因素。三种主要的APOE同种型等位基因ε2、ε3和ε4编码并产生仅相差1-2个氨基酸但具有不同结合伴侣相互作用的蛋白质。虽然APOE ε2相对于ε3对AD具有保护作用，但ε4与AD发展风险增加相关。然而，APOE在AD发病机制中的基因调控作用在很大程度上仍未确定。细胞外囊泡是由细胞释放的脂质双层界定的颗粒，用于处理不需要的物质并介导细胞间通讯，并且它们与AD病理生理学有关。脑源性EVs（bdEVs）可以局部作用于组织并反映细胞变化。为了揭示APOE基因型是否影响AD脑中的EV组分，从具有不同APOE基因型的AD患者中分离bdEV，用于平行的小RNA和蛋白质谱。使用我们公开的方案将来自具有APOE基因型ε2/3（n = 5）、ε3/3（n = 5）、ε3/4（n = 6）和ε4/4（n = 6）的患者的晚期AD脑（BRAAK阶段5-6）的bdEV分离成10，000 g沉淀的细胞外级分（10 K）和进一步纯化的EV级分。通过小RNA测序和蛋白质组学分析对10 K、EV和源组织进行计数、大小测定和多组学表征。比较APOE基因型，没有观察到bdEV总颗粒浓度或形态学的显着差异。10 K、EV和源组织的总体小RNA和蛋白质谱在不同APOE基因型之间也没有显著差异。然而，当比较最高和最低风险组（ε4/4和ε2/3）时，观察到10 K和EV中单个RNA（包括miRNA和tRNA）和蛋白质的几个差异。生物信息学分析和先前的出版物表明这些分子在AD中的潜在调节作用。对于本研究中的晚期AD患者，在APOE基因型之间仅观察到小RNA和蛋白质谱的一些中度差异。其中，一些新发现的10 K和EV相关分子可能在AD进展中发挥作用。可能存在较大的基因型相关差异，并且在疾病早期或之前更明显。
Variants of the apolipoprotein E (APOE) gene are the greatest known risk factors for sporadic Alzheimer disease (AD). Three major APOE isoform alleles, ε2, ε3, and ε4, encode and produce proteins that differ by only 1–2 amino acids but have different binding partner interactions. Whereas APOE ε2 is protective against AD relative to ε3, ε4 is associated with an increased risk for AD development. However, the role of APOE in gene regulation in AD pathogenesis has remained largely undetermined. Extracellular vesicles (EVs) are lipid bilayer–delimited particles released by cells to dispose of unwanted materials and mediate intercellular communication, and they are implicated in AD pathophysiology. Brain-derived EVs (bdEVs) could act locally in the tissue and reflect cellular changes. To reveal whether APOE genotype affects EV components in AD brains, bdEVs were separated from patients with AD with different APOE genotypes for parallel small RNA and protein profile. bdEVs from late-stage AD brains (BRAAK stages 5–6) from patients with APOE genotypes ε2/3 (n = 5), ε3/3 (n = 5), ε3/4 (n = 6), and ε4/4 (n = 6) were separated using our published protocol into a 10,000g pelleted extracellular fraction (10K) and a further purified EV fraction. Counting, sizing, and multiomic characterization by small RNA sequencing and proteomic analysis were performed for 10K, EVs, and source tissue. Comparing APOE genotypes, no significant differences in bdEV total particle concentration or morphology were observed. Overall small RNA and protein profiles of 10K, EVs, and source tissue also did not differ substantially between different APOE genotypes. However, several differences in individual RNAs (including miRNAs and tRNAs) and proteins in 10K and EVs were observed when comparing the highest and lowest risk groups (ε4/4 and ε2/3). Bioinformatic analysis and previous publications indicate a potential regulatory role of these molecules in AD. For patients with late-stage AD in this study, only a few moderate differences were observed for small RNA and protein profiles between APOE genotypes. Among these, several newly identified 10K and EV-associated molecules may play roles in AD progression. Possibly, larger genotype-related differences exist and are more apparent in or before earlier disease stages.