Department of Physiology, Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky 40536, USA.
J Neurosci. 2013 Aug 14;33(33):13320-5. doi: 10.1523/JNEUROSCI.1224-13.2013.
Genome-wide association studies are identifying novel Alzheimer's disease (AD) risk factors. Elucidating the mechanism underlying these polymorphisms is critical to the validation process and, by identifying rate-limiting steps in AD risk, may yield novel therapeutic targets. Here, we elucidate the mechanism of action of the AD-associated polymorphism rs3865444 in the promoter of CD33, a member of the sialic acid-binding Ig-superfamily of lectins (SIGLECs). Immunostaining established that CD33 is expressed in microglia in human brain. Consistent with this finding, CD33 mRNA expression correlated well with expression of the microglial genes CD11b and AIF-1 and was modestly increased with AD status and the rs3865444C AD-risk allele. Analysis of CD33 isoforms identified a common isoform lacking exon 2 (D2-CD33). The proportion of CD33 expressed as D2-CD33 correlated robustly with rs3865444 genotype. Because rs3865444 is in the CD33 promoter region, we sought the functional polymorphism by sequencing CD33 from the promoter through exon 4. We identified a single polymorphism that is coinherited with rs3865444, i.e., rs12459419 in exon 2. Minigene RNA splicing studies in BV2 microglial cells established that rs12459419 is a functional single nucleotide polymorphism (SNP) that modulates exon 2 splicing efficiency. Thus, our primary findings are that CD33 is a microglial mRNA and that rs3865444 is a proxy SNP for rs12459419 that modulates CD33 exon 2 splicing. Exon 2 encodes the CD33 IgV domain that typically mediates sialic acid binding in SIGLEC family members. In summary, these results suggest a novel model wherein SNP-modulated RNA splicing modulates CD33 function and, thereby, AD risk.
全基因组关联研究正在确定新的阿尔茨海默病（AD）风险因素。阐明这些多态性的机制对于验证过程至关重要，并且通过确定 AD 风险的限速步骤，可能产生新的治疗靶点。在这里，我们阐明了与 AD 相关的多态性 rs3865444 在 CD33 启动子中的作用机制，CD33 是唾液酸结合 Ig 超家族凝集素（SIGLECs）的成员。免疫染色确定 CD33 在人脑小胶质细胞中表达。与这一发现一致，CD33 mRNA 表达与小胶质细胞基因 CD11b 和 AIF-1 的表达密切相关，并且随着 AD 状态和 rs3865444C AD 风险等位基因适度增加。CD33 同工型分析确定了一种缺乏外显子 2 的常见同工型（D2-CD33）。表达为 D2-CD33 的 CD33 比例与 rs3865444 基因型密切相关。由于 rs3865444 位于 CD33 启动子区域，我们通过从启动子到外显子 4 对 CD33 进行测序来寻找功能多态性。我们确定了一个与 rs3865444 共遗传的单一多态性，即外显子 2 中的 rs12459419。BV2 小胶质细胞中的 minigene RNA 剪接研究表明，rs12459419 是一种调节外显子 2 剪接效率的功能性单核苷酸多态性（SNP）。因此，我们的主要发现是 CD33 是一种小胶质细胞 mRNA，而 rs3865444 是一种调节 CD33 外显子 2 剪接的 rs12459419 替代 SNP。外显子 2 编码 CD33 IgV 结构域，该结构域通常介导 SIGLEC 家族成员中的唾液酸结合。总之，这些结果表明了一种新的模型，即 SNP 调节的 RNA 剪接调节 CD33 功能，从而调节 AD 风险。
