Investigating CD33 function on microglia during Alzheimer’s disease using CRISPR nanoparticles

摘要 小胶质细胞是大脑的免疫细胞，在脑血管疾病的发生和发展中起着关键的细胞作用。 阿尔茨海默病(AD)。作为大脑的免疫细胞，小胶质细胞是主要参与的细胞类型。 在吞噬蛋白质聚集体中，如淀粉样蛋白b(Ab)，它聚集形成特征 AD中出现的斑块。小胶质细胞吞噬能力的降低，与促炎 表型，可能导致允许抗体斑块形成的脑环境。最新全基因组 关联研究发现了免疫细胞特异性基因CD33的突变，几乎表达了 仅限于大脑中的小胶质细胞，作为阿尔茨海默病发展的风险因素。CD33是Siglec的成员 受体家族，对唾液酸的检测有免疫抑制作用。减少了对 CD33通过刺激吞噬和改变促炎作用来保护抗体斑块的形成 小胶质细胞的表型。因此，CD33是治疗和预防慢性粒细胞白血病的重要靶点。 广告。然而，由于技术上的原因，抗CD33治疗的适当时间点尚未确定 在阿尔茨海默病转基因动物模型中操纵脑内CD33的挑战。在这里，我们建议使用 脑和小胶质细胞验证的方法将CRISPR输送到脑内以下调CD33在 在AD转基因动物模型的疾病进展过程中的不同时间。我们预测 CD33在疾病早期和晚期的下调将不同程度地改变小胶质细胞吞噬抗体的变化 斑块负荷，消除神经毒性，改善记忆缺陷。此外，我们的目标是了解 从单个细胞检测AD患者小胶质细胞CD33表达的异质性 转录组数据集。我们预测CD33降低的小胶质细胞将表现为吞噬和 神经保护基因签名。我们预测，这种基因特征也将在CD33-高和CD33- 转基因AD小鼠的小胶质细胞减少。最后，我们将测试唾液酸结合作为潜在的机制，通过它 CD33调节抗体的吞噬功能。总体而言，这些研究将增强我们目前对CD33如何 在体内改变小胶质细胞的功能，并将阐明早期(预防)与晚期(干预)的益处 CD33基因敲除，这将为未来CD33靶向治疗的潜在治疗提供信息。

Abstract Microglia, the immune cells of the brain, are key cellular players in the pathogenesis and progression of Alzheimer’s disease (AD). As the immune cells of the brain, microglia are the cell type predominantly involved in phagocytosis of protein aggregates such as amyloid b (Ab), which aggregates to form the characteristic plaques present in AD. A decrement of microglia phagocytic capability, combined with a pro-inflammatory phenotype, may lead to a brain environment permissive for Ab plaque formation. Recent genome-wide association studies have identified mutations in the immune-cell specific gene CD33, expressed almost exclusively on microglia in the brain, as a risk factor for development of AD. CD33 is a member of the Siglec family of receptors, and has an immunosuppressive effect upon detection sialic acids. Reduced expression of CD33 is protective against formation of Ab plaques by stimulating phagocytosis and altering pro-inflammatory phenotype in microglia. Thus, CD33 represents an important therapeutic target for treatment and prevention of AD. However, an appropriate timepoint for anti-CD33 treatments has not been established due to technical challenges with manipulating CD33 in the brain of transgenic animal models of AD. Here, we propose to use a brain- and microglia-validated method for delivery of CRISPR to the brain to knock down expression of CD33 at various times during the course of disease progression in a transgenic animal model of AD. We predict that downregulation of CD33 early and late in disease will differentially alter microglia phagocytosis of Ab, change plaque load, abrogate neurotoxicity, and improve memory deficits. Further, we aim to understand the heterogeneity of microglia CD33 expression in human AD patients by examining existing single-cell transcriptomics datasets. We predict that microglia with reduced CD33 will display a phagocytic and neuroprotective gene signature. We predict that this gene signature will also be evident in CD33-high and CD33- low microglia from transgenic AD mice. Finally, we will test sialic acid binding as a potential mechanism by which CD33 regulates phagocytosis of Ab. Overall, these studies will enhance our current understanding of how CD33 modifies microglia function in vivo and will elucidate the benefits of early (preventative) vs. late (interventional) CD33 knockdown which will inform potential future treatments of CD33-targeting therapeutics.