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

摘要 小胶质细胞是脑的免疫细胞，是脑胶质细胞瘤的发病机制和进展中的关键细胞参与者。 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 在蛋白质聚集体如淀粉样蛋白B（Ab）的吞噬中，其聚集形成特征性的 AD中存在斑块。小胶质细胞吞噬能力的降低，结合促炎性 表型，可能导致允许Ab斑块形成的脑环境。最近的全基因组 相关研究已经确定了免疫细胞特异性基因CD 33的突变，其表达几乎 专门针对大脑中的小胶质细胞，作为AD发展的风险因素。CD 33是Siglec的成员。 受体家族，并且在检测唾液酸时具有免疫抑制作用。的表达降低 CD 33通过刺激吞噬作用和改变促炎性细胞因子来保护抗体斑块的形成。 小胶质细胞中的表型。因此，CD 33代表了治疗和预防糖尿病的重要治疗靶点。 AD.然而，由于技术原因，尚未确定抗CD 33治疗的适当时间点。 在AD转基因动物模型的脑中操纵CD 33的挑战。在这里，我们建议使用 脑和小胶质细胞验证的方法，用于将CRISPR递送至脑以敲低CD 33的表达。 在AD的转基因动物模型中疾病进展过程中的不同时间。我们预测 疾病早期和晚期CD 33的下调将差异性地改变小胶质细胞对Ab的吞噬作用， 斑块负荷，消除神经毒性，并改善记忆缺陷。此外，我们的目标是了解 通过检查现有单细胞检测人类AD患者中小胶质细胞CD 33表达的异质性 转录组学数据集。我们预测，CD 33减少的小胶质细胞将表现出吞噬作用， 神经保护基因标记。我们预测这种基因标记在CD 33-high和CD 33-low中也是明显的。 转基因AD小鼠的低小胶质细胞。最后，我们将测试唾液酸结合作为一种潜在的机制， CD 33调节Ab的吞噬作用。总的来说，这些研究将加强我们目前对CD 33如何 改变体内小胶质细胞功能，并将阐明早期（预防性）与晚期（干预性）的益处 CD 33敲低，这将为CD 33靶向治疗剂的潜在未来治疗提供信息。

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.