CD33 and Alzheimer's Disease: from Biology to Therapy

描述(由申请人提供)：认知能力的保存是21世纪的主要医学挑战之一。我们目前无法预防或推迟阿尔茨海默病(AD)，预计AD患病率将增加，这将导致一场全球性的AD大流行。我们最近发现了一条新的淀粉样β蛋白(A�)清除途径，它与AD的发病机制高度相关。在一项非常大的基于家庭的全基因组关联研究中，我们确定CD33是一种新的晚发性AD危险因素。CD33编码一种跨膜唾液酸结合的免疫球蛋白样凝集素，调节先天免疫。我们发现CD33在小胶质细胞中特异性表达，并且在AD中的表达增加。CD33单核苷酸多态rs3865444的次要等位基因与AD脑中CD33表达和不溶性A�水平的降低有关。利用小胶质细胞培养，我们发现CD33抑制A�的摄取和清除，这一过程需要CD33的唾液酸结合结构域。最后，在AD小鼠模型中，CD33基因敲除导致不溶性A�水平和淀粉样斑块负荷显著降低。因此，小胶质细胞中的CD33活性通过抑制A�的摄取和清除而促进A�的病理改变。在这里，我们建议：1)识别和表征与晚发性AD相关的新的CD33突变；2)剖析CD33在小胶质细胞中作用的分子机制；3)出于治疗目的抑制CD33的活性。在Aim1中，我们将分析来自410个家庭(1376名受试者)的全基因组测序数据，以确定与AD相关的新的CD33突变。然后，我们将在150例AD病例(K99)的队列中检查这些突变对AD的多个病理指标的影响。最有希望的CD33变异体将在小胶质细胞(R00)中进行功能鉴定。在目标2中，我们将定义CD33介导的小胶质细胞信号转导通路。我们将表达特定结构域或预测的磷酸化位点已被移除或突变的突变CD33型，并评估它们对小胶质细胞转录组和A�清除的影响(K99)。我们还将使用质谱仪在体内鉴定CD33相互作用的蛋白质。为了探索CD33和TREM2在小胶质细胞中的功能相互作用，我们将使用小胶质细胞培养(R00)中的遗传分析。在目标3中，我们将通过鉴定从CD33基因失活的小鼠模型(K99)中分离的成年小胶质细胞的转录组，来确定CD33活性在小胶质细胞中的细胞特征。我们还将确定CD33基因失活是否能恢复AD小鼠模型的认知。最后，我们将通过筛选i)定制合成的基于唾液酸的拮抗剂和ii)CD33特异性抗体在基于小胶质细胞的分析中抑制CD33功能的能力来开发和验证有效的CD33抑制剂。成功的抑制剂将进行进一步测试 小鼠(R00)。总而言之，这些研究可能为了解衰老人脑中�学习的中心途径提供关键的见解，并可能导致一种新的、强大的AD治疗方法。

DESCRIPTION (provided by applicant): Preservation of cognitive abilities is one of the major medical challenges of the 21st century. Our current inability to prevent or delay Alzheimer's disease (AD) and the expected increase in the prevalence of AD are predicted to give rise to a global AD pandemic. We have recently identified a novel pathway for amyloid beta (A�clearance in the aging brain that is highly relevant to AD pathogenesis. In a very large family-based genome-wide association study, we identified CD33 as a novel late-onset AD risk factor. CD33 encodes a transmembrane sialic acid-binding immunoglobulin-like lectin that regulates innate immunity. We found that CD33 is specifically expressed in microglial cells and exhibits an increased expression in AD. The minor allele of the CD33 single nucleotide polymorphism rs3865444, which confers protection against AD, was associated with reductions in both CD33 expression and insoluble A� levels in AD brain. Using microglial cell cultures, we found that CD33 inhibits uptake and clearance of A�, a process that requires the sialic acid-binding domain of CD33. Finally, CD33 knock-out led to a marked reduction in insoluble A� levels and amyloid plaque burden in mouse models of AD. Thus, CD33 activity in microglial cells promotes A�athology by inhibiting A�ptake and clearance. Here, we propose to: 1) identify and characterize novel CD33 mutations linked to late-onset AD; 2) dissect the molecular mechanisms underlying the function of CD33 in microglia; and 3) inhibit CD33 activity for therapeutic purposes. In Aim1, we will analyze whole genome sequencing data from 410 families (1376 subjects) to identify novel CD33 mutations linked to AD. We will then examine the impact of these mutations on multiple pathological indices of AD in a cohort of 150 AD cases (K99). The most promising CD33 variants will be functionally characterized in microglial cells (R00). In Aim 2, we will define CD33-mediated signal transduction pathways in microglia. We will express mutant CD33 versions in which specific domains or predicted phosphorylation sites have been removed or mutated and assess their effects on the microglial transcriptome and A� clearance (K99). We will also identify CD33-interacting proteins in vivo using mass spectrometry. To explore the functional interaction between CD33 and TREM2 in microglia, we will use genetic analysis in microglial cell cultures (R00). In Aim 3, we will define the cellular signature of CD33 activity in microglia by characterizing the transcriptome of adult microglia isolated from mouse models of AD in which CD33 has been genetically inactivated (K99). We will also determine whether CD33 genetic inactivation restores cognition in mouse models of AD. Finally, we will develop and validate effective CD33 inhibitors by screening i) custom-synthesized sialic acid-based antagonists and ii) CD33-specific antibodies for their ability to inhibit CD33 function in microglial cell-based assays. Successful inhibitors will be further tested in mice (R00). Collectively, these studies might provide critical insights into a central pathway fr A�learance in the aging human brain and might result in a novel and powerful therapeutic approach for AD.