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世纪世纪的主要医学挑战之一。我们目前无法预防或延迟阿尔茨海默病（AD）和AD患病率的预期增加预计将导致全球AD大流行。我们最近发现了一种新的β淀粉样蛋白（A β）清除衰老大脑的途径，这与AD的发病机制高度相关。在一项非常大的基于家族的全基因组关联研究中，我们将CD33确定为一种新的迟发性AD风险因素。CD33编码调节先天免疫的跨膜唾液酸结合免疫球蛋白样凝集素。我们发现CD33在小胶质细胞中特异性表达，并且在AD中表达增加。CD33单核苷酸多态性rs3865444的次要等位基因，赋予对AD的保护作用，与AD脑中CD33表达和不溶性A β水平的降低相关。使用小胶质细胞培养，我们发现CD33抑制A β的摄取和清除，这一过程需要CD33的唾液酸结合结构域。最后，CD33基因敲除导致AD小鼠模型中不溶性A β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 β摄取和清除来促进A β病理学。在此，我们建议：1）鉴定和表征与迟发性AD相关的新型CD33突变; 2）剖析小胶质细胞中CD33功能的分子机制; 3）出于治疗目的抑制CD33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相关的新型CD33突变。然后，我们将在一个150例AD病例（K99）的队列中研究这些突变对AD多个病理指标的影响。最有希望的CD33变体将在小胶质细胞（R00）中进行功能表征。在目标2中，我们将定义小胶质细胞中CD33介导的信号转导途径。我们将表达突变的CD33版本，其中特定的结构域或预测的磷酸化位点已被去除或突变，并评估它们对小胶质细胞转录组和A β清除的影响（K99）。我们还将使用质谱法在体内鉴定CD33相互作用蛋白。为了探索小胶质细胞中CD33和TREM2之间的功能相互作用，我们将在小胶质细胞培养物中使用遗传分析（R00）。在目标3中，我们将通过表征从AD小鼠模型中分离的成年小胶质细胞的转录组来定义小胶质细胞中CD 33活性的细胞特征，其中CD 33已被遗传灭活（K99）。我们还将确定CD33基因失活是否能恢复AD小鼠模型的认知功能。最后，我们将通过筛选i）定制合成的唾液酸拮抗剂和ii）CD33特异性抗体来开发和验证有效的CD33抑制剂，以确定其在基于小胶质细胞的试验中抑制CD33功能的能力。成功的抑制剂将进一步测试 小鼠（R00）。总的来说，这些研究可能为衰老人脑中的中枢通路提供重要的见解，并可能为AD提供一种新的强大的治疗方法。

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.