Inhibition of CD33-sialic acid binding in Late-Onset Alzheimer's Disease

迟发性阿尔茨海默病中 CD33-唾液酸结合的抑制

• 批准号: 10752139
• 负责人: Jennifer Leigh Green
• 金额: $ 4.77万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752139
• 关键词: Affinity; Alanine; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease patient; Amyloid; Amyloid beta-Protein; Binding; Binding Proteins; Biological Assay; Brain; Cells; Development; Disease Progression; Gene Expression; Genes; Genetic study; Genotype; Glycoproteins; Human; Immune; Immune signaling; Immunoprecipitation; Impaired cognition; In Vitro; Individual; Inflammatory; Investigation; Late Onset Alzheimer Disease; Length; Ligands; Measures; Mediating; Methods; Microglia; Molecular; Morphology; Myeloid Cells; Neurodegenerative Disorders; PTPRC gene; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Peptides; Phase; Phosphoric Monoester Hydrolases; Predisposition; Process; Property; Protein Isoforms; Proteins; Risk; Risk Factors; Role; Senile Plaques; Sialic Acids; Signal Pathway; Signal Transduction; Solid; Specificity; Structure; Surface; TREM2 gene; Testing; Thick; Western Blotting; abeta accumulation; amyloid pathology; design; experimental study; genetic association; genetic risk factor; genome wide association study; glial activation; human model; human old age (65+); improved; in vitro Model; in vivo imaging; inhibitor; insight; monocyte; neuron loss; protein expression; receptor; response; risk variant; sialic acid binding Ig-like lectin; sialic acid receptor; targeted treatment; tau Proteins; uptake

PROJECT SUMMARY Alzheimer’s disease (AD) is a debilitating neurodegenerative disease characterized by progressive cognitive decline and pre-symptomatic accumulation of amyloid-beta (Aβ) and tau pathology with older age. AD pathogenesis is poorly understood, however genetic studies have clearly indicated a causal role for microglia, the innate immune cells of the CNS. Indeed, GWAS established the innate immune gene CD33 as a risk factor for Late-Onset AD (LOAD) in 2011. The CD33 LOAD associated rs3865444CC risk genotype is associated with diminished internalization of amyloid-β1-42 peptide, accumulation of neuritic amyloid pathology and fibrillar amyloid on in vivo imaging, and increased numbers of human microglia with small, thick processes, and a rounded morphology compared to the rs3865444AA protective genotype. Individuals with the LOAD associated rs3865444CC risk genotype have increased expression of full-length CD33, the isoform containing the sialic acid binding domain, compared to those with the rs3865444AA protective genotype, suggesting that the sialic- acid binding domain may be critical to the genetic association to AD. Based on the known inhibitory function of CD33, my hypothesis is that sialic acid mediated CD33 activation leads to microglial suppression, resulting in the inability to resolve inflammatory processes and mitigate pathogenic amyloid plaques, which may heighten susceptibility to neuronal loss and contribute to AD progression. To test this hypothesis, our lab developed an alternative approach for inhibiting CD33 function, by using peptides designed to bind to sialic acid to act as “decoy” receptors for sialic acid to reduce it’s binding to CD33 and inhibit CD33 signaling. I hypothesize that competitive inhibition of CD33 with sialic acid will reduce CD33 activation, restore microglial responsiveness, and increase Aβ uptake. The overarching objective of this proposal is to characterize the binding properties of these peptides and their inhibitory effects on CD33 activation and signaling (Aim 1) and then examine the functional responses of these peptides on immune pathways in vitro (Aim 2). In Aim 1, I will determine the potency and selectivity of peptides 1 and 2 in inhibiting CD33-sialic acid binding in a solid-phase binding assay I have optimized (Aim1a), determine the critical residues for efficacy through an alanine screen (Aim1b), and investigate the effects of the peptides on CD33 activation and signaling (Aim1c). In Aim 2, I will look at how human microglia functionally respond to treatment with peptides 1 and 2 in the context of AD using primary microglia-like cells from healthy donor and AD patients expressing the CD33 risk and protective genotypes by measuring gene expression changes in markers of microglial activation and amyloid uptake (Aim2a) and expression changes in key LOAD-associated immune-related proteins (Aim 2b).These experiments will provide insights to not only the mechanism of action of these peptides, but also to further clarify the role of CD33 and Siglec receptors in microglial activation, immune signaling pathways, and the pathogenesis of Alzheimer’s disease.

项目总结 阿尔茨海默病(AD)是一种以进行性认知为特征的衰弱神经退行性疾病 随着年龄的增长，淀粉样β蛋白(A-β)和tau病理的下降和症状前积聚。广告 发病机制尚不清楚，然而遗传学研究已明确指出小胶质细胞的因果作用， 中枢神经系统的先天免疫细胞。事实上，GWAs将先天免疫基因CD33确定为风险因素 2011年的晚发性AD(Load)。CD33载量相关的rs3865444CC风险基因型与 淀粉样蛋白-β1-42肽内化减少，神经性淀粉样蛋白病变和纤维堆积 体内成像中的淀粉样蛋白，以及具有小而粗的突起的人小胶质细胞数量的增加，以及 圆形形态与rs3865444AA保护性基因的比较。与负载相关联的个人 Rs3865444CC风险基因型增加了全长CD33的表达，CD33是含有唾液酸的亚型 与rs3865444AA保护性基因相比，提示唾液酸结合结构域与rs3865444AA保护性基因相比，唾液酸结合结构域与rs3865444AA保护性基因的同源性更高。 酸结合结构域可能是阿尔茨海默病基因关联的关键。基于已知的抑制功能 CD33，我的假设是唾液酸介导的CD33激活导致小胶质细胞抑制，从而导致 无法消解炎症过程和减轻致病的淀粉样斑块，这可能会加剧 易导致神经元丢失，并有助于AD的进展。为了验证这一假设，我们的实验室开发了一种 另一种抑制CD33功能的方法是使用旨在与唾液酸结合的多肽作为 唾液酸的“诱饵”受体，以减少它与CD33的结合，并抑制CD33信号。我假设 用唾液酸竞争性抑制CD33将减少CD33的激活，恢复小胶质细胞的反应性， 并增加Aβ的摄取。这项建议的首要目标是描述具有约束力的性质 以及它们对CD33激活和信号转导的抑制作用(Aim 1)，然后检测 这些多肽在体外免疫途径上的功能反应(目标2)。在目标1中，我将确定 固相结合实验中多肽1和2抑制CD33-唾液酸结合的效力和选择性 我已经优化了(Aim1a)，通过丙氨酸筛选(Aim1b)确定了疗效的关键残留物，以及 研究这些多肽对CD33激活和信号转导(Aim1c)的影响。在《目标2》中，我将了解如何 人小胶质细胞对阿尔茨海默病背景下肽1和2的功能反应 健康供者和AD患者小胶质细胞样细胞表达CD33风险和保护性基因 检测小胶质细胞活化和淀粉样蛋白摄取标记物(Aim2a)和 关键负荷相关免疫相关蛋白的表达变化(目标2b)。这些实验将提供 不仅深入了解这些多肽的作用机制，而且进一步阐明CD33和CD33的作用。 小胶质细胞激活、免疫信号通路中的Siglec受体与阿尔茨海默病的发病机制 疾病。