Screening for enhancers of secreted clusterin (sCLU) and evaluation in AD models

项目总结/摘要 该提议提供了一个独特的机会，以确定簇蛋白（CLU）的亚型特异性调节剂， 评估其在阿尔茨海默病（AD）中的作用。胆固醇，也称为载脂蛋白J（Apo J）是一种蛋白质， 最初于1979年发现。几种CLU基因变异与AD相关，SNP rs 11136000 C与AD相关。 晚发性阿尔茨海默病（LOAD）的第三大遗传风险因素。有证据表明 提高sCLU的脑水平将减缓或逆转AD发病的各种分子机制 病理生理学这是基于研究表明，sCLU促进清除致病 β淀粉样蛋白（Aβ）斑块，同时减少细胞应激反应，如氧化应激和炎症 已知导致激酶活性增加，如GSK-3β和参与 AD的进展。有趣的是，CLU基因变异增加了对LOAD的易感性， 降低sCLU水平，导致Aβ沉积增加和tau神经元缠结， 相对于非运营商而言，在这项建议中，将进行一项全面的研究计划， 鉴定增加sCLU水平和调节AD生物标志物的有效的脑渗透性小分子 模型这将通过高通量筛选（HTS）来鉴定具有强效sCLU的“命中”来实现 增强能力、药物样特性、药代动力学（PK）分析中的脑渗透性，以指导 用于在iPSC衍生的神经元中进一步测试和作用机制（MOA）研究的化合物选择， AD的离体模型。在Aim 1中，我们将筛选增加细胞外浓度的化合物， sCLU。加州大学洛杉矶分校的大型化合物库将通过使用全自动液体处理设备进行筛选， 分析仪器来识别增加sCLU水平的分子。对于这些实验，敏感和 基于AlphaLISA的易格式化免疫测定将被优化以检测不同的CLU同种型。在 目的2，我们将进行体外ADME/T测试和体内药代动力学（PK）分析，以指导命中 选择包括测定sCLU增强效力，表征理化性质 包括候选sCLU增强化合物的溶解度、脑渗透性和代谢稳定性。在 目的3，我们将在诱导多能干细胞（iPSC）衍生的神经元和离体脑中进行测试。 切片目的是评估具有足够口服脑生物利用度的sCLU增强剂的有效性， AD患者源性神经元以及来自AD患者的离体器官型脑切片培养物中的半衰期 AD小鼠模型的海马。在Aim 4中，我们将识别分子靶点和细胞信号通路， 受到优先打击的影响MOA研究涉及通过光亲和标记/纯化进行的靶标鉴定， 以及全局和磷酸化蛋白质组分析将使用最先进的质谱法来完成。 使用这些方法，我们将鉴定我们的sCLU增强化合物的分子靶标，以鉴定新的 参与调节AD中脑sCLU水平的机制。

PROJECT SUMMARY/ABSTRACT This proposal provides a unique opportunity to identify isoform-specific modulators of clusterin (CLU) and evaluate its role in Alzheimer's disease (AD). Clusterin, also known as apolipoprotein J (Apo J) is a protein originally identified in 1979. Several CLU gene variants are associated with AD and the SNP rs11136000C is the third strongest genetic risk factor for Late Onset Alzheimer's disease (LOAD). There is evidence that enhancing brain levels of sCLU will slow or reverse various molecular mechanisms underlying onset of the AD pathophysiology. This is based on studies that show sCLU promoting the clearance of disease-causing amyloid beta (Aβ) plaques, while reducing cellular stress responses such as oxidative stress and inflammation known to lead to increased kinase activity such as GSK-3β and increased hyperphosphorylated tau involved in progression of AD. Interestingly, the CLU gene variants that confer increased susceptibility to LOAD also reduce sCLU levels and result in increased Aβ deposition and tau neurofibrillary tangles and faster cognitive decline relative to non-carriers. In this proposal a comprehensive research program will be undertaken to identify potent, brain permeable, small molecules that increase levels of sCLU and modulate biomarkers in AD models. This will be accomplished using high throughput screening (HTS) to identify `hits' with potent sCLU enhancing ability, drug-like properties, brain permeability in pharmacokinetics (PK) analyses to guide compound selection for further testing and mechanism-of-action (MOA) studies in iPSC derived neurons and ex-vivo models of AD. In Aim1 we will screen for compounds that increase extracellular concentrations of sCLU. UCLA's large compound library will be screened by using fully automated liquid handling devices and analytical instruments to identify molecules that increase sCLU levels. For these experiments, sensitive and readily formattable AlphaLISA based immunoassays will be optimized to detect different CLU isoforms. In Aim2, we would conduct in-vitro ADME/T testing and in-vivo pharmacokinetics (PK) analyses to guide hit selection including determination of sCLU enhancing potency, characterizing the physiochemical properties including solubility, brain permeability and metabolic stability of candidate sCLU enhancing compounds. In Aim3, we would conduct testing in induced pluripotent stem cells (iPSCs) derived neurons and ex-vivo brain slices. The goal is to evaluate the effectiveness of sCLU enhancers with sufficient oral brain bioavailability and half-life in AD patient-derived neurons as well as in ex-vivo organotypic brain slice cultures from the hippocampi of an AD mouse model. In Aim4, we would identify molecular targets and cell signaling pathways affected by prioritized hits. MOA studies involving target identification by photoaffinity-labeling/purification as well as global and phosphoproteome analyses will be accomplished using state-of-the-art mass spectrometry. Using these approaches, we will identify molecular targets of our sCLU enhancing compounds to identify novel mechanisms involved in regulating brain sCLU levels in AD.