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

项目摘要/摘要 这一建议提供了一个独特的机会来确定聚集素(CLU)的异构体特异性调节子和 评估其在阿尔茨海默病(AD)中的作用。聚集素，又称载脂蛋白J(Apo J)，是一种蛋白质 最初发现于1979年。几个CLU基因变异与AD相关，SNP rs11136000C是 迟发性阿尔茨海默病(LOAD)的第三大遗传风险因素。有证据表明 提高大脑sCLU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促进了致病因素的清除。 淀粉样β蛋白(Aβ)斑块，同时减少氧化应激和炎症等细胞应激反应 已知会导致激酶活性增加，如葛兰素史克-3β和过度磷酸化的tau增加，参与 AD的进展。有趣的是，增加负荷易感性的CLU基因变异也 降低sCLU水平，导致Aβ沉积和tau神经原纤维缠结增加，认知速度加快 与非运营商相比有所下降。在这项提案中，将开展一项全面的研究计划，以 确定能增加阿尔茨海默病患者sCLU水平和调节生物标志物的有效的、大脑可穿透的小分子 模特们。这将使用高通量筛选(HTS)来识别具有强大sCLU的“命中” 增强药代动力学(PK)分析中的能力、类药物特性、脑通透性以指导 用于IPSC来源神经元的进一步测试和作用机制(MOA)研究的化合物选择 AD的体外模型。在Aim1中，我们将筛选能增加细胞外药物浓度的化合物 SCLU。加州大学洛杉矶分校的大型化合物图书馆将通过使用全自动液体处理设备和 用于识别增加sCLU水平的分子的分析仪器。对于这些实验，敏感和 易于格式化的基于AlphaLISA的免疫分析将被优化，以检测不同的CLU亚型。在……里面 AIM2，我们将进行体外ADME/T试验和体内药代动力学(PK)分析，以指导HIT 筛选包括sCLU增效剂的测定、理化性质的表征 包括候选sCLU增效剂的溶解度、脑渗透性和代谢稳定性。在……里面 AIM3，我们将在诱导多能干细胞(IPSCs)来源的神经元和体外脑中进行测试 切片。目标是评估具有足够口服脑生物利用度的sCLU增强剂的有效性和 AD患者来源的神经元的半衰期以及体外器官型脑片培养的半衰期 AD小鼠模型的海马区。在Aim4中，我们将识别分子靶点和细胞信号通路 受优先命中的影响。光亲和标记/纯化AS用于靶标识别的MOA研究 此外，全球和磷蛋白质组分析将使用最先进的质谱学完成。 使用这些方法，我们将识别我们的sclu增强型化合物的分子靶标，以识别新的 阿尔茨海默病中调节大脑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.