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-...