PROJECT SUMMARY Understanding how combinations of genetic risk factors influence risk for late-onset Alzheimer’s disease (LOAD) can lead to targeted strategies for therapeutic intervention. Apolipoprotein E4 (APOE4), a common variant of APOE, is the single largest genetic risk factor for developing LOAD.APOE4 status is linked to increased inflammation and higher β-amyloid burden in LOAD patients. Despite this increased genetic risk profile, APOE4 carriers do not always develop LOAD in the course of their lifetime. Several large-scale genetic studies have identified a common haplotype of the aging factor klotho that modify age of onset and reduce amyloid plaque deposition specifically in APOE4 carriers, suggesting that klotho variants can provide a protective effect against the development of LOAD by counteracting the negative effects of APOE4. In humans, klotho harbors two common missense variants (rs9536314, p.F352V; rs9527025, p.C370S). The combination of these two coding variants define the klotho V/S (KL-V/S) haplotype, which is protective against LOAD in APOE4 carriers, and the klotho F/C (KL-F/C) haplotype, which is not protective against LOAD. The overall objective of this proposal is to determine the physiological processes altered by klotho as an APOE4-specific protective factor in LOAD using a set of recently-created mouse models harboring combinations of relevant human variants in both klotho and APOE. Our central hypothesis is that the protective KL-V/S haplotype will significantly delay age-dependent inflammation and amyloid deposition while the reference KL-F/C haplotype will fail to attenuate these hallmark LOAD pathologies. We will assess multiple LOAD-relevant outcomes to validate and characterize this klotho- APOE genetic interaction with three specific aims: (1) Determine the effects of klotho haplotypes on age-related frailty and klotho isoform levels in blood and CSF in mice; (2) Determine changes in LOAD hallmark pathologies driven by the interaction between klotho and APOE alleles in mice; and (3) Identify molecular signatures shared in human LOAD stratified by klotho haplotype and the novel klotho mouse models. The outcome of this work will result in the characterization of new mouse models of human klotho haplotypes and identify the pathways which are differentially affected by klotho variants in an APOE-dependent manner. This information will provide a biological basis for the epistatic interaction observed in human genetic studies, thereby providing the necessary functional information to guide potential treatments based on KL-V/S protection for APOE4 carriers.