d at 1000006g to collect the supernatant for the b-secretase activity assay. b-Secretase Activity Assay Kit was used to measure b-secretase activity from the membrane TMS fraction according to the manufacturer’s instructions. Briefly, equal amounts of membrane protein fractions were incubated at +37uC for 1 hour with the b-secretase-specific fluorogenic substrate peptide conjugated to fluorescent reporter molecules EDANS and DABCYL. The b-secretase inhibitor provided in the assay kit was mixed with recombinant b-secretase and incubated for 1 hour. At the same time, another b-secretase inhibitor, GL189 was used to validate the specificity of the Statistical analysis All the statistical analyses were performed using SPSS/Win. One-way analysis of variance, followed by Fisher’s least significant difference post-hoc test was used in experiments with more than one variable. Haploview 4.2 program was used to determine the D’- and r2-values of SNP pairs within the single haplotype block at the 39-end of NR1H3 gene. The target gene option in the miRWalk database was used to assess the predicted miRNA binding sites in the NR1H3 gene. Correlations were assessed using Pearson’s two-tailed correlation analysis. All values are reported as means 6 standard deviation. The level of statistical significance was defined as p,0.05. Results Biochemical characterization of the temporal cortex tissue samples reveals augmented b-secretase activity and increased soluble Ab42 levels with respect to disease severity Before determining the genetic effects of APOE and NR1H3 gene variations in the brain, we first performed biochemical Effects of NR1H3 Gene Variation on LXRa and AD assessment of the brain sample set consisting of inferior temporal cortex samples from 87 15722457 subjects with neuropathologically welldefined AD neurofibrillary changes. For the subsequent analyses, the sample set was subdivided according to Braak staging into three severity groups: mild, moderate, and severe AD . Soluble Ab42 levels in the inferior 15168218 temporal cortex samples in the severe group of AD patients were significantly increased as compared to mild and moderate groups . Moreover, b-secretase activity was significantly increased in both severe and moderate groups as compared to the mild group. Additionally, a significant positive correlation between soluble Ab42 levels and b-secretase activity was detected . Ab42, total-tau and phospho-tau levels in the CSF were available for the subset of subjects. As expected, Ab42 levels in the CSF were decreased in both moderate and 
severe groups as compared to the mild group. A significant negative correlation between CSF Ab42 levels and soluble Ab42 levels in temporal cortex samples was observed . Furthermore, tot-tau levels in the CSF were increased in the severe group as compared to both moderate and mild groups . Also, p-tau levels in the CSF were significantly increased in the severe group as compared to the mild group. Overall, these biochemical measurements in relation to the neurofibrillary changes indicating disease progression and severity reinforce the validity of this brain sample cohort for determining the effects of APOE and NR1H3 gene variations on AD-related molecular events at different stages of AD. APOE e4 allele increases the soluble Ab42 levels in a dose-dependent manner in the AD brain tissue, but does not affect the expression status of APOE The frequency of APOE e2/3/4 alleles in the AD brain sample set was 0.03/0.64/0.33, respecti
