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Novel Alzheimer's disease (AD) risk factors have been identified by genome-wide association studies. Elucidating the mechanism underlying these factors is critical to the validation process and, by identifying rate-limiting steps in AD risk, may yield novel therapeutic targets. Here, we evaluated the association between the AD-associated polymorphism rs3851179 near PICALM, which encodes a clathrin-coated pit accessory protein. Immunostaining established that PICALM is expressed predominately in microvessels in human brain. Consistent with this finding, PICALM mRNA expression correlated with expression of the endothelial genes vWF and CD31. Additionally, we found that PICALM expression was modestly increased with the rs3851179A AD-protective allele. Analysis of PICALM isoforms found several isoforms lacking exons encoding elements previously identified as critical to PICALM function. Increased expression of the common isoform lacking exon 13 was also associated with the rs3851179A protective allele; this association was not apparent when this isoform was compared with total PICALM expression, indicating that the SNP is associated with total PICALM expression and not this isoform per se. Interestingly, PICALM lacking exons 2-4 was not associated with rs3851179 but was associated with rs592297, which is located in exon 5. Thus, our primary findings are that multiple PICALM isoforms are expressed in the human brain, that PICALM is robustly expressed in microvessels, and that expression of total PICALM is modestly correlated with the AD-associated SNP rs3851179. We interpret these results as suggesting that increased PICALM expression in the microvasculature may reduce AD risk.

The minor allele of rs11136000 within CLU is strongly associated with reduced Alzheimer's disease (AD) risk. The mechanism underlying this association is unclear. Here, we report that CLU1 and CLU2 are the two primary CLU isoforms in human brain; CLU1 and CLU2 share exons 2-9 but differ in exon 1 and proximal promoters. The expression of both CLU1 and CLU2 was increased in individuals with significant AD neuropathology. However, only CLU1 was associated with the rs11136000 genotype, with the minor "protective" rs11136000T allele being associated with increased CLU1 expression. Since CLU1 and CLU2 are predicted to encode intracellular and secreted proteins, respectively, we compared their expression; for both CLU1 and CLU2 transfected cells, clusterin is present in the secretory pathway, accumulates in the extracellular media, and is similar in size to clusterin in human brain. Overall, we interpret these results as indicating that the AD-protective minor rs11136000T allele is associated with increased CLU1 expression. Since CLU1 and CLU2 appear to produce similar proteins and are increased in AD, the AD-protection afforded by the rs11136000T allele may reflect increased soluble clusterin throughout life.