Project 5: PICALM, Oligomeric Abeta and Inflammation in Neurovascular Pathogenesis in AD In this renewal the emphasis will still be on how Abeta and inflammation affect cerebrovascular function, but will also take advantage of recent genome-wide association studies (GWAS) that have identified new AD risk genes that have been broadly linked to inflammation/endosomal processing. Because the blood brain barrier (BBB) is critically important for efficient transport of solutes in and out of the central nervous system (CNS), and phosphatidylinositol-binding clathrin assembly protein (PICALM) one of the genes identified with increased risk for AD is involved with initiating endocytosis/transcytosis of APP, ApoE and Abeta by endothelial cells, we have chosen to focus on how the PICALM risk variant affects endothelial function in the presence oligomeric Ap, angiotensin II, and inflammation (IL-1beta). Finally, because meta-analysis of AD risk loci has revealed interactions between PICALM and APOE genotypes, where PICALM conferred risk predominantiy in APOE e4-positive subjects, we propose to include APOE genotypes in our research strategy. Since PICALM knockout mice show deficits in erythroid maturation and transferrin internalization, we hypothesize that the PICALM risk variant represents either a deficit in endocytosis or an altered trafficking function where vesicle cargo is misrouted. We propose the following Specific Aims: Aim 1: To investigate the contribution of the PICALM AD risk variant on the level of parenchymal amyloid load, type-1 and type-2 cerebral amyloid angiopathy, Ap40 & 42 levels, tau, and inflammation in the UCI ADRC autopsy cases. Aim 2: We will investigate how human iPSCs-derived ECs homozygous PICALM risk or non-risk alleles affect endocytic function and vesicular trafficking in endothelial cells (APOE genotypes 3/3, versus ?3/?4). Aim 3: We will test endocytosis and transcytosis of transferrin, LRP1, ApoE and TrkB receptors, as well as APP and Ap using an in vitro transwell model of the BBB. We will use iPSCs-derived ECs homozygous for PICALM risk or non-risk coding variants and astrocytes from Down syndrome (DS) and non-DS that express APOE ?3/?3 or ApoE ?3/?4. Aim 4: To test the role of PICALM in EC BBB function on Ap vascular pathology in vivo. We propose to develop a transgenic mouse model with reduced expression of PICALM in endothelial cells using the Cre-LoxP System. The conditional EC PICALM knock-out mice will be crossed with APP Tg mice that develop Type 2 CAA or Type 1 CAA and the effect of PICALM knockdown on CAA, as well as parenchymal Abeta will be assessed.