PICALM, the gene encoding phosphatidylinositol binding clathrin assembly (PICALM) protein, plays a key role in endocytosis, a process which regulates the function of cell receptors and synaptic transmission. Several GWAS studies of AD have replicated the association of PICALM with AD and shown relationships with neurodegenerative processes underlying disease. Additionally, low levels of PICALM in brain and cerebral microvessels have been recently shown in late onset AD. The role of PICALM in AD pathogenesis remains, however, elusive. A genome-wide screen for modifiers of Ab toxicity in yeast has identified the yeast homologue of PICALM and some other endocytotic factors as a functional link between Ab toxicity, endocytosis, and AD risk. PICALM has been also shown to protect neurons against Ab toxicity by partially reversing Ab toxic effects on endocytotic trafficking. During YEAR 1, we showed that PICALM plays a central role in the molecular mechanism regulating Ab transcytosis and clearance across the blood-brain barrier (BBB). Using a human brain endothelial monolayer model of the BBB, we showed that PICALM binds to the cytoplasmic tail of LRP1 and is involved in clathrin-mediated Ab endocytosis and transport of Ab across the BBB controlled by Rab5 and Rab11 GTPases. We next showed that Picalmdeficiency diminishes Ab clearance across the murine BBB in vivo and reduces clearance of soluble Ab from the brain interstitial fluid (ISF) in APPsw/0 mice accelerating Ab pathology and cognitive decline. These findings have established that PICALM controls AbBBB transcytosis and clearance from brain. In YEAR 2, we propose to continue these studies and determine i) the cell-specific role of PICALM in brain endothelium (AIM 1) and neurons (AIM 2) in vivo in relation to neuronal dysfunction and neurodegeneration within the Ab pathway and Ab-independent pathway using novel murine models of Picalm deficiency; and ii) the effects of novel PICALMmutations on Ab BBB clearance using a human model of the BBB in vitro in collaboration with Dr. R. Tanzi. For AIMS 1 and 2, we will use Picalm lox/lox mice (generated by our collaborator Dr T. Maeda, Harvard Medical School) and will delete Picalmfrom endothelium and/or neurons to determine how these cell-specific deletions affect Ab metabolism, BBB integrity, neuronal function and neurodegeneration. We will use Ab clearance technique, multiphoton, confocal and light microscopy analysis, DTI MRI to evaluate brain structural and functional changes (tractography), DCE MRI for BBB integrity, and behavioral tests. The proposed studies should advance our knowledge about the role of PICALM as a risk for AD and how novel PICALM mutations affect Abclearanceand trafficking across the BBB. We expect that the present findings will identify PICALM as an important new therapeutic target for Ab clearancetherapyand treatment of Alzheimer’s neurovascular and neurodegenerative disorder.