s that prevent endocytotic Rocaglamide site uptake of AF633-Trf, 4uC and ATP depletion, did not affect F-Ab40 uptake. However, BBB endothelial cells, a major constituent of the neurovascular unit believed to play a critical role in neurodegenerative diseases like AD and vascular dementia, internalized F-Ab40 via endocytotic and energy dependent pathways. This inference was drawn from two crucial observations: a) in BBME cells, almost the entire amount of internalized F-Ab40 accumulated in the acidic cell compartments, which suggests endocytotic uptake; and b) like the uptake of endocytotic marker AF633-Trf, the uptake of F-Ab40 was inhibited at 4uC and under ATP depleted conditions. Energy independent uptake of cell 
penetrating peptides in various cell types has been proposed previously. But the energy independent uptake of proteins specific to a particular cell type is very unusual. Nevertheless, the possibility of Ab40 displaying 22315414 such attribute may not come as a surprise if the recent literature describing the biophysical and physiological behavior of this protein is carefully examined. In attempting to study the neuronal internalization of Ab40 and 42, researchers in the past have encountered non-saturable and energy independent uptake of these proteins. This atypical behavior has also been reported with other b-sheet forming proteins such as human calcitonin. It was argued that the bsheet structure could facilitate the interaction of the protein with the plasma membrane and enhance its passive diffusion across the cellular barrier. Several researchers have reported the ability of Ab40 to intercalate in the hydrocarbon core of the neuronal Cellular Uptake of Ab Proteins 10 Cellular Uptake of Ab Proteins membrane and increase its fluidity. After attaining higher concentrations in the neuronal membrane, Ab40 could passively diffuse to a region of lower concentration, most likely the neuroplasm. These biophysical interactions of Ab40 with the plasma membrane were reported to be influenced by the membrane lipid composition, which could change significantly with cell type. In addition to the expression of Ab40 receptors, differences in the plasma membrane lipid composition 11 Cellular Uptake of Ab Proteins 12150697 cally interact with the neuronal membrane. A significant proportion of internalized Ab40 is located outside of the endosomal or lysosomal compartments; as a consequence, the protein could accumulate in the neuroplasm without degradation and subsequently aggregate to form fibrils. In contrast, BBME cells exhibit energy dependent uptake of Ab40 and accumulate the protein in acidic cell organelle such as endosomes and lysosomes, which is indicative of endocytotic uptake. Such a phenomenal Cellular Uptake of Ab Proteins 13 Cellular Uptake of Ab Proteins difference in the internalization of Ab40 between neurons and BBB endothelial cells may provide essential clues to understanding how various cells can differentially regulate Ab proteins and help explain the vulnerability of cortical and hippocampal neurons to Ab toxicity. Materials and Methods Synthesis of fluorescein labeled human Ab40 F-Ab40 was synthesized on an ABI 433 peptide synthesizer with Val-NovaSyn TGA resin employing HBTU activation and synthesis protocols recommended by the manufacturer. After the final deprotection of the N-terminal Fmoc group, a two equivalent excess of NHS-fluorescein was dissolved in 6 ml of dimethylformamide and added to the resin saturated with 12% diisopropylethylamine/d