The goal of this proposal is the experimental elucidation of basic cellular mechanisms responsible for normal or abnormal function of the blood-barrier (BBB). The main hypothesis to be tested is that intentional modification of bovine serum albumin (BSA) molecules increases their absorption to the surface of endothelial cells (ECs) and consequently facilitates uptake (endocytosis) and eventual transport (transcytosis across the wall of brain blood microvessels (capillaries, arterioles and venules). Thus, such a modification can interfere with the barrier function of the brain microvasculature. The albumin was chosen as a model protein because it is one of the normal components of blood plasma that serves as a carrier for many important molecules and does not affect the brain function, it can be chemically modified, and after complexing with colloidal gold it is excellently visible by electron microscopy (EM). Thus, it meets all requirements as a tracer for ultrastructural study, enabling one to gain insight into cellular mechanisms governing the function of the BBB. To test this hypothesis, the following modified BSA compounds complexed with colloidal gold will be used for perfusion of brain vasculature, and their interaction with the Ecs will be examined by EM: (1) cationized BSA, (2) poly-L-lysine- BSA complexes, as cationic tracers acting through coulombic forces (attraction by anionic sites present on the EC surfaces, (3) WGA-BSA, (4) RCA-BSA complexes acting through the affinity of a given lectin to specific sugar residues known to be present on the EC surfaces, (5) carbohydrate-modified albumin (glucosyl-, mannosyl-, and galactosyl-BSA) complexes acting through affinity to specific sugar-binding molecules (putative sugar-recognition sites or lectin-like molecules), which have been found to be present on the surface of some non-BBB types of endothelia but have not yet been studied in the BBB, (6) fatty acid-BSA complexes, the uptake of which by non-BBB type of Ecs was observed to be doubled, as compared to native BSA-gold complex. All these experiments will be performed on normal adult mice and for comparative purposes on aged animals and also on newborn mice with undeveloped (immature) BBB. Scrapie-infected mice will be used as an animal model related to aging, human neurodegenerative diseases and slow viral infection, presumably affecting the normal function of the BBB. These studies can shed new light on the mechanisms of delivery of albumin-bound, pharmacologically active substances to brain parenchyma in any pathological conditions in which it may be therapeutically advantageous.
