(1) The blood-brain barrier (BBB) characteristics of cerebral endothelial cells are induced by organ-specific local signals. Brain endothelial cells lose their phenotype in cultures without cross-talk with neighboring cells. (2) In contrast to astrocytes, pericytes, another neighboring cell of endothelial cells in brain capillaries, are rarely used in BBB co-culture systems. (3) Seven different types of BBB models, mono-culture, double and triple co-cultures, were constructed from primary rat brain endothelial cells, astrocytes and pericytes on culture inserts. The barrier integrity of the models were compared by measurement of transendothelial electrical resistance and permeability for the small molecular weight marker fluorescein. (4) We could confirm that brain endothelial monolayers in mono-culture do not form tight barrier. Pericytes induced higher electrical resistance and lower permeability for fluorescein than type I astrocytes in co-culture conditions. In triple co-culture models the tightest barrier was observed when endothelial cells and pericytes were positioned on the two sides of the S. Nakagawa Á M. A. Deli Á S. Nakao Á R. Nakaoke Á M. Niwa Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan 687-694 DOI 10.1007/s10571-007-9195-4 123 porous filter membrane of the inserts and astrocytes at the bottom of the culture dish. (5) For the first time a rat primary culture based syngeneic triple co-culture BBB model has been constructed using brain pericytes beside brain endothelial cells and astrocytes. This model, mimicking closely the anatomical position of the cells at the BBB in vivo, was superior to the other BBB models tested. (6) The influence of pericytes on the BBB properties of brain endothelial cells may be as important as that of astrocytes and could be exploited in the construction of better BBB models.

The ␤-amyloid peptide, which forms extracellular cerebral deposits in Alzheimer's disease, is derived from a large membrane-spanning glycoprotein referred to as the ␤-amyloid precursor protein (APP). The APP is normally cleaved within the ␤-amyloid region by a putative proteinase (␣-secretase) to generate large soluble amino-terminal derivatives of APP, and this event prevents the ␤-amyloid peptide formation. It has been suggested that the gelatinase A (matrix metalloproteinase 2, a 72-kDa type IV collagenase) may act either as ␣-secretase or as ␤-secretase. Mice devoid of gelatinase A generated by gene targeting develop normally, except for a subtle delay in their growth, thus providing a useful system to examine the role of gelatinase A in the cleavage and secretion of APP in vivo. We show here that APP is cleaved within the ␤-amyloid region and secreted into the extracellular milieu of brain and cultured fibroblasts without gelatinase A activity. The data suggest that gelatinase A does not play an essential role in the generation and release of soluble derivatives of APP at physiological conditions. Amyloid precursor protein (APP) 1 is an integral membrane protein that is produced by most cells (1). Several isoforms ranging from 365 to 770 amino acids are generated by alternative splicing of transcripts from the APP gene on the long arm of chromosome 21 (2). Proteolytic cleavage of APP by enzymes termed ␣-, ␤-, and ␥-secretases generates various APP fragments that are released from APP expressing cells (3). ␤-Amyloid peptides (A␤) of 40 -43 amino acids are released by the action of ␤-and ␥-secretases cleaving at or near residues 671 and 713 (numbers refer to APP 770 ), respectively (3). Cleavage of APP at a membrane proximal site by an ␣-secretase releases larger APP fragments (sAPP) and prevents generation of A␤. The absolute and relative amounts of various APP fragments that are released in the brain are thought to be of importance in the formation of first amorphous (diffuse) and then filamentous (amyloid) plaques that are characteristic of Alzheimer's disease. Two larger forms sAPP also known as protease nexin II have a Kunitz type serine protease inhibitor domain (4, 5). In addition all forms of sAPP have in the carboxyl-terminal region a domain that inhibits gelatinase A (matrix metalloproteinase 2, a 72-kDa type IV collagenase) activity (6). On the other hand, gelatinase A has been suggested to act on APP either as ␣-secretase (6) or as ␤-secretase (7), although the hypothesis is controversial (8, 9). To clarify the putative role of this enzyme, we studied APP fragmentation and release in gelatinase A knockout mice. MATERIALS AND METHODSGeneration of Gelatinase A Gene-deficient Mice-A genomic DNA clone of mouse gelatinase A (Clg4a) was isolated from a 129/Sv genomic library. The fragments used for constructing the targeting vector were a 2.0-kb HindIII fragment of the distal region of the promoter and a 4.5-kb XbaI-SacI fragment. In the resulting targeting construct, 5.9 kb containing the exon 1 were r...

Activation of GSK-3β is presumed to be involved in various neurodegenerative diseases, including Alzheimer's disease (AD), which is characterized by memory disturbances during early stages of the disease. The normal function of GSK-3β in adult brain is not well understood. Here, we analyzed the ability of heterozygote GSK-3β knockout (GSK+/−) mice to form memories. In the Morris water maze (MWM), learning and memory performance of GSK+/− mice was no different from that of wild-type (WT) mice for the first 3 days of training. With continued learning on subsequent days, however, retrograde amnesia was induced in GSK+/− mice, suggesting that GSK+/− mice might be impaired in their ability to form long-term memories. In contextual fear conditioning (CFC), context memory was normally consolidated in GSK+/− mice, but once the original memory was reactivated, they showed reduced freezing, suggesting that GSK+/− mice had impaired memory reconsolidation. Biochemical analysis showed that GSK-3β was activated after memory reactivation in WT mice. Intraperitoneal injection of a GSK-3 inhibitor before memory reactivation impaired memory reconsolidation in WT mice. These results suggest that memory reconsolidation requires activation of GSK-3β in the adult brain.

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