Sang Gi Paik

ABSTRACT Alzheimer's disease (AD) occurs when neurons in the memory and cognition regions of the brain are accompanied by an accumulation of the long amyloid p‐proteins of the 39 to 43 amino acids derived from the amyloid precursor protein (APP) by cleavage with p‐and γ‐secretase. An increased production of Ap‐42 by mutation of PS2 genes promotes caspase expression and is associated with the Cox‐2 found in the brain of AD patients. To address this question in vivo, we expressed the human mutant PS2 (hPS2m ) (N141I) as well as wild PS2 (hPS2w) as a control in transgenic (Tg) mice under control of the neuron‐specific enolase ( NSE ) promoter. Water maze tests were used to demonstrate the behavioral defect; dot blot, Western blot, and immunohistochemical analyses were performed on the brain with the hPS2, Ap‐42, caspase‐3, and Cox‐2 antibody. We concluded that 1) Tg mice showed a behavioral dysfunction in the water maze test, 2) levels of hPS2, Ap‐42, caspase‐3, and Cox‐2 expression were modulated in the brains of both Tg mice, 3) dense staining with antibody to hPS2, Ap‐42, caspase‐3, and Cox‐2 was visible in the brains of Tg mice compared with age‐matched control mice, and 4) distinguishable AD phenotypes between hPS2w‐and hPS2m‐Tg mice did not appear. These results suggest that an elevation of Ap‐42 by overexpression of hPS2 and mutation of hPS2m might induce the behavioral deficit and caspase‐3 and Cox‐2 induction, which could be useful in the therapeutic testing of compounds to have considerable clinical effects.—Hwang, D. Y., Chae, K. R., Kang, T. S., Hwang, J. H., Lim, C. H., Kang, H. K., Goo, J. S., Lee, M. R., Lim, H. J., Min, S. H., Cho, J. Y., Hong, J. T., Song, C. W., Paik, S. G., Cho, J. S., Kim, Y. K. Alterations in behavior, amyloid p‐42, caspase‐3, and Cox‐2 in mutant PS2 transgenic mouse model of Alzheimer's disease. FASEB J. 16, 805–813 (2002)

UNLABELLED: CaBP-9k may be involved in the active calcium absorption and embryo implantation. Although we generated CaBP-9k KO mice to explore its function, no distinct phenotypes were observed in these KO mice. It can be hypothesized that TRPV5 and 6 and plasma membrane calcium ATPase 1b may play a role in the regulation of calcium transport to compensate CaBP-9k deficiency in its KO model. INTRODUCTION: Active calcium transport in the duodenum and kidney is carried in three steps: calcium entry through epithelial Ca2+ channels (TRPV5 and TRPV6), buffering and/or transport by calbindin-D9k (CaBP-9k) and -D28k (CaBP-28k), and extrusion through the plasma membrane calcium ATPase 1b (PMCA1b) and sodium/calcium exchanger 1. Although the molecular mechanism of calcium absorption has been studied using knockouts (KOs) of the vitamin D receptor and CaBP-28k in animals, the process is not fully understood. MATERIALS AND METHODS: We generated CaBP-9k KO mice and assessed the phenotypic characterization and the molecular regulation of active calcium transporting genes when the mice were fed different calcium diets during growth. RESULTS: General phenotypes showed no distinct abnormalities. Thus, the active calcium transport of CaBP-9k-null mice proceeded normally in this study. Therefore, the compensatory molecular regulation of this mechanism was elucidated. Duodenal TRPV6 and CaBP-9k mRNA of wildtype (WT) mice increased gradually during preweaning. CaBP-9k is supposed to be an important factor in active calcium transport, but its role is probably compensated for by other calcium transporter genes (i.e., intestinal TRPV6 and PMCA1b) during preweaning and renal calcium transporters in adult mice. CONCLUSIONS: Depletion of the CaBP-9k gene in a KO mouse model had little phenotypic effect, suggesting that its depletion may be compensated for by calcium transporter genes in the intestine of young mice and in the kidney of adult mice.

Numerous transgenic mouse models for Alzheimer's disease (AD) have been generated to recapitulate the histological pathogenesis and behavioral phenotypes of AD brain. However, none of the existing models exhibits the full spectrum of AD symptoms, nor have all of the traits mimicked by the developed animal models been successfully represented within a single mouse line, indicating that the development of transgenic lines showing new features of the AD-like brain should be explored. Here we report on a transgenic mouse line, named Tg-APP (Sw, V717F)/B6, that expresses the human amyloid precursor protein (APP) containing the Swedish and the V717F Indiana mutations in the brains of inbred C57BL/6 mice, designed to eliminate the potential phenotypic variations attributed to the compound genetic backgrounds adopted in most AD mouse models. The Tg-APP (Sw, V717F)/B6 mice expressed the transgene transcript, in the heterozygote state, at a level of 2.6 +/- 0.1 fold higher than that of endogenous mouse APP. However, no Abeta-plaque deposition was produced in the brain of the Tg-APP (Sw, V717F)/B6 mice up to 18 months of age. The Tg-APP(Sw, V717F)/B6 mice at 13-15 months showed reduced expression of calbindin and c-Fos in the brain. The Tg-APP (Sw, V717F)/B6 mice at 11-14 months displayed decreased motor coordination, learning and memory deficits, and severely increased anxiety. These phenotypes were not observed in the Tg-APP (Sw, V717F)/B6 mice at 5-7 months. Microarray analysis revealed altered expression, in the amygdala of the Tg-APP (Sw, V717F)/B6 mice, of genes previously implicated in anxiety. Taken together, these results suggest that the transgenic APP, or its derivatives, produces the age-dependent pathophysiology of the AD-like brain and that the progressive cognitive impairment and anxiety induction can proceed in the absence of visible Abeta-plaque deposition.

Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell-cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD(416)G and DSPD(748)G) in vitro, and point mutations substituting the Asp of DVPD(416)G and DSPD(748)G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD(416)G generated a 74-kDa fragment, which was in turn cleaved at DSPD(748)G, yielding 47- and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas-paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix.