Hwa J. Lim

Alterations in behavior, amyloid β-42, caspase-3, and Cox-2 in mutant PS2 transgenic mouse model of Alzheimer’s disease

Alzheimers 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 Alzheimers disease. FASEB J. 16, 805–813 (2002)

Aberrant expressions of pathogenic phenotype in Alzheimer's diseased transgenic mice carrying NSE-controlled APPsw

Mutations in the APP gene lead to enhanced cleavage by the beta- and gamma-secretase, and increased Abeta formation, which are tightly associated with Alzheimers disease (AD)-like neuropathological changes. To examine whether depositions of Abeta by APP mutations are increased, and if this is associated with potential pathogenic phenotypes, the APPsw was expressed in a transgenic line under the control of the neuron-specific enolase (NSE) promoter. A behavioral dysfunction was shown at 12 months, and intensive staining bands, with APP and Abeta-42 antibodies, were visible in the brains of transgenic mice. Of the MAPK family, both JNK and p38 were activated in the brains of transgenic mice, whereas there was no significant activation of the ERK. In parallel, tau phosphorylation was also enhanced in the transgenic relative to the control mice. Moreover, the Cox-2 levels, from Western blot and immunostaining, were increased in the brains of the transgenic line. Furthermore, there were significant caspase-3- and TUNEL-stained nuclei in the transgenic line compared to the age-matched control mice. Thus, these results suggest that NSE-controlled APPsw transgenic mice appear to be a more relevant model in neuropathological phenotypes of AD, and thus could be useful in developing new therapeutic treatments for targeting the aberrant phenotypes that appear in these mice.

Use of NSE/PS2m-transgenic mice in the study of the protective effect of exercise on Alzheimer's disease

In its late stage, Alzheimers disease results in progressive muscle weakness in the arms and legs. The aim of this study was to determine whether mice expressing the skeletal muscle-specific mutant PS2 gene (a model of Alzheimers disease) are a useful experimental system to study the protective effect of exercise on Aβ-42 reduction, improvement of behavioural function and changes in metabolic parameters. With this aim in mind, the transgenic mice were subjected to treadmill exercise for 3 months. The results showed that in transgenic mice, but not in normal mice, treadmill exercise resulted in a reduction of Aβ-42 deposits and an improvement in behavioural function, thereby restoring normal concentrations of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and triglyceride. Thus, exercise may represent a practical therapeutic strategy for use with human patients with Alzheimers disease.

Tau and GSK3β Dephosphorylations are Required for Regulating Pin1 Phosphorylation

Pin1 binds mitotically phosphorylated Thr231–Pro232 and Thr212–Pro213 sites on tau, and a Pin1 deficiency in mice leads to tau hyperphosphorylation. The aim of this study was to determine if the dephosphorylation or inhibition of tau and GSK3β phosphorylation induces the Pin1 phosphorylation. To test this, human SK-N-MC cells were stably transfected with a fusion gene containing neuron-specific enolase (NSE)-controlled APPsw gene(NSE/APPsw), to induce Aβ-42. The stable transfectants were then transiently transfected with NSE/Splice, lacking human tau (NSE/Splice), or NSE/hTau, containing human tau, into the cells. The NSE/Splice- and NSE/hTau-cells were then treated with lithium. We concluded that (i) there was more C99-β APP accumulation than C83-βAPP in APPsw-tansfectant and thereby promoted Aβ-42 production in transfectants. (ii) the inhibition of tau and GSK3β phosphorylations correlated with increase in Pin1 activation in NSE/hTau- cells. Thus, these observations suggest that Pin1 might have an inhibitive role in phosphorylating tau and GSK3β for protecting against Alzheimer’s disease.

Differential expression of the tetracycline-controlled transactivator-driven human CYP1B1 gene in double-transgenic mice is due to androgens: application for detecting androgens and antiandrogens.

Differential expression of the tetracycline-controlled transactivator (tTA)-driven human cytochrome p450 (CYP) 1B1 gene was found in the livers of male mice, at high levels in neonates, but at low levels in adults. The goals of this study were to determine whether the differential expression of the tTA-driven human CYP1B1 (hCYP1B1) gene in neonates and adults was testosterone dependent and whether flutamide, a representative potent antiandrogen, led to the induction of hCYP1B1. This was tested by treating castrated transgenic mice with testosterone propionate and musk extracts. It was concluded that: (i). the levels of expression of both tTA and hCYP1B1 gradually declined, with clear changes being apparent between 2 and 4 weeks of age, (ii). castration of adult males resulted in the increased expressions of both tTA and hCYP1B1 to levels similar to those found in adult females, (iii). treatment of castrated male and adult female mice with testosterone propionate and musk extracts led to the restoration of the levels of expression of hCYP1B1 in the adult males, and (iv). treatment of adult males with flutamide caused an increase in the levels of expression of hCYP1B1 in the adult females, as indicated by the antiandrogenic activity. Thus, the differential expression of the tTA-driven hCYP1B1 gene in the transgenic mice was caused by androgen, and it is possible that castrated male and adult female mice expressing the tTA-controlled hCYP1B1 could be used as the basis for a strategy for the detection of androgens and antiandrogens.

NSE-Controlled Carboxyl-Terminus of APP Gene Over-Expressing in Transgenic Mice Induces Altered Expressions in Behavior, Aβ-42, and GSK3β Binding Proteins

SummaryThe amyloid protein precursor (APP) is cleaved in its intramembranous domain by γ-secrease to generate amyloid β and a free carboxyl-terminal intracellular fragment. The carboxyl-terminal of 105 amino acids of APP (APP-C105) plays a crucial role in the neuropathology of Alzheimer’s disease (AD), but it is incompletely understand how APP-C105 overexpression interacts and regulates the brain function and Aβ-42 levels, and whether or not it is associated with the expressions of GSK3β-binding proteins. To test this, transgenic mice expressing NSE-controlled APP-C105 were produced and tested for their above phenotypes. A behavioral deficit was observed in the 9 months old transgenic mice, and western blot indicated that there was a predominant expression of APP-C105 in transgenic brains compared with those of non-transgenic brains. In parallel, APP-C105 overexpression resulted in the modulation of the Aβ-42 level, γ-secretase activity, GSK3β-binding proteins including PS1, tau, and β-catenin in the brains of the transgenic mice relative to the non-transgenic mice. Thus, altered expressions of these neuropathological phenotypes in APP-C105 transgenic mice could be useful targets in developing new therapeutic treatments.

An In Vitro Bioassay for Xenobiotics Using the SXR-Driven Human CYP3A4/lac Z Reporter Gene

The dose and time effect of nine xenobiotics, including 17β-estradiol, corticosterone, dexamethasone, progesterone, nifedipine, bisphenol A, rifampicin, methamphetamine, and nicotine were investigated, in vitro, using human steroid and xenobiotics receptor (SXR)-binding sites on the human CYP3A4 promoter, which can enhance the linked lac Z reporter gene transcription. To test this, liver-specific SAP (human serum amyloid P component)-SXR (SAP/SXR) and human CYP3A4 promoter-regulated lac Z (h CYP3A4/lac Z) constructs were transiently transfected into Hep G2 and NIH3T3 cells to compare the xenobiotic responsiveness between human and nonhuman cell lines. In the Hep G2 cells, rifampicin, followed by corticosterone, nicotine, methamphetamine, and dexamethasone, exhibited enhanced levels of the lac Z transcript, whereas those of bisphenol A and nifedipine were found to be reduced. No significant responses were observed with 17β-estradiol or progesterone. In addition, 17β-estradiol and progesterone did not change the levels of the lac Z transcripts in the Hep G2 cells, but did induce significant increases in the transcripts of the NIH3T3 cells. Treatment with corticosterone and dexamethasone, which were highly expressed in the Hep G2 cells, did not affect the levels of the lac Z transcript in NIH3T3 cells. These results show that lac Z transcripts can be measured, rapidly and reproducibly, using reverse transcriptase–polymerase chain reaction (RT-PCR) based on the expression of the h CYP3A4/lac Z reporter gene, and was mediated by the SXR. Thus, this in vitro reporter gene bioassay is useful for measuring xenobiotic activities, and is a means to a better relevant bioassay, using human cells, human genes and human promoters, in order to get a closer look at actual human exposure.