Sun B. Shim

Differentially expressed genes in transgenic mice carrying human mutant presenilin-2 (N141I): correlation of selenoprotein M with Alzheimer's disease.

Mutations in genes for Alzheimer’s disease (AD) result in a modulating of gene expressions in the brains of patients with AD. The aim of this study was to identify genes whose expression is modulated due to the over-expression of human mutant presenilin-2 (N141I) (hPS2m) in transgenic mice, which has previously been produced by us. To test this, GeneFishingTM DEG101 technique was performed on large-scale screen of mRNA from transgenic and non-transgenic brains. A total of 40 transcriptional products corresponding to cDNA were compared between two brains, and 17 showed a differential expression between the samples in all sets of experiments. However, all showed significant homology to known genes. Initially, a cloning corresponding to human selenoprotein M (hSelM) was chosen for investigation further because SelM induced by sodium selenite, a pro-oxidant, may have a functional role in catalyze the free radicals. We found that mouse SelM had significantly suppressed on its transcriptional products in transgenic brains. In parallel, suppression of endogenous was not observed in transgenic brains. Moreover, the levels of green fluorescence on hSelM fusion protein with EGFP were suppressed in the cells transfected with hPS2m, and its levels had actually increased by treatments of sodium selenite. Thus, the results indicate that SelM might play a suppressive or protective role in the pathology of patients with AD and it will be necessary to investigate further on functional roles of other up- and down-regulated gene in future.

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.

An In Vivo Bioassay for Detecting Antiandrogens Using Humanized Transgenic Mice Coexpressing the Tetracycline-Controlled Transactivator and Human CYP1B1 Gene

The typical strategy used in analysis of antiandrogens involves the morphological changes of a marker in castrated rats Hershberger assay for the prostate, seminal vesicle, levator ani plus bulbocavernosus muscles (LABC), Cowper’s gland, and glans penis. However, there are disadvantages to this approach, such as the time required, and the results may not correspond to those in actual human exposure. To evaluate its ability for detecting antiandrogens, in vivo the dose effect of di-(2-ethylhexyl) phthalate (DEHP) and time effect of five antiandrogens, DEHP, di-n-butyl phthalate (DBP), diethyl phthalate (DEP), linuron (3-(4-dichlorophenyl)-methoxy-1-methylurea), and 2,4′-DDE (1,1-dichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl)ethylene), were investigated using humanized transgenic mice coexpressing tetracycline-controlled transactivator (tTA) and the human cytochrome P450 (CYP) enzyme CYP1B1 (hCYP1B1). Adult transgenic males were treated with each of the five antiandrogens, and their tTA-driven hCYP1B1 expressions analyzed by real-time polymerase chain reaction (PCR) and/or Western blot and for O-debenzylation activity. Herein, the treatments of adult males with the five antiandrogens were shown to affect the increased levels of tTA-driven hCYP1B1 expression in both dose-dependent and repeated experiments. Thus, this novel in vivo bioassay, using humanized transgenic mice, is useful for measuring antiandrogens, and is a means to a more relevant bioas-say relating to actual human exposure.

Oligonucleotide-based Analysis of Differentially Expressed Genes in Hippocampus of Transgenic Mice Expressing NSE-controlled APPsw

The complexity of Alzheimer’s disease (AD) has made it difficult to examine its underlying mechanisms. A gene microarray offers a solution to the complexity through parallel analysis of most of the genes expressed in the hippocampal tissues from AD-transgenic and age-matched control littermates. This study examined the potential effect of APPsw over-expression on the modulation of genes for AD. To accomplish this, an oligonucleotide array was used with the large-scale screening of the hippocampus mRNA from 12-month-old APPsw-transgenic and control mice. There was a total of 116 differentially expressed genes, 59 up-regulated and 57 down-regulated, in the hippocampal region of the transgenic mice compared with the control mice. Initially, two of each of the down-regulated (Xlr3b and Mup3) and up-regulated genes (Serpina9 and Ccr6) were chosen for further investigation if the magnitude of change in these genes on the oligonucleotide array would correspond to those in the RT-PCR analysis from APPsw-transgenic mice. We also found that the changes in the differentially expressed genes are reliable. Thus, these genes might associate with AD neuropathology in neurodegenerative process of AD, although relevance of long lists altered genes should be evaluated in a future study.

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.

PEN-2 overexpression induces γ-secretase protein and its activity with amyloid β-42 production

PEN-2 is a component of the γ-secretase complex, which is involved in the cleavage of the β-amyloid precursor protein. The aim of this study was to determine the mechanism by which PEN-2 overexpression regulates γ-secretase expression and the production of Aβ-42. In order to determine this, a hybrid gene harboring human PEN-2 was constructed, and used in the transfection of SK-N-MC human neuroepitheliomal cells. This cell line was also co-transfected with a combination of human mutant presenilin 2 (hPS2m) and APPsw. Our results indicated that (i) human PEN-2 overexpression induced an increase in γ-secretase activity and its proteins, including PS1-CTF, APH-1, and nicastrin, thus production of Aβ-42, (ii) co-transfection of human PEN-2 with both hPS2m and APPsw exerted no more profound effects on the induction of γ-secretase proteins and its activity than did transfection with hPEN-2 alone. Thus, PEN-2 overexpression may facilitate assembly into the more active γ-secretase complex, and may also induce an increase in activity, thus affecting Aβ-42 production.

Early Changes in Behavior Deficits, Amyloid β-42 Deposits and MAPK Activation in Doubly Transgenic Mice Co-expressing NSE-Controlled Human Mutant PS2 and APPsw

Summary1.Doubly transgenic mice were some differences in the period proceeding of the development of Aβ-42 deposits and behavioral deficits. It was not characterized human mutant PS2 (hPS2) with APPsw in the brains of double transgenic mice. The aim of this study was to examine whether doubly transgenic mice co-expressing NSE-controlled APPsw and hPS2m develop AD-like phenotypes much earlier than singly APPsw or hPS2m alone.2.We produced doubly transgenic mice from a cross between our previously created NSE-controlled hPS2m and an APPsw transgenic line. This doubly transgenic line was quantitatively produced by cross with age-matched control mice, and the produced mice were separated into 5, 6, 7 and 8-month old age groups. At the age of 8 months, the four groups of mice were tested for behavioral function, levels of Aβ-42 deposition, and potential signaling events.3.It was shown that all the AD-like phenotypes, including behavior deficits, Aβ-42 levels, MAPK activation and ER expressions in doubly transgenic mice develop much earlier in the early time of AD development than their singly transgenic and non-transgenic littermates.4.The results suggest that elevated Aβ-42 levels, and MAPK activation in doubly transgenic mice are model for early diagnosis and treatment of AD with therapeutic drug.

Changes in presenilin 2-binding Wnt proteins, behavior, amyloid-β 42, γ-secretase activity, and testosterone sensitivity in transgenic mice coexpressing tetracycline-controlled transactivator and human mutant presenilin 2

Nonregulatable promoters have been mainly used to produce transgenic mice that express the human genes for Alzheimers disease (AD). The aim of this study was to produce doubly transgenic mice expressing the regulatable tet promoter-controlled transactivator (tTA) and human mutant presenilin 2 (N141I, hPS2m) genes in order to examine the AD-related phenotypes at the basal and inducible levels. To achieve this, the first lineage of the transgenic line, expressing Tet/tTA and the second lineage of transgenic mice, expressing Tet/hPS2m, were created, and the doubly transgenic mice were produced by crossing the Tet/tTA-transgenic mice with the Tet/hPS2m-transgenic mice. The doubly transgenic mice and nontransgenic littermates were then treated with or without doxycycline. The results showed that removing doxycycline from the transgenic mice resulted in the induction of the transgene, a Wnt signaling defect, behavioral impairment, elevated amyloid-β-42 and γ-secretase activity compared with in the group given doxycyline. Moreover, the expression levels of the hPS2m transgene decreased gradually in the transgenic males, with clear changes becoming apparent between 2 and 4 wk of age. Castrating these males resulted in an increased expression level of the hPS2m gene. This was restored to the normal levels by treatment with testosterone. Therefore, tetregulated transgenic mice can be used to examine the effect of the basal or inducible expression levels of hPS2m on the pathology of AD at the “on/off” states at any stage of development.

Differential Effect of 7,12-Dimethylbenz[a]anthracene on Human and Mouse CYP1B1 from Livers of Castrated Transgenic Mice

Humanized transgenic mice coexpressing tetracycline-controlled transactivator (tTA) and human cytochrome P450 1B1 (CYP1B1) (hCYP1B1) have been created by this group. The aims of this study was to determine if 7,12-dimethylbenz[a]anthracene (DMBA) functions as testosterone or doxycycline in its ability to induce or reduce expression of hCYP1B1 or endogenous mouse CYP1B1 (mCYP1B1). This was tested in the livers by treating castrated transgenic males and hCYP1B1/luciferase-transfected cells with DMBA. Herein, DMBA-treated group exhibited (i) gradual reduction of hCYP1B1 expression at the transcript, protein, and activity levels but gradually induced its transcript level during DMBA release; (ii) gradual reduction of hCYP1B1 at the transcript and protein levels, as in the case of doxycycline or testosterone; (iii) gradual induction of mCYP1B1 expression at the transcript and protein levels but gradually reduced its transcript level during DMBA release. In parallel, DMBA-treated transfected cells exhibited gradual increase in luciferase activity in a time- and dose-dependent manner. Thus, castrated transgenic males or in vitro system could be useful as models for the detection of polycyclic aromatic hydrocarbons (PAHs) or environmental toxicants by measuring either hCYP1B1 or mCYP1B1 expressions.