ABSTRACT
Selenium reportedly contribute to the modulation process of protein phosphorylation to regulate various cellular functions including growth, differentiation, proliferation and development. The aim of this study was to investigate whether selenium and Selenoprotein M (SelM) affects the mechanism of Alzheimer's disease. To achieve this, we determined the change of the MAPK pathway, secretase activity, and Tau phosphorylation in the transgenic rat overexpressing human selenoprotein M. Based on these results, we concluded that, i) CMV/GFP-hSelM Tg rats showed a high activity level of antioxidant enzyme in the brain tissues, ii) in response to selenium treatment, the ERK signaling pathway was significantly increased in Tg rats, but did not change in wild-type rats, iii) the activation of the ERK pathway by selenium treatment and SelM overexpression induced the inhibition of the alpha/gamma-secretase activity related to the protection of Abeta-42 production, iv) the activation of the ERK pathway by selenium treatment and SelM overexpression inhibited the phosphorylation in several sites of Tau protein. Therefore, these results provide strong evidence that selenium treatment and SelM activate the ERK pathway to attenuate alpha/gamma-secretase-mediated proteolysis and Tau phosphorylation to protect brain function.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Selenoproteins/metabolism , Sodium Selenite/pharmacology , tau Proteins/metabolism , Animals , Down-Regulation , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Phosphorylation , Rats , Rats, Transgenic , Selenoproteins/genetics , Signal TransductionABSTRACT
Nicotine is the principal psychoactive ingredient in cigarette smoke, and has been associated with health problems in humans. However, the pure form of nicotine may prove to be a valuable pharmaceutical agent for the treatment of AD. However, the beneficial effects of nicotine remain a matter of much controversy. In order to clarify this issue, 12-month-old transgenic mice, expressing neuron-specific enolase (NSE)-controlled APPsw, were treated with low, middle, and high doses of nicotine for 6 months. Herein, we have concluded that the nicotine-treated groups evidenced improvements in behavior and increases in the nicotine acetylcholine receptor, nAchRalpha7. These findings provide experimental evidence that nicotine effects an improvement in impaired memory, and that this improvement is associated with an increase in nAchRalpha7. Thus, nicotine may prove a good preventative or therapeutic modality for AD patients.
Subject(s)
Behavior, Animal/drug effects , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Receptors, Nicotinic/metabolism , Alzheimer Disease/drug therapy , Alzheimer Disease/pathology , Alzheimer Disease/physiopathology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Animals , Female , Humans , Male , Maze Learning/drug effects , Mice , Mice, Transgenic , Molecular Structure , Nicotine/chemistry , Nicotine/therapeutic use , Nicotinic Agonists/chemistry , Nicotinic Agonists/therapeutic use , Phosphopyruvate Hydratase/genetics , alpha7 Nicotinic Acetylcholine ReceptorABSTRACT
PEN-2 is a component of the gamma-secretase complex, which is involved in the cleavage of the beta-amyloid precursor protein. The aim of this study was to determine the mechanism by which PEN-2 overexpression regulates gamma-secretase expression and the production of Abeta-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 gamma-secretase activity and its proteins, including PS1-CTF, APH-1, and nicastrin, thus production of Abeta-42, (ii) co-transfection of human PEN-2 with both hPS2m and APPsw exerted no more profound effects on the induction of gamma-secretase proteins and its activity than did transfection with hPEN-2 alone. Thus, PEN-2 overexpression may facilitate assembly into the more active gamma-secretase complex, and may also induce an increase in activity, thus affecting Abeta-42 production.
Subject(s)
Amyloid Precursor Protein Secretases/biosynthesis , Amyloid beta-Peptides/biosynthesis , Peptide Fragments/biosynthesis , Presenilin-2/biosynthesis , Presenilin-2/genetics , Blotting, Western , Cell Line, Tumor , DNA/genetics , DNA Primers , Enzyme-Linked Immunosorbent Assay , Humans , Molecular Sequence Data , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
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.
Subject(s)
9,10-Dimethyl-1,2-benzanthracene/toxicity , Aryl Hydrocarbon Hydroxylases/metabolism , Carcinogens/toxicity , Cytochrome P-450 Enzyme System/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Orchiectomy , Animals , Anti-Bacterial Agents/pharmacology , Cytochrome P-450 CYP1B1 , Cytochrome P-450 Enzyme System/genetics , Doxycycline/pharmacology , Humans , Liver/drug effects , Liver/enzymology , Male , Mice , Mice, Transgenic , Testosterone Propionate/pharmacology , Tetracycline , Trans-Activators/geneticsABSTRACT
The complexity of Alzheimer's disease (AD) has made it difficult to examine its underlying mechanism. A gene microarray offers a solution to the complexity through a parallel analysis of most of the genes expressed in the brains from AD-transgenic mice. In our previous study, a total of 52 differentially expressed genes were identified in 18-month-old APPsw-transgenic mice compared to age-matched normal mice. We extended our work to better understand the relevant gene profiles from both early- and late-stage transgenic and normal mice. To accomplish this, cDNA microarray was used with the large-scale screening of the brain mRNA from transgenic and normal mice of 1 and 18 months of age. We identified a total of 48 genes, 6 up-regulated and 42 down-regulated, differentially expressed with a significant degree of induction and reduction in the brains from moderate 18-month-old transgenic mice compared to 1-month-old transgenic mice. In parallel, a total of 40 differentially expressed genes, 6 up-regulated and 34 down-regulated, were also found in the brains from moderate 18-month-old normal mice compared to 1-month-old normal mice. Thus, differentially expressed genes upon APPsw overexpression and the aging process are useful targets through which investigators can choose genes of particular interest. In the future, it will be necessary to study the function of differentially expressed genes, which are targets for developing drugs, using pharmacoproteomics.
Subject(s)
Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , Alzheimer Disease/pathology , Animals , Down-Regulation/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Up-Regulation/geneticsABSTRACT
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.
Subject(s)
Amyloid beta-Peptides/metabolism , Gene Expression Profiling , Gene Expression Regulation , Hippocampus/physiology , Oligonucleotides/metabolism , Peptide Fragments/metabolism , Phosphopyruvate Hydratase/genetics , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid beta-Peptides/genetics , Animals , Mice , Mice, Transgenic , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Peptide Fragments/geneticsABSTRACT
Nonregulatable promoters have been mainly used to produce transgenic mice that express the human genes for Alzheimer's 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-beta-42 and gamma-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.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/metabolism , Behavior, Animal/physiology , Peptide Fragments/metabolism , Presenilin-2/metabolism , Testosterone/metabolism , Wnt Proteins/metabolism , Animals , Brain/metabolism , Female , Gene Expression Regulation , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Presenilin-2/genetics , Protein Binding , Repressor Proteins/genetics , Repressor Proteins/metabolism , Tissue DistributionABSTRACT
Insulin-degrading enzyme (IDE) is a 110-kDa thiol zinc-methalloendopeptidase that can cleave small Abeta peptides and the APP intracellular domain (AICD). The aim of this study was to examine aging-related correlation of IDE with gamma-secretase-generated products involving insulin and glucose levels in transgenic brains expressing neuron-specific enolase (NSE)-controlled human mutant presenilin-2 (hPS2m). Herein, we concluded that the levels of IDE expression in transgenic brains were decreased relative to those of control mice at 15 months of age. In parallel, inhibition in the IDE expression at this age underlies to the levels-up of Abeta-42, AICD, gamma-secretase, and glucose with a level-down of insulin. Thus, IDE expression is critical target for the therapeutic trials.
Subject(s)
Aging/physiology , Blood Glucose/metabolism , Endopeptidases/metabolism , Insulin/metabolism , Insulysin/metabolism , Amyloid Precursor Protein Secretases , Amyloid beta-Peptides/metabolism , Animals , Aspartic Acid Endopeptidases , Brain/cytology , Brain/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Peptide Fragments/metabolism , Phosphopyruvate Hydratase/genetics , Phosphopyruvate Hydratase/metabolism , Presenilin-2ABSTRACT
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 GSK3beta 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 Abeta-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-beta APP accumulation than C83-betaAPP in APPsw-tansfectant and thereby promoted Abeta-42 production in transfectants. (ii) the inhibition of tau and GSK3beta 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 GSK3beta for protecting against Alzheimer's disease.
Subject(s)
Glycogen Synthase Kinase 3/metabolism , Peptidylprolyl Isomerase/metabolism , tau Proteins/metabolism , Amyloid beta-Protein Precursor/metabolism , Animals , Animals, Genetically Modified , Base Sequence , DNA Primers , Glycogen Synthase Kinase 3 beta , NIMA-Interacting Peptidylprolyl Isomerase , Rats , TransfectionABSTRACT
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, GeneFishing 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.
Subject(s)
Alzheimer Disease/genetics , Gene Expression Profiling , Gene Expression Regulation , Membrane Proteins/physiology , Selenoproteins/genetics , Animals , Base Sequence , DNA Primers , Humans , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Presenilin-2 , RNA, Messenger/genetics , Sodium Selenite/pharmacologyABSTRACT
The amyloid protein precursor (APP) is cleaved in its intramembranous domain by gamma-secrease to generate amyloid beta 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 Abeta-42 levels, and whether or not it is associated with the expressions of GSK3beta-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 Abeta-42 level, gamma-secretase activity, GSK3beta-binding proteins including PS1, tau, and beta-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.
Subject(s)
Alzheimer Disease/physiopathology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Behavior, Animal/physiology , Glycogen Synthase Kinase 3/metabolism , Peptide Fragments/metabolism , Phosphopyruvate Hydratase/genetics , Alzheimer Disease/genetics , Amino Acid Sequence , Amyloid beta-Protein Precursor/chemistry , Animals , Base Sequence , Disease Models, Animal , Female , Glycogen Synthase Kinase 3 beta , Male , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Molecular Sequence Data , Promoter Regions, Genetic/physiology , Protein Structure, Tertiary , Transgenes/physiologyABSTRACT
1. Doubly transgenic mice were some differences in the period proceeding of the development of Abeta-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 Abeta-42 deposition, and potential signaling events. 3. It was shown that all the AD-like phenotypes, including behavior deficits, Abeta-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 Abeta-42 levels, and MAPK activation in doubly transgenic mice are model for early diagnosis and treatment of AD with therapeutic drug.
Subject(s)
Alzheimer Disease/physiopathology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , MAP Kinase Signaling System/physiology , Membrane Proteins/genetics , Peptide Fragments/metabolism , Phosphopyruvate Hydratase/genetics , Alzheimer Disease/genetics , Animals , Behavior, Animal/physiology , Disease Models, Animal , Female , Humans , Male , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Presenilin-2 , Promoter Regions, Genetic/physiologyABSTRACT
cDNA microarray technique has been widely used for the detection and elucidation of differentially expressed genes on a large scale and at a speed never before possible. The aim of this study was to gain insight into the potentially overexpressed effects of APPsw on the modulation of genes for Alzheimer's disease (AD), which is central to understanding the complexity of AD. APPsw transgenic mice, which we previously produced, provide an important resource for identifying differentially expressed genes since this transgenic line was shown to have cognitive deficits along with Abeta-42 deposits at 12 months of age. To identify differentially expressed genes, cDNA microarray technique was conducted to get a large-scale screening of brain mRNA from 18 month-old NSE/APPsw transgenic and non-transgenic mice. A total of 52 differentially expressed genes, 10 up-regulated and 42 down-regulated, were found in the brains of moderately transgenic mice compared to non-transgenic littermates. Thus, the results suggest the need for future studies on gene functions, pathology, toxicogenomics, and pharmacogenomics.
Subject(s)
Amyloid beta-Protein Precursor/genetics , Brain/metabolism , Gene Expression Profiling , Oligonucleotide Array Sequence Analysis/methods , Animals , Down-Regulation/genetics , Female , Gene Expression Regulation/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phosphopyruvate Hydratase/genetics , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Up-Regulation/geneticsABSTRACT
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 bioassay relating to actual human exposure.
