Cell cycle aberrations by alpha-synuclein over-expression and cyclin B immunoreactivity in Lewy bodies.

alpha-Synuclein is a presynaptic protein that accumulates abnormally in Lewy bodies of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Its physiological function and role in neuronal death remain poorly understood. Recent immunohistochemical studies suggest that cell cycle-related phenomena may play a role in the pathogenesis of Alzheimer's disease and perhaps other neurodegenerative disorders. In this investigation, we examined the effects of alpha-synuclein expression levels on cell cycle indices in PC12 cells engineered to conditionally induce alpha-synuclein expression upon withdrawal of doxycycline. Over-expression of alpha-synuclein resulted in enhanced proliferation rate and enrichment of cells in the S phase of the cell cycle. This was associated with increased accumulation of the mitotic factor cyclin B and down-regulation of the tumor suppressor retinoblastoma 2. Additionally, ERK1/2, key molecules in proliferation signaling, were highly phosphorylated. Immunohistochemical studies on postmortem brains revealed intense cyclin B immunoreactivity in Lewy bodies in cases with DLB and to a lesser extent in PD. We propose that elevated expression of alpha-synuclein causes changes in cell cycle regulators through ERK activation leading to apoptosis of postmitotic neurons. These changes in cell cycle proteins are also associated with ectopic expression of cyclin B in Lewy bodies.

Apoptotic signaling in dopamine-induced cell death: the role of oxidative stress, p38 mitogen-activated protein kinase, cytochrome c and caspases.

Oxidative stress generated by dopamine (DA) oxidation could be one of the factors underlying the selective vulnerability of nigral dopaminergic neurons in Parkinson's diseases. Here we show that DA induces apoptosis in SH-SY5Y neuroblastoma cells demonstrated by activation of caspase-9 and caspase-3, cleavage of poly(ADP-ribose) polymerase as well as nuclear condensation. We also show that p38 mitogen-activated protein kinase is activated within 10 min of DA treatment, which precedes the onset of apoptosis because the potent p38 kinase inhibitor SB203580 protects against DA-induced cell death as well as against caspase-9 and caspase-3 activation. In addition, the antioxidant N-acetyl-L-cysteine (NAC) effectively blocks DA-induced p38 kinase activation, caspase-9 and caspase-3 cleavage and subsequent apoptosis, indicating that DA triggers apoptosis via a signaling pathway that is initiated by the generation of reactive oxygen species (ROS). Dopamine exerts its toxicity principally intracellularly as the DA uptake inhibitor, nomifensine significantly reduces DA-induced cell death as well as activation of p38 kinase and caspase-3. Furthermore, DA induces mitochondrial cytochrome c release, which is dependent on p38 kinase activation and precedes the cleavage of caspases. These observations indicate that DA induces apoptosis primarily by generating ROS, p38 kinase activation, cytochrome c release followed by caspase-9 and caspase-3 activation.

Characterization of the 5' flanking region of the rat D(3) dopamine receptor gene.

The D(3) dopamine receptor has a restricted regional distribution in brain and is regulated by dopaminergic agents. Additionally, the D(3) gene is implicated in the pathogenesis of several neuropsychiatric disorders or in their response to pharmacological agents. Elucidating its transcription control mechanisms is therefore of interest in order to explain these biological features of the D(3) gene. In this study, the 5' flanking region of the rat D(3) gene was characterized by isolating the 5' end of its cDNA as well as 4.6 kb of genomic sequence. Analysis of this region revealed the presence of two new exons 196-bp and 120-bp long, separated by an 855-bp intron, located several kilobases upstream of the previously published coding exons. Thus, current evidence indicates that the rat D(3) gene is organized into eight exons. Transcription initiation site was determined by primer extension analysis and repeated rounds of 5' RACE and was found to localize at a pyrimidine-rich consensus 'initiator' sequence, similar to the rat D(2) gene. The D(3) promoter lacks TATA or CAAT boxes but unlike that of other dopamine receptor genes has only 52% GC content. Functional analysis of D(3) promoter deletion mutants fused to a reporter gene in TE671 cells, which endogenously express this gene, revealed strong transcriptional activity localized within 36 nucleotides upstream of transcription start site, and a potent silencer between bases --37 and --537. The D(3) promoter is inactive in C6 and COS7 cells. We conclude that the D(3) gene, similar to the closely related D(2) gene, is transcribed from a tissue specific promoter which is under intense negative control.

ZIC2 and Sp3 repress Sp1-induced activation of the human D1A dopamine receptor gene.

The human D(1A) dopamine receptor is transcribed from a tissue-specific regulated gene under the control of two promoters. An activator region (AR1) located between nucleotides -1154 and -1136 (relative to the first ATG) enhances transcription from the upstream promoter that is active in the brain. In this investigation, we sought to identify the nuclear factors that regulate the D(1A) gene through their binding to AR1 using yeast one-hybrid screening. Sp3 and Zic2 were among the positive clones isolated. Although Sp1 was not isolated from this screening and purified Sp1 alone does not bind to AR1 in gel shift experiments, this general transcription factor binds to AR1 in the presence of D(1A) expressing NS20Y nuclear extract and activates the D(1A) promoter. Thus, Sp1 appears to require an unknown factor(s) or post-translational modification to interact with AR1. On the other hand, Zic2 and Sp3 inhibit Sp1-induced activation of the D(1A) gene in an AR1-dependent manner. Zic2 and D(1A) genes have reciprocal brain regional distributions; Zic2 is expressed primarily in the cerebellum, and D(1A) is highly expressed in corpus striatum. These observations collectively suggest that one of the physiologic functions of Zic2 is repression of D(1A) gene transcription and that the intracellular balance among Sp1, Sp3 and Zic2 is important for regulating the tissue-specific expression of this dopamine receptor.

Requirement of hydrogen peroxide generation in TGF-beta 1 signal transduction in human lung fibroblast cells: involvement of hydrogen peroxide and Ca2+ in TGF-beta 1-induced IL-6 expression.

Stimulation of human lung fibroblast cells with TGF-beta1 resulted in a transient burst of reactive oxygen species with maximal increase at 5 min after treatment. This reactive oxygen species increase was inhibited by the antioxidant, N-acetyl-l -cysteine (NAC). TGF-beta1 treatment stimulated IL-6 gene expression and protein synthesis in human lung fibroblast cells. Antioxidants including NAC, glutathione, and catalase reduced TGF-beta1-induced IL-6 gene expression, and direct H2O2 treatment induced IL-6 expression in a dose-dependent manner. NAC also reduced TGF-beta1-induced AP-1 binding activity, which is involved in IL-6 gene expression. It has been reported that Ca2+ influx is stimulated by TGF-beta1 treatment. EGTA suppressed TGF-beta1- or H2O2-induced IL-6 expression, and ionomycin increased IL-6 expression, with simultaneously modulating AP-1 activity in the same pattern. PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase/extracellular signal-related kinase kinase 1, suppressed TGF-beta1- or H2O2-induced IL-6 and AP-1 activation. In addition, TGF-beta1 or H2O2 increased MAPK activity which was reduced by EGTA and NAC, suggesting that MAPK is involved in TGF-beta1-induced IL-6 expression. Taken together, these results indicate that TGF-beta1 induces a transient increase of intracellular H2O2 production, which regulates downstream events such as Ca2+ influx, MAPK, and AP-1 activation and IL-6 gene expression.

PQBP-1/Npw38, a nuclear protein binding to the polyglutamine tract, interacts with U5-15kD/dim1p via the carboxyl-terminal domain.

PQBP-1 was identified as a binding protein to the polyglutamine tract present in various transcription-related factors and causative genes for neurodegenerative disorders. This novel gene contains at least two functional domains, WW domain and carboxyl-terminal domain (CTD), strictly conserved beyond species. Although human PQBP-1 additionally contains the polar amino acid-rich domain by which it binds to the polyglutamine tract, genuine physiological function(s) have not been clarified. In this study, we showed that U5-15kD, human homologue of fission yeast dim1p, is a partner molecule of PQBP-1 binding to CTD. This finding suggests physiological functions of PQBP-1 in splicing, cell cycle, and ubiquitination, through which we can speculate the pathological roles of PQBP-1 in triplet repeat diseases.

Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function.

As a result of identifying the regulatory proteins of thioredoxin (TRX), a murine homologue for human vitamin D3 up-regulated protein 1 (VDUP1) was identified from a yeast two-hybrid screen. Cotransfection into 293 cells and precipitation assays confirmed that mouse VDUP1 (mVDUP1) bound to TRX, but it failed to bind to a Cys32 and Cys35 mutant TRX, suggesting the redox-active site is critical for binding. mVDUP1 was ubiquitously expressed in various tissues and located in the cytoplasm. Biochemical analysis showed that mVDUP1 inhibited the insulin-reducing activity of TRX. When cells were treated with various stress stimuli such as H2O2 and heat shock, mVDUP1 was significantly induced. TRX is known to interact with other proteins such as proliferation-associated gene and apoptosis signal-regulating kinase 1. Coexpression of mVDUP1 interfered with the interaction between TRX and proliferation-associated gene or TRX and ASK-1, suggesting its roles in cell proliferation and oxidative stress. To investigate the roles of mVDUP1 in oxidative stress, mVDUP1 was overexpressed in NIH 3T3 cells. When cells were exposed to stress, cell proliferation was declined with elevated apoptotic cell death compared with control cells. In addition, c-Jun N-terminal kinase activation and IL-6 expression were elevated. Taken together, these results demonstrate that mVDUP1 functions as an oxidative stress mediator by inhibiting TRX activity.

Three-amino acid extension loop homeodomain proteins Meis2 and TGIF differentially regulate transcription.

Three-amino acid extension loop (TALE) homeobox proteins are highly conserved transcription regulators. We report that two members of this family, Meis2 and TGIF, which frequently have overlapping consensus binding sites on complementary DNA strands in opposite orientations, can function competitively. For example, in the D(1A) gene, which encodes the predominant dopamine receptor in the striatum, Meis2 and TGIF bind to the activator sequence ACT (-1174 to -1154) and regulate transcription differentially in a cell type-specific manner. Among the five cloned splice variants of Meis2, isoforms Meis2a-d activate the D(1A) promoter in most cell types tested, whereas TGIF competes with Meis2 binding to DNA and represses Meis2-induced transcription activation. Consequently, Meis2 cannot activate the D(1A) promoter in a cell that has abundant TGIF expression. The Meis2 message is highly co-localized with the D(1A) message in adult striatal neurons, whereas TGIF is barely detectable in the adult brain. Our observations provide in vitro and in vivo evidence that Meis2 and TGIF differentially regulate their target genes. Thus, the delicate ratio between Meis2 and TGIF expression in a given cell type determines the cell-specific expression of the D(1A) gene. We also found that splice variant Meis2e, which has a truncated homeodomain, cannot bind to the D(1A) ACT sequence or activate transcription. However, Meis2e is an effective dominant negative regulator by blocking Meis2d-induced transcription activation. Thus, truncated homeoproteins with no DNA binding domains can have important regulatory functions.

Repression of hepatitis B virus X gene expression by hammerhead ribozymes.

The X protein (HBx) of human hepatitis B virus (HBV) is a transcriptional activator protein. The HBx protein plays an important role in viral replication in HBV infected cells and the liver diseases including hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Therefore, the repression of HBx gene expression by hammerhead ribozymes may be a good way to inhibit HBV replication and cure HBV-related liver diseases. We designed two hammerhead ribozymes, RzA and RzB, to cleave target sites at nucleotides 114 and 309 in the HBx open reading frame (ORF), respectively. In vitro, RzA and RzB cleaved HBx RNAs at their target sites up to 52 and 75%, respectively; however, the disabled ribozymes (dRzs) which have mutations in the catalytic site did not cleave the target RNAs at all. When each of the ribozymes were cotransfected into HepG2 cells with HBx expression plasmid, RzA and RzB reduced the level of HBx mRNA to 40 and 57%, respectively. The transactivation activity of HBx protein was also reduced dramatically by the ribozymes. These results suggest that the hammerhead ribozymes, RzA and RzB, can be used for the gene therapy of liver diseases caused by HBV.

Detection of hammerhead ribozyme-mediated cleavage and reduced expression of LacZ' mRNA in E. coli.

Hammerhead ribozymes have been shown to specifically suppress the expression of target genes in various cells, but their in vivo cleavage products have seldom been directly detected. A hammerhead ribozyme sequence was designed to cleave the phosphodiester bond just 3' to the GUC of SalI site of M13mp 18. The ribozyme was inserted some base pairs upstream of the target region without disrupting the reading frame of the lacZ' gene and without introducing any translational stop codons. More than 90% RNAs synthesized in vitro were cleaved at the expected site after 1-h incubation in the presence of 10 mM magnesium ion at 37 and 50 degrees C. Inclusion of the designed ribozyme sequence was also shown to suppress the expression of the fused lacZ' gene in E. coli cells. When the cells were infected by the ribozyme-containing phage, they remained colourless in the presence of X-gal, and the cellular beta-galactosidase activity was reduced by more than 90%. Insertion of the same ribozyme sequence in reverse orientation showed little effect on beta-galactosidase activity. Furthermore, a primer extension by reverse transcriptase revealed a cleavage product that resulted from cleavage of LacZ' mRNA at the targeted site as designed. Thus, our data demonstrated that the designed hammerhead ribozyme cleaves and reduces the expression of a fused LacZ' mRNA in E. coli cells.

Development of analogical problem-solving skill.

3 experiments were performed to assess children's ability to solve a problem by analogy to a superficially dissimilar situation. Preschoolers and fifth and sixth graders were asked to solve a problem that allowed multiple solutions. Some subjects were first read a story that included an analogous problem and its solution. When the mapping between the relations involved in the corresponding solutions was relatively simple, and the corresponding instruments were perceptually and functionally similar, even preschoolers were able to use the analogy to derive a solution to the transfer problem (Experiment 1). Furthermore, salient similarity of the instruments was neither sufficient (Experiment 2) nor necessary (Experiment 3) for success by preschool subjects. When the story analog mapped well onto the transfer problem, 4-year-olds were often able to generate a solution that required transformation of an object with little perceptual or semantic similarity to the instrument used in the base analog (Experiment 3). The older children used analogies in a manner qualitatively similar to that observed in comparable studies with adults (Experiment 1), whereas the younger children exhibited different limitations.