Proliferation stage-dependent expression of DNA methyltransferase (Dnmt1) in mouse small intestine

In cultured cells, the maintenance-type DNA methyltransferase (Dnmt1) is highly expressed during the proliferation stage. In the present study, we detected significant expression of Dnmt1 protein in the nuclear fraction of mouse small intestine. From its mobility in SDS polyacrylamide gel electrophoresis and the specific antibodies against the somatic cell-type Dnmt1, Dnmt1 was determined as a somatic cell type. Immunofluorescence study revealed that the Dnmt1 was highly expressed in the proliferating stem cells in crypts, and was localized in the nuclei. The present results indicate that the expression of Dnmt1 in vivo is also under the control of cell proliferation as in cultured cells.     

Effects of Transient Cerebral Ischemia on the Expression of DNA Methyltransferase 1 in the Gerbil Hippocampal CA1 Region

DNA methylation is a key epigenetic modification of DNA that is catalyzed by DNA methyltransferases (Dnmt). Increasing evidences suggest that DNA methylation in neurons regulates synaptic plasticity as well as neuronal network activity. In the present study, we investigated the changes in DNA methyltransferases 1 (Dnmt1) immunoreactivity and its protein levels in the gerbil hippocampal CA1 region after 5 min of transient global cerebral ischemia. CA1 pyramidal neurons were well stained with NeuN (a neuron-specific soluble nuclear antigen) antibody in the sham-group, Four days after ischemia–reperfusion (I–R), NeuN-positive (⁺) cells were significantly decreased in the stratum pyramidale (SP) of the CA1 region, and many Fluro-Jade B (a marker for neuronal degeneration)⁺ cells were observed in the SP. Dnmt1 immunoreactivity was well detected in all the layers of the sham-group. Dnmt1 immunoreactivity was hardly detected only in the stratum pyramidale of the CA1 region from 4 days post-ischemia; however, at these times, Dnmt1 immunoreactivity was newly expressed in GABAergic interneurons or astrocytes in the ischemic CA1 region. In addition, the level of Dnmt1 was lowest at 4 days post-ischemia. In brief, both the Dnmt1 immunoreactivity and protein levels were distinctively decreased in the ischemic CA1 region 4 days after transient cerebral ischemia. These results indicate that the decrease of Dnmt1 expression at 4 days post-ischemia may be related to ischemia-induced delayed neuronal death.     

Expression of DNA methyltransferase (Dnmt1) in testicular germ cells during development of mouse embryo

The DNA methylation pattern is reprogrammed in embryonic germ cells. In female germ cells, the short-form DNA methyltransferase Dnmt1, which is an alternative isoform specifically expressed in growing oocytes, plays a crucial role in maintaining imprinted genes. To evaluate the contribution of Dnmt1 to the DNA methylation in male germ cells, the expression profiles of Dnmt1 in embryonic gonocytes were investigated. We detected a significant expression of Dnmt1 in primordial germ cells in 12.5-14.5 day postcoitum (dpc) embryos. The expression of Dnmt1 was downregulated after 14.5 dpc after which almost no Dnmt1 was detected in gonocytes prepared from 18.5 dpc embryos. The short-form Dnmt1 also was not detected in the 16.5-18.5 dpc gonocytes. On the other hand, Dnmt1 was constantly detected in Sertoli cells at 12.5-18.5 dpc. The expression profiles of Dnmt1 were similar to that of proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, suggesting that Dnmt1 was specifically expressed in the proliferating male germ cells. Inversely, genome-wide DNA methylation occurred after germ cell proliferation was arrested, when the Dnmt1 expression was downregulated. The present results indicate that not Dnmt1 but some other type of DNA methyltransferase contributes to the creation of DNA methylation patterns in male germ cells.     

Neuronal expression, cytosolic localization, and developmental regulation of the organic solute carrier partner 1 in the mouse brain

Organic solute carrier partner 1 (OSCP1) is a mammalian, transporter-related protein that is able to facilitate the uptake of structurally diverse organic compounds into the cell when expressed in Xenopus laevis oocytes. This protein has been implicated in testicular handling of organic solutes because its mRNA expression is almost exclusive in the testis. However, in this study, we demonstrated significant expression of OSCP1 protein in mouse brain, the level of which was rather higher than that in the testis, although the corresponding mRNA expression was one-tenth of the testicular level. Immunohistochemistry revealed that OSCP1 was broadly distributed throughout the brain, and various neuronal cells were immunostained, including pyramidal cells in the cerebral cortex and hippocampus. However, there was no evidence of OSCP1 expression in glia. In primary cultures of cerebral cortical neurons, double-labeling immunofluorescence localized OSCP1 to the cytosol throughout the cell body and neurites including peri-synaptic regions. This was consistent with the subcellular fractionation of brain homogenates, in which OSCP1 was mainly recovered after centrifugation both in the cytosolic fraction and the particulate fraction containing synaptosomes. Immunoelectron microscopy of brain sections also demonstrated OSCP1 in the cytosol near synapses. In addition, it was revealed that changes in the expression level of OSCP1 correlated with neuronal maturation during postnatal development of mouse brain. These results indicate that OSCP1 may have a role in the brain indirectly mediating substrate uptake into the neurons in adult animals.     

Angiotensin II regulation of neuromodulation: downstream signaling mechanism from activation of mitogen-activated protein kinase

Angiotensin II (Ang II) stimulates expression of tyrosine hydroxylase and norepinephrine transporter genes in brain neurons; however, the signal-transduction mechanism is not clearly defined. This study was conducted to determine the involvement of the mitogen-activated protein (MAP) kinase signaling pathway in Ang II stimulation of these genes. MAP kinase was localized in the perinuclear region of the neuronal soma. Ang II caused activation of MAP kinase and its subsequent translocation from the cytoplasmic to nuclear compartment, both effects being mediated by AT1 receptor subtype. Ang II also stimulated SRE- and AP1-binding activities and fos gene expression and its translocation in a MAP kinase-dependent process. These observations are the first demonstration of a downstream signaling pathway involving MAP kinase in Ang II-mediated neuromodulation in noradrenergic neurons.     

Cellular localization and stimulation-associated distribution dynamics of syntaxin-1 and syntaxin-3 in gastric parietal cells

Syntaxins are differentially localized in polarized cells and play an important role in vesicle trafficking and membrane fusion. These soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are believed to be involved in tubulovesicle trafficking and membrane fusion during the secretory cycle of the gastric parietal cell. We examined the cellular localization and distribution of syntaxin-1 and syntaxin-3 in rabbit parietal cells. Fractionation of gastric epithelial cell membranes showed that syntaxin-1 was more abundant in a fraction enriched in apical plasma membranes, whereas syntaxin-3 was found predominantly in the H,K-ATPase-rich tubulovesicle fraction. We also examined the cellular localization of syntaxins in cultured parietal cells. Parietal cells were infected with CFP-syntaxin-1 and CFP-syntaxin-3 adenoviral constructs. Fluorescence microscopy of live and fixed cells demonstrated that syntaxin-1 was primarily on the apical membrane vacuoles of infected cells, but there was also the expression of syntaxin-1 in a subadjacent cytoplasmic compartment. In resting, non-secreting parietal cells, syntaxin-3 was distributed throughout the cytoplasmic compartment; after stimulation, syntaxin-3 translocated to the apical membrane vacuoles, there co-localizing with H,K-ATPase, syntaxin-1 and F-actin. The differential location of these syntaxin isoforms in gastric parietal cells suggests that these proteins may be critical for maintaining membrane compartment identity and that they may play important, but somewhat different, roles in the membrane recruitment processes associated with secretory activation.     

Alpha-synuclein sequesters Dnmt1 from the nucleus: a novel mechanism for epigenetic alterations in Lewy body diseases

DNA methylation is a major epigenetic modification that regulates gene expression. Dnmt1, the maintenance DNA methylation enzyme, is abundantly expressed in the adult brain and is mainly located in the nuclear compartment, where it has access to chromatin. Hypomethylation of CpG islands at intron 1 of the SNCA gene has recently been reported to result in overexpression of α-synuclein in Parkinson disease (PD) and related disorders. We therefore investigated the mechanisms underlying altered DNA methylation in PD and dementia with Lewy bodies (DLB). We present evidence of reduction of nuclear Dnmt1 levels in human postmortem brain samples from PD and DLB patients as well as in the brains of α-synuclein transgenic mice models. Furthermore, sequestration of Dnmt1 in the cytoplasm results in global DNA hypomethylation in human and mouse brains, involving CpG islands upstream of SNCA, SEPW1, and PRKAR2A genes. We report that association of Dnmt1 and α-synuclein might mediate aberrant subcellular localization of Dnmt1. Nuclear Dnmt1 levels were partially rescued by overexpression of Dnmt1 in neuronal cell cultures and in α-synuclein transgenic mice brains. Our results underscore a novel mechanism for epigenetic dysregulation in Lewy body diseases, which might underlie the decrease in DNA methylation reported for PD and DLB.     

Methylation‐reprogrammed Wnt/β‐catenin signalling mediated prenatal hypoxia‐induced brain injury in foetal and offspring rats

Prenatal hypoxia (PH) is a common pregnancy complication, harmful to brain development. This study investigated whether and how PH affected Wnt pathway in the brain. Pregnant rats were exposed to hypoxia (10.5% O₂) or normoxia (21% O₂; Control). Foetal brain weight and body weight were decreased in the PH group, the ratio of brain weight to body weight was increased significantly. Prenatal hypoxia increased mRNA expression of Wnt3a, Wnt7a, Wnt7b and Fzd4, but not Lrp6. Activated β‐catenin protein and Fosl1 expression were also significantly up‐regulated. Increased Hif1a expression was found in the PH group associated with the higher Wnt signalling. Among 5 members of the Sfrp family, Sfrp4 was down‐regulated. In the methylation‐regulating genes, higher mRNA expressions of Dnmt1 and Dnmt3b were found in the PH group. Sodium bisulphite and sequencing revealed hyper‐methylation in the promoter region of Sfrp4 gene in the foetal brain, accounting for its decreased expression and contributing to the activation of the Wnt‐Catenin signalling. The study of PC12 cells treated with 5‐aza further approved that decreased methylation could result in the higher Sfrp4 expression. In the offspring hippocampus, protein levels of Hif1a and mRNA expression of Sfrp4 were unchanged, whereas Wnt signal pathway was inhibited. The data demonstrated that PH activated the Wnt pathway in the foetal brain, related to the hyper‐methylation of Sfrp4 as well as Hif1a signalling. Activated Wnt signalling might play acute protective roles to the foetal brain in response to hypoxia, also would result in disadvantageous influence on the offspring in long‐term.     

Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain

Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.     

Identification of BAIAP2 (BAI-associated protein 2), a novel human homologue of hamster IRSp53, whose SH3 domain interacts with the cytoplasmic domain of BAI1.

BAI1 (brain-specific angiogenesis inhibitor 1) was originally isolated as a p53-target gene specifically expressed in brain. To clarify its function, we have been searching for cellular proteins that associate with the cytoplasmic domain of BAI1. Using its intracellular carboxyl terminus as "bait" in a yeast two-hybrid system, we isolated a cDNA clone named BAIAP2 whose nucleotide sequence would encode a 521-amino acid protein showing significant homology to a 58/53-kDa substrate of insulin-receptor kinase in the hamster. As the expression profile of BAIAP2 examined by Northern blot analysis was almost identical to that of BAI1, BAIAP2 appears to be active mainly in neurons. In vitro binding assays confirmed that a proline-rich cytoplasmic fragment of BAI1 interacted with the Src homology 3 (SH3) domain of BAIAP2. Double-color immunofluorescent analysis revealed that BAIAP2 was localized at the cytoplasmic membrane when it was coexpressed with BAI1 in COS-7 cells; BAIAP2 not associated with BAI1 was diffused in the cytoplasm. Predominant localization of BAI1 protein in a sub-cellular fraction enriched in growth cones indicated a possible role of BAI1 as a cell adhesion molecule inducing growth cone guidance. As a protein partner of BAI1, BAIAP2 may represent an important link between membrane and cytoskeleton in the process of neuronal growth.     

Cloning,characterization and expression of two alternatively splicing isoforms of Ca2+/calmodulin-dependent protein kinase I gamma in the rat brain

Ca2+/calmodulin-dependent protein kinase I (CaMKI), originally identified as a protein kinase phosphorylating synapsin I, has been shown to constitute a family of closely related isoforms (alpha, beta and gamma). Here, we have isolated and determined the complete primary structures of two alternatively splicing isoforms of CaMKI termed CaMKI gamma 1 and -gamma 2. CaMKI gamma 1 and -gamma 2 contain an identical N-terminal catalytic domain with different C-terminal regions due to the deletion of the 425-bp nucleotide sequence of CaMKI gamma 1 in CaMKI gamma 2. In vitro kinase assay has demonstrated the marked enhancement of the Ca2+/CaM-dependent activity of CaMKI gamma 1 by the preincubation with Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), but no significant activation of CaMKI gamma 2. Northern blot analysis has demonstrated the predominant expression of CaMKI gamma in the brain. RT-PCR analysis has revealed similar expression patterns between CaMKI gamma 1 and CaMKI gamma 2 in various brain regions. In situ hybridization analysis has demonstrated that CaMKI gamma mRNA is expressed in a distinct pattern from other isoforms of CaMKI with predominant expression in some restricted brain regions such as the olfactory bulb, hippocampal pyramidal cell layer of CA3, central amygdaloid nuclei, ventromedial hypothalamic nucleus and pineal gland. In the primary hippocampal neurons and NG108-15 cells, transfected CaMKI gamma 1 and -gamma 2 are localized primarily in the cytoplasm and neurites but not in the nucleus. These findings suggest that both isoforms of CaMKI gamma may be involved in Ca2+ signal transduction in the cytoplasmic compartment of certain neuronal population.     

Complementary neuronal and glial expression of two high-affinity glutamate transporter GLT1/EAAT2 forms in rat cerebral cortex

The glutamate transporter GLT1 is essential in limiting transmitter signaling and restricting harmful receptor overstimulation. It has been shown recently that GLT1 exists in two forms, the generic GLT1 and a 3'-end-spliced variant of GLT1 (GLT1v), both with similar transport characteristics. To differentiate clearly the cellular distribution of both GLT1 forms in the cortex, specific cRNA probes for non-radioactive in situ hybridization were generated and applied to adult rat brain sections. The results were complemented by western and northern blot analyses and by immunocytochemical investigations using specific peptide antibodies against both GLT1 forms. The study confirmed that generic GLT1 mRNA was expressed predominantly in astrocytes and, to a small extent, in neurons, whereas GLT1 protein was detected only in cell membranes of astrocytes. On the other hand, GLT1v mRNA and protein were demonstrated predominantly in neurons and in non-astrocytic glial cells irrespective of the cortical areas studied. A cytoplasmic granular staining of neurons and astrocytes predominated in the demonstration of GLT1v protein. It is concluded that the cellular expression of the two GLT1 forms is complementary. The cytoplasmic vesicular distribution of GLT1v may represent an endogenous protective mechanism to limit glutamate-induced excitotoxicity.     

The C-terminal tail of the metabotropic glutamate receptor subtype 7 is necessary but not sufficient for cell surface delivery and polarized targeting in neurons and epithelia

Complex neuronal functions rely upon the precise sorting, targeting, and restriction of receptors to specific synaptic microdomains. Little is known, however, of the molecular signals responsible for mediating these selective distributions. Here we report that metabotropic glutamate receptor subtype 7a (mGluR7a) is polarized at the basolateral surface when expressed in Madin-Darby canine kidney (MDCK) epithelial cells but is not polarized when expressed in cultured hippocampal neurons. Truncation of the mGluR7 cytoplasmic tail produces a protein that is restricted to a perinuclear intracellular compartment in both neurons and MDCK cells, where this protein colocalizes with a trans-Golgi network antigen. The mGluR7 cytoplasmic domain appended to the transmembrane portion of the vesicular stomatitis virus G protein and the ectodomain of human placental alkaline phosphatase is distributed over the entire cell surface in cultured neurons. When expressed in MDCK cells, this construct remains in an intracellular compartment distinct from endosomes or lysosomes. Thus, the cytoplasmic tail domain of mGluR7 is necessary but not sufficient for polarized targeting in MDCK monolayers, whereas in neurons the cytoplasmic tail is sufficient for cell surface expression but not polarization. Additional mechanisms are likely required to mediate mGluR7 neuronal polarization and synaptic clustering.     

Phenotype dependent differential effects of interleukin-1beta and amyloid-beta on viability and cholinergic phenotype of T17 neuroblastoma cells

Amyloid-beta accumulation in brains of Alzheimer's disease (AD) victims is accompanied by glial inflammatory reactions and preferential loss of cholinergic neurons. Therefore, the aim of this study was to find out whether proinflamatory cytokine interleukin 1beta (IL1beta) modifies effects of amyloid-beta (Abeta) on viability and cholinergic phenotype of septum derived T17 cholinergic neuroblastoma cells. In nondifferentiated T17 cells (NC) Abeta(25-35) (1 microg/ml) caused no changes in choline acetyltransferase (ChAT) activity, acetylcholine (ACh) release, subcellular distribution of acetyl-CoA, but doubled content of trypan blue positive cells. IL1beta (10 ng/ml) increased ACh release (125%) but did not change other parameters of NC. In the presence of Abeta IL1beta also increased ChAT activity (47%), ACh release (100%) but had no effect on acetyl-CoA distribution and cell viability. Differentiation with retinoic acid and dibutyryl cyclic AMP caused over two-fold increase of ChAT activity and ACh content, four-fold increase of ACh release and about 50% decrease of acetyl-CoA level in the mitochondria. In differentiated cells (DC), Abeta decreased ChAT activity (31%), ACh release (47%) and content of acetyl-CoA (80%) in cell cytoplasmic compartment, whereas IL1beta elevated ChAT activity (54%) and ACh release (32%). IL1beta totally reversed Abeta-evoked inhibition of ChAT activity and ACh release and restored control level of cytoplasmic acetyl-CoA but increased fraction of nonviable cells to 25%. Thus, IL1beta could compensate Abeta-evoked cholinergic deficits through the restoration of adequate expression of ChAT and provision of acetyl-CoA to cytoplasmic compartment in cholinergic neurons that survive under such pathologic conditions. These data indicate that IL1beta possess independent cholinotrophic and cholinotoxic activities that may modify Abeta effects on cholinergic neurons.     

Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer's disease-related gene seladin-1

Seladin-1 (SELective Alzheimer's Disease INdicator-1) is an anti-apoptotic gene, which is down-regulated in brain regions affected by Alzheimer's disease (AD). In addition, seladin-1 catalyzes the conversion of desmosterol into cholesterol. Disruption of cholesterol homeostasis in neurons may increase cell susceptibility to toxic agents. Because the hippocampus and the subventricular zone, which are affected in AD, are the unique regions containing stem cells with neurogenic potential in the adult brain, it might be hypothesized that this multipotent cell compartment is the predominant source of seladin-1 in normal brain. In the present study, we isolated and characterized human mesenchymal stem cells (hMSC) as a model of cells with the ability to differentiate into neurons. hMSC were then differentiated toward a neuronal phenotype (hMSC-n). These cells were thoroughly characterized and proved to be neurons, as assessed by molecular and electrophysiological evaluation. Seladin-1 expression was determined and found to be significantly reduced in hMSC-n compared to undifferentiated cells. Accordingly, the total content of cholesterol was decreased after differentiation. These original results demonstrate for the first time that seladin-1 is abundantly expressed by stem cells and appear to suggest that reduced expression in AD might be due to an altered pool of multipotent cells.     

Association of syntaxin 3 and vesicle-associated membrane protein (VAMP) with H+/K(+)-ATPase-containing tubulovesicles in gastric parietal cells.

H+/K(+)-ATPase is the proton pump in the gastric parietal cell that is responsible for gastric acid secretion. Stimulation of acid secretion is associated with a reorganization of the parietal cells resulting in the incorporation of H+/K(+)-ATPase from a cytoplasmic membrane pool, the tubulovesicle compartment, into the apical canalicular membrane. To better characterize the role of membrane trafficking events in the morphological and physiological changes associated with acid secretion from parietal cells, we have characterized the expression and localization of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in these cells. Each of the six different SNARE proteins examined [syntaxins 1 through 4 of 25-kDa synaptosome-associated protein, and vesicle-associated membrane protein] were found to be expressed in parietal cells. Furthermore, two of these SNAREs, vesicle-associated membrane protein and syntaxin 3, were associated with H+/K(+)-ATPase-containing tubulovesicles while the remainder were excluded from this compartment. The expression of syntaxin 1 and synaptosome-associated protein of 25 kDa in parietal cells, two SNAREs previously thought to be restricted to neuroendocrine tissues, suggests that parietal cells may utilize membrane trafficking machinery that is similar to that utilized for regulated exocytosis in neurons. Furthermore, the localization of syntaxin 3, a putative target membrane SNARE, to the tubulovesicle compartment indicates that syntaxin 3 may have an alternative function. These observations support a role for intracellular membrane trafficking events in the regulated recruitment of H+/K(+)-ATPase to the plasma membrane after parietal cell stimulation.     

5-Aza-dC在mES细胞向生殖细胞分化中的DNA甲基化调控机制

目的: 探讨小鼠胚胎干细胞(mouse embryonic stem cells, mESCs)向生殖细胞(Embryonic germ cells,EG)分化过程中5-杂氮-2'-脱氧胞苷(5-Aza-2'-deoxycytidine,5-Aza-dC) 对DNA甲基化转移酶Dnmt1和Dnmt3a及生殖细胞特征基因Mvh表达变化的DNA甲基化调控机制。方法:将mES细胞分化形成拟胚体(embryoid bodies, EBs) 作为向生殖细胞分化的启动步骤,采用不同浓度(0.05μmol/L,0.1μmol/L,0.5μmol/L,1μmol/L,3μmol/L)处理EBs,RT-PCR实时荧光定量RT-PCR和Western blot分别检测检测在5-Aza-dC处理前后Dnmt1和Dnmt3a在ES细胞和EBs中的表达,甲基化特异性PCR(MSP)检测原始生殖细胞分化特征基因Mvh启动子甲基化状态。结果: 5-Aza-dC的浓度在0.05 μmol/L~1 μmol/L之间时,EBs保持较高的存活率而EBs的形态明显发生了变化;5-Aza-dC 处理后, Dnmt1和Dnmt3a在EBs中mRNA表达量明显降低,其变化特点与WB结果相一致。MSP和测序结果显示, Mvh启动子区表现为部分甲基化,5-Aza-dC 处理后的4d EBs中Mvh CpG岛有4个CG位点发生突变,而mES细胞中未见突变。结论: EBs经5-Aza-dC处理后,Dnmt1和Dnmt3a的表达明显下调;同时,Mvh启动子发生部分甲基化,有可能启动了向生殖细胞的分化进程。