小鼠成骨细胞Wnt信号通路相关因子表达与胰岛素样生长因子1的影响

背景：胰岛素样生长因子1可促进细胞增殖、分化,但其具体作用机制尚不清楚。 目的：观察胰岛素样生长因子1对小鼠成骨细胞增殖﹑分化的影响,及Wnt信号通路相关因子mRNA的表达。 方法：向体外培养的小鼠成骨细胞中加入25 μg/L的胰岛素样生长因子1,分别于培养的第1,2,3,4,5天用CCK-8比色法检测细胞增殖率;于培养的第3,6,9天应用酶联免疫法检测细胞内碱性磷酸酶活性;细胞培养第3天提取总RNA,采用Real-time RT-PCR检测Wnt-3a,低密度脂蛋白受体相关蛋白5,β-catenin mRNA的表达。 结果与结论：25 μg/L胰岛素样生长因子1能够促进成骨细胞增殖,提高碱性磷酸酶活性,而且明显增加成骨细胞中Wnt-3a、低密度脂蛋白受体相关蛋白5、β-catenin mRNA的表达(P < 0.05)。说明胰岛素样生长因子1能够促进成骨细胞的增殖和分化,Wnt信号通路参与了该调控过程。     

Identification of genes showing differential expression in anorexia mutant mouse

Differential display (DD) PCR was applied to identify genes regulated by anorexia (anx) in mouse brain. Of the four apparently differentially expressed genes, three (H2-H4) had high homology with known genes such as apoptotic protease activating factor 1 (Apaf 1), adenylate cyclase 6 (Adcy 6), and myelin proteolipid protein (PLP). H1, designated as HIPA1, had a homology with unknown function gene fragments. Northern blot analysis confirmed HIPA1 expression induced by anorexia. A 1579 bp full-length cDNA of HIPA1 was isolated from a mouse brain cDNA library using a probe from the differentially displayed H1 fragment. Sequence analysis showed that HIPA1 had 98.7% homology to mouse hippocampus cDNA. In situ hybridization demonstrated that the HIPA1 mRNA was highly up-regulated in the hippocampus of anx/anx mouse brain.     

Postnatal developmental expression of regulator of G protein signaling 14 (RGS14) in the mouse brain

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and mitogen‐activated protein kinase (MAPK) signaling pathways. In the adult mouse brain, RGS14 mRNA and protein are found almost exclusively in hippocampal CA2 neurons. We have shown that RGS14 is a natural suppressor of CA2 synaptic plasticity and hippocampal‐dependent learning and memory. However, the protein distribution and spatiotemporal expression patterns of RGS14 in mouse brain during postnatal development are unknown. Here, using a newly characterized monoclonal anti‐RGS14 antibody, we demonstrate that RGS14 protein immunoreactivity is undetectable at birth (P0), with very low mRNA expression in the brain. However, RGS14 protein and mRNA are upregulated during early postnatal development, with protein first detected at P7, and both increasing over time until reaching highest sustained levels throughout adulthood. Our immunoperoxidase data demonstrate that RGS14 protein is expressed in regions outside of hippocampal CA2 during development including the primary olfactory areas, the anterior olfactory nucleus and piriform cortex, and the olfactory associated orbital and entorhinal cortices. RGS14 is also transiently expressed in neocortical layers II/III and V during postnatal development. Finally, we show that RGS14 protein is first detected in the hippocampus at P7, with strongest immunoreactivity in CA2 and fasciola cinerea and sporadic immunoreactivity in CA1; labeling intensity in hippocampus increases until adulthood. These results show that RGS14 mRNA and protein are upregulated throughout postnatal mouse development, and RGS14 protein exhibits a dynamic localization pattern that is enriched in hippocampus and primary olfactory cortex in the adult mouse brain. J. Comp. Neurol. 522:186–203, 2014. © 2013 Wiley Periodicals, Inc.     

Distinct localization of SAPK isoforms in neurons of adult mouse brain implies multiple signaling modes of SAPK pathway.

Various cellular and environmental stresses lead to the activation of stress-activated protein kinase (SAPK), which is also referred to as c-Jun N-terminal kinase (JNK). In mammals, multiple SAPK isoforms, encoded by three independent genes, were identified. To gain insight into the roles of SAPK pathway in adult mouse brain, detailed expression patterns of three SAPK isoforms in brain were examined by using immunohistochemical and cell biological analyses. SAPKbeta was heavily expressed in almost all regions of brain as previously reported. Interestingly, SAPKgamma was also widely expressed at high levels. SAPKgamma expression was generally overlapped with SAPKbeta although there were some exceptions such as in hippocampus, where SAPKgamma was restricted to CA3 and CA4 regions while SAPKbeta was evenly expressed. SAPKalpha was widely expressed, but at low levels. It is particularly intriguing to note the differential subcellular localization of SAPK isoforms in neurons. In brain of normally reared mice, SAPKbeta was identified in nucleus as well as in cytoplasm of neurons, while SAPKgamma was detected mainly in cytoplasm and dendrites. Biochemical and immunological analyses revealed extraordinarily high basal activities of all SAPK isoforms in brain compared to peripheral organs, indicating that SAPK pathway may play a role in normal brain physiology. In addition, differential regional and subcellular localizations of SAPK isoforms allow us to speculate multiple signaling modes for SAPK activation in brain.     

探讨一种新型Wnt替代蛋白激活脑血管Wnt信号通路对血脑屏障的保护作用

目的探讨一种新型Wnt替代蛋白(Wnt-S)对人脑血管内皮细胞中Wnt/β-catenin信号通路的作用,可能提供一种保护血脑屏障的新途径。方法用分子克隆技术构建Wnt-S in pcDNA3. 1(+)质粒,在HEK293F细胞中瞬时转染该质粒,表达纯化Wnt-S蛋白; Western blot检测含5x His-tag的Wnt-S蛋白;免疫荧光染色检测人源脑血管内皮细胞(hCMEC/D3)标记物CD31、VE-cadherin、GLUT1和MFSD2A;实时荧光定量PCR检测HEK293和hCMEC/D3细胞的Wnt蛋白受体FZD1-10和LRP5/6的表达情况,检测加药处理后HEK293和hCMEC/D3细胞中Wnt/β-catenin信号通路下游靶基因AXIN2的表达情况。结果核酸电泳结果显示Wnt-S质粒构建完成; Western blot结果显示检测到了Wnt-S蛋白; HEK293中FZD1-7和LRP6高表达,hCMEC/D3中FZD2,4,6和LRP6高表达,FZD1,3低表达;实时荧光定量PCR结果显示,加Wnt-S蛋白处理后,HEK293和hCMEC/D3细胞中AXIN2基因表达水平显著上调(P 0. 001),说明Wnt-S蛋白有生物活性,并且激活了Wnt/β-catenin信号通路。结论 Wnt-S蛋白具有与天然Wnt3a蛋白类似的生物功能,能够激活人脑血管内皮细胞中的经典Wnt信号通路,以保护血脑屏障。     

Genetic deletion of regulator of G-protein signaling 4 (RGS4) rescues a subset of fragile X related phenotypes in the FMR1 knockout mouse

Fragile X syndrome (FXS), the most common cause of inherited mental retardation, is caused by the loss of the mRNA binding protein, FMRP. Persons with FXS also display epileptic seizures, social anxiety, hyperactivity, and autistic behaviors. The metabotropic glutamate receptor theory of FXS postulates that in the absence of FMRP, enhanced signaling though G-protein coupled group I metabotropic glutamate receptors in the brain contributes to many of the abnormalities observed in the disorder. However, recent evidence suggests that alterations in cellular signaling through additional G-protein coupled receptors may also be involved in the pathogenesis of FXS, thus providing impetus for examining downstream molecules. One group of signaling molecules situated downstream of the receptors is the regulator of G-protein signaling (RGS) proteins. Notably, RGS4 is highly expressed in brain and has been shown to negatively regulate signaling through Group I mGluRs and GABA(B) receptors. To examine the potential role for RGS4 in the pathogenesis of FXS, we generated FXS/RGS4 double knockout mice. Characterization of these mice revealed that a subset of FXS related phenotypes, including increased body weight, altered synaptic protein expression, and abnormal social behaviors, were rescued in the double knockout mice. Other phenotypes, such as hyperactivity and macroorchidism, were not affected by the loss of RGS4. These findings suggest that tissue and cell-type specific differences in GPCR signaling and RGS function may contribute to the spectrum of phenotypic differences observed in FXS.     

Differential expression of C-protein isoforms in developing and degenerating mouse striated muscles

With the aim of clarifying the roles of C-protein isoforms in developing mammalian skeletal muscle, we cloned the complementary DNA (cDNAs) encoding mouse fast (F) and slow (S) skeletal muscle C-proteins and determined their entire sequences. Northern blotting with these cDNAs together with mouse cardiac (C) C-protein cDNA was performed. It revealed that in adult mice, C, F, and S isoforms are expressed in a tissue-specific fashion, although the messages for both F and S isoforms are transcribed in extensor digitorum longus muscle, which has been categorized as a fast muscle. In addition, although C isoform is expressed first and transiently during development of chicken skeletal muscles, C isoform is not expressed in mouse skeletal muscles at all through the developmental stages; S isoform is first expressed, followed by the appearance of F isoform. Finally, in dystrophic mouse skeletal muscles, the expression of S isoform is increased as it is in dystrophic chicken muscle. These observations suggest that mutations in C isoform (MyBP-C) do not lead to any disturbance in skeletal muscle, although they may lead to familial hypertrophic cardiomyopathy. We also suggest that the expression of S isoform may be stimulated in degenerating human dystrophic muscles.     

Differential expression and tissue distribution of parkin isoforms during mouse development.

Mutations of the parkin gene are a cause of autosomal recessive juvenile parkinsonism. Although the parkin gene has been isolated from mouse, rat, and human, little is known about its expression in neural and nonneural tissues during development. In this study, we used a polyclonal antibody to a peptide downstream of the parkin ubiquitin domain to investigate (1) the differential expression of parkin isoforms in protein extracts from fetal and adult mouse tissues, and (2) the distribution of parkin in mouse fetal tissues at different developmental stages and in adult CNS tissues. By Western blot analyses, at least three isoforms of parkin of 22, 50, and 55 kDa were differentially expressed in mouse tissues. The p22 and p50 isoforms were found in fetal and adult mouse CNS tissues, while the p55 isoform was found only in adult tissues. The p50 isoform is the predominant form in both fetal and adult tissues. Immunolocalization in mouse fetuses showed that parkin was expressed only after neuronal differentiation. Although parkin was localized throughout the cytoplasm, the highest level of parkin was found in the neurites of both fetal and adult neurons.     

Unraveling the differential expression of the two isoforms of myelin-associated glycoprotein in a mouse expressing GFP-tagged S-MAG specifically regulated and targeted into the different myelin compartments

The two myelin-associated glycoprotein (MAG) isoforms are cell adhesion molecules that differ only in their cytoplasmic domains, but their specific roles are not well understood. In this study, we present a transgenic mouse line that specifically expresses GFP-tagged S-MAG correctly regulated and targeted into the myelin sheath allowing the specific discrimination of L- and S-MAG on the subcellular level. Here, we describe the differential expression pattern and spatial distribution of L- and S-MAG during development as well as in the adult central and peripheral nervous system. In peripheral nerves, where S-MAG is the sole isoform, we observed S-MAG concentrated in different ring-like structures such as periaxonal and abaxonal rings, and discs spanning through the compact myelin sheath perpendicular to the axon. In summary, our data provide new insight in the subcellular distribution of the two isoforms fundamental for the understanding of their specific functions in myelin formation and maintenance.     

Calcitonin gene-related peptide (CGRP), a potent regulator of biliary flow

This study was designed to investigate the effect of porcine calcitonin gene-related peptide (CGRP) on the motility of the porcine biliary tract in vivo. We measured the pressure in the gallbladder and sphincter of Oddi and, in separate experiments, the biliary flow into the duodenum during local intra-arterial infusions of CGRP. To determine if the observed effect could be caused by release of cholecystokinin (CCK), we measured the CCK release. The basal pressure in the sphincter of Oddi increased dose-dependently from 5.9 ± 0.5 mmHg to 11.5 ± 2.1 mmHg and the motility index of phasic contractions (amplitude × frequency) from 47 ± 8 to 347 ± 64 mmHg s⁻¹, at an infusion rate of 32.6 pmol kg⁻¹ min⁻¹. No effect was observed on the gallbladder pressure. CGRP at 6.5 pmol kg⁻¹ min⁻¹ significantly reduced the biliary flow into the duodenum to 47.7 ± 6% of the basal level. Atropine, injected intravenously, completely abolished the contractile effect of CGRP. CGRP had no effect on the release of CCK.
We conclude that CGRP increases biliary motility and hereby reduces bile flow, an effect which involves cholinergic but not cholecystokininergic mechanisms.     

Developmental and plasticity-related differential expression of two SNAP-25 isoforms in the rat brain

In this article we study the relationship between the expression pattern of two recently identified isoforms of the 25-kD synaptosomal-associated protein (SNAP-25a and SNAP-25b) and the morphological changes inherent to neuronal plasticity during development and kainic acid treatment. SNAP-25 has been involved in vescicle fusion in the nerve terminal, and most likely participates in different membrane fusion-related processes, such as those involved in neurotransmitter release and axonal growth. In the adult brain, SNAP-25b expression exceeded SNAP-25a in distribution and intensity, being present in most brain structures. Moderate or high levels of SNAP-25a hybridization signal were found in neurons of the olfactory bulb, the layer Va of the frontal and parietal cortices, the piriform cortex, the subiculum and the hippocampal CA4 field, the substantia nigra/pars compacta, and the pineal gland, partially overlapping SNAP-25b mRNA distribution. In restricted regions of cerebral cortex, thalamus, mammillary bodies, substantia nigra, and pineal glands the two isoforms were distributed in reciprocal fashion. During development SNAP-25a mRNA was the predominant isoform, whereas SNAP-25b expression increased postnatally. The early expression of SNAP-25a in the embryo and the decrease after P21 is suggestive of a potential involvement of this isoform in axonal growth and/or synaptogenesis. This conclusion is indirectly supported by the observation that SNAP-25a mRNA, but not SNAP-25b mRNA, was upregulated in the granule cells of the adult dentate gyrus 48 hours after kainate-induced neurotoxic damage of the hippocampal CA3-CA4 regions. Increase of SNAP-25 immunoreactivity was observed as early as 4 days after kainate injection within the mossy fiber terminals of the CA3 region, and in the newly formed mossy fiber aberrant terminals of the supragranular layer. These data suggest an isoform-specific role of SNAP-25 in neural plasticity. © 1996 Wiley-Liss, Inc.     

Opposing mitogenic regulation by PACAP in sympathetic and cerebral cortical precursors correlates with differential expression of PACAP receptor (PAC1-R) isoforms

Neurogenesis in the peripheral and central nervous systems proceeds in region-specific fashion, although underlying mechanisms remain undefined. Emerging evidence indicates that the neuropeptide PACAP and its G-protein–coupled receptor are expressed widely in the embryonic brain, suggesting that the ligand/receptor system plays a role in development. We found previously that PAC₁-R activation elicited opposing mitogenic effects in neurogenetic cultures, stimulating peripheral sympathetic neuroblasts while inhibiting cerebral cortical precursors. We have now defined the expression of PAC₁-R mRNA isoforms and activation of second-messenger pathways in these model populations. Sympathetic neuroblasts express the “hop” receptor isoform, through which PACAP elicits increased levels of cAMP and activation of the PI signaling pathway. In contrast, cerebral cortical precursors express primarily the “short” (non-insert) receptor isoform and exhibit increased cAMP levels alone following PACAP treatment. Thus, opposing mitogenic regulation in sympathetic and cortical precursors correlates with differential receptor isoform expression and distinct second-messenger signaling. In addition to receptor, PACAP ligand mRNA was expressed by both populations, suggesting that the peptide is produced and acts locally to regulate precursor proliferation. These observations indicate that the PACAP ligand/receptor system is expressed in both the peripheral and central nervous system during development. More generally, these studies suggest that widely expressed extracellular factors mediate region-specific neurogenesis by activating lineage-restricted receptor isoforms and intracellular pathways. J. Neurosci. Res. 53:651–662, 1998. © 1998 Wiley-Liss, Inc.     

TCR signaling and environment affect vasoactive intestinal peptide receptor-1 (VPAC-1) expression in primary mouse CD4 T cells

Strict regulation of T cell function is imperative to control adaptive immunity, and dysregulation of T cell activation can contribute to infectious and autoimmune diseases. Vasoactive intestinal peptide receptor-1 (VPAC-1), an anti-inflammatory G-protein coupled receptor, has been reported to be downregulated during T cell activation. However, the regulatory mechanisms controlling the expression of VPAC-1 in T cells are not well understood. Therefore, mouse splenic CD4 T cells were treated in complete media+/-anti-CD3 for 24h, total RNA isolated and VPAC-1 levels measured by qPCR. Surprisingly, we discovered that T cells incubated in complete media steadily upregulated VPAC-1 mRNA levels over time (24h). Importantly, CD4 T cells isolated from blood also showed elevated VPAC-1 expression compared to splenic T cells. Collectively, these data support that the vascular environment positively influences VPAC-1 mRNA expression that is negatively regulated by TCR signaling. This research was supported by a national service award (1KO1 DK064828) to G.D., the Center for Protease Research (2P20RR015566), and INBRE (P20 RR016741).     

Pannexin 1-based channels activity as a novel regulator of multiple sclerosis progression

Multiple sclerosis(MS):MS is a neurodegenerative disease affecting around 2.5 million people worldwide,representing the second cause of disabilities in the young adult population.MS is a demyelinating pathology which originates in the autoimmune attack of T and B lymphocytes against myelin.This lack of myelin leads,in turn,to axonal degeneration,neuronal death and the consequent neurological disabilities(Franklin and Ffrench-Constant,2017).A main hallmark of MS is a preserved local neuroinflammatory environment.It is now acknowledged that this persistent inflammatory scenario is a central and common condition in almost all neurodegenerative pathologies(as in Parkinson’s and Alzheimer’s diseases,among others)controlling and modulating the regulatory responses of the system to the triggering insult.In the case of MS,this original insult corresponds to the loss of myelin(Chitnis and Weiner,2017;Franklin and Ffrench-Constant,2017).     

The cyclin-dependent kinase inhibitor p27(Kip1) is a positive regulator of Schwann cell differentiation in vitro

Schwann cell precursors differentiating into a myelinating phenotype are critical for peripheral nerve development and regeneration. However, little is known about the underlying molecular mechanisms of Schwann cell differentiation. In the present study, we performed a cyclic adenosine monophosphate-induced Schwann cell differentiation model in vitro. Western blot analysis showed that p27^Kip1 expression was upregulated during the differentiation of Schwann cell, while the inhibition of p27^Kip1 expression by short hairpin RNA-mediated knockdown significantly abolished the expression of promyelinating markers and the alteration of cellular morphology. In addition, immunofluorescence revealed a decrease of p27^Kip1 nuclear staining and a concomitant increase of cytoplasmic staining in differentiated Schwann cells. In summary, our data indicated that p27^Kip1 was a positive regulator of Schwann cell differentiation in vitro.     

Wnt信号与神经干细胞分化

背景：研究表明，移植入宿主体内的神经干细胞可分化为神经元或神经胶质细胞。Wnt信号通路与神经干细胞的分化密切相关。通过调节Wnt信号通路可控制神经干细胞的定向分化。 目的：对神经干细胞分化及其与Wnt信号通路的关系进行综述. 方法：应用计算机检索2002-02/2010-03 Medline数据库、Ovid数据库、CNKI、EBSCO数据库与神经干细胞相关文献。检索词为“神经干细胞，神经再生，Wnt信号，神经元，分化”。纳入与神经干细胞分化及Wnt信号系统相关文献，排除重复性研究，保留30篇文献进行综述。 结果与结论：神经干细胞是一类具有自我更新和多向分化潜能的细胞，能分化形成机体中枢神经系统几乎所有类型的细胞。Wnt信号通路在神经干细胞的分化中起重要作用。文章从神经干细胞、Wnt信号通路、Wnt信号通路与神经干细胞的分化等方面分别进行了叙述。然而，Wnt信号通路控制神经干细胞分化的具体机制还不是很清楚。     

Spatial, temporal and subcellular localization of islet-brain 1 (IB1), a homologue of JIP-1, in mouse brain

Islet-brain 1 (IB1) was recently identified as a DNA-binding protein of the GLUT2 gene promoter. The mouse IB1 is the rat and human homologue of the Jun-interacting protein 1 (JIP-1) which has been recognized as a key player in the regulation of c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. JIP-1 is involved in the control of apoptosis and may play a role in brain development and aging. Here, IB1 was studied in adult and developing mouse brain tissue by in situ hybridization, Northern and Western blot analysis at cellular and subcellular levels, as well as by immunocytochemistry in brain sections and cell cultures. IB1 expression was localized in the synaptic regions of the olfactory bulb, retina, cerebral and cerebellar cortex and hippocampus in the adult mouse brain. IB1 was also detected in a restricted number of axons, as in the mossy fibres from dentate gyrus in the hippocampus, and was found in soma, dendrites and axons of cerebellar Purkinje cells. After birth, IB1 expression peaks at postnatal day 15. IB1 was located in axonal and dendritic growth cones in primary telencephalon cells. By biochemical and subcellular fractionation of neuronal cells, IB1 was detected both in the cytosolic and membrane fractions. Taken together with previous data, the restricted neuronal expression of IB1 in developing and adult brain and its prominent localization in synapses suggest that the protein may be critical for cell signalling in developing and mature nerve terminals.