Sanbing Shen

The VPAC2 Receptor Is Essential for Circadian Function in the Mouse Suprachiasmatic Nuclei

The neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are implicated in the photic entrainment of circadian rhythms in the suprachiasmatic nuclei (SCN). We now report that mice carrying a null mutation of the VPAC(2) receptor for VIP and PACAP (Vipr2(-/-)) are incapable of sustaining normal circadian rhythms of rest/activity behavior. These mice also fail to exhibit circadian expression of the core clock genes mPer1, mPer2, and mCry1 and the clock-controlled gene arginine vasopressin (AVP) in the SCN. Moreover, the mutants fail to show acute induction of mPer1 and mPer2 by nocturnal illumination. This study highlights the role of intercellular neuropeptidergic signaling in maintenance of circadian function within the SCN.

Ectopic expression of Hoxb-8 causes duplication of the ZPA in the forelimb and homeotic transformation of axial structures.

Transgenic embryos were generated carrying a Hoxb-8 transgene under control of the mouse RAR beta 2 promoter, which extends the normal expression domain to more anterior regions of the embryo. These embryos showed mirror-image duplications in the forelimb, analogous to the duplications observed in chick in response to transplantation of a ZPA to the anterior margin of the limb bud. Examination of Sonic hedgehog, Fgf-4, and Hoxd-11 gene expression confirmed that a second ZPA had been generated at the anterior side of the limb bud. Besides other alterations, posterior homeotic transformations of axial structures were observed, involving the first spinal (Frorieps) ganglion and several cervical vertebrae.

Schizophrenia-related neural and behavioral phenotypes in transgenic mice expressing truncated Disc1.

Disrupted-in-Schizophrenia-1 (DISC1), identified by positional cloning of a balanced translocation (1;11) with the breakpoint in intron 8 of a large Scottish pedigree, is associated with a range of neuropsychiatric disorders including schizophrenia. To model this mutation in mice, we have generated Disc1tr transgenic mice expressing 2 copies of truncated Disc1 encoding the first 8 exons using a bacterial artificial chromosome (BAC). With this partial simulation of the human situation, we have discovered a range of phenotypes including a series of novel features not previously reported. Disc1tr transgenic mice display enlarged lateral ventricles, reduced cerebral cortex, partial agenesis of the corpus callosum, and thinning of layers II/III with reduced neural proliferation at midneurogenesis. Parvalbumin GABAergic neurons are reduced in the hippocampus and medial prefrontal cortex, and displaced in the dorsolateral frontal cortex. In culture, transgenic neurons grow fewer and shorter neurites. Behaviorally, transgenic mice exhibit increased immobility and reduced vocalization in depression-related tests, and impairment in conditioning of latent inhibition. These abnormalities in Disc1tr transgenic mice are consistent with findings in severe schizophrenia.

Overexpression of the 5-Hydroxytryptamine Transporter Gene Effect on Pulmonary Hemodynamics and Hypoxia-Induced Pulmonary Hypertension

Background—Increased serotonin (5-hydroxytryptamine, 5-HT) transporter activity has been observed in human familial pulmonary hypertension. Methods and Results—We investigated pulmonary hemodynamics and the development of hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling in mice overexpressing the gene for the 5-HT transporter (5-HTT+ mice). Right ventricular pressure was elevated 3-fold in normoxic 5-HTT+ mice compared with their wild-type controls. Hypoxia-induced increases in right ventricular hypertrophy and pulmonary vascular remodeling were also potentiated in the 5-HTT+ mice. 5-HTT–like immunoreactivity, protein, and binding sites were markedly increased in the lungs from the 5-HTT+ mice. Hypoxia, however, decreased 5-HT transporter immunoreactivity, mRNA transcription, protein, and binding sites in both wild-type and 5-HTT+ mice. Conclusions—Increased 5-HT transporter expression causes elevated right ventricular pressures, and this occurs before the onset of right ventricular hypertrophy or pulmonary arterial remodeling. Hypoxia-induced remodeling is, however, increased in 5-HTT+ mice, whereas hypoxia inhibits 5-HTT expression. This provides a unique model that demonstrates differential mechanisms for familial pulmonary arterial hypertension and pulmonary arterial hypertension with hypoxemia.

The mouse VPAC2 receptor confers suprachiasmatic nuclei cellular rhythmicity and responsiveness to vasoactive intestinal polypeptide in vitro

Expression of coherent and rhythmic circadian (≈ 24 h) variation of behaviour, metabolism and other physiological processes in mammals is governed by a dominant biological clock located in the hypothalamic suprachiasmatic nuclei (SCN). Photic entrainment of the SCN circadian clock is mediated, in part, by vasoactive intestinal polypeptide (VIP) acting through the VPAC2 receptor. Here we used mice lacking the VPAC2 receptor (Vipr2−/−) to examine the contribution of this receptor to the electrophysiological actions of VIP on SCN neurons, and to the generation of SCN electrical firing rate rhythms SCN in vitro. Compared with wild‐type controls, fewer SCN cells from Vipr2−/− mice responded to VIP and the VPAC2 receptor‐selective agonist Ro 25‐1553. By contrast, similar proportions of Vipr2−/− and wild‐type SCN cells responded to gastrin‐releasing peptide, arginine vasopressin or N‐methyl‐d‐aspartate. Moreover, VIP‐evoked responses from control SCN neurons were attenuated by the selective VPAC2 receptor antagonist PG 99‐465. In firing rate rhythm experiments, the midday peak in activity observed in control SCN cells was lost in Vipr2−/− mice. The loss of electrical activity rhythm in Vipr2−/− mice was mimicked in control SCN slices by chronic treatment with PG 99‐465. These results demonstrate that the VPAC2 receptor is necessary for the major part of the electrophysiological actions of VIP on SCN cells in vitro, and is of fundamental importance for the rhythmic and coherent expression of circadian rhythms governed by the SCN clock. These findings suggest a novel role of VPAC2 receptor signalling, and of cell‐to‐cell communication in general, in the maintenance of core clock function in mammals, impacting on the cellular physiology of SCN neurons.

Increased Expression of the 5-HT Transporter Confers a Low- Anxiety Phenotype Linked to Decreased 5-HT Transmission

A commonly occurring polymorphic variant of the human 5-hydroxytryptamine (5-HT) transporter (5-HTT) gene that increases 5-HTT expression has been associated with reduced anxiety levels in human volunteer and patient populations. However, it is not known whether this linkage between genotype and anxiety relates to variation in 5-HTT expression and consequent changes in 5-HT transmission. Here we test this hypothesis by measuring the neurochemical and behavioral characteristics of a mouse genetically engineered to overexpress the 5-HTT. Transgenic mice overexpressing the human 5-HTT (h5-HTT) were produced from a 500 kb yeast artificial chromosome construct. These transgenic mice showed the presence of h5-HTT mRNA in the midbrain raphe nuclei, as well as a twofold to threefold increase in 5-HTT binding sites in the raphe nuclei and a range of forebrain regions. The transgenic mice had reduced regional brain whole-tissue levels of 5-HT and, in microdialysis experiments, decreased brain extracellular 5-HT, which reversed on administration of the 5-HTT inhibitor paroxetine. Compared with wild-type mice, the transgenic mice exhibited a low-anxiety phenotype in plus maze and hyponeophagia tests. Furthermore, in the plus maze test, the low-anxiety phenotype of the transgenic mice was reversed by acute administration of paroxetine, suggesting a direct link between the behavior, 5-HTT overexpression, and low extracellular 5-HT. In toto, these findings demonstrate that associations between increased 5-HTT expression and anxiety can be modeled in mice and may be specifically mediated by decreases in 5-HT transmission.

Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC(2) receptor for vasoactive intestinal peptide

Vasoactive intestinal peptide (VIP) and its G protein-coupled receptors, VPAC1R and VPAC2R, are prominent in the immune system and regulate many aspects of T cell-dependent immunity. In mouse T cells, VPAC1R is expressed constitutively, whereas VPAC2R is induced by immune stimuli. VPAC2R-null (VPAC2R−/−) mice on a C57BL/6 background are shown here to have normal basic immune characteristics, including serum Ig concentrations, blood levels of all leukocytes, and spleen number of total T cells (CD3+) and T cells bearing CD4, CD8, and CD28. Hapten-evoked cutaneous delayed-type hypersensitivity (DTH) was significantly enhanced in VPAC2R-null mice compared with age- and sex-matched wild-type mice. In contrast, generation of IgE anti-hapten antibodies and active cutaneous anaphylaxis were ≥70% lower in VPAC2R-null mice than in wild-type controls. Cytokine production by splenic CD4+ T cells, stimulated with adherent anti-CD3 plus anti-CD28 antibodies, revealed higher levels of IL-2 (mean = 3-fold) and IFN-γ (mean = 3-fold), and lower levels of IL-4 (mean = one-fifth) in VPAC2R-null mice than wild-type controls. Loss of VIP-VPAC2R maintenance of the normal ratio of Th2/Th1 cytokines thus leads to a state of enhanced DTH and depressed immediate-type hypersensitivity, which may alter both host defense and susceptibility to immune-mediated diseases.

Characteristics of embryonic stem cell differentiation: a comparison with two embryonal carcinoma cell lines.

Embryonic stem (ES) cells isolated from late blastocysts can now be maintained in culture in an undifferentiated state provided they are grown in the presence of a specific differentiation inhibitor, known variously as leukaemia inhibiting factor (LIF) or differentiation inhibiting activity (DIA), found at high concentrations in medium conditioned by Buffalo rat liver (BRL) cells. ES cells acquired a differentiated phenotype in monolayer, either when in the absence of LIF/DIA or in the presence of retinoic acid (RA). We have now characterized this bipotential differentiation of ES cells in terms of a series of extracellular matrix and cell surface proteins as well as cytokeratin expression, and compared it with the changes observed during the differentiation of two embryonal carcinoma (EC) cell lines, P19 and F9. ES cells exposed to RA in the presence of LIF/DIA largely resembled F9 EC + RA after 5 days, while ES cells deprived of LIF/DIA formed a culture with mixed phenotype resembling P19 EC + RA. This study therefore establishes the predominantly parietal endoderm-like phenotype of cells derived from ES by RA induction, and suggests that a mixed population of endoderm- and ill-defined mesoderm-like cells are formed after removal of specific inhibitor(s) of differentiation.

Deregulation of EIF4E: a novel mechanism for autism

Background: Autism is a common childhood onset neurodevelopmental disorder, characterised by severe and sustained impairment of social interaction and social communication, as well as a notably restricted repertoire of activities and interests. Its aetiology is multifactorial with a strong genetic basis. EIF4E is the rate limiting component of eukaryotic translation initiation, and plays a key role in learning and memory through its control of translation within the synapse. EIF4E mediated translation is the final common process modulated by the mammalian target of rapamycin (mTOR), PTEN and fragile X mental retardation protein (FMRP) pathways, which are implicated in autism. Linkage of autism to the EIF4E region on chromosome 4q has been found in genome wide linkage studies. Methods and results: The authors present evidence that directly implicates EIF4E in autism. In a boy with classic autism, the authors observed a de novo chromosome translocation between 4q and 5q and mapped the breakpoint site to within a proposed alternative transcript of EIF4E. They then screened 120 autism families for mutations and found two unrelated families where in each case both autistic siblings and one of the parents harboured the same single nucleotide insertion at position −25 in the basal element of the EIF4E promoter. Electrophoretic mobility shift assays and reporter gene studies show that this mutation enhances binding of a nuclear factor and EIF4E promoter activity. Conclusions: These observations implicate EIF4E, and more specifically control of EIF4E activity, directly in autism. The findings raise the exciting possibility that pharmacological manipulation of EIF4E may provide therapeutic benefit for those with autism caused by disturbance of the converging pathways controlling EIF4E activity.

Presence of Multiple Functional Polyadenylation Signals and a Single Nucleotide Polymorphism in the 3′ Untranslated Region of the Human Serotonin Transporter Gene

Abstract: The human serotonin transporter (hSERT) gene is a candidate for involvement in the aetiology of affective disorders. In humans, multiple transcripts of the gene have been detected by northern blot analysis of brain and other tissues. We performed 3′ rapid amplification of cDNA ends to identify the common sites of polyadenylation in hSERT mRNA from human JAR cells and whole blood. Two major polyadenylation sites were identified: one 567 bp downstream of the stop codon, consistent with the usage of the polyadenylation signal AATGAA, and a second site 690 bp downstream of the stop codon. The putative polyadenylation signal upstream of this site contained a single nucleotide polymorphism (AG/TTAAC). However, allelic variation at this site did not influence polyadenylation site usage, and there were no significant differences in the abundance of the two alleles of this polymorphism between 329 control subjects, 158 individuals with major depression, and 130 individuals with bipolar affective disorder. This single nucleotide polymorphism in the 3′ untranslated region of the hSERT gene should provide a useful genetic marker in the evaluation of hSERT as a candidate gene influencing susceptibility to mood disorders.