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Norepinephrine transport-mediated gene expression in noradrenergic neurogenesis.

ABSTRACT: We have identified a differential gene expression profile in neural crest stem cells that is due to deletion of the norepinephrine transporter (NET) gene. NET is the target of psychotropic substances, such as tricyclic antidepressants and the drug of abuse, cocaine. NET mutations have been implicated in depression, anxiety, orthostatic intolerance and attention deficit hyperactivity disorder (ADHD). NET function in adult noradrenergic neurons of the peripheral and central nervous systems is to internalize norepinephrine from the synaptic cleft. By contrast, during embryogenesis norepinephrine (NE) transport promotes differentiation of neural crest stem cells and locus ceruleus progenitors into noradrenergic neurons, whereas NET inhibitors block noradrenergic differentiation. While the structure of NET und the regulation of NET function are well described, little is known about downstream target genes of norepinephrine (NE) transport.We have prepared gene expression profiles of in vitro differentiating wild type and norepinephrine transporter-deficient (NETKO) mouse neural crest cells using long serial analysis of gene expression (LongSAGE). Comparison analyses have identified a number of important differentially expressed genes, including genes relevant to neural crest formation, noradrenergic neuron differentiation and the phenotype of NETKO mice. Examples of differentially expressed genes that affect noradrenergic cell differentiation include genes in the bone morphogenetic protein (BMP) signaling pathway, the Phox2b binding partner Tlx2, the ubiquitin ligase Praja2, and the inhibitor of Notch signaling, Numbl. Differentially expressed genes that are likely to contribute to the NETKO phenotype include dopamine-beta-hydroxylase (Dbh), tyrosine hydroxylase (Th), the peptide transmitter 'cocaine and amphetamine regulated transcript' (Cart), and the serotonin receptor subunit Htr3a. Real-time PCR confirmed differential expression of key genes not only in neural crest cells, but also in the adult superior cervical ganglion and locus ceruleus. In addition to known genes we have identified novel differentially expressed genes and thus provide a valuable database for future studies.Loss of NET function during embryonic development in the mouse deregulates signaling pathways that are critically involved in neural crest formation and noradrenergic cell differentiation. The data further suggest deregulation of signaling pathways in the development and/or function of the NET-deficient peripheral, central and enteric nervous systems.

SUBMITTER: Hu YF

PROVIDER: S-EPMC2679758 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Norepinephrine transport-mediated gene expression in noradrenergic neurogenesis.

Hu Yao Fei YF Caron Marc G MG Sieber-Blum Maya M

BMC genomics 20090408

<h4>Background</h4>We have identified a differential gene expression profile in neural crest stem cells that is due to deletion of the norepinephrine transporter (NET) gene. NET is the target of psychotropic substances, such as tricyclic antidepressants and the drug of abuse, cocaine. NET mutations have been implicated in depression, anxiety, orthostatic intolerance and attention deficit hyperactivity disorder (ADHD). NET function in adult noradrenergic neurons of the peripheral and central nerv ...[more]

PMID: 19356247

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Project description:The norepinephrine transporter (NET) mediates the clearance of norepinephrine (NE) from the extracellular space and is a target of therapeutic antidepressants and psychostimulants. Previously we identified a MAP kinase phosphatase 3 (MKP3), as an important modulator of protein kinase C (PKC) mediated internalization of the related dopamine transporter (DAT). Here we show that MKP3 decreases PKC-mediated down regulation of NET expressed in PC12 cells. We demonstrate that this process involves a PKC-stimulated decrease of NET surface expression that is dependent on dynamin. Surprisingly, MAP kinase inhibitors have no effect on the PKC-mediated regulation of NET activity, suggesting that, like PKC-mediated regulation of the DAT, the acute activation of MAP kinases is not likely to be involved. To elucidate potential mechanisms we used a substrate trap-based assay to identify extracellular-signal-regulated kinase (ERK)1/2 as the predominant substrate of MKP3. Furthermore we also established that brief chemical stabilization of a modified destabilized MKP3 does not alter PKC-mediated down regulation of NET. Finally, the expression of a dominant negative version of H-Ras, an upstream activator of ERK1/2, abolishes phorbol 12-myristate 13-acetate (PMA)-mediated down regulation of NET in a manner similar to MKP3. Taken together we propose that chronic MKP3 expression regulates surface NET through the sustained inhibition of ERK1/2 MAP kinase signaling that alters gene expression in PC12 cells. This is supported by gene expression data from naïve and MKP3-expressing PC12 cells that reveal robust decreases in gene expression of several genes in the MKP3-tranfected cells. Interestingly, caveolin-1, a protein with a critical role in membrane protein trafficking is down regulated by MKP3 expression. We further show that selective silencing of the caveolin-1 gene in naïve PC12 cells attenuates PKC-mediated downregulation of NET activity, consistent with a potential role for caveolins in regulating NET surface expression. In summary, these results suggest that chronic MKP3 expression alters the expression of genes in PC12 cells that are involved in the regulation of NET surface expression.

Project description:Objective- Perivascular adipose tissue (PVAT) contains an independent adrenergic system that can take up, metabolize, release, and potentially synthesize the vasoactive catecholamine norepinephrine. Norepinephrine has been detected in PVAT, but the mechanism of its protection within this tissue is unknown. Here, we investigate whether PVAT adipocytes can store norepinephrine using VMAT (vesicular monoamine transporter). Approach and Results- High-performance liquid chromatography identified norepinephrine in normal male Sprague Dawley rat aortic, superior mesenteric artery, and mesenteric resistance vessel PVATs, and retroperitoneal fat. Real-time polymerase chain reaction revealed VMAT1 and VMAT2 mRNA expression in the adipocytes and stromal vascular fraction of mesenteric resistance vessel PVAT. Immunofluorescence demonstrated the presence of VMAT1 and VMAT2, and the colocalization of VMAT2 with norepinephrine, in the cytoplasm of adipocytes in mesenteric resistance vessel PVAT. A protocol was developed to capture real-time uptake of Mini 202-a functional and fluorescent VMAT probe-in live rat PVAT adipocytes. Mini 202 was taken up by freshly isolated and differentiated adipocytes from mesenteric resistance vessel PVAT and adipocytes from thoracic aortic and superior mesenteric artery PVATs. In adipocytes freshly isolated from mesenteric resistance vessel PVAT, addition of rose bengal (VMAT inhibitor), nisoxetine (norepinephrine transporter inhibitor), or corticosterone (organic cation 3 transporter inhibitor) significantly reduced Mini 202 signal. Immunofluorescence supports that neither VMAT1 nor VMAT2 is present in retroperitoneal adipocytes, suggesting that PVAT adipocytes may be unique in storing norepinephrine. Conclusions- This study supports a novel function of PVAT adipocytes in storing amines in a VMAT-dependent manner. It provides a foundation for future studies exploring the purpose and mechanisms of norepinephrine storage by PVAT in normal physiology and obesity-related hypertension.

Dataset Information

Norepinephrine transport-mediated gene expression in noradrenergic neurogenesis.

Publications

Norepinephrine transport-mediated gene expression in noradrenergic neurogenesis.

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