MicroRNAs 29b and 181a down-regulate the expression of the norepinephrine transporter and glucocorticoid receptors in PC12 cells.

MicroRNAs are short non-coding RNAs that provide global regulation of gene expression at the post-transcriptional level. Such regulation has been found to play a role in stress-induced epigenetic responses in the brain. The norepinephrine transporter (NET) and glucocorticoid receptors are closely related to the homeostatic integration and regulation after stress. Our previous studies demonstrated that NET mRNA and protein levels in rats are regulated by chronic stress and by administration of corticosterone, which is mediated through glucocorticoid receptors. Whether miRNAs are intermediaries in the regulation of these proteins remains to be elucidated. This study was undertaken to determine possible regulatory effects of miRNAs on the expression of NET and glucocorticoid receptors in the noradrenergic neuronal cell line. Using computational target prediction, we identified several candidate miRNAs potentially targeting NET and glucocorticoid receptors. Western blot results showed that over-expression of miR-181a and miR-29b significantly repressed protein levels of NET, which is accompanied by a reduced [3 H] norepinephrine uptake, and glucocorticoid receptors in PC12 cells. Luciferase reporter assays verified that both miR-181a and miR-29b bind the 3'UTR of mRNA of NET and glucocorticoid receptors. Furthermore, exposure of PC12 cells to corticosterone markedly reduced the endogenous levels of miR-29b, which was not reversed by the application of glucocorticoid receptor antagonist mifepristone. These observations indicate that miR-181a and miR-29b can function as the negative regulators of NET and glucocorticoid receptor translation in vitro. This regulatory effect may be related to stress-induced up-regulation of the noradrenergic phenotype, a phenomenon observed in stress models and depressive patients. This study demonstrated that miR-29b and miR-181a, two short non-coding RNAs that provide global regulation of gene expression, markedly repressed protein levels of norepinephrine (NE) transporter and glucocorticoid receptor (GR), as well as NE uptake by binding the 3'UTR of their mRNAs in PC12 cells. Also, exposure of cells to corticosterone significantly reduced miR-29b levels through a GR-independent way.

Comprehensive phenotype/genotype analyses of the norepinephrine transporter gene (SLC6A2) in ADHD: relation to maternal smoking during pregnancy.

OBJECTIVE: Despite strong pharmacological evidence implicating the norepinephrine transporter in ADHD, genetic studies have yielded largely insignificant results. We tested the association between 30 tag SNPs within the SLC6A2 gene and ADHD, with stratification based on maternal smoking during pregnancy, an environmental factor strongly associated with ADHD. METHODS: Children (6-12 years old) diagnosed with ADHD according to DSM-IV criteria were comprehensively evaluated with regard to several behavioral and cognitive dimensions of ADHD as well as response to a fixed dose of methylphenidate (MPH) using a double-blind placebo controlled crossover trial. Family-based association tests (FBAT), including categorical and quantitative trait analyses, were conducted in 377 nuclear families. RESULTS: A highly significant association was observed with rs36021 (and linked SNPs) in the group where mothers smoked during pregnancy. Association was noted with categorical DSM-IV ADHD diagnosis (Z=3.74, P=0.0002), behavioral assessments by parents (CBCL, P=0.00008), as well as restless-impulsive subscale scores on Conners'-teachers (P=0.006) and parents (P=0.006). In this subgroup, significant association was also observed with cognitive deficits, more specifically sustained attention, spatial working memory, planning, and response inhibition. The risk allele was associated with significant improvement of behavior as measured by research staff (Z=3.28, P=0.001), parents (Z=2.62, P=0.009), as well as evaluation in the simulated academic environment (Z=3.58, P=0.0003). CONCLUSIONS: By using maternal smoking during pregnancy to index a putatively more homogeneous group of ADHD, highly significant associations were observed between tag SNPs within SLC6A2 and ADHD diagnosis, behavioral and cognitive measures relevant to ADHD and response to MPH. This comprehensive phenotype/genotype analysis may help to further understand this complex disorder and improve its treatment. Clinical trial registration information - Clinical and Pharmacogenetic Study of Attention Deficit with Hyperactivity Disorder (ADHD); www.clinicaltrials.gov; NCT00483106.

Angiotensin-(1-7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways.

As angiotensin (Ang) (1-7) decreases norepinephrine (NE) content in the synaptic cleft, we investigated the effect of Ang-(1-7) on NE neuronal uptake in spontaneously hypertensive rats. [(3)H]-NE neuronal uptake was measured in isolated hypothalami. NE transporter (NET) expression was evaluated in hypothalamic neuronal cultures by western-blot. Ang-(1-7) lacked an acute effect on neuronal NE uptake. Conversely, Ang-(1-7) caused an increase in NET expression after 3 h incubation (40 ± 7%), which was blocked by the Mas receptor antagonist, a PI3-kinase inhibitor or a MEK1/2 inhibitor suggesting the involvement of Mas receptor and the PI3-kinase/Akt and MEK1/2-ERK1/2 pathways in the Ang-(1-7)-stimulated NET expression. Ang-(1-7) through Mas receptors stimulated Akt and ERK1/2 activities in spontaneously hypertensive rat neurons. Cycloheximide attenuated Ang-(1-7) stimulation of NET expression suggesting that Ang-(1-7) stimulates NET synthesis. In fact, Ang-(1-7) increased NET mRNA levels. Thus, we evaluated the long-term effect of Ang-(1-7) on neuronal NE uptake after 3 h incubation. Under this condition, Ang-(1-7) increased neuronal NE uptake by 60 ± 14% which was blocked by cycloheximide and the Mas receptor antagonist. Neuronal NE uptake and NET expression were decreased after 3 h incubation with an anti-Ang-(1-7) antibody. Ang-(1-7) induces a chronic stimulatory effect on NET expression. In this way, Ang-(1-7) may regulate a pre-synaptic mechanism in maintaining appropriate synaptic NE levels during hypertensive conditions.

Dexamethasone-induced up-regulation of the human norepinephrine transporter involves the glucocorticoid receptor and increased binding of C/EBP-ß to the proximal promoter of norepinephrine transporter.

Previously, we have found glucocorticoids up-regulate norepinephrine (NE) transporter (NET) expression in vitro. However, the underlying transcriptional mechanism is poorly understood. In this study, the role of glucocorticoids on the transcriptional regulation of NET was investigated. Exposure of neuroblastoma SK-N-BE(2)M17 cells to dexamethasone (Dex) significantly increased NET mRNA and protein levels in a time- and dose-dependent manner. This effect was attenuated by glucocorticoid receptor (GR) antagonist mifepristone, suggesting that up-regulation of NET by Dex was mediated by the GR. In reporter gene assays, exposure of cells to Dex resulted in dose-dependent increases of luciferase activity that were also prevented by mifepristone. Serial deletions of the NET promoter delineated Dex-responsiveness to a -301 to -148 bp region containing a CCAAT/enhancer binding protein-ß (C/EBP-ß) response element. Co-immunoprecipitation experiments demonstrated that Dex treatment caused the interaction of the GR with C/EBP-ß. Chromatin immunoprecipitation (ChIP) assay revealed that Dex exposure resulted in binding of both GR and C/EBP-ß to the NET promoter. Further experiments showed that mutation of the C/EBP-ß response element abrogated C/EBP-ß- and GR-mediated transactivation of NET. These findings demonstrate that Dex-induced increase in NET expression is mediated by the GR via a non-conventional transcriptional mechanism involving interaction of C/EBP-ß with a C/EBP-ß response element.

Effects of transcription factors Phox2 on expression of norepinephrine transporter and dopamine beta-hydroxylase in SK-N-BE(2)C cells.

Phox2a and Phox2b are two homeodomain proteins that control the differentiation of noradrenergic neurons during embryogenesis. In the present study, we examined the possible effect of Phox2a/2b on the in vitro expression of the norepinephrine transporter (NET) and dopamine beta-hydroxylase (DBH), two important markers of the noradrenergic system. SK-N-BE(2)C cells were transfected with cDNAs or short hairpin RNAs specific to the human Phox2a and Phox2b genes. Transfection of 0.1 to 5 mug of cDNAs of Phox2a or Phox2b significantly increased mRNA and protein levels of NET and DBH in a concentration-dependent manner. As a consequence of the enhanced expression of NET after transfection, there was a parallel increase in the uptake of [(3)H]norepinephrine. Co-transfection of Phox2a and Phox2b did not further increase the expression of noradrenergic markers when compared with transfection of either Phox2a or Phox2b alone. Transfection of shRNAs specific to Phox2a or Phox2b genes significantly reduced mRNA and protein levels of NET and DBH after shutdown of endogenous Phox2, which was accompanied by a decreased [(3)H]norepinephrine uptake. Furthermore, there was an additive effect after cotransfection with both shRNAs specific to Phox2a or Phox2b genes on NET mRNA levels. Finally, the reduced DBH expression caused by the shRNA specific to Phox2a could be reversed by transfection with Phox2b cDNA and vice versa. The present findings verify the determinant role of Phox2a and Phox2b on the expression and function of NET and DBH in vitro. Further clarifying the regulatory role of these two transcription factors on key proteins of the noradrenergic system may open a new avenue for therapeutics of aging-caused dysfunction of the noradrenergic system.

[Construction and expression of replication-deficient recombinant adenovirus vector with hNET gene by adeasy-1 system].

OBJECTIVE: To construct and identify the recombinant replication deficient adenovirus vector which codes for human Norepinephrine Transporter (hNET) gene by using the method of homogenous recombination in bacteria. METHODS: hNET gene was obtained from the recombinant plasmid pCMV5 via Kpn I + Xba I digestion, and subcloned into E1 deleted expression plasmid pAdtrack-CMV shuttle vector, forming transfer vector pAdtrack-CMV-hNET. Then it was linearized with Pme I followed by homologous recombination with bone plasmid pAdEasy-1 in BJ5183 cells to generate recombinant plasmid Ad-hNET. The DNA of identified Ad-hNET was digested with Pac I and transfected to HEK293 cells by liposome-mediated method to package recombinant adenovirus. The PCR technique was applied to detect the target gene and Western Blotting to verify the expression of hNET. The titre of the Ad-hNET was measured with the aid of green fluorescence protein (GFP) expression after multiplication and purification. RESULTS: By sequencing, it was confirmed that the product was the gene of hNET. PCR test, restriction endonuclease digestion and Western Blotting confirmed the successful construction of the recombinants Ad-hNET. The titre of purified recombinant adenovirus Ad-hNET was 1.2 X 10(10) pfu/mL. CONCLUSION: The recombinant adenovirus with the hNET gene was constructed successfully. It will be helpful for the further investigation of its potentiality to be applied in the tumors targeted therapeutic strategy.

Regulation of the norepinephrine transporter by alpha-synuclein-mediated interactions with microtubules.

alpha-Synuclein (alpha-Syn) regulates catecholaminergic neurotransmission. We demonstrate that alpha-Syn regulates the activity and surface expression of the norepinephrine transporter (NET), depending on its expression levels. In cells co-transfected with NET and low amounts of alpha-Syn, NET activity and cell surface expression were increased and protein interactions with alpha-Syn decreased, compared with cells transfected with NET alone. Converse effects were observed at higher levels of alpha-Syn expression. Treatment with nocodazole and other microtubule (MT) destabilizers abolished the expression-dependent bimodal regulation of NET by alpha-Syn. At low alpha-Syn levels, nocodazole had no effect on NET surface expression or protein interactions, while inducing increases in these measures at higher levels. Cells that were transfected with NET alone displayed no sensitivity to nocodazole, indicating that alpha-Syn expression was necessary for the MT-dependent changes in NET activity. MT destabilizers also caused a significant increase in [(3)H]-NE uptake in brainstem primary neurons and synaptosomes from the frontal cortex, but not striatal synaptosomes. These findings suggest that the surface localization and activity of NET is modulated by alpha-Syn in a manner that is both dependent on interactions with the MT cytoskeleton and varies across brain regions.

Expression of the noradrenaline transporter and phenylethanolamine N-methyltransferase in normal human adrenal gland and phaeochromocytoma.

Expression of the noradrenaline transporter (NAT) was examined in normal human adrenal medulla and phaeochromocytoma by using immunohistochemistry and confocal microscopy. The enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were used as catecholamine biosynthetic markers and chromogranin A (CGA) as a marker for secretory granules. Catecholamine content was measured by using high performance liquid chromatography (HPLC). In normal human adrenal medulla (n=5), all chromaffin cells demonstrated strong TH, PNMT and NAT immunoreactivity. NAT was co-localized with PNMT and was located within the cytoplasm with a punctate appearance. Human phaeochromocytomas demonstrated strong TH expression (n=20 samples tested) but variable NAT and PNMT expression (n=24). NAT immunoreactivity ranged from absent (n=3) to weak (n=10) and strong (n=11) and, in some cases, occupied an apparent nuclear location. Unlike the expression seen in normal human adrenal medullary tissue, NAT expression was not consistently co-localized with PNMT. PNMT also showed highly variable expression that was poorly correlated with tumour adrenaline content. Immunoreactivity for CGA was colocalized with NAT within the cytoplasm of normal human chromaffin cells (n=4). This co-localization was not consistent in phaeochromocytoma tumour cells (n=7). The altered pattern of expression for both NAT and PNMT in phaeochromocytoma indicates a significant disruption in the regulation and possibly in the function of these proteins in adrenal medullary tumours.

Comparison of radiohaloanalogues of meta-iodobenzylguanidine (MIBG) for a combined gene- and targeted radiotherapy approach to bladder carcinoma.

Targeted radiotherapy using radiolabelled meta-iodobenzylguanidine (MIBG) is a promising treatment option for bladder cancer, restricting the effects of radiotherapy to malignant cells thereby increasing efficacy and decreasing morbidity of radiotherapy. We investigated the efficacy of a combined gene therapy and targeted radiotherapy approach for bladder cancer using radiolabelled MIBG. The effectiveness of alternative radiohalogens and alternative preparations of radiolabelled MIBG for this therapeutic strategy were compared. Bladder cancer cells, EJ138, were transfected with a gene encoding the noradrenaline transporter (NAT) under the control of a tumour specific telomerase promoter, enabling them to actively take up radiolabelled MIBG. This resulted in tumour-specific cell kill. Uptake and retention of radioactivity in cells transfected with the NAT gene were compared with that obtained in cells transfected with the sodium iodide symporter (NIS) gene. Substantially greater uptake and longer retention of radioactivity in NAT-transfected cells was observed. Carrier-added (c.a.) [131I]MIBG, no-carrier added (n.c.a.) [131I]MIBG, and [211At]-labelled benzylguanidine (i.e. [211At] meta-astatobenzylguanidine (MABG)) were compared with respect to efficiency of induction of cell kill. N.c.a[(131)I]MIBG was more cytotoxic than c.a.[131I]MIBG. However, the alpha-emitter [211At]MABG was, by three orders of magnitude, more effective in causing tumour cell kill than the beta-emitter [131I]MIBG. We conclude that NAT gene transfer combined with the administration of n.c.a.[131I]MIBG or [211At]MABG, is a promising novel treatment approach for bladder cancer therapy.