The expression of dopamine beta-hydroxylase, tyrosine hydroxylase, and Phox2 transcription factors in sympathetic neurons: evidence for common regulation during noradrenergic induction and diverging regulation later in development

During differentiation of sympathetic neurons in chick embryos, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) mRNAs become detectable during the same developmental period and are both induced by BMP 4. Later during sympathetic ganglion development, DBH is detectable in TH-positive and -negative cells. Moreover, BMPs reduce DBH mRNA in cultures of sympathetic neurons while leaving TH unaffected. The data provide evidence for a common regulation of TH and DBH early during sympathetic neuron differentiation and indicate that BMPs promote their initial expression but not the maintenance during later development. The time course of Phox2a and 2b expression suggests an evolutionary conserved role in noradrenergic induction. In addition, Phox2a, Phox2b, and c-ret may be involved in the differentiation of cholinergic sympathetic neurons.     

Differential Expression of SNAP-25 Isoforms and SNAP-23 in the Adrenal Gland

Abstract : In the rat adrenal gland, we previously observed that SNAP-25 is not restricted to the plasmalemma in noradrenergic cells as it is in adrenergic cells, and hypothesized that SNAP-25 isoform expression is different in the two phenotypes. Expression of SNAP-25 isoforms and SNAP-23 was examined by immunoblotting, immunofluorescence, and RT-PCR. Amplifications of SNAP-25 mRNAs were combined with Southern hybridization, restriction enzyme analysis, and sequencing of cloned PCR products to compare SNAP-25 isoform expression in rat and bovine adrenal glands. SNAP-25 and SNAP-23 mRNA and protein are expressed in the glands ; SNAP-23 is enriched in the adrenal cortex, whereas SNAP-25 is restricted to the adrenal medulla. Furthermore, high levels of SNAP-25 and low levels of SNAP-23 are observed in the PC12 cells, whereas both SNAP-25 and SNAP-23 are expressed in adrenal medullary cultures. In all extracts, the SNAP-23 mRNA corresponded to SNAP-23a. SNAP-25a is the major form expressed in rat adrenal glands (75%), as it is in PC12 cells (80%), but both SNAP-25a and SNAP-25b (40% vs. 60%) are expressed in bovine adrenal medulla in situ and in culture. In addition, an enriched population of adrenergic cells (93%) expressed a higher level of SNAP-25b (70%), suggesting that this isoform may not be restricted to fast neurotransmission.     

Corticosterone administration up‐regulated expression of norepinephrine transporter and dopamine β‐hydroxylase in rat locus coeruleus and its terminal regions

Stress has been reported to activate the locus coeruleus (LC)–noradrenergic system. In this study, corticosterone (CORT) was orally administrated to rats for 21 days to mimic stress status. In situ hybridization measurements showed that CORT ingestion significantly increased mRNA levels of norepinephrine transporter (NET) and dopamine β‐hydroxylase (DBH) in the LC region. Immunofluorescence staining and western blotting revealed that CORT treatment also increased protein levels of NET and DBH in the LC, as well as NET protein levels in the hippocampus, the frontal cortex and the amygdala. However, CORT‐induced increase in DBH protein levels only appeared in the hippocampus and the amygdala. Elevated NET and DBH expression in most of these areas (except for NET protein levels in the LC) was abolished by simultaneous treatment with combination of corticosteroid receptor antagonist mifepristone and spironolactone (s.c. for 21 days). Also, treatment with mifepristone alone prevented CORT‐induced increases of NET expression and DBH protein levels in the LC. In addition, behavioral tasks showed that CORT ingestion facilitated escape in avoidance trials using an elevated T‐maze, but interestingly, there was no significant effect on the escape trial. Corticosteroid receptor antagonists failed to counteract this response in CORT‐treated rats. In the open‐field task, CORT treatment resulted in less activity in a defined central zone compared to controls and corticosteroid receptor antagonist treatment alleviated this increase. In conclusion, this study demonstrates that chronic exposure to CORT results in a phenotype that mimics stress‐induced alteration of noradrenergic phenotypes, but the effects on behavior are task dependent. As the sucrose consumption test strongly suggests CORT ingestion‐induced depression‐like behavior, further elucidation of underlying mechanisms may improve our understanding of the correlation between stress and the development of depression.

     

Quantitation of changes in gene expression of norepinephrine biosynthetic enzymes in rat stellate ganglia induced by stress

Enzymes involved in catecholamine synthesis are present in the highest concentration in the adrenal medulla, however they were found also in other, mainly nervous tissues. The aim of our study was to quantify the exact concentration of tyrosine hydroxylase (TH) and dopamine-ss-hydroxylase (DBH) mRNA in rat stellate ganglia under control conditions and at different intervals after exposure to immobilization stress (IMO). In rats immobilized once for 2h, we determined TH and DBH mRNA in different time intervals up to 22 h after the end of the stress stimulus. TH immunoreactive protein levels were also determined in stellate ganglia. TH and DBH mRNA levels were quantified by RT-competitive-PCR.In stellate ganglia, the concentration of TH mRNA was 17+/-1.6 amol/microg of total RNA, which is approximately 30-times lower than in the adrenal medulla. The concentration of DBH mRNA in the stellate ganglia was 2601+/-203 amol/microg of total RNA, which is the concentration similar to adrenal medulla, but is 150-times higher than concentration of TH mRNA in stellate ganglia. After a single 2-h immobilization the highest elevation of TH and DBH mRNA levels was measured 22 h after the termination of the stress stimulus. Repeated immobilization (7 days, 2h daily) did not produce further increase in TH and DBH mRNA levels compared to already elevated levels in adapted control group (immobilized for 6 days, 2h daily and decapitated 22 h later). Levels of TH protein were significantly changed only after the repeated immobilization.This study compared for the first time the precise amounts of TH and DBH mRNA in rat stellate ganglia under control conditions and after immobilization stress, and indicates large differences in their concentration. TH and DBH mRNA concentrations in stellate ganglia are markedly elevated for a prolonged period of time after termination of the stress stimuli.     

Norepinephrine transporter expression and function in noradrenergic cell differentiations

The classical view of norepinephrine transporter (NET) function is the re-uptake of released norepinephrine (NE) by mature sympathetic neurons and noradrenergic neurons of the locus ceruleus (LC; [1-3]). In this report we review previous data and present new results that show that NET is expressed in the young embryo in a wide range of neuronal and non-neuronal tissues and that NET has additional functions during embryonic development. Sympathetic neurons are derived from neural crest stem cells. Fibroblast growth factor-2 (FGF-2), neurotrophin-3 (NT-3) and transforming growth factor-1 (TGF-1) regulate NET expression in cultured quail neural crest cells by causing an increase in NET mRNA levels. They also promote NET function in both neural crest cells and presumptive noradrenergic cells of the LC. The growth factors are synthesized by the neural crest cells and therefore are likely to have autocrine function. In a subsequent stage of development, NE transport regulates differentiation of noradrenergic neurons in the peripheral nervous system and the LC by promoting expression of tyrosine hydroxylase (TH) and dopamine--hydroxylase (DBH). Conversely, uptake inhibitors, such as the tricyclic antidepressant, desipramine, and the drug of abuse, cocaine, inhibit noradrenergic differentiation in both tissues. Taken together, our data indicate that NET is expressed early in embryonic development, NE transport is involved in regulating expression of the noradrenergic phenotype in the peripheral and central nervous systems, and norepinephrine uptake inhibitors can disturb noradrenergic cell differentiation in the sympathetic ganglion (SG) and LC.     

Development of noradrenergic neurons in the zebrafish hindbrain requires BMP, FGF8, and the homeodomain protein soulless/Phox2a

We report that the zebrafish mutation soulless, in which the development of locus coeruleus (LC) noradrenergic (NA) neurons failed to occur, disrupts the homeodomain protein Phox2a. Phox2a is not only necessary but also sufficient to induce Phox2b+ dopamine-beta-hydroxylase+ and tyrosine hydroxylase+ NA neurons in ectopic locations. Phox2a is first detected in LC progenitors in the dorsal anterior hindbrain, and its expression there is dependent on FGF8 from the mid/hindbrain boundary and on optimal concentrations of BMP signal from the epidermal ectoderm/future dorsal neural plate junction. These findings suggest that Phox2a coordinates the specification of LC in part through the induction of Phox2b and in response to cooperating signals that operate along the mediolateral and anteroposterior axes of the neural plate.     

Catecholamine Metabolism in Paraganglioma and Pheochromocytoma: Similar Tumors in Different Sites?

Pheochromocytoma (PHEO) and paraganglioma (PGL) are catecholamine-producing neuroendocrine tumors that arise respectively inside or outside the adrenal medulla. Several reports have shown that adrenal glucocorticoids (GC) play an important regulatory role on the genes encoding the main enzymes involved in catecholamine (CAT) synthesis i.e. tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). To assess the influence of tumor location on CAT metabolism, 66 tissue samples (53 PHEO, 13 PGL) and 73 plasma samples (50 PHEO, 23 PGL) were studied. Western blot and qPCR were performed for TH, DBH and PNMT expression. We found a significantly lower intra-tumoral concentration of CAT and metanephrines (MNs) in PGL along with a downregulation of TH and PNMT at both mRNA and protein level compared with PHEO. However, when PHEO were partitioned into noradrenergic (NorAd) and mixed tumors based on an intra-tumoral CAT ratio (NE/E >90%), PGL and NorAd PHEO sustained similar TH, DBH and PNMT gene and protein expression. CAT concentration and composition were also similar between NorAd PHEO and PGL, excluding the use of CAT or MNs to discriminate between PGL and PHEO on the basis of biochemical tests. We observed an increase of TH mRNA concentration without correlation with TH protein expression in primary cell culture of PHEO and PGL incubated with dexamethasone during 24 hours; no changes were monitored for PNMT and DBH at both mRNA and protein level in PHEO and PGL. Altogether, these results indicate that long term CAT synthesis is not driven by the close environment where the tumor develops and suggest that GC alone is not sufficient to regulate CAT synthesis pathway in PHEO/PGL.