Tyrosine Hydroxylase Gene Expression Research Articles

Blood Levels of Monoamine Precursors and Smoking in Patients with Schizophrenia.

Smoking is highly prevalent in patients with schizophrenia and exerts a negative impact on cardiovascular mortality in these patients. Smoking has complex interactions with monoamine metabolism through the ability of cigarette smoke to suppress Type 1 T helper cell (Th1) type immunity, the immunophenotype that is implicated in phenylalanine hydroxylase (PAH) dysfunction and tryptophan (Trp) breakdown to kynurenine (Kyn) via indoleamine 2,3-dioxygenase. Nicotine also induces tyrosine hydroxylase (TH) gene expression, leading to increased synthesis of catecholamines. Furthermore, there is evidence for PAH dysfunction in schizophrenia. This study aimed to compare the plasma levels of selected monoamine precursors and their metabolites in smokers vs. non-smokers in a large sample of patients with schizophrenia. We measured plasma phenylalanine (Phe), tyrosine (Tyr), Trp, and Kyn levels using high-performance liquid chromatography and calculated Phe:Tyr and Kyn:Trp ratios in 920 patients with schizophrenia. Analysis of variance and linear regression analyses were used to compare these endpoints between three groups of patients with schizophrenia: (1) current smokers, (2) past smokers, and (3) non-smokers. There were significant differences among the three groups with regards to Tyr levels [F(2,789) = 3.77, p = 0.02], with current smokers having lower Tyr levels when compared with non-smokers (p = 0.02). Kyn levels and Kyn:Trp ratio were different among the three groups [F(2,738) = 3.17, p = 0.04, F(2,738) = 3.61, p = 0.03] with current smokers having lower Kyn levels (p = 0.04) and higher Kyn:Trp ratio (p = 0.02) when compared with past smokers. These findings need to be replicated with protocols that include healthy controls to further elucidate the neurobiological underpinnings of altered Tyr and Kyn levels in smokers. Results do suggest potential molecular links between schizophrenia and smoking that may represent biomarkers and treatment targets for reducing an important modifiable cause of general morbidity and mortality in patients with schizophrenia.

Erratum to: Noggin Along with a Self-Assembling Peptide Nanofiber Containing Long Motif of Laminin Induces Tyrosine Hydroxylase Gene Expression.

Erratum to: Noggin Along with a Self-Assembling Peptide Nanofiber Containing Long Motif of Laminin Induces Tyrosine Hydroxylase Gene Expression.

Noggin Along with a Self-Assembling Peptide Nanofiber Containing Long Motif of Laminin Induces Tyrosine Hydroxylase Gene Expression.

Tyrosine hydroxylase (TH), a rate-limiting step in catecholamine synthesis in which its activity influences Alzheimer disease, Parkinson disease, and IQ of schizophrenia patients, has been studied for a long time. In the meantime, the present investigation assessed the effect of noggin and type of self-assembling nanofibers in TH gene over-expression by neuron-like cells derived from human endometrial-derived stromal cells (hEnSCs). Neuroblastoma cells and hEnSCs encapsulated into nanofibers including Matrigel, (RADA)4, laminin, and BMHP-1 motif bounded to (RADA)4 and their cell viability were studied for 48h and 18days in basal and neurogenic media, respectively, in noggin-rich media. Then, expression of neural genes and proteins has been investigated by immunocytochemistry (ICC) and real-time PCR methods, respectively. The results indicated that neuroblastoma cell and hEnSC viability is in good agreement with the level of Bcl2 and β-tubulin III gene expression; however, -BMHP-1 and -laminin nanofibers exhibited significantly higher cell viability eventually through Wnt/β-catenin signaling pathway as compared to others, respectively. The gene expression analysis of nanofibers showed that none of them induced gamma-aminobutyric acid (GABA) gene expression while glial fibrillary acidic protein (GFAP) gene just over-expressed in cells encapsulated into Matrigel with a low level of Bcl2 gene expression. However, the TH gene just had been over-expressed in cells encapsulated into -laminin nanofiber and 2D cell culture. In the absence of noggin with -laminin nanofibers, TH gene expression was suppressed. It might be concluded that although noggin through anti-BMP pathways resulted in GFAP decrement and TH gene increment, the type of scaffold that defined the final fate of cells and -laminin accompaniment might be useful for the recovery of Alzheimer and Parkinson disease patients.

Exaggerated phosphorylation of brain tau protein in CRH KO mice exposed to repeated immobilization stress

Neuroendocrine and behavioral stress responses are orchestrated by corticotropin-releasing hormone (CRH) and norepinephrine (NE) synthesizing neurons. Recent findings indicate that stress may promote development of neurofibrillary pathology in Alzheimer's disease. Therefore, we investigated relationships among stress, tau protein phosphorylation, and brain NE using wild-type (WT) and CRH-knockout (CRH KO) mice. We assessed expression of phosphorylated tau (p-tau) at the PHF-1 epitope and NE concentrations in the locus coeruleus (LC), A1/C1 and A2/C2 catecholaminergic cell groups, hippocampus, amygdala, nucleus basalis magnocellularis, and frontal cortex of unstressed, singly stressed or repeatedly stressed mice. Moreover, gene expression and protein levels of tyrosine hydroxylase (TH) and CRH receptor mRNA were determined in the LC. Plasma corticosterone levels were also measured. Exposure to a single stress increases tau phosphorylation throughout the brain in WT mice when compared to singly stressed CRH KO animals. In contrast, repeatedly stressed CRH KO mice showed exaggerated tau phosphorylation relative to WT controls. We also observed differences in extent of tau phosphorylation between investigated structures, e.g. the LC and hippocampus. Moreover, CRH deficiency leads to different responses to stress in gene expression of TH, NE concentrations, CRH receptor mRNA, and plasma corticosterone levels. Our data indicate that CRH effects on tau phosphorylation are dependent on whether stress is single or repeated, and differs between brain regions. Our findings indicate that CRH attenuates mechanisms responsible for development of stress-induced tau neuropathology, particularly in conditions of chronic stress. However, the involvement of central catecholaminergic neurons in these mechanisms remains unclear and is in need of further investigation.

Free-choice high-fat diet alters circadian oscillation of energy intake in adolescent mice: role of prefrontal cortex.

Our aim was to characterize the effect of an unfamiliar high-fat diet (HFD) on circadian feeding behaviour, plasma parameters, body weight (BW), and gene expression in the prefrontal cortex (PFC) of adolescent male mice. To this end, mice were allowed to consume a HFD during 48h, but one group was allowed a free choice of HFD or normal chow (FC-HFD), while the other was restricted to a non-optional unfamiliar HFD feeding (NOP-HFD). Energy intake was monitored at 6-h intervals during 48h. Mice cohorts were killed at 6-h intervals after 48-h dietary treatment, and PFC samples dissected for RT-PCR analysis. Mice on the FC-HFD protocol avoided eating the standard chow, showed lower energy intake and lower BW increase than NOP-HFD mice. All animals with access to HFD exhibited nocturnal overeating, but diurnal hyperphagia was more prominent in the FC-HFD cohort. A robust increase in tyrosine hydroxylase (Th) gene expression was detected specifically during the light period of the circadian cycle in FC-HFD mice. In contrast, both protocols similarly up-regulated the expression of cytosolic malic enzyme (Me1), which is very sensitive to HFD. Our data show that the PFC participates in driving motivational feeding during HFD-evoked hyperphagia and also suggest that sensory neural pathways might be relevant for the onset of eating disorders and overweight. Moreover, we have observed that animals that had the possibility of choosing between standard chow and HFD were more hyperphagic and specifically displayed an overexpression of the tyrosine hydroxylase gene.

MP89-16 INCREASE IN TNF-ALPHA LEADS TO APOPTOSIS OF NITRERGIC NEURONS IN MAJOR PELVIC GANGLION AFTER BILATERAL CAVERNOUS NERVE INJURY

You have accessJournal of UrologySexual Function/Dysfunction: Basic Research & Pathophysiology II1 Apr 2016MP89-16 INCREASE IN TNF-ALPHA LEADS TO APOPTOSIS OF NITRERGIC NEURONS IN MAJOR PELVIC GANGLION AFTER BILATERAL CAVERNOUS NERVE INJURY Hotaka Matsui, Nikolai A. Sopko, Johanna L. Hannan, Allison A. Reinhardt, Emmanuel Weyne, Max Kates, Xiaopu Liu, Maarten Albersen, and Trinity J. Bivalacqua Hotaka MatsuiHotaka Matsui More articles by this author , Nikolai A. SopkoNikolai A. Sopko More articles by this author , Johanna L. HannanJohanna L. Hannan More articles by this author , Allison A. ReinhardtAllison A. Reinhardt More articles by this author , Emmanuel WeyneEmmanuel Weyne More articles by this author , Max KatesMax Kates More articles by this author , Xiaopu LiuXiaopu Liu More articles by this author , Maarten AlbersenMaarten Albersen More articles by this author , and Trinity J. BivalacquaTrinity J. Bivalacqua More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2477AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Erectile dysfunction (ED) is induced by inflammation in cavernous nerve (CN) after radical prostatectomy (RP). This study examines changes of inflammatory cytokine: tumor necrosis factor-alpha (TNF-α) after bilateral CN injury (BCNI) in vivo and its effect on neurite outgrowth from major pelvic ganglion (MPG) ex vivo. METHODS Male rats underwent BCNI or sham surgery. MPGs were harvested 2, 7 and 14 days after surgery (n=10/group). MPGs were processed for qPCR, Western blot (WB), and immunofluorescence (IF) for TNF-α. MPGs were also harvested from untouched rats and cultured in Matrigel with exogenous TNF-α (eTNF-α) in concentrations of 0, 10, 20, 30 ng/mL (n=5/group). Neurite lengths were measured 3 days after culture. MPGs were then processed for qPCR to evaluate gene expression of neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), and caspase (CASP)-1 (n=5/group). Additional MPGs were cultured in Matrigel with/without eTNF-α 20 ng/mL to perform IF for TH and nNOS and to evaluate protein amount of CASP3 by WB (n=3/group). RESULTS BCNI increased gene and protein expression of TNF-α (p<0.05). Maximal increase was at 2 days for gene and at 7 days for protein expression of TNF-α. IF demonstrated that TNF-α mainly localized in periphery at 2 days, cytoplasm of soma at 7 days, and interstitial space at 14 days after BCNI. eTNF-α inhibited neurite outgrowth from MPG in a dose-dependent fashion (neurite lengths: 586±20.5, 455±11.5, 408±28.7, 361±2.65 μm; eTNF-α 0, 10, 20, 30 ng/mL, respectively: p<0.01). MPGs cultured with eTNF-α showed increased gene expression of CASP1 (p=0.03) and decreased gene expression of nNOS (p<0.05). Gene expression of TH remained unchanged. WB demonstrated increased protein amount of total CASP3 in MPG cultured with eTNF-α (p=0.03). However, there was no significant difference in ratio of cleaved and total CASP3. IF demonstrated that nNOS+ neurites were shorter than TH+ neurites in MPGs cultured with eTNF-α (p<0.01), while there was no difference in neurite lengths between nNOS+ neurites and TH+ neurites in control (p=0.29). CONCLUSIONS This study demonstrates that TNF-α is increased after BCNI and impairs neurite outgrowth from MPG in a dose-dependent fashion. Furthermore, eTNF-α upregulated gene and protein expression of apoptosis markers and downregluated gene and protein expression of nNOS, suggesting that TNF-α selectively induces apoptosis of nitrergic neurons. These results suggest that TNF-α antagonists may prevent post-RP ED. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1142-e1143 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Hotaka Matsui More articles by this author Nikolai A. Sopko More articles by this author Johanna L. Hannan More articles by this author Allison A. Reinhardt More articles by this author Emmanuel Weyne More articles by this author Max Kates More articles by this author Xiaopu Liu More articles by this author Maarten Albersen More articles by this author Trinity J. Bivalacqua More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Medicinal plant Combretum leprosum mart ameliorates motor, biochemical and molecular alterations in a Parkinson's disease model induced by MPTP

Ethnopharmacological relevanceCombretum leprosum is a popular medicinal plant distributed in north and northeastern regions of Brazil. Many different parts of this plant are used in traditional medicine to treat several inflammatory diseases. Parkinson's disease (PD) is a disorder associated with inflammatory toxic factors and the treatments available provide merely a delay of the neurodegeneration. Aim of the studyWe investigated the potential neuroprotective properties of the C. leprosum ethanolic extract (C.l.EE) in a murine model of PD using the toxin 1-methyl-4 phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Materials and methodsThe mice were split into four groups: V/S (vehicle/saline), E/S (extract/saline), V/M (vehicle/MPTP) and E/M (extract/ MPTP). Mice received MPTP (30mg/kg, i.p.) or vehicle (10ml/kg, i.p.) once a day for 5 consecutive days and vehicle (10ml/kg) or C.l.EE (100mg/kg) orally by intra-gastric gavage (i.g.) during a 14-d period, starting 3 days before the first MPTP injection. All groups were assessed for behavioural impairments (amphetamine-induced locomotor activity and muscle strength), dopamine content in striatum using high performance liquid chromatography (HPLC), tyrosine hydroxylase (TH) and dopamine transporter (DAT) gene expressions using qPCR. ResultsAnimals were injected with d-amphetamine (2mg/kg) and the activity was recorded. Amphetamine-induced hyperlocomotion was observed in all groups; however animals treated with MPTP showed exacerbated hyperlocomotion (approximately 3 fold increase compared to control groups). By contrast, mice treated with MPTP that received C.l.EE exhibited attenuation of the hyperlocomotion and did not differ from control groups. Muscle strength test pointed that C.l.EE strongly avoided muscular deficits caused by MPTP (approximately 2 fold increase compared to V/M group). Dopamine and its metabolites were measured in the striatum. The V/M group presented a dopamine reduction of 80%. On the other hand, the E/M group exhibited an increase in dopamine and its metabolites levels (approximately 3 fold increase compared to V/M group). Tyrosine hydroxylase (TH) and dopamine transporter (DAT) gene expressions were significantly reduced in the V/M group (60%). Conversely, C.l.EE treatment was able to increase the mRNA levels of those genes in the E/M group (approximately 2 fold for TH and DAT). ConclusionsThese data show, for the first time, that C. leprosum ethanolic extract prevented motor and molecular changes induced by MPTP, and partially reverted dopamine deficit. Thus, our results demonstrate that C.l.EE has potential for the treatment and prevention of PD.

Tauopathy in transgenic (SHR72) rats impairs function of central noradrenergic system and promotes neuroinflammation.

BackgroundBrain norepinephrine (NE) plays an important role in the modulation of stress response and neuroinflammation. Recent studies indicate that in Alzheimer’s disease (AD), the tau neuropathology begins in the locus coeruleus (LC) which is the main source of brain NE. Therefore, we investigated the changes in brain NE system and also the immune status under basal and stress conditions in transgenic rats over-expressing the human truncated tau protein.MethodsBrainstem catecholaminergic cell groups (LC, A1, and A2) and forebrain subcortical (nucleus basalis of Meynert), hippocampal (cornu ammonis, dentate gyrus), and neocortical areas (frontal and temporal association cortices) were analyzed for NE and interleukin 6 (IL-6) mRNA levels in unstressed rats and also in rats exposed to single or repeated immobilization. Moreover, gene expression of NE-biosynthetic enzyme, tyrosine hydroxylase (TH), and several pro- and anti-inflammatory mediators were determined in the LC.ResultsIt was found that tauopathy reduced basal NE levels in forebrain areas, while the gene expression of IL-6 was increased in all selected areas at the same time. The differences between wild-type and transgenic rats in brain NE and IL-6 mRNA levels were observed in stressed animals as well. Tauopathy increased also the gene expression of TH in the LC. In addition, the LC exhibited exaggerated expression of pro- and anti-inflammatory mediators (IL-6, TNFα, inducible nitric oxide synthases 2 (iNOS2), and interleukin 10 (IL-10)) in transgenic rats suggesting that tauopathy affects also the immune background in LC. Positive correlation between NE and IL-6 mRNA levels in cornu ammonis in stressed transgenic animals indicated the reduction of anti-inflammatory effect of NE.ConclusionsOur data thus showed that tauopathy alters the functions of LC further leading to the reduction of NE levels and exaggeration of neuroinflammation in forebrain. These findings support the assumption that tau-related dysfunction of LC activates the vicious circle perpetuating neurodegeneration leading to the development of AD.

Updates in the pathophysiological mechanisms of Parkinson's disease: Emerging role of bone marrow mesenchymal stem cells.

To explore the approaches exerted by mesenchymal stem cells (MSCs) to improve Parkinson's disease (PD) pathophysiology. MSCs were harvested from bone marrow of femoral bones of male rats, grown and propagated in culture. Twenty four ovariectomized animals were classified into 3 groups: Group (1) was control, Groups (2) and (3) were subcutaneously administered with rotenone for 14 d after one month of ovariectomy for induction of PD. Then, Group (2) was left untreated, while Group (3) was treated with single intravenous dose of bone marrow derived MSCs (BM-MSCs). SRY gene was assessed by PCR in brain tissue of the female rats. Serum transforming growth factor beta-1 (TGF-β1), monocyte chemoattractant protein-1 (MCP-1) and brain derived neurotrophic factor (BDNF) levels were assayed by ELISA. Brain dopamine DA level was assayed fluorometrically, while brain tyrosine hydroxylase (TH) and nestin gene expression were detected by semi-quantitative real time PCR. Brain survivin expression was determined by immunohistochemical procedure. Histopathological investigation of brain tissues was also done. BM-MSCs were able to home at the injured brains and elicited significant decrease in serum TGF-β1 (489.7 ± 13.0 vs 691.2 ± 8.0, P < 0.05) and MCP-1 (89.6 ± 2.0 vs 112.1 ± 1.9, P < 0.05) levels associated with significant increase in serum BDNF (3663 ± 17.8 vs 2905 ± 72.9, P < 0.05) and brain DA (874 ± 15.0 vs 599 ± 9.8, P < 0.05) levels as well as brain TH (1.18 ± 0.004 vs 0.54 ± 0.009, P < 0.05) and nestin (1.29 ± 0.005 vs 0.67 ± 0.006, P < 0.05) genes expression levels. In addition to, producing insignificant increase in the number of positive cells for survivin (293.2 ± 15.9 vs 271.5 ± 15.9, P > 0.05) expression. Finally, the brain sections showed intact histological structure of the striatum as a result of treatment with BM-MSCs. The current study sheds light on the therapeutic potential of BM-MSCs against PD pathophysiology via multi-mechanistic actions.

Alcohol consumption induces global gene expression changes in VTA dopaminergic neurons

Alcoholism is associated with dysregulation in the neural circuitry that mediates motivated and goal-directed behaviors. The dopaminergic (DA) connection between the ventral tegmental area (VTA) and the nucleus accumbens is viewed as a critical component of the neurocircuitry mediating alcohol's rewarding and behavioral effects. We sought to determine the effects of binge alcohol drinking on global gene expression in VTA DA neurons. Alcohol-preferring C57BL/6J × FVB/NJ F1 hybrid female mice were exposed to a modified drinking in the dark (DID) procedure for 3 weeks, while control animals had access to water only. Global gene expression of laser-captured tyrosine hydroxylase (TH)-positive VTA DA neurons was measured using microarrays. A total of 644 transcripts were differentially expressed between the drinking and nondrinking mice, and 930 transcripts correlated with alcohol intake during the last 2 days of drinking in the alcohol group. Bioinformatics analysis of alcohol-responsive genes identified molecular pathways and networks perturbed in DA neurons by alcohol consumption, which included neuroimmune and epigenetic functions, alcohol metabolism and brain disorders. The majority of genes with high and specific expression in DA neurons were downregulated by or negatively correlated with alcohol consumption, suggesting a decreased activity of DA neurons in high drinking animals. These changes in the DA transcriptome provide a foundation for alcohol-induced neuroadaptations that may play a crucial role in the transition to addiction.

Dynamic Trk and G Protein Signalings Regulate Dopaminergic Neurodifferentiation in Human Trophoblast Stem Cells.

Understanding the mechanisms in the generation of neural stem cells from pluripotent stem cells is a fundamental step towards successful management of neurodegenerative diseases in translational medicine. Albeit all-trans retinoic acid (RA) has been associated with axon outgrowth and nerve regeneration, the maintenance of differentiated neurons, the association with degenerative disease like Parkinson's disease, and its regulatory molecular mechanism from pluripotent stem cells to neural stem cells remain fragmented. We have previously reported that RA is capable of differentiation of human trophoblast stem cells to dopamine (DA) committed progenitor cells. Intracranial implantation of such neural progenitor cells into the 6-OHDA-lesioned substantia nigra pars compacta successfully regenerates dopaminergic neurons and integrity of the nigrostriatal pathway, ameliorating the behavioral deficits in the Parkinson’s disease rat model. Here, we demonstrated a dynamic molecular network in systematic analysis by addressing spatiotemporal molecular expression, intracellular protein-protein interaction and inhibition, imaging study, and genetic expression to explore the regulatory mechanisms of RA induction in the differentiation of human trophoblast stem cells to DA committed progenitor cells. We focused on the tyrosine receptor kinase (Trk), G proteins, canonical Wnt2B/β-catenin, genomic and non-genomic RA signaling transductions with Tyrosine hydroxylase (TH) gene expression as the differentiation endpoint. We found that at the early stage, integration of TrkA and G protein signalings aims for axonogenesis and morphogenesis, involving the novel RXRα/Gαq/11 and RARβ/Gβ signaling pathways. While at the later stage, five distinct signaling pathways together with epigenetic histone modifications emerged to regulate expression of TH, a precursor of dopamine. RA induction generated DA committed progenitor cells in one day. Our results provided substantial mechanistic evidence that human trophoblast stem cell-derived neural stem cells can potentially be used for neurobiological study, drug discovery, and as an alternative source of cell-based therapy in neurodegenerative diseases like Parkinson’s disease.

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.

Impact of DOCA‐Salt Hypertension on Gene Expression in the Dorsal Pons: a Microarray Analysis

We had previously reported that induction of DOCA/salt hypertension altered the cardiovascular response to stress and this was associated with altered gene expression in the amygdala. Since a major source of afferent input to the amygdala originates from the dorsal pons, the present study was undertaken to evaluate how DOCA/salt hypertension alters gene expression in this brain region. Differential gene expression was measured by high‐density cDNA microarray analysis (Affymetrix Rat Gene Chip) and validated with real‐time PCR in DOCA‐salt hypertensive versus normotensive controls (n=4/group). Microanalysis identified 934 genes that were up‐regulated, including genes related to catecholamine synthesis and transport, inflammation, taste and smell. Solute carrier family 6 (dopamine neurotransmitter transporter) and tyrosine hydroxylase (TH) were the two most highly upregulated genes in DOCA/salt vs normotensive controls. Real‐time PCR analysis confirmed a 5.3 fold increase in dopamine transporter and a 10 fold increase in TH gene expression in the dorsal pons of the DOCA/salt treated animals vs controls. There was also evidence of up‐regulation of GFAP and IL‐10 and down regulation of glutamate receptors. Since changes in catecholamine release in both the amygdala and parabrachial nucleus can alter salt intake and catecholamine receptor activation in the lateral hypothalamus has been linked to salt intake gratification, our results raise the new possibility that alterations in catecholamine synthesis in the dorsal pons plays a significant role in DOCA driven salt intake.

Antidepressant-like effects of rosmarinic acid through mitogen-activated protein kinase phosphatase-1 and brain-derived neurotrophic factor modulation

Rosmarinic acid (RA) is one of the major bioactive compounds of Rosmarinus officinalis, a culinary and edible aromatic plant and was demonstrated to have several neuroprotective properties including anti-depressive-like effect. The current study was designed to contribute to understanding the molecular mechanism of RA beneficial effects in tail suspension test (TST)-induced depression in mice with bupropion as positive control, and mice without TST as negative control. Changes in serum corticosterone (CORT) level and cerebrum level of catecholamines: dopamine (DOP), noradrenaline (NAD), and adrenaline (ADR) were investigated. Moreover, brain-derived neurotrophic factor (Bdnf), mitogen-activated protein kinase phosphatase-1 (Mkp-1), tyrosine hydroxylase (Th) and pyruvate carboxylase (Pc) gene expression in mice cerebrum were investigated using real-time PCR. RA was demonstrated to show anti-depressant-like effect by significant reduction of immobility time in the TST in mice. This effect was accompanied by a downregulation of Mkp-1 to reach the unstressed group level, an upregulation of Bdnf, Th and Pc in the limbic system. Furthermore, RA significantly decreased serum corticosterone level and increased dopamine level in the limbic system in mice brain. Our study provides the first evidence that RA has anti-depressant activity via downregulation of Mkp-1, upregulation of Bdnf and modulation of dopamine and corticosterone synthesis.

Maternal vitamin D deficiency alters fetal brain development in the BALB/c mouse

Prenatal exposure to vitamin D is thought to be critical for optimal fetal neurodevelopment, yet vitamin D deficiency is apparent in a growing proportion of pregnant women. The aim of this study was to determine whether a mouse model of vitamin D-deficiency alters fetal neurodevelopment. Female BALB/c mice were placed on either a vitamin D control (2195IU/kg) or deficient (0IU/kg) diet for 5 weeks prior to and during pregnancy. Fetal brains were collected at embryonic day (E) 14.5 or E17.5 for morphological and gene expression analysis. Vitamin D deficiency during pregnancy reduced fetal crown–rump length and head size. Moreover, lateral ventricle volume was reduced in vitamin D-deficient foetuses. Expression of neurotrophin genes brain-derived neurotrophic factor (Bdnf) and transforming growth factor-β1 (Tgf-β1) was altered, with Bdnf reduced at E14.5 and increased at E17.5 following vitamin D deficiency. Brain expression of forkhead box protein P2 (Foxp2), a gene known to be important in human speech and language, was also altered. Importantly, Foxp2 immunoreactive cells in the developing cortex were reduced in vitamin D-deficient female foetuses. At E17.5, brain tyrosine hydroxylase (TH) gene expression was reduced in females, as was TH protein localization (to identify dopamine neurons) in the substantia nigra of vitamin D-deficient female foetuses. Overall, we show that prenatal vitamin D-deficiency leads to alterations in fetal mouse brain morphology and genes related to neuronal survival, speech and language development, and dopamine synthesis. Vitamin D appears to play an important role in mouse neurodevelopment.

Posttranscriptional regulation of adrenal TH gene expression contributes to the maladaptive responses triggered by insulin-induced recurrent hypoglycemia.

Acute metabolic stress such as insulin-induced hypoglycemia triggers a counterregulatory response during which the release of catecholamines (epinephrine), the activation of tyrosine hydroxylase (TH) enzyme and subsequent compensatory catecholamine biosynthesis occur in the adrenal medulla. However, recurrent exposure to hypoglycemia (RH), a consequence of tight glycemic control in individuals with type 1 and type 2 diabetes compromises this physiological response. The molecular mechanisms underlying the maladaptive response to repeated glucose deprivation are incompletely understood. We hypothesize that impaired epinephrine release following RH reflects altered regulation of adrenal catecholamine biosynthesis. To test this hypothesis, we compared the effect of single daily (RH) and twice-daily episodes of insulin-induced hypoglycemia (2RH) on adrenal epinephrine release and production in normal rats. Control animals received saline injections under similar conditions (RS and 2RS, respectively). Following 3 days of treatment, we assessed the counterregulatory hormonal responses during a hypoglycemic clamp. Changes in adrenal TH gene expression were also analyzed. The counterregulatory responses, relative TH transcription and TH mRNA levels and Ser40-TH phosphorylation (marker for enzyme activation) were induced to a similar extent in RS, 2RS, and RH groups. In contrast, epinephrine and glucagon responses were attenuated in the 2RH group and this was associated with a limited elevation of adrenal TH mRNA, rapid inactivation of TH enzyme and no significant changes in TH protein. Our results suggest that novel posttranscriptional mechanisms controlling TH mRNA and activated TH enzyme turnover contribute to the impaired epinephrine responses and may provide new therapeutic targets to prevent HAAF.

No Detection of Potential Cancer Risk for Free-Viral Reprogrammed Stem Cell-Derived Dopaminergic Neurons from Adult Mice Fibroblasts

Objective: Stem cell replacement therapy through the reprogramming of somatic cells is a significant prospective therapy for neurodegenerative diseases and personalized medicine. As virus-carrying reprogramming genes impart a considerable risk for tumor formation, current approaches tend to replace the viral pattern with a non-viral system. However, there is still great concern regarding the oncogenic properties of these reprogramming genes. At present no direct evidence can verify that non-viral systems do not influence genomic DNA integration and mutagenesis events in the final products after reprogramming. Methods: We evaluated the potential cancer risk of reprogrammed stem cell-derived dopaminergic neurons using a unique non-viral vector containing four reprogramming genes. Reprogrammed stem cell-derived dopaminergic neurons were the final products after 50 days of cell culture from adult mice fibroblasts. After 6 months of culture, these cells were assessed for cancer risk. Results: Overall cancer risk assessments were first examined using multiple stem cell biomarkers. We did not detect any overexpression of these biomarkers. Moreover, we assessed whether or not the most important neurotransmission factors were expressed after 6 months in mass cell culture. Our results determined that only robust gene expression of tyrosine hydroxylase (TH), as the rate-limiting enzyme in dopamine synthesis, was detected. Furthermore, the final product of TH-positive dopaminergic neurons was confirmed by sequencing TH genomic DNA. Our data suggested that none of the four reprogramming genes integrated into the host cell’s genomic DNA and that no other mutagenesis events occurred after gene sequencing TH genomic DNA related complete 13 coding exons with two-ended partial untranslated regions. Conclusion: Re-programmed adult mouse fibroblasts-derived dopaminergic neurons were proven to be a safe technology and approach as a potential therapy for neurodegenerative diseases.

Enhancement of catecholamine release from PC12 cells by the traditional Japanese medicine, rikkunshito.

BackgroundRikkunshito is a traditional Japanese herbal medicine that is used to treat appetite loss associated with cancer and other disorders. The formulation contains various constituents that influence cell signaling, and rikkunshito may accordingly affect human homeostasis through multiple regulatory pathways, including those governed by the endocrine system. We investigated the actions of rikkunshito on catecholamine release from PC12 cells, an adrenal chromaffin cell line.MethodsThe actions of rikkunshito on PC12 cells were evaluated by measuring intracellular cAMP levels, tyrosine hydroxylase (TH) and vasoactive intestinal peptide (VIP) mRNA expression levels, and catecholamine levels in the culture medium. The transcriptional activation of VIP gene by rikkunshito was assessed by using a VIP promoter-driven reporter gene assay.ResultsRikkunshito dose-dependently enhanced forskolin-induced elevations in cAMP in PC12 cells, and also increased the gene expression of TH and VIP. The transcriptional activation of VIP gene by rikkunshito was confirmed. Norepinephrine and dopamine secretion into the culture medium of PC12 cells were also dose-dependently augmented by rikkunshito and/or forskolin, but experiments with a protein kinase C (PKC) activator and a phosphodiesterase inhibitor revealed that the effects of rikkunshito were not simply due to the modulation of PKC or phosphodiesterase activity.ConclusionsThese findings suggest that rikkunshito enhances the release of catecholamines by a novel mechanism involving cAMP.

Long term exendin-4 treatment reduces food intake and body weight and alters expression of brain homeostatic and reward markers.

Repeated administration of the long-acting glucagon-like peptide 1 receptor agonist exendin-4 (EX-4) has been shown to reduce food intake and body weight and do so without a rebound increase in food intake after treatment termination. The current study examines the neural mechanisms underlying these actions. After 6 weeks of maintenance on a standard chow or a high-fat (HF) diet, male Sprague Dawley rats were treated with EX-4 (3.2 μg/kg, i.p., twice a day) or vehicle for 9 consecutive days. Food intake and body weight (BW) were monitored daily. Expression of the genes for the hypothalamic arcuate nucleus (ARC) peptides proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti gene-related protein was determined. Expression of the dopamine precursor tyrosine hydroxylase (TH) gene in the ventral tegmental area and genes for dopamine receptors 1 (D1R) and dopamine receptor 2 in the nucleus accumbens were also determined. Pair-fed groups were included to control for the effects of reduced food intake and BW. Treatment with EX-4 significantly decreased food intake and BW over the 9-day period in both the standard chow and HF groups. HF feeding decreased POMC without changing NPY/agouti gene-related protein gene expression in the ARC. Treatment with EX-4 increased POMC and decreased NPY expression independent of the reduction of food intake and BW. Mesolimbic TH and D1R gene expression were decreased significantly in chronic HF diet-fed rats, and these changes were reversed in both EX-4 and pair-fed conditions. These results suggest a role for increased POMC and decreased NPY expression in the ARC in the effects of EX-4 on food intake and BW. Our findings also suggest that EX-4 induced the recovery of mesolimbic TH and D1R expression in HF diet-fed rats may be secondary to HF intake reduction and/or weight loss.

Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells

Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.