Neural crest origin of sympathetic neurons at the dawn of vertebrates.

The neural crest is an embryonic stem cell population unique to vertebrates1 whose expansion and diversification are thought to have promoted vertebrate evolution by enabling emergence of new cell types and structures such as jaws and peripheral ganglia2. Although jawless vertebrates have sensory ganglia, convention has it that trunk sympathetic chain ganglia arose only in jawed vertebrates3-8. Here, by contrast, we report the presence of trunk sympathetic neurons in the sea lamprey, Petromyzon marinus, an extant jawless vertebrate. These neurons arise from sympathoblasts near the dorsal aorta that undergo noradrenergic specification through a transcriptional program homologous to that described in gnathostomes. Lamprey sympathoblasts populate the extracardiac space and extend along the length of the trunk in bilateral streams, expressing the catecholamine biosynthetic pathway enzymes tyrosine hydroxylase and dopamine ß-hydroxylase. CM-DiI lineage tracing analysis further confirmed that these cells derive from the trunk neural crest. RNA sequencing of isolated ammocoete trunk sympathoblasts revealed gene profiles characteristic of sympathetic neuron function. Our findings challenge the prevailing dogma that posits that sympathetic ganglia are a gnathostome innovation, instead suggesting that a late-developing rudimentary sympathetic nervous system may have been characteristic of the earliest vertebrates.

Brainstem Dbh+ neurons control allergen-induced airway hyperreactivity.

Exaggerated airway constriction triggered by repeated exposure to allergen, also called hyperreactivity, is a hallmark of asthma. Whereas vagal sensory neurons are known to function in allergen-induced hyperreactivity1-3, the identity of downstream nodes remains poorly understood. Here we mapped a full allergen circuit from the lung to the brainstem and back to the lung. Repeated exposure of mice to inhaled allergen activated the nuclei of solitary tract (nTS) neurons in a mast cell-, interleukin-4 (IL-4)- and vagal nerve-dependent manner. Single-nucleus RNA sequencing, followed by RNAscope assay at baseline and allergen challenges, showed that a Dbh+ nTS population is preferentially activated. Ablation or chemogenetic inactivation of Dbh+ nTS neurons blunted hyperreactivity whereas chemogenetic activation promoted it. Viral tracing indicated that Dbh+ nTS neurons project to the nucleus ambiguus (NA) and that NA neurons are necessary and sufficient to relay allergen signals to postganglionic neurons that directly drive airway constriction. Delivery of noradrenaline antagonists to the NA blunted hyperreactivity, suggesting noradrenaline as the transmitter between Dbh+ nTS and NA. Together, these findings provide molecular, anatomical and functional definitions of key nodes of a canonical allergen response circuit. This knowledge informs how neural modulation could be used to control allergen-induced airway hyperreactivity.

Norepinephrine Neurons in the Nucleus of the Solitary Tract Suppress Luteinizing Hormone Secretion in Female Mice.

Stress impairs fertility, at least in part, via inhibition of gonadotropin secretion. Luteinizing hormone (LH) is an important gonadotropin that is released in a pulsatile pattern in males and in females throughout the majority of the ovarian cycle. Several models of stress, including acute metabolic stress, suppress LH pulses via inhibition of neurons in the arcuate nucleus of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (termed KNDy cells) which form the pulse generator. The mechanism for inhibition of KNDy neurons during stress, however, remains a significant outstanding question. Here, we investigated a population of catecholamine neurons in the nucleus of the solitary tract (NTS), marked by expression of the enzyme dopamine beta-hydroxylase (DBH), in female mice. First, we found that a subpopulation of DBH neurons in the NTS is activated (express c-Fos) during metabolic stress. Then, using chemogenetics, we determined that activation of these cells is sufficient to suppress LH pulses, augment corticosterone secretion, and induce sickness-like behavior. In subsequent studies, we identified evidence for suppression of KNDy cells (rather than downstream signaling pathways) and determined that the suppression of LH pulses was not dependent on the acute rise in glucocorticoids. Together these data support the hypothesis that DBH cells in the NTS are important for regulation of neuroendocrine and behavioral responses to stress.

Norepinephrine Drives Sleep Fragmentation Activation of Asparagine Endopeptidase, Locus Ceruleus Degeneration, and Hippocampal Amyloid-ß42 Accumulation.

Chronic sleep disruption (CSD), from insufficient or fragmented sleep and is an important risk factor for Alzheimer's disease (AD). Underlying mechanisms are not understood. CSD in mice results in degeneration of locus ceruleus neurons (LCn) and CA1 hippocampal neurons and increases hippocampal amyloid-ß42 (Aß42), entorhinal cortex (EC) tau phosphorylation (p-tau), and glial reactivity. LCn injury is increasingly implicated in AD pathogenesis. CSD increases NE turnover in LCn, and LCn norepinephrine (NE) metabolism activates asparagine endopeptidase (AEP), an enzyme known to cleave amyloid precursor protein (APP) and tau into neurotoxic fragments. We hypothesized that CSD would activate LCn AEP in an NE-dependent manner to induce LCn and hippocampal injury. Here, we studied LCn, hippocampal, and EC responses to CSD in mice deficient in NE [dopamine ß-hydroxylase (Dbh)-/-] and control male and female mice, using a model of chronic fragmentation of sleep (CFS). Sleep was equally fragmented in Dbh -/- and control male and female mice, yet only Dbh -/- mice conferred resistance to CFS loss of LCn, LCn p-tau, and LCn AEP upregulation and activation as evidenced by an increase in AEP-cleaved APP and tau fragments. Absence of NE also prevented a CFS increase in hippocampal AEP-APP and Aß42 but did not prevent CFS-increased AEP-tau and p-tau in the EC. Collectively, this work demonstrates AEP activation by CFS, establishes key roles for NE in both CFS degeneration of LCn neurons and CFS promotion of forebrain Aß accumulation, and, thereby, identifies a key molecular link between CSD and specific AD neural injuries.

Dopamine ß-hydroxylase shapes intestinal inflammation through modulating T cell activation.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic and relapsing disease characterized by immune-mediated dysfunction of intestinal homeostasis. Alteration of the enteric nervous system and the subsequent neuro-immune interaction are thought to contribute to the initiation and progression of IBD. However, the role of dopamine beta-hydroxylase (DBH), an enzyme converting dopamine into norepinephrine, in modulating intestinal inflammation is not well defined. METHODS: CD4+CD45RBhighT cell adoptive transfer, and 2,4-dinitrobenzene sulfonic acid (DNBS) or dextran sodium sulfate (DSS)-induced colitis were collectively conducted to uncover the effects of DBH inhibition by nepicastat, a DBH inhibitor, in mucosal ulceration, disease severity, and T cell function. RESULTS: Inhibition of DBH by nepicastat triggered therapeutic effects on T cell adoptive transfer induced chronic mouse colitis model, which was consistent with the gene expression of DBH in multiple cell populations including T cells. Furthermore, DBH inhibition dramatically ameliorated the disease activity and colon shortening in chemically induced acute and chronic IBD models, as evidenced by morphological and histological examinations. The reshaped systemic inflammatory status was largely associated with decreased pro-inflammatory mediators, such as TNF-α, IL-6 and IFN-γ in plasma and re-balanced Th1, Th17 and Tregs in mesenteric lymph nodes (MLNs) upon colitis progression. Additionally, the conversion from dopamine (DA) to norepinephrine (NE) was inhibited resulting in increase in DA level and decrease in NE level and DA/NE showed immune-modulatory effects on the activation of immune cells. CONCLUSION: Modulation of neurotransmitter levels via inhibition of DBH exerted protective effects on progression of murine colitis by modulating the neuro-immune axis. These findings suggested a promising new therapeutic strategy for attenuating intestinal inflammation.

Changes in Noradrenergic Synthesis and Dopamine Beta-Hydroxylase Activity in Response to Oxidative Stress after Iron-induced Brain Injury.

Noradrenaline (NA) levels are altered during the first hours and several days after cortical injury. NA modulates motor functional recovery. The present study investigated whether iron-induced cortical injury modulated noradrenergic synthesis and dopamine beta-hydroxylase (DBH) activity in response to oxidative stress in the brain cortex, pons and cerebellum of the rat. Seventy-eight rats were divided into two groups: (a) the sham group, which received an intracortical injection of a vehicle solution; and (b) the injured group, which received an intracortical injection of ferrous chloride. Motor deficits were evaluated for 20 days post-injury. On the 3rd and 20th days, the rats were euthanized to measure oxidative stress indicators (reactive oxygen species (ROS), reduced glutathione (GSH) and oxidized glutathione (GSSG)) and catecholamines (NA, dopamine (DA)), plus DBH mRNA and protein levels. Our results showed that iron-induced brain cortex injury increased noradrenergic synthesis and DBH activity in the brain cortex, pons and cerebellum at 3 days post-injury, predominantly on the ipsilateral side to the injury, in response to oxidative stress. A compensatory increase in contralateral noradrenergic activity was observed, but without changes in the DBH mRNA and protein levels in the cerebellum and pons. In conclusion, iron-induced cortical injury increased the noradrenergic response in the brain cortex, pons and cerebellum, particularly on the ipsilateral side, accompanied by a compensatory response on the contralateral side. The oxidative stress was countered by antioxidant activity, which favored functional recovery following motor deficits.

Norepinephrine innervation of the supraoptic nucleus contributes to increased copeptin and dilutional hyponatremia in male rats.

Dilutional hyponatremia associated with liver cirrhosis is due to inappropriate release of arginine vasopressin (AVP). Elevated plasma AVP causes water retention resulting in a decrease in plasma osmolality. Cirrhosis, in this study caused by ligation of the common bile duct (BDL), leads to a decrease in central vascular blood volume and hypotension, stimuli for nonosmotic AVP release. The A1/A2 neurons stimulate the release of AVP from the supraoptic nucleus (SON) in response to nonosmotic stimuli. We hypothesize that the A1/A2 noradrenergic neurons support chronic release of AVP in cirrhosis leading to dilutional hyponatremia. Adult, male rats were anesthetized with 2-3% isoflurane (mixed with 95% O2/5% CO2) and injected in the SON with anti-dopamine ß-hydroxylase (DBH) saporin (DSAP) or vehicle followed by either BDL or sham surgery. Plasma copeptin, osmolality, and hematocrit were measured. Brains were processed for ΔFosB, dopamine ß-hydroxylase (DBH), and AVP immunohistochemistry. DSAP injection: 1) significantly reduced the number of DBH immunoreactive A1/A2 neurons (A1, P < 0.0001; A2, P = 0.0014), 2) significantly reduced the number of A1/A2 neurons immunoreactive to both DBH and ΔFosB positive neurons (A1, P = 0.0015; A2, P < 0.0001), 3) reduced the number of SON neurons immunoreactive to both AVP and ΔFosB (P < 0.0001), 4) prevented the increase in plasma copeptin observed in vehicle-injected BDL rats (P = 0.0011), and 5) normalized plasma osmolality and hematocrit (plasma osmolality, P = 0.0475; hematocrit, P = 0.0051) as compared with vehicle injection. Our data suggest that A1/A2 neurons contribute to increased plasma copeptin and hypoosmolality in male BDL rats.

A5 noradrenergic-projecting C1 neurons activate sympathetic and breathing outputs in anaesthetized rats.

NEW FINDINGS: What is the central question of this study? C1 neurons innervate pontine noradrenergic cell groups, including the A5 region: do A5 noradrenergic neurons contribute to the activation of sympathetic and respiratory responses produced by selective activation of the C1 group of neurons. What is the main finding and its importance? The increase in sympathetic and respiratory activities elicited by selective stimulation of C1 neurons is reduced after blockade of excitatory amino acid within the A5 region, suggesting that the C1-A5 pathway might be important for sympathetic-respiratory control. ABSTRACT: Adrenergic C1 neurons innervate and excite pontine noradrenergic cell groups, including the ventrolateral pontine noradrenergic region (A5). Here, we tested the hypothesis that C1 activates A5 neurons through the release of glutamate and this effect is important for sympathetic and respiratory control. Using selective tools, we restricted the expression of channelrhodopsin2 under the control of the artificial promoter PRSx8 to C1 neurons (69%). Transduced catecholaminergic terminals within the A5 region are in contact with noradrenergic A5 neurons and the C1 terminals within the A5 region are predominantly glutamatergic. In a different group of animals, we performed retrograde lesion of C1 adrenergic neurons projecting to the A5 region with unilateral injection of the immunotoxin anti-dopamine ß-hydroxylase-saporin (anti-DßH-SAP) directly into the A5 region during the hypoxic condition. As expected, hypoxia (8% O2 , 3 h) induced a robust increase in fos expression within the catecholaminergic C1 and A5 regions of the brainstem. Depletion of C1 cells projecting to the A5 regions reduced fos immunoreactivity induced by hypoxia within the C1 region. Physiological experiments showed that bilateral injection of kynurenic acid (100 mM) into the A5 region reduced the rise in mean arterial pressure, and sympathetic and phrenic nerve activities produced by optogenetic stimulation of C1 cells. In conclusion, the C1 neurons activate the ventrolateral pontine noradrenergic neurons (A5 region) possibly via the release of glutamate and might be important for sympathetic and respiratory outputs in anaesthetized rats.

Norepinephrine and dopamine contribute to distinct repetitive behaviors induced by novel odorant stress in male and female mice.

Exposure to unfamiliar odorants induces an array of repetitive defensive and non-defensive behaviors in rodents which likely reflect adaptive stress responses to the uncertain valence of novel stimuli. Mice genetically deficient for dopamine ß-hydroxylase (Dbh-/-) lack the enzyme required to convert dopamine (DA) into norepinephrine (NE), resulting in globally undetectable NE and supranormal DA levels. Because catecholamines modulate novelty detection and reactivity, we investigated the effects of novel plant-derived odorants on repetitive behaviors in Dbh-/- mice and Dbh+/- littermate controls, which have catecholamine levels comparable to wild-type mice. Unlike Dbh+/- controls, which exhibited vigorous digging in response to novel odorants, Dbh-/- mice displayed excessive grooming. Drugs that block NE synthesis or neurotransmission suppressed odorant-induced digging in Dbh+/- mice, while a DA receptor antagonist attenuated grooming in Dbh-/- mice. The testing paradigm elicited high circulating levels of corticosterone regardless of Dbh genotype, indicating that NE is dispensable for this systemic stress response. Odorant exposure increased NE and DA abundance in the prefrontal cortex (PFC) of Dbh+/- mice, while Dbh-/- animals lacked NE and had elevated PFC DA levels that were unaffected by novel smells. Together, these findings suggest that novel odorant-induced increases in central NE tone contribute to repetitive digging and reflect psychological stress, while central DA signaling contributes to repetitive grooming. Further, we have established a simple method for repeated assessment of stress-induced repetitive behaviors in mice, which may be relevant for modeling neuropsychiatric disorders like Tourette syndrome or obsessive-compulsive disorder that are characterized by stress-induced exacerbation of compulsive symptoms.

Distribution of the Noradrenaline Innervation and Adrenoceptors in the Macaque Monkey Thalamus.

Noradrenaline (NA) in the thalamus has important roles in physiological, pharmacological, and pathological neuromodulation. In this work, a complete characterization of NA axons and Alpha adrenoceptors distributions is provided. NA axons, revealed by immunohistochemistry against the synthesizing enzyme and the NA transporter, are present in all thalamic nuclei. The most densely innervated ones are the midline nuclei, intralaminar nuclei (paracentral and parafascicular), and the medial sector of the mediodorsal nucleus (MDm). The ventral motor nuclei and most somatosensory relay nuclei receive a moderate NA innervation. The pulvinar complex receives a heterogeneous innervation. The lateral geniculate nucleus (GL) has the lowest NA innervation. Alpha adrenoceptors were analyzed by in vitro quantitative autoradiography. Alpha-1 receptor densities are higher than Alpha-2 densities. Overall, axonal densities and Alpha adrenoceptor densities coincide; although some mismatches were identified. The nuclei with the highest Alpha-1 values are MDm, the parvocellular part of the ventral posterior medial nucleus, medial pulvinar, and midline nuclei. The nucleus with the lowest Alpha-1 receptor density is GL. Alpha-2 receptor densities are highest in the lateral dorsal, centromedian, medial and inferior pulvinar, and midline nuclei. These results suggest a role for NA in modulating thalamic involvement in consciousness, limbic, cognitive, and executive functions.

Intermittent Hypoxia Increased the Expression of DBH and PNMT in Neuroblastoma Cells via MicroRNA-375-Mediated Mechanism.

Sleep apnea syndrome (SAS), characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia (IH)), is a risk factor for hypertension and insulin resistance. We report a correlation between IH and insulin resistance/diabetes. However, the reason why hypertension is induced by IH is elusive. Here, we investigated the effect of IH on the expression of catecholamine-metabolizing enzymes using an in vitro IH system. Human and mouse neuroblastoma cells (NB-1 and Neuro-2a) were exposed to IH or normoxia for 24 h. Real-time RT-PCR revealed that IH significantly increased the mRNA levels of dopamine ß-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in both NB-1 and Neuro-2a. Western blot showed that the expression of DBH and PNMT in the NB-1 cells was significantly increased by IH. Reporter assays revealed that promoter activities of DBH and PNMT were not increased by IH. The miR-375 level of IH-treated cells was significantly decreased relative to that of normoxia-treated cells. The IH-induced up-regulation of DBH and PNMT was abolished by the introduction of the miR-375 mimic, but not by the control RNA. These results indicate that IH stress increases levels of DBH and PNMT via the inhibition of miR-375-mediated mRNA degradation, potentially playing a role in the emergence of hypertension in SAS patients.

Cannabinoid CB1 Receptor Deletion from Catecholaminergic Neurons Protects from Diet-Induced Obesity.

High-calorie diets and chronic stress are major contributors to the development of obesity and metabolic disorders. These two risk factors regulate the activity of the sympathetic nervous system (SNS). The present study showed a key role of the cannabinoid type 1 receptor (CB1) in dopamine ß-hydroxylase (dbh)-expressing cells in the regulation of SNS activity. In a diet-induced obesity model, CB1 deletion from these cells protected mice from diet-induced weight gain by increasing sympathetic drive, resulting in reduced adipogenesis in white adipose tissue and enhanced thermogenesis in brown adipose tissue. The deletion of CB1 from catecholaminergic neurons increased the plasma norepinephrine levels, norepinephrine turnover, and sympathetic activity in the visceral fat, which coincided with lowered neuropeptide Y (NPY) levels in the visceral fat of the mutant mice compared with the controls. Furthermore, the mutant mice showed decreased plasma corticosterone levels. Our study provided new insight into the mechanisms underlying the roles of the endocannabinoid system in regulating energy balance, where the CB1 deletion in dbh-positive cells protected from diet-induced weight gain via multiple mechanisms, such as increased SNS activity, reduced NPY activity, and decreased basal hypothalamic-pituitary-adrenal (HPA) axis activity.

Pain Modulation from the Locus Coeruleus in a Model of Hydrocephalus: Searching for Oxidative Stress-Induced Noradrenergic Neuroprotection.

Pain transmission at the spinal cord is modulated by noradrenaline (NA)-mediated actions that arise from supraspinal areas. We studied the locus coeruleus (LC) to evaluate the expression of the cathecolamine-synthetizing enzyme tyrosine hydroxylase (TH) and search for local oxidative stress and possible consequences in descending pain modulation in a model of hydrocephalus, a disease characterized by enlargement of the cerebral ventricular system usually due to the obstruction of cerebrospinal fluid flow. Four weeks after kaolin injection into the cisterna magna, immunodetection of the catecholamine-synthetizing enzymes TH and dopamine-ß-hydroxylase (DBH) was performed in the LC and spinal cord. Colocalization of the oxidative stress marker 8-OHdG (8-hydroxyguanosine; 8-OHdG), with TH in the LC was performed. Formalin was injected in the hindpaw both for behavioral nociceptive evaluation and the immunodetection of Fos expression in the spinal cord. Hydrocephalic rats presented with a higher expression of TH at the LC, of TH and DBH at the spinal dorsal horn along with decreased nociceptive behavioral responses in the second (inflammatory) phase of the formalin test, and formalin-evoked Fos expression at the spinal dorsal horn. The expression of 8-OHdG was increased in the LC neurons, with higher co-localization in TH-immunoreactive neurons. Collectively, the results indicate increased noradrenergic expression at the LC during hydrocephalus. The strong oxidative stress damage at the LC neurons may lead to local neuroprotective-mediated increases in NA levels. The increased expression of catecholamine-synthetizing enzymes along with the decreased nociception-induced neuronal activation of dorsal horn neurons and behavioral pain signs may indicate that hydrocephalus is associated with alterations in descending pain modulation.

Tyrosine Hydroxylase Neurons Regulate Growth Hormone Secretion via Short-Loop Negative Feedback.

Classical studies suggest that growth hormone (GH) secretion is controlled by negative-feedback loops mediated by GH-releasing hormone (GHRH)- or somatostatin-expressing neurons. Catecholamines are known to alter GH secretion and neurons expressing TH are located in several brain areas containing GH-responsive cells. However, whether TH-expressing neurons are required to regulate GH secretion via negative-feedback mechanisms is unknown. In the present study, we showed that between 50% and 90% of TH-expressing neurons in the periventricular, paraventricular, and arcuate hypothalamic nuclei and locus ceruleus of mice exhibited STAT5 phosphorylation (pSTAT5) after an acute GH injection. Ablation of GH receptor (GHR) from TH cells or in the entire brain markedly increased GH pulse secretion and body growth in both male and female mice. In contrast, GHR ablation in cells that express the dopamine transporter (DAT) or dopamine ß-hydroxylase (DBH; marker of noradrenergic/adrenergic cells) did not affect body growth. Nevertheless, less than 50% of TH-expressing neurons in the hypothalamus were found to express DAT. Ablation of GHR in TH cells increased the hypothalamic expression of Ghrh mRNA, although very few GHRH neurons were found to coexpress TH- and GH-induced pSTAT5. In summary, TH neurons that do not express DAT or DBH are required for the autoregulation of GH secretion via a negative-feedback loop. Our findings revealed a critical and previously unidentified group of catecholaminergic interneurons that are apt to sense changes in GH levels and regulate the somatotropic axis in mice.SIGNIFICANCE STATEMENT Textbooks indicate until now that the pulsatile pattern of growth hormone (GH) secretion is primarily controlled by GH-releasing hormone and somatostatin neurons. The regulation of GH secretion relies on the ability of these cells to sense changes in circulating GH levels to adjust pituitary GH secretion within a narrow physiological range. However, our study identifies a specific population of tyrosine hydroxylase-expressing neurons that is critical to autoregulate GH secretion via a negative-feedback loop. The lack of this mechanism in transgenic mice results in aberrant GH secretion and body growth. Since GH plays a key role in cell proliferation, body growth, and metabolism, our findings provide a major advance to understand how the brain regulates the somatotropic axis.

Sympathetic innervation of the mouse kidney and liver arising from prevertebral ganglia.

The sympathetic nervous system (SNS) plays a crucial role in the regulation of renal and hepatic functions. Although sympathetic nerves to the kidney and liver have been identified in many species, specific details are lacking in the mouse. In the absence of detailed information of sympathetic prevertebral innervation of specific organs, selective manipulation of a specific function will remain challenging. Despite providing major postganglionic inputs to abdominal organs, limited data are available about the mouse celiac-superior mesenteric complex. We used tyrosine hydroxylase (TH) and dopamine ß-hydroxylase (DbH) reporter mice to visualize abdominal prevertebral ganglia. We found that both the TH and DbH reporter mice are useful models for identification of ganglia and nerve bundles. We further tested if the celiac-superior mesenteric complex provides differential inputs to the mouse kidney and liver. The retrograde viral tracer, pseudorabies virus (PRV)-152 was injected into the cortex of the left kidney or the main lobe of the liver to identify kidney-projecting and liver-projecting neurons in the celiac-superior mesenteric complex. iDISCO immunostaining and tissue clearing were used to visualize unprecedented anatomical detail of kidney-related and liver-related postganglionic neurons in the celiac-superior mesenteric complex and aorticorenal and suprarenal ganglia compared with TH-positive neurons. Kidney-projecting neurons were restricted to the suprarenal and aorticorenal ganglia, whereas only sparse labeling was observed in the celiac-superior mesenteric complex. In contrast, liver-projecting postganglionic neurons were observed in the celiac-superior mesenteric complex and aorticorenal and suprarenal ganglia, suggesting spatial separation between the sympathetic innervation of the mouse kidney and liver.

Clioquinol inhibits dopamine-ß-hydroxylase secretion and noradrenaline synthesis by affecting the redox status of ATOX1 and copper transport in human neuroblastoma SH-SY5Y cells.

Clioquinol (5-chloro-7-indo-8-quinolinol), a chelator and ionophore of copper/zinc, was extensively used as an amebicide to treat indigestion and diarrhea in the mid-1900s. However, it was withdrawn from the market in Japan because its use was epidemiologically linked to an increase in the incidence of subacute myelo-optic neuropathy (SMON). SMON is characterized by the subacute onset of sensory and motor disturbances in the lower extremities with occasional visual impairments, which are preceded by abdominal symptoms. Although pathological studies demonstrated axonopathy of the spinal cord and optic nerves, the underlying mechanisms of clioquinol toxicity have not been elucidated in detail. In the present study, a reporter assay revealed that clioquinol (20-50 µM) activated metal response element-dependent transcription in human neuroblastoma SH-SY5Y cells. Clioquinol significantly increased the cellular level of zinc within 1 h, suggesting zinc influx due to its ionophore effects. On the other hand, clioquinol (20-50 µM) significantly increased the cellular level of copper within 24 h. Clioquinol (50 µM) induced the oxidation of the copper chaperone antioxidant 1 (ATOX1), suggesting its inactivation and inhibition of copper transport. The secretion of dopamine-ß-hydroxylase (DBH) and lysyl oxidase, both of which are copper-dependent enzymes, was altered by clioquinol (20-50 µM). Noradrenaline levels were reduced by clioquinol (20-50 µM). Disruption of the ATOX1 gene suppressed the secretion of DBH. This study suggested that the disturbance of cellular copper transport by the inactivation of ATOX1 is one of the mechanisms involved in clioquinol-induced neurotoxicity in SMON.

Changes in the Neurochemical Coding of the Anterior Pelvic Ganglion Neurons Supplying the Male Pig Urinary Bladder Trigone after One-Sided Axotomy of Their Nerve Fibers.

The present study investigated the effect of unilateral axotomy of urinary bladder trigone (UBT)-projecting nerve fibers from the right anterior pelvic ganglion (APG) on changes in the chemical coding of their neuronal bodies. The study was performed using male pigs with immunohistochemistry and quantitative real-time PCR (qPCR). The animals were divided into a control (C), a morphological (MG) or a molecular biology group (MBG). APG neurons supplying UBT were revealed using the retrograde tracing technique with Fast Blue (FB). Unilateral axotomy resulted in an over 50% decrease in the number of FB+ neurons in both APG ganglia. Immunohistochemistry revealed significant changes in the chemical coding of FB+ cells only in the right ganglion: decreased expression of dopamine-B-hydroxylase (DBH)/tyrosine hydroxylase (TH) and up-regulation of the vesicular acetylcholine transporter (VAChT)/choline acetyltransferase (ChAT), galanin (GAL), vasoactive intestinal polypeptide (VIP) and brain nitric oxide synthase (bNOS). The qPCR results partly corresponded with immunofluorescence findings. In the APGs, genes for VAChT and ChAT, TH and DBH, VIP, and NOS were distinctly down-regulated, while the expression of GAL was up-regulated. Such data may be the basis for further studies concerning the plasticity of these ganglia under experimental or pathological conditions.

New Inhibitors of Laccase and Tyrosinase by Examination of Cross-Inhibition between Copper-Containing Enzymes.

Coppers play crucial roles in the maintenance homeostasis in living species. Approximately 20 enzyme families of eukaryotes and prokaryotes are known to utilize copper atoms for catalytic activities. However, small-molecule inhibitors directly targeting catalytic centers are rare, except for those that act against tyrosinase and dopamine-ß-hydroxylase (DBH). This study tested whether known tyrosinase inhibitors can inhibit the copper-containing enzymes, ceruloplasmin, DBH, and laccase. While most small molecules minimally reduced the activities of ceruloplasmin and DBH, aside from known inhibitors, 5 of 28 tested molecules significantly inhibited the function of laccase, with the Ki values in the range of 15 to 48 µM. Enzyme inhibitory kinetics classified the molecules as competitive inhibitors, whereas differential scanning fluorimetry and fluorescence quenching supported direct bindings. To the best of our knowledge, this is the first report on organic small-molecule inhibitors for laccase. Comparison of tyrosinase and DBH inhibitors using cheminformatics predicted that the presence of thione moiety would suffice to inhibit tyrosinase. Enzyme assays confirmed this prediction, leading to the discovery of two new dual tyrosinase and DBH inhibitors.

The Influence of an Adrenergic Antagonist Guanethidine on the Distribution Pattern and Chemical Coding of Caudal Mesenteric Ganglion Perikarya and Their Axons Supplying the Porcine Bladder.

This study was aimed at disclosing the influence of intravesically instilled guanethidine (GUA) on the distribution, relative frequency and chemical coding of both the urinary bladder intramural sympathetic nerve fibers and their parent cell bodies in the caudal mesenteric ganglion (CaMG) in juvenile female pigs. GUA instillation led to a profound decrease in the number of perivascular nerve terminals. Furthermore, the chemical profile of the perivascular innervation within the treated bladder also distinctly changed, as most of axons became somatostatin-immunoreactive (SOM-IR), while in the control animals they were found to be neuropeptide Y (NPY)-positive. Intravesical treatment with GUA led not only to a significant decrease in the number of bladder-projecting tyrosine hydroxylase (TH) CaMG somata (94.3 ± 1.8% vs. 73.3 ± 1.4%; control vs. GUA-treated pigs), but simultaneously resulted in the rearrangement of their co-transmitters repertoire, causing a distinct decrease in the number of TH+/NPY+ (89.6 ± 0.7% vs. 27.8 ± 0.9%) cell bodies and an increase in the number of SOM-(3.6 ± 0.4% vs. 68.7 ± 1.9%), calbindin-(CB; 2.06 ± 0.2% vs. 9.1 ± 1.2%) or galanin-containing (GAL; 1.6 ± 0.3% vs. 28.2 ± 1.3%) somata. The present study provides evidence that GUA significantly modifies the sympathetic innervation of the porcine urinary bladder wall, and thus may be considered a potential tool for studying the plasticity of this subdivision of the bladder innervation.

Dopamine beta-hydroxylase and its genetic variants in human health and disease.

Dopamine beta-hydroxylase (DßH) is an essential neurotransmitter-synthesizing enzyme that catalyzes the formation of norepinephrine (NE) from dopamine and has been extensively studied since its discovery in the 1950s. NE serves as a neurotransmitter in both the central and peripheral nervous systems and is the precursor to epinephrine synthesis in the brain and adrenal medulla. Alterations in noradrenergic signaling have been linked to both central nervous system and peripheral pathologies. DßH protein, which is found in circulation, can, therefore, be evaluated as a marker of norepinephrine function in a plethora of different disorders and diseases. In many of these diseases, DßH protein availability and activity are believed to contribute to disease presentation or select symptomology and are believed to be under strong genetic control. Alteration in the DßH protein by genetic polymorphisms may result in DßH becoming rate-limiting and directly contributing to lower NE and epinephrine levels and disease. With the completion of the human genome project and the advent of next-generation sequencing, new insights have been gained into the existence of naturally occurring DßH sequencing variants (genetic polymorphisms) in disease. Also, biophysical tools coupled with genetic sequences are illuminating structure-function relationships within the enzyme. In this review, we discuss the role of genetic variants in DßH and its role in health and disease.