Comparisons of neuroinflammation, microglial activation, and degeneration of the locus coeruleus-norepinephrine system in APP/PS1 and aging mice.

BACKGROUND: The role of microglia in Alzheimer's disease (AD) pathogenesis is becoming increasingly important, as activation of these cell types likely contributes to both pathological and protective processes associated with all phases of the disease. During early AD pathogenesis, one of the first areas of degeneration is the locus coeruleus (LC), which provides broad innervation of the central nervous system and facilitates norepinephrine (NE) transmission. Though the LC-NE is likely to influence microglial dynamics, it is unclear how these systems change with AD compared to otherwise healthy aging. METHODS: In this study, we evaluated the dynamic changes of neuroinflammation and neurodegeneration in the LC-NE system in the brain and spinal cord of APP/PS1 mice and aged WT mice using immunofluorescence and ELISA. RESULTS: Our results demonstrated increased expression of inflammatory cytokines and microglial activation observed in the cortex, hippocampus, and spinal cord of APP/PS1 compared to WT mice. LC-NE neuron and fiber loss as well as reduced norepinephrine transporter (NET) expression was more evident in APP/PS1 mice, although NE levels were similar between 12-month-old APP/PS1 and WT mice. Notably, the degree of microglial activation, LC-NE nerve fiber loss, and NET reduction in the brain and spinal cord were more severe in 12-month-old APP/PS1 compared to 12- and 24-month-old WT mice. CONCLUSION: These results suggest that elevated neuroinflammation and microglial activation in the brain and spinal cord of APP/PS1 mice correlate with significant degeneration of the LC-NE system.

Mapping Astrocyte Transcriptional Signatures in Response to Neuroactive Compounds.

Astrocytes play central roles in normal brain function and are critical components of synaptic networks that oversee behavioral outputs. Despite their close affiliation with neurons, how neuronal-derived signals influence astrocyte function at the gene expression level remains poorly characterized, largely due to difficulties associated with dissecting neuron- versus astrocyte-specific effects. Here, we use an in vitro system of stem cell-derived astrocytes to identify gene expression profiles in astrocytes that are influenced by neurons and regulate astrocyte development. Furthermore, we show that neurotransmitters and neuromodulators induce distinct transcriptomic and chromatin accessibility changes in astrocytes that are unique to each of these neuroactive compounds. These findings are highlighted by the observation that noradrenaline has a more profound effect on transcriptional profiles of astrocytes compared to glutamate, gamma-aminobutyric acid (GABA), acetylcholine, and serotonin. This is demonstrated through enhanced noradrenaline-induced transcriptomic and chromatin accessibility changes in vitro and through enhanced calcium signaling in vivo. Taken together, our study reveals distinct transcriptomic and chromatin architecture signatures in astrocytes in response to neuronal-derived neuroactive compounds. Since astrocyte function is affected in all neurological disorders, this study provides a new entry point for exploring genetic mechanisms of astrocyte-neuron communication that may be dysregulated in disease.

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.

Strategies to Treat Chronic Pain and Strengthen Impaired Descending Noradrenergic Inhibitory System.

Gabapentinoids (gabapentin and pregabalin) and antidepressants (tricyclic antidepressants and serotonin noradrenaline reuptake inhibitors) are often used to treat chronic pain. The descending noradrenergic inhibitory system from the locus coeruleus (LC) to the dorsal horn of the spinal cord plays an important role in the analgesic mechanisms of these drugs. Gabapentinoids activate the LC by inhibiting the release of γ-aminobutyric acid (GABA) and inducing the release of glutamate, thereby increasing noradrenaline levels in the spinal cord. Antidepressants increase noradrenaline levels in the spinal cord by inhibiting reuptake, and accumulating noradrenaline inhibits chronic pain through α2-adrenergic receptors in the spinal cord. Recent animal studies, however, revealed that the function of the descending noradrenergic inhibitory system is impaired in chronic pain states. Other recent studies found that histone deacetylase inhibitors and antidepressants restore the impaired noradrenergic descending inhibitory system acting on noradrenergic neurons in the LC.

Role of α1-adrenoceptor subtypes on corneal epithelial thickness and cell proliferation in mice.

Adrenergic stimuli are important for corneal epithelial structure and healing. The purpose of the present study was to examine the hypothesis that the lack of a single α1-adrenoceptor (α1-AR) subtype affects corneal epithelial thickness and cell proliferation. Expression levels of α1-AR mRNA were determined in mouse cornea using real-time PCR. In mice devoid of one of the three α1-AR subtypes (α1A-AR-/-, α1B-AR-/-, α1D-AR-/-) and in wild-type controls, thickness of individual corneal layers, the number of epithelial cell layers, and average epithelial cell size were determined in cryosections. Endothelial cell density and morphology were calculated in corneal explants, and epithelial cell proliferation rate was determined with immunofluorescence microscopy. Moreover, the ultrastructure of the corneal epithelium was examined by transmission electron microscopy. Messenger RNA for all three α1-AR subtypes was expressed in whole cornea and in corneal epithelium from wild-type mice with a rank order of abundance of α1A ≥ α1B > α1D. In contrast, no α1-AR mRNA was detected in the stroma, and only α1B-AR mRNA was found in the Descemet endothelial complex. Remarkably, corneal epithelial thickness and mean epithelial cell size were reduced in α1A-AR-/- mice. Our findings suggest that the α1A-AR exerts growth effects in mouse corneal epithelial cells.

Neuroendocrine regulation of inflammation.

The interaction between the sympathetic nervous system and the immune system has been documented over the last several decades. In this review, the neuroanatomical, cellular, and molecular evidence for neuroimmune regulation in the maintenance of immune homeostasis will be discussed, as well as the potential impact of neuroimmune dysregulation in health and disease.

Direct effect of hypothalamic neuropeptides on the release of catecholamines by adrenal medulla in sheep - study ex vivo.

Stress causes the activation of both the hypothalamic-pituitary-adrenocortical axis and sympatho-adrenal system, thus leading to the release from the adrenal medulla of catecholamines: adrenaline and, to a lesser degree, noradrenaline. It has been established that in addition to catecholamines, the adrenomedullary cells produce a variety of neuropeptides, including corticoliberine (CRH), vasopressin (AVP), oxytocin (OXY) and proopiomelanocortine (POMC) - a precursor of the adrenocorticotropic hormone (ACTH). The aim of this study was to investigate adrenal medulla activity in vitro depending, on a dose of CRH, AVP and OXY on adrenaline and noradrenaline release. Pieces of sheep adrenal medulla tissue (about 50 mg) were put on 24-well plates and were incubated in 1 mL of Eagle medium without hormone (control) or supplemented only once with CRH, AVP and OXY in three doses (10-7, 10-8 and 10-9 M) in a volume of 10 µL. The results showed that CRH stimulates adrenaline and noradrenaline release from the adrenal medulla tissue. The stimulating influence of AVP on adrenaline release was visible after the application of the two lower doses of this neuropeptide; however, AVP reduced noradrenaline release from the adrenal medulla tissue. A strong, inhibitory OXY effect on catecholamine release was observed, regardless of the dose of this hormone. Our results indicate the important role of OXY in the inhibition of adrenal gland activity and thus a better adaptation to stress on the adrenal gland level.

Neurogenetic variations in norepinephrine availability enhance perceptual vividness.

Emotionally salient aspects of the world are experienced with greater perceptual vividness than mundane ones; however, such emotionally enhanced vividness (EEV) may be experienced to different degrees for different people. We examined whether BOLD activity associated with a deletion variant of the ADRA2b gene coding for the α2b adrenoceptor modulates EEV in humans. Relative to noncarriers, ADRA2b deletion carriers showed higher levels of perceptual vividness, with the ventromedial prefrontal cortex (VMPFC) showing greater modulation by EEV. Deletion carriers were also more sensitive to the featural salience of the images, suggesting a more pervasive role of norepinephrine in perceptual encoding. Path analysis revealed that, whereas a simple model by which the amygdala modulated the lateral occipital complex best characterized EEV-related activity in noncarriers, contributions of an additional VMPFC pathway best characterized deletion carriers. Thus, common norepinephrine-related neurogenetic differences enhance the subjective vividness of perceptual experience and its emotional enhancement.

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

BACKGROUND: Brain 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. METHODS: Brainstem 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. RESULTS: It 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. CONCLUSIONS: Our 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.

Variation in key genes of serotonin and norepinephrine function predicts gamma-band activity during goal-directed attention.

Recent evidence shows that genetic variations in key regulators of serotonergic (5-HT) signaling explain variance in executive tasks, which suggests modulatory actions of 5-HT on goal-directed selective attention as one possible underlying mechanism. To investigate this link, 130 volunteers were genotyped for the 5-HT transporter gene-linked polymorphic region (5-HTTLPR) and for a variation (TPH2-703 G/T) of the TPH2 gene coding for the rate-limiting enzyme of 5-HT synthesis in the brain. Additionally, a functional polymorphism of the norepinephrine transporter gene (NET -3081 A/T) was considered, which was recently found to predict attention and working memory processes in interaction with serotonergic genes. The flanker-based Attention Network Test was used to assess goal-directed attention and the efficiency of attentional networks. Event-related gamma-band activity served to indicate selective attention at the intermediate phenotype level. The main findings were that 5-HTTLPR s allele and TPH2 G-allele homozygotes showed increased induced gamma-band activity during target processing when combined with the NET A/A genotype compared with other genotype combinations, and that gamma activity mediates the genotype-specific effects on task performance. The results further support a modulatory role of 5-HT and NE function in the top-down attentional selection of motivationally relevant over competing or irrelevant sensory input.

Extracellular norepinephrine clearance by the norepinephrine transporter is required for skeletal homeostasis.

Changes in bone remodeling induced by pharmacological and genetic manipulation of ß-adrenergic receptor (ßAR) signaling in osteoblasts support a role of sympathetic nerves in the regulation of bone remodeling. However, the contribution of endogenous sympathetic outflow and nerve-derived norepinephrine (NE) to bone remodeling under pathophysiological conditions remains unclear. We show here that differentiated osteoblasts, like neurons, express the norepinephrine transporter (NET), exhibit specific NE uptake activity via NET and can catabolize, but not generate, NE. Pharmacological blockade of NE transport by reboxetine induced bone loss in WT mice. Similarly, lack of NE reuptake in norepinephrine transporter (Net)-deficient mice led to reduced bone formation and increased bone resorption, resulting in suboptimal peak bone mass and mechanical properties associated with low sympathetic outflow and high plasma NE levels. Last, daily sympathetic activation induced by mild chronic stress was unable to induce bone loss, unless NET activity was blocked. These findings indicate that the control of endogenous NE release and reuptake by presynaptic neurons and osteoblasts is an important component of the complex homeostatic machinery by which the sympathetic nervous system controls bone remodeling. These findings also suggest that drugs antagonizing NET activity, used for the treatment of hyperactivity disorders, may have deleterious effects on bone accrual.

Norepinephrine deficiency is caused by combined abnormal mRNA processing and defective protein trafficking of dopamine beta-hydroxylase.

Human norepinephrine (NE) deficiency (or dopamine ß-hydroxylase (DBH) deficiency) is a rare congenital disorder of primary autonomic failure, in which neurotransmitters NE and epinephrine are undetectable. Although potential pathogenic mutations, such as a common splice donor site mutation (IVS1+2T→C) and various missense mutations, in NE deficiency patients were identified, molecular mechanisms underlying this disease remain unknown. Here, we show that the IVS1+2T→C mutation results in a non-detectable level of DBH protein production and that all three missense mutations tested lead to the DBH protein being trapped in the endoplasmic reticulum (ER). Supporting the view that mutant DBH induces an ER stress response, exogenous expression of mutant DBH dramatically induced expression of BiP, a master ER chaperone. Furthermore, we found that a pharmacological chaperone, glycerol, significantly rescued defective trafficking of mutant DBH proteins. Taken together, we propose that NE deficiency is caused by the combined abnormal processing of DBH mRNA and defective protein trafficking and that this disease could be treated by a pharmacological chaperone(s).

Effect of aging on norepinephrine-related proliferative response in primary cultured periportal and perivenous hepatocytes.

Norepinephrine (NE) amplifies the mitogenic effect of EGF in a rat liver through the adrenergic receptor coupled with G protein, Ghα. Ghα is also known as a transglutaminase 2 (TG2), whose cross-linking activity is implicated in hepatocyte growth. Recently, we found that NE-induced amplification of EGF-induced DNA synthesis in hepatocytes obtained from perivenous regions of liver is caused by inhibiting the downregulation of EGF receptor (EGFR) by TG2. In the present study, we investigated the effect of aging on NE-related proliferative response. Hepatocytes were obtained from the liver of 7- and 90-wk-old rats. To examine this in detail, periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated using the digitonin/collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, G protein activity, and TG2 activity were measured. NE slightly potentiated [125I]EGF binding to EGFR, and EGF-induced DNA synthesis in PVH but not in PPH. [3H]NE binding studies indicated that PVH have a greater number of receptors than PPH, and that the number of receptors in both subpopulations increased with aging. NE-induced changes in G protein activity and TG2 activity in 90-wk-old rats were slight compared with 7-wk-old rats. These results suggest that NE results in a slight recovery effect on the age-related decline in EGF-induced DNA synthesis because of incomplete switching of the function from TG2 to Ghα.

Catecholaminergic signalling through thymic nerve fibres, thymocytes and stromal cells is dependent on both circulating and locally synthesized glucocorticoids.

Glucocorticoids have been shown to modulate the expression of noradrenaline metabolizing enzymes and ß(2)- and α(1B)-adrenoceptors in a tissue- and cell- specific manner. In the thymus, apart from extensive sympathetic innervation, a regulatory network has been identified that encompasses catecholamine-containing non-lymphoid and lymphoid cells. We examined a putative role of adrenal- and thymus-derived glucocorticoids in modulation of rat thymic noradrenaline levels and adrenoceptor expression. Seven days postadrenalectomy, the thymic levels of mRNAs encoding tyrosine hydroxylase, dopamine ß-hydroxylase, monoamine oxidase-A and, consequently, noradrenaline were decreased. Catecholamine content was diminished in autofluorescent nerve fibres (judging by the intensity of fluorescence) and thymocytes (considering HPLC measurements of noradrenaline and the frequency of tyrosine hydroxylase-positive cells), while it remained unaltered in non-lymphoid autofluorescent cells. In addition, adrenalectomy diminished the thymocyte expression of ß(2)- and α(1B)-adrenoceptors at both mRNA and protein levels. Administration of ketoconazole (an inhibitor of glucocorticoid synthesis/action; 25 mg kg(-1) day(-1), s.c.) to glucocorticoid-deprived rats increased the thymic levels of tyrosine hydroxylase, dopamine ß-hydroxylase and, consequently, noradrenaline. The increased intensity of the autofluorescent cell fluorescence in ketoconazole-treated rats indicated an increase in their catecholamine content, and suggested differential glucocorticoid-mediated regulation of catecholamines in thymic lymphoid and non-lymphoid cells. In addition, ketoconazole increased the thymocyte expression of α(1B)-adrenoceptors. Thus, this study indicates that in the thymus, as in some other tissues, glucocorticoids not only act in concert with cateholamines, but they may modulate catecholamine action by tuning thymic catecholamine metabolism and adrenoceptor expression in a cell-specific manner. Additionally, the study indicates a role of thymus-derived glucocorticoids in this modulation.

Weight-gain propensity and morphine withdrawal alters locomotor behavior and regional norepinephrine-related gene expression in male and female mice.

Interactions between obesity and opioid use are poorly understood. The objective of this study was to determine whether phenotypic differences in diet-induced weight gain altered morphine withdrawal responses. Male and female C57BL/6J mice were characterized as obese prone (OP) or obese resistant (OR) based on median split in body weights following exposure to high-fat diet (45% fat). After classification into OP or OR, all mice were fed a low-fat diet (10% fat) for the remainder of the study (≥5 weeks) to remain weight matched. Mice were treated with a 7-day escalating dosing scheme of morphine (20-100 mg/kg; IP) or saline and underwent a spontaneous withdrawal. Morphine-induced weight loss was restored by withdrawal day 7. On withdrawal day 8, male OP demonstrated less total time mobile in the open field test (OFT). In females, OR-morphine traveled less distance than OR-saline, and OR-morphine spent less time mobile compared with all other groups in the OFT. Female OP also increased time spent in the center of the apparatus, regardless of treatment. On withdrawal day 8, relative gene expression was measured by qPCR. For males, expression of dopamine beta-hydroxylase (dbh), alpha-adrenergic receptor 2 a (adra2a), and orexin receptor 1 (orx1) were increased in the locus coeruleus (LC) region of OP mice, regardless of treatment. In comparison, in females, dbh and adra2a were decreased in the LC region of OP mice, regardless of treatment. Also, in the LC region of females, OP-morphine had lower expression of alpha-adrenergic receptor 1 a (adra1a) than OR-morphine and OP-saline. In the hypothalamic paraventricular nucleus (PVN) of females, adra2a was increased in OP-morphine compared with OP-saline and OR-morphine. Our findings suggest morphine withdrawal responses and regional expression of noradrenergic-related genes are differentially influenced by weight gain propensity.

Noradrenergic control of cortico-striato-thalamic and mesolimbic cross-structural synchrony.

Although normal dopaminergic tone has been shown to be essential for the induction of cortico-striatal and mesolimbic theta oscillatory activity, the influence of norepinephrine on these brain networks remains relatively unknown. To address this question, we simultaneously recorded local field potentials and single-neuron activity across 10 interconnected brain areas (ventral striatum, frontal association cortex, hippocampus, primary motor cortex, orbital frontal cortex, prelimbic cortex, dorsal lateral striatum, medial dorsal nucleus of thalamus, substantia nigra pars reticularis, and ventral tegmental area) in a combined genetically and pharmacologically induced mouse model of hyponoradrenergia. Our results show that norepinephrine (NE) depletion induces a novel state in male mice characterized by a profound disruption of coherence across multiple cortico-striatal circuits and an increase in mesolimbic cross-structural coherence. Moreover, this brain state is accompanied by a complex behavioral phenotype consisting of transient hyperactivity, stereotypic behaviors, and an acute 12-fold increase in grooming. Notably, treatment with a norepinephrine precursors (l-3,4-dihydroxyphenylalanine at 100 mg/kg or l-threo-dihydroxyphenylserine at 5 mg/kg) or a selective serotonin reuptake inhibitor (fluoxetine at 20 mg/kg) attenuates the abnormal behaviors and selectively reverses the circuit changes observed in NE-depleted mice. Together, our results demonstrate that norepinephrine modulates the dynamic tuning of coherence across cortico-striato-thalamic circuits, and they suggest that changes in coherence across these circuits mediate the abnormal generation of hyperactivity and repetitive behaviors.

Cognitive effects of genetic variation in monoamine neurotransmitter systems: a population-based study of COMT, MAOA, and 5HTTLPR.

Individual differences in cognitive function are highly heritable and most likely driven by multiple genes of small effect. Well-characterized common functional polymorphisms in the genes MAOA, COMT, and 5HTTLPR each have predictable effects on the availability of the monoamine neurotransmitters dopamine, noradrenaline, and serotonin. We hypothesized that 5HTTLPR genotype would show little association with prefrontal cognitive performance, but that COMT and MAOA would have interacting effects on cognition through their shared influence on prefrontal catecholamine availability. We assessed the individual and epistatic effects of functional polymorphisms in COMT, MAOA, and 5HTTLPR on children's prefrontal cognitive function in nearly 6,000 children from the population-based Avon Longitudinal Study of Parents and Children (ALSPAC). Neither MAOA nor 5HTTLPR polymorphisms showed significant effects on cognitive function. In boys but not girls, there was a modest but statistically significant interaction between MAOA and COMT genotypes such that increased prefrontal catecholamine availability was associated with better working memory. These results suggest that assessment of multiple genes within functionally related systems may improve our understanding of the genetic basis of cognition.

Fatty Acids Rescue the Thermogenic Function of Sympathetically Denervated Brown Fat.

Sympathetic nervous system (SNS) innervation into brown adipose tissue (BAT) has been viewed as an impetus for brown fat thermogenesis. However, we surprisingly discovered that BAT SNS innervation is dispensable for mice to maintain proper body temperature during a prolonged cold exposure. Here we aimed to uncover the physiological factors compensating for maintaining brown fat thermogenesis in the absence of BAT innervation. After an initial decline of body temperature during cold exposure, mice with SNS surgical denervation in interscapular BAT gradually recovered their temperature comparable to that of sham-operated mice. The surgically denervated BAT also maintained a sizable uncoupling protein 1 (UCP1) protein along with basal norepinephrine (NE) at a similar level to that of sham controls, which were associated with increased circulating NE. Furthermore, the denervated mice exhibited increased free fatty acid levels in circulation. Indeed, surgical denervation of mice with CGI-58 deletion in adipocytes, a model lacking lipolytic capacity to release fatty acids from WAT, dramatically reduced BAT UCP1 protein and rendered the mice susceptible to cold. We conclude that circulating fatty acids and NE may serve as key factors for maintaining BAT thermogenic function and body temperature in the absence of BAT sympathetic innervation.