Superior Cervical Ganglion Neurons Research Articles

Sympathetic Hyperactivity and Age Affect Segregation and Expression of Neurotransmitters

Sympathetic neurons of the rat superior cervical ganglion (SCG) can segregate their neurotransmitters and co-transmitters to separate varicosities of single axons. We have shown that transmitter segregation is a plastic phenomenon and that it is correlated with the strength of synaptic transmission. Here, we determined whether sympathetic dysfunction occurring in stress and hypertension was correlated with plastic changes of neurotransmitter segregation. We characterized the expression of the markers, L-glutamic acid decarboxylase of 67 kDa (GAD67) and vesicular acetylcholine (ACh) transporter (VAChT) in the SCG of cold stressed and spontaneously hypertensive rats (SHR). Considering that the SCG comprises a heterogeneous neuronal population, we explored whether the expression and segregation of neurotransmitters would also have an intraganglionic heterogeneous distribution in ganglia of stressed and hypertensive rats. Furthermore, since hypertension in SHR is detected around 8–10 weeks, we evaluated expression and segregation of ACh and GABA in adult hypertensive (12-week old (wo)) and young pre-hypertensive (6-wo) SHR. We found an increase in segregation of ACh and GABA with no change in transmitter expression in ganglia of stressed animals. In contrast, in SHR, there was an increase in GABA expression, although segregation did not vary. Segregation showed a caudo-rostral gradient in controls but not in the ganglia of stressed animals. GABA expression showed a rostro-caudal gradient in adult SHR, which was not present in young 6-wo rats. In young SHR, ACh increased and, unexpectedly, segregation of ACh and GABA was higher than in adults. Data suggest that ACh and GABA segregation increases in acute sympathetic hyperactivity like stress, but does not vary in chronic hyperactivity such as in hypertension. Changes in segregation are age-dependent and might be involved in the mechanisms underlying stress and hypertension.

P7C3-A20 neuroprotection is independent of Wallerian degeneration in primary neuronal culture.

The antiapoptotic, neuroprotective compound P7C3-A20 reduces neurological deficits when administered to murine in-vivo models of traumatic brain injury. P7C3-A20 is thought to exert its activity through small-molecule activation of the enzyme nicotinamide phosphoribosyltransferase. This enzyme converts nicotinamide to nicotinamide mononucleotide, the precursor to nicotinamide adenine dinucleotide synthesis. Alterations to this bioenergetic pathway have been shown to induce Wallerian degeneration (WD) of the distal neurite following injury. This study aimed to establish whether P7C3-A20, through induction of nicotinamide phosphoribosyltransferase activity, would affect the rate of WD. The model systems used were dissociated primary cortical neurons, dissociated superior cervical ganglion neurons and superior cervical ganglion explants. P7C3-A20 failed to show any protection against WD induced by neurite transection or vincristine administration. Furthermore, there was a concentration-dependent neurotoxicity. These findings are important in understanding the mechanism by which P7C3-A20 mediates its effects – a key step before moving to human clinical trials.

Low levels of NMNAT2 compromise axon development and survival.

Nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) is an endogenous axon maintenance factor that preserves axon health by blocking Wallerian-like axon degeneration. Mice lacking NMNAT2 die at birth with severe axon defects in both the peripheral nervous system and central nervous system so the complete absence of NMNAT2 in humans is likely to be similarly harmful but probably rare. However, there is evidence of widespread natural variation in human NMNAT2 mRNA expression so it is important to establish whether reduced levels of NMNAT2 have consequences that impact health. While mice that express reduced levels of NMNAT2, either those heterozygous for a silenced Nmnat2 allele or compound heterozygous for one silenced and one partially silenced Nmnat2 allele, remain overtly normal into old age, we now report that Nmnat2 compound heterozygote mice present with early and age-dependent peripheral nerve axon defects. Compound heterozygote mice already have reduced numbers of myelinated sensory axons at 1.5months and lose more axons, likely motor axons, between 18 and 24months and, crucially, these changes correlate with early temperature insensitivity and a later-onset decline in motor performance. Slower neurite outgrowth and increased sensitivity to axonal stress are also evident in primary cultures of Nmnat2 compound heterozygote superior cervical ganglion neurons. These data reveal that reducing NMNAT2 levels below a particular threshold compromises the development of peripheral axons and increases their vulnerability to stresses. We discuss the implications for human neurological phenotypes where axons are longer and have to be maintained over a much longer lifespan.

Modulation of Ion Channels in the Superior Cervical Ganglion Neurons by Myocardial Ischemia and Fluvastatin Treatment.

Background: The superior cervical ganglion (SCG) of the autonomic nervous system plays an important role in different cardiovascular diseases. In this study, we investigated the effects of ischemia and fluvastatin treatment on the ion channel characteristics of SCG neurons in a rabbit myocardial ischemia (MI) model.Methods: MI was induced by abdominal subcutaneous injections of isoproterenol (ISO). The properties of the delayed rectifier potassium channel current (IK), sodium channel current (INa), and action potential (APs) on isolated SCG neurons in the control, MI-7d, MI-14d, fluvastatin-7d (fluvastatin pretreated 14 days and treated 7 days after ISO-induced MI), and fluvastatin-14d (fluvastatin pretreated 14 days and treated 14 days after ISO-induced MI) groups were studied. In addition, the RNA expressions of KCNQ3 and SCN9A in the SCG tissue were determined by performing real-time PCR. Intracellular calcium concentration was monitored using laser scanning confocal microscopy.Results: Compared with the control group, the current amplitude of IK and INa were increased in the MI-7d and MI-14d groups. KCNQ3 RNA (corresponding to channel proteins of IK) expression and SCN9A RNA (corresponding to channel proteins of INa) expression were also increased in MI groups. Activation and inactivation curves for INa in the two MI groups shifted negatively compared with the control group. These changes were reversed by fluvastatin treatment. Intracellular calcium concentration in SCG neurons was not altered significantly by MI or fluvastatin treatment. By contrast, increased AP amplitude and shortened APD90 were observed in the MI-7d and MI-14d groups. These changes were reversed in the fluvastatin-treated MI group.Conclusion: Fluvastatin treatment partly reversed the characteristics of SCG neurons in MI. The ion channel of SCG neurons could be one of the potential targets of fluvastatin in treating coronary heart diseases.

The mechanism of botulinum A on Raynaud syndrome.

BackgroundBotulinum neurotoxin type A (BoNT/A) is emerging as a treatment modality for Raynaud’s phenomenon (RP). However, the mechanism of the role of BoNT/A in antagonizing the constriction of arteriola in RP remains unclear.Materials and methodsWe tested the constriction of arteriole diameter and the distribution of adrenergic receptors on the rat cremaster modle. Moreover, we measured the secretion of norepinephrine (NE), protein level changes and related receptors on cultured rat superior cervical ganglia neurons(SCGNs), a model of sympathetic neuron.ResultsBased on our results, the inhibition of arteriole vasoconstriction was increased with increasing doses of BoNT/A. BoNT/A, prazosin, and BQ123 treatment can result in significant inhibition of arteriole vasoconstriction with the same electrical stimulation. The inhibition effect of prazosin was equivalent to BoNT/A, while BQ123 has a synergistic effect with BoNT/A. After treating SCGNs using BoNT/A for 30 min, the decrease in fluorescence intensity of FM1-43 slowed down which was correlated with the doses of BoNT/A. Furthermore, release of NE in the supernatant was significantly decreased as measured by enzyme-linked immunosorbent assay, 24 h after a high dose of BoNT/A (25 µ/mL). Cleaved-SNAP-25 was detected by Western blotting 24 h following BoNT/A (50 µ/mL) treatment. Moreover, receptor SV2C, GM1, and FGFR3 were detected on sympathetic neurons, similarly to cholinergic neurons.ConclusionOur study showed that BoNT/A could significantly inhibit electrical stimulation-induced arteriole vasoconstriction through the sympathetic pathway. The mechanism was similar to the cholinergic one, in which the vesicle release of sympathetic neurons could be inhibited by cleavage of SNAP-25. The end result was blocked vesicle fusion with the presynaptic membrane after BoNT/A treatment, inhibiting the release of the NE.

A Calcium-Dependent Chloride Current Increases Repetitive Firing in Mouse Sympathetic Neurons.

Ca2+-activated ion channels shape membrane excitability in response to elevations in intracellular Ca2+. The most extensively studied Ca2+-sensitive ion channels are Ca2+-activated K+ channels, whereas the physiological importance of Ca2+-activated Cl- channels has been poorly studied. Here we show that a Ca2+-activated Cl- currents (CaCCs) modulate repetitive firing in mouse sympathetic ganglion cells. Electrophysiological recording of mouse sympathetic neurons in an in vitro preparation of the superior cervical ganglion (SCG) identifies neurons with two different firing patterns in response to long depolarizing current pulses (1 s). Neurons classified as phasic (Ph) made up 67% of the cell population whilst the remainders were tonic (T). When a high frequency train of spikes was induced by intracellular current injection, SCG sympathetic neurons reached an afterpotential mainly dependent on the ratio of activation of two Ca2+-dependent currents: the K+ [IK(Ca)] and CaCC. When the IK(Ca) was larger, an afterhyperpolarization was the predominant afterpotential but when the CaCC was larger, an afterdepolarization (ADP) was predominant. These afterpotentials can be observed after a single action potential (AP). Ph and T neurons had similar ADPs and hence, the CaCC does not seem to determine the firing pattern (Ph or T) of these neurons. However, inhibition of Ca2+-activated Cl- channels with anthracene-9′-carboxylic acid (9AC) selectively inhibits the ADP, reducing the firing frequency and the instantaneous frequency without affecting the characteristics of single- or first-spike firing of both Ph and T neurons. Furthermore, we found that the CaCC underlying the ADP was significantly larger in SCG neurons from males than from females. Furthermore, the CaCC ANO1/TMEM16A was more strongly expressed in male than in female SCGs. Blocking ADPs with 9AC did not modify synaptic transmission in either Ph or T neurons. We conclude that the CaCC responsible for ADPs increases repetitive firing in both Ph and T neurons, and it is more relevant in male mouse sympathetic ganglion neurons.

Immunohistochemical localization of angiotensin AT1 receptors in the rat carotid body

The carotid body (CB) is a major peripheral arterial chemoreceptor that initiates respiratory and cardiovascular adjustments to maintain homeostasis. Recent evidence suggests that circulating or locally produced hormones like angiotensin II acting via AT1 receptors modulate its activity in a paracrine-autocrine manner. The aim of this study was to examine the immunohistochemical localization of AT1 receptor in the CB of adult rats and to compare its expression in vehicle-treated animals, and after the long-term application of its selective blocker losartan. Immunohistochemistry revealed that a subset of CB glomeruli and the vast majority of neurons in the adjacent superior cervical ganglion (SCG) were strongly AT1 receptor-immunoreactive. In the CB immunostaining was observed in the chemosensory glomus cells typically aggregated in cell clusters while the nerve fibers in-between and large capillaries around them were immunonegative. Exogenous administration of losartan for a prolonged time significantly reduces the intensity of AT1 receptor immunostaining in the CB glomus cells and SCG neurons. Our results show that AT1 receptors are largely expressed in the rat CB under physiological conditions, and their expression is down-regulated by losartan treatment.

Expression of CGRP neurotransmitter and vascular genesis marker mRNA is age-dependent in superior cervical ganglia of senescence-accelerated prone mice.

Calcitonin gene-related peptide (CGRP) is a neurotransmitter that is released from the superior cervical ganglion (SCG) and causes head and neck pain. The morphological properties of human SCG neurons, including neurotransmitter content, are altered during aging. However, morphological changes in CGRP in the SCG during aging are not known. Therefore, we investigated CGRP and other markers in the SCG during aging in an aging model of senescence-accelerated prone mouse (SAMP8) and senescence-accelerated resistant mice (SAMR1) using real-time RT-PCR mRNA analyses and in situ hybridization. The abundance of neurotransmitter (CGRP, NPY, TRPV1), vascular genesis marker (CD31, LYVE-1), and cytochrome C mRNA differed between 12-week-old and 24-week-old SAMP8 and SAMR1. Abundance of TRPV1, CD31 and cytochrome C mRNAs of SAMP8 decreased between 12- and 24-week-old. The ratio of CGRP mRNA positive cells and CGRP mRNA abundance levels of the SCG of aging mouse such as SAMP8 have already been also higher than that of SAMR1 at 12-week-old. The CGRP positive shrunken ganglion cells was increased from 12- to 24-weeks-old mouse in SAMR1 and SAMP8. The SCG primarily affected the internal and external carotid arteries, larynx thyroid gland, and pharyngeal muscle during aging.

Fast Inactivation of CaV2.2 Channels Is Prevented by the Gβ1 Subunit in Rat Sympathetic Neurons.

Voltage-dependent regulation of CaV2.2 channels by G-proteins is performed by the β (Gβ) subunit. Most studies of regulation by G-proteins have focused on channel activation; however, little is known regarding channel inactivation. This study investigated inactivation of CaV2.2 channels in superior cervical ganglion neurons that overexpressed Gβ subunits. CaV2.2 currents were recorded by whole-cell patch clamping configuration. We found that the Gβ1 subunit reduced inactivation, while Gβ5 subunit did not alter at all inactivation kinetics compared to control recordings. CaV2.2 current decay in control neurons consisted of both fast and slow inactivation; however, Gβ1-overexpressing neurons displayed only the slow inactivation. Fast inactivation was restored by a strong depolarization of Gβ1-overexpressing neurons, therefore, through a voltage-dependent mechanism. The Gβ1 subunit shifted the voltage dependence of inactivation to more positive voltages and reduced the fraction of CaV2.2 channels resting in the inactivated state. These results support that the Gβ1 subunit inhibits the fast inactivation of CaV2.2 channels in SCG neurons. They explain the long-observed sustained Ca2+ current under G-protein modulation.

CaV2 channel subtype expression in rat sympathetic neurons is selectively regulated by α2δ subunits

ABSTRACTType two voltage gated calcium (CaV2) channels are the primary mediators of neurotransmission at neuronal presynapses, but their function at neural soma is also important in regulating excitability.1 Mechanisms that regulate CaV2 channel expression at synapses have been studied extensively, which motivated us to perform similar studies in the soma. Rat sympathetic neurons from the superior cervical ganglion (SCG) natively express CaV2.2 and CaV2.3.2 We noted previously that heterologous expression of CaV2.1 but not CaV2.2 results in increased calcium current in SCG neurons.3 In the present study, we extended these observations to show that both CaV2.1 and CaV2.3 expression resulted in increased calcium currents while CaV2.2 expression did not. Further, CaV2.1 could displace native CaV2.2 channels, but CaV2.3 expression could not. Heterologous expression of the individual accessory subunits α2δ-1, α2δ-2, α2δ-3, or β4 alone failed to increase current density, suggesting that the calcium current ceiling when CaV2.2 was over-expressed was not due to lack of these subunits. Interestingly, introduction of recombinant α2δ subunits produced surprising effects on displacement of native CaV2.2 by recombinant channels. Both α2δ-1 and α2δ-2 seemed to promote CaV2.2 displacement by recombinant channel expression, while α2δ-3 appeared to protect CaV2.2 from displacement. Thus, we observe a selective prioritization of CaV channel functional expression in neurons by specific α2δ subunits. These data highlight a new function for α2δ subtypes that could shed light on subtype selectivity of CaV2 membrane expression.

The amyloid precursor protein modulates α2A-adrenergic receptor endocytosis and signaling through disrupting arrestin 3 recruitment.

The amyloid precursor protein (APP) has long been appreciated for its role in Alzheimer's disease (AD) pathology. However, less is known about the physiologic function of APP outside of AD. Particularly, whether and how APP may regulate functions of cell surface receptors, including GPCRs, remains largely unclear. In this study, we identified a novel direct interaction between APP and the α2A-adrenergic receptor (α2AAR) that occurs at the intracellular domains of both proteins. The APP interaction with α2AAR is promoted by agonist stimulation and competes with arrestin 3 binding to the receptor. Consequently, the presence of APP attenuates α2AAR internalization and desensitization, which are arrestin-dependent processes. Furthermore, in neuroblastoma neuro-2A cells and primary superior cervical ganglion neurons, where APP is highly expressed, the lack of APP leads to a dramatic increase in plasma membrane recruitment of endogenous arrestin 3 following α2AAR activation. Concomitantly, agonist-induced internalization of α2AAR is significantly enhanced in these neuronal cells. Our study provided the first evidence that APP fine tunes GPCR signaling and trafficking. Given the important role of α2AAR in controlling norepinephrine release and response, this novel regulation of α2AAR by APP may have an impact on modulation of noradrenergic activity and sympathetic tone.-Zhang, F., Gannon, M., Chen, Y., Zhou, L., Jiao, K., Wang, Q. The amyloid precursor protein modulates α2A-adrenergic receptor endocytosis and signaling through disrupting arrestin 3 recruitment.

Gene expression analyses reveal metabolic specifications in acute O2 -sensing chemoreceptor cells.

Glomus cells in the carotid body (CB) and chromaffin cells in the adrenal medulla (AM) are essential for reflex cardiorespiratory adaptation to hypoxia. However, the mechanisms whereby these cells detect changes in O2 tension are poorly understood. The metabolic properties of acute O2 -sensing cells have been investigated by comparing the transcriptomes of CB and AM cells, which are O2 -sensitive, with superior cervical ganglion neurons, which are practically O2 -insensitive. In O2 -sensitive cells, we found a characteristic prolyl hydroxylase 3 down-regulation and hypoxia inducible factor 2α up-regulation, as well as overexpression of genes coding for three atypical mitochondrial electron transport subunits and pyruvate carboxylase, an enzyme that replenishes tricarboxylic acid cycle intermediates. In agreement with this observation, the inhibition of succinate dehydrogenase impairs CB acute O2 sensing. The responsiveness of peripheral chemoreceptor cells to acute hypoxia depends on a 'signature metabolic profile'. Acute O2 sensing is a fundamental property of cells in the peripheral chemoreceptors, e.g. glomus cells in the carotid body (CB) and chromaffin cells in the adrenal medulla (AM), and is necessary for adaptation to hypoxia. These cells contain O2 -sensitive ion channels, which mediate membrane depolarization and transmitter release upon exposure to hypoxia. However, the mechanisms underlying the detection of changes in O2 tension by cells are still poorly understood. Recently, we suggested that CB glomus cells have specific metabolic features that favour the accumulation of reduced quinone and the production of mitochondrial NADH and reactive oxygen species during hypoxia. These signals alter membrane ion channel activity. To investigate the metabolic profile characteristic of acute O2 -sensing cells, we used adult mice to compare the transcriptomes of three cell types derived from common sympathoadrenal progenitors, but exhibiting variable responsiveness to acute hypoxia: CB and AM cells, which are O2 -sensitive (glomus cells > chromaffin cells), and superior cervical ganglion neurons, which are practically O2 -insensitive. In the O2 -sensitive cells, we found a characteristic mRNA expression pattern of prolyl hydroxylase 3/hypoxia inducible factor 2α and up-regulation of several genes, in particular three atypical mitochondrial electron transport subunits and some ion channels. In addition, we found that pyruvate carboxylase, an enzyme fundamental to tricarboxylic acid cycle anaplerosis, is overexpressed in CB glomus cells. We also observed that the inhibition of succinate dehydrogenase impairs CB acute O2 sensing. Our data suggest that responsiveness to acute hypoxia depends on a 'signature metabolic profile' in chemoreceptor cells.

Presence of Functional Neurotrophin TrkB Receptors in the Rat Superior Cervical Ganglion

Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity.

Physiological approaches to assess diminished sympathetic activity in the conscious rat

The purpose of this study was to evaluate functional measures of diminished sympathetic activity after postganglionic neuronal loss in the conscious rat. To produce variable degrees of sympathetic postganglionic neuronal loss, adult rats were treated daily with toxic doses of guanethidine (100mg/kg) for either 5days or 11days, followed by a recovery period of at least 18days. Heart rate, blood pressure, cardiac baroreflex responsiveness, urinalysis (for catecholamine metabolite, 3-methoxy-4-hydroxyphenylethylenglycol; MHPG), and pupillometry were performed during the recovery period. At the end of the recovery period stereology of superior cervical ganglia (SCG) was performed to determine the degree of neuronal loss. Total number of SCG neurons was correlated to physiological outcomes using regression analysis. Whereas guanethidine treatment for 11days caused significant reduction in the number of neurons (15,646±1460 vs. 31,958±1588), guanethidine treatment for 5days caused variable levels of neuronal depletion (26,009±3518). Regression analysis showed that only changes in urinary MHPG levels and systolic blood pressure significantly correlated with reduction of SCG neurons (r2=0.45 and 0.19, both p<0.05). Although cardiac baroreflex-induced reflex tachycardia (345.7±19.6 vs. 449.7±20.3) and pupil/iris ratio (0.50±0.03% vs. 0.61±0.02%) were significantly attenuated in the 11-day guanethidine treated rats there was no significant relationship between these measurements and the number of remaining SCG neurons after treatment (p>0.05). These data suggest that basal systolic blood pressure and urinary MHPG levels predict drug-induced depletion of sympathetic activity in vivo.

Injury-induced gp130 cytokine signaling in peripheral ganglia is reduced in diabetes mellitus

Neuropathy is a major diabetic complication. While the mechanism of this neuropathy is not well understood, it is believed to result in part from deficient nerve regeneration. Work from our laboratory established that gp130 family of cytokines are induced in animals after axonal injury and are involved in the induction of regeneration-associated genes (RAGs) and in the conditioning lesion response. Here, we examine whether a reduction of cytokine signaling occurs in diabetes. Streptozotocin (STZ) was used to destroy pancreatic β cells, leading to chronic hyperglycemia. Mice were injected with either low doses of STZ (5×60mg/kg) or a single high dose (1×200mg/kg) and examined after three or one month, respectively. Both low and high dose STZ treatment resulted in sustained hyperglycemia and functional deficits associated with the presence of both sensory and autonomic neuropathy. Diabetic mice displayed significantly reduced intraepidermal nerve fiber density and sudomotor function. Furthermore, low and high dose diabetic mice showed significantly reduced tactile touch sensation measured with Von Frey monofilaments. To look at the regenerative and injury-induced responses in diabetic mice, neurons in both superior cervical ganglia (SCG) and the 4th and 5th lumbar dorsal root ganglia (DRG) were unilaterally axotomized. Both high and low dose diabetic mice displayed significantly less axonal regeneration in the sciatic nerve, when measured in vivo, 48h after crush injury. Significantly reduced induction of two gp130 cytokines, leukemia inhibitory factor and interleukin-6, occurred in diabetic animals in SCG 6h after injury compared to controls. Injury-induced expression of interleukin-6 was also found to be significantly reduced in the DRG at 6h after injury in low and high dose diabetic mice. These effects were accompanied by reduced phosphorylation of signal transducer and activator of transcription 3 (STAT3), a downstream effector of the gp130 signaling pathway. We also found decreased induction of several gp130-dependent RAGs, including galanin and vasoactive intestinal peptide. Together, these data suggest a novel mechanism for the decreased response of diabetic sympathetic and sensory neurons to injury.

Stress Hormones Epinephrine and Corticosterone Selectively Modulate Herpes Simplex Virus 1 (HSV-1) and HSV-2 Productive Infections in Adult Sympathetic, but Not Sensory, Neurons

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect and establish latency in peripheral neurons, from which they can reactivate to cause recurrent disease throughout the life of the host. Stress is associated with the exacerbation of clinical symptoms and the induction of recurrences in humans and animal models. The viruses preferentially replicate and establish latency in different subtypes of sensory neurons, as well as in neurons of the autonomic nervous system that are highly responsive to stress hormones. To determine if stress-related hormones modulate productive HSV-1 and HSV-2 infections within sensory and autonomic neurons, we analyzed viral DNA and the production of viral progeny after treatment of primary adult murine neuronal cultures with the stress hormones epinephrine and corticosterone. Both sensory trigeminal ganglion (TG) and sympathetic superior cervical ganglion (SCG) neurons expressed adrenergic receptors (activated by epinephrine) and the glucocorticoid receptor (activated by corticosterone). Productive HSV infection colocalized with these receptors in SCG but not in TG neurons. In productively infected neuronal cultures, epinephrine treatment significantly increased the levels of HSV-1 DNA replication and production of viral progeny in SCG neurons, but no significant differences were found in TG neurons. In contrast, corticosterone significantly decreased the levels of HSV-2 DNA replication and production of viral progeny in SCG neurons but not in TG neurons. Thus, the stress-related hormones epinephrine and corticosterone selectively modulate acute HSV-1 and HSV-2 infections in autonomic, but not sensory, neurons.IMPORTANCE Stress exacerbates acute disease symptoms resulting from HSV-1 and HSV-2 infections and is associated with the appearance of recurrent skin lesions in millions of people. Although stress hormones are thought to impact HSV-1 and HSV-2 through immune system suppression, sensory and autonomic neurons that become infected by HSV-1 and HSV-2 express stress hormone receptors and are responsive to hormone fluctuations. Our results show that autonomic neurons are more responsive to epinephrine and corticosterone than are sensory neurons, demonstrating that the autonomic nervous system plays a substantial role in HSV pathogenesis. Furthermore, these results suggest that stress responses have the potential to differentially impact HSV-1 and HSV-2 so as to produce divergent outcomes of infection.

Disruption of the Axonal Trafficking of Tyrosine Hydroxylase mRNA Impairs Catecholamine Biosynthesis in the Axons of Sympathetic Neurons.

Tyrosine hydroxylase (TH) is the enzyme that catalyzes the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. In a previous communication, evidence was provided that TH mRNA is trafficked to the axon, where it is locally translated. In addition, a 50-bp sequence element in the 3′untranslated region (3’UTR) of TH mRNA was identified that directs TH mRNA to distal axons (i.e., zip-code). In the present study, the hypothesis was tested that local translation of TH plays an important role in the biosynthesis of the catecholamine neurotransmitters in the axon and/or presynaptic nerve terminal. Toward this end, a targeted deletion of the axonal transport sequence element was developed, using the lentiviral delivery of the CRISPR/Cas9 system, and two guide RNA (gRNA) sequences flanking the 50-bp cis-acting regulatory element in rat superior cervical ganglion (SCG) neurons. Deletion of the axonal transport element reduced TH mRNA levels in the distal axons and reduced the axonal protein levels of TH and TH activity as measured by phosphorylation of SER40 in SCG neurons. Moreover, deletion of the zip-code diminished the axonal levels of dopamine (DA) and norepinephrine (NE). Conversely, the local translation of exogenous TH mRNA in the distal axon enhanced TH levels and activity, and elevated axonal NE levels. Taken together, these results provide direct evidence to support the hypothesis that TH mRNA trafficking and local synthesis of TH play an important role in the synthesis of catecholamines in the axon and presynaptic terminal.

Dynamic Regulation of Homer Binding to Group I Metabotropic Glutamate Receptors by Preso1 and Converging Kinase Cascades.

In rat sympathetic neurons from the superior cervical ganglia (SCG) expressing metabotropic glutamate receptor mGluR1 or mGluR5, overexpression of scaffolding Homer proteins, which bind to a Homer ligand in their C termini, cause receptor clustering and uncoupling from ion channel modulation. In the absence of recombinant Homer protein overexpression, uncoupling of mGluRs from voltage-dependent channels can be induced by expression of Preso1, an adaptor of proline-directed kinases that phosphorylates the Homer ligand and recruits binding of endogenous Homer proteins. Here we show that in SCG neurons expressing mGluR1 and the tyrosine receptor kinase B, treatment with brain-derived neurotrophic factor (BDNF) produces a similar uncoupling of the receptors from calcium channels. We investigated the pathways that mediate this uncoupling and compared it with uncoupling observed with Preso1 expression. Both BDNF- and Preso1-induced uncoupling require residues T1151 and S1154 in the mGluR1 Homer ligand (TPPSPF). Uncoupling via Preso1 but not BDNF was prevented by expression of a dominant negative Cdk5, suggesting that endogenous Cdk5 mediates Preso1-dependent phosphorylation of mGluR1. Dominant negative Cdk5 did not block the BDNF effect but this was sensitive to inhibitors of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase cascade. Interestingly, the BDNF pathway appeared to require native Preso1 binding to mGluR, because overexpression of the Preso1 FERM domain, which mediates the Preso1-mGluR interaction, prevented BDNF-induced uncoupling. These data suggest that the BDNF/tyrosine receptor kinase B and Cdk5 pathways converge at the level of mGluR to similarly induce Homer ligand phosphorylation, recruit Homer binding, and uncouple mGluRs from channel regulation.

NGF-dependent axon growth and regeneration are altered in sympathetic neurons of dystrophic mdx mice.

Duchenne muscular dystrophy (DMD) is a lethal disease, determined by lack of dystrophin (Dp427), a muscular cytoskeletal protein also expressed by selected neuronal populations. Consequently, besides muscular wasting, both human patients and DMD animal models suffer several neural disorders. In previous studies on the superior cervical ganglion (SCG) of wild type and dystrophic mdx mice (Lombardi et al. 2008), we hypothesized that Dp427 could play some role in NGF-dependent axonal growth, both during development and adulthood. To address this issue, we first analyzed axon regeneration potentials of SCG neurons of both genotypes after axotomy in vivo. While noradrenergic innervation of mdx mouse submandibular gland, main source of nerve growth factor (NGF), recovered similarly to wild type, iris innervation (muscular target) never did. We, therefore, evaluated whether dystrophic SCG neurons were poorly responsive to NGF, especially at low concentration. Following in vitro axotomy in the presence of either 10 or 50ng/ml NGF, the number of regenerated axons in mdx mouse neuron cultures was indeed reduced, compared to wild type, at the lower concentration. Neurite growth parameters (i.e. number, length), growth cone dynamics and NGF/TrkA receptor signaling in differentiating neurons (not injured) were also significantly reduced when cultured with 10ng/ml NGF, but also with higher NGF concentrations. In conclusion, we propose a role for Dp427 in NGF-dependent cytoskeletal dynamics associated to growth cone advancement, possibly through indirect stabilization of TrkA receptors. Considering NGF activity in nervous system development/remodeling, this aspect could concur in some of the described DMD-associated neural dysfunctions.

Molecular determinants of cytochrome C oxidase IV mRNA axonal trafficking.

In previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3' untranslated region of the cytochrome c oxidase subunit IV (COXIV) mRNA that was necessary and sufficient for the axonal localization of the message in primary superior cervical ganglion (SCG) neurons. However, little is known about the proteins that interact with the COXIV-zipcode and regulate the axonal trafficking and local translation of the COXIV message. To identify proteins involved in the axonal transport of the COXIV mRNA, we used the biotinylated 38-nucleotide COXIV RNA zipcode as bait in the affinity purification of COXIV zipcode binding proteins. Gel-shift assays of the biotinylated COXIV zipcode indicated that the putative stem-loop structure functions as a nucleation site for the formation of ribonucleoprotein complexes. Mass spectrometric analysis of the COXIV zipcode ribonucleoprotein complex led to the identification of a large number RNA binding proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), and Y-box protein 1 (YB-1). Validation experiments, using western analyses, confirmed the presence of the candidate proteins in the COXIV zipcode affinity purified complexes obtained from SCG axons. Immunohistochemical studies show that FUS, and YB-1 are present in SCG axons. Importantly, RNA immunoprecipitation studies show that FUS, and YB-1 interact with endogenous axonal COXIV transcripts. siRNA-mediated downregulation of the candidate proteins FUS and YB-1 expression in the cell-bodies diminishes the levels of COXIV mRNA in the axon, suggesting functional roles for these proteins in the axonal trafficking of COXIV mRNA.