Evidence for TGF-ß1/Nrf2 Signaling Crosstalk in a Cuprizone Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic and degenerative disease that impacts central nervous system (CNS) function. One of the major characteristics of the disease is the presence of regions lacking myelin and an oxidative and inflammatory environment. TGF-ß1 and Nrf2 proteins play a fundamental role in different oxidative/inflammatory processes linked to neurodegenerative diseases such as MS. The evidence from different experimental settings has demonstrated a TGF-ß1-Nrf2 signaling crosstalk under pathological conditions. However, this possibility has not been explored in experimental models of MS. Here, by using the cuprizone-induced demyelination model of MS, we report that the in vivo pharmacological blockage of the TGF-ß1 receptor reduced Nrf2, catalase, and TGFß-1 protein levels in the demyelination phase of cuprizone administration. In addition, ATP production, locomotor function and cognitive performance were diminished by the treatment. Altogether, our results provide evidence for a crosstalk between TGF-ß1 and Nrf2 signaling pathways under CNS demyelination, highlighting the importance of the antioxidant cellular response of neurodegenerative diseases such as MS.

A preclinical mice model of multiple sclerosis based on the toxin-induced double-site demyelination of callosal and cerebellar fibers.

BACKGROUND: Multiple sclerosis (MS) is an irreversible progressive CNS pathology characterized by the loss of myelin (i.e. demyelination). The lack of myelin is followed by a progressive neurodegeneration triggering symptoms as diverse as fatigue, motor, locomotor and sensory impairments and/or bladder, cardiac and respiratory dysfunction. Even though there are more than fourteen approved treatments for reducing MS progression, there are still no cure for the disease. Thus, MS research is a very active field and therefore we count with different experimental animal models for studying mechanisms of demyelination and myelin repair, however, we still lack a preclinical MS model assembling demyelination mechanisms with relevant clinical-like signs. RESULTS: Here, by inducing the simultaneous demyelination of both callosal and cerebellar white matter fibers by the double-site injection of lysolecithin (LPC), we were able to reproduce CNS demyelination, astrocyte recruitment and increases levels of proinflammatory cytokines levels along with motor, locomotor and urinary impairment, as well as cardiac and respiratory dysfunction, in the same animal model. Single site LPC-injections either in corpus callosum or cerebellum only, fails in to reproduce such a complete range of MS-like signs. CONCLUSION: We here report that the double-site LPC injections treatment evoke a complex MS-like mice model. We hope that this experimental approach will help to deepen our knowledge about the mechanisms of demyelinated diseases such as MS.

Neuroinflammation in Demyelinating Diseases: Oxidative Stress as a Modulator of Glial Cross-Talk.

Myelin is a specialized membrane allowing for saltatory conduction of action potentials in neurons, an essential process to achieve the normal communication across the nervous system. Accordingly, in diseases characterized by the loss of myelin and myelin forming cells -oligodendrocytes in the CNS-, patients show severe neurological disabilities. After a demyelinated insult, microglia, astrocytes and oligodendrocyte precursor cells invade the lesioned area initiating a spontaneous process of myelin repair (i.e. remyelination). A preserved hallmark of this neuroinflammatory scenario is a local increase of oxidative stress, where several cytokines and chemokines are released by glial and other cells. This generates an environment that determines cell interaction resulting in oligodendrocyte maturity and the ability to synthesize new myelin. Herein we review the main features of the regulatory aspect of these molecules based on recent findings and propose new putative signal molecules involved in the remyelination process, focused in the etiology of Multiple Sclerosis, one of the main demyelinating diseases causing disabilities in the population.

Carotid Body Type-I Cells Under Chronic Sustained Hypoxia: Focus on Metabolism and Membrane Excitability.

Chronic sustained hypoxia (CSH) evokes ventilatory acclimatization characterized by a progressive hyperventilation due to a potentiation of the carotid body (CB) chemosensory response to hypoxia. The transduction of the hypoxic stimulus in the CB begins with the inhibition of K+ currents in the chemosensory (type-I) cells, which in turn leads to membrane depolarization, Ca2+ entry and the subsequent release of one- or more-excitatory neurotransmitters. Several studies have shown that CSH modifies both the level of transmitters and chemoreceptor cell metabolism within the CB. Most of these studies have been focused on the role played by such putative transmitters and modulators of CB chemoreception, but less is known about the effect of CSH on metabolism and membrane excitability of type-I cells. In this mini-review, we will examine the effects of CSH on the ion channels activity and excitability of type-I cell, with a particular focus on the effects of CSH on the TASK-like background K+ channel. We propose that changes on TASK-like channel activity induced by CSH may contribute to explain the potentiation of CB chemosensory activity.

Characterization of a Novel Drosophila SERT Mutant: Insights on the Contribution of the Serotonin Neural System to Behaviors.

A better comprehension on how different molecular components of the serotonergic system contribute to the adequate regulation of behaviors in animals is essential in the interpretation on how they are involved in neuropsychiatric and pathological disorders. It is possible to study these components in "simpler" animal models including the fly Drosophila melanogaster, given that most of the components of the serotonergic system are conserved between vertebrates and invertebrates. Here we decided to advance our understanding on how the serotonin plasma membrane transporter (SERT) contributes to serotonergic neurotransmission and behaviors in Drosophila. In doing this, we characterized for the first time a mutant for Drosophila SERT (dSERT) and additionally used a highly selective serotonin-releasing drug, 4-methylthioamphetamine (4-MTA), whose mechanism of action involves the SERT protein. Our results show that dSERT mutant animals exhibit an increased survival rate in stress conditions, increased basal motor behavior, and decreased levels in an anxiety-related parameter, centrophobism. We also show that 4-MTA increases the negative chemotaxis toward a strong aversive odorant, benzaldehyde. Our neurochemical data suggest that this effect is mediated by dSERT and depends on the 4-MTA-increased release of serotonin in the fly brain. Our in silico data support the idea that these effects are explained by specific interactions between 4-MTA and dSERT. In sum, our neurochemical, in silico, and behavioral analyses demonstrate the critical importance of the serotonergic system and particularly dSERT functioning in modulating several behaviors in Drosophila.

Multiple binding sites in the nicotinic acetylcholine receptors: An opportunity for polypharmacolgy.

For decades, the development of selective compounds has been the main goal for chemists and biologists involved in drug discovery. However, diverse lines of evidence indicate that polypharmacological agents, i.e. those that act simultaneously at various protein targets, might show better profiles than selective ligands, regarding both efficacy and side effects. On the other hand, the availability of the crystal structure of different receptors allows a detailed analysis of the main interactions between drugs and receptors in a specific binding site. Neuronal nicotinic acetylcholine receptors (nAChRs) constitute a large and diverse family of ligand-gated ion channels (LGICs) that, as a product of its modulation, regulate neurotransmitter release, which in turns produce a global neuromodulation of the central nervous system. nAChRs are pentameric protein complexes in such a way that expression of compatible subunits can lead to various receptor assemblies or subtypes. The agonist binding site, located at the extracellular region, exhibits different properties depending on the subunits that conform the receptor. In the last years, it has been recognized that nAChRs could also contain one or more allosteric sites which could bind non-classical nicotinic ligands including several therapeutically useful drugs. The presence of multiple binding sites in nAChRs offers an interesting possibility for the development of novel polypharmacological agents with a wide spectrum of actions.

Wnt5a inhibits K(+) currents in hippocampal synapses through nitric oxide production.

Hippocampal synapses play a key role in memory and learning processes by inducing long-term potentiation and depression. Wnt signaling is essential in the development and maintenance of synapses via several mechanisms. We have previously found that Wnt5a induces the production of nitric oxide (NO), which modulates NMDA receptor expression in the postsynaptic regions of hippocampal neurons. Here, we report that Wnt5a selectively inhibits a voltage-gated K(+) current (Kv current) and increases synaptic activity in hippocampal slices. Further supporting a specific role for Wnt5a, the soluble Frizzled receptor protein (sFRP-2; a functional Wnt antagonist) fully inhibits the effects of Wnt5a. We additionally show that these responses to Wnt5a are mediated by activation of a ROR2 receptor and increased NO production because they are suppressed by the shRNA-mediated knockdown of ROR2 and by 7-nitroindazole, a specific inhibitor of neuronal NOS. Together, our results show that Wnt5a increases NO production by acting on ROR2 receptors, which in turn inhibit Kv currents. These results reveal a novel mechanism by which Wnt5a may regulate the excitability of hippocampal neurons.

Startup and oxygen concentration effects in a continuous granular mixed flow autotrophic nitrogen removal reactor.

The startup and performance of the completely autotrophic nitrogen removal over nitrite (CANON) process was tested in a continuously fed granular bubble column reactor (BCR) with two different aeration strategies: controlling the oxygen volumetric flow and oxygen concentration. During the startup with the control of oxygen volumetric flow, the air volume was adjusted to 60mL/h and the CANON reactor had volumetric N loadings ranging from 7.35 to 100.90mgN/Ld with 36-71% total nitrogen removal and high instability. In the second stage, the reactor was operated at oxygen concentrations of 0.6, 0.4 and 0.2mg/L. The best condition was 0.2 mgO2/L with a total nitrogen removal of 75.36% with a CANON reactor activity of 0.1149gN/gVVSd and high stability. The feasibility and effectiveness of CANON processes with oxygen control was demonstrated, showing an alternative design tool for efficiently removing nitrogen species.

Serotonin receptors expressed in Drosophila mushroom bodies differentially modulate larval locomotion.

Drosophila melanogaster has been successfully used as a simple model to study the cellular and molecular mechanisms underlying behaviors, including the generation of motor programs. Thus, it has been shown that, as in vertebrates, CNS biogenic amines (BA) including serotonin (5HT) participate in motor control in Drosophila. Several evidence show that BA systems innervate an important association area in the insect brain previously associated to the planning and/or execution of motor programs, the Mushroom Bodies (MB). The main objective of this work is to evaluate the contribution of 5HT and its receptors expressed in MB to motor behavior in fly larva. Locomotion was evaluated using an automated tracking system, in Drosophila larvae (3(rd)-instar) exposed to drugs that affect the serotonergic neuronal transmission: alpha-methyl-L-dopa, MDMA and fluoxetine. In addition, animals expressing mutations in the 5HT biosynthetic enzymes or in any of the previously identified receptors for this amine (5HT1AR, 5HT1BR, 5HT2R and 5HT7R) were evaluated in their locomotion. Finally, RNAi directed to the Drosophila 5HT receptor transcripts were expressed in MB and the effect of this manipulation on motor behavior was assessed. Data obtained in the mutants and in animals exposed to the serotonergic drugs, suggest that 5HT systems are important regulators of motor programs in fly larvae. Studies carried out in animals pan-neuronally expressing the RNAi for each of the serotonergic receptors, support this idea and further suggest that CNS 5HT pathways play a role in motor control. Moreover, animals expressing an RNAi for 5HT1BR, 5HT2R and 5HT7R in MB show increased motor behavior, while no effect is observed when the RNAi for 5HT1AR is expressed in this region. Thus, our data suggest that CNS 5HT systems are involved in motor control, and that 5HT receptors expressed in MB differentially modulate motor programs in fly larvae.

Neonicotinic analogues: selective antagonists for α4ß2 nicotinic acetylcholine receptors.

Nicotine is an agonist of nicotinic acetylcholine receptors (nAChRs) that has been extensively used as a template for the synthesis of α4ß2-preferring nAChRs. Here, we used the N-methyl-pyrrolidine moiety of nicotine to design and synthesise novel α4ß2-preferring neonicotinic ligands. We increased the distance between the basic nitrogen and aromatic group of nicotine by introducing an ester functionality that also mimics acetylcholine (Fig. 2). Additionally, we introduced a benzyloxy group linked to the benzoyl moiety. Although the neonicotinic compounds fully inhibited binding of both [α-(125)I]bungarotoxin to human α7 nAChRs and [(3)H]cytisine to human α4ß2 nAChRs, they were markedly more potent at displacing radioligand binding to human α4ß2 nAChRs than to α7 nAChRs. Functional assays showed that the neonicotinic compounds behave as antagonists at α4ß2 and α4ß2α5 nAChRs. Substitutions on the aromatic ring of the compounds produced compounds that displayed marked selectivity for α4ß2 or α4ß2α5 nAChRs. Docking of the compounds on homology models of the agonist binding site at the α4/ß2 subunit interfaces of α4ß2 nAChRs suggested the compounds inhibit function of this nAChR type by binding the agonist binding site.

nAChR-induced octopamine release mediates the effect of nicotine on a startle response in Drosophila melanogaster.

Biogenic amines (BAs) play a central role in the generation of complex behaviors in vertebrates and invertebrates, including the fly Drosophila melanogaster. The comparative advantages of Drosophila as a genetic model to study the contribution of BAs to behaviors stumble upon the difficulty to access the fly brain to ask relevant physiological questions. For instance, it is not known whether the activation of nicotinic acetylcholine receptors (nAChRs) induces the release of BAs in fly brain, a phenomenon associated to several behaviors in vertebrates. Here, we describe a new preparation to study the efflux of BAs in the adult fly brain by in vitro chronoamperometry. Using this preparation we show that nAChR agonists including nicotine induce a fast, transient, dose-dependent efflux of endogenous BAs, an effect mediated by α-bungarotoxin-sensitive nAChRs. By using different genetic tools we demonstrate that the BA whose efflux is induced by nAChR activation is octopamine (Oct). Furthermore, we show that the impairment of a mechanically induced startle response after nicotine exposure is not observed in flies deficient in Oct transmission. Thus, our data show that the efflux of BAs in Drosophila brain is increased by nAChR activation as in vertebrates, and that then AChR-induced Oct release could have implications in a nicotine-induced behavioral response.

Inhibition of rat carotid body glomus cells TASK-like channels by acute hypoxia is enhanced by chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea, enhances carotid body (CB) chemosensory responses to acute hypoxia. In spite of that, the primary molecular target of CIH in the CB remains unknown. A key step of the hypoxic response in the CB is the chemoreceptor cell depolarization elicited by the inhibition of K(+) channels. Thus, we tested the hypothesis that CIH potentiates the hypoxic-induced depolarization of rat CB chemoreceptor cells by enhancing the inhibition of a background K(+) TASK-like channel. Membrane potential, single channel and macroscopic currents were recorded in the presence of TEA and 4-aminopyridine in CB chemoreceptor cells isolated from adult rats exposed to CIH. The CIH treatment did not modify the resting membrane properties but the hypoxic-evoked depolarization increased by 2-fold. In addition, the hypoxic inhibition of the TASK-like channel current was larger and faster in glomus cells from CIH-treated animals. This novel effect of CIH may contribute to explain the enhancing effect of CIH on CB oxygen chemoreception.

Contribution of TASK-like potassium channels to the enhanced rat carotid body responsiveness to hypoxia.

A major hallmark of obstructive sleep apnea is the potentiation of the carotid body (CB) chemosensory response to acute hypoxia, as result of the chronic intermittent hypoxia (CIH) exposition. Several mechanisms have been involved in this CB chemosensory potentiation, but the primary target of CIH remains elusive. In physiological conditions, hypoxia depolarized CB chemoreceptor cells, trigger an increase of intracellular Ca(2+), and the subsequent transmitter's release. Since the depolarization is initiated by the inhibition of a TASK-like K(+) channel, we studied if CIH may increase the amplitude of the hypoxic-induced depolarization in the chemoreceptor cells, due to an enhanced inhibition of the TASK-like current.CBs obtained from adult rats exposed to CIH (5% O2, 12 times/hr for 8 hr/day) for 7 days were acute dissociated, and the membrane potential and TASK-like current were recorded from isolated chemoreceptor cells. Resting membrane properties were not modified by CIH, but the amplitude of the hypoxic-evoked depolarization increases ∼2-fold. The same result was obtained when all the voltage-dependent K(+) currents were pharmacologically blocked. Accordingly, the inhibition of the TASK-like current induced by acute hypoxia (PO(2) ∼5 torr) increased from ∼62% in control cells to ∼96% in the CIH cells.Present results show that acute hypoxic inhibition of TASK-like K(+) channel is potentiated by CIH exposure, suggesting that the enhancing effect of CIH on CB chemosensory responsiveness to hypoxia occurs at the initial step of the oxygen transduction in the CB chemoreceptor cells.

Functional expression of the α7 and α4-containing nicotinic acetylcholine receptors on the neonatal rat carotid body.

The carotid bodies (CBs) are chemosensory organs that respond to hypoxemia with transmitter neurosecretion, leading to a respiratory reflex response. It has been proposed that acetylcholine is a key regulator of transmitter release through activation of presynaptic nicotinic acetylcholine receptors (nAChRs). In the present work, we studied the identity of such nAChRs and their contribution to catecholamine release from CBs. Neonatal rat CBs were placed in a recording chamber for electrochemical recordings or disassociated for voltage-clamp studies on isolated cells. Fast nicotine superfusion increases catecholamine release from intact CBs. This response was diminished reversibly by the non-selective nAChR blocker hexamethonium, by the selective α7 blocker α-bungarotoxin and by the α4-containing nAChR blocker erysodine. In isolated CB cells the nAChR agonists nicotine, acetylcholine and cytisine all evoke inward currents with similar potencies. The nicotine-evoked current was fully blocked by mecamylamine and partially inhibited by α-bungarotoxin or erysodine. However, the combination of both α-bungarotoxin an erysodine failed to suppress this response. Immunodetection studies confirm the presence of α7 and α4 subunits in isolated dopaminergic CB cells. Our results show that activation of α7 and/or α4-containing nAChR subtypes have the ability to regulate catecholamine release from intact CB due to activation of fast inward currents expressed in chemoreceptor cells. Therefore, our results suggest that both nAChR subtypes contribute to the cholinergic nicotinic regulation of catecholamine signaling in the carotid body system.

Muscarinic modulation of TASK-like background potassium channel in rat carotid body chemoreceptor cells.

The carotid body is the main peripheral arterial chemoreceptor and it is essential to initiate the cardiovascular and respiratory compensatory reflex responses to a decrease in the arterial oxygen. The carotid body chemoreceptor (type-I) cells respond to hypoxia with membrane depolarization, voltage-gated Ca(2+) entry and secretion of transmitters. A key step in this response is the inhibition of a TASK-like background K(+) current. It has been reported that TASK-K(+) channels can be modulated by G-protein coupled receptors, such as the muscarinic acetylcholine receptor (mAChRs). Since there is a proposed role for ACh as an autocrine/paracrine modulator of the carotid body function, we have investigated the possible regulation of the background K(+) current by mAChRs. In identified type-I cells, methacholine (100microM) or muscarine (50microM) increased intracellular Ca(2+) levels. In cell-attached patch recordings, TASK-K(+) background channel activity was reduced by approximately 50% during mAChR activation and by the diacylglycerol analogue oleoylacetylglycerol (OAG, 20microM). The co-application of both metacholine and OAG do not further inhibit K(+) channel activity. In addition, two chemically different inhibitors of protein kinase C activity, calphostin C (100nM) and chelerythrine (50microM) are both able to suppress the muscarinic inhibition of the TASK-like K(+) channel and to increase channel activity in the absence of mAChR agonists. Our results suggest a muscarinic regulation of the TASK-like K(+) current in rat carotid body type-I cells through a PLC/PKC-dependent pathway. Additionally, our findings are consistent with an autocrine/paracrine role for cholinergic autoreceptors present within the carotid body.

Modulation of TASK-like background potassium channels in rat arterial chemoreceptor cells by intracellular ATP and other nucleotides.

The carotid body's physiological role is to sense arterial oxygen, CO(2) and pH. It is however, also powerfully excited by inhibitors of oxidative phosphorylation. This latter observation is the cornerstone of the mitochondrial hypothesis which proposes that oxygen is sensed through changes in energy metabolism. All of these stimuli act in a similar manner, i.e. by inhibiting a background TASK-like potassium channel (K(B)) they induce membrane depolarization and thus neurosecretion. In this study we have evaluated the role of ATP in modulating K(B) channels. We find that K(B) channels are strongly activated by MgATP (but not ATP(4)(-)) within the physiological range (K(1/2) = 2.3 mm). This effect was mimicked by other Mg-nucleotides including GTP, UTP, AMP-PCP and ATP-gamma-S, but not by PP(i) or AMP, suggesting that channel activity is regulated by a Mg-nucleotide sensor. Channel activation by MgATP was not antagonized by either 1 mm AMP or 500 microm ADP. Thus MgATP is probably the principal nucleotide regulating channel activity in the intact cell. We therefore investigated the effects of metabolic inhibition upon both [Mg(2+)](i), as an index of MgATP depletion, and channel activity in cell-attached patches. The extent of increase in [Mg(2+)](i) (and thus MgATP depletion) in response to inhibition of oxidative phosphorylation were consistent with a decline in [MgATP](i) playing a prominent role in mediating inhibition of K(B) channel activity, and the response of arterial chemoreceptors to metabolic compromise.

Electrical and pharmacological properties of petrosal ganglion neurons that innervate the carotid body.

The petrosal ganglion (PG) contains the somata of primary afferent neurons that innervate the chemoreceptor (glomus) cells in the carotid body (CB). The most accepted model of CB chemoreception states that natural stimuli trigger the release of one or more transmitters from glomus cells, which in turn acting on specific post-synaptic receptors increases the rate of discharge in the nerve endings of PG neurons. However, PG neurons that project to the CB represent only small fraction (roughly 20%) of the whole PG and their identification is not simple since their electrophysiological and pharmacological properties are not strikingly different as compared with other PG neurons, which project to the carotid sinus or the tongue. In addition, differences reported on the actions of putative transmitters on PG neurons may reflect true species differences. Nevertheless, some experimental strategies have contributed to identify and characterize the properties of PG neurons that innervate the CB. In this review, we examined the electrophysiological properties and pharmacological responses of PG neurons to putative CB excitatory transmitters, focusing on the methods of study and species differences. The evidences suggest that ACh and ATP play a major role in the fast excitatory transmission between glomus cells and chemosensory nerve endings in the cat, rat and rabbit. However, the role of other putative transmitters such as dopamine, 5-HT and GABA is less clear and depends on the specie studied.

ATP- and ACh-induced responses in isolated cat petrosal ganglion neurons.

Chemoreceptor (glomus) cells of the carotid body are synaptically connected to the sensory nerve endings of petrosal ganglion (PG) neurons. In response to natural stimuli, the glomus cells release transmitters, which acting on the nerve terminals of petrosal neurons increases the chemosensory afferent discharge. Among several transmitter molecules present in glomus cells, acetylcholine (ACh) and adenosine 5'-triphosphate (ATP) are considered to act as excitatory transmitter in this synapse. To test if ACh and ATP play a role as excitatory transmitters in the cat CB, we recorded the electrophysiological responses from PG neurons cultured in vitro. Under voltage clamp, ATP induces a concentration-dependent inward current that partially desensitizes during 20-30 s application pulses. The ATP-induced current has a threshold near 100 nM and saturates between 20-50 muM. ACh induces a fast, inactivating inward current, with a threshold between 10-50 muM, and saturates around 1 mM. A large part of the population of PG neurons (60%) respond to both ATP and ACh. Present results support the hypothesis that ACh and ATP act as excitatory transmitters between cat glomus cells and PG neurons.

Electrophysiological characterization of nicotinic acetylcholine receptors in cat petrosal ganglion neurons in culture: effects of cytisine and its bromo derivatives.

Petrosal ganglion neurons are depolarized and fire action potentials in response to acetylcholine and nicotine. However, little is known about the subtype(s) of nicotinic acetylcholine receptors involved, although alpha4 and alpha7 subunits have been identified in petrosal ganglion neurons. Cytisine, an alkaloid unrelated to nicotine, and its bromo derivatives are agonists exhibiting different affinities, potencies and efficacies at nicotinic acetylcholine receptors containing alpha4 or alpha7 subunits. To characterize the receptors involved, we studied the effects of these agonists and the nicotinic acetylcholine receptor antagonists hexamethonium and alpha-bungarotoxin in isolated petrosal ganglion neurons. Petrosal ganglia were excised from anesthetized cats and cultured for up to 16 days. Using patch-clamp technique, we recorded whole-cell currents evoked by 5-10 s applications of acetylcholine, cytisine or its bromo derivatives. Agonists and antagonists were applied by gravity from a pipette near the neuron surface. Neurons responded to acetylcholine, cytisine, 3-bromocytisine and 5-bromocytisine with fast inward currents that desensitized during application of the stimuli and were reversibly blocked by 1 microM hexamethonium or 10 nM alpha-bungarotoxin. The order of potency of the agonists was 3-bromocytisine >> acetylcholine approximately = cytisine >> 5-bromocytisine, suggesting that homomeric alpha7 neuronal nicotinic receptors predominate in cat petrosal ganglion neurons in culture.