Nicotine-mediated effects in neuronal and mouse models of synucleinopathy.

Introduction: Alpha-synuclein (α-Syn) aggregation, transmission, and contribution to neurotoxicity represent central mechanisms underlying Parkinson's disease. The plant alkaloid "nicotine" was reported to attenuate α-Syn aggregation in different models, but its precise mode of action remains unclear. Methods: In this study, we investigated the effect of 2-week chronic nicotine treatment on α-Syn aggregation, neuroinflammation, neurodegeneration, and motor deficits in D-line α-Syn transgenic mice. We also established a novel humanized neuronal model of α-Syn aggregation and toxicity based on treatment of dopaminergic neurons derived from human induced pluripotent stem cells (iPSC) with α-Syn preformed fibrils (PFF) and applied this model to investigate the effects of nicotine and other compounds and their modes of action. Results and discussion: Overall, our results showed that nicotine attenuated α-Syn-provoked neuropathology in both models. Moreover, when investigating the role of nicotinic acetylcholine receptor (nAChR) signaling in nicotine's neuroprotective effects in iPSC-derived dopaminergic neurons, we observed that while α4-specific antagonists reduced the nicotine-induced calcium response, α4 agonists (e.g., AZD1446 and anatabine) mediated similar neuroprotective responses against α-Syn PFF-provoked neurodegeneration. Our results show that nicotine attenuates α-Syn-provoked neuropathology in vivo and in a humanized neuronal model of synucleinopathy and that activation of α4ß2 nicotinic receptors might mediate these neuroprotective effects.

Differentiating the Neuropharmacological Properties of Nicotinic Acetylcholine Receptor-Activating Alkaloids.

Alkaloids that target nicotinic acetylcholine receptors (nAChR) are of great interest because of the critical role they play in mood and anxiety. However, understanding of the neuropharmacological effects of nicotinic alkaloids, such as cotinine and anatabine, is very limited. In this study, we investigated the neuropharmacological effects of three naturally occurring alkaloids-nicotine, cotinine, and anatabine-in vitro and in vivo. A single injection of nicotine induced anxiolytic-like behavioral features in mice by using the SmartCube® behavioral profiling system, while cotinine and anatabine had no detectable effect. The results were corroborated by using the zebrafish novel tank test (NTT), which showed a profound anxiolytic-like effect induced by multiple doses of nicotine after a single 20-min treatment. When the regulation of dopamine and norepinephrine release-the neurotransmitter systems relevant for anxiety-were examined in vitro, we found that nicotine stimulated the release of both norepinephrine and dopamine, while cotinine and anatabine mainly stimulated the dopamine release. The molecular targets of nicotine were confirmed to be nAChRs with its most potent activities against α4ß2 and α6/3ß2ß3 subtypes in vitro. Anatabine was a weaker agonist for these receptors than nicotine. Cotinine was the least potent nAChR compound, only being able to activate α4ß2 and α6/3ß2ß3 subtypes at high doses and no detectable activities against α3ß4 and α7 subtypes at the concentrations tested. The observed effects were unlikely due to the off-target effect, because these alkaloids did not bind or regulate >160 other molecular targets in vitro. Thus, the present results suggest that natural nicotinic alkaloids can induce an anxiolytic-like behavior in nonclinical animal models, potency of which may depend on the activation of various nAChRs and regulation of various neurotransmitter systems. Further investigations would help understand their effects on humans, because non-clinical studies should not be taken as a direct indication for human behavior and nicotine is not risk free.

Changes in H+, K+, and Ca2+ Concentrations, as Observed in Seizures, Induce Action Potential Signaling in Cortical Neurons by a Mechanism That Depends Partially on Acid-Sensing Ion Channels.

Acid-sensing ion channels (ASICs) are activated by extracellular acidification. Because ASIC currents are transient, these channels appear to be ideal sensors for detecting the onset of rapid pH changes. ASICs are involved in neuronal death after ischemic stroke, and in the sensation of inflammatory pain. Ischemia and inflammation are associated with a slowly developing, long-lasting acidification. Recent studies indicate however that ASICs are unable to induce an electrical signaling activity under standard experimental conditions if pH changes are slow. In situations associated with slow and sustained pH drops such as high neuronal signaling activity and ischemia, the extracellular K+ concentration increases, and the Ca2+ concentration decreases. We hypothesized that the concomitant changes in H+, K+, and Ca2+ concentrations may allow a long-lasting ASIC-dependent induction of action potential (AP) signaling. We show that for acidification from pH7.4 to pH7.0 or 6.8 on cultured cortical neurons, the number of action potentials and the firing time increased strongly if the acidification was accompanied by a change to higher K+ and lower Ca2+ concentrations. Under these conditions, APs were also induced in neurons from ASIC1a-/- mice, in which a pH of ≤ 5.0 would be required to activate ASICs, indicating that ASIC activation was not required for the AP induction. Comparison between neurons of different ASIC genotypes indicated that the ASICs modulate the AP induction under such changed ionic conditions. Voltage-clamp measurements of the Na+ and K+ currents in cultured cortical neurons showed that the lowering of the pH inhibited Na+ and K+ currents. In contrast, the lowering of the Ca2+ together with the increase in the K+ concentration led to a hyperpolarizing shift of the activation voltage dependence of voltage-gated Na+ channels. We conclude that the ionic changes observed during high neuronal activity mediate a sustained AP induction caused by the potentiation of Na+ currents, a membrane depolarization due to the changed K+ reversal potential, the activation of ASICs, and possibly effects on other ion channels. Our study describes therefore conditions under which slow pH changes induce neuronal signaling by a mechanism involving ASICs.

Effects of nicotinic acetylcholine receptor-activating alkaloids on anxiety-like behavior in zebrafish.

Alkaloids are a structurally complex group of natural products that have a diverse range of biological activities and significant therapeutic applications. In this study, we examined the acute, anxiolytic-like effects of nicotinic acetylcholine receptor (nAChR)-activating alkaloids with reported neuropharmacological effects but whose effects on anxiety are less well understood. Because α4ß2 nAChRs can regulate anxiety, we first demonstrated the functional activities of alkaloids on these receptors in vitro. Their effects on anxiety-like behavior in zebrafish were then examined using the zebrafish novel tank test (NTT). The NTT is a relatively high-throughput behavioral paradigm that takes advantage of the natural tendency of fish to dive down when stressed or anxious. We report for the first time that cotinine, anatabine, and methylanatabine may suppress this anxiety-driven zebrafish behavior after a single 20-min treatment. Effective concentrations of these alkaloids were well above the concentrations naturally found in plants and the concentrations needed to induce anxiolytic-like effect by nicotine. These alkaloids showed good receptor interactions at the α4ß2 nAChR agonist site as demonstrated by in vitro binding and in silico docking model, although somewhat weaker than that for nicotine. Minimal or no significant effect of other compounds may have been due to low bioavailability of these compounds in the brain, which is supported by the in silico prediction of blood-brain barrier permeability. Taken together, our findings indicate that nicotine, although not risk-free, is the most potent anxiolytic-like alkaloid tested in this study, and other natural alkaloids may regulate anxiety as well.

Slowing of the Time Course of Acidification Decreases the Acid-Sensing Ion Channel 1a Current Amplitude and Modulates Action Potential Firing in Neurons.

Acid-sensing ion channels (ASICs) are H+-activated neuronal Na+ channels. They are involved in fear behavior, learning, neurodegeneration after ischemic stroke and in pain sensation. ASIC activation has so far been studied only with fast pH changes, although the pH changes associated with many roles of ASICs are slow. It is currently not known whether slow pH changes can open ASICs at all. Here, we investigated to which extent slow pH changes can activate ASIC1a channels and induce action potential signaling. To this end, ASIC1a current amplitudes and charge transport in transfected Chinese hamster ovary cells, and ASIC-mediated action potential signaling in cultured cortical neurons were measured in response to defined pH ramps of 1-40 s duration from pH 7.4 to pH 6.6 or 6.0. A kinetic model of the ASIC1a current was developed and integrated into the Hodgkin-Huxley action potential model. Interestingly, whereas the ASIC1a current amplitude decreased with slower pH ramps, action potential firing was higher upon intermediate than fast acidification in cortical neurons. Indeed, fast pH changes (<4 s) induced short action potential bursts, while pH changes of intermediate speed (4-10 s) induced longer bursts. Slower pH changes (>10 s) did in many experiments not generate action potentials. Computer simulations corroborated these observations. We provide here the first description of ASIC function in response to defined slow pH changes. Our study shows that ASIC1a currents, and neuronal activity induced by ASIC1a currents, strongly depend on the speed of pH changes. Importantly, with pH changes that take >10 s to complete, ASIC1a activation is inefficient. Therefore, it is likely that currently unknown modulatory mechanisms allow ASIC activity in situations such as ischemia and inflammation.

An electrophysiological characterization of naturally occurring tobacco alkaloids and their action on human α4ß2 and α7 nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptor (nAChR) subtype-selective pharmacological profiles of tobacco alkaloids are essential for understanding the physiological effects of tobacco products. In this study, automated electrophysiology was used to functionally characterize the effects of distinct groups of tobacco alkaloids on human α4ß2 and α7 nAChRs. We found that, in tobacco alkaloids, pyridine as a hydrogen bond acceptor and a basic nitrogen atom at a distance of 4-7 Šare pharmacophoric elements necessary for molecular recognition by α4ß2 and α7 nAChRs with various degrees of selectivity, potency, and efficacy. While four alkaloids-nicotine, nornicotine, anabasine and R-anatabine-potently activated α4ß2, they were also weak agonists of α7 nAChRs. Nicotine was the most potent agonist of α4ß2, while anabasine elicited the highest activation of α7. None of the tobacco alkaloids enhanced nAChR activity elicited by the endogenous ligand acetylcholine; therefore, none was considered to be a positive allosteric modulator (PAM) of either α4ß2 or α7 nAChRs. In contrast, we identified tobacco alkaloids, such as the tryptophan metabolite 6-hydroxykynurenic acid, that decreased the activity of both α4ß2 and α7 nAChRs. Our study identified a class of alkaloids with positive and negative effects against human α4ß2 and α7 nAChRs. It also revealed human α4ß2 to be the principal receptor for sensing the most abundant alkaloids in tobacco leaves.

Accelerated Current Decay Kinetics of a Rare Human Acid-Sensing ion Channel 1a Variant That Is Used in Many Studies as Wild Type.

Acid-sensing ion channels (ASICs) are neuronal Na+-permeable ion channels that are activated by extracellular acidification and are involved in fear sensing, learning, neurodegeneration after ischemia, and in pain sensation. We have recently found that the human ASIC1a (hASIC1a) wild type (WT) clone which has been used by many laboratories in recombinant expression studies contains a point mutation that occurs with a very low frequency in humans. Here, we compared the function and expression of ASIC1a WT and of this rare variant, in which the highly conserved residue Gly212 is substituted by Asp. Residue 212 is located at a subunit interface that undergoes changes during channel activity. We show that the modulation of channel function by commonly used ASIC inhibitors and modulators, and the pH dependence, are the same or only slightly different between hASIC1a-G212 and -D212. hASIC1a-G212 has however a higher current amplitude per surface-expressed channel and considerably slower current decay kinetics than hASIC1a-D212, and its current decay kinetics display a higher dependency on the type of anion present in the extracellular solution. We demonstrate for a number of channel mutants previously characterized in the hASIC1a-D212 background that they have very similar effects in the hASIC1a-G212 background. Taken together, we show that the variant hASIC1a-D212 that has been used as WT in many studies is, in fact, a mutant and that the properties of hASIC1a-D212 and hASIC1a-G212 are sufficiently close that the conclusions made in previous pharmacology and structure-function studies remain valid.

Heteroarylguanidines as Allosteric Modulators of ASIC1a and ASIC3 Channels.

Acid-sensing ion channels (ASICs) are neuronal Na+-selective ion channels that open in response to extracellular acidification. They are involved in pain, fear, learning, and neurodegeneration after ischemic stroke. 2-Guanidine-4-methylquinazoline (GMQ) was recently discovered as the first nonproton activator of ASIC3. GMQ is of interest as a gating modifier and pore blocker of ASICs. It has however a low potency, and exerts opposite effects on ASIC1a and ASIC3. To further explore the molecular mechanisms of GMQ action, we have used the guanidinium moiety of GMQ as a scaffold and tested the effects of different GMQ derivatives on the ASIC pH dependence and maximal current. We report that GMQ derivatives containing quinazoline and quinoline induced, as GMQ, an alkaline shift of the pH dependence of activation in ASIC3 and an acidic shift in ASIC1a. Another group of 2-guanidinopyridines shifted the pH dependence of both ASIC1a and ASIC3 to more acidic values. Several compounds induced an alkaline shift of the pH dependence of ASIC1a/2a and ASIC2a/3 heteromers. Compared to GMQ, guanidinopyridines showed a 20-fold decrease in the IC50 for ASIC1a and ASIC3 current inhibition at pH 5. Strikingly, 2-guanidino-quinolines and -pyridines showed a concentration-dependent biphasic effect that resulted at higher concentrations in ASIC1a and ASIC3 inhibition (IC50 > 100 µM), while causing at lower concentration a potentiation of ASIC1a, but not ASIC3 currents (EC50 ≈ 10 µM). In conclusion, we describe a new family of small molecules as ASIC ligands and identify an ASIC subtype-specific potentiation by a subgroup of these compounds.

The function and regulation of acid-sensing ion channels (ASICs) and the epithelial Na(+) channel (ENaC): IUPHAR Review 19.

Acid-sensing ion channels (ASICs) and the epithelial Na(+) channel (ENaC) are both members of the ENaC/degenerin family of amiloride-sensitive Na(+) channels. ASICs act as proton sensors in the nervous system where they contribute, besides other roles, to fear behaviour, learning and pain sensation. ENaC mediates Na(+) reabsorption across epithelia of the distal kidney and colon and of the airways. ENaC is a clinically used drug target in the context of hypertension and cystic fibrosis, while ASIC is an interesting potential target. Following a brief introduction, here we will review selected aspects of ASIC and ENaC function. We discuss the origin and nature of pH changes in the brain and the involvement of ASICs in synaptic signalling. We expose how in the peripheral nervous system, ASICs cover together with other ion channels a wide pH range as proton sensors. We introduce the mechanisms of aldosterone-dependent ENaC regulation and the evidence for an aldosterone-independent control of ENaC activity, such as regulation by dietary K(+) . We then provide an overview of the regulation of ENaC by proteases, a topic of increasing interest over the past few years. In spite of the profound differences in the physiological and pathological roles of ASICs and ENaC, these channels share many basic functional and structural properties. It is likely that further research will identify physiological contexts in which ASICs and ENaC have similar or overlapping roles.

Calcium entry via TRPV1 but not ASICs induces neuropeptide release from sensory neurons.

Inflammatory mediators induce neuropeptide release from nociceptive nerve endings and cell bodies, causing increased local blood flow and vascular leakage resulting in edema. Neuropeptide release from sensory neurons depends on an increase in intracellular Ca(2+) concentration. In this study we investigated the role of two types of pH sensors in acid-induced Ca(2+) entry and neuropeptide release from dorsal root ganglion (DRG) neurons. The transient receptor potential vanilloid 1 channel (TRPV1) and acid-sensing ion channels (ASICs) are both H(+)-activated ion channels present in these neurons, and are therefore potential pH sensors for this process. We demonstrate with in situ hybridization and immunocytochemistry that TRPV1 and several ASIC subunits are co-expressed with neuropeptides in DRG neurons. The activation of ASICs and of TRPV1 led to an increase in intracellular Ca(2+) concentration. While TRPV1 has a high Ca(2+) permeability and allows direct Ca(2+) entry when activated, we show here that ASICs of DRG neurons mediate Ca(2+) entry mostly by depolarization-induced activation of voltage-gated Ca(2+) channels and only to a small extent via the pore of Ca(2+)-permeable ASICs. Extracellular acidification led to the release of the neuropeptide calcitonin gene-related peptide from DRG neurons. The pH dependence and the pharmacological profile indicated that TRPV1, but not ASICs, induced neuropeptide secretion. In conclusion, this study shows that although both TRPV1 and ASICs mediate Ca(2+) influx, TRPV1 is the principal sensor for acid-induced neuropeptide secretion from sensory neurons.

Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures.

The epithelial sodium channel (ENaC) is responsible for Na(+) and fluid absorption across colon, kidney, and airway epithelia. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is a secreted, innate defense protein and an autocrine inhibitor of ENaC that is highly expressed in airway epithelia. While SPLUNC1 has a bactericidal permeability-increasing protein (BPI)-type structure, its NH2-terminal region lacks structure. Here we found that an 18 amino acid peptide, S18, which corresponded to residues G22-A39 of the SPLUNC1 NH2 terminus inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold), while SPLUNC1 protein lacking this region was without effect. S18 did not inhibit the structurally related acid-sensing ion channels, indicating specificity for ENaC. However, S18 preferentially bound to the ßENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Unlike control, CF human bronchial epithelial cultures (HBECs) where airway surface liquid (ASL) height was abnormally low (4.2 ± 0.6 µm), addition of S18 prevented ENaC-led ASL hyperabsorption and maintained CF ASL height at 7.9 ± 0.6 µm, even in the presence of neutrophil elastase, which is comparable to heights seen in normal HBECs. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, suggesting that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the S18 peptide suggests that this peptide may be suitable for treating CF lung disease.

Molecular determinants of desensitization in an ENaC/degenerin channel.

Epithelial Na(+) channel (ENaC)/degenerin family members are involved in mechanosensation, blood pressure control, pain sensation, and the expression of fear. Several of these channel types display a form of desensitization that allows the channel to limit Na(+) influx during prolonged stimulation. We used site-directed mutagenesis and chemical modification, functional analysis, and molecular dynamics simulations to investigate the role of the lower palm domain of the acid-sensing ion channel 1, a member of the ENaC/degenerin family. The lower palm domains of this trimeric channel are arranged around a central vestibule, at ∼20 Šabove the plasma membrane and are covalently linked to the transmembrane channel parts. We show that the lower palm domains approach one another during desensitization. Residues in the palm co-determine the pH dependence of desensitization, its kinetics, and the stability of the desensitized state. Mutations of palm residues impair desensitization by preventing the closing movement of the palm. Overexpression of desensitization-impaired channel mutants in central neurons allowed--in contrast to overexpression of wild type--a sustained signaling response to rapid pH fluctuations. We identify and describe here the function of an important regulatory domain that most likely has a conserved role in ENaC/degenerin channels.

Subtype-specific modulation of acid-sensing ion channel (ASIC) function by 2-guanidine-4-methylquinazoline.

Acid-sensing ion channels (ASICs) are neuronal Na(+)-selective channels that are transiently activated by extracellular acidification. ASICs are involved in fear and anxiety, learning, neurodegeneration after ischemic stroke, and pain sensation. The small molecule 2-guanidine-4-methylquinazoline (GMQ) was recently shown to open ASIC3 at physiological pH. We have investigated the mechanisms underlying this effect and the possibility that GMQ may alter the function of other ASICs besides ASIC3. GMQ shifts the pH dependence of activation to more acidic pH in ASIC1a and ASIC1b, whereas in ASIC3 this shift goes in the opposite direction and is accompanied by a decrease in its steepness. GMQ also induces an acidic shift of the pH dependence of inactivation of ASIC1a, -1b, -2a, and -3. As a consequence, the activation and inactivation curves of ASIC3 but not other ASICs overlap in the presence of GMQ at pH 7.4, thereby creating a window current. At concentrations >1 mM, GMQ decreases maximal peak currents by reducing the unitary current amplitude. Mutation of residue Glu-79 in the palm domain of ASIC3, previously shown to be critical for channel opening by GMQ, disrupted the GMQ effects on inactivation but not activation. This suggests that this residue is involved in the consequences of GMQ binding rather than in the binding interaction itself. This study describes the mechanisms underlying the effects of a novel class of ligands that modulate the function of all ASICs as well as activate ASIC3 at physiological pH.