Regulation of nicotinic acetylcholine receptors by post-translational modifications.

Nicotinic acetylcholine receptors (nAChRs) comprise a family of pentameric ligand-gated ion channels widely distributed in the central and peripheric nervous system and in non-neuronal cells. nAChRs are involved in chemical synapses and are key actors in vital physiological processes throughout the animal kingdom. They mediate skeletal muscle contraction, autonomic responses, contribute to cognitive processes, and regulate behaviors. Dysregulation of nAChRs is associated with neurological, neurodegenerative, inflammatory and motor disorders. In spite of the great advances in the elucidation of nAChR structure and function, our knowledge about the impact of post-translational modifications (PTMs) on nAChR functional activity and cholinergic signaling has lagged behind. PTMs occur at different steps of protein life cycle, modulating in time and space protein folding, localization, function, and protein-protein interactions, and allow fine-tuned responses to changes in the environment. A large body of evidence demonstrates that PTMs regulate all levels of nAChR life cycle, with key roles in receptor expression, membrane stability and function. However, our knowledge is still limited, restricted to a few PTMs, and many important aspects remain largely unknown. There is thus a long way to go to decipher the association of aberrant PTMs with disorders of cholinergic signaling and to target PTM regulation for novel therapeutic interventions. In this review we provide a comprehensive overview of what is known about how different PTMs regulate nAChR.

Cannabidiol as a modulator of α7 nicotinic receptors.

Cannabidiol (CBD), an important terpenoid compound from marijuana with no psychoactive effects, has become of great pharmaceutical interest for several health conditions. As CBD is a multitarget drug, there is a need to establish the molecular mechanisms by which CBD may exert therapeutic as well as adverse effects. The α7 nicotinic acetylcholine receptor (α7 nAChR) is a cation-permeable ACh-gated channel present in the nervous system and in non-neuronal cells. It is involved in different pathological conditions, including neurological and neurodegenerative disorders, inflammation, and cancer. By high-resolution single-channel recordings and confocal microscopy, we here reveal how CBD modulates α7 nAChR ionotropic and metabotropic functions. CBD leads to a profound concentration-dependent decrease of α7 nAChR single-channel activity with an IC50 in the sub-micromolar range. The inhibition of α7 nAChR activity, which takes place through a membrane pathway, is neither mediated by receptor phosphorylation nor overcome by positive allosteric modulators and is compatible with CBD stabilization of resting or desensitized α7 nAChR conformational states. CBD modulation is complex as it also leads to the later appearance of atypical, low-frequency α7 nAChR channel openings. At the cellular level, CBD inhibits the increase in intracellular calcium triggered by α7 nAChR activation, thus decreasing cell calcium responses. The modulation of α7 nAChR is of pharmacological relevance and should be considered in the evaluation of CBD potential therapeutic uses. Thus, our study provides novel molecular information of CBD multiple actions and targets, which is required to set the basis for prospective applications in human health.

Vigilancia epidemiológica de COVID-19 mediante testeos agrupados focalizada en trabajadores de salud asintomáticos en Bahía Blanca / Epidemiological surveillance of COVID-19 through group testing focused on asymptomatic health workers in Bahía Blanca

INTRODUCCIÓN: Uno de los desafíos más relevantes al comienzo de la pandemia consistió en implementar estrategias dirigidas a prevenir la transmisión del virus SARS-CoV-2 y mitigar el impacto de la COVID-19. El propósito de este estudio fue contribuir a reducir la transmisión comunitaria a través de una iniciativa interinstitucional, cuyos objetivos fueron validar un método de detección de ARN del SARS-CoV-2 e implementar y evaluar la vigilancia en trabajadores de salud asintomáticos de instituciones de salud pública de Bahía Blanca. MÉTODOS: Se validó una prueba de detección del gen E del coronavirus mediante RT-PCR a partir de ARN aislado de hisopados nasofaríngeos. Para aumentar la capacidad de testeo se validó la detección del ARN viral en muestras agrupadas (pooles). Se realizó un estudio de cohorte prospectiva entre el 15/09/20 y el 15/09/21. RESULTADOS: La sensibilidad y especificidad de la prueba en muestras individuales fue del 95% (IC 95%: 85%-100%). La sensibilidad de la detección en pooles fue del 73% (IC 95%: 46%-99%) y la especificidad, del 100%. A lo largo de la vigilancia se incluyeron 855 trabajadores y 1764 hisopados, con una incidencia acumulada anual de 2,3% (IC 95%: 1,2%-3,4%). Se detectaron 20 casos asintomáticos positivos. DISCUSIÓN: El tamizaje de trabajadores de salud asintomáticos en la pandemia contribuyó a reducir el riesgo de brotes hospitalarios. Asimismo, se generó un marco de trabajo interdisciplinario aplicable a otros problemas de salud.

COVID-19 epidemiological surveillance through pool testing focused on asymptomatic healthcare workers in the city of Bahía Blanca / Vigilancia epidemiológica de covid-19 mediante testeos agrupados focalizada en trabajadores de salud asintomáticos en bahía blanca

RESUMEN INTRODUCCIÓN: Uno de los desafíos más relevantes al comienzo de la pandemia consistió en implementar estrategias dirigidas a prevenir la transmisión del virus SARS-CoV-2 y mitigar el impacto de la COVID-19. El propósito de este estudio fue contribuir a reducir la transmisión comunitaria a través de una iniciativa interinstitucional, cuyos objetivos fueron validar un método de detección de ARN del SARS-CoV-2 e implementar y evaluar la vigilancia en trabajadores de salud asintomáticos de instituciones de salud pública de Bahía Blanca. MÉTODOS: Se validó una prueba de detección del gen E del coronavirus mediante RT-PCR a partir de ARN aislado de hisopados nasofaríngeos. Para aumentar la capacidad de testeo se validó la detección del ARN viral en muestras agrupadas (pooles). Se realizó un estudio de cohorte prospectiva entre el 15/09/20 y el 15/09/21. RESULTADOS: La sensibilidad y especificidad de la prueba en muestras individuales fue del 95% (IC 95%: 85%-100%). La sensibilidad de la detección en pooles fue del 73% (IC 95%: 46%-99%) y la especificidad, del 100%. A lo largo de la vigilancia se incluyeron 855 trabajadores y 1764 hisopados, con una incidencia acumulada anual de 2,3% (IC 95%: 1,2%-3,4%). Se detectaron 20 casos asintomáticos positivos. DISCUSIÓN: El tamizaje de trabajadores de salud asintomáticos en la pandemia contribuyó a reducir el riesgo de brotes hospitalarios. Asimismo, se generó un marco de trabajo interdisciplinario aplicable a otros problemas de salud.

ABSTRACT INTRODUCTION: One of the most important challenges at the beginning of the pandemic was to implement strategies to prevent SARS-CoV-2 virus transmission and reduce the impact of COVID-19. The purpose of this study was to contribute to the reduction of community transmission through an interinstitutional initiative, aimed at validating a SARS-CoV-2 RNA detection method, and at implementing and assessing the surveillance of asymptomatic infected healthcare workers (HCWs) at public health institutions in the city of Bahía Blanca. METHODS: To validate a coronavirus RNA detection method, RNA was extracted from nasopharyngeal swabs and identification of the viral E gene was done by RT-PCR. Validation of sample pooling was performed to increase the testing capacity. A prospective cohort study was conducted from 15 September 2020 to 15 September 2021. RESULTS: The sensitivity and specificity of the test in individual samples was 95% (CI 95%: 85%-100%). The sensitivity of the pooling strategy was 73% (CI 95%: 46%-99%) and the specificity was 100%. A total of the 855 HCWs were included in the surveillance and 1764 swabs were performed, with an annual cumulative incidence of 2.3% (CI 95%: 1,2%-3,4%), and 20 positive asymptomatic cases were detected. DISCUSSION: The screening of asymptomatic HCWs during the pandemic contributed to reduce the risk of outbreaks in hospital settings. Moreover, an interdisciplinary team framework applicable to other health problems was generated.

Tyrosine phosphorylation differentially fine-tunes ionotropic and metabotropic responses of human α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor is involved in neurological, neurodegenerative, and inflammatory disorders. It operates both as a ligand-gated cationic channel and as a metabotropic receptor in neuronal and non-neuronal cells. As protein phosphorylation is an important cell function regulatory mechanism, deciphering how tyrosine phosphorylation modulates α7 dual ionotropic/metabotropic molecular function is required for understanding its integral role in physiological and pathological processes. α7 single-channel activity elicited by ACh appears as brief isolated openings and less often as episodes of few openings in quick succession. The reduction of phosphorylation by tyrosine kinase inhibition increases the duration and frequency of activation episodes, whereas the inhibition of phosphatases has the opposite effect. Removal of two tyrosine residues at the α7 intracellular domain recapitulates the effects mediated by tyrosine kinase inhibition. The tyrosine-free mutant receptor shows longer duration-activation episodes, reduced desensitization rate and significantly faster recovery from desensitization, indicating that phosphorylation decreases α7 channel activity by favoring the desensitized state. However, the mutant receptor is incapable of triggering ERK1/2 phosphorylation in response to the α7-agonist. Thus, while tyrosine phosphorylation is absolutely required for α7-triggered ERK pathway, it negatively modulates α7 ionotropic activity. Overall, phosphorylation/dephosphorylation events fine-tune the integrated cell response mediated by α7 activation, thus having a broad impact on α7 cholinergic signaling.

Alterations in the memory of rat offspring exposed to low levels of fluoride during gestation and lactation: Involvement of the α7 nicotinic receptor and oxidative stress.

Daily exposure to fluoride (F) depends mainly on the intake of this element with drinking water. When administered during gestation and lactation, F has been associated with cognitive deficits in the offspring. However, the mechanisms underlying the neurotoxicity of F remain obscure. In the current study, we investigated the effects of oral exposure to low levels of F during the gestational and lactation periods, on the memory of adult female rat offspring. We also considered a possible underlying neurotoxic mechanism. Our results showed that this exposure reduced step-down latency in the inhibitory avoidance task, and decreased both mRNA expression of the α7 nicotinic receptor (nAChR) and catalase activity in hippocampus. Our data indicates that low F concentrations administrated during gestation and lactation decrease the memory of 90-day-old female offspring. This suggests that the mechanism might be connected with an α7 nAChR deficit in the hippocampus, induced by oxidative stress.

A human-specific, truncated α7 nicotinic receptor subunit assembles with full-length α7 and forms functional receptors with different stoichiometries.

The cholinergic α7 nicotinic receptor gene, CHRNA7, encodes a subunit that forms the homopentameric α7 receptor, involved in learning and memory. In humans, exons 5-10 in CHRNA7 are duplicated and fused to the FAM7A genetic element, giving rise to the hybrid gene CHRFAM7A Its product, dupα7, is a truncated subunit lacking part of the N-terminal extracellular ligand-binding domain and is associated with neurological disorders, including schizophrenia, and immunomodulation. We combined dupα7 expression on mammalian cells with patch clamp recordings to understand its functional role. Transfected cells expressed dupα7 protein, but they exhibited neither surface binding of the α7 antagonist α-bungarotoxin nor responses to acetylcholine (ACh) or to an allosteric agonist that binds to the conserved transmembrane region. To determine whether dupα7 assembles with α7, we generated receptors comprising α7 and dupα7 subunits, one of which was tagged with conductance substitutions that report subunit stoichiometry and monitored ACh-elicited channel openings in the presence of a positive allosteric α7 modulator. We found that α7 and dupα7 subunits co-assemble into functional heteromeric receptors, which require at least two α7 subunits for channel opening, and that dupα7's presence in the pentameric arrangement does not affect the duration of the potentiated events compared with that of α7. Using an α7 subunit mutant, we found that activation of (α7)2(dupα7)3 receptors occurs through ACh binding at the α7/α7 interfacial binding site. Our study contributes to the understanding of the modulation of α7 function by the human specific, duplicated subunit, associated with human disorders.

Low arsenic concentrations impair memory in rat offpring exposed during pregnancy and lactation: Role of α7 nicotinic receptor, glutamate and oxidative stress.

Inorganic arsenic (iAs) is an important natural pollutant. Millions of individuals worldwide drink water with high levels of iAs. Arsenic exposure has been associated to cognitive deficits. However, the underlying mechanisms remain unknown. In the present work we investigated in female adult offspring the effect of the exposure to low arsenite sodium levels through drinking water during pregnancy and lactation on short- and long-term memory. We also considered a possible underlying neurotoxic mechanism. Pregnant rats were exposed during pregnancy and lactation to environmentally relevant iAs concentrations (0.05 and 0.10 mg/L). In 90-day-old female offspring, short-term memory (STM) and long-term memory (LTM) were evaluated using a step-down inhibitory avoidance task. In addition, we evaluated the α7 nicotinic receptor (α7-nAChR) expression, the transaminases and the oxidative stress levels in hippocampus. The results showed that the exposure to 0.10 mg/L iAs in this critical period produced a significant impairment in the LTM retention. This behavioral alteration might be associated with several events that occur in the hippocampus: decrease in α7-nAChR expression, an increase of glutamate levels that may produce excitotoxicity, and a decrease in the antioxidant enzyme catalase (CAT) activity.

Molecular function of α7 nicotinic receptors as drug targets.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels involved in many physiological and pathological processes. In vertebrates, there are seventeen different nAChR subunits that combine to yield a variety of receptors with different pharmacology, function, and localization. The homomeric α7 receptor is one of the most abundant nAChRs in the nervous system and it is also present in non-neuronal cells. It plays important roles in cognition, memory, pain, neuroprotection, and inflammation. Its diverse physiological actions and associated disorders have made of α7 an attractive novel target for drug modulation. Potentiation of the α7 receptor has emerged as a novel therapeutic strategy for several neurological diseases, such as Alzheimer's and Parkinson's diseases, and inflammatory disorders. In contrast, increased α7 activity has been associated with cancer cell proliferation. The presence of different drug target sites offers a great potential for α7 modulation in different pathological contexts. In particular, compounds that target allosteric sites offer significant advantages over orthosteric agonists due to higher selectivity and a broader spectrum of degrees and mechanisms of modulation. Heterologous expression of α7, together with chaperone proteins, combined with patch clamp recordings have provided important advances in our knowledge of the molecular basis of α7 responses and their potential modulation for pathological processes. This review gives a synthetic view of α7 and its molecular function, focusing on how its unique activation and desensitization features can be modified by pharmacological agents. This fundamental information offers insights into therapeutic strategies.

Neurotransmitter GABA activates muscle but not α7 nicotinic receptors.

Cys-loop receptors are neurotransmitter-activated ion channels involved in synaptic and extrasynaptic transmission in the brain and are also present in non-neuronal cells. As GABAA and nicotinic receptors (nAChR) belong to this family, we explored by macroscopic and single-channel recordings whether the inhibitory neurotransmitter GABA has the ability to activate excitatory nAChRs. GABA differentially activates nAChR subtypes. It activates muscle nAChRs, with maximal peak currents of about 10% of those elicited by acetylcholine (ACh) and 15-fold higher EC50 with respect to ACh. At the single-channel level, the weak agonism is revealed by the requirement of 20-fold higher concentration of GABA for detectable channel openings, a major population of brief openings, and absence of clusters of openings when compared with ACh. Mutations at key residues of the principal binding-site face of muscle nAChRs (αY190 and αG153) affect GABA activation similarly as ACh activation, whereas a mutation at the complementary face (εG57) shows a selective effect for GABA. Studies with subunit-lacking receptors show that GABA can activate muscle nAChRs through the α/δ interface. Interestingly, single-channel activity elicited by GABA is similar to that elicited by ACh in gain-of-function nAChR mutants associated to congenital myasthenic syndromes, which could be important in the progression of the disorders due to steady exposure to serum GABA. In contrast, GABA cannot elicit single-channel or macroscopic currents of α7 or the chimeric α7-serotonin-type 3 receptor, a feature important for preserving an adequate excitatory/inhibitory balance in the brain as well as for avoiding activation of non-neuronal receptors by serum GABA.

GABAA receptor plasticity in Jurkat T cells.

GABAA receptors (GABAAR) mediate inhibitory neurotransmission in the human brain. Neurons modify subunit expression, cellular distribution and function of GABAAR in response to different stimuli, a process named plasticity. Human lymphocytes have a functional neuronal-like GABAergic system with GABAAR acting as inhibitors of proliferation. We here explore if receptor plasticity occurs in lymphocytes. To this end, we analyzed human T lymphocyte Jurkat cells exposed to different physiological stimuli shown to mediate plasticity in neurons: GABA, progesterone and insulin. The exposure to 100 µM GABA differently affected the expression of GABAAR subunits measured at both the mRNA and protein level, showing an increase of α1, ß3, and γ2 subunits but no changes in δ subunit. Exposure of Jurkat cells to different stimuli produced different changes in subunit expression: 0.1 µM progesterone decreased δ and 0.5 µM insulin increased ß3 subunits. To identify the mechanisms underlying plasticity, we evaluated the Akt pathway, which is involved in the phosphorylation of ß subunits and receptor translocation to the membrane. A significant increase of phosphorylated Akt and on the expression of ß3 subunit in membrane occurred in cells exposed 15 h to GABA. To determine if plastic changes are translated into functional changes, we performed whole cell recordings. After 15 h GABA-exposure, a significantly higher percentage of cells responded to GABA application when compared to 0 and 40 h exposure, thus indicating that the detected plastic changes may have a role in GABA-modulated lymphocyte function. Our results reveal that lymphocyte GABAAR are modified by different stimuli similarly and by similar mechanisms to those in neurons. This property is of significance for the development of future therapies involving pharmacological modulation of the immune response.

An intrinsic GABAergic system in human lymphocytes.

γ-amino butyric acid (GABA) is an ubiquitous neurotransmitter in the central nervous system and it is also present in non-neuronal cells. In this study we investigated the presence of neuronal components of the GABAergic system in lymphocytes and its functional significance. By using RT-PCR we detected mRNA expression of different components of the GABAergic system in resting and mitogen-activated lymphocytes: i) GAD67, an isoform of the enzyme that synthetizes GABA; ii) VIAAT, the vesicular protein involved in GABA storage; iii) GABA transporters (GAT-1 and GAT-2); iv) GABA-T, the enzyme that catabolizes GABA; and v) subunits that conform ionotropic GABA receptors. The presence of VIAAT protein in resting and activated cells was confirmed by immunocytochemistry. The functionality of GABA transporters was evaluated by measuring the uptake of radioactive GABA. The results show that [(3)H]GABA uptake is 5-fold higher in activated than in resting lymphocytes. To determine if GABA subunits assemble into functional channels, we performed whole-cell recordings in activated lymphocytes. GABA and muscimol, a specific agonist of ionotropic GABA receptors, elicit macroscopic currents in about 10-15% of the cells. Finally, by using [(3)H]thymidine incorporation assays, we determined that the presence of agonists of GABA receptor during activation inhibits lymphocyte proliferation. Our results reveal that lymphocytes have a functional GABAergic system, similar to the neuronal one, which may operate as a modulator of T-cell activation. Pharmacological modulation of this system may provide new approaches for regulation of T-cell response.

Alpha 7 nicotinic acetylcholine receptor modulates lymphocyte activation.

AIMS: Even though the presence of alpha7 nicotinic receptor (nAChR) in lymphocytes has been demonstrated, its functional role still remains elusive. The aim of our study was to characterize alpha7 nAChRs in human lymphocytes upon phytohemagglutinin (PHA) stimulation. MAIN METHODS: Lymphocytes were activated with the mitogen PHA. alpha7 nAChRs were studied by reverse transcription-polymerase chain reaction (RT-PCR), real time PCR, flow cytometry and confocal laser scanning microscopy. The effects of nicotinic drugs on PHA-induced proliferation was evaluated by the [(3)H]-thymidine incorporation assay. KEY FINDINGS: We show that the expression of functional alpha7 receptors increases after PHA stimulation. The activation of peripheral lymphocytes by PHA increases 2.2-fold the alpha7 subunit mRNA expression and 4-fold the binding of the antagonist alpha-bungarotoxin (alpha-BTX) with respect to non activated lymphocytes. By measuring the increase of intracellular calcium in response to nicotine we determine that alpha7 receptors in lymphocytes are functional. Nicotinic drugs differentially modulate T cell activation. While nicotine tends to inhibit proliferative responses, specific alpha7 antagonists, such as alpha-BTX and methyllycaconitine, enhance cell division. SIGNIFICANCE: This study reveals that the alpha7 receptor modulates lymphocyte activation and contributes to clarifying the role of the non neuronal cholinergic system in the immune response.

Relationship between alpha 7 nAChR and apoptosis in human lymphocytes.

The presence of nicotinic receptors (nAChRs) in blood cells has been demonstrated. However, little is known about their functional roles. We have detected mRNA of alpha7 nAChR in peripheral human lymphocytes and determined that its expression is highly variable among individuals and within the same individual at different times. Upregulation of alpha7 is systematically observed after incubation of lymphocytes with nicotine or alpha-bungarotoxin. In addition, the incubation with these drugs decreases the percentage of apoptotic cells induced by the exposure to cortisol. Our results suggest that alpha7 nAChRs are involved in the modulation of cortisol-induced apoptosis.