Metabolic Rewiring and Altered Glial Differentiation in an iPSC-Derived Astrocyte Model Derived from a Nonketotic Hyperglycinemia Patient.

The pathophysiology of nonketotic hyperglycinemia (NKH), a rare neuro-metabolic disorder associated with severe brain malformations and life-threatening neurological manifestations, remains incompletely understood. Therefore, a valid human neural model is essential. We aimed to investigate the impact of GLDC gene variants, which cause NKH, on cellular fitness during the differentiation process of human induced pluripotent stem cells (iPSCs) into iPSC-derived astrocytes and to identify sustainable mechanisms capable of overcoming GLDC deficiency. We developed the GLDC27-FiPS4F-1 line and performed metabolomic, mRNA abundance, and protein analyses. This study showed that although GLDC27-FiPS4F-1 maintained the parental genetic profile, it underwent a metabolic switch to an altered serine-glycine-one-carbon metabolism with a coordinated cell growth and cell cycle proliferation response. We then differentiated the iPSCs into neural progenitor cells (NPCs) and astrocyte-lineage cells. Our analysis showed that GLDC-deficient NPCs had shifted towards a more heterogeneous astrocyte lineage with increased expression of the radial glial markers GFAP and GLAST and the neuronal markers MAP2 and NeuN. In addition, we detected changes in other genes related to serine and glycine metabolism and transport, all consistent with the need to maintain glycine at physiological levels. These findings improve our understanding of the pathology of nonketotic hyperglycinemia and offer new perspectives for therapeutic options.

Functional crosstalk of the glycine transporter GlyT1 and NMDA receptors.

NMDA-type glutamate receptors (NMDARs) constitute one of the main glutamate (Glu) targets in the central nervous system and are involved in synaptic plasticity, which is the molecular substrate of learning and memory. Hypofunction of NMDARs has been associated with schizophrenia, while overstimulation causes neuronal death in neurodegenerative diseases or in stroke. The function of NMDARs requires coincidental binding of Glu along with other cellular signals such as neuronal depolarization, and the presence of other endogenous ligands that modulate their activity by allosterism. Among these allosteric modulators are zinc, protons and Gly, which is an obligatory co-agonist. These characteristics differentiate NMDARs from other receptors, and their structural bases have begun to be established in recent years. In this review we focus on the crosstalk between Glu and glycine (Gly), whose concentration in the NMDAR microenvironment is maintained by various Gly transporters that remove or release it into the medium in a regulated manner. The GlyT1 transporter is particularly involved in this task, and has become a target of great interest for the treatment of schizophrenia since its inhibition leads to an increase in synaptic Gly levels that enhances the activity of NMDARs. However, the only drug that has completed phase III clinical trials did not yield the expected results. Notwithstanding, there are additional drugs that continue to be investigated, and it is hoped that knowledge gained from the recently published 3D structure of GlyT1 may allow the rational design of more effective new drugs. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".

Experimental and Bioinformatic Insights into the Effects of Epileptogenic Variants on the Function and Trafficking of the GABA Transporter GAT-1.

In this article, we identified a novel epileptogenic variant (G307R) of the gene SLC6A1, which encodes the GABA transporter GAT-1. Our main goal was to investigate the pathogenic mechanisms of this variant, located near the neurotransmitter permeation pathway, and compare it with other variants located either in the permeation pathway or close to the lipid bilayer. The mutants G307R and A334P, close to the gates of the transporter, could be glycosylated with variable efficiency and reached the membrane, albeit inactive. Mutants located in the center of the permeation pathway (G297R) or close to the lipid bilayer (A128V, G550R) were retained in the endoplasmic reticulum. Applying an Elastic Network Model, to these and to other previously characterized variants, we found that G307R and A334P significantly perturb the structure and dynamics of the intracellular gate, which can explain their reduced activity, while for A228V and G362R, the reduced translocation to the membrane quantitatively accounts for the reduced activity. The addition of a chemical chaperone (4-phenylbutyric acid, PBA), which improves protein folding, increased the activity of GAT-1WT, as well as most of the assayed variants, including G307R, suggesting that PBA might also assist the conformational changes occurring during the alternative access transport cycle.

Proximity labeling methods for proteomic analysis of membrane proteins.

Membrane proteins constitute the filter that controls the cellular traffic of nutrients, ions and other essential molecules, as well as the transmission of signals across the membrane. These proteins interact with other proteins in the cytosol, cytoskeleton or the extracellular side of the membrane, giving rise to complex interactomes that are distributed throughout the various lipid microdomains of the membrane plane. In this manner, complex networks of protein-protein and protein-lipid interactions are formed which regulate the most diverse biological functions, and disturbance of these networks can lead to disease. Therefore, characterization of these interactomes is a priority for current biomedical sciences. Traditionally, such studies have largely depended on solubilization/dissociation of the essential components of multiprotein complexes with detergents of various strength. However, this technique may result in the loss of certain components of such complexes, especially those whose binding is weak or transient. Moreover, protein solubilization can lead to the formation of non-native spurious interactions. As an alternative, proximity labelling (PL) techniques have been developed in recent years that can identify interactors of the protein of interest in a native cellular environment, prior to solubilization. In this article, we review the recent advances in PL and explore the new possibilities they offer for the characterization of membrane interactomes. SIGNIFICANCE: Membranes establish a series of complex protein-protein and protein-lipid interactions that are essential for cell physiology. For decades, they have been one of the central objects of study in Cell and Molecular Biology. However, knowledge of the structure of membrane proteins and their respective interactomes lags far behind that of soluble proteins, mainly due to technical difficulties in their handling and characterization caused by their insolubility. Recent research has developed various techniques to study these proteins in their native cellular environment. In this review article we address the application to membrane proteins of the so-called 'proximity labeling methods', which allows neighborhood relationships to be established between proteins in intact cells. The scarcity of alternatives for study of the components of membrane complexes make these methods especially attractive for analyzing this type of membrane associated supramolecular structures.

Regulation of the Glycine Transporter GLYT1 by microRNAs.

The glycine transporter GLYT1 participates in inhibitory and excitatory neurotransmission by controlling the reuptake of this neuroactive substance from synapses. Over the past few years, microRNAs have emerged as potent negative regulators of gene expression. In this report, we investigate the possible regulation of GLYT1 by microRNAs. TargetScan software predicted the existence of multiple targets for microRNAs within the 3' UTR of the human GLYT1 (miR-7, miR-30, miR-96, miR-137 and miR-141), and as they are all conserved among mammalian orthologues, their effects on GLYT1 expression were determined experimentally. Dual reporter bioluminescent assays showed that only miR-96 and miR-137 down-regulated expression of the Renilla reporter fused to the 3' UTR of GLYT1. Mutations introduced into the target sequences blocked this inhibitory effect. Consistently, these two microRNAs downregulated the uptake of [3H]glycine into glial C6 cells, a cell line where GLYT1 is the main carrier for glycine. Moreover, the expression of endogenous GLYT1 in primary mixed cultures from rat spinal cord was decreased upon lentiviral expression of miR-96 and miR-137. Although the bulk of GLYT1 is glial, it is abundantly expressed in glycinergic neurons of the retina and in smaller amounts in glutamatergic neurons though the brain. Since miR-96 in the retina is strongly downregulated by light exposure, when rats were maintained in darkness for a few hours we observed a concomitant increase of GLYT1 expression, suggesting that at least miR-96 might be an important negative regulator of GLYT1 under physiological conditions.

Cross-cultural adaptation and validation of the Spanish version of the Johns Hopkins Fall Risk Assessment Tool.

PURPOSE: To perform a cross-cultural adaptation and validation of the Johns Hopkins Fall Risk Assessment Tool to develop its Spanish version (JHFRAT-Sp). MATERIAL AND METHODS: Two hundred eleven participants aged 60 years or older participated in this observational study. After translation and transcultural adaptation of the JHFRAT-Sp, the internal consistency, criterion validity and construct validity were calculated using the Falls Efficacy Scale International, Foot Health Status Questionnaire (FHSQ), Health Questionnaire EuroQol (5Dimensions and VAS), Short Form-12v2 and Health Assessment Questionnaire. RESULTS: The internal consistency was 0.986. The test-retest analysis ranged from 0.971 to 0.983. The error measures presented values in MDC90 and SEM of 0.602 and 1.404%, respectively. The chi-Square value was 120.662 (p < 0.001). The extraction method by principal components showed a solution of four factors. Regarding the criterion validity, the correlation value ranged from r = 0.200 (FHSQ-Vigour) to r = 0.891 (EuroQol-VAS). CONCLUSIONS: The JHFRAT was translated and adapted culturally from the original version to Spanish. The psychometric analysis carried out in the JHFRAT-Sp showed excellent reliability, as well as satisfactory results both in the measurement error analysis and in the construct and criterion validities. Spanish researchers and clinicians may use this tool to analyse the risk of falling.IMPLICATIONS FOR REHABILITATIONA transcultural translation and adaptation of the JHFRAT questionnaire into Spanish (JHFRAT-Sp) has been carried out.The JHFRAT-Sp questionnaire is shown as a tool with very satisfactory psychometric characteristics, which would allow its use by both researchers and clinicians for the evaluation and monitoring of patients at risk of falls.The results that can be extracted from the use of JHFRAT-Sp, can be compared with the same type of patients who have used the same questionnaire but in other clinical or research environments that have the validated version of JHFRAT in their native language, such as English, Chinese or Portuguese (Brazilian).

Identification by proximity labeling of novel lipidic and proteinaceous potential partners of the dopamine transporter.

Dopamine (DA) transporters (DATs) are regulated by trafficking and modulatory processes that probably rely on stable and transient interactions with neighboring proteins and lipids. Using proximity-dependent biotin identification (BioID), we found novel potential partners for DAT, including several membrane proteins, such as the transmembrane chaperone 4F2hc, the proteolipid M6a and a potential membrane receptor for progesterone (PGRMC2). We also detected two cytoplasmic proteins: a component of the Cullin1-dependent ubiquitination machinery termed F-box/LRR-repeat protein 2 (FBXL2), and the enzyme inositol 5-phosphatase 2 (SHIP2). Immunoprecipitation (IP) and immunofluorescence studies confirmed either a physical association or a close spatial proximity between these proteins and DAT. M6a, SHIP2 and the Cullin1 system were shown to increase DAT activity in coexpression experiments, suggesting a functional role for their association. Deeper analysis revealed that M6a, which is enriched in neuronal protrusions (filopodia or dendritic spines), colocalized with DAT in these structures. In addition, the product of SHIP2 enzymatic activity (phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]) was tightly associated with DAT, as shown by co-IP and by colocalization of mCherry-DAT with a specific biosensor for this phospholipid. PI(3,4)P2 strongly stimulated transport activity in electrophysiological recordings, and conversely, inhibition of SHIP2 reduced DA uptake in several experimental systems including striatal synaptosomes and the dopaminergic cell line SH-SY5Y. In summary, here we report several potential new partners for DAT and a novel regulatory lipid, which may represent new pharmacological targets for DAT, a pivotal protein in dopaminergic function of the brain.

Regulation of the voltage-dependent sodium channel NaV1.1 by AKT1.

The voltage-sensitive sodium channel NaV1.1 plays a critical role in regulating excitability of GABAergic neurons and mutations in the corresponding gene are associated to Dravet syndrome and other forms of epilepsy. The activity of this channel is regulated by several protein kinases. To identify novel regulatory kinases we screened a library of activated kinases and we found that AKT1 was able to directly phosphorylate NaV1.1. In vitro kinase assays revealed that the phosphorylation site was located in the C-terminal part of the large intracellular loop connecting domains I and II of NaV1.1, a region that is known to be targeted by other kinases like PKA and PKC. Electrophysiological recordings revealed that activated AKT1 strongly reduced peak Na+ currents and displaced the inactivation curve to more negative potentials in HEK-293 cell stably expressing NaV1.1. These alterations in current amplitude and steady-state inactivation were mimicked by SC79, a specific activator of AKT1, and largely reverted by triciribine, a selective inhibitor. Neurons expressing endogenous NaV1.1 in primary cultures were identified by expressing a fluorescent protein under the NaV1.1 promoter. There, we also observed a strong decrease in the current amplitude after addition of SC79, but small effects on the inactivation parameters. Altogether, we propose a novel mechanism that might regulate the excitability of neural networks in response to AKT1, a kinase that plays a pivotal role under physiological and pathological conditions, including epileptogenesis.

Microglia and Inhibitory Circuitry in the Medullary Dorsal Horn: Laminar and Time-Dependent Changes in a Trigeminal Model of Neuropathic Pain.

Craniofacial neuropathic pain affects millions of people worldwide and is often difficult to treat. Two key mechanisms underlying this condition are a loss of the negative control exerted by inhibitory interneurons and an early microglial reaction. Basic features of these mechanisms, however, are still poorly understood. Using the chronic constriction injury of the infraorbital nerve (CCI-IoN) model of neuropathic pain in mice, we have examined the changes in the expression of GAD, the synthetic enzyme of GABA, and GlyT2, the membrane transporter of glycine, as well as the microgliosis that occur at early (5 days) and late (21 days) stages post-CCI in the medullary and upper spinal dorsal horn. Our results show that CCI-IoN induces a down-regulation of GAD at both postinjury survival times, uniformly across the superficial laminae. The expression of GlyT2 showed a more discrete and heterogeneous reduction due to the basal presence in lamina III of 'patches' of higher expression, interspersed within a less immunoreactive 'matrix', which showed a more substantial reduction in the expression of GlyT2. These patches coincided with foci lacking any perceptible microglial reaction, which stood out against a more diffuse area of strong microgliosis. These findings may provide clues to better understand the neural mechanisms underlying allodynia in neuropathic pain syndromes.

Outcome implication of sex-related effective orifice area normalized to body size in aortic stenosis.

INTRODUCTION: Although several echocardiographic parameters have different values according to sex, there are no studies in echocardiographic variables of aortic stenosis (AS) severity. Our aim was to evaluate the sex-related prognosis of several echocardiographic parameters in AS. METHODS: Two hundred and twenty-five patients with at least moderate AS (effective orifice area [EOA] ≤ 1.50 cm2 ) were prospectively enrolled. EOA was normalized to body surface area (BSA), height, and body mass index (BMI). Receiver operating characteristic curves, in women and men separately, were plotted to determine the best cutoff value for predicting cardiovascular death. RESULTS: The largest area under the curve (AUC) to predict cardiovascular death was EOA in men (AUC 0.74, P < .001) and EOA/height in women (AUC 0.81, P < .001). An EOA/height cutoff value of 0.55 cm2 /m in women had a sensitivity of 100% and specificity of 61%; a cutoff of 0.50 cm2 /m in men obtained a sensitivity of 92% and a specificity of 56%. During a mean follow-up of 247 ± 183 days, there were 33 cardiovascular deaths. Women with EOA/height ≤ 0.55 cm2 /m had higher cardiovascular mortality (22% vs 0%, P < .001) and men with EOA/height ≤ 0.50 cm2 /m (21% vs 2%, P < .001). One-year survival in women with EOA/height ≤ 0.55 cm2 /m was 67 ± 8% and 100 ± 0% in EOA/height > 0.55 cm2 /m (P < .001). In men, 1-year survival was 70 ± 8% in EOA/height ≤ 0.50 cm2 /m, and 93 ± 6% in EOA/height > 0.50 cm2 /m (P = .004). CONCLUSIONS: Normalization of EOA is useful in AS, especially in women. We recommend using an EOA/height cutoff value of 0.55 cm2 /m in women, and 0.50 cm2 /m in men to identify a subgroup with higher cardiovascular risk.

Resección quirúrgica de papilomatosis glandular endobronquial / Surgical Resection of Endobronchial Glandular Papilloma

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Identification of potassium channel proteins Kv7.2/7.3 as common partners of the dopamine and glutamate transporters DAT and GLT-1.

Dopamine and glutamate transporters (DAT and GLT-1, respectively) share some biophysical characteristics, as both are secondary active carriers coupled to electrochemical ion gradients. In order to identify common or specific components of their respective proteomes, we performed a proximity labelling assay (BioID) in the hippocampal cell line HT22. While most of the identified proteins were specific for each transporter (and will be analyzed elsewhere), we detected two membrane proteins in the shared interactome of GLT-1 and DAT: the transmembrane protein 263 (Tmem263) and the potassium channel protein Kv7.3. However, only Kv7.3 formed immunoprecipitable complexes with GLT-1 and DAT in lysates of transfected HEK293 cells. Moreover, either DAT or GLT-1 co-clustered with Kv7.2/7.3 along the axonal tracts in co-transfected primary neurons, indicating a close spatial proximity between these proteins. Kv7.3, forming heterotetramers with the closely related subunit Kv7.2, underlies the M-currents that control the resting membrane potential and spiking activity in neurons. To investigate whether the presence of the potassium channel affected DAT or GLT-1 function, we performed uptake determinations using radioactive substrate and electrophysiological measurements. Uptake through both transporters was mildly stimulated by the presence of the channel, an effect that was reversed by the potassium channel blocker XE-991. Electrophysiological recording (in transfected HT22 and differentiated SH-SY5Y cells) indicated that the depolarizing effect induced by the presence of the neurotransmitter was reverted by the activity of the potassium channel. Altogether, these data suggest a tight spatial and functional relationship between the DAT/GLT-1 transporters and the Kv7.2/7.3 potassium channel that immediately readjusts the membrane potential of the neuron, probably to limit the neurotransmitter-mediated neuronal depolarization. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

Activity dependent internalization of the glutamate transporter GLT-1 requires calcium entry through the NCX sodium/calcium exchanger.

GLT-1 is the main glutamate transporter in the brain and its trafficking controls its availability at the cell surface, thereby shaping glutamatergic neurotransmission under physiological and pathological conditions. Extracellular glutamate is known to trigger ubiquitin-dependent GLT-1 internalization from the surface of the cell to the intracellular compartment, yet here we show that internalization also requires the participation of calcium ions. Consistent with previous studies, the addition of glutamate (1 mM) to mixed primary cultures (containing neurons and astrocytes) promotes GLT-1 internalization, an effect that was suppressed in the absence of extracellular Ca2+. The pathways of Ca2+ mobilization by astrocytes were analyzed in these mixed cultures using the genetically encoded calcium sensor GCaMP6f. A complex pattern of calcium entry was activated by glutamate, with a dramatic and rapid rise in the intracellular Ca2+ concentration partially driven by glutamate transporters, especially in the initial stages after exposure to glutamate. The Na+/Ca2+ exchanger (NCX) plays a dominant role in this Ca2+ mobilization and its blockade suppresses the glutamate induced internalization of GLT-1, both in astrocytes and in a more straightforward experimental system like HEK293 cells transiently transfected with GLT-1. This regulatory mechanism might be relevant to control the amount of GLT-1 transporter at the cell surface in conditions like ischemia or traumatic brain injury, where extracellular concentrations of glutamate are persistently elevated and they promote rapid Ca2+ mobilization.

Fisiopatología de los transportadores de glutamato y de glicina: nuevas dianas terapéuticas / Pathophysiology of the glutamate and the glycine transporters: new therapeutic targets

Introducción. Los aminoácidos glutamato y glicina, aparte de su papel en la síntesis de proteínas, son dos neurotransmisores fundamentales en el sistema nervioso central de los mamíferos. El primero es ubicuo y está implicado en vías excitatorias de la neocorteza, la retina y el cerebelo, y el segundo está asociado a vías inhibitorias de zonas caudales del cerebro. Sin embargo, ambos comparten su manera de actuar al integrarse en el funcionamiento de los receptores de glutamato del tipo NMDA, fundamentales en la regulación de sistemas motores, sensitivos y cognitivos. Objetivo. Evidenciar la necesidad de una regulación exquisita de las concentraciones de glutamato y de glicina en los espacios intra y extracelulares del sistema nervioso mediante la actuación de transportadores muy específicos para ambos neurotransmisores localizados en la membrana plasmática de las neuronas y de las células de la glía. Desarrollo. Se describe el papel de los transportadores de glutamato y glicina en la neurotransmisión glutamatérgica y glicinérgica, y en el funcionamiento del sistema nervioso. Se señalan las consecuencias patológicas de los desequilibrios en estas vías de señalización. También se describe su participación en patologías como la esquizofrenia, el dolor crónico, la isquemia cerebral, la hiperplexia hereditaria, la hiperglicinemia no cetósica o trastornos neurodegenerativos. Conclusiones. El conocimiento de la forma molecular de actuar de los transportadores de glutamato y de glicina está permitiendo la identificación y el desarrollo de nuevas estrategias terapéuticas para patologías como las descritas y el desarrollo de nuevos fármacos

Introduction. The amino acids glutamate and glycine, apart from their role in protein synthesis, are two fundamental neurotransmitters in the central nervous system of mammals. The first one is ubiquitous and is involved in excitatory pathways of the neocortex, the retina and the cerebellum, and the second is involved in inhibitory pathways of brain caudal areas. However, both share their way of acting by integrating into the functioning of glutamate receptors of the NMDA type fundamentals in the regulation of motor, sensory and cognitive systems. Aim. To highlight the need for a fine regulation of glutamate and glycine concentrations in the intracellular and extracellular spaces of the nervous system through the action of very specific transporters for both neurotransmitters located in the plasma membrane of neurons and glial cells. Development. The role of the glutamate and glycine transporters in glutamatergic and glycinergic neurotransmission and in the functioning of the nervous system is described. The pathological consequences of imbalances in these signaling pathways are pointed out. We also describe its involvement in pathologies such as schizophrenia, chronic pain, cerebral ischemia, diseases such as hereditary hyperekplexia and the non-ketotic hyperglycinemia, and neurodegenerative disorders. Conclusions. The knowledge at molecular level of the way of acting of these transporters for glutamate and glycine is allowing the identification and development of new therapeutic strategies for pathologies such as those described above and the development of new drugs

Identification of novel regulatory partners of the glutamate transporter GLT-1.

We used proximity-dependent biotin identification (BioID) to find proteins that potentially interact with the major glial glutamate transporter, GLT-1, and we studied how these interactions might affect its activity. GTPase Rac1 was one protein identified, and interfering with its GTP/GDP cycle in mixed primary rat brain cultures affected both the clustering of GLT-1 at the astrocytic processes and the transport kinetics, increasing its uptake activity at low micromolar glutamate concentrations in a manner that was dependent on the effector kinase PAK1 and the actin cytoskeleton. Interestingly, the same manipulations had a different effect on another glial glutamate transporter, GLAST, inhibiting its activity. Importantly, glutamate acts through metabotropic receptors to stimulate the activity of Rac1 in astrocytes, supporting the existence of cross-talk between extracellular glutamate and the astrocytic form of the GLT-1 regulated by Rac1. CDC42EP4/BORG4 (a CDC42 effector) was also identified in the BioID screen, and it is a protein that regulates the assembly of septins and actin fibers, influencing the organization of the cytoskeleton. We found that GLT-1 interacts with septins, which reduces its lateral mobility at the cell surface. Finally, the G-protein subunit GNB4 dampens the activity of GLT-1, as revealed by its response to the activator peptide mSIRK, both in heterologous systems and in primary brain cultures. This effect occurs rapidly and thus, it is unlikely to depend on cytoskeletal dynamics. These novel interactions shed new light on the events controlling GLT-1 activity, thereby helping us to better understand how glutamate homeostasis is maintained in the brain.

Interaction of the α7-nicotinic subunit with its human-specific duplicated dupα7 isoform in mammalian cells: Relevance in human inflammatory responses.

The α7 nicotinic receptor subunit and its partially duplicated human-specific dupα7 isoform are coexpressed in neuronal and non-neuronal cells. In these cells, α7 subunits form homopentameric α7 nicotinic acetylcholine receptors (α7-nAChRs) implicated in numerous pathologies. In immune cells, α7-nAChRs are essential for vagal control of inflammatory response in sepsis. Recent studies show that the dupα7 subunit is a dominant-negative regulator of α7-nAChR activity in Xenopus oocytes. However, its biological significance in mammalian cells, particularly immune cells, remains unexplored, as the duplicated form is indistinguishable from the original subunit in standard tests. Here, using immunocytochemistry, confocal microscopy, coimmunoprecipitation, FRET, flow cytometry, and ELISA, we addressed this challenge in GH4C1 rat pituitary cells and RAW264.7 murine macrophages transfected with epitope- and fluorescent protein-tagged α7 or dupα7. We used quantitative RT-PCR of dupα7 gene expression levels in peripheral blood mononuclear cells (PBMCs) from patients with sepsis to analyze its relationship with PBMC α7 mRNA levels and with serum concentrations of inflammatory markers. We found that a physical interaction between dupα7 and α7 subunits in both cell lines generates heteromeric nAChRs that remain mainly trapped in the endoplasmic reticulum. The dupα7 sequestration of α7 subunits reduced membrane expression of functional α7-nAChRs, attenuating their anti-inflammatory capacity in lipopolysaccharide-stimulated macrophages. Moreover, the PBMC's dupα7 levels correlated inversely with their α7 levels and directly with the magnitude of the patients' inflammatory state. These results indicate that dupα7 probably reduces human vagal anti-inflammatory responses and suggest its involvement in other α7-nAChR-mediated pathophysiological processes.

Glycine Transporters and Its Coupling with NMDA Receptors.

Glycine plays two roles in neurotransmission. In caudal areas like the spinal cord and the brainstem, it acts as an inhibitory neurotransmitter, but in all regions of the CNS, it also works as a co-agonist with L-glutamate at N-methyl-D-aspartate receptors (NMDARs). The glycine fluxes in the CNS are regulated by two specific transporters for glycine, GlyT1 and GlyT2, perhaps with the cooperation of diverse neutral amino acid transporters like Asc-1 or SNAT5/SN2. While GlyT2 and Asc-1 are neuronal proteins, GlyT1 and SNAT5 are mainly astrocytic, although neuronal forms of GlyT1 also exist. GlyT1 has attracted considerable interest from the medical community and the pharmaceutical industry since compelling evidence indicates a clear association with the functioning of NMDARs, whose activity is decreased in various psychiatric illnesses. By controlling extracellular glycine, transporter inhibitors might potentiate the activity of NMDARs without activating excitotoxic processes. Physiologically, GlyT1 is a central actor in the cross talk between glutamatergic, glycinergic, dopaminergic, and probably other neurotransmitter systems. Many of these relationships begin to be unraveled by studies performed in recent years using genetic and pharmacological models. These studies are also clarifying the interactions between glycine, glycine transporters, and other co-agonists of the glycine site of NMDARs like D-serine. These findings are also relevant to understand the pathophysiology of devastating diseases like schizophrenia, depression, anxiety, epilepsy, stroke, and chronic pain.

P2X receptors up-regulate the cell-surface expression of the neuronal glycine transporter GlyT2.

Glycinergic inhibitory neurons of the spinal dorsal horn exert critical control over the conduction of nociceptive signals to higher brain areas. The neuronal glycine transporter 2 (GlyT2) is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft and its activity modulates intra and extracellular glycine concentrations. In this report we show that the stimulation of P2X purinergic receptors with ßγ-methylene adenosine 5'-triphosphate induces the up-regulation of GlyT2 transport activity by increasing total and plasma membrane expression and reducing transporter ubiquitination. We identified the receptor subtypes involved by combining pharmacological approaches, siRNA-mediated protein knockdown, and dorsal root ganglion cell enrichment in brainstem and spinal cord primary cultures. Up-regulation of GlyT2 required the combined stimulation of homomeric P2X3 and P2X2 receptors or heteromeric P2X2/3 receptors. We measured the spontaneous glycinergic currents, glycine release and GlyT2 uptake concurrently in response to P2X receptor agonists, and showed that the impact of P2X3 receptor activation on glycinergic neurotransmission involves the modulation of GlyT2 expression or activity. The recognized pro-nociceptive action of P2X3 receptors suggests that the fine-tuning of GlyT2 activity may have consequences in nociceptive signal conduction.