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.

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.

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.

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.

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.

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.

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.

Presynaptic control of glycine transporter 2 (GlyT2) by physical and functional association with plasma membrane Ca2+-ATPase (PMCA) and Na+-Ca2+ exchanger (NCX).

Fast inhibitory glycinergic transmission occurs in spinal cord, brainstem, and retina to modulate the processing of motor and sensory information. After synaptic vesicle fusion, glycine is recovered back to the presynaptic terminal by the neuronal glycine transporter 2 (GlyT2) to maintain quantal glycine content in synaptic vesicles. The loss of presynaptic GlyT2 drastically impairs the refilling of glycinergic synaptic vesicles and severely disrupts neurotransmission. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans. Here, we show a novel endogenous regulatory mechanism that can modulate GlyT2 activity based on a compartmentalized interaction between GlyT2, neuronal plasma membrane Ca(2+)-ATPase (PMCA) isoforms 2 and 3, and Na(+)/Ca(2+)-exchanger 1 (NCX1). This GlyT2·PMCA2,3·NCX1 complex is found in lipid raft subdomains where GlyT2 has been previously found to be fully active. We show that endogenous PMCA and NCX activities are necessary for GlyT2 activity and that this modulation depends on lipid raft integrity. Besides, we propose a model in which GlyT2·PMCA2-3·NCX complex would help Na(+)/K(+)-ATPase in controlling local Na(+) increases derived from GlyT2 activity after neurotransmitter release.

Endocytosis of the neuronal glycine transporter GLYT2: role of membrane rafts and protein kinase C-dependent ubiquitination.

Glycinergic neurotransmission is terminated by sodium- and chloride-dependent plasma membrane transporters. The neuronal glycine transporter 2 (GLYT2) supplies the terminal with substrate to refill synaptic vesicles containing glycine. This crucial process is defective in human hyperekplexia, a condition that can be caused by mutations in GLYT2. Inhibitory glycinergic neurotransmission is modulated by the GLYT2 exocytosis/endocytosis equilibrium, although the mechanisms underlying the turnover of this transporter remain elusive. We studied GLYT2 internalization pathways and the role of ubiquitination and membrane raft association of the transporter in its endocytosis. Using pharmacological tools, dominant-negative mutants and small-interfering RNAs, we show that the clathrin-mediated pathway is the primary mechanism for constitutive and regulated GLYT2 endocytosis in heterologous cells and neurons. We show that GLYT2 is constitutively internalized from cell surface lipid rafts, remaining associated with rafts in subcellular recycling structures. Protein kinase C (PKC) negatively modulates GLYT2 via rapid and dynamic redistribution of GLYT2 from raft to non-raft membrane subdomains and increasing ubiquitinated GLYT2 endocytosis. This biphasic mechanism is a versatile means to modulate GLYT2 behavior and hence, inhibitory glycinergic neurotransmission. These findings may reveal new therapeutic targets to address glycinergic pathologies associated with alterations in GLYT2 trafficking.

Protein kinase C (PKC)-promoted endocytosis of glutamate transporter GLT-1 requires ubiquitin ligase Nedd4-2-dependent ubiquitination but not phosphorylation.

Glutamate transporter-1 (GLT-1) is the main glutamate transporter in the central nervous system, and its concentration severely decreases in neurodegenerative diseases. The number of transporters in the plasma membrane reflects the balance between their insertion and removal, and it has been reported that the regulated endocytosis of GLT-1 depends on its ubiquitination triggered by protein kinase C (PKC) activation. Here, we identified serine 520 of GLT-1 as the primary target for PKC-dependent phosphorylation, although elimination of this serine did not impair either GLT-1 ubiquitination or endocytosis in response to phorbol esters. In fact, we present evidence indicating that the ubiquitin ligase Nedd4-2 mediates the PKC-dependent ubiquitination and down-regulation of GLT-1. Overexpression of Nedd4-2 increased the ubiquitination of the transporter and promoted its degradation. Moreover, phorbol myristate acetate enhanced Nedd4-2 phosphorylation and the formation of GLT-1·Nedd4-2 complexes, whereas siRNA knockdown of Nedd4-2 prevented ubiquitination, endocytosis, and the concomitant decrease in GLT-1 activity triggered by PKC activation. These results indicate that GLT-1 endocytosis is independent of its phosphorylation and that Nedd4-2 mediates PKC-dependent down-regulation of the transporter.

A novel dominant hyperekplexia mutation Y705C alters trafficking and biochemical properties of the presynaptic glycine transporter GlyT2.

Hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, producing hypertonia and apnea episodes. Although rare, this orphan disorder can have serious consequences, including sudden infant death. Dominant and recessive mutations in the human glycine receptor (GlyR) α1 gene (GLRA1) are the major cause of this disorder. However, recessive mutations in the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 gene (SLC6A5) are rapidly emerging as a second major cause of startle disease. In this study, systematic DNA sequencing of SLC6A5 revealed a new dominant GlyT2 mutation: pY705C (c.2114A→G) in transmembrane domain 11, in eight individuals from Spain and the United Kingdom. Curiously, individuals harboring this mutation show significant variation in clinical presentation. In addition to classical hyperekplexia symptoms, some individuals had abnormal respiration, facial dysmorphism, delayed motor development, or intellectual disability. We functionally characterized this mutation using molecular modeling, electrophysiology, [(3)H]glycine transport, cell surface expression, and cysteine labeling assays. We found that the introduced cysteine interacts with the cysteine pair Cys-311-Cys-320 in the second external loop of GlyT2. This interaction impairs transporter maturation through the secretory pathway, reduces surface expression, and inhibits transport function. Additionally, Y705C presents altered H(+) and Zn(2+) dependence of glycine transport that may affect the function of glycinergic neurotransmission in vivo.

A comparative study of bivalirudin plus clopidogrel versus bivalirudin plus prasugrel in primary angioplasty using propensity score matching.

INTRODUCTION AND OBJECTIVES: In primary angioplasty, bivalirudin is superior to treatment with heparin plus glycoprotein inhibitors for reducing cardiovascular events, although bivalirudin increases the risk of stent thrombosis. Our hypothesis is that the use of prasugrel plus bivalirudin in primary angioplasty would reduce stent thrombosis and cardiovascular events. METHOD: Consecutive patients with acute ST-segment elevation myocardial infarction who were treated by primary angioplasty within 12 hours of the onset of symptoms received bivalirudin plus clopidogrel (Group A) or bivalirudin plus prasugrel (Group B). We compared the groups using propensity score matching. The combined end-point was cardiac death, thrombosis, acute myocardial infarction, and cerebrovascular accident at 30 days. RESULTS: We assessed 168 patients. The approach was preferentially radial (95.7%). No differences in baseline characteristics were observed between Groups A (n = 70) and B (n = 70). The total mortality and rate of major bleeding complications at 30 days were 0% for both of the groups. The rate of acute and subacute thrombosis was 4.3% in Group A and 0% in Group B (P = 0.08). We observed an increased rate of events in Group A (5.7%) versus Group B (0%) (P = 0.042). CONCLUSIONS: The administration of bivalirudin plus prasugrel in primary percutaneous coronary intervention reduces cardiovascular effects compared to bivalirudin plus clopidogrel without increasing major bleeding complications during the first 30 days following primary angioplasty performed with a preferentially radial approach.

[Surgical management of descending necrotizing mediastinitis]. / Tratamiento quirúrgico de la mediastinitis necrosante descendente.

INTRODUCTION: Descending necrotizing mediastinitis (DNM) is a serious infection which occurs as a complication of oropharyngeal infection. Its surgical management and the routine transthoracic approach remain controversial. In this article we report our experience in the management of this disease, and review the different surgical approaches that have been reported in the medical literature. MATERIAL AND METHODS: A retrospective review was made of the clinical records of 29 patients treated between 1988 and 2009. Several demographic variables were analyzed, origin of the initial infection, stage of the disease according to Endo's classification, surgical technique and outcome. RESULTS: Surgical treatment consisted of both cervical and mediastinal drainage and radical debridement. The mediastinal drainage was made through a transcervical approach in 10 cases and transthoracic in 19, depending on the extent of the mediastinitis. The outcome was satisfactory in 24 patients and 5 died (mortality 17.2%). CONCLUSIONS: According to our results and the conclusions of the main authors, we recommend a prompt and aggressive surgery with a transthoracic approach in cases of widespread DNM.

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".

P2Y purinergic regulation of the glycine neurotransmitter transporters.

The sodium- and chloride-coupled glycine neurotransmitter transporters (GLYTs) control the availability of glycine at glycine-mediated synapses. The mainly glial GLYT1 is the key regulator of the glycine levels in glycinergic and glutamatergic pathways, whereas the neuronal GLYT2 is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft. In this study, we report that stimulation of P2Y purinergic receptors with 2-methylthioadenosine 5'-diphosphate in rat brainstem/spinal cord primary neuronal cultures and adult rat synaptosomes leads to the inhibition of GLYT2 and the stimulation of GLYT1 by a paracrine regulation. These effects are mainly mediated by the ADP-preferring subtypes P2Y(1) and P2Y(13) because the effects are partially reversed by the specific antagonists N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate and pyridoxal-5'-phosphate-6-azo(2-chloro-5-nitrophenyl)-2,4-disulfonate and are totally blocked by suramin. P2Y(12) receptor is additionally involved in GLYT1 stimulation. Using pharmacological approaches and siRNA-mediated protein knockdown methodology, we elucidate the molecular mechanisms of GLYT regulation. Modulation takes place through a signaling cascade involving phospholipase C activation, inositol 1,4,5-trisphosphate production, intracellular Ca(2+) mobilization, protein kinase C stimulation, nitric oxide formation, cyclic guanosine monophosphate production, and protein kinase G-I (PKG-I) activation. GLYT1 and GLYT2 are differentially sensitive to NO/cGMP/PKG-I both in brain-derived preparations and in heterologous systems expressing the recombinant transporters and P2Y(1) receptor. Sensitivity to 2-methylthioadenosine 5'-diphosphate by GLYT1 and GLYT2 was abolished by small interfering RNA (siRNA)-mediated knockdown of nitric-oxide synthase. Our data may help define the role of GLYTs in nociception and pain sensitization.

Cell surface turnover of the glutamate transporter GLT-1 is mediated by ubiquitination/deubiquitination.

The main glutamate transporter in the brain, GLT-1, mediates glutamatergic neurotransmission in both physiological and pathological conditions. GLT-1 activity is controlled by both constitutive and regulated trafficking, and although recent evidence indicates that the turnover of this protein in the plasma membrane is accelerated by protein kinase C via an ubiquitin-dependent process, the mechanisms driving the constitutive trafficking of GLT-1 remain unexplored. Here, we used a heterologous system and primary astrocytes to investigate the turnover of GLT-1 and the role of ubiquitin attachment in this process. We show that GLT-1 is endocytosed constitutively in a clathrin-dependent manner, recycling the transporter into endosomes containing EEA1 and Rab4, a marker of rapidly recycling endosomes, and not Rab11 or Rab7, markers of the slow recycling and late endosomal compartments, respectively. We also show that this process is dependent on ubiquitination, because the inhibitor of the ubiquitin-activating enzyme E1, 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester, promotes the retention of GLT-1 at the plasma membrane. Moreover, site-directed mutagenesis demonstrated the involvement of lysines 517 and 526 of GLT-1 in the constitutive internalization of the transporter. The translocation of GLT-1 from the recycling endosomes to the plasma membrane was blocked by LDN-57444, a specific inhibitor to the deubiquitinating enzyme (DUB) ubiquitin C-terminal hydrolase-L1, but not by an inhibitor of the related DUB ubiquitin C-terminal hydrolase-L3, supporting the existence of specific ubiquitination/deubiquitination cycles that ensure the correct concentrations of GLT-1 at the cell surface.

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.

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).

Subcellular localization of the neuronal glycine transporter GLYT2 in brainstem.

The neuronal glycine transporter GLYT2 belongs to the neurotransmitter:sodium:symporter (NSS) family and removes glycine from the synaptic cleft, thereby aiding the termination of the glycinergic signal and achieving the reloading of the presynaptic terminal. The task fulfilled by this transporter is fine tuned by regulating both transport activity and intracellular trafficking. Different stimuli such as neuronal activity or protein kinase C (PKC) activation can control GLYT2 surface levels although the intracellular compartments where GLYT2 resides are largely unknown. Here, by biochemical and immunological techniques in combination with electron and confocal microscopy, we have investigated the subcellular distribution of GLYT2 in rat brainstem tissue, and characterized the vesicles that contain the transporter. GLYT2 is shown to be present in small and larger vesicles that contain the synaptic vesicle protein synaptophysin, the recycling endosome small GTPase Rab11, and in the larger vesicle population, the vesicular inhibitory amino acid transporter VIAAT. Rab5A, the GABA transporter GAT1, synaptotagmin2 and synaptobrevin2 (VAMP2) were not present. Coexpression of a Rab11 dominant negative mutant with recombinant GLYT2 impaired transporter trafficking and glycine transport. Dual immunogold labeling of brainstem synaptosomes showed a very close proximity of GLYT2 and Rab11. Therefore, the intracellular GLYT2 resides in a subset of endosomal membranes and may traffic around several compartments, mainly Rab11-positive endosomes.