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 Ca. The pathways of Ca 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 Ca concentration partially driven by glutamate transporters, especially in the initial stages after exposure to glutamate. The Na/Ca exchanger (NCX) plays a dominant role in this Ca 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 Ca mobilization.This work was supported by grants from the Spanish MINECO (SAF2014- 55686-R) and the “Fundación Ramón Areces

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 brainOpen Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by grants from the Spanish MINECO (RTI2018-098712-B-100) and the “Fundación Ramón Areces”, the latter also providing an institutional grant to CBMS

The role of ultrasound as a diagnostic and therapeutic tool in experimental animal models of stroke: A review

Ultrasound is a noninvasive technique that provides real-time imaging with excellent resolution, and several studies demonstrated the potential of ultrasound in acute ischemic stroke monitoring. However, only a few studies were performed using animal models, of which many showed ultrasound to be a safe and effective tool also in therapeutic applications. The full potential of ultrasound application in experimental stroke is yet to be explored to further determine the limitations of this technique and to ensure the accuracy of translational research. This review covers the current status of ultrasound applied to monitoring and treatment in experimental animal models of stroke and examines the safety, limitations, and future perspectives.This research was funded by the Carlos III Health Institute Health Care Research Fund grant number FIS PI16/01052 and cofunded by the European Regional Development Fund (ERDF)–Miguel Servet (CP15/00069, CPII20/00002 to María Gutiérrez–Fernández; CP20/00024 to Laura Otero–Ortega) and predoctoral fellowship (FI17/00188 to Mari Carmen Gómez–de Frutos, FI18/00026 to Fernando Laso–García) and the INVICTUS PLUS Spanish Network (RD16/0019/0005) of the Carlos III Health Institute (ISCIII)

Brain and immune system-derived extracellular vesicles mediate regulation of complement system, extracellular matrix remodeling, brain repair and antigen tolerance in multiple sclerosis

Multiple sclerosis (MS) is an immune-mediated central nervous system disease whose course is unpredictable. Finding biomarkers that help to better comprehend the disease’s pathogenesis is crucial for supporting clinical decision-making. Blood extracellular vesicles (EVs) are membrane-bound particles secreted by all cell types that contain information on the disease’s pathological processes. Purpose: To identify the immune and nervous system-derived EV profile from blood that could have a specific role as biomarker in MS and assess its possible correlation with disease state. Results: Higher levels of T cell-derived EVs and smaller size of neuron-derived EVs were associated with clinical relapse. The smaller size of the oligodendrocyte-derived EVs was related with motor and cognitive impairment. The proteomic analysis identified mannose-binding lectin serine protease 1 and complement factor H from immune system cell-derived EVs as autoimmune disease-associated proteins. We observed hepatocyte growth factor-like protein in EVs from T cells and inter-alpha-trypsin inhibitor heavy chain 2 from neurons as white matter injury-related proteins. In patients with MS, a specific protein profile was found in the EVs, higher levels of alpha-1-microglobulin and fibrinogen β chain, lower levels of C1S and gelsolin in the immune system-released vesicles, and Talin-1 overexpression in oligodendrocyte EVs. These specific MS-associated proteins, as well as myelin basic protein in oligodendrocyte EVs, correlated with disease activity in the patients with MS. Conclusion: Neural-derived and immune-derived EVs found in blood appear to be good specific biomarkers in MSfor reflecting the disease stateWe greatly appreciate the support of Morote Traducciones S.L. for their editing assistance. This work was sponsored by a grant from Miguel Servet (CP20/00024 to Laura Otero-Ortega), Miguel Servet (CPII20/00002 to María Guti´errez-Fernández), a predoctoral fellowship (FI18/00026 to Fernando Laso-García), a Río-Hortega grant (CM22/00065 to Gabriel Torres Iglesias and CM20/00047 to Elisa Alonso-Lopez) and byResearch Project (PI21/00918) from the Instituto de Salud Carlos III and co-funded by the European Union and by a grant CA1/RSUE/2021-00753 to Dolores Piniella funded by Ministerio de Universidades, Plan de Recuperacion, ´ Transformacion ´ y Resiliencia y la Universidad Autónoma de Madri

Protein content of blood-derived extracellular vesicles: An approach to the pathophysiology of cerebral hemorrhage

Introduction: Extracellular vesicles (EVs) participate in cell-to-cell paracrine signaling and can be biomarkers of the pathophysiological processes underlying disease. In intracerebral hemorrhage, the study of the number and molecular content of circulating EVs may help elucidate the biological mechanisms involved in damage and repair, contributing valuable information to the identification of new therapeutic targets.Methods: The objective of this study was to describe the number and protein content of blood-derived EVs following an intracerebral hemorrhage (ICH). For this purpose, an experimental ICH was induced in the striatum of Sprague-Dawley rats and EVs were isolated and characterized from blood at baseline, 24 h and 28 days. The protein content in the EVs was analyzed by mass spectrometric data-dependent acquisition; protein quantification was obtained by sequential window acquisition of all theoretical mass spectra data and compared at pre-defined time points.Results: Although no differences were found in the number of EVs, the proteomic study revealed that proteins related to the response to cellular damage such as deubiquitination, regulation of MAP kinase activity (UCHL1) and signal transduction (NDGR3), were up-expressed at 24 h compared to baseline; and that at 28 days, the protein expression profile was characterized by a higher content of the proteins involved in healing and repair processes such as cytoskeleton organization and response to growth factors (COR1B) and the regulation of autophagy (PI42B).Discussion: The protein content of circulating EVs at different time points following an ICH may reflect evolutionary changes in the pathophysiology of the disease

Brain and immune system-derived extracellular vesicles mediate regulation of complement system, extracellular matrix remodeling, brain repair and antigen tolerance in Multiple sclerosis

Background: Multiple sclerosis (MS) is an immune-mediated central nervous system disease whose course is unpredictable. Finding biomarkers that help to better comprehend the disease's pathogenesis is crucial for supporting clinical decision-making. Blood extracellular vesicles (EVs) are membrane-bound particles secreted by all cell types that contain information on the disease's pathological processes. Purpose: To identify the immune and nervous system-derived EV profile from blood that could have a specific role as biomarker in MS and assess its possible correlation with disease state. Results: Higher levels of T cell-derived EVs and smaller size of neuron-derived EVs were associated with clinical relapse. The smaller size of the oligodendrocyte-derived EVs was related with motor and cognitive impairment. The proteomic analysis identified mannose-binding lectin serine protease 1 and complement factor H from immune system cell-derived EVs as autoimmune disease-associated proteins. We observed hepatocyte growth factor-like protein in EVs from T cells and inter-alpha-trypsin inhibitor heavy chain 2 from neurons as white matter injury-related proteins. In patients with MS, a specific protein profile was found in the EVs, higher levels of alpha-1-microglobulin and fibrinogen β chain, lower levels of C1S and gelsolin in the immune system-released vesicles, and Talin-1 overexpression in oligodendrocyte EVs. These specific MS-associated proteins, as well as myelin basic protein in oligodendrocyte EVs, correlated with disease activity in the patients with MS. Conclusion: Neural-derived and immune-derived EVs found in blood appear to be good specific biomarkers in MS for reflecting the disease state.This work was sponsored by a grant from Miguel Servet (CP20/00024 to Laura Otero-Ortega), Miguel Servet (CPII20/00002 to María Gutiérrez-Fernández), a predoctoral fellowship (FI18/00026 to Fernando Laso-García), a Río-Hortega grant (CM22/00065 to Gabriel Torres Iglesias and CM20/00047 to Elisa Alonso-López) and by Research Project (PI21/00918) from the Instituto de Salud Carlos III and co-funded by the European Union and by a grant CA1/RSUE/2021-00753 to Dolores Piniella funded by Ministerio de Universidades, Plan de Recuperación, Transformación y Resiliencia y la Universidad Autónoma de Madrid.S

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

Identificación de nuevas proteínas que interaccionan con los transportadores de glutamato y dopamina (GLT-1 y DAT) mediante alteraciones en sus respectivos entornos

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 16-04-2021Esta tesis tiene embargado el acceso al texto completo hasta el 16-10-2022El glutamato es el principal neurotransmisor excitador del sistema nervioso central y está involucrado en funciones tales como el aprendizaje o la memoria, y en situaciones patológicas como el ictus o las enfermedades neurodegenerativas. La concentración extracelular de este neurotransmisor se regula por varios transportadores, aunque uno de ellos, GLT-1, es el responsable de la recaptura de más del 90% del glutamato en el cerebro anterior, modulando la transmisión glutamatérgica y evitando de esta manera sus efectos excitotóxicos. La dopamina es otro neurotransmisor y, aunque su distribución es mucho mas restringida que la de glutamato, controla funciones importantes que van desde la actividad motora al comportamiento, la motivación o la recompensa, y está involucrada en patologías tales como la esquizofrenia o la enfermedad de Parkinson. En la sinapsis dopaminérgica el transportador de dopamina, DAT, es el encargado de recapturar este neurotransmisor y devolverlo a los terminales presinápticos. DAT es la diana de drogas de abuso como la cocaína o las anfetaminas, y de fármacos como los antidepresivos tricíclicos. Los transportadores GLT-1 y DAT pertenecen a la superfamilia SLC (Solute Carriers), y aunque difieran en sus estructuras y lugares de expresión, ambos son transportadores activos secundarios acoplados al gradiente electroquímico de iones, especialmente de sodio. Las propiedades de estos transportadores se regulan por modificaciones postraduccionales, así como por proteínas y lípidos que interaccionan con ellos y modulan sus cinéticas y su tráfico intracelular. En esta tesis, con el fin de identificar nuevos componentes de los interactomas de GLT-1 y DAT, se realizaron ensayos de marcaje por proximidad mediante la técnica BioID, que, por la acción del enzima BirA*, marca con biotina las proteínas que residan de manera estable o transitoria en las proximidades de los transportadores, a los que BirA* se había fusionado previamente. Las proteínas marcadas se identificaron mediante espectrometría de masas. De esta manera hemos definido una serie de posibles interactores de GLT-1 (la GTPasa Rac1, el efector de CDC42 CDC42EP4/BORG4 y la subunidad beta de las proteínas G-triméricas asociadas a receptores de membrana GNB4). Entre los potenciales interactores de DAT se encuentra la chaperona 4F2hc (Slc3a2), el receptor de membrana PGMRC2, el proteolípido de la membrana neuronal M6a, la proteína F-Box FBXL2 y la fosfatasa de fosfoinosítidos SHIP2 (Inppl1). Además, se encontró que el canal de potasio Kv7 es capaz de interaccionar tanto con GLT-1 como con DAT. En esta Tesis se han caracterizado algunas de estas interacciones mediante el uso de técnicas de inmunoprecipitación, microscopía confocal, electrofisiología y transporte de sustratos radioactivos. Se ha puesto de manifiesto la importancia para esos transportadores de las interacciones con el citoesqueleto (Rac1, BORG4) y con los fosfolípidos de membrana (SHIP2), así como con los reguladores de los gradientes iónicos (Kv7). El conocimiento de estos interactomas ayuda a comprender el mecanismo de funcionamiento de estos transportadores, de su tráfico intracelular y regulación, y podría proporcionar nuevas dianas de intervención farmacológica para la modulación de sus respectivas actividade

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 voltage-dependent sodium channel NaV1.1 by AKT1

The voltage-sensitive sodium channel Na1.1 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 Na1.1. 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 Na1.1, 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 Na1.1. 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 Na1.1 in primary cultures were identified by expressing a fluorescent protein under the Na1.1 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.“Fundación Ramón Areces”, MINECO (RTI2018-098712-B-I00) and “Fundación Síndrome de Drave