The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Raul Estevez - One of the best experts on this subject based on the ideXlab platform.
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Megalencephalic Leukoencephalopathy with Subcortical Cysts Protein-1 (MLC1) Counteracts Astrocyte Activation in Response to Inflammatory Signals
Molecular Neurobiology, 2019Co-Authors: Maria Stefania Brignone, Angela Lanciotti, Cinzia Mallozzi, Marco Sbriccoli, Caterina Veroni, Paola Molinari, Xabier Elorza-vidal, Tamara Corinna Petrucci, Barbara Serafini, Raul EstevezAbstract:Megalencephalic leukoencephalopathy with subcortical cysts protein-1 (MLC1) is a membrane protein expressed by perivascular astrocytes. MLC1 mutations cause MLC, an incurable leukodystrophy characterized by macrocephaly, brain edema, cysts, myelin vacuolation, and astrocytosis, leading to cognitive/motor impairment and epilepsy. Although its function is unknown, MLC1 favors regulatory volume decrease after astrocyte osmotic swelling and down-regulates intracellular signaling pathways controlling astrocyte activation and proliferation. By combining analysis of human brain tissues with in vitro experiments, here we investigated MLC1 role in astrocyte activation during neuroinflammation, a pathological condition exacerbating patient symptoms. MLC1 upregulation was observed in brain tissues from multiple sclerosis, Alzheimer’s, and Creutzfeld-Jacob disease, all pathologies characterized by strong astrocytosis and release of inflammatory cytokines, particularly IL-1β. Using astrocytoma lines overexpressing wild-type (WT) or mutated MLC1 and astrocytes from control and MLC1 knock-out (KO) mice, we found that IL-1β stimulated WT-MLC1 plasma membrane expression in astrocytoma cells and control primary astrocytes. In astrocytoma, WT-MLC1 inhibited the activation of IL-1β–induced inflammatory signals (pERK, pNF-kB) that, conversely, were constitutively activated in mutant expressing cells or abnormally upregulated in KO astrocytes. WT-MLC1^+ cells also expressed reduced levels of the astrogliosis marker pSTAT3. We then monitored MLC1 expression timing in a demyelinating/remyelinating murine cerebellar organotypic culture model where, after the demyelination and release of inflammatory cytokines, recovery processes occur, revealing MLC1 upregulation in these latter phases. Altogether, these findings suggest that by modulating specific pathways, MLC1 contributes to restore astrocyte homeostasis after inflammation, providing the opportunity to identify drug target molecules to slow down disease progression.
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postnatal development of the astrocyte perivascular MLC1 glialcam complex defines a temporal window for the gliovascular unit maturation
Brain Structure & Function, 2019Co-Authors: Alice Gilbert, Raul Estevez, Xabier Elorza Vidal, Martine Cohensalmon, Annececile BoulayAbstract:Astrocytes, the most abundant glial cells of the central nervous system are morphologically complex. They display numerous processes interacting with synapses and blood vessels. At the vascular interface, astrocyte endfeet-terminated processes almost entirely cover the blood vessel surface and participate to the gliovascular unit where important vascular properties of the brain are set such as the blood-brain barrier (BBB) integrity. How specific morphological and functional interactions between astrocytes and the vascular compartment develop has not been fully investigated. Here, we elaborated an original experimental strategy to study the postnatal development of astrocyte perivascular endfeet. Using purified gliovascular units, we focused on the postnatal expression of MLC1 and GlialCAM, two transmembrane proteins forming a complex enriched at the junction between mature astrocyte perivascular endfeet. We showed that MLC1 and GlialCAM were enriched and assembled into mature complexes in astrocyte perivascular endfeet between postnatal days 10 and 15, after the formation of astrocyte perivascular Aquaporin 4 water channels. These events correlated with the increased expression of Claudin-5 and P-gP, two endothelial-specific BBB components. These results illustrate for the first time that astrocyte perivascular endfeet differentiation is a complex and progressive process which correlates with BBB maturation. Moreover, our results suggest that maturation of the astrocyte endfeet MLC1/GlialCAM complex between postnatal days 10 and 15 might be a key event in the gliovascular unit maturation.
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structural determinants of interaction trafficking and function in the clc 2 MLC1 subunit glialcam involved in leukodystrophy
The Journal of Physiology, 2015Co-Authors: Xavier Capdevilanortes, Xabier Elorzavidal, Alejandro Barrallogimeno, Michael Pusch, Elena Jeworutzki, Raul EstevezAbstract:Key points The extracellular domain of GlialCAM is necessary for its targeting to cell junctions, as well as for interactions with itself and MLC1 and ClC-2. The C-terminus of GlialCAM is not necessary for interaction but is required for targeting to cell junctions. The first three residues of the transmembrane segment of GlialCAM are required for GlialCAM-mediated ClC-2 activation. Abstract Mutations in the genes encoding the astrocytic protein MLC1, the cell adhesion molecule GlialCAM or the Cl− channel ClC-2 underlie human leukodystrophies. GlialCAM binds to itself, to MLC1 and to ClC-2, and directs these proteins to cell–cell contacts. In addition, GlialCAM dramatically activates ClC-2 mediated currents. In the present study, we used mutagenesis studies combined with functional and biochemical analyses to determine which parts of GlialCAM are required to perform these cellular functions. We found that the extracellular domain of GlialCAM is necessary for cell junction targeting and for mediating interactions with itself or with MLC1 and ClC-2. The C-terminus is also necessary for proper targeting to cell–cell junctions but is not required for the biochemical interaction. Finally, we identified the first three amino acids of the transmembrane segment of GlialCAM as being essential for the activation of ClC-2 currents but not for targeting or biochemical interaction. Our results provide new mechanistic insights concerning the regulation of the cell biology and function of MLC1 and ClC-2 by GlialCAM.
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megalencephalic leukoencephalopathy with subcortical cysts chronic white matter oedema due to a defect in brain ion and water homoeostasis
Lancet Neurology, 2012Co-Authors: Marjo S Van Der Knaap, Ilja Boor, Raul EstevezAbstract:Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is characterised by chronic white matter oedema. The disease has an infantile onset and leads to slow neurological deterioration in most cases, but, surprisingly, some patients recover. The first disease gene, MLC1, identified in 2001, is mutated in 75% of patients. At that time, nothing was known about MLC1 protein function and the pathophysiology of MLC. More recently, HEPACAM (also called GLIALCAM) has been identified as a second disease gene. GlialCAM serves as an escort for MLC1 and the chloride channel CLC2. The defect in MLC1 has been shown to hamper the cell volume regulation of astrocytes. One of the most important consequences involves the potassium siphoning process, which is essential in brain ion and water homoeostasis. An understanding of the mechanisms of white matter oedema in MLC is emerging. Further insight into the specific function of MLC1 is necessary to find treatment targets.
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expression patterns of MLC1 protein in the central and peripheral nervous systems
Neurobiology of Disease, 2007Co-Authors: Oscar Teijido, Ricardo P Casarolimarano, Tatjana Kharkovets, Fernando Aguado, Antonio Zorzano, Manuel Palacin, Eduardo Soriano, Albert Martinez, Raul EstevezAbstract:Mutations in MLC1 cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), a disorder characterized clinically by macrocephaly, deterioration of motor functions, epilepsy and mental decline. Recent studies have detected MLC1 mRNA and protein in astroglial processes. In addition, our group previously reported MLC1 expression in some neurons in the adult mouse brain. Here we performed an exhaustive study of the expression pattern of MLC1 in the developing mouse brain by means of optic and electron microscopy. In the central nervous system, MLC1 was detected mainly in axonal tracts early in development. In addition, MLC1 was also observed in the peripheral nervous system and in several sensory epithelia, as retina or saccula maculae. Post-embedding immunogold experiments indicated that MLC1 is localized in astrocyte-astrocyte junctions, but not in the perivascular membrane, indicating that MLC1 is not a component of the dystrophin-glycoprotein complex. In neurons, MLC1 is located at the plasma membrane and vesicular structures. Our data provide a mouse MLC1 expression map that could be useful to understand the phenotype of MLC patients, and suggested that MLC disease is caused by an astrocytic and a neuronal dysfunction.
Angela Lanciotti - One of the best experts on this subject based on the ideXlab platform.
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megalencephalic leukoencephalopathy with subcortical cysts disease linked MLC1 protein favors gap junction intercellular communication by regulating connexin 43 trafficking in astrocytes
Cells, 2020Co-Authors: Angela Lanciotti, Maria Stefania Brignone, Cinzia Mallozzi, Paola Molinari, Tamara C Petrucci, Sergio Visentin, Marcello Belfiore, Sandra Columbacabezas, Olimpia Vincentini, Elena AmbrosiniAbstract:Astrocytes, the most numerous cells of the central nervous system, exert critical functions for brain homeostasis. To this purpose, astrocytes generate a highly interconnected intercellular network allowing rapid exchange of ions and metabolites through gap junctions, adjoined channels composed of hexamers of connexin (Cx) proteins, mainly Cx43. Functional alterations of Cxs and gap junctions have been observed in several neuroinflammatory/neurodegenerative diseases. In the rare leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), astrocytes show defective control of ion/fluid exchanges causing brain edema, fluid cysts, and astrocyte/myelin vacuolation. MLC is caused by mutations in MLC1, an astrocyte-specific protein of elusive function, and in GlialCAM, a MLC1 chaperon. Both proteins are highly expressed at perivascular astrocyte end-feet and astrocyte-astrocyte contacts where they interact with zonula occludens-1 (ZO-1) and Cx43 junctional proteins. To investigate the possible role of Cx43 in MLC pathogenesis, we studied Cx43 properties in astrocytoma cells overexpressing wild type (WT) MLC1 or MLC1 carrying pathological mutations. Using biochemical and electrophysiological techniques, we found that WT, but not mutated, MLC1 expression favors intercellular communication by inhibiting extracellular-signal-regulated kinase 1/2 (ERK1/2)-mediated Cx43 phosphorylation and increasing Cx43 gap-junction stability. These data indicate MLC1 regulation of Cx43 in astrocytes and Cx43 involvement in MLC pathogenesis, suggesting potential target pathways for therapeutic interventions.
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Megalencephalic Leukoencephalopathy with Subcortical Cysts Protein-1 (MLC1) Counteracts Astrocyte Activation in Response to Inflammatory Signals
Molecular Neurobiology, 2019Co-Authors: Maria Stefania Brignone, Angela Lanciotti, Cinzia Mallozzi, Marco Sbriccoli, Caterina Veroni, Paola Molinari, Xabier Elorza-vidal, Tamara Corinna Petrucci, Barbara Serafini, Raul EstevezAbstract:Megalencephalic leukoencephalopathy with subcortical cysts protein-1 (MLC1) is a membrane protein expressed by perivascular astrocytes. MLC1 mutations cause MLC, an incurable leukodystrophy characterized by macrocephaly, brain edema, cysts, myelin vacuolation, and astrocytosis, leading to cognitive/motor impairment and epilepsy. Although its function is unknown, MLC1 favors regulatory volume decrease after astrocyte osmotic swelling and down-regulates intracellular signaling pathways controlling astrocyte activation and proliferation. By combining analysis of human brain tissues with in vitro experiments, here we investigated MLC1 role in astrocyte activation during neuroinflammation, a pathological condition exacerbating patient symptoms. MLC1 upregulation was observed in brain tissues from multiple sclerosis, Alzheimer’s, and Creutzfeld-Jacob disease, all pathologies characterized by strong astrocytosis and release of inflammatory cytokines, particularly IL-1β. Using astrocytoma lines overexpressing wild-type (WT) or mutated MLC1 and astrocytes from control and MLC1 knock-out (KO) mice, we found that IL-1β stimulated WT-MLC1 plasma membrane expression in astrocytoma cells and control primary astrocytes. In astrocytoma, WT-MLC1 inhibited the activation of IL-1β–induced inflammatory signals (pERK, pNF-kB) that, conversely, were constitutively activated in mutant expressing cells or abnormally upregulated in KO astrocytes. WT-MLC1^+ cells also expressed reduced levels of the astrogliosis marker pSTAT3. We then monitored MLC1 expression timing in a demyelinating/remyelinating murine cerebellar organotypic culture model where, after the demyelination and release of inflammatory cytokines, recovery processes occur, revealing MLC1 upregulation in these latter phases. Altogether, these findings suggest that by modulating specific pathways, MLC1 contributes to restore astrocyte homeostasis after inflammation, providing the opportunity to identify drug target molecules to slow down disease progression.
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glialcam MLC1 modulates lrrc8 vrac currents in an indirect manner implications for megalencephalic leukoencephalopathy
Neurobiology of Disease, 2018Co-Authors: Xabier Elorzavidal, Maria Stefania Brignone, Angela Lanciotti, Sonia Sirisi, Hector Gaitanpenas, Carla Perezrius, Marta Alonsogardon, Mercedes Armandugon, Esther Prat, Virginia NunesAbstract:Abstract Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM genes. Previous work indicated that chloride currents mediated by the volume-regulated anion channel (VRAC) and ClC-2 channels were affected in astrocytes deficient in either MLC1 or Glialcam. ClC-2 forms a ternary complex with GlialCAM and MLC1. LRRC8 proteins have been identified recently as the molecular components of VRAC, but the relationship between MLC and LRRC8 proteins is unknown. Here, we first demonstrate that LRRC8 and MLC1 are functionally linked, as MLC1 cannot potentiate VRAC currents when LRRC8A, the main subunit of VRAC, is knocked down. We determine that LRRC8A and MLC1 do not co-localize or interact and, in Xenopus oocytes, MLC1 does not potentiate LRRC8-mediated VRAC currents, indicating that VRAC modulation in astrocytes by MLC1 may be indirect. Investigating the mechanism of modulation, we find that a lack of MLC1 does not influence either mRNA or total and plasma membrane protein levels of LRRC8A; and neither does it affect LRRC8A subcellular localization. In agreement with recent results that indicated that overexpression of MLC1 decreases the phosphorylation of extracellular signal-regulated kinases (ERK), we find that astrocytes lacking MLC1 show an increase in ERK phosphorylation. In astrocytes with reduced or increased levels of MLC1 we observe changes in the phosphorylation state of the VRAC subunit LRRC8C. Our results thus reinforce previous suggestions that indicated that GlialCAM/MLC1 might modify signal transduction pathways that influence the activity of different proteins, such as VRAC.
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MLC1 protein a likely link between leukodystrophies and brain channelopathies
Frontiers in Cellular Neuroscience, 2015Co-Authors: Maria Stefania Brignone, Angela Lanciotti, Tamara C Petrucci, Serena Camerini, Chiara De Nuccio, Sergio Visentin, Elena AmbrosiniAbstract:Megalencephalic leukoencephalopathy with subcortical cysts (MLC) disease is a rare inherited, autosomal recessive form of childhood-onset spongiform leukodystrophy characterized by macrocephaly, deterioration of motor functions, epileptic seizures and mental decline. Brain edema, subcortical fluid cysts, myelin and astrocyte vacuolation are the histopathological hallmarks of MLC. Mutations in either the MLC1 gene (>75% of patients) or the GlialCAM gene (<20% of patients) are responsible for the disease. Recently, the GlialCAM adhesion protein was found essential for the membrane expression and function of the chloride channel ClC-2 indicating MLC disease caused by mutation in GlialCAM as the first channelopathy among leukodystrophies.. These results may explain the phenotypic convergence of ClC-2 KO mice showing brain edema and myelin vacuolation and human MLC pathology, despite mutations in the ClC-2 gene were not found in patients affected by this leukodystrophy. On the contrary, the function of MLC1 protein, which binds GlialCAM, its functional relationship with ClC-2 and the molecular mechanisms underlying MLC1 mutation-induced functional defects are not fully understood yet. The human MLC1 gene encodes a 377-amino acid membrane protein with eight predicted transmembrane domains which shows very low homology with voltage-dependent potassium K+ channel subunits. The high expression of MLC1 in brain astrocytes contacting blood vessels and meninges and brain alterations observed in MLC patients have led to hypothesize a role for MLC1 in the regulation of ion and water homeostasis. Recent studies have shown that MLC1 establishes structural and/or functional interactions with several ion/water channels and transporters and ion channel accessory proteins, and that these interactions are affected by MLC1 mutations causing MLC. Here, we review data on MLC1 functional properties obtained in in vitro and in vivo models and discuss evidence linking the effects of MLC1 mut
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the β1 subunit of the na k atpase pump interacts with megalencephalic leucoencephalopathy with subcortical cysts protein 1 MLC1 in brain astrocytes new insights into mlc pathogenesis
Human Molecular Genetics, 2011Co-Authors: Maria Stefania Brignone, Angela Lanciotti, Tamara C Petrucci, Francesca Aloisi, Gianfranco Macchia, Pompeo Macioce, Matteo Gaetani, Elena AmbrosiniAbstract:Megalencephalic leucoencephalopathy with subcortical cysts (MLC) is a rare congenital leucodystrophy caused by mutations in MLC1, a membrane protein of unknown function. MLC1 expression in astrocyte end-feet contacting blood vessels and meninges, along with brain swelling, fluid cysts and myelin vacuolation observed in MLC patients, suggests a possible role for MLC1 in the regulation of fluid and ion homeostasis and cellular volume changes. To identify MLC1 direct interactors and dissect the molecular pathways in which MLC1 is involved, we used NH2-MLC1 domain as a bait to screen a human brain library in a yeast two-hybrid assay. We identified the β1 subunit of the Na,K-ATPase pump as one of the interacting clones and confirmed it by pull-downs, co-fractionation assays and immunofluorescence stainings in human and rat astrocytes in vitro and in brain tissue. By performing ouabain-affinity chromatography on astrocyte and brain extracts, we isolated MLC1 and the whole Na,K-ATPase enzyme in a multiprotein complex that included Kir4.1, syntrophin and dystrobrevin. Because Na,K-ATPase is involved in intracellular osmotic control and volume regulation, we investigated the effect of hypo-osmotic stress on MLC1/Na,K-ATPase relationship in astrocytes. We found that hypo-osmotic conditions increased MLC1 membrane expression and favoured MLC1/Na,K-ATPase-β1 association. Moreover, hypo-osmosis induced astrocyte swelling and the reversible formation of endosome-derived vacuoles, where the two proteins co-localized. These data suggest that through its interaction with Na,K-ATPase, MLC1 is involved in the control of intracellular osmotic conditions and volume regulation in astrocytes, opening new perspectives for understanding the pathological mechanisms of MLC disease.
Xavier Capdevilanortes - One of the best experts on this subject based on the ideXlab platform.
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depolarization causes the formation of a ternary complex between glialcam MLC1 and clc 2 in astrocytes implications in megalencephalic leukoencephalopathy
Human Molecular Genetics, 2017Co-Authors: Sonia Sirisi, Xabier Elorzavidal, Mercedes Armandugon, Alejandro Barrallogimeno, Tania Lopezhernandez, Xavier Capdevilanortes, Gerard Callejo, Tanit Arnedo, Uwe Schulte, Virginia NunesAbstract:Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM. GlialCAM is necessary for the correct targeting of MLC1, but also for the targeting of the Cl- channel ClC-2. Furthermore, GlialCAM modifies ClC-2 functional properties in vitro. However, in vivo studies in GlialCAM-/- mice have shown that the modification of ClC-2 activity only occurs in oligodendrocytes, despite GlialCAM and ClC-2 being expressed in astrocytes. Thus, the relationship between GlialCAM, MLC1 and ClC-2 in astrocytes is unknown. Here, we show that GlialCAM, ClC-2 and MLC1 can form a ternary complex in cultured astrocytes, but only under depolarizing conditions. We also provide biochemical evidences that this ternary complex exists in vivo. The formation of this complex changes ClC-2 localization in the membrane and its functional properties. ClC-2 association with GlialCAM/MLC1 depends on calcium flux through L-type calcium channels and activation of calcium-dependent calpain proteases. Based on these studies, we propose that the chloride influx mediated by GlialCAM/MLC1/ClC-2 in astrocytes may be needed to compensate an excess of potassium, as occurs in conditions of high neuronal activity. We suggest that a defect in this compensation may contribute to the pathogenesis of MLC disease.
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structural determinants of interaction trafficking and function in the clc 2 MLC1 subunit glialcam involved in leukodystrophy
The Journal of Physiology, 2015Co-Authors: Xavier Capdevilanortes, Xabier Elorzavidal, Alejandro Barrallogimeno, Michael Pusch, Elena Jeworutzki, Raul EstevezAbstract:Key points The extracellular domain of GlialCAM is necessary for its targeting to cell junctions, as well as for interactions with itself and MLC1 and ClC-2. The C-terminus of GlialCAM is not necessary for interaction but is required for targeting to cell junctions. The first three residues of the transmembrane segment of GlialCAM are required for GlialCAM-mediated ClC-2 activation. Abstract Mutations in the genes encoding the astrocytic protein MLC1, the cell adhesion molecule GlialCAM or the Cl− channel ClC-2 underlie human leukodystrophies. GlialCAM binds to itself, to MLC1 and to ClC-2, and directs these proteins to cell–cell contacts. In addition, GlialCAM dramatically activates ClC-2 mediated currents. In the present study, we used mutagenesis studies combined with functional and biochemical analyses to determine which parts of GlialCAM are required to perform these cellular functions. We found that the extracellular domain of GlialCAM is necessary for cell junction targeting and for mediating interactions with itself or with MLC1 and ClC-2. The C-terminus is also necessary for proper targeting to cell–cell junctions but is not required for the biochemical interaction. Finally, we identified the first three amino acids of the transmembrane segment of GlialCAM as being essential for the activation of ClC-2 currents but not for targeting or biochemical interaction. Our results provide new mechanistic insights concerning the regulation of the cell biology and function of MLC1 and ClC-2 by GlialCAM.
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insights into mlc pathogenesis glialcam is an MLC1 chaperone required for proper activation of volume regulated anion currents
Human Molecular Genetics, 2013Co-Authors: Xavier Capdevilanortes, Sonia Sirisi, Virginia Nunes, Tania Lopezhernandez, Miguel Lopez De Heredia, Pirjo M Apaja, Gerard Callejo, Tanit Arnedo, Gergely L Lukacs, Xavier GasullAbstract:Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM genes and is associated with myelin and astrocyte vacuolation. It has been suggested that MLC is caused by impaired cell volume regulation as a result of defective activation of astrocytic volume-regulated anion currents (VRAC). GlialCAM brings MLC1 and the ClC-2 Cl(-) channel to cell-cell junctions, even though the role of ClC-2 in MLC disease remains incompletely understood. To gain insights into the biological role of GlialCAM in the pathogenesis of MLC disease, here we analyzed the gain- and loss-of-function phenotypes of GlialCAM in Hela cells and primary astrocytes, focusing on its interaction with the MLC1 protein. Unexpectedly, GlialCAM ablation provoked intracellular accumulation and reduced expression of MLC1 at the plasma membrane. Conversely, over-expression of GlialCAM increased the cellular stability of mutant MLC1 variants. Reduction in GlialCAM expression resulted in defective activation of VRAC and augmented vacuolation, phenocopying MLC1 mutations. Importantly, over-expression of GlialCAM together with MLC1 containing MLC-related mutations was able to reactivate VRAC currents and to reverse the vacuolation caused in the presence of mutant MLC1. These results indicate a previously unrecognized role of GlialCAM in facilitating the biosynthetic maturation and cell surface expression of MLC1, and suggest that pharmacological strategies aimed to increase surface expression of MLC1 and/or VRAC activity may be beneficial for MLC patients.
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molecular mechanisms of MLC1 and glialcam mutations in megalencephalic leukoencephalopathy with subcortical cysts
Human Molecular Genetics, 2011Co-Authors: Tania Lopezhernandez, Sonia Sirisi, Xavier Capdevilanortes, Marisol Montolio, Victor Fernandezduenas, Gert C Scheper, Marjo S Van Der Knaap, Pilar Casquero, Francisco Ciruela, Isidre FerrerAbstract:Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in MLC1 or GLIALCAM. The GLIALCAM gene product functions as an MLC1 beta-subunit. We aim to further clarify the molecular mechanisms of MLC caused by mutations in MLC1 or GLIALCAM. For this purpose, we analyzed a human post-mortem brain obtained from an MLC patient, who was homozygous for a missense mutation (S69L) in MLC1. We showed that this mutation affects the stability of MLC1 in vitro and reduces MLC1 protein levels in the brain to almost undetectable. However, the amount of GlialCAM and its localization were nearly unaffected, indicating that MLC1 is not necessary for GlialCAM expression or tar- geting. These findings were supported by experiments in primary astrocytes and in heterologous cells. In addition, we demonstrated that MLC1 and GlialCAM form homo- and hetero-complexes and that MLC-causing mutations in GLIALCAM mainly reduce the formation of GlialCAM homo-complexes, leading to a defect in the trafficking of GlialCAM alone to cell junctions. GLIALCAM mutations also affect the trafficking of its associ- ated molecule MLC1, explaining why GLIALCAM and MLC1 mutations lead to the same disease: MLC. © The Author 2011. Published by Oxford University Press.
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knockdown of MLC1 in primary astrocytes causes cell vacuolation a mlc disease cell model
Neurobiology of Disease, 2011Co-Authors: Anna Duarri, Tania Lopezhernandez, Xavier Capdevilanortes, Marisol Montolio, Miguel Lopez De Heredia, Margreet C Ridder, Ilja Boor, Chunfu Lien, Tracy L Hagemann, Albee MessingAbstract:Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, in the majority of cases caused by mutations in the MLC1 gene. MRI from MLC patients shows diffuse cerebral white matter signal abnormality and swelling, with evidence of increased water content. Histopathology in a MLC patient shows vacuolation of myelin, which causes the cerebral white matter swelling. MLC1 protein is expressed in astrocytic processes that are part of blood- and cerebrospinal fluid-brain barriers. We aimed to create an astrocyte cell model of MLC disease. The characterization of rat astrocyte cultures revealed MLC1 localization in cell-cell contacts, which contains other proteins described typically in tight and adherent junctions. MLC1 localization in these contacts was demonstrated to depend on the actin cytoskeleton; it was not altered when disrupting the microtubule or the GFAP networks. In human tissues, MLC1 and the protein Zonula Occludens 1 (ZO-1), which is linked to the actin cytoskeleton, co-localized by EM immunostaining and were specifically co-immunoprecipitated. To create an MLC cell model, knockdown of MLC1 in primary astrocytes was performed. Reduction of MLC1 expression resulted in the appearance of intracellular vacuoles. This vacuolation was reversed by the co-expression of human MLC1. Re-examination of a human brain biopsy from an MLC patient revealed that vacuoles were also consistently present in astrocytic processes. Thus, vacuolation of astrocytes is also a hallmark of MLC disease.
Maria Stefania Brignone - One of the best experts on this subject based on the ideXlab platform.
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megalencephalic leukoencephalopathy with subcortical cysts disease linked MLC1 protein favors gap junction intercellular communication by regulating connexin 43 trafficking in astrocytes
Cells, 2020Co-Authors: Angela Lanciotti, Maria Stefania Brignone, Cinzia Mallozzi, Paola Molinari, Tamara C Petrucci, Sergio Visentin, Marcello Belfiore, Sandra Columbacabezas, Olimpia Vincentini, Elena AmbrosiniAbstract:Astrocytes, the most numerous cells of the central nervous system, exert critical functions for brain homeostasis. To this purpose, astrocytes generate a highly interconnected intercellular network allowing rapid exchange of ions and metabolites through gap junctions, adjoined channels composed of hexamers of connexin (Cx) proteins, mainly Cx43. Functional alterations of Cxs and gap junctions have been observed in several neuroinflammatory/neurodegenerative diseases. In the rare leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), astrocytes show defective control of ion/fluid exchanges causing brain edema, fluid cysts, and astrocyte/myelin vacuolation. MLC is caused by mutations in MLC1, an astrocyte-specific protein of elusive function, and in GlialCAM, a MLC1 chaperon. Both proteins are highly expressed at perivascular astrocyte end-feet and astrocyte-astrocyte contacts where they interact with zonula occludens-1 (ZO-1) and Cx43 junctional proteins. To investigate the possible role of Cx43 in MLC pathogenesis, we studied Cx43 properties in astrocytoma cells overexpressing wild type (WT) MLC1 or MLC1 carrying pathological mutations. Using biochemical and electrophysiological techniques, we found that WT, but not mutated, MLC1 expression favors intercellular communication by inhibiting extracellular-signal-regulated kinase 1/2 (ERK1/2)-mediated Cx43 phosphorylation and increasing Cx43 gap-junction stability. These data indicate MLC1 regulation of Cx43 in astrocytes and Cx43 involvement in MLC pathogenesis, suggesting potential target pathways for therapeutic interventions.
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Megalencephalic Leukoencephalopathy with Subcortical Cysts Protein-1 (MLC1) Counteracts Astrocyte Activation in Response to Inflammatory Signals
Molecular Neurobiology, 2019Co-Authors: Maria Stefania Brignone, Angela Lanciotti, Cinzia Mallozzi, Marco Sbriccoli, Caterina Veroni, Paola Molinari, Xabier Elorza-vidal, Tamara Corinna Petrucci, Barbara Serafini, Raul EstevezAbstract:Megalencephalic leukoencephalopathy with subcortical cysts protein-1 (MLC1) is a membrane protein expressed by perivascular astrocytes. MLC1 mutations cause MLC, an incurable leukodystrophy characterized by macrocephaly, brain edema, cysts, myelin vacuolation, and astrocytosis, leading to cognitive/motor impairment and epilepsy. Although its function is unknown, MLC1 favors regulatory volume decrease after astrocyte osmotic swelling and down-regulates intracellular signaling pathways controlling astrocyte activation and proliferation. By combining analysis of human brain tissues with in vitro experiments, here we investigated MLC1 role in astrocyte activation during neuroinflammation, a pathological condition exacerbating patient symptoms. MLC1 upregulation was observed in brain tissues from multiple sclerosis, Alzheimer’s, and Creutzfeld-Jacob disease, all pathologies characterized by strong astrocytosis and release of inflammatory cytokines, particularly IL-1β. Using astrocytoma lines overexpressing wild-type (WT) or mutated MLC1 and astrocytes from control and MLC1 knock-out (KO) mice, we found that IL-1β stimulated WT-MLC1 plasma membrane expression in astrocytoma cells and control primary astrocytes. In astrocytoma, WT-MLC1 inhibited the activation of IL-1β–induced inflammatory signals (pERK, pNF-kB) that, conversely, were constitutively activated in mutant expressing cells or abnormally upregulated in KO astrocytes. WT-MLC1^+ cells also expressed reduced levels of the astrogliosis marker pSTAT3. We then monitored MLC1 expression timing in a demyelinating/remyelinating murine cerebellar organotypic culture model where, after the demyelination and release of inflammatory cytokines, recovery processes occur, revealing MLC1 upregulation in these latter phases. Altogether, these findings suggest that by modulating specific pathways, MLC1 contributes to restore astrocyte homeostasis after inflammation, providing the opportunity to identify drug target molecules to slow down disease progression.
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glialcam MLC1 modulates lrrc8 vrac currents in an indirect manner implications for megalencephalic leukoencephalopathy
Neurobiology of Disease, 2018Co-Authors: Xabier Elorzavidal, Maria Stefania Brignone, Angela Lanciotti, Sonia Sirisi, Hector Gaitanpenas, Carla Perezrius, Marta Alonsogardon, Mercedes Armandugon, Esther Prat, Virginia NunesAbstract:Abstract Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM genes. Previous work indicated that chloride currents mediated by the volume-regulated anion channel (VRAC) and ClC-2 channels were affected in astrocytes deficient in either MLC1 or Glialcam. ClC-2 forms a ternary complex with GlialCAM and MLC1. LRRC8 proteins have been identified recently as the molecular components of VRAC, but the relationship between MLC and LRRC8 proteins is unknown. Here, we first demonstrate that LRRC8 and MLC1 are functionally linked, as MLC1 cannot potentiate VRAC currents when LRRC8A, the main subunit of VRAC, is knocked down. We determine that LRRC8A and MLC1 do not co-localize or interact and, in Xenopus oocytes, MLC1 does not potentiate LRRC8-mediated VRAC currents, indicating that VRAC modulation in astrocytes by MLC1 may be indirect. Investigating the mechanism of modulation, we find that a lack of MLC1 does not influence either mRNA or total and plasma membrane protein levels of LRRC8A; and neither does it affect LRRC8A subcellular localization. In agreement with recent results that indicated that overexpression of MLC1 decreases the phosphorylation of extracellular signal-regulated kinases (ERK), we find that astrocytes lacking MLC1 show an increase in ERK phosphorylation. In astrocytes with reduced or increased levels of MLC1 we observe changes in the phosphorylation state of the VRAC subunit LRRC8C. Our results thus reinforce previous suggestions that indicated that GlialCAM/MLC1 might modify signal transduction pathways that influence the activity of different proteins, such as VRAC.
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MLC1 protein a likely link between leukodystrophies and brain channelopathies
Frontiers in Cellular Neuroscience, 2015Co-Authors: Maria Stefania Brignone, Angela Lanciotti, Tamara C Petrucci, Serena Camerini, Chiara De Nuccio, Sergio Visentin, Elena AmbrosiniAbstract:Megalencephalic leukoencephalopathy with subcortical cysts (MLC) disease is a rare inherited, autosomal recessive form of childhood-onset spongiform leukodystrophy characterized by macrocephaly, deterioration of motor functions, epileptic seizures and mental decline. Brain edema, subcortical fluid cysts, myelin and astrocyte vacuolation are the histopathological hallmarks of MLC. Mutations in either the MLC1 gene (>75% of patients) or the GlialCAM gene (<20% of patients) are responsible for the disease. Recently, the GlialCAM adhesion protein was found essential for the membrane expression and function of the chloride channel ClC-2 indicating MLC disease caused by mutation in GlialCAM as the first channelopathy among leukodystrophies.. These results may explain the phenotypic convergence of ClC-2 KO mice showing brain edema and myelin vacuolation and human MLC pathology, despite mutations in the ClC-2 gene were not found in patients affected by this leukodystrophy. On the contrary, the function of MLC1 protein, which binds GlialCAM, its functional relationship with ClC-2 and the molecular mechanisms underlying MLC1 mutation-induced functional defects are not fully understood yet. The human MLC1 gene encodes a 377-amino acid membrane protein with eight predicted transmembrane domains which shows very low homology with voltage-dependent potassium K+ channel subunits. The high expression of MLC1 in brain astrocytes contacting blood vessels and meninges and brain alterations observed in MLC patients have led to hypothesize a role for MLC1 in the regulation of ion and water homeostasis. Recent studies have shown that MLC1 establishes structural and/or functional interactions with several ion/water channels and transporters and ion channel accessory proteins, and that these interactions are affected by MLC1 mutations causing MLC. Here, we review data on MLC1 functional properties obtained in in vitro and in vivo models and discuss evidence linking the effects of MLC1 mut
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the β1 subunit of the na k atpase pump interacts with megalencephalic leucoencephalopathy with subcortical cysts protein 1 MLC1 in brain astrocytes new insights into mlc pathogenesis
Human Molecular Genetics, 2011Co-Authors: Maria Stefania Brignone, Angela Lanciotti, Tamara C Petrucci, Francesca Aloisi, Gianfranco Macchia, Pompeo Macioce, Matteo Gaetani, Elena AmbrosiniAbstract:Megalencephalic leucoencephalopathy with subcortical cysts (MLC) is a rare congenital leucodystrophy caused by mutations in MLC1, a membrane protein of unknown function. MLC1 expression in astrocyte end-feet contacting blood vessels and meninges, along with brain swelling, fluid cysts and myelin vacuolation observed in MLC patients, suggests a possible role for MLC1 in the regulation of fluid and ion homeostasis and cellular volume changes. To identify MLC1 direct interactors and dissect the molecular pathways in which MLC1 is involved, we used NH2-MLC1 domain as a bait to screen a human brain library in a yeast two-hybrid assay. We identified the β1 subunit of the Na,K-ATPase pump as one of the interacting clones and confirmed it by pull-downs, co-fractionation assays and immunofluorescence stainings in human and rat astrocytes in vitro and in brain tissue. By performing ouabain-affinity chromatography on astrocyte and brain extracts, we isolated MLC1 and the whole Na,K-ATPase enzyme in a multiprotein complex that included Kir4.1, syntrophin and dystrobrevin. Because Na,K-ATPase is involved in intracellular osmotic control and volume regulation, we investigated the effect of hypo-osmotic stress on MLC1/Na,K-ATPase relationship in astrocytes. We found that hypo-osmotic conditions increased MLC1 membrane expression and favoured MLC1/Na,K-ATPase-β1 association. Moreover, hypo-osmosis induced astrocyte swelling and the reversible formation of endosome-derived vacuoles, where the two proteins co-localized. These data suggest that through its interaction with Na,K-ATPase, MLC1 is involved in the control of intracellular osmotic conditions and volume regulation in astrocytes, opening new perspectives for understanding the pathological mechanisms of MLC disease.
Tania Lopezhernandez - One of the best experts on this subject based on the ideXlab platform.
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depolarization causes the formation of a ternary complex between glialcam MLC1 and clc 2 in astrocytes implications in megalencephalic leukoencephalopathy
Human Molecular Genetics, 2017Co-Authors: Sonia Sirisi, Xabier Elorzavidal, Mercedes Armandugon, Alejandro Barrallogimeno, Tania Lopezhernandez, Xavier Capdevilanortes, Gerard Callejo, Tanit Arnedo, Uwe Schulte, Virginia NunesAbstract:Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM. GlialCAM is necessary for the correct targeting of MLC1, but also for the targeting of the Cl- channel ClC-2. Furthermore, GlialCAM modifies ClC-2 functional properties in vitro. However, in vivo studies in GlialCAM-/- mice have shown that the modification of ClC-2 activity only occurs in oligodendrocytes, despite GlialCAM and ClC-2 being expressed in astrocytes. Thus, the relationship between GlialCAM, MLC1 and ClC-2 in astrocytes is unknown. Here, we show that GlialCAM, ClC-2 and MLC1 can form a ternary complex in cultured astrocytes, but only under depolarizing conditions. We also provide biochemical evidences that this ternary complex exists in vivo. The formation of this complex changes ClC-2 localization in the membrane and its functional properties. ClC-2 association with GlialCAM/MLC1 depends on calcium flux through L-type calcium channels and activation of calcium-dependent calpain proteases. Based on these studies, we propose that the chloride influx mediated by GlialCAM/MLC1/ClC-2 in astrocytes may be needed to compensate an excess of potassium, as occurs in conditions of high neuronal activity. We suggest that a defect in this compensation may contribute to the pathogenesis of MLC disease.
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insights into mlc pathogenesis glialcam is an MLC1 chaperone required for proper activation of volume regulated anion currents
Human Molecular Genetics, 2013Co-Authors: Xavier Capdevilanortes, Sonia Sirisi, Virginia Nunes, Tania Lopezhernandez, Miguel Lopez De Heredia, Pirjo M Apaja, Gerard Callejo, Tanit Arnedo, Gergely L Lukacs, Xavier GasullAbstract:Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM genes and is associated with myelin and astrocyte vacuolation. It has been suggested that MLC is caused by impaired cell volume regulation as a result of defective activation of astrocytic volume-regulated anion currents (VRAC). GlialCAM brings MLC1 and the ClC-2 Cl(-) channel to cell-cell junctions, even though the role of ClC-2 in MLC disease remains incompletely understood. To gain insights into the biological role of GlialCAM in the pathogenesis of MLC disease, here we analyzed the gain- and loss-of-function phenotypes of GlialCAM in Hela cells and primary astrocytes, focusing on its interaction with the MLC1 protein. Unexpectedly, GlialCAM ablation provoked intracellular accumulation and reduced expression of MLC1 at the plasma membrane. Conversely, over-expression of GlialCAM increased the cellular stability of mutant MLC1 variants. Reduction in GlialCAM expression resulted in defective activation of VRAC and augmented vacuolation, phenocopying MLC1 mutations. Importantly, over-expression of GlialCAM together with MLC1 containing MLC-related mutations was able to reactivate VRAC currents and to reverse the vacuolation caused in the presence of mutant MLC1. These results indicate a previously unrecognized role of GlialCAM in facilitating the biosynthetic maturation and cell surface expression of MLC1, and suggest that pharmacological strategies aimed to increase surface expression of MLC1 and/or VRAC activity may be beneficial for MLC patients.
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molecular mechanisms of MLC1 and glialcam mutations in megalencephalic leukoencephalopathy with subcortical cysts
Human Molecular Genetics, 2011Co-Authors: Tania Lopezhernandez, Sonia Sirisi, Xavier Capdevilanortes, Marisol Montolio, Victor Fernandezduenas, Gert C Scheper, Marjo S Van Der Knaap, Pilar Casquero, Francisco Ciruela, Isidre FerrerAbstract:Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in MLC1 or GLIALCAM. The GLIALCAM gene product functions as an MLC1 beta-subunit. We aim to further clarify the molecular mechanisms of MLC caused by mutations in MLC1 or GLIALCAM. For this purpose, we analyzed a human post-mortem brain obtained from an MLC patient, who was homozygous for a missense mutation (S69L) in MLC1. We showed that this mutation affects the stability of MLC1 in vitro and reduces MLC1 protein levels in the brain to almost undetectable. However, the amount of GlialCAM and its localization were nearly unaffected, indicating that MLC1 is not necessary for GlialCAM expression or tar- geting. These findings were supported by experiments in primary astrocytes and in heterologous cells. In addition, we demonstrated that MLC1 and GlialCAM form homo- and hetero-complexes and that MLC-causing mutations in GLIALCAM mainly reduce the formation of GlialCAM homo-complexes, leading to a defect in the trafficking of GlialCAM alone to cell junctions. GLIALCAM mutations also affect the trafficking of its associ- ated molecule MLC1, explaining why GLIALCAM and MLC1 mutations lead to the same disease: MLC. © The Author 2011. Published by Oxford University Press.
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knockdown of MLC1 in primary astrocytes causes cell vacuolation a mlc disease cell model
Neurobiology of Disease, 2011Co-Authors: Anna Duarri, Tania Lopezhernandez, Xavier Capdevilanortes, Marisol Montolio, Miguel Lopez De Heredia, Margreet C Ridder, Ilja Boor, Chunfu Lien, Tracy L Hagemann, Albee MessingAbstract:Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, in the majority of cases caused by mutations in the MLC1 gene. MRI from MLC patients shows diffuse cerebral white matter signal abnormality and swelling, with evidence of increased water content. Histopathology in a MLC patient shows vacuolation of myelin, which causes the cerebral white matter swelling. MLC1 protein is expressed in astrocytic processes that are part of blood- and cerebrospinal fluid-brain barriers. We aimed to create an astrocyte cell model of MLC disease. The characterization of rat astrocyte cultures revealed MLC1 localization in cell-cell contacts, which contains other proteins described typically in tight and adherent junctions. MLC1 localization in these contacts was demonstrated to depend on the actin cytoskeleton; it was not altered when disrupting the microtubule or the GFAP networks. In human tissues, MLC1 and the protein Zonula Occludens 1 (ZO-1), which is linked to the actin cytoskeleton, co-localized by EM immunostaining and were specifically co-immunoprecipitated. To create an MLC cell model, knockdown of MLC1 in primary astrocytes was performed. Reduction of MLC1 expression resulted in the appearance of intracellular vacuoles. This vacuolation was reversed by the co-expression of human MLC1. Re-examination of a human brain biopsy from an MLC patient revealed that vacuoles were also consistently present in astrocytic processes. Thus, vacuolation of astrocytes is also a hallmark of MLC disease.
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molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts mutations in MLC1 cause folding defects
Human Molecular Genetics, 2008Co-Authors: Anna Duarri, Oscar Teijido, Fernando Aguado, Antonio Zorzano, Manuel Palacin, Tania Lopezhernandez, Gert C Scheper, Ilja Boor, Herve Barriere, Albert MartinezAbstract:Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, most often caused by mutations in the MLC1 gene. MLC1 is an oligomeric plasma membrane (PM) protein of unknown function expressed mainly in glial cells and neurons. Most disease-causing missense mutations dramatically reduced the total and PM MLC1 expression levels in Xenopus oocytes and mammalian cells. The impaired expression of the mutants was verified in primary cultures of rat astrocytes, as well as human monocytes, cell types that endogenously express MLC1, demonstrating the relevance of the tissue culture models. Using a combination of biochemical, pharmacological and imaging methods, we also demonstrated that increased endoplasmatic reticulum-associated degradation and endo-lysosomal-associated degradation can contribute to the cell surface expression defect of the mutants. Based on these results, we suggest that MLC1 mutations reduce protein levels in vivo. Since the expression defect of the mutants could be rescued by exposing the mutant-protein expressing cells to low temperature and glycerol, a chemical chaperone, we propose that MLC belongs to the class of conformational diseases. Therefore, we suggest the use of pharmacological strategies that improve MLC1 expression to treat MLC patients.