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1.
Biomolecules ; 14(4)2024 Apr 04.
Article in English | MEDLINE | ID: mdl-38672460

ABSTRACT

A considerable effort has been spent in the past decades to develop targeted therapies for the treatment of demyelinating diseases, such as multiple sclerosis (MS). Among drugs with free radical scavenging activity and oligodendrocyte protecting effects, Edaravone (Radicava) has recently received increasing attention because of being able to enhance remyelination in experimental in vitro and in vivo disease models. While its beneficial effects are greatly supported by experimental evidence, there is a current paucity of information regarding its mechanism of action and main molecular targets. By using high-throughput RNA-seq and biochemical experiments in murine oligodendrocyte progenitors and SH-SY5Y neuroblastoma cells combined with molecular docking and molecular dynamics simulation, we here provide evidence that Edaravone triggers the activation of aryl hydrocarbon receptor (AHR) signaling by eliciting AHR nuclear translocation and the transcriptional-mediated induction of key cytoprotective gene expression. We also show that an Edaravone-dependent AHR signaling transduction occurs in the zebrafish experimental model, associated with a downstream upregulation of the NRF2 signaling pathway. We finally demonstrate that its rapid cytoprotective and antioxidant actions boost increased expression of the promyelinating Olig2 protein as well as of an Olig2:GFP transgene in vivo. We therefore shed light on a still undescribed potential mechanism of action for this drug, providing further support to its therapeutic potential in the context of debilitating demyelinating conditions.


Subject(s)
Antioxidants , Edaravone , Receptors, Aryl Hydrocarbon , Signal Transduction , Animals , Humans , Mice , Antioxidants/pharmacology , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Line, Tumor , Edaravone/pharmacology , Molecular Docking Simulation , Molecular Dynamics Simulation , NF-E2-Related Factor 2/metabolism , Receptors, Aryl Hydrocarbon/drug effects , Receptors, Aryl Hydrocarbon/metabolism , Signal Transduction/drug effects , Zebrafish/metabolism
2.
Cells ; 11(17)2022 08 26.
Article in English | MEDLINE | ID: mdl-36078064

ABSTRACT

Astrocytes, the main glial cells of the central nervous system, play a key role in brain volume control due to their intimate contacts with cerebral blood vessels and the expression of a distinctive equipment of proteins involved in solute/water transport. Among these is MLC1, a protein highly expressed in perivascular astrocytes and whose mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, chronic brain edema, cysts, myelin vacuolation, and astrocyte swelling. Although, in astrocytes, MLC1 mutations are known to affect the swelling-activated chloride currents (ICl,swell) mediated by the volume-regulated anion channel (VRAC), and the regulatory volume decrease, MLC1's proper function is still unknown. By combining molecular, biochemical, proteomic, electrophysiological, and imaging techniques, we here show that MLC1 is a Ca2+/Calmodulin-dependent protein kinase II (CaMKII) target protein, whose phosphorylation, occurring in response to intracellular Ca2+ release, potentiates VRAC-mediated ICl,swell. Overall, these findings reveal that MLC1 is a Ca2+-regulated protein, linking volume regulation to Ca2+ signaling in astrocytes. This knowledge provides new insight into the MLC1 protein function and into the mechanisms controlling ion/water exchanges in the brain, which may help identify possible molecular targets for the treatment of MLC and other pathological conditions caused by astrocyte swelling and brain edema.


Subject(s)
Brain Edema , Cysts , Astrocytes/metabolism , Brain Edema/pathology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Chlorides/metabolism , Cysts/metabolism , Hereditary Central Nervous System Demyelinating Diseases , Humans , Membrane Proteins/metabolism , Proteomics , Voltage-Dependent Anion Channels/metabolism , Water/metabolism
3.
Int J Mol Sci ; 23(1)2021 Dec 27.
Article in English | MEDLINE | ID: mdl-35008700

ABSTRACT

Astrocytes are very versatile cells, endowed with multitasking capacities to ensure brain homeostasis maintenance from brain development to adult life. It has become increasingly evident that astrocytes play a central role in many central nervous system pathologies, not only as regulators of defensive responses against brain insults but also as primary culprits of the disease onset and progression. This is particularly evident in some rare leukodystrophies (LDs) where white matter/myelin deterioration is due to primary astrocyte dysfunctions. Understanding the molecular defects causing these LDs may help clarify astrocyte contribution to myelin formation/maintenance and favor the identification of possible therapeutic targets for LDs and other CNS demyelinating diseases. To date, the pathogenic mechanisms of these LDs are poorly known due to the rarity of the pathological tissue and the failure of the animal models to fully recapitulate the human diseases. Thus, the development of human induced pluripotent stem cells (hiPSC) from patient fibroblasts and their differentiation into astrocytes is a promising approach to overcome these issues. In this review, we discuss the primary role of astrocytes in LD pathogenesis, the experimental models currently available and the advantages, future evolutions, perspectives, and limitations of hiPSC to study pathologies implying astrocyte dysfunctions.


Subject(s)
Astrocytes/pathology , Induced Pluripotent Stem Cells/cytology , Leukoencephalopathies/pathology , Cell Differentiation , Humans , Models, Biological , Myelin Sheath/pathology
4.
Int J Mol Sci ; 21(19)2020 Sep 29.
Article in English | MEDLINE | ID: mdl-33003644

ABSTRACT

An adequate protection from oxidative and inflammatory reactions, together with the promotion of oligodendrocyte progenitor (OP) differentiation, is needed to recover from myelin damage in demyelinating diseases. Mitochondria are targets of inflammatory and oxidative insults and are essential in oligodendrocyte differentiation. It is known that nuclear factor-erythroid 2-related factor/antioxidant responsive element (NRF2/ARE) and peroxisome proliferator-activated receptor gamma/PPAR-γ response element (PPAR-γ/PPRE) pathways control inflammation and overcome mitochondrial impairment. In this study, we analyzed the effects of activators of these pathways on mitochondrial features, protection from inflammatory/mitochondrial insults and cell differentiation in OP cultures, to depict the specificities and similarities of their actions. We used dimethyl-fumarate (DMF) and pioglitazone (pio) as agents activating NRF2 and PPAR-γ, respectively, and two synthetic hybrids acting differently on the NRF2/ARE pathway. Only DMF and compound 1 caused early effects on the mitochondria. Both DMF and pio induced mitochondrial biogenesis but different antioxidant repertoires. Moreover, pio induced OP differentiation more efficiently than DMF. Finally, DMF, pio and compound 1 protected from tumor necrosis factor-alpha (TNF-α) insult, with pio showing faster kinetics of action and compound 1 a higher activity than DMF. In conclusion, NRF2 and PPAR-γ by inducing partially overlapping pathways accomplish complementary functions aimed at the preservation of mitochondrial function, the defense against oxidative stress and the promotion of OP differentiation.


Subject(s)
Mitochondria/genetics , NF-E2-Related Factor 2/genetics , Oligodendroglia/drug effects , PPAR gamma/genetics , Animals , Antioxidants/pharmacology , Cell Differentiation/drug effects , Dimethyl Fumarate/pharmacology , Humans , Mitochondria/drug effects , Neurogenesis/drug effects , Neurogenesis/genetics , Oligodendrocyte Precursor Cells/drug effects , Oligodendrocyte Precursor Cells/metabolism , Oligodendroglia/metabolism , Organelle Biogenesis , Oxidative Stress/drug effects , Oxidative Stress/genetics , Pioglitazone/pharmacology , Rats , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/genetics
5.
Cells ; 9(6)2020 06 08.
Article in English | MEDLINE | ID: mdl-32521795

ABSTRACT

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.


Subject(s)
Astrocytes/metabolism , Cell Communication , Connexin 43/metabolism , Cysts/metabolism , Cysts/pathology , Gap Junctions/metabolism , Hereditary Central Nervous System Demyelinating Diseases/metabolism , Hereditary Central Nervous System Demyelinating Diseases/pathology , Membrane Proteins/metabolism , Cell Line, Tumor , Cytosol/metabolism , Humans , MAP Kinase Signaling System , Membrane Proteins/genetics , Models, Biological , Mutation/genetics , Phosphorylation , Protein Stability , Protein Transport
6.
Mol Neurobiol ; 56(12): 8237-8254, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31209783

ABSTRACT

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-1ß. Using astrocytoma lines overexpressing wild-type (WT) or mutated MLC1 and astrocytes from control and Mlc1 knock-out (KO) mice, we found that IL-1ß stimulated WT-MLC1 plasma membrane expression in astrocytoma cells and control primary astrocytes. In astrocytoma, WT-MLC1 inhibited the activation of IL-1ß-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.


Subject(s)
Astrocytes/pathology , Inflammation/pathology , Membrane Proteins/metabolism , Signal Transduction , Adult , Aged , Alzheimer Disease/pathology , Animals , Astrocytes/metabolism , Cell Membrane/metabolism , Demyelinating Diseases/pathology , Disease Models, Animal , ErbB Receptors/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , Interleukin-1beta/metabolism , Male , Membrane Proteins/deficiency , Mice, Knockout , Middle Aged , Models, Biological , Mutation/genetics , NF-kappa B/metabolism , Phosphorylation , Rats , Up-Regulation
7.
Neurobiol Dis ; 119: 88-99, 2018 11.
Article in English | MEDLINE | ID: mdl-30076890

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.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Astrocytes/metabolism , Cysts/metabolism , Hereditary Central Nervous System Demyelinating Diseases/metabolism , Membrane Proteins/metabolism , Proteins/metabolism , Adaptor Proteins, Signal Transducing/analysis , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Animals , Astrocytes/chemistry , Astrocytes/pathology , Cell Cycle Proteins , Cells, Cultured , Cysts/pathology , HeLa Cells , Hereditary Central Nervous System Demyelinating Diseases/pathology , Humans , Membrane Proteins/analysis , Membrane Proteins/genetics , Proteins/analysis , Proteins/genetics , Rats , Xenopus
8.
Sci Rep ; 6: 34325, 2016 Sep 28.
Article in English | MEDLINE | ID: mdl-27677466

ABSTRACT

Dysfunction of the inwardly-rectifying potassium channels Kir4.1 (KCNJ10) represents a pathogenic mechanism contributing to Autism-Epilepsy comorbidity. To define the role of Kir4.1 variants in the disorder, we sequenced KCNJ10 in a sample of affected individuals, and performed genotype-phenotype correlations. The effects of mutations on channel activity, protein trafficking, and astrocyte function were investigated in Xenopus laevis oocytes, and in human astrocytoma cell lines. An in vivo model of the disorder was also explored through generation of kcnj10a morphant zebrafish overexpressing the mutated human KCNJ10. We detected germline heterozygous KCNJ10 variants in 19/175 affected children. Epileptic spasms with dysregulated sensory processing represented the main disease phenotype. When investigated on astrocyte-like cells, the p.R18Q mutation exerted a gain-of-function effect by enhancing Kir4.1 membrane expression and current density. Similarly, the p.R348H variant led to gain of channel function through hindrance of pH-dependent current inhibition. The frequent polymorphism p.R271C seemed, instead, to have no obvious functional effects. Our results confirm that variants in KCNJ10 deserve attention in autism-epilepsy, and provide insight into the molecular mechanisms of autism and seizures. Similar to neurons, astrocyte dysfunction may result in abnormal synaptic transmission and electrical discharge, and should be regarded as a possible pharmacological target in autism-epilepsy.

9.
Hum Mol Genet ; 25(8): 1543-58, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-26908604

ABSTRACT

Mutations in the MLC1 gene, which encodes a protein expressed in brain astrocytes, are the leading cause of MLC, a rare leukodystrophy characterized by macrocephaly, brain edema, subcortical cysts, myelin and astrocyte vacuolation. Although recent studies indicate that MLC1 protein is implicated in the regulation of cell volume changes, the exact role of MLC1 in brain physiology and in the pathogenesis of MLC disease remains to be clarified. In preliminary experiments, we observed that MLC1 was poorly expressed in highly proliferating astrocytoma cells when compared with primary astrocytes, and that modulation of MLC1 expression influenced astrocyte growth. Because volume changes are key events in cell proliferation and during brain development MLC1 expression is inversely correlated to astrocyte progenitor proliferation levels, we investigated the possible role for MLC1 in the control of astrocyte proliferation. We found that overexpression of wild type but not mutant MLC1 in human astrocytoma cells hampered cell growth by favoring epidermal growth factor receptor (EGFR) degradation and by inhibiting EGF-induced Ca(+) entry, ERK1/2 and PLCγ1 activation, and calcium-activated KCa3.1 potassium channel function, all molecular pathways involved in astrocyte proliferation stimulation. Interestingly, MLC1 did not influence AKT, an EGFR-stimulated kinase involved in cell survival. Moreover, EGFR expression was higher in macrophages derived from MLC patients than from healthy individuals. Since reactive astrocytes proliferate and re-express EGFR in response to different pathological stimuli, the present findings provide new information on MLC pathogenesis and unravel an important role for MLC1 in other brain pathological conditions where astrocyte activation occurs.


Subject(s)
Astrocytes/cytology , Cysts/pathology , ErbB Receptors/metabolism , Hereditary Central Nervous System Demyelinating Diseases/pathology , Membrane Proteins/metabolism , Animals , Astrocytes/metabolism , Astrocytoma/genetics , Astrocytoma/pathology , Cell Line, Tumor , Cell Proliferation , Cysts/genetics , Gene Expression Regulation , Hereditary Central Nervous System Demyelinating Diseases/genetics , Humans , Membrane Proteins/genetics , Mutation , Rats , Signal Transduction
10.
Brain Behav Immun ; 55: 225-235, 2016 07.
Article in English | MEDLINE | ID: mdl-26593276

ABSTRACT

Repeated stimulation of TLR4 signaling by lipopolysaccharide (LPS) in microglia induces a state of tolerance/sensitization consisting in the reprogramming of the expression of pro-inflammatory genes in favor of anti-inflammatory ones. The molecular mechanisms underlying this adaptive response are far to be elucidated. Glycogen synthase kinase 3 (GSK3) has emerged as crucial regulator of TLR signaling, mediating the balance between pro- and anti-inflammatory functions in both periphery and central nervous system. The present study extends this notion identifying GSK3 as part of the molecular machinery regulating the LPS-adaptive response in microglial cells, by using primary microglial cultures and organotypic hippocampal slices (OHSCs). We found that lithium chloride (LiCl), a widely used GSK3 inhibitor and the mainstay treatment for bipolar disorder, reinforced the LPS adaptive response by enhancing both downregulation of pro-inflammatory genes (inducible nitric oxide synthase, interleukin 1ß, interleukin 6, tumor necrosis factor α), and upregulation of genes typically associated to anti-inflammatory functions (interleukin 10 and MRC1). The effects of GSK3 inhibition were mimicked by Wnt3a, added exogenously, and reversed by Inhibitor of Wnt-Response-1-endo, a pharmacological disruptor of the canonical Wnt/ß-catenin pathway, and GW9662, a selective peroxisome proliferator activated receptor γ antagonist, suggesting that these two pathways are involved in the regulation of LPS-tolerance/sensitization by GSK. Finally, LiCl treatment of OHSCs enhanced the protective functional consequences of the microglial adaptive response to LPS on oligodendrocyte maturation, as indicated by MBP mRNA upregulation. These results further indicate GSK3 as key component in the orchestration of neuroinflammation and target for neuroprotective strategies.


Subject(s)
Endotoxins/metabolism , Glycogen Synthase Kinase 3/metabolism , Hippocampus/metabolism , Inflammation/metabolism , Lipopolysaccharides/pharmacology , Lithium Chloride/pharmacology , Microglia/metabolism , PPAR gamma/metabolism , Protein Kinase Inhibitors/pharmacology , Wnt3A Protein/metabolism , Animals , Glycogen Synthase Kinase 3/antagonists & inhibitors , Rats , Rats, Wistar
11.
Transl Neurosci ; 4(2)2013 Jun 01.
Article in English | MEDLINE | ID: mdl-24340223

ABSTRACT

Astrocytes are the predominant glial cell population in the central nervous system (CNS). Once considered only passive scaffolding elements, astrocytes are now recognised as cells playing essential roles in CNS development and function. They control extracellular water and ion homeostasis, provide substrates for energy metabolism, and regulate neurogenesis, myelination and synaptic transmission. Due to these multiple activities astrocytes have been implicated in almost all brain pathologies, contributing to various aspects of disease initiation, progression and resolution. Evidence is emerging that astrocyte dysfunction can be the direct cause of neurodegeneration, as shown in Alexander's disease where myelin degeneration is caused by mutations in the gene encoding the astrocyte-specific cytoskeleton protein glial fibrillary acidic protein. Recent studies point to a primary role for astrocytes in the pathogenesis of other genetic leukodystrophies such as megalencephalic leukoencephalopathy with subcortical cysts and vanishing white matter disease. The aim of this review is to summarize current knowledge of the pathophysiological role of astrocytes focusing on their contribution to the development of the above mentioned leukodystrophies and on new perspectives for the treatment of neurological disorders.

12.
Mol Cell Neurosci ; 56: 307-21, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23851226

ABSTRACT

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy characterized by macrocephaly, subcortical cysts and demyelination. The majority of patients harbor mutations in the MLC1 gene encoding for a membrane protein with largely unknown function. Mutations in MLC1 hamper its normal trafficking and distribution in cell membranes, leading to enhanced degradation. MLC1 protein is highly expressed in brain astrocytes and in circulating blood cells, particularly monocytes. We used these easily available cells and monocyte-derived macrophages from healthy donors and MLC1-mutated patients to study MLC1 expression and localization, and to investigate how defective MLC1 mutations may affect macrophage functions. RT-PCR, western blot and immunofluorescence analyses show that MLC1 is expressed in both monocytes and macrophages, and its biosynthesis follows protein trafficking between endoplasmic reticulum and trans-Golgi network and the secretory pathway to the cell surface. MLC1 is transported along the endosomal recycling pathway passing through Rab5+ and Rab11A+vesicles before lysosomal degradation. Alterations in MLC1 trafficking and distribution were observed in macrophages from MLC1-mutated patients, which also showed changes in the expression and localization of several proteins involved in plasma membrane permeability, ion and water homeostasis and ion-regulated exocytosis. As a consequence of these alterations, patient-derived macrophages show abnormal cell morphology and intracellular calcium influx and altered response to hypo-osmotic stress. Our results suggest that blood-derived macrophages may give relevant information on MLC1 function and may be considered as valid biomarkers for MLC diagnosis and for investigating therapeutic strategies aimed to restore MLC1 trafficking in patient cells.


Subject(s)
Cysts/metabolism , Hereditary Central Nervous System Demyelinating Diseases/metabolism , Macrophages/metabolism , Membrane Proteins/metabolism , Monocytes/metabolism , Adolescent , Adult , Biomarkers/metabolism , Case-Control Studies , Cell Membrane/metabolism , Child , Cysts/diagnosis , Cysts/genetics , Endoplasmic Reticulum/metabolism , Hereditary Central Nervous System Demyelinating Diseases/diagnosis , Hereditary Central Nervous System Demyelinating Diseases/genetics , Humans , Membrane Proteins/genetics , Middle Aged , Mutation , Protein Transport , Secretory Pathway , trans-Golgi Network/metabolism
13.
Neurobiol Dis ; 37(3): 581-95, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19931615

ABSTRACT

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy caused by mutations in the MLC1 gene that encodes a membrane protein of unknown function. In the brain MLC1 protein is mainly expressed in astrocyte end-feet, localizes in lipid rafts and associates with the dystrophin glycoprotein complex (DGC). Using pull-down and co-fractionation assays in cultured human and rat astrocytes, we show here that MLC1 intracellular domains pull-down the DGC proteins syntrophin, dystrobrevin, Kir4.1 and caveolin-1, the structural protein of caveolae, thereby supporting a role for DGC and caveolar structures in MLC1 function. By immunostaining and subcellular fractionation of cultured rat or human astrocytes treated with agents modulating caveolin-mediated trafficking, we demonstrate that MLC1 is also expressed in intracellular vesicles and endoplasmic reticulum and undergoes caveolae/raft-mediated endocytosis. Inhibition of endocytosis, cholesterol lowering and protein kinases A- and C-mediated MLC1 phosphorylation favour the expression of membrane-associated MLC1. Because pathological mutations prevent MLC1 membrane expression, the identification of substances regulating MLC1 intracellular trafficking is potentially relevant for the therapy of MLC.


Subject(s)
Astrocytes/metabolism , Brain/metabolism , Caveolae/metabolism , Caveolin 1/metabolism , Leukoencephalopathies/metabolism , Membrane Proteins/metabolism , Animals , Animals, Newborn , Brain/pathology , Brain/physiopathology , Caveolae/ultrastructure , Cell Line, Tumor , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cells, Cultured , Cholesterol/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytoplasmic Vesicles/metabolism , Cytoplasmic Vesicles/ultrastructure , Dystrophin-Associated Protein Complex/metabolism , Endocytosis/physiology , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Humans , Leukoencephalopathies/genetics , Leukoencephalopathies/physiopathology , Membrane Microdomains/metabolism , Membrane Microdomains/ultrastructure , Phosphorylation , Protein Kinase C/metabolism , Protein Transport/physiology , Rats
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