Megalencephalic leukoencephalopathy with subcortical cysts protein 1 regulates glial surface localization of GLIALCAM from fish to humans

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by myelin vacuolization and caused by mutations in MLC1 or GLIALCAM. Patients with recessive mutations in either MLC1 or GLIALCAM show the same clinical phenotype. It has been shown that GLIALCAM is necessary for the correct targeting of MLC1 to the membrane at cell junctions, but its own localization was independent of MLC1 in vitro. However, recent studies in Mlc1−/− mice have shown that GlialCAM is mislocalized in glial cells. In order to investigate whether the relationship between Mlc1 and GlialCAM is species-specific, we first identified MLC-related genes in zebrafish and generated an mlc1−/− zebrafish. We have characterized mlc1−/− zebrafish both functionally and histologically and compared the phenotype with that of the Mlc1−/− mice. In mlc1−/− zebrafish, as in Mlc1−/− mice, Glialcam is mislocalized. Re-examination of a brain biopsy from an MLC patient indicates that GLIALCAM is also mislocalized in Bergmann glia in the cerebellum. In vitro, impaired localization of GlialCAM was observed in astrocyte cultures from Mlc1−/− mouse only in the presence of elevated potassium levels, which mimics neuronal activity. In summary, here we demonstrate an evolutionary conserved role for MLC1 in regulating glial surface levels of GLIALCAM, and this interrelationship explains why patients with mutations in either gene (MLC1 or GLIALCAM) share the same clinical phenotyp

Megalencephalic leukoencephalopathy with subcortical cysts: a personal biochemical retrospective

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by dysfunction of the role of glial cells in controlling brain fluid and ion homeostasis. Patients affected by MLC present macrocephaly, cysts and white matter vacuolation, which lead to motor and cognitive impairments. To date, there is no treatment for MLC, only supportive care. MLC is caused by mutations in the MLC1 and GLIALCAM genes. MLC1 is a membrane protein with low identity to the Kv1.1 potassium channel and GlialCAM belongs to an adhesion molecule family. Both proteins form a complex with an as-yet-unknown function that is expressed mainly in the astrocytes surrounding the blood-brain barrier and in Bergmann glia. GlialCAM also acts as an auxiliary subunit of the chloride channel ClC-2, thus regulating its localization at cell-cell junctions and modifying its functional properties by affecting the common gate of ClC-2. Recent studies in Mlc1-,GlialCAM-and Clcn2-knockout mice or Mlc1- knockout zebrafish have provided fresh insight into the pathophysiology of MLC and further details about the molecular interactions between these three proteins. Additional studies have shown that GlialCAM/MLC1 also regulates other ion channels (TRPV4, VRAC) or transporters (Na+/K+-ATPase) in a not-understood manner. Furthermore, it has been shown that GlialCAM/ MLC1 may influence signal transduction mechanisms, thereby affecting other proteins not related with transport such as the EGFreceptor. Here, we offer a personal biochemical retrospective of the work that has been performed to gain knowledge of the pathophysiology of MLC, and we discuss future strategies that may be used to identify therapeutic solutions for MLC patients

Estudis bioquímics i funcionals de les proteïnes implicades en la Leucoencefalopatia Megalencefàlica amb Quists subcorticals

[cat]La Leucoencefalopatia Megalencefàlica amb Quists subcorticals (MLC) és un tipus rar de leucodistròfia vacuolitzant de progressió lenta, que presenta com a principals característiques clíniques macrocefàlia acusada durant els primers anys de vida, deteriorament de les funcions motores, epilèpsia i retard mental de grau mig. L’any 2001 es va identificar el primer gen responsable de la malaltia en humans denominat MLC1, tot i que existien pacients de MLC que no presentaven mutacions en MLC1 ni lligació amb el seu locus. Això suggeria que hi havia com a mínim un altre gen involucrat en la malaltia. MLC1 codifica per una proteïna de membrana que porta el mateix nom que s’expressa en cèl•lules glials. La funció de MLC1 és desconeguda però estudis bioquímics van mostrar que mutacions en aquesta proteïna provoquen una reducció dels nivells de proteïna a la membrana i una forta retenció al reticle endoplasmàtic. L’objectiu general d’aquesta Tesi és avançar en la comprensió del possible mecanisme d’acció i la funció de la proteïna MLC1 i així aprofundir en el coneixement de la fisiopatologia de la malaltia. Per realitzar aquest objectiu es va seguir l’aproximació experimental d’identificar i analitzar l’interactoma de MLC1. Per poder validar interaccions entre proteïnes de membrana es va utilitzar el mètode PCA de Split-TEV. Es va observar que el mètode no presentava suficient especificitat i per això, es van realitzar una sèrie de modificacions en la tècnica per obtenir una nova variant de la metodologia amb alta sensibilitat i especificitat. Paral•lelament, es van realitzar estudis de genòmica i proteòmica que van permetre obtenir un llistat de proteïnes candidates a interaccionar amb MLC1. Es van seleccionar les proteïnes més interessants i es va validar la interacció amb MLC1 per coimmunolocalització en cultius primaris d’astròcits de rata i pel mètode de Split-TEV. Mitjançant col•laboracions del grup es va realitzar un estudi genètic dels candidats favorables a interaccionar amb MLC1 i així es va identificar a GlialCAM com a segon gen implicat en MLC. GlialCAM és una proteïna amb una estructura similar a les proteïnes d’adhesió que s’expressa en cèl•lules glials. Estudis del grup van descriure a GlialCAM com a subunitat β de MLC1 i subunitat auxiliar del canal de clorur ClC-2, ja que és capaç d’interaccionar i dirigir aquestes proteïnes a les zones de contacte entre cèl•lules i provocar canvis funcional en el canal. El descobriment de GlialCAM va fer focalitzar els objectius de la Tesi en l’estudi d’aquesta proteïna. Es van realitzar estudis d’estructura-funció de GlialCAM mitjançant la utilització de proteïnes quimèriques i la generació de delecions. Aquests estudis van identificar el domini extracel•lular de GlialCAM com a domini responsable de la interacció en cis i en trans de la proteïna. El domini citoplasmàtic es va relacionar amb la localització de la proteïna a les unions cel•lulars i finalment, es van identificar els primers aminoàcids del domini transmembrana de GlialCAM com els responsables dels canvis funcionals de ClC-2. També es van generar i caracteritzar els models knock-down tant de MLC1 com de GlialCAM en cultius primaris d’astròcits de rata. L’estudi d’aquests models i de complementacions dels models amb l’expressió de proteïnes humanes o diferents mutacions de MLC1 i GlialCAM, van permetre descriure GlialCAM com a xaperona necessària per la sortida del reticle endoplasmàtic i la localització de MLC1 a les unions astrocitàries. Estudis funcionals van permetre relacionar MLC1 amb l’activitat VRAC i el control del volum cel•lular i es va identificar com a proteïna responsable de l’aparició de vacuoles astrocitàries en els pacients de MLC. Finalment, es va observar que les mutacions en MLC1 eren capaces d’activar VRAC per se i causaven el defecte en trobar-se retingudes. La coexpressió de GlialCAM amb aquestes mutacions de MLC1 provocava una recuperació de la localització de MLC1 i una disminució del fenotip vacuolitzant astrocitari. Aquest resultat podria ser el principi d’una possible teràpia pels pacients amb MLC basada en l’augment de l’expressió en superfície de les variants mutants de MLC1.[eng]Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a rare type of leukodystrophy characterized by early-onset macrocephaly and delayed-onset neurological deterioration. In 2001 MLC1 was identified as the first gen involved in 75% of human MLC patients. These results suggested that there was at least another gen involved in MLC pathogenesis. MLC1 is a membrane protein with an unknown function that is expressed in glial cells. Biochemical studies showed a decrease of surface expression and retention in the endoplasmatic reticulum in MLC1 mutations. The principal aim of this study is to advance in the knowledge of the MLC pathogenesis and the MLC1 function. To accomplish the study, the group performed the identification and analysis of the MLC1 interactome as experimental approach. The Split-TEV method was used to identify interactions between membrane proteins. However this method showed a low specificity and some modifications were developed to obtain a new variant of Split-TEV method with high sensibility and specificity. At the same time, using genomic and proteomic studies we identified a list of interaction proteins that were candidates to belong to the MLC1 interactome. The interaction candidates were validated by coimmunolocalization in astrocyte primary cultures and by Split-TEV method. A genetic study was performed with the most favourable candidates and GlialCAM was identified as the second gene involved in MLC disease. GlialCAM is a membrane protein with a similar structure to the adhesion proteins that is expressed in glial cells. Biochemical studies identified GlialCAM as a β subunit of MLC1 and as an auxiliary subunit of the ClC-2 chloride channel. The aim of this study was focused then to advance in the knowledge of the GlialCAM protein. We conducted structure-function studies using chimaeric proteins and GlialCAM deletions. These studies identified the extracellular domain of GlialCAM as the responsible domain of the cis and trans protein interaction. The citoplasmatic domain was related to the correct location of the protein in the cell-cell junctions, and the first aminoacids of the transmembrane domain were responsible for the functional changes in ClC-2. Finally, we developed and characterized MLC1 and GlialCAM knock-down models in astrocyte primary cultures. The study of these models and its complementation with MLC1 and GlialCAM human variants or/and MLC1 and GlialCAM mutant variants, identified a new function of GlialCAM as a chaperone needed for MLC1 endoplasmatic reticulum exit and correct localization in astrocytic junctions. Functional studies implicated MLC1 with the VRAC activation and cell volume regulation. Moreover the coexpression of MLC1 mutant variants with GlialCAM caused an increase of surface expression of MLC1 mutant variants and an activation of VRAC function. These results may be the beginning of a possible pharmacological strategy to obtain a therapy for MLC patients

Knockdown of MLC1 in primary astrocytes causes cell vacuolation: A MLC disease cell model

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. (C) 2011 Elsevier Inc. All rights reserve

Knockdown of Mlc1 in Primary Astrocytes Causes Cell Vacuolation: A Mlc Disease Cell Model

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. (C) 2011 Elsevier Inc. All rights reserved

Knockdown of Mlc1 in Primary Astrocytes Causes Cell Vacuolation: A Mlc Disease Cell Model

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. (C) 2011 Elsevier Inc. All rights reserved

Mutant GlialCAM Causes Megalencephalic Leukoencephalopathy with Subcortical Cysts, Benign Familial Macrocephaly, and Macrocephaly with Retardation and Autism

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by early-onset macrocephaly and delayed-onset neurological deterioration. Recessive MLC1 mutations are observed in 75% of patients with MLC. Genetic-linkage studies failed to identify another gene. We recently showed that some patients without MLC1 mutations display the classical phenotype; others improve or become normal but retain macrocephaly. To find another MLC-related gene, we used quantitative proteomic analysis of affinity-purified MLC1 as an alternative approach and found that GlialCAM, an IgG-like cell adhesion molecule that is also called HepaCAM and is encoded by HEPACAM, is a direct MLC1-binding partner. Analysis of 40 MLC patients without MLC1 mutations revealed multiple different HEPACAM mutations. Ten patients with the classical, deteriorating phenotype had two mutations, and 18 patients with the improving phenotype had one mutation. Most parents with a single mutation had macrocephaly, indicating dominant inheritance. In some families with dominant HEPACAM mutations, the clinical picture and magnetic resonance imaging normalized, indicating that HEPACAM mutations can cause benign familial macrocephaly. In other families with dominant HEPACAM mutations, patients had macrocephaly and mental retardation with or without autism. Further experiments demonstrated that GlialCAM and MLC1 both localize in axons and colocalize in junctions between astrocytes. GlialCAM is additionally located in myelin. Mutant GlialCAM disrupts the localization of MLC1-GlialCAM complexes in astrocytic junctions in a manner reflecting the mode of inheritance. In conclusion, GlialCAM is required for proper localization of MLC1. HEPACAM is the second gene found to be mutated in MLC. Dominant HEPACAM mutations can cause either macrocephaly and mental retardation with or without autism or benign familial macrocephaly

Molecular mechanisms of MLC1 and GLIALCAM mutations in megalencephalic leukoencephalopathy with subcortical cysts

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