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

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Abstract

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.

Research highlights

► Mutations in MLC1 cause Megalencephalic Leukoencephalopathy with Cysts. ► Characterization of endogenous MLC1 in cultures of astrocytes has been performed. ► This study indicates factors important for MLC1 localization in astrocytes. ► Knockdown of MLC1 in primary astrocytes resulted in cell vacuolation. ► Re-examination of a brain biopsy revealed vacuoles at astrocytic endfeet.

Introduction

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) (OMIM 604004) is an unusual leukodystrophy (van der Knaap et al., 1995a, van der Knaap et al., 1995b) characterized by infantile onset macrocephaly, diffuse signal abnormality, swelling of the cerebral white matter and the presence of cysts in the anterior temporal regions (van der Knaap et al., 1995a, van der Knaap et al., 1995b). Diagnosis is based on clinical and MRI criteria. A brain biopsy from an MLC patient showed that myelin was vacuolated (van der Knaap et al., 1996).

The first disease chromosome locus was found in 2000 (Topcu et al., 2000) and the first disease gene in 2001 (Leegwater et al., 2001). Mutations in the MLC1 gene are found in approximately 80% of the MLC patients (Ilja Boor et al., 2006, Leegwater et al., 2001, Leegwater et al., 2002, Montagna et al., 2006); there is evidence that other unknown genes are also involved (Blattner et al., 2003, Patrono et al., 2003). MLC1 (the protein product of MLC1) is an oligomeric membrane protein with some degree of homology to ion channels (Leegwater et al., 2001, Teijido et al., 2004). Mutations found in MLC patients reduce MLC1 protein expression (Duarri et al., 2008). The physiological role of MLC1 is unknown. Based on the myelin vacuolation present in MLC patients and the low homology of MLC1 to ion channels, it has been suggested that MLC1 could have a role in ion transport processes linked to water movements. Unfortunately, functional evidence is lacking, because no ion channel activity has been detected after expression of MLC1 in heterologous systems (Kaganovich et al., 2004, Teijido et al., 2004).

Within the brain, MLC1 is located in two populations: neurons and astrocytes (Boor et al., 2005, Schmitt et al., 2003, Teijido et al., 2004, Teijido et al., 2007). In astrocytes, MLC1 is mainly present in the processes that are in contact with blood- and cerebrospinal fluid-brain barriers (CSF). Electron microscopic immunohistochemistry indicated that, in mouse tissue, MLC1 is located in astrocyte–astrocyte contacts and not in astrocyte–endothelial contacts (Teijido et al., 2007).

Here, we used rat primary astrocytes to understand the pathophysiological mechanisms of MLC. First, we characterized the endogenous MLC1 protein; and second, we reduced MLC1 expression and analyzed the consequences of this reduction. We then re-examined the human MLC brain tissue by EM to confirm the relevance of our findings.

Section snippets

Animal experimentation and human samples

All the animal experimental protocols have been approved by the Animal Care and Ethics Committee of the University of Barcelona and conformed to the rules set by the Government of Catalunya.

Human brain samples have been examined, registered, classified and stored following general consensus of the European Brain Bank Network at the Institute of Neuropathology Brain Bank at the Bellvitge Hospital. The anonymized register included age and gender, minimal clinical data set, post-mortem delay and

Expression of MLC1 in primary astrocyte cultures

Previous studies on brain tissue identified MLC1 in astrocyte–astrocyte plasma membrane contact regions in Bergmann glia and astrocytic processes surrounding blood vessels (Teijido et al., 2007). We aimed to study the role of MLC1 in astrocyte physiology to understand the molecular pathogenesis of MLC. In primary rat astrocytes, endogenous MLC1 was detected mostly in a diffuse intracellular localization through the cytoplasm. Since expression of other transporters or channels in astrocytes

Discussion

The present study extends our knowledge on the important factors for MLC1 localization in astrocytes and provides a cell model to study the pathophysiology of MLC disease.

Results obtained from rat and mouse astrocytes and from mouse and human tissue localize MLC1 in astrocyte–astrocyte contacts. Colocalization experiments between MLC1 and other junction proteins indicate that these contain components typically described in tight (Occludin, ZO-1), adherent (β-Catenin) and gap junctions (Cx43).

Conflict of interest statement

The authors declare no conflict of interest.

Role of funding source

This study was supported in part by SAF 2009–07014 (RE), PS09/02672-ERARE to RE, Fundación Ramon Areces project (RE), ELA Foundation 2007-017 C4 project (RE and MSvdK), 2009 SGR 719 to RE, SAF 2009-12606-C02-02 (VN), CIBERER INTRA08/750 (RE and VN), 2009 SGR01490 to VN. RE is a recipient of an ICREA Academia prize. MCR, IB, GSC and MSvdK are supported by the Dutch Organization for Scientific Research ZonMw (TOP grant 9120.6002), the Hersenstichting (grants 10 F02(2).02, 13 F05.04 and 15 F07.30) and

Acknowledgments

We thank Miguel Morales, Xavier Gasull and Soledad Alcántara for initial help with primary astrocyte cultures, Logopharm GmbH for initial proteomic studies, Elena Ambrosini for the gift of antibodies against DGC proteins, Thomas J Jentsch for the gift of antibodies against ClC-2 and KCC1, Nienke Postma for performing the immunohistochemical stainings in brain tissue from a patient with Alexander disease, Gajja S. Salomons for performing mutational analysis of the GFAP gene in the patient with

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    These authors contributed equally to this study.

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