Megalencephalic leucoencephalopathy with cysts: Defect in chloride currents and cell volume regulation

Department of Child Neurology, VU University Medical Center, 1081 HV Amsterdam, The Netherlands.
Brain (Impact Factor: 9.2). 11/2011; 134(Pt 11):3342-54. DOI: 10.1093/brain/awr255
Source: PubMed


Megalencephalic leucoencephalopathy with subcortical cysts is a genetic brain disorder with onset in early childhood. Affected infants develop macrocephaly within the first year of life, after several years followed by slowly progressive, incapacitating cerebellar ataxia and spasticity. From early on, magnetic resonance imaging shows diffuse signal abnormality and swelling of the cerebral white matter, with evidence of highly increased white matter water content. In most patients, the disease is caused by mutations in the gene MLC1, which encodes a plasma membrane protein almost exclusively expressed in brain and at lower levels in leucocytes. Within the brain, MLC1 is mainly located in astrocyte-astrocyte junctions adjacent to the blood-brain and cereborspinal fluid-brain barriers. Thus far, the function of MLC1 has remained unknown. We tested the hypothesis that MLC1 mutations cause a defect in ion currents involved in water and ion homeostasis, resulting in cerebral white matter oedema. Using whole-cell patch clamp studies we demonstrated an association between MLC1 expression and anion channel activity in different cell types, most importantly astrocytes. The currents were absent in chloride-free medium and in cells with disease-causing MLC1 mutations. MLC1-dependent currents were greatly enhanced by hypotonic pretreatment causing cell swelling, while ion channel blockers, including Tamoxifen, abolished the currents. Down regulation of endogenous MLC1 expression in astrocytes by small interfering RNA greatly reduced the activity of this channel, which was rescued by overexpression of normal MLC1. The current-voltage relationship and the pharmacological profiles of the currents indicated that the channel activated by MLC1 expression is a volume-regulated anion channel. Such channels are involved in regulatory volume decrease. We showed that regulatory volume decrease was hampered in lymphoblasts from patients with megalencephalic leucoencephalopathy. A similar trend was observed in astrocytes with decreased MLC1 expression; this effect was rescued by overexpression of normal MLC1. In the present study, we show that absence or mutations of the MLC1 protein negatively impact both volume-regulated anion channel activity and regulatory volume decrease, indicating that megalencephalic leucoencephalopathy is caused by a disturbance of cell volume regulation mediated by chloride transport.

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    • "od yet . MLC1 protein structural features , molecular interactors ( see below ) and brain alterations observed in MLC patients ( edema , fluid cysts , astrocyte , and myelin vacuolation ) suggest that MLC1 can play a role in the regulation of ion and water fluxes and cell volume . However , although several recent studies support this hypothesis ( Ridder et al . , 2011 ; Lanciotti et al . , 2012 ; Brignone et al . , 2014 ; Hoegg - Beiler et al . , 2014 ; Dubey et al . , 2015 ) , the exact function of MLC1 is still unknown ."
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    ABSTRACT: Megalencephalic leukoencephalopathy with subcortical cysts (MLCs) 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. 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 mutations to brain channelopathies.
    Full-text · Article · May 2015 · Frontiers in Cellular Neuroscience
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    • "In this context, the cytoskeleton alterations observed in M/M of mutated patients may reflect variations in volume-sensing mechanisms, as observed in other cell types (Pasantes-Morales et al., 2006). An association between MLC1 expression and chloride currents has recently been demonstrated in patient-derived lymphoblasts and MLC1-silenced astrocytes, but it remains unclear whether MLC1 corresponds to, or may be a component or a direct/indirect activator, of a volume-regulated anion channel (Ridder et al., 2011). Although Cl − channel ClC-2 and MLC1 proteins did not interact with each other (Duarri et al., 2011), the recent discovery that GlialCAM, the second MLC gene (Lopez-Hernandez et al., 2011) interacts with the Cl − channel ClC-2 in several glial cell types, and targets ClC-2 to cell junctions increasing its conductance (Jeworutzki et al., 2012), suggests that we should soon investigate the behavior of Cl − channels in M/M of mutated patients. "
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    ABSTRACT: Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy characterized by macrocephaly, subcortical cysts and demyelination. The majority of patients harbour 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.
    Full-text · Article · Jul 2013 · Molecular and Cellular Neuroscience
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    • "Pericapillary astrocyte endfeet are also enriched in the water channel aquaporin 4 (AQP4), which is anchored to the membrane by the dystrophin-associated protein complex and functionally cooperates with Kir4.1 to regulate water and K + exchange at these sites [52] [53] [54]. Other ion channels involved in the control of cell volume [55] [56] [57], like the chloride channel ClC2, the calcium channel transient receptor potential vanilloid-4 cation channel (TRPV4) and the putative ion channel MLC1 are enriched in astrocyte endfeet contacting blood vessels and the pial membrane [55] [58] [59]. "
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    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.
    Full-text · Article · Jun 2013
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