Depolarization causes the formation of a ternary complex between GlialCAM, MLC1 and ClC-2 in astrocytes: implications in megalencephalic leukoencephalopathy.

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.

Leukoencephalopathy-causing CLCN2 mutations are associated with impaired Cl- channel function and trafficking.

KEY POINTS: Characterisation of most mutations found in CLCN2 in patients with CC2L leukodystrophy show that they cause a reduction in function of the chloride channel ClC-2. GlialCAM, a regulatory subunit of ClC-2 in glial cells and involved in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), increases the activity of a ClC-2 mutant by affecting ClC-2 gating and by stabilising the mutant at the plasma membrane. The stabilisation of ClC-2 at the plasma membrane by GlialCAM depends on its localisation at cell-cell junctions. The membrane protein MLC1, which is defective in MLC, also contributes to the stabilisation of ClC-2 at the plasma membrane, providing further support for the view that GlialCAM, MLC1 and ClC-2 form a protein complex in glial cells. ABSTRACT: Mutations in CLCN2 have been recently identified in patients suffering from a type of leukoencephalopathy involving intramyelinic oedema. Here, we characterised most of these mutations that reduce the function of the chloride channel ClC-2 and impair its plasma membrane (PM) expression. Detailed biochemical and electrophysiological analyses of the Ala500Val mutation revealed that defective gating and increased cellular and PM turnover contributed to defective A500V-ClC-2 functional expression. Co-expression of the adhesion molecule GlialCAM, which forms a tertiary complex with ClC-2 and megalencephalic leukoencephalopathy with subcortical cysts 1 (MLC1), rescued the functional expression of the mutant by modifying its gating properties. GlialCAM also restored the PM levels of the channel by impeding its turnover at the PM. This rescue required ClC-2 localisation to cell-cell junctions, since a GlialCAM mutant with compromised junctional localisation failed to rescue the impaired stability of mutant ClC-2 at the PM. Wild-type, but not mutant, ClC-2 was also stabilised by MLC1 overexpression. We suggest that leukodystrophy-causing CLCN2 mutations reduce the functional expression of ClC-2, which is partly counteracted by GlialCAM/MLC1-mediated increase in the gating and stability of the channel.

Insights into MLC pathogenesis: GlialCAM is an MLC1 chaperone required for proper activation of volume-regulated anion currents.

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.

Functional analyses of mutations in HEPACAM causing megalencephalic leukoencephalopathy.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by white matter edema. Autosomal-recessive mutations in MLC1 cause MLC type 1, and autosomal-recessive or dominant mutations in HEPACAM (also called GLIALCAM) cause MLC type 2A and type 2B, respectively. The role of MLC1 and HEPACAM is unknown, although they have been related with the processes of cell-volume regulation and potassium siphoning by astrocytes. Previous studies with some of the mutations identified in HEPACAM showed that most of them are associated with a trafficking defect. Here, we analyzed biochemically and functionally most mutations identified up-to-date in HEPACAM. Our results allow classifying the effect of mutations in different subtypes and we indicate different cellular mechanisms that lead to MLC pathogenesis.

Expanding the spectrum of megalencephalic leukoencephalopathy with subcortical cysts in two patients with GLIALCAM mutations.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a heterogeneous neurodegenerative leukodystrophy caused by recessive mutations in MLC1 or GLIALCAM (types MLC1 and MLC2A) of by dominant mutations in GLIALCAM (MLC2B). GlialCAM functions as an auxiliary subunit of both MLC1 and ClC-2 chloride channel, increasing and modifying the function of the latter. Dominant mutations in GLIALCAM cause transient features of MLC but lacks clinical deterioration. Most recessive and dominant mutations in GLIALCAM studied so far affect the targeting of GlialCAM and its associated subunits. Here, we have investigated two patients with MLC2. The first patient has MLC2B disease, as shown by the improvement in MRI and clinical parameters. In this case, we identified a novel GLIALCAM mutation (p.Q56P) which affected the localization of GlialCAM and its associated subunits, however activating ClC-2 function as the wild-type protein. The second patient has MLC2A disease, as indicated by the lack of clinical improvement, even though, interestingly, the MRI of this patient shows a partial improvement. In this case, we found a recessive mode of inheritance, as the patient harbors two compound heterozygous mutations in GLIALCAM. One of them introduces a stop codon (p.Q56X), whereas the second mutation is a missense mutation (p.R73W), for which we could not identify any trafficking defect or an altered functional effect on ClC-2 in vitro.

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 EGF receptor. 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.

GlialCAM, a protein defective in a leukodystrophy, serves as a ClC-2 Cl(-) channel auxiliary subunit.

Ion fluxes mediated by glial cells are required for several physiological processes such as fluid homeostasis or the maintenance of low extracellular potassium during high neuronal activity. In mice, the disruption of the Cl(-) channel ClC-2 causes fluid accumulation leading to myelin vacuolation. A similar vacuolation phenotype is detected in humans affected with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a leukodystrophy which is caused by mutations in MLC1 or GLIALCAM. We here identify GlialCAM as a ClC-2 binding partner. GlialCAM and ClC-2 colocalize in Bergmann glia, in astrocyte-astrocyte junctions at astrocytic endfeet around blood vessels, and in myelinated fiber tracts. GlialCAM targets ClC-2 to cell junctions, increases ClC-2 mediated currents, and changes its functional properties. Disease-causing GLIALCAM mutations abolish the targeting of the channel to cell junctions. This work describes the first auxiliary subunit of ClC-2 and suggests that ClC-2 may play a role in the pathology of MLC disease.