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

Abstract

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 phenotype.

Identification and characterization of the zebrafish ClC-2 chloride channel orthologs

“…As we have previously described [47], there are two GlialCAM paralogs (glialcama and glialcamb) in zebrafish. Similarly to GlialCAM, glialcama could be predominantly detected in cell junctions, while glialcamb was not, and is probably being retained intracellularly.…”

“…We co-expressed clc-2c with human GlialCAM or with the zebrafish GlialCAM paralogs [47] but no currents could be detected (data not shown). Co-expression of clc-2c with human Barttin [10] also did not result in functional currents (data not shown).…”

“…We have previously generated a zebrafish MLC model, in which some of the molecular features of the disease are conserved [47]. In this work, we have identified the zebrafish clcn2 orthologous genes, and characterized their expression patterns, subcellular localization, interaction with GLIALCAM homologues, and electrophysiological properties in Xenopus oocytes.…”

Megalencephalic leukoencephalopathy with cysts: theGlialcam‐null mouse model

“…The finding, however, of an abolished expression in Bergmann glia from different MLC mouse and zebrafish models could be compatible with a selective vulnerability of this specific astrocyte type to loss of MLC1 function 18, 26, 46. The recent report of GlialCAM mislocalization in the Bergmann glia of an MLC patient supports this possibility also in humans 46. Bergmann glia are a highly diversified cell type that also subserve an important role in extracellular ion homeostasis 47, 48…”

“…However, from an overall clinical standpoint, this hypothesis conflicts with the observation that recessive mutations in MLC1 and GLIALCAM on one hand and in CLCN2 on the other cause two distinct phenotypes in humans with different MRI abnormalities 7, 17. The finding, however, of an abolished expression in Bergmann glia from different MLC mouse and zebrafish models could be compatible with a selective vulnerability of this specific astrocyte type to loss of MLC1 function 18, 26, 46. The recent report of GlialCAM mislocalization in the Bergmann glia of an MLC patient supports this possibility also in humans 46.…”