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

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 contain 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. Reexamination 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.

Megalencephalic leukoencephalopathy with cysts: theGlialcam‐null mouse model

“…Expression of proteins allegedly associated or interacting with MLC113, 30, 31, 32, 33, 34 was evaluated by immunohistochemistry in 7‐month‐old mice. Compared to wild‐type animals, expression of the water channel aquaporin4 was redistributed along the cell bodies and processes of Glialcam ‐null astrocytes (Fig.…”

“…Most previous studies on MLC1 function are based on patient leukocytes, artificial cell systems and scarcely available patient brain tissue obtained from biopsies and one autopsy 5, 6, 8, 9, 10, 15, 21, 30, 31, 32, 33, 34, 37. To allow research on MLC1 dysfunction in the intact brain, we previously developed an Mlc1 ‐null mouse that models the human disease 18.…”

Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction

“…It thus appears possible that GlialCAM mutations cause leukodystrophy by mislocalizing ClC-2 and/or by affecting its currents. By contrast, no effects of MLC1 on ClC-2 function, localization or abundance were found 12,16 . Thus, it remains unclear why patients with MLC1 mutations have the same symptoms as patients with recessive GLIALCAM mutations, who, based on these cell culture data, would be expected to be more severely affected.…”

The role of the surface on microglia function: implications for central nervous system tissue engineering

“…Comparable results were obtained when astrocyte metabolic activity was assessed after 72 h in contact with microglia-conditioned media (data not shown). Figure 5c shows the typical morphology [38] of the astrocytic cell culture. No alterations were identified after incubating astrocytes with the different microglia-conditioned media under investigation.…”