Ilja Boor

Leukoencephalopathy with vanishing white matter: a review.

Vanishing white matter (VWM) is one of the most prevalent inherited childhood leukoencephalopathies, but this may affect people ofall ages, including neonates and adults. It is a progressive disorder clinically dominated by cerebellar ataxia and in which minor stress conditions, such as fever or mild trauma, provoke major episodes of neurologic deterioration. Typical pathological findings include increasing white matter rarefaction and cystic degeneration, oligodendrocytosis with highly characteristic foamy oligodendrocytes, meager astrogliosis with dysmorphic astrocytes, and loss of oligodendrocytesby apoptosis. Vanishing white matter is caused by mutations in any of the genes encoding the 5 subunits of the eukaryotic translation initiation factor 2B (eIF2B), EIF2B1 through EIF2B5. eIF2B is a ubiquitously expressed protein complex that plays a crucial role in regulating the rate of protein synthesis. Vanishing white matter mutations reduce the activity of eIF2B and impair its function to couple protein synthesis to the cellular demands in basal conditions and during stress. Reduced eIF2B activity leads to sustained improper activation of the unfolded protein response, resulting in concomitant expression of proliferation, prosurvival, and proapoptotic downstream effectors. Consequently, VWM cells are constitutively predisposed and hyperreactive to stress. In view of the fact that VWM genes are housekeeping genes, it is surprising that the disease is primarily a leukoencephalopathy. The pathophysiology of selective glial vulnerability in VWM remains poorly understood.

Megalencephalic leukoencephalopathy with subcortical cysts: chronic white matter oedema due to a defect in brain ion and water homoeostasis

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is characterised by chronic white matter oedema. The disease has an infantile onset and leads to slow neurological deterioration in most cases, but, surprisingly, some patients recover. The first disease gene, MLC1, identified in 2001, is mutated in 75% of patients. At that time, nothing was known about MLC1 protein function and the pathophysiology of MLC. More recently, HEPACAM (also called GLIALCAM) has been identified as a second disease gene. GlialCAM serves as an escort for MLC1 and the chloride channel CLC2. The defect in MLC1 has been shown to hamper the cell volume regulation of astrocytes. One of the most important consequences involves the potassium siphoning process, which is essential in brain ion and water homoeostasis. An understanding of the mechanisms of white matter oedema in MLC is emerging. Further insight into the specific function of MLC1 is necessary to find treatment targets.

Molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts: mutations in MLC1 cause folding defects

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, most often caused by mutations in the MLC1 gene. MLC1 is an oligomeric plasma membrane (PM) protein of unknown function expressed mainly in glial cells and neurons. Most disease-causing missense mutations dramatically reduced the total and PM MLC1 expression levels in Xenopus oocytes and mammalian cells. The impaired expression of the mutants was verified in primary cultures of rat astrocytes, as well as human monocytes, cell types that endogenously express MLC1, demonstrating the relevance of the tissue culture models. Using a combination of biochemical, pharmacological and imaging methods, we also demonstrated that increased endoplasmatic reticulum-associated degradation and endo-lysosomal-associated degradation can contribute to the cell surface expression defect of the mutants. Based on these results, we suggest that MLC1 mutations reduce protein levels in vivo. Since the expression defect of the mutants could be rescued by exposing the mutant-protein expressing cells to low temperature and glycerol, a chemical chaperone, we propose that MLC belongs to the class of conformational diseases. Therefore, we suggest the use of pharmacological strategies that improve MLC1 expression to treat MLC patients.

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

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.

MLC1 is associated with the Dystrophin-Glycoprotein Complex at astrocytic endfeet

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a progressive cerebral white matter disease with onset in childhood, caused by mutations in the MLC1 gene. MLC1 is a protein with unknown function that is mainly expressed in the brain in astrocytic endfeet at the blood–brain and cerebrospinal fluid–brain barriers. It shares its localization at astrocytic endfeet with the dystrophin-associated glycoprotein complex (DGC). The objective of the present study was to investigate the possible association of MLC1 with the DGC. To test this hypothesis, (co)-localization of DGC-proteins and MLC1 was analyzed by immunohistochemical stainings in gliotic brain tissue from a patient with multiple sclerosis, in glioblastoma tissue and in brain tissue from an MLC patient. In control tissue, a direct protein interaction was tested by immunoprecipitation. Results revealed that MLC1 is co-localized with DGC-proteins in gliotic brain tissue. We demonstrated that both MLC1 and aquaporin-4, a member of the DGC, were redistributed in glioblastoma cells. In MLC brain tissue, we showed absence of MLC1 and altered expression of several DGC-proteins. We demonstrated a direct protein interaction between MLC1 and Kir4.1. From these results we conclude that MLC1 is associated with the DGC at astrocytic endfeet.

Hyaluronan accumulation and arrested oligodendrocyte progenitor maturation in vanishing white matter disease.

Vanishing white matter disease is a genetic leukoencephalopathy caused by mutations in eukaryotic translation initiation factor 2B. Patients experience a slowly progressive neurological deterioration with episodes of rapid clinical worsening triggered by stress. The disease may occur at any age and leads to early death. Characteristic neuropathological findings include cystic degeneration of the white matter with feeble, if any, reactive gliosis, dysmorphic astrocytes and paucity of myelin despite an increase in oligodendrocytic density. These features have been linked to a maturation defect of astrocytes and oligodendrocytes. However, the nature of the link between glial immaturity and the observed neuropathological features is unclear. We hypothesized that the defects in maturation and function of astrocytes and oligodendrocytes are related. Brain tissue of seven patients with genetically proven vanishing white matter disease was investigated using immunohistochemistry, western blotting, quantitative polymerase chain reaction and size exclusion chromatography. The results were compared with those obtained from normal brain tissue of age-matched controls, from chronic demyelinated multiple sclerosis lesions and from other genetic and acquired white matter disorders. We found that the white matter of patients with vanishing white matter disease is enriched in CD44-expressing astrocyte precursor cells and accumulates the glycosaminoglycan hyaluronan. Hyaluronan is a major component of the extracellular matrix, and CD44 is a hyaluronan receptor. We found that a high molecular weight form of hyaluronan is overabundant, especially in the most severely affected areas. Comparison between the more severely affected frontal white matter and the relatively spared cerebellum confirms that high molecular weight hyaluronan accumulation is more pronounced in the frontal white matter than in the cerebellum. High molecular weight hyaluronan is known to inhibit astrocyte and oligodendrocyte precursor maturation and can explain the arrested glial progenitor maturation observed in vanishing white matter disease. In conclusion, high molecular weight species of hyaluronan accumulate in the white matter of patients with vanishing white matter disease, and by inhibiting glial maturation and proper function, they may be a major determinant of the white matter pathology and lack of repair.

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

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.

Mice with megalencephalic leukoencephalopathy with cysts: a developmental angle

Megalencephalic leukoencephalopathy with cysts (MLC) is a genetic disease characterized by infantile onset white matter edema and delayed onset neurological deterioration. Loss of MLC1 function causes MLC. MLC1 is involved in ion–water homeostasis, but its exact role is unknown. We generated Mlc1‐null mice for further studies.