Megalencephalic Leukoencephalopathy with Subcortical Cysts: Chronic White Matter Oedema Due to a Defect in Brain Ion and Water Homoeostasis

Review

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

Marjo S van der Knaap, Ilja Boor, Raúl Estévez

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is characterised by chronic white matter Lancet Neurol 2012; 11: 973–85 oedema. The disease has an infantile onset and leads to slow neurological deterioration in most cases, but, Department of Pediatrics/Child surprisingly, some patients recover. The fi rst disease gene, MLC1, identifi ed in 2001, is mutated in 75% of Neurology, VU University patients. At that time, nothing was known about MLC1 protein function and the pathophysiology of MLC. More Medical Centre, Amsterdam, Netherlands recently, HEPACAM (also called GLIALCAM) has been identifi ed as a second disease gene. GlialCAM serves as (Prof M S van der Knaap MD, an escort for MLC1 and the chloride channel CLC2. The defect in MLC1 has been shown to hamper the cell I Boor PhD); and Physiology volume regulation of astrocytes. One of the most important consequences involves the potassium siphoning Section, Department of process, which is essential in brain ion and water homoeostasis. An understanding of the mechanisms of white Physiological Sciences II, School of Medicine, Barcelona, Spain matter oedema in MLC is emerging. Further insight into the specifi c function of MLC1 is necessary to fi nd and U-750, Centro de treatment targets. Investigación Biomédica en Red de Enfermedades Raras Introduction associated genes: MLC1 and HEPACAM (also called (CIBERER), ISCIII, Barcelona, Spain (Prof R Estévez PhD) Megalencephalic leukoencephalopathy with subcortical GLIALCAM). Classic MLC related to recessive MLC1 Correspondence to: cysts (MLC) is an infantile-onset inherited disease of the mutations has been called MLC1 (MIM number Prof Marjo S van der Knaap, brain white matter. The disease was recognised inde- 604004).12 Classic MLC related to recessive HEPACAM Department of Child Neurology, pendently in India and the Netherlands.1,2 MLC has also mutations has been called MLC2A (MIM number VU University Medical Centre, been called van der Knaap disease,3–6 but if one prefers 613925).12 PO Box 7057, 1007 MB Amsterdam, eponyms, van der Knaap–Singhal disease would be more At birth, infants with classic MLC are healthy. In the Netherlands appropriate. Two aff ected siblings with a severe variant fi rst year of life, patients develop macrocephaly in the [email protected] had been described before,7 in whom we later confi rmed absence of other neurological signs.1,2,13,14 After the fi rst the diagnosis by DNA analysis. The core phenotype was year, head growth rate becomes normal, with a plot of described in the fi rst two reports of MLC.1,2 Patients head circumference that runs parallel to and above the develop macrocephaly in infancy. Brain MRI shows 98th percentile for healthy children.1 Some patients have diff use cerebral white matter signal abnormalities and a borderline macrocephaly,2,15 but more often the swelling of the abnormal white matter. By contrast, most macrocephaly is striking.1 Most children are mildly patients initially have no or only mild neurological signs. delayed in achieving unsupported walking and their gait MLC is very rare and there have been no formal studies is unstable.1,2,13,14 After several years, patients with MLC of its incidence, which seems to be highest in the Turkish develop slowly progressive cerebellar ataxia and, to a population1,8 and Indian Agrawal community.2,9–11 As is lesser degree, spasticity.1,2,13,14 They most often become the case for diseases with a low carrier frequency, wheelchair-dependent as teenagers. Cognitive capacities consanguinity and inbreeding contribute to its are initially either normal or mildly decreased. 1,2,13,14 occurrence. Autism is observed in some patients.2 Mild cognitive During the past few years, insight into the molecular deterioration becomes apparent over the years, but basis and disease mechanisms of MLC has increased communication and social skills remain relatively rapidly. MLC is now known to be a disease with a defect unaff ected.1,2 Some patients develop extrapyramidal in brain ion and water homoeostasis and, with that, a movement abnormalities with dystonia and athetosis.15 defect in brain volume regulation. The result is chronic Almost all children have occasional epileptic seizures, white matter oedema and slowly progressive neuro- easily controlled with drugs,1 but status epilepticus can logical deterioration, but some patients have a surprising occur.2,16 Minor head trauma can induce temporary recovery. In this Review we discuss new research fi ndings deterioration, most often with seizures or status and provide background information that is necessary to epilepticus, prolonged unconsciousness lasting days to understand which processes are aff ected by the MLC- months, or acute motor deterioration with gradual related defect. Understanding of disease mechanisms of improvement.15,17,18 No systematic study of the lifespan of MLC is pertinent for insight into brain oedema. patients with MLC has been done. The earliest death known to us is at age 15 years due to status epilepticus Clinical phenotypes: classic and remitting (personal observation), but many living patients in their phenotypes and variation 40s and 50s are known.5,9,10,19 The classic phenotype is most common and has an Some patients have a more severe clinical course and autosomal recessive mode of inheritance. There are two maintain the ability to walk without support for only a www.thelancet.com/neurology Vol 11 November 2012 973 Review

short time or never achieve it.7 Other patients have a might have slightly lower signal intensity than the more benign clinical course. As a young adult they can cortex, related to the very high water content of the have normal cognitive and motor function and only white matter.25 Diff usion-weighted imaging shows borderline macrocephaly. Some patients pursue a career increased diff usivity with high apparent diff usion and still walk without support in their 40s.9,10,19 Striking coeffi cient values in the cerebral white matter and diff erences in disease severity can occur among patients reduced anisotropy, indicative of increased size of the with the same mutation2,9–11 and sibling patients.9,10,19,20 water spaces.4,26 Subcortical cysts are invariably present Unaff ected siblings and parents are healthy and do not in the anterior temporal region, frequently also in have macrocephaly. frontal and parietal regions (fi gure 1A).1 In infants, the Patients have been recognised who initially have a anterior temporal white matter can be rarefi ed, but not typical clinical picture, but subsequently improve and yet cystic. lack signs of deteri oration.21,22 This phenotype is related to Central structures including the corpus callosum and autosomal dominant mutations in HEPACAM, has been anterior limb of the internal capsule are usually called remitting MLC or MLC2B, and has been assigned preserved.1 The occipital periventricular and subcortical MIM number 613926.12 white matter is better preserved than the rest of the Patients with MLC2B develop macrocephaly within the cerebral hemispheric white matter.1 The posterior limb of fi rst year of life. Their initial development is normal or the internal capsule is usually partly aff ected, either in the mildly delayed. Subsequently, motor capabilities can lateral part or as two lines with a spared line in the middle. become normal or patients can remain clumsy, but not The brainstem and cerebellar white matter are typically spastic or ataxic. IQ can be normal or mildly decreased. mildly abnormal in signal and never swollen.1 Cortical About half of the patients with a cognitive defi cit have and central grey matter structures are not aff ected.1 autism.21 Most patients retain macrocephaly, but in some Over time, the abnormal cerebral white matter becomes head circumference becomes normal.22 Often one of the less severely swollen and atrophy ensues, leading to parents has macrocephaly, sometimes a history of widened lateral ventricles and subarachnoid spaces transient macrocephaly as a child.21,22 A similar patient (fi gure 1B, D).1 Prominent atrophy is usually seen only in was described in the 1980s, before MLC was recognised adults. The subcortical cysts can become larger and more as a disease.23 numerous over the years.1 Contrast enhancement is not a feature of the disease at any stage, indicating that the Investigations blood–brain barrier is grosso modo intact. Laboratory fi ndings We have observed less prominent white matter abnor- Laboratory investigations are unrevealing.1,2,13 No bio- malities in exceptional patients with DNA-confi rmed chemical marker of the disease has been found in urine, classic MLC and clinically mild disease (personal blood, or CSF. observations). In such children, early MRI shows the typical diff use cerebral white matter abnormalities with Neurophysiology swelling, whereas follow-up MRI shows improvement Peripheral motor and sensory nerve conduction vel- or even normalisation of part of the cerebral white ocities and electromyograms are normal.1,2,13,14 Evoked matter and decreased swelling. The subcortical white potentials are initially normal. Brainstem auditory matter remains abnormal and the characteristic evoked potentials usually remain normal.1,13 In classic subcortical cysts remain. No systematic study has been MLC, somatosensory and visual evoked potentials done on the subject, but we and others10 have the deteriorate over the years, with prolonged latencies and impression that more severe changes on MRI, especially abnormal cortical responses.1 Electroencephalograms are in patients older than a few years, are associated with initially normal. Subsequently, in the classic phenotype, more severe disease. background slowing occurs and sharp waves, spikes, and In patients with the remitting clinical phenotype, spike-wave complexes with variable location are seen.1,24 striking improvement and normalisation of the initial Abnormal photoparoxysmal responses are present in MRI abnormalities occur (fi gure 2).21–23 In retrospect, some patients,7,13 but not in all.1,7,16 No systematic early MRIs also show less severe abnormalities than do neurophysiological data are available for patients with those of patients with the classic phenotype. Cysts in the remitting phenotype. the anterior temporal region are typically but not invariably present, but there are no subcortical cysts MRI and spectroscopy elsewhere. The cerebellar white matter is typically Most patients undergo MRI of the brain in the fi rst normal from the outset.21 Subsequently, the signal year because of rapidly increasing macrocephaly. MRI abnormality and swelling decrease, fi rst in the shows diff use cerebral white matter abnormality with pericentral and occipital regions, spreading to the swelling of the abnormal white matter, compressing remainder of the cerebral white matter. The last white the ventricles and subarachnoid spaces (fi gure 1A, C).1 matter abnormalities are seen in subcortical regions On FLAIR images, part of the cerebral white matter (fi gure 2F).21 Subcortical cysts decrease in size and

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disappear (ﬁ gure 2A–C).21 Most improvement occurs A B between 1 year and 4 years. We have documented completely normal MRIs on long-term follow-up, although follow-up of some patients has not been long enough to document normalisation.21 We suspect that, in the end, normalisation occurs in all patients with the remitting phenotype. Proton magnetic resonance spectroscopy (MRS) of the cerebral white matter has been reported only in patients with the classic phenotype. It reveals a reduction of all signals per volume,1,4,25–27 indicative of high water content. In severely aﬀ ected white matter, C D metabolites might be undetectable.15,16 Relative to creatine, N-acetylaspartate is decreased and myoinositol is increased, indicating axonal damage and gliosis. With increasing age, the abnormalities become more pronounced.1

Pathology Few pathological data are available for MLC. Most information comes from four brain biopsies.7,19,28,29 In all biopsies, the cortex was normal, apart from astrocytosis of the molecular layer, a common fi nding in patients with chronic epilepsy.7,28 A brain biopsy Figure 1: MRI in classic MLC from the frontal lobe in a 13-year-old patient revealed Sagittal T1-weighted (A, B) and axial T2-weighted (C, D) images in two patients with classic MLC and mutations in MLC1 at the ages of 6 years (A, C) and countless small or larger vacuoles in the white matter, 30 years (B, D). At age 6 years, the cerebral white matter is diff usely abnormal accompanied by fi brillary astrogliosis, without signs of and swollen (A, C), with relative sparing of the corpus callosum, internal capsule infl ammation.28 This brain biopsy was carefully (C), and occipital white matter (A). Subcortical cysts are present in the anterior prepared to avoid artifacts of swelling or shrinking. temporal and parietal regions (arrows in A). At age 30 years, the cerebral white matter has become atrophic (D). A subcortical cyst is present in the anterior The lining of the vacuoles was positive for myelin temporal region (arrow in B). MLC=megalencephalic leukoencephalopathy with basic protein. Electron microscopy revealed that the subcortical cysts. vacuoles were covered by membranes with a layered structure with major dense lines and intraperiod lines A B C (fi gure 3A, B), confi rming their myelinic nature. The separation of the myelin lamellae was invariably at the intraperiod line that is continuous with the extracellular space. Most vacuoles were covered by fi ve layers and some had a multilamellar border over part of their extent with the typical periodicity of myelin, suggesting that the splitting occurred at the intraperiod line in the outer part of myelin sheaths. Vacuoles covered by multiple myelin lamellae over their entire extent, located in the middle of myelin sheaths, and D E F vacuoles bordering axons, located in the inner part of myelin sheaths, were not observed. No appreciable decrease in myelin content was observed. In the same brain biopsy, vacuoles were also found in the endfeet of astrocytic processes ending on capillaries (fi gure 3C).30 Intramyelinic vacuoles within the white matter have also been reported by others.29 Two studies mention enlarged extracellular spaces.7,19 Myelin sheaths were abnormally thin in three biopsies.7,19,29 Figure 2: MRI in remitting MLC Genetics Sagittal T1-weighted (A–C) and axial T2-weighted (D–F) images in a patient with remitting MLC and a single mutation in HEPACAM at the ages of 9 months (A, D), 1·5 years (B, E), and 3·5 years (C, F). The fi rst MRI fulfi ls the Association of MLC1 and HEPACAM with MLC criteria for MLC, but on follow-up the white matter abnormalities improved and the anterior temporal cyst became In 2000, genetic linkage studies revealed that MLC smaller. The temporal subcortical cysts are shown by arrows in A, B, and C. At 3·5 years, only the subcortical white 31 mapped to chromosome 22qtel. In 2001, the related matter is still slightly T2-hyperintense (F). MLC=megalencephalic leukoencephalopathy with subcortical cysts. www.thelancet.com/neurology Vol 11 November 2012 975 Review

disease gene was found, called KIAA0027 or WKL1, encoding the chloride channel CLC2 was analysed. No which was renamed MLC1.32 Many diff erent mutations pathogenic mutations were found.41 See Online for appendix have been published (appendix), which are distributed Recently, two diff erent phenotypes were shown among over the entire gene. There are several founder eff ects non-MLC1-mutated patients with MLC, substantiating for the MLC1 gene. The most common founder the idea of genetic heterogeneity.21 Some of these mutation is in the Indian Agrawal community, in patients had the classic phenotype, but more than half which all patients share an insertion of one base-pair had the remitting phenotype. A proteomic approach (c.135dupC), causing a frame shift and premature stop showed a consistent association between MLC1 and a (p.Cys46LeufsX34).11,33,34 Founder eff ects have also been protein called GlialCAM.22 Analysis of the gene described in Jewish,35 Lebanese,36 and Japanese5,37 HEPACAM revealed two mutations with recessive patients (appendix). There is no evidence of a inheritance in patients with the classic pheno type, and genotype–phenotype correlation.32,33,38,39 The full one mutation with dominant inheritance in patients phenotypic range is seen among patients from the with the remitting phenotype (appendix).22 Recessive Agrawal community, who are homozygous for the HEPACAM mutations were spread over the entire same founder mutation.11 extracellular region of GlialCAM, whereas dominant From the beginning, it was clear that MLC1 mutations mutations were clustered in the fi rst immunoglobulin were found in only 70–80% of patients with MLC. domain.22 There was no overlap between dominant and Several informative MLC families in whom no MLC1 recessive mutations; they were consistently diff erent, mutations were found excluded linkage of the disease but could aff ect the same residue.22 20,33,40 with the locus on chromosome 22qtel, indicating There is no evidence that there is another gene for that there had to be at least one other gene for MLC. MLC.22 No informative MLC families have been reported Studies in several laboratories failed to identify a that exclude linkage of the disease with both the MLC1 second disease gene, suggesting further genetic and HEPACAM loci.22 heterogeneity. In a candidate gene approach, CLCN2 Association of MLC1 and HEPACAM with psychiatric disorders A B C In 2001, an association between a p.Leu309Met mutation in MLC1 and autosomal dominant catatonic schizo phrenia (MIM 605419) was reported in one large pedigree.42 Several subsequent studies did not confi rm an association between heterozygous MLC1 variants and schizophrenia.43–46 In 2005, however, an association of the MLC1 gene with schizophrenia and bipolar disorder was shown,47 again suggesting an involvement of MLC1 as a susceptibility gene for schizophrenia and bipolar disorder. A further study suggested an association between MLC1 variants and periodic catatonia, a non-remitting subtype of schizophrenia with bipolar course, but not with schizophrenia or bipolar disorder in general.48 Interestingly, blood gene expression profi les of unmedicated patients with a major depressive disorder led to identifi cation of a set of seven genes that serves as a molecular signature of D E AST major depressive disorder, and MLC1 was one of these.49 AST AST Also interesting is that patients with the remitting phenotype of MLC and heterozygous HEPACAM 22 AST mutations frequently have autism. BL Proteins, their functions, and eff ect of mutations Figure 3: Electron microscopy MLC1 protein In a brain biopsy sample from a patient with classic MLC (A–C), countless membrane-bound vacuoles are found in the subcortical white matter (A). The vacuoles are lined by membranes with the typical periodicity of myelin, When MLC1 was identifi ed as a gene associated with with alternating major dense lines and intraperiod lines (B). Vacuoles are also present in astrocytic endfeet MLC, nothing was known about the function of the around capillaries (asterisks in C). Immunogold electron microscopy with double labelling for MLC1 (18 nm gold MLC1 protein. All species that produce myelin have the particles in D and E) and β dystroglycan (12 nm gold particles in D) or GlialCAM (12 nm gold particles in E) show gene, whereas species that do not produce myelin do that MLC1 is present in astrocyte–astrocyte contacts, whereas β dystroglycan is present along the basal lamina 50 (D). MLC1 and GlialCAM colocalise in astrocyte–astrocyte contacts (E). AST=astrocyte. BL=basal lamina. not have the gene. MLC1 is a plasma membrane MLC=megalencephalic leukoencephalopathy with subcortical cysts. protein.50 Northern blot shows that it is expressed

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mainly in the brain and white blood cells.51 The A B expression level is much higher in the brain.52 Immunohistochemical staining of brain tissue shows that MLC1 is located at the blood–brain and CSF–brain barriers: in distal astroglial processes around blood vessels in the cortex and white matter (fi gure 4A), in distal astroglial processes at the glial limiting membrane of the pia, and in ependymal cells.50,53,54 Astrocytic endfeet form a continuous lining around blood vessels, separated from the endothelial cells by the basal lamina. MLC1 expression has also been Normal brain MLC1 MLC1-mutated brain MLC1 shown in processes of Bergmann glia in the 50,53,54 cerebellum. Oligodendrocytes and microglia do C D E not express MLC1.50,53,54 The striking similarity in brain MRI abnormalities between MLC and congenital muscular dystrophy with merosin defi ciency and the histological evidence of myelin vacuolation in both disorders drew our atten- tion.1,55,56 Merosin is part of the dystrophin-glycoprotein complex that links cytoskeletal proteins to the extracellular matrix. In the brain, one of its locations is the GlialCAM merge MLC1 + GlialCAM merge CLC2 + GlialCAM interface between astrocytic endfeet and the perivascular basal lamina, where it anchors endfeet to the basal F G H lamina.57 Evidence of colocalisation of MLC1 and members of the dystrophin-glycoprotein complex was found in immunohistochemistry at light microscopic level55 and in coimmunoprecipitation using cross-linking reagents,55,58 but coimmunopre cipitation studies without cross-linking failed to confi rm the interaction.30 Electron microscopy with immunogold staining for MLC1 showed GlialCAM G89D MLC1 (+ GlialCAM G89D) CLC2 (+ GlialCAM G89D) that the protein is present in astrocytic membranes between endfeet, but not at the vascular basal lamina Figure 4: Immunohistochemistry and immunocytochemistry MLC1 immunoreactivity (A, B) is found in astrocytic processes surrounding blood vessels in the normal human 30,59 (fi gure 3D). In these astrocyte junctions, MLC1 has brain (A), but no MLC1 immunoreactivity is found in the brain from a patient with MLC (B). been shown to colocalise with components of tight Immunocytochemistry in astrocytes shows that normal localisation of GlialCAM (red, C), MLC1 (green, (ZO-1) and adherent (β-catenin) junctions.30 Double coexpressed with GlialCAM, D), and CLC2 (green, coexpressed with GlialCAM, E) is seen in junctions of astrocytic immunogold electron microscopy for MLC1 and processes (overlap in yellow, D, E). By contrast, the presence of an MLC-causing HEPACAM mutation (G89D, p.Gly89Asp) leads to mislocalisation of GlialCAM (red, F), MLC1 (green, G), and CLC2 (green, H). β dystroglycan, a member of the dystrophin-glycoprotein MLC=megalencephalic leukoencephalopathy with subcortical cysts. complex, provided fi nal proof that MLC1 does not colocalise with the dystrophin-glycoprotein complex at osmoregulatory process.60–63 Even small osmotic per- astrocytic endfeet (fi gure 3D).30 turbations induce a transmembrane fl ow of ions and Aminoacid sequence analysis reveals a weak similarity water in astrocytes that rapidly restores the osmotic with several channels and transporters.32,50,53 MLC1 equilibrium and induces temporary swelling or contains an internal aminoacid sequence repeat, as shrinkage of astrocytes, followed by regulatory volume found in several ion channel proteins.53 These data, in decrease or increase, respectively, to normalise cell combination with the localisation of MLC1 at astrocytic volume. Regulatory volume decrease involves activation endfeet at blood–brain and CSF–brain barriers and the of ion channels and transporters that allow effl uxes of highly increased water content of the cerebral white potassium, chloride, organic osmolytes, and water.64 matter in MLC,26 suggested that MLC1 could have a Volume-regulated anion channels (VRACs) play an channel or transporter function, involved in brain water important part in the regulatory volume decrease. and ion homoeostasis.32 VRACs are activated by water fl uxes and changes in cell Water homoeostasis and osmotic balance are vital in shape and volume.65,66 VRAC function is probably the brain. Within the bony casket of the skull, any dependent on multiple channels, which could be signifi cant brain swelling leads to herniation of the brain diff erent for diff erent cell types. The molecular identity and death of the patient. Astrocytes are central in the of most of these channels is unknown.67,68 process of brain volume regulation. They are highly An association has been found between MLC1 sensitive to changes in extracellular osmolarity and can expression and chloride currents in diff erent cell types, display prominent cell volume changes as part of the including lymphoblasts and astrocytes, the main cell www.thelancet.com/neurology Vol 11 November 2012 977 Review

types that express MLC1 (fi gure 5A–D).52 These currents current is a VRAC activity.52 Further studies of the are greatly increased by cell swelling induced by hypo- regulatory volume decrease after cell swelling in osmotic pretreatment (fi gure 5C, D).52 Ion substitution lymphoblasts and astrocytes confi rm that a defect in experiments, current–voltage profi le, the activation of MLC1 reduces the rate of the regulatory volume the currents by hypo-osmotic pretreatment, and decrease (fi gure 5E, F).52 Notably, the regulatory volume sensitivity to inhibition by specifi c ion channel decrease is slower and not abolished in the absence of blockers67–70 indicate that the MLC1-related chloride normal MLC1, indicating that it is not exclusively dependent on MLC1 function. MLC is not an immediately life-threat ening disease. Complete A Astrocyctes B Astrocyctes abolition of the regulatory volume decrease would not 3000 Control 1000 Control be compatible with life. These studies left open the MLC1 MLC1 siRNA 800 question of whether MLC1 is a VRAC, a component of a 2000 600 VRAC, or a protein that activates a VRAC, either directly † * 52 1000 400 or indirectly. Recent studies suggest that MLC1 could be part of a

Current (pA) 200 0 † Current (pA) 0 multiprotein complex that interacts with TRPV4, a –1000 –200 channel that mediates hypo-osmosis-induced cytosolic –100 –50 500 100 –100 500–50 100 calcium increase, which is essential for activation of the Voltage (mV) Voltage (mV) regulatory volume decrease after cell swelling.71–73 MLC1 C Lymphoblasts D Lymphoblasts mutations have been suggested to reduce MLC1 inter- 300 Control ISO 300 Patient 1 ISO Control HYPO Patient 1 HYPO actions with the complex and abolish the TRPV4- 200 200 mediated intracellular calcium inﬂ ux in astrocytes 72 100 † 100 induced by hypo-osmosis. The decreased inﬂ ux of calcium in response to cell swelling could account for 0 * 0

Current (pA) the decreased VRAC activation and decreased regulatory Current (pA) –100 –100 volume decrease in MLC. Importantly, these studies –200 –200 were not based on speciﬁ c coimmunoprecipitation –100 500–50 100 –100 500–50 100 using MLC1 antibodies, but on less speciﬁ c ouabain- Voltage (mV) Voltage (mV) chromatography. Additionally, all experiments were E Astrocytes F Lymphoblasts done with overexpression of normal and mutant MLC1 1·2 Control 1·4 Control in astrocytoma tumour cells, and overexpression can MLC1 siRNA 1·3 Patient 1·1 MLC1 siRNA cause artifactual results. Further studies are necessary 1·2 0 + wt MLC1 to clarify the possible role of TRPV4 in MLC, including 0 /F t 1·0 1·1 F /S t

S 1·0 experiments in cells with reduced MLC1 expression 0·9 0·9 and diﬀ erent cell types, in particular cultured primary

0·8 Hypo-osmotic 0·8 Hypo-osmotic astrocytes. 0·7 0 10 20 30 40 0 302010 40 GlialCAM protein Time (min) Time (min) In 2005, a novel gene was identiﬁ ed, designated Figure 5: Volume-regulated chloride channel currents and regulatory volume decrease HEPACAM, encoding a hepatic cell adhesion molecule MLC1-expression-related chloride currents in primary rat astrocytes (A, B). In astrocytes overexpressing (HEPACAM).74 In 2008, the protein was renamed glial human MLC1 (A, blue trace), chloride currents have increased compared with astrocytes overexpressing LacZ cell adhesion molecule (GlialCAM), because liver as a control (A, red trace) after mild trypsin treatment to activate chloride currents. Chloride currents are lower in astrocytes with decreased expression of MLC1 due to treatment with small interfering RNAs (siRNA) expression is very low and it is predominantly expressed 75 directed against MLC1 (B, green trace) than in astrocytes treated with non-targeted siRNA as a control (B, red in glia of the nervous system. Cell adhesion molecules trace) after mild trypsin treatment. Chloride currents in control and MLC patient lymphoblasts (C, D). In (CAMs) are transmembrane proteins at the cell control lymphoblasts, currents are activated by hypo-osmotic pretreatment (compare hypo-osmotic surface.75–77 They interact with other cell adhesion pretreatment [HYPO], green trace, to iso-osmotic treatment [ISO], red trace). Chloride currents are decreased in MLC patient lymphoblasts and do not respond to hypo-osmotic pretreatment (D, compare ISO, green molecules on the adjacent cell surface and with proteins trace, to HYPO, purple trace). MLC1-dependent regulatory volume decrease in astrocytes and lymphoblasts in the extracellular matrix. Inside the cell, they interact (E–F). Rat astrocytes (E) and human lymphoblasts (F) were bathed in a chloride-selective iso-osmotic with the actin cytoskeleton, which is the key structure in solution that was switched to a hypo-osmotic chloride-selective solution for 30 min (black bar in E and F). the maintenance of cell integrity and shape and in the Average relative fluorescence was assessed in astrocytes (E) and average relative cell surface in lymphoblasts (F), as measures for cell volume. In rat astrocytes expressing siRNA against MLC1 (blue), the regulatory movement of cells. Cell adhesion is crucial for the decrease is slower than in astrocytes expressing non-targeted siRNA as a control (E, red). The slowing of the formation and maintenance of cellular architecture and regulatory volume decrease is rescued by expression of human wild-type (wt) MLC1 (purple). The regulatory for processes such as migration, proliferation, diﬀ eren- volume decrease was slower in lymphoblasts from patients with MLC (blue) than in lymphoblasts from tiation, apoptosis, and survival. CAMs also play a crucial controls (red). Modified from figures 4, 5, and 6 of Ridder and colleagues,52 by permission of Oxford University Press. See the original figures for numbers of cells and individuals investigated and the standard part in modulating cytoplasmic signalling cascades by errors of the mean. Significant differences are shown by asterisks and daggers (*p<0·05, †p<0·01). capturing and integrating signals from the extracellular MLC=megalencephalic leukoencephalopathy with subcortical cysts. environment.77

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GlialCAM has the typical structure of immuno- infertility.92 Additionally, they have highly increased globulin-like cell adhesion molecules (IgCAMs), with brain white matter water content with vacuole two extracellular immunoglobulin-like domains, a formation in the outer myelin lamellae, similar to what trans membrane segment, and a cytoplasmic tail.74,75 is seen in the brains of patients with MLC.91 IgCAMs are particularly abundant in the nervous Homozygous Clcn2 mutant mice also display myelin system.78 During development, IgCAMs are involved in vacuolation.93 Strikingly and similar to the earlier stages neuronal and glial migration, process guidance, target of MLC in patients, the mice lack overt neurological recognition, and synapse formation.79 In the mature defi cits despite extensive myelin vacuolation of the nervous system, they participate in the maintenance brain white matter.91 and function of neuronal networks, the integrity of the No CLCN2 mutations were found in patients with ependymal lining, and in the formation and stability of MLC who did not have MLC1 mutations.41,91 No direct myelin sheaths around axons.80,81 protein interaction was found between MLC1 and Within the human brain, GlialCAM expression is CLC2.30 The Clcn2 knockout mouse showed no changes seen mainly within white matter tracts and ependymal in GlialCAM or MLC1 expression and localisation.90 cells.75 Within the white matter, it is present mainly in GlialCAM has been shown to be a CLC2 binding astrocytic endfeet around blood vessels.22 Additionally, partner.90 GlialCAM targets CLC2 to astrocyte–astrocyte GlialCAM is localised inside axons, in contact regions junctions, whereas HEPACAM mutations, as identifi ed between myelin and axons, on the outside of myelin in patients with MLC, abolish this targeting (fi gure 4).88 sheaths, and in oligodendrocytes.22,75 Immunogold elec- GlialCAM greatly increases CLC2-mediated chloride tron microscopy with double staining for GlialCAM currents, changing properties of activation and and MLC1 show that they colocalise in astrocyte– rectifi cation (appendix).90 GlialCAM has a much more astrocyte junctions at astrocytic endfeet (fi gure 3E).22 restricted expression pattern than does CLC294 and Both MLC158,82 and GlialCAM83 are associated with GlialCAM is clearly not an obligate binding partner of caveolae, which are small, fl ask-like invaginations of CLC2, but an auxiliary subunit that associates with the plasma membrane, present in most adherent cells. CLC2 only in glial cells. Caveolae are important in the compartmentalisation of lipid and protein components that function in signal Mutations and their eff ects transduction, biosynthetic transport functions, endo- MLC1 mutations, both missense and null, lead to the cytosis, and transcytosis.84 same result: major reduction or absence of the plasma The function of GlialCAM is unknown. It has been membrane expression of MLC1 (fi gure 4B).53,95 This studied mainly in the context of diff erent types of similarity probably accounts for the absence of genotype– cancer.85–87 Studies of MLC indicate that one of the phenotype correlation. GlialCAM functions is to act as an MLC1 β subunit All known MLC-causing HEPACAM mutations, both necessary for its traffi cking to cell–cell junctions.88 dominant and recessive, aff ect the extracellular part of GlialCAM is necessary for the correct localisation of GlialCAM and not its transmembrane and intracellular MLC1, but the expression and localisation of GlialCAM part.22 MLC-causing HEPACAM mutations lead to a are independent of MLC1.88 In the presence of mutant GlialCAM-traffi cking defect.88 Partial or complete GlialCAM, MLC1 is mislocalised (fi gure 4), but mutant deletion of the intracellular GlialCAM domain does not MLC1 does not change GlialCAM localisation.88 The change the plasma membrane localisation.96 Apparently, facts that GlialCAM is not obligatorily associated with the intracellular domain is needed for cell-matrix MLC1 and that GlialCAM is expressed in cell types that modulation, but is dispensable for the membrane do not express MLC1, such as oligodendrocytes, suggest localisation;96 only disruption of membrane localisation that it has other functions.88 leads to MLC. The eff ect of dominant versus recessive GlialCAM Chloride channel CLC2 mutations has been studied at the cellular level. The defects The chloride channel encoded by CLCN2 belongs to the in traffi cking of MLC1 and GlialCAM caused by recessive CLC family of chloride channels or transporters.89 It is mutations in GlialCAM can be rescued by coexpression of almost ubiquitously expressed and is also abundant in the normal protein, but the traffi cking defects caused by the brain. Within the brain, CLC2 is, like MLC1 and dominant mutations in GlialCAM cannot be rescued by GlialCAM, localised in astrocyte–astrocyte junctions at coexpression of the normal protein.22 These results suggest astrocytic endfeet around blood vessels and in Bergmann that the diff erence between the recessive progressive MLC glia.13,90 Additionally, CLC2 is, like GlialCAM, located in phenotype and dominant remitting phenotype is not a myelinated fi bre tracts.91 Hypotonic cell swelling can protein dosage eff ect. At present, why some GlialCAM activate CLC2.67 Glial CLC2 is important in main taining mutations behave as recessive and others as dominant, and extracellular ion homoeostasis in the brain.91 why dominant mutations are associated with a much more Clcn2 knockout mice display severe degeneration of benign disease course than recessive mutations, is unclear. the retina and testes, leading to blindness and male All dominant mutations identifi ed thus far are located in a www.thelancet.com/neurology Vol 11 November 2012 979 Review

H2O K+

+ H2O Na K+ K+

H2O K+ Cx

Cx47 Cx32

AQP4 H2O Kir4.1 K+ MLC1

Kir4.1 AQP4 MLC1 GlialCAM Connexin 32 Connexin 47 Connexins 26, 30, 43

Figure 6: Action-potential-driven potassium and water ﬂ uxes Entry of sodium at nodes of Ranvier; exit of potassium at the paranodal axonal plasma membrane; and intracellular and intercellular pathways for potassium and water through connexin32 (C×32) gap junctions linking successive layers of myelin, into the astrocytic syncytium via gap junctions, constituted by C×32 and connexin47 (C×47), and ultimately to the astrocytic endfeet, to be released into pericapillary and subpial spaces through the potassium channel Kir4.1 and the water channel aquaporin-4 (AQP4). Drawing courtesy of Gert C Scheper, modiﬁ ed from Rash,98 by permission of Elsevier.

putative pocket of the immunoglobulin variable domain The role of CLC2 in this context is unclear. There is and have been suggested to disrupt interactions with no evidence that CLCN2 mutations underlie MLC or GlialCAM itself and other molecules, but recessive any other human disease.41,52 In view of the widespread mutations might aff ect the same domain.22 expression of CLC2 in the human body, CLCN2 The clinical and MRI phenotypes related to recessive mutations could lead to a disease dominated by non- MLC1 and HEPACAM mutations are indistinguish- neurological manifestations and a brain white matter able,21,22 suggesting that the molecular basis of MLC is disease could be present, but overlooked because of either in all cases dependent on defi ciency of cell-surface lack of overt neurological signs, as happened in the expression and function of MLC1 or on a defect in the Clcn2 knockout mouse. HEPACAM mutations, as interaction of MLC1 and GlialCAM or the interaction of identifi ed in patients with MLC, disrupt the localisation these proteins with a third partner, such as CLC2. In view of both MLC1 and CLC2. The data available at present of the presumed functional roles of GlialCAM as a cell suggest that MLC1–GlialCAM and CLC2–GlialCAM are adhesion molecule in cell growth, diff erentiation, diff erent complexes, although they could be functionally motility, and adhesion and its widespread expression related. In view of the functional association of compared with that of MLC1, that there is no evidence GlialCAM with both MLC1 and CLC2, why the disease that these functions are aff ected in patients with MLC associated with HEPACAM mutations is not more and HEPACAM mutations is surprising. A possible severe than the disease associated with MLC1 mutations explanation is that apart from bringing MLC1 to is unclear. astrocyte–astrocyte junctions, GlialCAM functions are redundant and can be executed by other proteins. Pathophysiology Another possibility is that other HEPACAM mutations MLC is a disease of chronic brain white matter oedema or mutations in other domains are associated with a with accumulation of water in vacuoles between myelin diff erent, as yet unidentifi ed, disease. lamellae and astrocytic endfeet. MLC1 has recently been

980 www.thelancet.com/neurology Vol 11 November 2012 Review

astrocytes. The astrocytic syncytium allows rapid Panel: Diagnostic criteria for megalencephalic dispersion of potassium and water between myelin and leukoencephalopathy with subcortical cysts astrocytic endfeet, which release potassium by the 1 Macrocephaly within the fi rst year of life potassium channel Kir4.1 and water by the water 2 No or slow neurological deterioration channel aquaporin-4 into pericapillary and subpial 3 Diff use cerebral white matter abnormalities with signs of spaces (fi gure 6).98 Electrical and osmotic gradients are swelling of the abnormal white matter the driving forces for this process, which prevents 4 Relative sparing of central white matter structures such potassium-induced osmotic swelling of myelin.98 as corpus callosum, internal capsule, and brainstem If any molecular component of the panglial 5 Anterior temporal cysts syncytium is compromised, hampered siphoning of 6 No grey matter abnormalities excess potassium related to neuronal activity leads to 7 On follow-up, either atrophy of the cerebral white matter osmotic myelin swelling.98 Kir4.1 knockout mice99 and or improvement to complete normalisation of the white double-knockout mice for gap junction proteins matter abnormalities; no other abnormalities arise connexin32 and connexin47100 display myelin vacuolation. Disruption of CLC2 similarly leads to brain myelin vacuolation in mice.91,93 Interestingly, no shown to have a role in the regulatory volume decrease myelin vacuolation in the optic nerve is observed in after cell swelling.52 Kir4.1 knockout mice, if action potential generation is Action potential fi ring causes continuous shifts of blocked with tetrodotoxin,99 and in Clcn2 knockout ions and associated osmotic water, necessitating tight mice, which are blind because of retinal degeneration,91 control of water and ion homoeostasis in the brain. emphasising the importance of the siphoning process Astrocytes are central to this process. They are the during neuronal activity. In human beings, mutations most abundant cell type of the so-called panglial in connexin32 are associated with stress-provoked syncytium. This is a vast network of interconnected episodes of brain dysfunction with MRI evidence of glia, consisting of astrocytes, oligodendrocytes, and acute, reversible myelin vacuolation.101,102 We hypo- ependymal cells, which are extensively interlinked by thesise that in MLC one of the most important gap junctions. Gap junctions are composed of connexin consequences of the defect in the astrocytic regulatory proteins and form channels across the intercellular volume decrease, caused by disrupted volume- space. The panglial syncytium is essential for long- regulated chloride channel activity, is that the disposal distance disposal of excess axonally derived potassium of excess potassium and water associated with action and water in a process called potassium siphoning.97,98 potential fi ring through the astrocytic syncytium is In myelinated axons, depolarisation-related sodium hampered, necessitating slightly steeper electrical and infl ux occurs at nodes of Ranvier; potassium effl ux osmotic gradients, explaining the presence of occurs in paranodal and internodal axonal regions extra water in the form of vacuoles in myelin and covered by myelin (fi gure 6).98 From the periaxonal astrocytic endfeet. space, the excess potassium and osmotic water enters the inner myelin layers. Gap junctions allow potassium Diagnosis and diff erential diagnosis and water transport between myelin layers at successive In MLC, rapidly increasing macrocephaly is the most paranodal loops and link myelin to surrounding common reason for presentation in the fi rst or second

Onset Typical disease course Typical MRI features Defi cient enzyme Mutated gene Megalencephalic Infancy Mild and slow Diff use cerebral leukoencephalopathy, no grey matter ·· MLC1, HEPACAM leukoencephalopathy with lesions, anterior temporal cysts subcortical cysts Merosin-defi cient congenital Infancy Severe muscle weakness Diff use cerebral leukoencephalopathy, no grey matter ·· LAMA2 muscular dystrophy lesions, occasionally anterior temporal cysts Alexander disease Infancy to Early onset associated with severe course; Leukoencephalopathy with frontal preponderance, ·· GFAP adulthood later onset associated with slower course abnormalities of basal nuclei, contrast enhancement, sometimes cystic white matter degeneration Canavan disease Infancy to Early onset associated with severe course; Diff use cerebral leukoencephalopathy, abnormalities of Aspartoacylase ASPA childhood later onset associated with slower course globus pallidus and thalamus L-2-hydroxyglutaric aciduria Infancy to Mild and slow Subcortical cerebral leukoencephalopathy, which becomes L-2-hydroxyglutarate L2HGDH childhood diff use over time, abnormalities of basal nuclei and dentate dehydrogenase nucleus

GM1 and GM2 gangliosidoses, Infancy Severe course Diﬀ use cerebral leukoencephalopathy, abnormalities of basal β galactosidase and GLB1 and HEXA, infantile form nuclei and thalamus β hexosaminidase HEXB, or GM2A

Table: Diﬀ erential diagnosis of megalencephalic leukodystrophies

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regulation by astrocytes. The defect results in chronic Search strategy and selection criteria brain white matter oedema. The excess water is located References for this Review were identifi ed by searches of in vacuoles within myelin sheaths and, to a lesser PubMed between 1969 and May, 2012, and references from extent, astrocytic endfeet. Most patients have slow relevant articles. The search terms “leukoencephalopathy”, neurological deterioration, but some patients show “MLC”, “MLC1”, “HEPACAM”, “GlialCAM”, “ClC-2”, and surprising improvement or recovery, clinically and on “CLCN2” were used. There were no language restrictions. The MRI, suggesting that the disease might be accessible fi nal reference list was generated on the basis of relevance to for treatment. the topics covered in this Review. Brain oedema is a major problem in neurology. Whereas other organs have space to swell without causing a life-threatening situation, brain oedema year of life. Such patients undergo CT or MRI, which rapidly leads to coma and death. Combating of brain rules out hydrocephalus and shows profound cerebral oedema is of major importance in many acute and white matter abnormalities with swelling. The severity subacute situations, including cerebral infarction, of the white matter disease contrasts with the presence trauma, and tumours. MLC can be regarded as a natural of no or only mild clinical signs at this age. The disease model of brain oedema, and the discovery of diagnosis of MLC is not diffi cult in typical cases and successful strategies for the treatment of MLC will can be made on the basis of clinical and MRI criteria probably have implications for the treatment of certain (panel). If no early MRI is available in patients with types of brain oedema. Further studies should focus on remitting MLC, the MRI might show only limited or no the precise role of MLC1 dysfunction in the development white matter abnormalities and the diagnosis can be of oedema. Such studies could reveal therapeutic easily missed. options for patients with MLC and yield information The combination of macrocephaly and diff use that is relevant for other conditions. cerebral white matter abnormalities can be seen in Contributors Alexander disease, Canavan disease, L-2-hydroxyglutaric MSvdK designed, organised, and undertook the literature review. IB and aciduria, congenital muscular dystrophy with merosin RE contributed to the literature review. MSvdK wrote the fi rst draft. defi ciency, and sometimes in infantile-onset GM and MSvdK, IB, and RE wrote the subsequent drafts together, reviewed and 1 critiqued the report, and agreed on the fi nal version. All personal 1,2,12,101,103 GM2 ganglio sidoses. These disorders can be observations, cited in the text, were those of MSvdK. diff erentiated by clinical signs, MRI fi ndings, and Confl icts of interest 1,2,12,101,103 laboratory tests (table). We declare that we have no confl icts of interest. Acknowledgments Treatment We thank all patients, families, and colleagues who contributed to our Understanding of MLC disease mechanisms is in- studies for their cooperation; Gert C Scheper (molecular scientist, creasing, but is still incomplete. The most important Crucell Holland NV, Leiden, Netherlands) for design of fi gure 6; and James M Powers (neuropathologist, University of Rochester, missing link is that, although it has become clear that Rochester, NY, USA) for critical reading of the report. Financial MLC1 is involved in brain volume regulation through support for the work of MSvdK and IB was provided by the Dutch astrocytes, its exact function in this process has not been Organisation for Scientifi c Research (ZonMw, TOP grants 903-42-097 identifi ed. There is no easy treatment option. Trials with and 9120.6002), E-Rare (grant 11-330-1024), the Hersenstichting (grants 10F02(2).02, 13F05.04, 15F07.30 and 2009(2)-14), and the diuretic drugs have failed (unpublished observations). Optimix Foundation for Scientifi c Research. Financial support to RE The transient MLC phenotype in patients with a is provided by SAF 2009-07014, PS09/02672-ERARE, ELA Foundation dominant HEPACAM mutation suggests that there is a 2009-07014, 2009 SGR 719, and the ICREA Academia Prize. The window of time in which rescue of MLC1 function can funding sources had no direct involvement with the contents, design, or conduct of the authors’ studies. prevent the disease or modify its course. With our present knowledge, two therapeutic strategies References 1 Van der Knaap MS, Barth PG, Stroink H, et al. could be envisioned. Pharmacological strategies aimed at Leukoencephalopathy with swelling and a discrepantly mild clinical increasing cell membrane expression of MLC1 and course in eight children. Ann Neurol 1995; 37: 324–34. GlialCAM could be benefi cial, although whether ex- 2 Singhal BS, Gursahani RD, Udani VP, Biniwale AA. Megalencephalic leukodystrophy in an Asian Indian ethnic group. pression of mutant proteins would compensate for the Pediatr Neurol 1996; 14: 291–96. functional defi cit is not known. Alternatively, activation 3 Thelle T, Balslev T, Christenen T. Van der Knaap’s vacuolating of ion channels that have a reduced function in MLC, leukoencephalopathy: two additional cases. Eur J Pediatr Neurol 1999; 3: 83–86. such as VRACs, or perhaps TRPV4 or CLC2, could be 4 Gelal F, Call C, Apaydin M, Erdem G. Van der Knaap’s useful. Additional experiments are needed to investigate leukoencephalopathy: report of fi ve new cases with emphasis on these hypotheses. diff usion-weighted MRI fi ndings. Neuroradiology 2002; 4: 625–30. 5 Saijo H, Nakayama H, Ezoe T, et al. A case of megalencephalic leukoencephalopathy with subcortical cysts (van der Knaap disease): Conclusions molecular genetic study. Brain Dev 2003; 25: 362–66. MLC is the fi rst known human disease with a genetic 6 Van der Knaap MS. Forget about “van der Knaap syndrome”. defect in brain ion and water homoeostasis and volume AJNR Am J Neuroradiol 2003; 24: 1030.

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