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Megalencephalic Leukoencephalopathy with Subcortical Cysts Boor, P.K.I.

2008

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citation for published version (APA) Boor, P. K. I. (2008). Megalencephalic Leukoencephalopathy with Subcortical Cysts: from disease gene to protein function.

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Summary, Discussion, and regel 8

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Prospects regel 10

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regel 39 Summary, Discussion, and Prospects

regel 1 1. Clinical Description regel 2 regel 3 The studies described in this thesis all started when a ten-year-old boy with macrocephaly regel 4 and cerebellar ataxia visited the out-patient department of the VU University Medical T regel 5 Center in 1991. MRI analysis revealed that this boy had a disease involving the white matter regel 6 of the brain. Soon after this patient other patients with strikingly similar clinical and MRI 1 regel 7 features were detected, as reported by Van der Knaap et al . Since then several other 2-10 regel 8 groups have described similar cases . In none of these children a specifi c diagnosis regel 9 could be established or a basic biochemical defect could be identifi ed. The homogeneous regel 10 clinical picture and MRI fi ndings were suggestive of a ‘new’ disease entity, now known regel 11 as ‘Megalencephalic Leukoencephalopathy with subcortical Cysts’ (MLC). Characteristic regel 12 clinical features of the disease are a macrocephaly that arises in the course of the fi rst regel 13 year of life and a delayed onset of motor deterioration, dominated by cerebellar ataxia. regel 14 Characteristic MRI features include diff use cerebral white matter signal abnormalities, 1 regel 15 swelling of the aff ected white matter, and the typical subcortical cysts . regel 16 regel 17 regel 18 2. Discovery of the MLC1 gene regel 19 regel 20 The high frequency of consanguinity of the parents and occurrence of families with regel 21 two aff ected siblings suggested an autosomal recessive mode of inheritance. In the late regel 22 nineties we had collected enough informative families (four consanguineous families regel 23 and one family with two aff ected children) to start a genetic linkage study with micro- regel 24 satellite markers. In the mean time, a Turkish-French consortium detected the locus for 11 regel 25 the disease gene at the very end of the long arm of chromosome 22 (22qtel) . The critical regel 26 region, however, was too large to identify the disease gene. We used the information of regel 27 our families to limit the critical region to a DNA stretch that only contained four genes. regel 28 Mutation analysis showed mutations in the second gene investigated, KIAA0027, which regel 29 we renamed into MLC1. The discovery of this gene is described in chapter 2. regel 30 regel 31 regel 32 3. Characterization of the MLC1 gene T regel 33 regel 34 3a. Types of mutations 12-22 regel 35 Over the years many diff erent mutations in the MLC1 gene have been found . These regel 36 mutations are distributed along the whole gene and include all diff erent types: splice- regel 37 site mutations, nonsense mutations, missense mutations, deletions, and insertions. regel 38 regel 39 114 Novel mutations in our patient population are described in chapter 3. In chapter 4 an regel 1 update is given describing all mutations found up to 2005. Although most families have regel 2 13,16,18,20 unique mutations, evidence for a founder eff ect is present in four communities . regel 3

The strongest evidence for a founder eff ect is found in the Indian Agarwal community regel 4 13,20 . All patients within this community share the same insertion (c.135_136insC) causing a regel 5 frameshift (p.Cys46LeufsX34) and the appearance of a premature stop. regel 6

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3b. Extended mutation analysis regel 8

In about 20% of the typical MLC cases, diagnosed by clinical and MRI criteria, no or only regel 9 one MLC1 mutation can be found. One reason for not fi nding mutation(s) is the standard regel 10 mutation analysis at genomic level, which may miss heterozygous deletions, mutations regel 11 in the promoter or 3’- and 5’-untranslated regions, and intron mutations that may aff ect regel 12 splicing of the mRNA. For this reason, we performed extended mutation analysis in regel 13 patients that have only one or no mutations in the MLC1 gene. This analysis included regel 14 sequencing of cDNA derived from lymphoblasts of the patients to show any missed regel 15 splice-site mutations, and qPCR to elucidate diff erences in expression levels of the MLC1 regel 16 gene. Chapter 4 provides, in addition to the mutation update, information on mutations regel 17 found with the extended mutation analysis. regel 18

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3c. Evidence for additional genes involved in MLC regel 20

Several families, in which no mutations are found, also do not show linkage with the regel 21

MLC1 locus. This indicates that there must be more than one gene associated with MLC regel 22 14,21,23 . Therefore linkage analysis was performed in four informative families, without regel 23 mutations in the MLC1 gene and without linkage on the MLC1 locus, which resulted in regel 24 several candidate regions. Some promising genes in these regions were sequenced, regel 25 but unfortunately no mutations have been found. Since there are too many candidate regel 26 regions, this study will only be continued when DNA from additional informative families regel 27 will become available that can possibly reduce this number. It should be noted that the regel 28 identifi cation of multiple candidate regions may not only be due to the limited number of regel 29 informative families, but can also be the result of several additional genes being involved regel 30 in MLC. regel 31

The consequence of the above fi ndings is that the diagnosis remains primarily based on regel 32 clinical and MRI fi ndings. Thus, if the MRI shows the typical features of MLC in an ataxic 8 regel 33 patient with macrocephaly, the diagnosis is MLC even if no mutations are found in the regel 34

MLC1 gene. regel 35

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regel 39 115 Summary, Discussion, and Prospects

regel 1 4. Genotype-phenotype correlation regel 2 regel 3 No genotype-phenotype correlation has been observed. So, the severity of the phenotype regel 4 does not appear to correlate with the type and position of the mutation(s) in the gene. regel 5 There is no phenotypic diff erence between MLC patients with mutations in MLC1 and regel 6 patients without. Patients from the Agarwal community, who are all homozygous for the regel 7 same mutation, exhibit a wide phenotypic variation. regel 8 regel 9 regel 10 5. Localization of the MLC1 protein regel 11 regel 12 The MLC1 gene encodes a 377 amino acid plasma membrane protein, called MLC1. In regel 13 chapter 5 we report that this protein contains eight transmembrane domains and that regel 14 it is highly conserved throughout evolution in a variety of myelin-producing vertebrates regel 15 (see Figure 1). Moreover we show that, in the brain, MLC1 is expressed in distal processes regel 16 of astrocytes at the blood-brain and CSF-brain barriers and in the cellular processes of regel 17 Bergmann glia. This specifi c localization of MLC1 strongly suggests that MLC1 is localized regel 18 in astroglial endfeet, but higher-resolution electron microscopic (EM) studies are necessary regel 19 to show the exact localization. In support of this suggestion, Teijdo et al. already showed 24 regel 20 that mouse MLC1 is expressed in astrocytic endfeet . Besides the expression in brain, regel 21 MLC1 is also expressed in all types of leukocytes, as shown in chapter 5. The signifi cance regel 22 of this expression remains unclear. There is no evidence that mutations in the MLC1 gene regel 23 cause a defect in leukocyte function in patients. 25 regel 24 Recently, Teijido et al. published a paper about the expression of MLC1 in mice. They regel 25 showed that MLC1 is expressed in neurons in the adult mouse brain. In addition, EM regel 26 studies showed a more precise localization of MLC1 in the plasma membrane and in regel 27 vesicular structures of neurons. In the developing brain, MLC1 was mainly expressed regel 28 in axonal tracts during early stages of development. So far, we have not been able to regel 29 confi rm these results in humans. We, however, did not immuno-stain the complete regel 30 human brain systematically yet. Another contradiction with the human situation is MLC1 regel 31 expression in the peripheral nervous system. The lack of expression in humans is in line regel 32 with the absence of ‘peripheral dysfunction’ in MLC patients. Teijido et al. also described regel 33 EM studies showing that MLC1 is localized in astrocyte-astrocyte junctions, and not in regel 34 the perivascular membrane. They therefore concluded that MLC1 can not be a part of the regel 35 dystrophin-associated glycoprotein complex (DGC) (see chapter 6 for further discussion). regel 36 To confi rm or reject these fi ndings between species it is necessary to perform EM-studies regel 37 in the human brain. regel 38 regel 39 116 It is important to note that MLC is the second genetic defect that specifi cally involves an regel 1 astrocytic protein. The fi rst genetic defect was described in 2001 by Brenner et al. who regel 2 demonstrated that Alexander disease is caused by mutations in GFAP, a cytoskeleton regel 3 26 protein that is only present in astrocytes . Interestingly, both MLC and Alexander disease regel 4 are childhood leukoencephalopathies. regel 5

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Figure 1: Alignment of the protein products of the MLC1 genes of several vertebrates. Gray block Conservative regel 27 diff erences black blocks highly conserved. The eight predicted transmembrane domains are boxed. regel 28

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6. Binding partners of MLC1 regel 31

regel 32 A multi-subunit complex called the dystrophin-glycoprotein complex (DGC) is expressed 8 regel 33 27 in astrocytic endfeet . This complex is expressed in diff erent tissues and has been regel 34 characterized best in skeletal muscle where it connects the cytoskeleton of a muscle fi ber regel 35 28 to its surrounding extracellular matrix . Mutations in diff erent components of the DGC regel 36 disrupt this complex and lead to various muscular dystrophies. In the congenital muscular regel 37

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regel 39 117 Summary, Discussion, and Prospects

regel 1 dystrophies (CMDs), the muscular phenotype is often combined with brain abnormalities, regel 2 including white matter abnormalities. regel 3 From early on, a striking similarity in MRI features of MLC and CMD with merosin defi ciency 1,29 regel 4 (MDC1A) was noted . MDC1A is caused by mutations in the LAMA2 gene, encoding the 30 regel 5 laminin-α2 chain of merosin , a member of the DGC. Patients with MDC1A also have regel 6 diff usely abnormal, mildly swollen cerebral white matter and in some cases there are 29 regel 7 anterior temporal cysts . In addition, microscopic examination of the brain revealed 31 regel 8 myelin vacuolation in MDC1A, similar to MLC . The striking similarities in MRI features regel 9 and pathology between MLC and MDC1A, together with the specifi c localization of both regel 10 MLC1 and merosin at astrocytic endfeet, led to the hypothesis that the MLC1 protein regel 11 might be associated with the DGC in the brain (chapter 6). regel 12 To test this hypothesis, a general feature of the DGC was used: if one of its composing T regel 13 proteins is mutant or missing, the complex can be instable and other DGC-members 32-38 regel 14 can show reduced or altered expression . We, therefore, tested the (co-) localization regel 15 of MLC1 and several DGC-members in control, glioblastoma and MLC brain tissue. The regel 16 results in chapter 6 show an almost perfect co-localization of MLC1 and members of regel 17 the DGC. Altered expression of MLC1 and aquaporin-4 was found in glioblastoma tissue. regel 18 Additionally, the absence of MLC1 and altered expression of both agrin, Kir4.1 and D- regel 19 dystroglycan in brain tissue of MLC patients was demonstrated. Furthermore, a direct regel 20 protein interaction between MLC1 and Kir4.1 was shown. Unfortunately, we were not regel 21 able to immunoprecipitate multiple DGC-members with MLC1 antibodies, despite great regel 22 eff orts of two laboratories (Amsterdam and Barcelona). This might be due to the inability regel 23 of these antibodies to immunoprecipitate or because immunoprecipitation is not regel 24 possible between these proteins. Together the above fi ndings provide strong evidence 39 regel 25 for an association between MLC1 and DGC-members. Ambrosini et al. recently provided regel 26 additional evidence for functional and structural relationships between MLC1 and the regel 27 DGC. regel 28 Virtually all known defects in DGC-proteins have been associated with a muscular regel 29 dystrophy, which is absent in MLC patients. This may be due to a diff erence in DGC regel 30 composition for muscle compared to brain tissue. Whereas merosin is expressed both in regel 31 muscle and brain, MLC1 is not expressed in skeletal muscle. regel 32 Another striking similarity between MLC and MDC1A is in that both diseases the water regel 33 content of the aff ected white matter is abnormally high due to intramyelinic vacuole regel 34 formation. The DGC is crucial for anchoring of water and potassium channels at the regel 35 perivascular endfeet. Disruption of the DGC may lead to altered ion and water homeostasis regel 36 of the brain and result in an increased water content within myelin. regel 37 regel 38 regel 39 118 7. Functional studies on the MLC1 protein regel 1

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Several observations prompted us to state the hypothesis that MLC1 has a transporter regel 3 or channel function. First of all, amino acid sequence analysis reveals a weak, similarity regel 4 between MLC1 and potassium channel Kv1.1, ABC-2 type transporters, and sodium: regel 5 galactoside symporters. Secondly, MLC1 contains an internal repeat, found in several ion regel 6 channel proteins. Based on these theoretical fi ndings, several papers have suggested a regel 7 20,24,40,41 possible transporter function of the MLC1 protein . Thirdly, as described in chapter regel 8

5, MLC1 has eight transmembrane domains. At the time, the Swissprot database contained regel 9

22 human proteins with eight transmembrane domains of which the majority (17 proteins) regel 10 has a transporter or channel function. regel 11

To test this hypothesis we applied the whole-cell patch clamp technique and regel 12 demonstrated that the MLC1 expression-induced current is carried by chloride (chapter regel 13

7). In chapter 7 we also described that this chloride transport is associated with volume regel 14 regulation. The chloride channel activity shows similarities with the activity of the so- regel 15 called volume-regulated anion channels (VRACs). The modulation of this VRAC has been regel 16 42 hypothesized to infl uence astrocytic cell volume and ion homeostasis in the brain . regel 17

Although the biophysical characteristics of VRACs have been investigated extensively regel 18 43,44 , the related proteins have not been identifi ed. Specifi c criteria for VRAC activity have regel 19 been published: (I) VRAC is activated by hypo-osmotic shock and associated cell swelling; regel 20

(II) currents through VRAC are outwardly rectifying and display only time-dependent regel 21 inactivation at high positive potentials; (III) VRAC currents are mainly carried by chloride; regel 22

(IV) VRAC is inhibited by anti-estrogens (for example Tamoxifen); and (V) VRAC activation regel 23 44,45 requires intracellular ATP . In our study we show chloride channel activity associated regel 24 with the presence of wild-type MLC1. Based on these results we conclude that MLC1 is regel 25 either a chloride channel involved in volume regulation or a protein indispensable for the regel 26 function of such channel. To elucidate the latter issue, we studied Sf9 insect cells, which regel 27 are evolutionary very distant from human cells. They do not contain a MLC1 ortholoque regel 28 and therefore most likely also lack the natural binding partners of MLC1. The fi nding that regel 29

MLC1 expression induced a chloride current profi le associated with volume regulation regel 30 in Sf9 cells strongly suggests that MLC1 harbors a channel function itself. Interestingly, regel 31 46 recently Blanz et al. published a paper about chloride channel CLC-2 knock-out mice, regel 32 that show brain pathology that is highly similar to MLC brain pathology. This is another 8 regel 33 hint that MLC1 may indeed be involved in chloride transport. regel 34

MLC patientderived lymphoblasts lack the chloride current profi le as well as cell swelling regel 35 under hypo-osmotic conditions. Strikingly, the lack of cell swelling under hypo-osmotic regel 36 conditions is also seen in control lymphoblasts, when chloride channel function is regel 37

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regel 39 119 Summary, Discussion, and Prospects

regel 1 inactivated by Tamoxifen. Hence, water infl ux and the activity of this MLC1-related regel 2 chloride channel must either be mutually dependent, co-regulated, or use the same regel 3 channel in these cells. In astrocytes a functional interdependence between MLC1 and a regel 4 water channel could involve the water channel Aquaporin-4 (AQP4), which is, like MLC1, 34,47 regel 5 localized in astrocytic endfeet . In addition, both MLC1 and AQP4 are associated with regel 6 the DGC (chapter 6). Interestingly, Benfenati et al. demonstrated that knockdown of 48 regel 7 AQP4 in rat astrocytes leads to a decrease of VRAC-mediated currents , supporting such regel 8 functional interdependence. In addition, Amiry-Moghaddam et al. showed an example of regel 9 functional interdependence between AQP4 and another ion channel (Kir4.1) in astrocytic regel 10 endfeet, which is also part of the DGC. Disruption of the AQP4 localization severely aff ected 49 regel 11 the Kir4.1 function, but its localization was unchanged . So far, we have been unable regel 12 to confi rm a physical interaction between MLC1 and AQP4 despite extensive immuno- regel 13 precipitation studies (data not shown). Alternatively, one may consider the possibility that 50 regel 14 activated chloride channels themselves are able to transport water . Further detailed regel 15 studies are required to unravel the relationship between this MLC1-associated chloride regel 16 channel and water homeostasis. regel 17 The complete absence of chloride currents in cells from patients with a frame-shift regel 18 mutation is indicative of a non-functional channel in these patients. Similar fi ndings, regel 19 however, were obtained in cells from a MLC patient with missense mutations. Absence regel 20 of function cannot be solely explained by a complete absence of MLC1 in the plasma 24 regel 21 membrane, because Teijido et al. showed that a signifi cant amount of MLC1 with amino regel 22 acid substitutions is expressed in the plasma membrane. Amino acid changes probably regel 23 aff ect the conformation of the protein and abolish its function. regel 24 In chapter 7 evidence was provided that increased white matter water content and regel 25 vacuolation of myelin and astrocytic endfeet are the cellular consequences of MLC1 regel 26 dysfunction. In chapter 5 it was reported that MLC1 is almost exclusively present in regel 27 leukocytes and brain tissue. There is, however, no evidence for abnormal leukocyte regel 28 morphology or function. In addition, it was shown in chapter 7 that MLC1 expression regel 29 is much higher in brain than in leukocytes, indicating that the implications of our regel 30 electrophysiological fi ndings are much more important for astrocytes than for leukocytes regel 31 and negligible for other cell types. The diff erential expression of MLC1 would explain the regel 32 exclusive involvement of the cerebral white matter in MLC. Chloride channel activity regel 33 associated with cell swelling has been documented in many diff erent cell types. This regel 34 activity in cells other than astrocytes is probably attributable to proteins other than MLC1. regel 35 In line with this, VRAC kinetics and pharmacology have been shown to be diff erent for 45 regel 36 diff erent tissues, indicative of the molecular diversity of VRAC . regel 37 In summary, our data suggest that MLC1 with a normal function is an absolute prerequisite regel 38 for chloride channel activity associated with cell swelling, given the total lack of hypo- regel 39 120 osmotically induced chloride currents in MLC patient-derived cells. The ultimate test regel 1 to show whether MLC1 is a volume-regulated chloride channel is to isolate the MLC1 regel 2 protein in a lipid bilayer, containing no other proteins than MLC1, and to record the regel 3 response to whole-cell chloride currents in both iso- and hypotonic conditions, or to regel 4 show anion-selectivity changes by using mutant MLC1 constructs. It might, however, regel 5 be diffi cult to fi nd the amino acid changes that are important for pore properties and regel 6 gating. Alternatively, MLC1 may be a component of a complex in which another channel regel 7 protein has chloride channel activity. In astrocytes, proper MLC1 function would then be regel 8 an absolute requirement for activity of this complex. regel 9

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8. Remaining questions regel 12

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One of the most important remaining question of this thesis is how dysfunction of chloride regel 14 channel activity, associated with volume regulation in astrocytes, leads to inclusion of regel 15 vacuoles in the outer layers of the myelin sheaths and in astrocytic endfeet. This question regel 16 is not easy to answer. Patient data indicate that the head circumference and neurological regel 17 condition are normal at birth. The head circumference increases rapidly over the fi rst year regel 18 of life and by 9-12 months all patients have a prominent macrocephaly. At birth, the brain regel 19 contains hardly any myelin and brain development during the fi rst year of life is dominated regel 20 by rapid myelin deposition in the white matter. This suggests that the deposited myelin regel 21 contains countless vacuoles from the beginning, explaining that the megalencephaly and regel 22 associated macrocephaly arise at the same time as the myelin is deposited. regel 23

During this process of myelination, substantial amounts of water need to be removed regel 24 for a proper compact myelin sheath formation. Kamasawa et al. 2005 suggests two regel 25 pathways to transport excess ions and water to the external layers of the myelin sheath: regel 26

(I) cytoplasmic diff usion via the cytoplasmic loops of myelin at paranodes and Schmidt- regel 27

Lanterman incisures, or (II) more direct radial diff usion to the outer layer by interlamellar regel 28 connexin32-containing gap junctions. From the outer lamellae of the myelin sheath, regel 29 ions and water are transported through oligodendrocyte-to-astrocyte gap junctions regel 30

(containing both connexin32 and connexin46.6 (same as connexin47 in mice)) into the regel 31 astrocytic syncytium and then passed on to astocytic endfeet at capillaries and the regel 32 51 glia limitans . Insuffi cient clearance of water and ions due to dysfunctional chloride 8 regel 33 channel activity may lead to inclusion of vacuoles in myelin sheaths during deposition. regel 34

Once trapped, the vacuoles may be permanent. The likelihood of this interpretation is regel 35 substantiated by the Cx32/Cx47 double knockout mouse that displays vacuoles in the regel 36 52 innermost and outermost myelin layers . regel 37

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regel 39 121 Summary, Discussion, and Prospects

regel 1 9. Possible treatment for MLC patients regel 2 regel 3 The phenomenon that MLC patient-derived lymphoblasts are not able to swell under T regel 4 hypotonic conditions can be used in the search for treatment of MLC. By high throughput regel 5 screening of large libraries of chemicals, the ability of individual chemicals to increase cell regel 6 swelling in patient-derived lymphoblasts can be tested. The ideal is to fi nd a molecule 53 regel 7 that is specifi c and will not interfere with other, related targets . regel 8 Recently Welch et al. published a paper about a new drug with potential therapeutic regel 9 options for patients with nonsense mutations. In this paper a new chemical entity, PTC124, regel 10 was described that selectively induces ribosomal read-through of premature terminations 54 regel 11 codons by performing throughput screenings . Table 1 in chapter 5 describes two MLC regel 12 patients who have a premature stop-codon mutation in one or both alleles (p.Tyr71X). regel 13 Eventhough stop-codon read-through will produce mutant MLC1, this mutant protein regel 14 might have residual chloride channel activity and, therefore, might still help to alter the regel 15 disease course. Studies in muscle cell cultures of Duchenne muscular dystrophy patients or regel 16 mdx mice (mouse model for Duchenne muscular dystrophy) already showed that PTC124 regel 17 can be useful as therapeutic agent. If PTC124 is able to cross the blood brain barrier, this regel 18 drug might also be benefi cial in MLC patients with nonsense mutations. regel 19 Curcumin is the principal curcuminoid of the Indian curry spice turmeric. Curcumin is regel 20 known for its anti-tumor, antioxidant, anti-amyloid and anti-infl ammatory properties. regel 21 In 2004 Egan et al. reported on the potential use of curcumin as a novel drug for the 55 regel 22 treatment of Cystic Fibrosis (CF) patients . Curcumin increases the expression of regel 23 membrane proteins in the plasma membrane. CF is caused by mutations in the gene regel 24 encodingthe cystic fi brosis transmembrane conductance regulator (CFTR) that functions regel 25 as a chloride channel and controls the regulation of other transport pathways. The most regel 26 common mutation (p.Phe508del) in CF patients results in the production of a misfolded regel 27 protein that is retained in the endoplasmicreticulum and targeted for degradation. Egan regel 28 et al. demonstrated that oral curcumin treatment leads to successful targeting of mutant regel 29 CFTR to the plasma membrane and partially restoring CFTR function in CFTR mice and 55 regel 30 baby hamster kidney cells . The data presented in this paper suggest that curcumin regel 31 might also prove to be useful as a therapy for MLC patients with mutations that cause regel 32 protein misfolding and reduced membrane expression. Preliminary results of cells treated regel 33 with curcumin and overexpressing mutant MLC1 (Ser280Leu) show indeed increased regel 34 membrane expression (personal communication Raul Estevez). Further experiments, regel 35 however, are needed to test whether increased membrane expression of MLC1 also leads regel 36 to partial recovery of the function. regel 37 regel 38 regel 39 122 10. Future experiments with animal models regel 1

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To unravel the exact mechanism behind myelin and astrocytic endfeet vacuolation and to regel 3 test possible therapies, the use of animal models will be indispensable. If the knock-out regel 4 of MLC1 in the mouse leads to vacuolation of myelin and astrocytic endfeet, this model regel 5 can be used to study the development of vacuoles during the deposition of myelin by regel 6 sacrifi cing the animals at diff erent embryonic and postnatal stages. regel 7

An animal model can also be used to further support the existence of a functional regel 8 relationship between aquaporins and MLC1. In embryonic brain the presence of a large regel 9 extracellular volume allows the diff usion of water and ions through vessels not surrounded regel 10 by astrocytes. Later during development and blood-brain barrier diff erentiation, the glial regel 11 processes envelop endothelial cells and form a perivascular sheath. When this process regel 12 56 comes to an end, the extracellular space is dramatically reduced . To reach a volume regel 13 fraction typical of adulthood the development of specifi c transporter mechanisms for regel 14 water and ions is required. Interestingly, rodents start to buff er extracellular potassium regel 15 during the fi rst 2-3 weeks of life in correspondence with a marked increase in AQP4 levels regel 16 56 . If there is indeed a functional relationship between MLC1 and AQP4, we can hypothesize regel 17 that the expression levels of MLC1 are in correspondence with the increased levels of both regel 18

AQP4 and Kir4.1. This hypothesis can be tested by investigating the expression levels of regel 19

MLC1, AQP4 and Kir4.1 in mice by sacrifi cing the animals at diff erent time points during regel 20 development and especially at the time the blood-brain barrier is formed. regel 21

More distant future experiments with a MLC1 knock-out mouse model can be MLC1 gene regel 22 transfer and cell transplantation. The adeno-associated virus mediated gene transfer regel 23 57 to the brain has been used in an ASPA knock-out mouse , which is a model for the regel 24 leukodystrophy known as Canavan disease. In this knock-out mouse the improvements regel 25 observed in MRI of the brain were sustained till 3–5 months after injection. A disadvantage regel 26 of this approach is that the eff ect of the gene transfer is predominantly present close regel 27 to the injected site. It is doubtful whether the gene transfer occurs in the entire central regel 28 nervous system, and if the disease is modifi ed in all areas. regel 29

Another approach to recover the lost MLC1 function is cell therapy. This method has regel 30 been already been used with some success in the ASPA knock-out mouse brain, in which regel 31 the implanted neural progenitor cells have been shown to be able to diff erentiate into regel 32 58 oligodendrocytes and astrocytes . Implantation of neural progenitor cells is a promising 8 regel 33 option to recover the lost MLC1 function. regel 34

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regel 39 123 Summary, Discussion, and Prospects

regel 1 References regel 2 regel 3 1 M. S. van der Knaap, P. G. Barth, H. Stroink et al., Ann. Neurol. 37 (3), 324 (1995). regel 4 2 F. Goutières, J. Boulloche, M. Bourgeois et al., J. Child Neurol. 11 (6), 439 (1996). regel 5 3 T. Koeda and K. Takeshita, Brain Dev. 20 (4), 245 (1998). regel 6 4 V. Mejaski-Bosnjak, N. Besenski, K. Brockmann et al., Pediatr. Neurol. 16 (4), 347 (1997). regel 7 5 M. Olivier, H. G. Lenard, F. Aksu et al., Neuropediatrics 29 (5), 225 (1998). regel 8 6 B. S. Singhal, R. D. Gursahani, V. P. Udani et al., Pediatr. Neurol. 14 (4), 291 (1996). regel 9 7 J. Takanashi, K. Sugita, and Y. Kohno, J. Neurol. Sci. 165 (1), 90 (1999). regel 10 8 T. Thelle, T. Balslev, and T. Christensen, Eur. J. Paediatr. Neurol. 3 (2), 83 (1999). regel 11 9 M. Topçu, I. Saatci, M. A. Topcuoglu et al., Brain Dev. 20 (3), 142 (1998). regel 12 10 C. Yalcinkaya, S. Comu, N. Kocer et al., J. Child Neurol. 15 (10), 690 (2000). regel 13 11 M. Topçu, C. Gartioux, F. Ribierre et al., Am. J. Hum. Genet. 66 (2), 733 (2000). regel 14 12 B. Ben-Zeev, V. Gross, T. Kushnir et al., J. Child Neurol. 16 (2), 93 (2001). regel 15 13 J. R. Gorospe, B. S. Singhal, T. Kainu et al., Neurology 62 (6), 878 (2004). regel 16 14 C. Patrono, G. Di Giacinto, E. Eymard-Pierre et al., Neurology 61 (4), 534 (2003). regel 17 15 C. Rubie, P. Lichtner, J. Gartner et al., Hum. Mutat. 21 (1), 45 (2003). regel 18 16 B. Ben-Zeev, E. Levy-Nissenbaum, H. Lahat et al., Hum. Genet. 111 (2), 214 (2002). regel 19 17 S. Koussa, H. Roukoz, T. Rizk et al., Rev. Neurol. (Paris) 161 (2), 183 (2005).

18 regel 20 S. Tsujino, N. Kanazawa, H. Yoneyama et al., J. Hum. Genet. 48 (12), 605 (2003).

19 regel 21 H. Saijo, H. Nakayama, T. Ezoe et al., Brain Dev. 25 (5), 362 (2003).

20 regel 22 P. A. Leegwater, P. K. Boor, B. Q. Yuan et al., Hum. Genet. 110 (3), 279 (2002).

21 regel 23 P. A. Leegwater, B. Q. Yuan, J. van der Steen et al., Am. J. Hum. Genet. 68 (4), 831 (2001).

22 regel 24 P. K. Boor, K. de Groot, V. Mejaski-Bosnjak et al., Hum. Mutat. 27 (6), 505 (2006).

23 regel 25 R. Blattner, A. Von Moers, P. A. Leegwater et al., Neuropediatrics 34 (4), 215 (2003).

24 regel 26 O. Teijido, A. Martinez, M. Pusch et al., Hum. Mol. Genet. 13 (21), 2581 (2004).

25 regel 27 O. Teijido, R. Casaroli-Marano, T. Kharkovets et al., Neurobiol. Dis. (2007).

26 regel 28 M. Brenner, A. B. Johnson, O. Boespfl ug-Tanguy et al., Nat. Genet. 27 (1), 117 (2001).

27 regel 29 M. Tian, C. Jacobson, S. H. Gee et al., Eur. J. Neurosci. 8 (12), 2739 (1996).

28 regel 30 F. Muntoni, M. Brockington, S. Torelli et al., Curr. Opin. Neurol. 17 (2), 205 (2004).

29 regel 31 M. S. van der Knaap, L. M. Smit, P. G. Barth et al., Ann. Neurol. 42 (1), 50 (1997).

30 regel 32 A. Helbling-Leclerc, X. Zhang, H. Topaloglu et al., Nat. Genet. 11 (2), 216 (1995).

31 regel 33 B. Echenne, M. Pages, and C. Marty-Double, Brain Dev. 6 (5), 491 (1984).

32 regel 34 A. Warth, S. Kroger, and H. Wolburg, Acta Neuropathol. (Berl.) 107 (4), 311 (2004).

33 regel 35 D. E. Michele, R. Barresi, M. Kanagawa et al., Nature 418 (6896), 417 (2002). regel 36 regel 37 regel 38 regel 39 124 34 J. D. Neely, M. Amiry-Moghaddam, O. P. Ottersen et al., Proc. Natl. Acad. Sci. U. S. A. 98 (24), 14108 regel 1

(2001). regel 2

35 Y. Wakayama, M. Inoue, H. Kojima et al., Histol. Histopathol. 21 (1), 23 (2006). regel 3

36 N. Tachi, K. Ohya, S. Chiba et al., Neurology 49 (2), 579 (1997). regel 4

37 N. Tachi, S. Chiba, M. Matsuo et al., Pediatr. Neurol. 24 (5), 373 (2001). regel 5

38 M. Brockington, D. J. Blake, P. Prandini et al., Am. J. Hum. Genet. 69 (6), 1198 (2001). regel 6

39 E. Ambrosini, B. Serafi ni, A. Lanciotti et al., Mol. Cell. Neurosci. (2007). regel 7

40 J. Meyer, A. Huberth, G. Ortega et al., Mol. Psychiatry 6 (3), 302 (2001). regel 8

41 M. Kaganovich, A. Peretz, M. Ritsner et al., Am. J. Med. Genet. 125B (1), 31 (2004). regel 9

42 C. D. Lascola and R. P. Kraig, J. Neurosci. 16 (8), 2532 (1996). regel 10

43 A. Sardini, J. S. Amey, K. H. Weylandt et al., Biochim. Biophys. Acta 1618 (2), 153 (2003). regel 11

44 B. Nilius and G. Droogmans, Acta Physiol. Scand. 177 (2), 119 (2003). regel 12

45 T. J. Jentsch, V. Stein, F. Weinreich et al., Physiol. Rev. 82 (2), 503 (2002). regel 13

46 J. Blanz, M. Schweizer, M. Auberson et al., J. Neurosci. 27 (24), 6581 (2007). regel 14

47 P. K. Boor, K. de Groot, Q. Waisfi sz et al., J. Neuropathol. Exp. Neurol. 64 (5), 412 (2005). regel 15

48 V. Benfenati, G. P. Nicchia, M. Svelto et al., J. Neurochem. 100 (1), 87 (2007). regel 16

49 M.Amiry-Moghaddam, A. Williamson, M. Palomba et al., Proc. Natl. Acad. Sci. U. S. A. 100 (23), 13615 regel 17

(2003). regel 18

50 B. Nilius, Neurochem. Res. 29 (1), 3 (2004). regel 19

51 N. Kamasawa, A. Sik, M. Morita et al., Neuroscience 136 (1), 65 (2005). regel 20

52 D. M. Menichella, D. A. Goodenough, E. Sirkowski et al., J. Neurosci. 23 (13), 5963 (2003). regel 21

53 H. Ma and K. Y. Horiuchi, Drug Discov Today 11 (13-14), 661 (2006). regel 22

54 E. M. Welch, E. R. Barton, J. Zhuo et al., Nature 447 (7140), 87 (2007). regel 23

55 M. E. Egan, M. Pearson, S. A. Weiner et al., Science 304 (5670), 600 (2004). regel 24

56 G. P. Nicchia, B. Nico, L. M. Camassa et al., Neuroscience 129 (4), 935 (2004). regel 25

57 R. Matalon, S. Surendran, P. L. Rady et al., Mol. Ther. 7 (5 Pt 1), 580 (2003). regel 26

58 S. Surendran, L. S. Shihabuddin, J. Clarke et al., Brain Res. Dev. Brain Res. 153 (1), 19 (2004). regel 27

regel 28

regel 29

regel 30

regel 31

regel 32 8 regel 33 regel 34

regel 35

regel 36

regel 37

regel 38

regel 39 125