MLC1 Expression Research Articles

MLC1 Overexpression Inhibits Tumor Progression through PI3K/AKT Signal Pathway in Prostate Cancer.

Prostate cancer (PC) is a prevalent malignancy in males, characterized by high morbidity and mortality. Despite MLC1 being established as a key mediator in tumor progression, its role in PC remains unexplored. This study aims to validate MLC1's anti-tumor effects and uncover potential mechanisms. MLC1's clinical significance is assessed using data from The Cancer Genome Atlas and the Genotype-Tissue Expression databases. MLC1 expression is significantly reduced in PC samples compared with the adjacent normal tissues. MLC1 expression correlates negatively with tumor metastasis and positively with the survival of patients with PC. In vitro, up-regulating MLC1 effectively inhibits tumor progression by curtailing proliferation, infestation, and migration through the deactivation of the PI3K/AKT signaling pathway. Conversely, down-regulating MLC1 promotes PC progression, a phenomenon alleviated by the PI3K/AKT inhibitor, Gefitinib. Furthermore, the anti-tumor function of MLC1 is corroborated by a reduction in tumor volume compared with the negative control in vivo. This study confirms the anti-tumor effects of MLC1 via in vitro and in vivo experiments, demonstrating its potential mechanism of inhibiting the PI3K/AKT signaling pathway.

Top-down proteomics of myosin light chain isoforms define chamber-specific expression in the human heart

Myosin functions as the “molecular motor” of the sarcomere and generates the contractile force necessary for cardiac muscle contraction. Myosin light chains 1 and 2 (MLC-1 and -2) play important functional roles in regulating the structure of the hexameric myosin molecule. Each of these light chains has an ‘atrial’ and ‘ventricular’ isoform, so called because they are believed to exhibit chamber-restricted expression in the heart. However, recently the chamber-specific expression of MLC isoforms in the human heart has been questioned. Herein, we analyzed the expression of MLC-1 and -2 atrial and ventricular isoforms in each of the four cardiac chambers in adult non-failing donor hearts using top-down mass spectrometry (MS)-based proteomics. Strikingly, we detected an isoform thought to be ventricular, MLC-2v (gene: MYL2), in the atria and confirmed the protein sequence using tandem MS (MS/MS). For the first time, a putative deamidation post-translation modification (PTM) located on MLC-2v in atrial tissue was localized to amino acid N13. MLC-1v (MYL3) and MLC-2a (MYL7) were the only MLC isoforms exhibiting chamber-restricted expression patterns across all donor hearts. Importantly, our results unambiguously show that MLC-1v, not MLC-2v, is ventricle-specific in adult human hearts. Moreover, we found elevated MLC-2 phosphorylation in male hearts compared to female hearts across each cardiac chamber. Overall, top-down proteomics allowed an unbiased analysis of MLC isoform expression throughout the human heart, uncovering previously unexpected isoform expression patterns and PTMs.

Control of membrane protein homeostasis by a chaperone-like glial cell adhesion molecule at multiple subcellular locations

The significance of crosstalks among constituents of plasma membrane protein clusters/complexes in cellular proteostasis and protein quality control (PQC) remains incompletely understood. Examining the glial (enriched) cell adhesion molecule (CAM), we demonstrate its chaperone-like role in the biosynthetic processing of the megalencephalic leukoencephalopathy with subcortical cyst 1 (MLC1)-heteromeric regulatory membrane protein complex, as well as the function of the GlialCAM/MLC1 signalling complex. We show that in the absence of GlialCAM, newly synthesized MLC1 molecules remain unfolded and are susceptible to polyubiquitination-dependent proteasomal degradation at the endoplasmic reticulum. At the plasma membrane, GlialCAM regulates the diffusional partitioning and endocytic dynamics of cluster members, including the ClC-2 chloride channel and MLC1. Impaired folding and/or expression of GlialCAM or MLC1 in the presence of diseases causing mutations, as well as plasma membrane tethering compromise the functional expression of the cluster, leading to compromised endo-lysosomal organellar identity. In addition, the enlarged endo-lysosomal compartments display accelerated acidification, ubiquitinated cargo-sorting and impaired endosomal recycling. Jointly, these observations indicate an essential and previously unrecognized role for CAM, where GliaCAM functions as a PQC factor for the MLC1 signalling complex biogenesis and possess a permissive role in the membrane dynamic and cargo sorting functions with implications in modulations of receptor signalling.

Megalencephalic Leukoencephalopathy: Insights Into Pathophysiology and Perspectives for Therapy.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare genetic disorder belonging to the group of vacuolating leukodystrophies. It is characterized by megalencephaly, loss of motor functions, epilepsy, and mild mental decline. In brain biopsies of MLC patients, vacuoles were observed in myelin and in astrocytes surrounding blood vessels. It is mainly caused by recessive mutations in MLC1 and HEPACAM (also called GLIALCAM) genes. These disease variants are called MLC1 and MLC2A with both types of patients sharing the same clinical phenotype. Besides, dominant mutations in HEPACAM were also identified in a subtype of MLC patients (MLC2B) with a remitting phenotype. MLC1 and GlialCAM proteins form a complex mainly expressed in brain astrocytes at the gliovascular interface and in Bergmann glia at the cerebellum. Both proteins regulate several ion channels and transporters involved in the control of ion and water fluxes in glial cells, either directly influencing their location and function, or indirectly regulating associated signal transduction pathways. However, the MLC1/GLIALCAM complex function and the related pathological mechanisms leading to MLC are still unknown. It has been hypothesized that, in MLC, the role of glial cells in brain ion homeostasis is altered in both physiological and inflammatory conditions. There is no therapy for MLC patients, only supportive treatment. As MLC2B patients show an MLC reversible phenotype, we speculated that the phenotype of MLC1 and MLC2A patients could also be mitigated by the re-introduction of the correct gene even at later stages. To prove this hypothesis, we injected in the cerebellar subarachnoid space of Mlc1 knockout mice an adeno-associated virus (AAV) coding for human MLC1 under the control of the glial-fibrillary acidic protein promoter. MLC1 expression in the cerebellum extremely reduced myelin vacuolation at all ages in a dose-dependent manner. This study could be considered as the first preclinical approach for MLC. We also suggest other potential therapeutic strategies in this review.

Megalencephalic Leukoencephalopathy with Subcortical Cysts Disease-Linked MLC1 Protein Favors Gap-Junction Intercellular Communication by Regulating Connexin 43 Trafficking in Astrocytes.

Astrocytes, the most numerous cells of the central nervous system, exert critical functions for brain homeostasis. To this purpose, astrocytes generate a highly interconnected intercellular network allowing rapid exchange of ions and metabolites through gap junctions, adjoined channels composed of hexamers of connexin (Cx) proteins, mainly Cx43. Functional alterations of Cxs and gap junctions have been observed in several neuroinflammatory/neurodegenerative diseases. In the rare leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), astrocytes show defective control of ion/fluid exchanges causing brain edema, fluid cysts, and astrocyte/myelin vacuolation. MLC is caused by mutations in MLC1, an astrocyte-specific protein of elusive function, and in GlialCAM, a MLC1 chaperon. Both proteins are highly expressed at perivascular astrocyte end-feet and astrocyte-astrocyte contacts where they interact with zonula occludens-1 (ZO-1) and Cx43 junctional proteins. To investigate the possible role of Cx43 in MLC pathogenesis, we studied Cx43 properties in astrocytoma cells overexpressing wild type (WT) MLC1 or MLC1 carrying pathological mutations. Using biochemical and electrophysiological techniques, we found that WT, but not mutated, MLC1 expression favors intercellular communication by inhibiting extracellular-signal-regulated kinase 1/2 (ERK1/2)-mediated Cx43 phosphorylation and increasing Cx43 gap-junction stability. These data indicate MLC1 regulation of Cx43 in astrocytes and Cx43 involvement in MLC pathogenesis, suggesting potential target pathways for therapeutic interventions.

Cerebellar Astrocyte Transduction as Gene Therapy for Megalencephalic Leukoencephalopathy.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare genetic disorder belonging to the group of vacuolating leukodystrophies. It is characterized by megalencephaly, loss of motor functions, epilepsy, and mild mental decline. In brain biopsies of MLC patients, vacuoles were observed in myelin and in astrocytes surrounding blood vessels. There is no therapy for MLC patients, only supportive treatment. We show here a preclinical gene therapy approach for MLC using the Mlc1 knock-out mouse. An adeno-associated virus coding for human MLC1 under the control of the glial fibrillary acidic protein promoter was injected in the cerebellar subarachnoid space of Mlc1 knock-out and wild-type animals at 2months of age, before the onset of the disease, as a preventive approach. We also tested a therapeutic strategy by injecting the animals at 5months, once the histopathological abnormalities are starting, or at 15months, when they have progressed to a more severe pathology. MLC1 expression in the cerebellum restored the adhesion molecule GlialCAM and the chloride channel ClC-2 localization in Bergmann glia, which both are mislocalized in Mlc1 knock-out model. More importantly, myelin vacuolation was extremely reduced in treated mice at all ages and correlated with the amount of expressed MLC1 in Bergmann glia, indicating not only the preventive potential of this strategy but also its therapeutic capacity. In summary, here we provide the first therapeutic approach for patients affected with MLC. This work may have also implications to treat other diseases affecting motor function such as ataxias.

Plasma membrane localization of MLC1 regulates cellular morphology and motility

BackgroundMegalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare form of infantile-onset leukodystrophy. The disorder is caused primarily by mutations of MLC1 that leads to a series of phenotypic outcomes including vacuolation of myelin and astrocytes, subcortical cysts, brain edema, and macrocephaly. Recent studies have indicated that functional interactions among MLC1, GlialCAM, and ClC-2 channels play key roles in the regulation of neuronal, glial and vascular homeostasis. However, the physiological role of MLC1 in cellular homeostatic communication remains poorly understood. In the present study, we investigated the cellular function of MLC1 and its effects on cell–cell interactions.MethodsMLC1-dependent cellular morphology and motility were analyzed by using confocal and live cell imaging technique. Biochemical approaches such as immunoblotting, co-immunoprecipitation, and surface biotinylation were conducted to support data.ResultsWe found that the altered MLC1 expression and localization led to a great alteration in cellular morphology and motility through actin remodeling. MLC1 overexpression induced filopodia formation and suppressed motility. And, MLC1 proteins expressed in patient-derived MLC1 mutants resulted in trapping in the ER although no changes in morphology or motility were observed. Interestingly knockdown of Mlc1 induced Arp3-Cortactin interaction, lamellipodia formation, and increased the membrane ruffling of the astrocytes. These data indicate that subcellular localization of expressed MLC1 at the plasma membrane is critical for changes in actin dynamics through ARP2/3 complex. Thus, our results suggest that misallocation of pathogenic mutant MLC1 may disturbs the stable cell-cell communication and the homeostatic regulation of astrocytes in patients with MLC.

Comparison of zebrafish and mice knockouts for Megalencephalic Leukoencephalopathy proteins indicates that GlialCAM/MLC1 forms a functional unit

BackgroundMegalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a rare type of leukodystrophy characterized by astrocyte and myelin vacuolization, epilepsy and early-onset macrocephaly. MLC is caused by mutations in MLC1 or GLIALCAM, coding for two membrane proteins with an unknown function that form a complex specifically expressed in astrocytes at cell-cell junctions. Recent studies in Mlc1−/− or Glialcam−/− mice and mlc1−/− zebrafish have shown that MLC1 regulates glial surface levels of GlialCAM in vivo and that GlialCAM is also required for MLC1 expression and localization at cell-cell junctions.MethodsWe have generated and analysed glialcama−/− zebrafish. We also generated zebrafish glialcama−/−mlc1−/− and mice double KO for both genes and performed magnetic resonance imaging, histological studies and biochemical analyses.Resultsglialcama−/− shows megalencephaly and increased fluid accumulation. In both zebrafish and mice, this phenotype is not aggravated by additional elimination of mlc1. Unlike mice, mlc1 protein expression and localization are unaltered in glialcama−/− zebrafish, possibly because there is an up-regulation of mlc1 mRNA. In line with these results, MLC1 overexpressed in Glialcam−/− mouse primary astrocytes is located at cell-cell junctions.ConclusionsThis work indicates that the two proteins involved in the pathogenesis of MLC, GlialCAM and MLC1, form a functional unit, and thus, that loss-of-function mutations in these genes cause leukodystrophy through a common pathway.

Megalencephalic Leukoencephalopathy with Subcortical Cysts Protein-1 (MLC1) Counteracts Astrocyte Activation in Response to Inflammatory Signals.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 (MLC1) is a membrane protein expressed by perivascular astrocytes. MLC1 mutations cause MLC, an incurable leukodystrophy characterized by macrocephaly, brain edema, cysts, myelin vacuolation, and astrocytosis, leading to cognitive/motor impairment and epilepsy. Although its function is unknown, MLC1 favors regulatory volume decrease after astrocyte osmotic swelling and down-regulates intracellular signaling pathways controlling astrocyte activation and proliferation. By combining analysis of human brain tissues with in vitro experiments, here we investigated MLC1 role in astrocyte activation during neuroinflammation, a pathological condition exacerbating patient symptoms. MLC1 upregulation was observed in brain tissues from multiple sclerosis, Alzheimer's, and Creutzfeld-Jacob disease, all pathologies characterized by strong astrocytosis and release of inflammatory cytokines, particularly IL-1β. Using astrocytoma lines overexpressing wild-type (WT) or mutated MLC1 and astrocytes from control and Mlc1 knock-out (KO) mice, we found that IL-1β stimulated WT-MLC1 plasma membrane expression in astrocytoma cells and control primary astrocytes. In astrocytoma, WT-MLC1 inhibited the activation of IL-1β-induced inflammatory signals (pERK, pNF-kB) that, conversely, were constitutively activated in mutant expressing cells or abnormally upregulated in KO astrocytes. WT-MLC1+ cells also expressed reduced levels of the astrogliosis marker pSTAT3. We then monitored MLC1 expression timing ina demyelinating/remyelinating murine cerebellar organotypic culture model where, after the demyelination and release of inflammatory cytokines, recovery processes occur, revealing MLC1 upregulation in these latter phases. Altogether, these findings suggest that by modulating specific pathways, MLC1 contributes to restore astrocyte homeostasis after inflammation, providing the opportunity to identify drug target molecules to slow down disease progression.

Postnatal development of the astrocyte perivascular MLC1/GlialCAM complex defines a temporal window for the gliovascular unit maturation.

Astrocytes, the most abundant glial cells of the central nervous system are morphologically complex. They display numerous processes interacting with synapses and blood vessels. At the vascular interface, astrocyte endfeet-terminated processes almost entirely cover the blood vessel surface and participate to the gliovascular unit where important vascular properties of the brain are set such as the blood-brain barrier (BBB) integrity. How specific morphological and functional interactions between astrocytes and the vascular compartment develop has not been fully investigated. Here, we elaborated an original experimental strategy to study the postnatal development of astrocyte perivascular endfeet. Using purified gliovascular units, we focused on the postnatal expression of MLC1 and GlialCAM, two transmembrane proteins forming a complex enriched at the junction between mature astrocyte perivascular endfeet. We showed that MLC1 and GlialCAM were enriched and assembled into mature complexes in astrocyte perivascular endfeet between postnatal days 10 and 15, after the formation of astrocyte perivascular Aquaporin 4 water channels. These events correlated with the increased expression of Claudin-5 and P-gP, two endothelial-specific BBB components. These results illustrate for the first time that astrocyte perivascular endfeet differentiation is a complex and progressive process which correlates with BBB maturation. Moreover, our results suggest that maturation of the astrocyte endfeet MLC1/GlialCAM complex between postnatal days 10 and 15 might be a key event in the gliovascular unit maturation.

Issue Cover (November 2018)

Journal of NeurochemistryVolume 147, Issue 3 Issue CoverFree Access Issue Cover (November 2018) First published: 29 October 2018 https://doi.org/10.1111/jnc.14199 Read the full article: ‘Ectopic positioning of Bergmann glia and impaired cerebellar wiring in Mlc1-over-expressing mice’ by S. Kikuchihara, S. Sugio, K.-F. Tanaka, T. Watanabe, M. Kano, Y. Yamazaki, M. Watanabe, K. Ikenaka (J. Neurochem. 2018, vol. 147(3), pp. 344–360) on doi: 10.1111/jnc.14486 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Front cover: Background: Mlc1 is a causative gene for megalencephalic leukoencephalopathy with subcortical cysts (MLC), which represents astrocytic swelling followed by myelin membrane splitting. In the cerebellum, Mlc1 is highly expressed in Bergmann glia (BG), the astrocytes associated with Purkinje cells (PCs). Mlc1-overexpressing mice were generated to see the effect of Mlc1 expression level on the development of the brain. In continuous Mlc1 overexpression mice, alignment of BG was disturbed (BG ectopia). Manipulation of Mlc1-overexpressing period resulted in mice with and without BG ectopia, depending on the overexpressing period. Main finding: Ectopic BG poorly wrapped around somatodendritic elements of PCs and exhibited reduced expression of the glutamate transporter, GLAST. This research indicates that there is a critical time window for positioning of BG in the PC layer. Once BG impairments were established, synaptic wiring on PCs were affected by glutamate spillover via the insufficient BG association to PCs. Implications: Once normal positioning of BG is affected, the differentiation of BG is impaired, leading to insufficient glial wrapping, exacerbated glutamate spillover, and aberrant synaptic wiring in PCs. Image Content: In this image of Purkinje cell layer in the Mlc1-overexpressiong mouse cerebellum, insufficient glial wrapping is observed. BG membrane (GLAST, green) only partially surrounded the soma of PCs (Calbindin, red). Read the full article ‘Ectopic positioning of Bergmann glia and impaired cerebellar wiring in Mlc1-over-expressing mice’ by S. Kikuchihara, S. Sugio, K.-F. Tanaka, T. Watanabe, M. Kano, Y. Yamazaki, M. Watanabe, K. Ikenaka (J. Neurochem. 2018, vol. 147(3), pp. 344–360) on doi: 10.1111/jnc.14486 Volume147, Issue3November 2018 RelatedInformation

Altered Right Ventricular Mechanical Properties Are Afterload Dependent in a Rodent Model of Bronchopulmonary Dysplasia.

Infants born premature are at increased risk for development of bronchopulmonary dysplasia (BPD), pulmonary hypertension (PH), and ultimately right ventricular (RV) dysfunction, which together carry a high risk of neonatal mortality. However, the role alveolar simplification and abnormal pulmonary microvascular development in BPD affects RV contractile properties is unknown. We used a rat model of BPD to examine the effect of hyperoxia-induced PH on RV contractile properties. We measured in vivo RV pressure as well as passive force, maximum Ca2+ activated force, calcium sensitivity of force (pCa50) and rate of force redevelopment (ktr) in RV skinned trabeculae isolated from hearts of 21-and 35-day old rats pre-exposed to 21% oxygen (normoxia) or 85% oxygen (hyperoxia) for 14 days after birth. Systolic and diastolic RV pressure were significantly higher at day 21 in hyperoxia exposed rats compared to normoxia control rats, but normalized by 35 days of age. Passive force, maximum Ca2+ activated force, and calcium sensitivity of force were elevated and cross-bridge cycling kinetics depressed in 21-day old hyperoxic trabeculae, whereas no differences between normoxic and hyperoxic trabeculae were seen at 35 days. Myofibrillar protein analysis revealed that 21-day old hyperoxic trabeculae had increased levels of beta-myosin heavy chain (β-MHC), atrial myosin light chain 1 (aMLC1; often referred to as essential light chain), and slow skeletal troponin I (ssTnI) compared to age matched normoxic trabeculae. On the other hand, 35-day old normoxic and hyperoxic trabeculae expressed similar level of α- and β-MHC, ventricular MLC1 and predominantly cTnI. These results suggest that neonatal exposure to hyperoxia increases RV afterload and affect both the steady state and dynamic contractile properties of the RV, likely as a result of hyperoxia-induced expression of β-MHC, delayed transition of slow skeletal TnI to cardiac TnI, and expression of atrial MLC1. These hyperoxia-induced changes in contractile properties are reversible and accompany the resolution of PH with further developmental age, underscoring the importance of reducing RV afterload to allow for normalization of RV function in both animal models and humans with BPD.

Megalencephalic leukoencephalopathy with cysts: theGlialcam‐null mouse model

ObjectiveMegalencephalic leukoencephalopathy with cysts (MLC) is a genetic infantile‐onset disease characterized by macrocephaly and white matter edema due to loss of MLC1 function. Recessive mutations in either MLC1 or GLIALCAM cause the disease. MLC1 is involved in astrocytic volume regulation; GlialCAM ensures the correct membrane localization of MLC1. Their exact role in brain ion‐water homeostasis is only partly defined. We characterized Glialcam‐null mice for further studies.MethodsWe investigated the consequences of loss of GlialCAM in Glialcam‐null mice and compared GlialCAM developmental expression in mice and men.Results Glialcam‐null mice had early‐onset megalencephaly and increased brain water content. From 3 weeks, astrocytes were abnormal with swollen processes abutting blood vessels. Concomitantly, progressive white matter vacuolization developed due to intramyelinic edema. Glialcam‐null astrocytes showed abolished expression of MLC1, reduced expression of the chloride channel ClC‐2 and increased expression and redistribution of the water channel aquaporin4. Expression of other MLC1‐interacting proteins and the volume regulated anion channel LRRC8A was unchanged. In mice, GlialCAM expression increased until 3 weeks and then stabilized. In humans, GlialCAM expression was highest in the first 3 years to then decrease and stabilize from approximately 5 years.Interpretation Glialcam‐null mice replicate the early stages of the human disease with early‐onset intramyelinic edema. The earliest change is astrocytic swelling, further substantiating that a defect in astrocytic volume regulation is the primary cellular defect in MLC. GlialCAM expression affects expression of MLC1, ClC‐2 and aquaporin4, indicating that abnormal interplay between these proteins is a disease mechanism in megalencephalic leukoencephalopathy with cysts.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 regulates epidermal growth factor receptor signaling in astrocytes.

Mutations in the MLC1 gene, which encodes a protein expressed in brain astrocytes, are the leading cause of MLC, a rare leukodystrophy characterized by macrocephaly, brain edema, subcortical cysts, myelin and astrocyte vacuolation. Although recent studies indicate that MLC1 protein is implicated in the regulation of cell volume changes, the exact role of MLC1 in brain physiology and in the pathogenesis of MLC disease remains to be clarified. In preliminary experiments, we observed that MLC1 was poorly expressed in highly proliferating astrocytoma cells when compared with primary astrocytes, and that modulation of MLC1 expression influenced astrocyte growth. Because volume changes are key events in cell proliferation and during brain development MLC1 expression is inversely correlated to astrocyte progenitor proliferation levels, we investigated the possible role for MLC1 in the control of astrocyte proliferation. We found that overexpression of wild type but not mutant MLC1 in human astrocytoma cells hampered cell growth by favoring epidermal growth factor receptor (EGFR) degradation and by inhibiting EGF-induced Ca(+) entry, ERK1/2 and PLCγ1 activation, and calcium-activated KCa3.1 potassium channel function, all molecular pathways involved in astrocyte proliferation stimulation. Interestingly, MLC1 did not influence AKT, an EGFR-stimulated kinase involved in cell survival. Moreover, EGFR expression was higher in macrophages derived from MLC patients than from healthy individuals. Since reactive astrocytes proliferate and re-express EGFR in response to different pathological stimuli, the present findings provide new information on MLC pathogenesis and unravel an important role for MLC1 in other brain pathological conditions where astrocyte activation occurs.

MLC1 protein: a likely link between leukodystrophies and brain channelopathies.

Megalencephalic leukoencephalopathy with subcortical cysts (MLCs) disease is a rare inherited, autosomal recessive form of childhood-onset spongiform leukodystrophy characterized by macrocephaly, deterioration of motor functions, epileptic seizures and mental decline. Brain edema, subcortical fluid cysts, myelin and astrocyte vacuolation are the histopathological hallmarks of MLC. Mutations in either the MLC1 gene (>75% of patients) or the GlialCAM gene (<20% of patients) are responsible for the disease. Recently, the GlialCAM adhesion protein was found essential for the membrane expression and function of the chloride channel ClC-2 indicating MLC disease caused by mutation in GlialCAM as the first channelopathy among leukodystrophies. On the contrary, the function of MLC1 protein, which binds GlialCAM, its functional relationship with ClC-2 and the molecular mechanisms underlying MLC1 mutation-induced functional defects are not fully understood yet. The human MLC1 gene encodes a 377-amino acid membrane protein with eight predicted transmembrane domains which shows very low homology with voltage-dependent potassium (K+) channel subunits. The high expression of MLC1 in brain astrocytes contacting blood vessels and meninges and brain alterations observed in MLC patients have led to hypothesize a role for MLC1 in the regulation of ion and water homeostasis. Recent studies have shown that MLC1 establishes structural and/or functional interactions with several ion/water channels and transporters and ion channel accessory proteins, and that these interactions are affected by MLC1 mutations causing MLC. Here, we review data on MLC1 functional properties obtained in in vitro and in vivo models and discuss evidence linking the effects of MLC1 mutations to brain channelopathies.

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. We investigated which brain cell types express MLC1, compared developmental expression in mice and men, and studied the consequences of loss of MLC1 in Mlc1-null mice. Like humans, mice expressed MLC1 only in astrocytes, especially those facing fluid-brain barriers. In mice, MLC1 expression increased until 3 weeks and then stabilized. In humans, MLC1 expression was highest in the first year, decreased, and stabilized from approximately 5 years. Mlc1-null mice had early onset megalencephaly and increased brain water content. From 3 weeks, abnormal astrocytes were present with swollen processes abutting fluid-brain barriers. From 3 months, widespread white matter vacuolization with intramyelinic edema developed. Mlc1-null astrocytes showed slowed regulatory volume decrease and reduced volume-regulated anion currents, which increased upon MLC1 re-expression. Mlc1-null astrocytes showed reduced expression of adhesion molecule GlialCAM and chloride channel ClC-2, but no substantial changes in other known MLC1-interacting proteins. Mlc1-null mice replicate early stages of the human disease with early onset intramyelinic edema. The cellular functional defects, described for human MLC, were confirmed. The earliest change was astrocytic swelling, substantiating that in MLC the primary defect is in volume regulation by astrocytes. MLC1 expression affects expression of GlialCAM and ClC-2. Abnormal interplay between these proteins is part of the pathomechanisms of MLC.

Monocytes and macrophages as biomarkers for the diagnosis of megalencephalic leukoencephalopathy with subcortical cysts

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy characterized by macrocephaly, subcortical cysts and demyelination. The majority of patients harbor mutations in the MLC1 gene encoding for a membrane protein with largely unknown function. Mutations in MLC1 hamper its normal trafficking and distribution in cell membranes, leading to enhanced degradation. MLC1 protein is highly expressed in brain astrocytes and in circulating blood cells, particularly monocytes. We used these easily available cells and monocyte-derived macrophages from healthy donors and MLC1-mutated patients to study MLC1 expression and localization, and to investigate how defective MLC1 mutations may affect macrophage functions. RT-PCR, western blot and immunofluorescence analyses show that MLC1 is expressed in both monocytes and macrophages, and its biosynthesis follows protein trafficking between endoplasmic reticulum and trans-Golgi network and the secretory pathway to the cell surface. MLC1 is transported along the endosomal recycling pathway passing through Rab5+ and Rab11A+vesicles before lysosomal degradation. Alterations in MLC1 trafficking and distribution were observed in macrophages from MLC1-mutated patients, which also showed changes in the expression and localization of several proteins involved in plasma membrane permeability, ion and water homeostasis and ion-regulated exocytosis. As a consequence of these alterations, patient-derived macrophages show abnormal cell morphology and intracellular calcium influx and altered response to hypo-osmotic stress. Our results suggest that blood-derived macrophages may give relevant information on MLC1 function and may be considered as valid biomarkers for MLC diagnosis and for investigating therapeutic strategies aimed to restore MLC1 trafficking in patient cells.

Insights into MLC pathogenesis: GlialCAM is an MLC1 chaperone required for proper activation of volume-regulated anion currents

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM genes and is associated with myelin and astrocyte vacuolation. It has been suggested that MLC is caused by impaired cell volume regulation as a result of defective activation of astrocytic volume-regulated anion currents (VRAC). GlialCAM brings MLC1 and the ClC-2 Cl(-) channel to cell-cell junctions, even though the role of ClC-2 in MLC disease remains incompletely understood. To gain insights into the biological role of GlialCAM in the pathogenesis of MLC disease, here we analyzed the gain- and loss-of-function phenotypes of GlialCAM in Hela cells and primary astrocytes, focusing on its interaction with the MLC1 protein. Unexpectedly, GlialCAM ablation provoked intracellular accumulation and reduced expression of MLC1 at the plasma membrane. Conversely, over-expression of GlialCAM increased the cellular stability of mutant MLC1 variants. Reduction in GlialCAM expression resulted in defective activation of VRAC and augmented vacuolation, phenocopying MLC1 mutations. Importantly, over-expression of GlialCAM together with MLC1 containing MLC-related mutations was able to reactivate VRAC currents and to reverse the vacuolation caused in the presence of mutant MLC1. These results indicate a previously unrecognized role of GlialCAM in facilitating the biosynthetic maturation and cell surface expression of MLC1, and suggest that pharmacological strategies aimed to increase surface expression of MLC1 and/or VRAC activity may be beneficial for MLC patients.

Expression and functional analyses of two essential myosin light chains from the fast white muscle of the spotted mandarin fish Siniperca scherzeri

Objective: The essential myosin light chains (EMLC) are the major structural and regulatory proteins that control actin-myosin interaction in striated muscles. The purpose of this work is to provide a better understanding about the EMLC properties and their expression pattern during muscle development in the mandarin fish. Material and Methods: The cDNA library was constructed to isolate and characterize MLC sequences. The nucleotide sequences and inferred amino acid sequences were analyzed. Whole-mount in situ hybridization was carried out to investigate the gene expression pattern. To assess their biological function in calcium binding, MLC recombinant proteins were purified to analyze their calcium binding activity in vitro. Results: Two cDNA clones encoding MLC1 and MLC3 were identified from the fast white muscles of the spotted mandarin fish, Siniperca scherzeri. Sequence analysis revealed that MLC1 and MLC3 in the spotted mandarin were not produced by alternative splicing as reported in avians and rodents but were encoded by two different genes. MLC1 and MLC3 proteins contained two EF-hand domains, which are helix-loop-helix structural domains found in a large family of calcium-binding proteins. Conclusion: These two EMLC genes exhibited a muscle-specific expression. Moreover, the distinct patterns of expression of MLC1 and MLC3 had overlapped during the skeletal muscle development in the early embryonic stage. MLC1 and MLC3 recombinant proteins were able to bind Ca2+ in vitro, which is a typical characteristic of EF-hand superfamily proteins.

Megalencephalic leukoencephalopathy with subcortical cysts protein 1 functionally cooperates with the TRPV4 cation channel to activate the response of astrocytes to osmotic stress: dysregulation by pathological mutations

Megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare leukodystrophy characterized by macrocephaly, subcortical fluid cysts and myelin vacuolation, has been linked to mutations in the MLC1 gene. This gene encodes a membrane protein that is highly expressed in astrocytes. Based on MLC pathological features, it was proposed that astrocyte-mediated defects in ion and fluid homeostasis could account for the alterations observed in MLC-affected brains. However, the role of MLC1 and the effects of pathological mutations on astrocyte osmoregulatory functions have still to be demonstrated. Using human astrocytoma cells stably overexpressing wild-type MLC1 or three known MLC-associated pathological mutations, we investigated MLC1 involvement in astrocyte reaction to osmotic changes using biochemical, dynamic video imaging and immunofluorescence techniques. We have found that MLC1 overexpressed in astrocytoma cells is mainly localized in the plasma membrane, is part of the Na,K-ATPase-associated molecular complex that includes the potassium channel Kir4.1, syntrophin and aquaporin-4 and functionally interacts with the calcium permeable channel TRPV4 (transient receptor potential vanilloid-4 cation channel) which mediates swelling-induced cytosolic calcium increase and volume recovery in response to hyposmosis. Pathological MLC mutations cause changes in MLC1 expression and intracellular localization as well as in the astrocyte response to osmotic changes by altering MLC1 molecular interactions with the Na,K-ATPase molecular complex and abolishing the increase in calcium influx induced by hyposmosis and treatment with the TRPV4 agonist 4αPDD. These data demonstrate, for the first time, that MLC1 plays a role in astrocyte osmo-homeostasis and that defects in intracellular calcium dynamics may contribute to MLC pathogenesis.