Unique variants in CLCN3, encoding an endosomal anion/proton exchanger, underlie a spectrum of neurodevelopmental disorders.

The genetic causes of global developmental delay (GDD) and intellectual disability (ID) are diverse and include variants in numerous ion channels and transporters. Loss-of-function variants in all five endosomal/lysosomal members of the CLC family of Cl- channels and Cl-/H+ exchangers lead to pathology in mice, humans, or both. We have identified nine variants in CLCN3, the gene encoding CIC-3, in 11 individuals with GDD/ID and neurodevelopmental disorders of varying severity. In addition to a homozygous frameshift variant in two siblings, we identified eight different heterozygous de novo missense variants. All have GDD/ID, mood or behavioral disorders, and dysmorphic features; 9/11 have structural brain abnormalities; and 6/11 have seizures. The homozygous variants are predicted to cause loss of ClC-3 function, resulting in severe neurological disease similar to the phenotype observed in Clcn3-/- mice. Their MRIs show possible neurodegeneration with thin corpora callosa and decreased white matter volumes. Individuals with heterozygous variants had a range of neurodevelopmental anomalies including agenesis of the corpus callosum, pons hypoplasia, and increased gyral folding. To characterize the altered function of the exchanger, electrophysiological analyses were performed in Xenopus oocytes and mammalian cells. Two variants, p.Ile607Thr and p.Thr570Ile, had increased currents at negative cytoplasmic voltages and loss of inhibition by luminal acidic pH. In contrast, two other variants showed no significant difference in the current properties. Overall, our work establishes a role for CLCN3 in human neurodevelopment and shows that both homozygous loss of ClC-3 and heterozygous variants can lead to GDD/ID and neuroanatomical abnormalities.

Structural basis for the dominant or recessive character of GLIALCAM mutations found in leukodystrophies.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a type of leukodystrophy characterized by white matter edema, and it is caused mainly by recessive mutations in MLC1 and GLIALCAM genes. These variants are called MLC1 and MLC2A with both types of patients sharing the same clinical phenotype. In addition, dominant mutations in GLIALCAM have also been identified in a subtype of MLC patients with a remitting phenotype. This variant has been named MLC2B. GLIALCAM encodes for an adhesion protein containing two immunoglobulin (Ig) domains and it is needed for MLC1 targeting to astrocyte-astrocyte junctions. Most mutations identified in GLIALCAM abolish GlialCAM targeting to junctions. However, it is unclear why some mutations behave as recessive or dominant. Here, we used a combination of biochemistry methods with a new developed anti-GlialCAM nanobody, double-mutants and cysteine cross-links experiments, together with computer docking, to create a structural model of GlialCAM homo-interactions. Using this model, we suggest that dominant mutations affect different GlialCAM-GlialCAM interacting surfaces in the first Ig domain, which can occur between GlialCAM molecules present in the same cell (cis) or present in neighbouring cells (trans). Our results provide a framework that can be used to understand the molecular basis of pathogenesis of all identified GLIALCAM mutations.

Drosophila ClC-a is required in glia of the stem cell niche for proper neurogenesis and wiring of neural circuits.

Glial cells form part of the neural stem cell niche and express a wide variety of ion channels; however, the contribution of these channels to nervous system development is poorly understood. We explored the function of the Drosophila ClC-a chloride channel, since its mammalian ortholog CLCN2 is expressed in glial cells, and defective channel function results in leukodystrophies, which in humans are accompanied by cognitive impairment. We found that ClC-a was expressed in the niche in cortex glia, which are closely associated with neurogenic tissues. Characterization of loss-of-function ClC-a mutants revealed that these animals had smaller brains and widespread wiring defects. We showed that ClC-a is required in cortex glia for neurogenesis in neuroepithelia and neuroblasts, and identified defects in a neuroblast lineage that generates guidepost glial cells essential for photoreceptor axon guidance. We propose that glia-mediated ionic homeostasis could nonautonomously affect neurogenesis, and consequently, the correct assembly of neural circuits.

Role of zebrafish ClC-K/barttin channels in apical kidney chloride reabsorption.

KEY POINTS: We have characterized the zebrafish clc-k and barttin proteins, demonstrating that they form a protein complex mediating chloride flux in a similar manner to their mammalian counterparts. As in mammals, in zebrafish, clc-k and barttin are basically expressed in the kidney. Contrary to what is found in mammals, in zebrafish both proteins show an apical localization in the kidney. We have generated the first knockout in zebrafish of a CLC protein. Lack of clc-k in zebrafish resulted in embryonic lethality, possibly caused by a reduction in total chloride content. As a consequence, there is an up-regulation of other chloride channels and other regulatory mechanisms such as renin or the uro-guanylin receptor in the kidney. barttin is mislocalized in vivo when clc-k is not present, indicating that there is a mutual dependence of the protein expression and localization between barttin and clc-k proteins. ABSTRACT: ClC-K/barttin channels are very important for salt transport in the kidney. This function can be clearly seen since mutations in CLCNKB or BSND cause Bartter's syndrome types III and IV, respectively. Working with the freshwater teleost zebrafish, we characterized the genes homologous to the mammalian chloride channel ClC-K and its obligate subunit barttin and we obtained and studied clc-k knockout zebrafish. The zebrafish clc-k/barttin proteins are very similar to their mammalian counterparts, and both proteins are necessary to mediate chloride currents. Localization studies indicated that both proteins are exclusively expressed in the apical membranes of zebrafish kidney tubules. Knockout of clc-k resulted in embryonic lethality. These animals showed barttin mislocalization and a reduction in whole-body chloride concentration, as well as up-regulation of the expression of other chloride channels and renin, and an increase in the kidney expression of the uroguanylin receptor. Our results indicate that apical kidney chloride reabsorption through clc-k/barttin channels is crucial for chloride homeostasis in zebrafish as it is in humans. The zebrafish model could be used as a new in vivo system to study ClC-K function.

Chloride Channels in Astrocytes: Structure, Roles in Brain Homeostasis and Implications in Disease.

Astrocytes are the most abundant cell type in the CNS (central nervous system). They exert multiple functions during development and in the adult CNS that are essential for brain homeostasis. Both cation and anion channel activities have been identified in astrocytes and it is believed that they play key roles in astrocyte function. Whereas the proteins and the physiological roles assigned to cation channels are becoming very clear, the study of astrocytic chloride channels is in its early stages. In recent years, we have moved from the identification of chloride channel activities present in astrocyte primary culture to the identification of the proteins involved in these activities, the determination of their 3D structure and attempts to gain insights about their physiological role. Here, we review the recent findings related to the main chloride channels identified in astrocytes: the voltage-dependent ClC-2, the calcium-activated bestrophin, the volume-activated VRAC (volume-regulated anion channel) and the stress-activated Maxi-Cl-. We discuss key aspects of channel biophysics and structure with a focus on their role in glial physiology and human disease.

GlialCAM/MLC1 modulates LRRC8/VRAC currents in an indirect manner: Implications for megalencephalic leukoencephalopathy.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM genes. Previous work indicated that chloride currents mediated by the volume-regulated anion channel (VRAC) and ClC-2 channels were affected in astrocytes deficient in either Mlc1 or Glialcam. ClC-2 forms a ternary complex with GlialCAM and MLC1. LRRC8 proteins have been identified recently as the molecular components of VRAC, but the relationship between MLC and LRRC8 proteins is unknown. Here, we first demonstrate that LRRC8 and MLC1 are functionally linked, as MLC1 cannot potentiate VRAC currents when LRRC8A, the main subunit of VRAC, is knocked down. We determine that LRRC8A and MLC1 do not co-localize or interact and, in Xenopus oocytes, MLC1 does not potentiate LRRC8-mediated VRAC currents, indicating that VRAC modulation in astrocytes by MLC1 may be indirect. Investigating the mechanism of modulation, we find that a lack of MLC1 does not influence either mRNA or total and plasma membrane protein levels of LRRC8A; and neither does it affect LRRC8A subcellular localization. In agreement with recent results that indicated that overexpression of MLC1 decreases the phosphorylation of extracellular signal-regulated kinases (ERK), we find that astrocytes lacking MLC1 show an increase in ERK phosphorylation. In astrocytes with reduced or increased levels of MLC1 we observe changes in the phosphorylation state of the VRAC subunit LRRC8C. Our results thus reinforce previous suggestions that indicated that GlialCAM/MLC1 might modify signal transduction pathways that influence the activity of different proteins, such as VRAC.

Deficient LRRC8A-dependent volume-regulated anion channel activity is associated with male infertility in mice.

Ion channel-controlled cell volume regulation is of fundamental significance to the physiological function of sperm. In addition to volume regulation, LRRC8A-dependent volume-regulated anion channel (VRAC) activity is involved in cell cycle progression, insulin signaling, and cisplatin resistance. Nevertheless, the contribution of LRRC8A and its dependent VRAC activity in the germ cell lineage remain unknown. By utilizing a spontaneous Lrrc8a mouse mutation (c.1325delTG, p.F443*) and genetically engineered mouse models, we demonstrate that LRRC8A-dependent VRAC activity is essential for male germ cell development and fertility. Lrrc8a-null male germ cells undergo progressive degeneration independent of the apoptotic pathway during postnatal testicular development. Lrrc8a-deficient mouse sperm exhibit multiple morphological abnormalities of the flagella (MMAF), a feature commonly observed in the sperm of infertile human patients. Importantly, we identified a human patient with a rare LRRC8A hypomorphic mutation (c.1634G>A, p.Arg545His) possibly linked to Sertoli cell-only syndrome (SCOS), a male sterility disorder characterized by the loss of germ cells. Thus, LRRC8A is a critical factor required for germ cell development and volume regulation in the mouse, and it might serve as a novel diagnostic and therapeutic target for SCOS patients.

Expression of LRRC8/VRAC Currents in Xenopus Oocytes: Advantages and Caveats.

Volume-regulated anion channels (VRACs) play a role in controlling cell volume by opening upon cell swelling. Apart from controlling cell volume, their function is important in many other physiological processes, such as transport of metabolites or drugs, and extracellular signal transduction. VRACs are formed by heteromers of the pannexin homologous protein LRRC8A (also named Swell1) with other LRRC8 members (B, C, D, and E). LRRC8 proteins are difficult to study, since they are expressed in all cells of our body, and the channel stoichiometry can be changed by overexpression, resulting in non-functional heteromers. Two different strategies have been developed to overcome this issue: complementation by transient transfection of LRRC8 genome-edited cell lines, and reconstitution in lipid bilayers. Alternatively, we have used Xenopus oocytes as a simple system to study LRRC8 proteins. Here, we have reviewed all previous experiments that have been performed with VRAC and LRRC8 proteins in Xenopus oocytes. We also discuss future strategies that may be used to perform structure-function analysis of the VRAC in oocytes and other systems, in order to understand its role in controlling multiple physiological functions.

CLCN1 Myotonia congenita mutation with a variable pattern of inheritance suggests a novel mechanism of dominant myotonia.

INTRODUCTION: Mutations in CLCN1 cause recessive or dominant forms of myotonia congenita (MC). Some mutations have been found to exhibit both patterns of inheritance but the mechanism explaining this behavior is unknown. METHODS: A known recessive missense mutation, A493E, was identified in a family with dominant MC. The mutant p.A493E alone or in co-expression with wild-type (WT) ClC-1 was expressed in Xenopus oocytes. Currents were measured and biochemical assays were performed. RESULTS: The mutant showed no significant activity and reduced total and plasma membrane (PM) protein levels. Co-expression with the mutant reduced the activity and PM levels of an engineered lower expression variant of ClC-1, whereas no effect was observed on a higher expression variant. DISCUSSION: Our results suggest that the dominant effect of some CLCN1 mutations showing recessive or dominant inheritance patterns may be due to a dose-dependent defect in PM delivery of the WT channel. Muscle Nerve, 2018.

Megalencephalic leukoencephalopathy with subcortical cysts: A personal biochemical retrospective.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by dysfunction of the role of glial cells in controlling brain fluid and ion homeostasis. Patients affected by MLC present macrocephaly, cysts and white matter vacuolation, which lead to motor and cognitive impairments. To date, there is no treatment for MLC, only supportive care. MLC is caused by mutations in the MLC1 and GLIALCAM genes. MLC1 is a membrane protein with low identity to the Kv1.1 potassium channel and GlialCAM belongs to an adhesion molecule family. Both proteins form a complex with an as-yet-unknown function that is expressed mainly in the astrocytes surrounding the blood-brain barrier and in Bergmann glia. GlialCAM also acts as an auxiliary subunit of the chloride channel ClC-2, thus regulating its localization at cell-cell junctions and modifying its functional properties by affecting the common gate of ClC-2. Recent studies in Mlc1-, GlialCAM- and Clcn2-knockout mice or Mlc1-knockout zebrafish have provided fresh insight into the pathophysiology of MLC and further details about the molecular interactions between these three proteins. Additional studies have shown that GlialCAM/MLC1 also regulates other ion channels (TRPV4, VRAC) or transporters (Na+/K+-ATPase) in a not-understood manner. Furthermore, it has been shown that GlialCAM/MLC1 may influence signal transduction mechanisms, thereby affecting other proteins not related with transport such as the EGF receptor. Here, we offer a personal biochemical retrospective of the work that has been performed to gain knowledge of the pathophysiology of MLC, and we discuss future strategies that may be used to identify therapeutic solutions for MLC patients.

Leukoencephalopathy-causing CLCN2 mutations are associated with impaired Cl- channel function and trafficking.

KEY POINTS: Characterisation of most mutations found in CLCN2 in patients with CC2L leukodystrophy show that they cause a reduction in function of the chloride channel ClC-2. GlialCAM, a regulatory subunit of ClC-2 in glial cells and involved in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), increases the activity of a ClC-2 mutant by affecting ClC-2 gating and by stabilising the mutant at the plasma membrane. The stabilisation of ClC-2 at the plasma membrane by GlialCAM depends on its localisation at cell-cell junctions. The membrane protein MLC1, which is defective in MLC, also contributes to the stabilisation of ClC-2 at the plasma membrane, providing further support for the view that GlialCAM, MLC1 and ClC-2 form a protein complex in glial cells. ABSTRACT: Mutations in CLCN2 have been recently identified in patients suffering from a type of leukoencephalopathy involving intramyelinic oedema. Here, we characterised most of these mutations that reduce the function of the chloride channel ClC-2 and impair its plasma membrane (PM) expression. Detailed biochemical and electrophysiological analyses of the Ala500Val mutation revealed that defective gating and increased cellular and PM turnover contributed to defective A500V-ClC-2 functional expression. Co-expression of the adhesion molecule GlialCAM, which forms a tertiary complex with ClC-2 and megalencephalic leukoencephalopathy with subcortical cysts 1 (MLC1), rescued the functional expression of the mutant by modifying its gating properties. GlialCAM also restored the PM levels of the channel by impeding its turnover at the PM. This rescue required ClC-2 localisation to cell-cell junctions, since a GlialCAM mutant with compromised junctional localisation failed to rescue the impaired stability of mutant ClC-2 at the PM. Wild-type, but not mutant, ClC-2 was also stabilised by MLC1 overexpression. We suggest that leukodystrophy-causing CLCN2 mutations reduce the functional expression of ClC-2, which is partly counteracted by GlialCAM/MLC1-mediated increase in the gating and stability of the channel.

Cisplatin activates volume sensitive LRRC8 channel mediated currents in Xenopus oocytes.

LRRC8 proteins have been shown to underlie the ubiquitous volume regulated anion channel (VRAC). VRAC channels are composed of the LRRC8A subunit and at least one among the LRRC8B-E subunits. In addition to their role in volume regulation, LRRC8 proteins have been implicated in the uptake of chemotherapeutic agents. We had found that LRRC8 channels can be conveniently expressed in Xenopus oocytes, a system without endogenous VRAC activity. The fusion with fluorescent proteins yielded constitutive activity for A/C, A/D and A/E heteromers. Here we tested the effect of the anticancer drug cisplatin on LRRC8A-VFP/8E-mCherry and LRRC8A-VFP/8D-mCherry co-expressing oocytes. Incubation with cisplatin dramatically activated currents for both subunit combinations, confirming that VRAC channels provide an uptake pathway for cisplatin and that intracellular cisplatin accumulation strongly activates the channels. Thus, specific activators of LRRC8 proteins might be useful tools to counteract chemotherapeutic drug resistance.

Investigation of LRRC8-Mediated Volume-Regulated Anion Currents in Xenopus Oocytes.

Volume-regulated anion channels (VRACs) play an important role in controlling cell volume by opening upon cell swelling. Recent work has shown that heteromers of LRRC8A with other LRRC8 members (B, C, D, and E) form the VRAC. Here, we used Xenopus oocytes as a simple system to study LRRC8 proteins. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity. Using these constructs, we reproduced previous findings indicating that LRRC8 heteromers mediate anion and osmolyte flux with subunit-dependent kinetics and selectivity. Additionally, we found that LRRC8 heteromers mediate glutamate and ATP flux and that the inhibitor carbenoxolone acts from the extracellular side, binding to probably more than one site. Our results also suggest that the stoichiometry of LRRC8 heteromers is variable, with a number of subunits ≥6, and that the heteromer composition depends on the relative expression of different subunits. The system described here enables easy structure-function analysis of LRRC8 proteins.

Identification and Functional Characterization of CLCN1 Mutations Found in Nondystrophic Myotonia Patients.

Mutations in the gene coding for the skeletal muscle Cl(-) channel (CLCN1) lead to dominant or recessive myotonia. Here, we identified and characterized CLCN1 mutations in Costa Rican patients, who had been clinically diagnosed with myotonic dystrophy type 1 but who were negative for DM1 mutations. CLCN1 mutations c.501C>G, p.F167L and c.1235A>C, p.Q412P appeared to have recessive inheritance but patients had atypical clinical phenotypes; c.313C>T, p.R105C was found in combination with c.501C>G, p.F167L in an apparently recessive family and the c.461A>G, p.Q154R variant was associated with a less clear clinical picture. In Xenopus oocytes, none of the mutations exhibited alterations of fast or slow gating parameters or single channel conductance, and mutations p.R105C, p.Q154R, and p.F167L were indistinguishable from wild-type (WT). p.Q412P displayed a dramatically reduced current density, surface expression and exerted no dominant negative effect in the context of the homodimeric channel. Fluorescently tagged constructs revealed that p.Q412P is expressed inefficiently. Our study confirms p.F167L and p.R105C as myotonia mutations in the Costa Rican population, whereas p.Q154R may be a benign variant. p.Q412P most likely induces a severe folding defect, explaining the lack of dominance in patients and expression systems, but has WT properties once expressed in the plasma membrane.

Circadian and Sex Differences After Acute High-Altitude Exposure: Are Early Acclimation Responses Improved by Blue Light?

OBJECTIVE: The possible effects of blue light during acute hypoxia and the circadian rhythm on several physiological and cognitive parameters were studied. METHODS: Fifty-seven volunteers were randomly assigned to 2 groups: nocturnal (2200-0230 hours) or diurnal (0900-1330 hours) and exposed to acute hypoxia (4000 m simulated altitude) in a hypobaric chamber. The participants were illuminated by blue LEDs or common artificial light on 2 different days. During each session, arterial oxygen saturation (Spo2), blood pressure, heart rate variability, and cognitive parameters were measured at sea level, after reaching the simulated altitude of 4000 m, and after 3 hours at this altitude. RESULTS: The circadian rhythm caused significant differences in blood pressure and heart rate variability. A 4% to 9% decrease in waking nocturnal Spo2 under acute hypoxia was observed. Acute hypoxia also induced a significant reduction (4%-8%) in systolic pressure, slightly more marked (up to 13%) under blue lighting. Women had significantly increased systolic (4%) and diastolic (12%) pressures under acute hypoxia at night compared with daytime pressure; this was not observed in men. Some tendencies toward better cognitive performance (d2 attention test) were seen under blue illumination, although when considered together with physiological parameters and reaction time, there was no conclusive favorable effect of blue light on cognitive fatigue suppression after 3 hours of acute hypobaric hypoxia. CONCLUSIONS: It remains to be seen whether longer exposure to blue light under hypobaric hypoxic conditions would induce favorable effects against fatigue.

Identification and characterization of the zebrafish ClC-2 chloride channel orthologs.

ClC-2 is a Cl(-) channel that belongs to the CLC family of chloride channel/transport proteins. ClC-2 molecular role is not clear, and Clcn2 knockout mice develop blindness, sterility, and leukodystrophy by unknown reasons. ClC-2 is associated in the brain with the adhesion molecule GlialCAM, which is defective in a type of leukodystrophy, involving ClC-2 in the homeostasis of myelin. To get more insight into the functions of ClC-2, we have identified in this work the three ClC-2 orthologs in zebrafish. clcn2a and clcn2b resulted from the teleost-specific whole genome duplication, while clcn2c arose from a gene duplication from clcn2b. The expression patterns in adult tissues and embryos of zebrafish clcn2 paralogs support their subfunctionalization after the duplications, with clcn2a being enriched in excitable tissues and clcn2c in ionocytes. All three zebrafish clc-2 proteins interact with human GLIALCAM, that is able to target them to cell junctions, as it does with mammalian ClC-2. We could detect clc-2a and clc-2b inward rectified chloride currents with different voltage-dependence and kinetics in Xenopus oocytes, while clc-2c remained inactive. Interestingly, GlialCAM proteins did not modify clc-2b inward rectification. Then, our work extends the repertoire of ClC-2 proteins and provides new tools for structure-function and physiology studies.

Megalencephalic leukoencephalopathy with subcortical cysts protein 1 regulates glial surface localization of GLIALCAM from fish to humans.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by myelin vacuolization and caused by mutations in MLC1 or GLIALCAM. Patients with recessive mutations in either MLC1 or GLIALCAM show the same clinical phenotype. It has been shown that GLIALCAM is necessary for the correct targeting of MLC1 to the membrane at cell junctions, but its own localization was independent of MLC1 in vitro. However, recent studies in Mlc1(-/-) mice have shown that GlialCAM is mislocalized in glial cells. In order to investigate whether the relationship between Mlc1 and GlialCAM is species-specific, we first identified MLC-related genes in zebrafish and generated an mlc1(-/-) zebrafish. We have characterized mlc1(-/-) zebrafish both functionally and histologically and compared the phenotype with that of the Mlc1(-/-) mice. In mlc1(-/-) zebrafish, as in Mlc1(-/-) mice, Glialcam is mislocalized. Re-examination of a brain biopsy from an MLC patient indicates that GLIALCAM is also mislocalized in Bergmann glia in the cerebellum. In vitro, impaired localization of GlialCAM was observed in astrocyte cultures from Mlc1(-/-) mouse only in the presence of elevated potassium levels, which mimics neuronal activity. In summary, here we demonstrate an evolutionary conserved role for MLC1 in regulating glial surface levels of GLIALCAM, and this interrelationship explains why patients with mutations in either gene (MLC1 or GLIALCAM) share the same clinical phenotype.