GPR37 Receptors and Megalencephalic Leukoencephalopathy with Subcortical Cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of vacuolating leukodystrophy (white matter disorder), which is mainly caused by defects in MLC1 or glial cell adhesion molecule (GlialCAM) proteins. In addition, autoantibodies to GlialCAM are involved in the pathology of multiple sclerosis. MLC1 and GLIALCAM genes encode for membrane proteins of unknown function, which has been linked to the regulation of different ion channels and transporters, such as the chloride channel VRAC (volume regulated anion channel), ClC-2 (chloride channel 2), and connexin 43 or the Na+/K+-ATPase pump. However, the mechanisms by which MLC proteins regulate these ion channels and transporters, as well as the exact function of MLC proteins remain obscure. It has been suggested that MLC proteins might regulate signalling pathways, but the mechanisms involved are, at present, unknown. With the aim of answering these questions, we have recently described the brain GlialCAM interactome. Within the identified proteins, we could validate the interaction with several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptors GPR37L1 and GPR37. In this review, we summarize new aspects of the pathophysiology of MLC disease and key aspects of the interaction between GPR37 receptors and MLC proteins.

Ubr1-induced selective endophagy/autophagy protects against the endosomal and Ca2+-induced proteostasis disease stress.

The cellular defense mechanisms against cumulative endo-lysosomal stress remain incompletely understood. Here, we identify Ubr1 as a protein quality control (QC) E3 ubiquitin-ligase that counteracts proteostasis stresses by facilitating endosomal cargo-selective autophagy for lysosomal degradation. Astrocyte regulatory cluster membrane protein MLC1 mutations cause endosomal compartment stress by fusion and enlargement. Partial lysosomal clearance of mutant endosomal MLC1 is accomplished by the endosomal QC ubiquitin ligases, CHIP and Ubr1 via ESCRT-dependent route. As a consequence of the endosomal stress, a supportive QC mechanism, dependent on both Ubr1 and SQSTM1/p62 activities, targets ubiquitinated and arginylated MLC1 mutants for selective endosomal autophagy (endophagy). This QC pathway is also activated for arginylated Ubr1-SQSTM1/p62 autophagy cargoes during cytosolic Ca2+-assault. Conversely, the loss of Ubr1 and/or arginylation elicited endosomal compartment stress. These findings underscore the critical housekeeping role of Ubr1 and arginylation-dependent endophagy/autophagy during endo-lysosomal proteostasis perturbations and suggest a link of Ubr1 to Ca2+ homeostasis and proteins implicated in various diseases including cancers and brain disorders.

Dynamic expression of homeostatic ion channels in differentiated cortical astrocytes in vitro.

The capacity of astrocytes to adapt their biochemical and functional features upon physiological and pathological stimuli is a fundamental property at the basis of their ability to regulate the homeostasis of the central nervous system (CNS). It is well known that in primary cultured astrocytes, the expression of plasma membrane ion channels and transporters involved in homeostatic tasks does not closely reflect the pattern observed in vivo. The individuation of culture conditions that promote the expression of the ion channel array found in vivo is crucial when aiming at investigating the mechanisms underlying their dynamics upon various physiological and pathological stimuli. A chemically defined medium containing growth factors and hormones (G5) was previously shown to induce the growth, differentiation, and maturation of primary cultured astrocytes. Here we report that under these culture conditions, rat cortical astrocytes undergo robust morphological changes acquiring a multi-branched phenotype, which develops gradually during the 2-week period of culturing. The shape changes were paralleled by variations in passive membrane properties and background conductance owing to the differential temporal development of inwardly rectifying chloride (Cl-) and potassium (K+) currents. Confocal and immunoblot analyses showed that morphologically differentiated astrocytes displayed a large increase in the expression of the inward rectifier Cl- and K+ channels ClC-2 and Kir4.1, respectively, which are relevant ion channels in vivo. Finally, they exhibited a large diminution of the intermediate filaments glial fibrillary acidic protein (GFAP) and vimentin which are upregulated in reactive astrocytes in vivo. Taken together the data indicate that long-term culturing of cortical astrocytes in this chemical-defined medium promotes a quiescent functional phenotype. This culture model could aid to address the regulation of ion channel expression involved in CNS homeostasis in response to physiological and pathological challenges.

Megalencephalic leukoencephalopathy with subcortical cysts is a developmental disorder of the gliovascular unit.

Absence of the astrocyte-specific membrane protein MLC1 is responsible for megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare type of leukodystrophy characterized by early-onset macrocephaly and progressive white matter vacuolation that lead to ataxia, spasticity, and cognitive decline. During postnatal development (from P5 to P15 in the mouse), MLC1 forms a membrane complex with GlialCAM (another astrocytic transmembrane protein) at the junctions between perivascular astrocytic processes. Perivascular astrocytic processes along with blood vessels form the gliovascular unit. It was not previously known how MLC1 influences the physiology of the gliovascular unit. Here, using the Mlc1 knock-out mouse model of MLC, we demonstrated that MLC1 controls the postnatal development and organization of perivascular astrocytic processes, vascular smooth muscle cell contractility, neurovascular coupling, and intraparenchymal interstitial fluid clearance. Our data suggest that MLC is a developmental disorder of the gliovascular unit, and perivascular astrocytic processes and vascular smooth muscle cell maturation defects are primary events in the pathogenesis of MLC and therapeutic targets for this disease.

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.

Muscarinic acetylcholine receptor M1 mutations causing neurodevelopmental disorder and epilepsy.

De novo rare damaging variants in genes involved in critical developmental pathways, notably regulation of synaptic transmission, have emerged as a frequent cause of neurodevelopmental disorders (NDD). NDD show great locus heterogeneity and for many of the associated genes, there is substantial phenotypic diversity, including epilepsy, intellectual disability, autism spectrum disorder, movement disorders, and combinations thereof. We report two unrelated patients, a young girl with early-onset refractory epilepsy, severe disability, and progressive cerebral and cerebellar atrophy, and a second girl with mild dysmorphism, global developmental delay, and moderate intellectual disability in whom trio-based whole-exome sequencing analysis uncovered de novo missense variants in CHRM1. Biochemical analyses of one of the NDD-associated variants proved that it caused a reduction in protein levels and impaired cellular trafficking. In addition, the mutated receptor showed defective activation of intracellular signaling pathways. Our data strengthen the concept that brain-reduced muscarinic signaling lowers the seizure threshold and severely impairs neurodevelopment.

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.

HepaCAM controls astrocyte self-organization and coupling.

Astrocytes extensively infiltrate the neuropil to regulate critical aspects of synaptic development and function. This process is regulated by transcellular interactions between astrocytes and neurons via cell adhesion molecules. How astrocytes coordinate developmental processes among one another to parse out the synaptic neuropil and form non-overlapping territories is unknown. Here we identify a molecular mechanism regulating astrocyte-astrocyte interactions during development to coordinate astrocyte morphogenesis and gap junction coupling. We show that hepaCAM, a disease-linked, astrocyte-enriched cell adhesion molecule, regulates astrocyte competition for territory and morphological complexity in the developing mouse cortex. Furthermore, conditional deletion of Hepacam from developing astrocytes significantly impairs gap junction coupling between astrocytes and disrupts the balance between synaptic excitation and inhibition. Mutations in HEPACAM cause megalencephalic leukoencephalopathy with subcortical cysts in humans. Therefore, our findings suggest that disruption of astrocyte self-organization mechanisms could be an underlying cause of neural pathology.

Identification of the GlialCAM interactome: the G protein-coupled receptors GPRC5B and GPR37L1 modulate megalencephalic leukoencephalopathy proteins.

Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a type of vacuolating leukodystrophy, which is mainly caused by mutations in MLC1 or GLIALCAM. The two MLC-causing genes encode for membrane proteins of yet unknown function that have been linked to the regulation of different chloride channels such as the ClC-2 and VRAC. To gain insight into the role of MLC proteins, we have determined the brain GlialCAM interacting proteome. The proteome includes different transporters and ion channels known to be involved in the regulation of brain homeostasis, proteins related to adhesion or signaling as several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptor GPR37L1. Focusing on these two GPCRs, we could validate that they interact directly with MLC proteins. The inactivation of Gpr37l1 in mice upregulated MLC proteins without altering their localization. Conversely, a reduction of GPRC5B levels in primary astrocytes downregulated MLC proteins, leading to an impaired activation of ClC-2 and VRAC. The interaction between the GPCRs and MLC1 was dynamically regulated upon changes in the osmolarity or potassium concentration. We propose that GlialCAM and MLC1 associate with different integral membrane proteins modulating their functions and acting as a recruitment site for various signaling components as the GPCRs identified here. We hypothesized that the GlialCAM/MLC1 complex is working as an adhesion molecule coupled to a tetraspanin-like molecule performing regulatory effects through direct binding or influencing signal transduction events.

Split-Tobacco Etch Virus (Split-TEV) Method in G Protein-Coupled Receptor Interacting Proteins.

Split-TEV assay enables the identification of protein-protein interaction in mammalian cells. This method is based on the split of tobacco etch virus (TEV) protease in two fragments, where each fragment is fused to the candidate proteins predicted to interact. If there is indeed an interaction between both proteins, TEV protease reconstitutes its proteolytic activity and this activity is used to induce the expression of some reporter genes. However, some studies have detected unspecific interaction between membrane proteins due to its higher tendency to aggregate. Here we describe a variation of the Split-TEV method developed with the aim to increase the specificity in the study of G protein-coupled receptor (GPCR) interacting proteins. This approach for monitoring interactions between GPCRs is an easy and robust assay and offers good perspectives in drug discovery.

Author Correction: The LRRC8-mediated volume-regulated anion channel is altered in glaucoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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 2 months of age, before the onset of the disease, as a preventive approach. We also tested a therapeutic strategy by injecting the animals at 5 months, once the histopathological abnormalities are starting, or at 15 months, 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.

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.

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.

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

BACKGROUND: Megalencephalic 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. METHODS: We 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. RESULTS: glialcama-/- 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. CONCLUSIONS: This 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.

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.

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-1ß. Using astrocytoma lines overexpressing wild-type (WT) or mutated MLC1 and astrocytes from control and Mlc1 knock-out (KO) mice, we found that IL-1ß stimulated WT-MLC1 plasma membrane expression in astrocytoma cells and control primary astrocytes. In astrocytoma, WT-MLC1 inhibited the activation of IL-1ß-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 in a 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.

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.

The LRRC8-mediated volume-regulated anion channel is altered in glaucoma.

Regulation of cellular volume is an essential process to balance volume changes during cell proliferation and migration or when intracellular osmolality increases due to transepithelial transport. We previously characterized the key role of volume-regulated anion channels (VRAC) in the modulation of the volume of trabecular meshwork (TM) cells and, in turn, the aqueous humour (AH) outflow from the eye. The balance between the secretion and the drainage of AH determines the intraocular pressure (IOP) that is the major casual risk factor for glaucoma. Glaucoma is an ocular disease that causes irreversible blindness due to the degeneration of retinal ganglion cells. The recent identification of Leucine-Rich Repeat-Containing 8 (LRRC8A-E) proteins as the molecular components of VRAC opens the field to elucidate their function in the physiology of TM and glaucoma. Human TM cells derived from non-glaucomatous donors and from open-angle glaucoma patients were used to determine the expression and the functional activity of LRRC8-mediated channels. Expression levels of LRRC8A-E subunits were decreased in HTM glaucomatous cells compared to normotensive HTM cells. Consequently, the activity of VRAC currents and volume regulation of TM cells were significantly affected. Impaired cell volume regulation will likely contribute to altered aqueous outflow and intraocular pressure.