ABSTRACT
The function of the chloride channel ClC-1 is crucial for the control of muscle excitability. Thus, reduction of ClC-1 functions by CLCN1 mutations leads to myotonia congenita. Many different animal models have contributed to understanding the myotonia pathophysiology. However, these models do not allow in vivo screening of potentially therapeutic drugs, as the zebrafish model does. In this work, we identified and characterized the two zebrafish orthologues (clc-1a and clc-1b) of the ClC-1 channel. Both channels are mostly expressed in the skeletal muscle as revealed by RT-PCR, western blot, and electrophysiological recordings of myotubes, and clc-1a is predominantly expressed in adult stages. Characterization in Xenopus oocytes shows that the zebrafish channels display similar anion selectivity and voltage dependence to their human counterparts. However, they show reduced sensitivity to the inhibitor 9-anthracenecarboxylic acid (9-AC), and acidic pH inverts the voltage dependence of activation. Reduction of clc-1a/b expression hampers spontaneous and mechanically stimulated movement, which could be reverted by expression of human ClC-1 but not by some ClC-1 containing myotonia mutations. Treatment of clc-1-depleted zebrafish with mexiletine, a typical drug used in human myotonia, improves the motor behaviour. Our work extends the repertoire of ClC channels to evolutionary structure-function studies and proposes the zebrafish clcn1 crispant model as a simple tool to find novel therapies for myotonia. KEY POINTS: We have identified two orthologues of ClC-1 in zebrafish (clc-1a and clc-1b) which are mostly expressed in skeletal muscle at different developmental stages. Functional characterization of the activity of these channels reveals many similitudes with their mammalian counterparts, although they are less sensitive to 9-AC and acidic pH inverts their voltage dependence of gating. Reduction of clc-1a/b expression hampers spontaneous and mechanically stimulated movement which could be reverted by expression of human ClC-1. Myotonia-like symptoms caused by clc-1a/b depletion can be reverted by mexiletine, suggesting that this model could be used to find novel therapies for myotonia.
Subject(s)
Chloride Channels , Zebrafish , Chloride Channels/genetics , Chloride Channels/metabolism , Chloride Channels/physiology , Animals , Humans , Disease Models, Animal , Myotonia/genetics , Muscle, Skeletal/physiology , Muscle, Skeletal/metabolism , Muscle, Skeletal/drug effects , Xenopus laevis , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Myotonia Congenita/genetics , AnthracenesABSTRACT
Vestibular sensory epithelia contain type I and type II sensory hair cells (HCI and HCII). Recent studies have revealed molecular markers for the identification of these cells, but the precise composition of each vestibular epithelium (saccule, utricle, lateral crista, anterior crista, posterior crista) and their postnatal maturation have not been described in detail. Moreover, in vitro methods to study this maturation are not well developed. We obtained total HCI and HCII counts in adult rats and studied the maturation of the epithelia from birth (P0) to postnatal day 28 (P28). Adult vestibular epithelia hair cells were found to comprise â¼65% HCI expressing osteopontin and PMCA2, â¼30% HCII expressing calretinin, and â¼4% HCII expressing SOX2 but neither osteopontin nor calretinin. At birth, immature HCs express both osteopontin and calretinin. P28 epithelia showed an almost adult-like composition but still contained 1.3% of immature HCs. In addition, we obtained free-floating 3D cultures of the epithelia at P1, which formed a fluid-filled cyst, and studied their survival and maturation in vitro up to day 28 (28 DIV). These cultures showed good HC resiliency and maturation. Using an enriched medium for the initial 4 days, a HCI/calretinin+-HCII ratio close to the in vivo ratio was obtained. These cultures are suitable to study HC maturation and mature HCs in pharmacological, toxicological and molecular research.
ABSTRACT
Hair cell (HC) loss by epithelial extrusion has been described to occur in the rodent vestibular system during chronic 3,3'-iminodipropionitrile (IDPN) ototoxicity. This is preceded by dismantlement of the calyceal junction in the contact between type I HC (HCI) and calyx afferent terminals. Here, we evaluated whether these phenomena have wider significance. First, we studied rats receiving seven different doses of streptomycin, ranging from 100 to 800 mg/kg/day, for 3-8 weeks. Streptomycin caused loss of vestibular function associated with partial loss of HCI and decreased expression of contactin-associated protein (CASPR1), denoting calyceal junction dismantlement, in the calyces encasing the surviving HCI. Additional molecular and ultrastructural data supported the conclusion that HC-calyx detachment precede HCI loss by extrusion. Animals allowed to survive after the treatment showed functional recuperation and rebuilding of the calyceal junction. Second, we evaluated human sensory epithelia obtained during therapeutic labyrinthectomies and trans-labyrinthine tumour excisions. Some samples showed abnormal CASPR1 label strongly suggestive of calyceal junction dismantlement. Therefore, reversible dismantlement of the vestibular calyceal junction may be a common response triggered by chronic stress, including ototoxic stress, before HCI loss. This may partly explain clinical observations of reversion in function loss after aminoglycoside exposure.
Subject(s)
Hair Cells, Vestibular , Vestibule, Labyrinth , Humans , Rats , Animals , Streptomycin/toxicity , Vestibule, Labyrinth/pathology , Epithelium/pathology , Hair Cells, Vestibular/pathology , Hair Cells, Auditory/pathologyABSTRACT
The vestibular ganglion contains primary sensory neurons that are postsynaptic to the transducing hair cells (HC) and project to the central nervous system. Understanding the response of these neurons to HC stress or loss is of great interest as their survival and functional competence will determine the functional outcome of any intervention aiming at repair or regeneration of the HCs. We have shown that subchronic exposure to the ototoxicant 3,3'-iminodipropionitrile (IDPN) in rats and mice causes a reversible detachment and synaptic uncoupling between the HCs and the ganglion neurons. Here, we used this paradigm to study the global changes in gene expression in vestibular ganglia using RNA-seq. Comparative gene ontology and pathway analyses of the data from both model species indicated a robust downregulation of terms related to synapses, including presynaptic and postsynaptic functions. Manual analyses of the most significantly downregulated transcripts identified genes with expressions related to neuronal activity, modulators of neuronal excitability, and transcription factors and receptors that promote neurite growth and differentiation. For choice selected genes, the mRNA expression results were replicated by qRT-PCR, validated spatially by RNA-scope, or were demonstrated to be associated with decreased expression of the corresponding protein. We conjectured that decreased synaptic input or trophic support on the ganglion neurons from the HC was triggering these expression changes. To support this hypothesis, we demonstrated decreased expression of BDNF mRNA in the vestibular epithelium after subchronic ototoxicity and also downregulated expression of similarly identified genes (e.g Etv5, Camk1g, Slc17a6, Nptx2, Spp1) after HC ablation with another ototoxic compound, allylnitrile. We conclude that vestibular ganglion neurons respond to decreased input from HCs by decreasing the strength of all their synaptic contacts, both as postsynaptic and presynaptic players.
Subject(s)
Ototoxicity , Rodentia , Rats , Mice , Animals , Rodentia/metabolism , Ototoxicity/metabolism , Neurons/metabolism , Transcription Factors/metabolism , RNA, Messenger/metabolism , DNA-Binding Proteins/metabolismABSTRACT
Hearing or balance loss are disabling conditions that have a serious impact in those suffering them, especially when they appear in children. Their ultimate cause is frequently the loss of function of mechanosensory hair cells in the inner ear. Hair cells can be damaged by environmental insults, like noise or chemical agents, known as ototoxins. Two of the most common ototoxins are life-saving medications: cisplatin against solid tumors, and aminoglycoside antibiotics to treat infections. However, due to their localization inside the temporal bone, hair cells are difficult to study in mammals. As an alternative animal model, zebrafish larvae have hair cells similar to those in mammals, some of which are located in a fish specific organ on the surface of the skin, the lateral line. This makes them easy to observe in vivo and readily accessible for ototoxins or otoprotective substances. These features have made possible advances in the study of the mechanisms mediating ototoxicity or identifying new potential ototoxins. Most importantly, the small size of the zebrafish larvae has allowed screening thousands of molecules searching for otoprotective agents in a scale that would be highly impractical in rodent models. The positive hits found can then start the long road to reach clinical settings to prevent hearing or balance loss.
ABSTRACT
The tail-lift reflex and the air-righting reflex in rats are anti-gravity reflexes that depend on vestibular function. To begin identifying their cellular basis, this study examined the relationship between reflex loss and the graded lesions caused in the vestibular sensory epithelia by varying doses of an ototoxic compound. After ototoxic exposure, we recorded these reflexes using high speed video. The movies were used to obtain objective measures of the reflexes: the minimum angle formed by the nose, the back of the neck and the base of the tail during the tail-lift maneuver and the time to right in the air-righting test. The vestibular sensory epithelia were then collected from the rats and used to estimate the loss of type I (HCI), type II (HCII) and all hair cells (HC) in both central and peripheral parts of the crista, utricle, and saccule. As expected, tail-lift angles decreased, and air-righting times increased, while the numbers of HCs remaining in the epithelia decreased in a dose-dependent manner. The results demonstrated greater sensitivity of HCI compared to HCII to the IDPN ototoxicity, as well as a relative resiliency of the saccule compared to the crista and utricle. Comparing the functional measures with the cell counts, we observed that loss of the tail-lift reflex associates better with HCI than with HCII loss. In contrast, most HCI in the crista and utricle were lost before air-righting times increased. These data suggest that these reflexes depend on the function of non-identical populations of vestibular HCs.
Subject(s)
Hair Cells, Vestibular , Animals , Hair Cells, Auditory , Ototoxicity , Rats , Reflex , Saccule and Utricle , Vestibule, LabyrinthABSTRACT
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.
Subject(s)
Cell Adhesion Molecules, Neuron-Glia/metabolism , Membrane Proteins/metabolism , Myelin Sheath/metabolism , Nerve Tissue Proteins/metabolism , Animals , Astrocytes/metabolism , Cell Adhesion Molecules, Neuron-Glia/genetics , Loss of Function Mutation/genetics , Membrane Proteins/genetics , Mice , Mice, Knockout , Mutation , Myelin Sheath/genetics , Nerve Tissue Proteins/genetics , Zebrafish , Zebrafish Proteins/metabolismABSTRACT
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.
Subject(s)
Chloride Channels/physiology , Kidney/metabolism , Renal Reabsorption , Zebrafish Proteins/physiology , Animals , Chloride Channels/genetics , Chlorides/metabolism , HEK293 Cells , Humans , Mutation , Protein Transport , Zebrafish , Zebrafish Proteins/geneticsABSTRACT
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.
Subject(s)
Cysts/genetics , Hereditary Central Nervous System Demyelinating Diseases/genetics , Proteins/genetics , Animals , Brain/metabolism , Cell Cycle Proteins , Cysts/pathology , Hereditary Central Nervous System Demyelinating Diseases/pathology , Humans , Membrane Proteins/metabolism , Protein Binding , Proteins/chemistry , Proteins/metabolismABSTRACT
Introducción. El grado de atención de los estudiantes en el aula, por lo general, va disminuyendo a lo largo de la hora lectiva. Normalmente, el planteamiento de preguntas para repasar contenidos y romper con el ritmo de la explicación favorece la atención. En este sentido, en la asignatura de Fisiología Humana del Grado de Ciencias Biomédicas de la Universitat de Barcelona se optó por dinamizar la clase a través de la aplicación Socrative, que permite plantear preguntas y conocer la respuesta de los estudiantes a tiempo real a través de ordenadores y dispositivos móviles. Material y métodos. Al inicio de cada clase, se hacían preguntas para repasar contenidos del día anterior o para introducir aspectos aún no tratados. A continuación, se revisaban las respuestas globales y se aclaraban las dudas que habían surgido. Al final de cada tema o bloque temático también se planteaban preguntas para integrar y revisar los contenidos. Resultados. La valoración de esta experiencia se recogió mediante encuestas de opinión a los estudiantes. Estos evaluaron positivamente la experiencia, al considerarla globalmente como satisfactoria, con una puntuación de 4,3 (5 es el máximo grado de acuerdo). También indicaron que era una herramienta fácil e intuitiva (4,6/5), que había sido motivadora y que había favorecido que las clases fueran más dinámicas y amenas (4,4/5). Además, manifestaron que había resultado útil para reforzar el aprendizaje de los contenidos (4,4/5). Conclusiones. El uso de preguntas de refuerzo en clase a través de Socrative hizo que los estudiantes se mostraran atentos y participaran activamente en las clases (AU)
Introduction. In general, the level of attention of students in the classroom is decreasing throughout the class time. Normally, asking questions to review content and breaking the rhythm of the explanation favours attention. In this sense, in the subject of Human Physiology of the Degree of Biomedical Sciences of the University of Barcelona it was decided to add dynamism to the class through the Socrative application, which allows to raise questions and to know the response of the students in real time through computers and mobile devices. Material and methods. At the beginning of each class, questions were asked to review contents of the previous day or to introduce aspects not presented yet. The global responses were then reviewed and the doubts that had emerged were clarified. At the end of each topic or thematic block, questions were also asked to review the contents. Results. The assessment of this experience was collected through student opinion surveys. They evaluated positively the experience, considering it globally as satisfactory, with a score of 4.3 (5 being the highest degree of agreement). They also indicated that it was an easy and intuitive tool (4.6 / 5), which had been motivating and had favoured that classes were more dynamic and enjoyable (4.4 / 5). In addition, they stated that it had been useful to reinforce the learning of contents (4.4 / 5). Conclusions. The use of reinforcement questions in class through Socrative caused students to be attentive and to actively participate in the clases (AU)
Subject(s)
Humans , Physiology/education , Problem-Based Learning/methods , Problem-Based Learning/trends , Education, Medical/organization & administration , Problem-Based Learning/organization & administration , Problem-Based Learning/standards , Students/statistics & numerical data , Surveys and Questionnaires , Cell Phone , Personal SatisfactionABSTRACT
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM. GlialCAM is necessary for the correct targeting of MLC1, but also for the targeting of the Cl- channel ClC-2. Furthermore, GlialCAM modifies ClC-2 functional properties in vitro. However, in vivo studies in GlialCAM-/- mice have shown that the modification of ClC-2 activity only occurs in oligodendrocytes, despite GlialCAM and ClC-2 being expressed in astrocytes. Thus, the relationship between GlialCAM, MLC1 and ClC-2 in astrocytes is unknown. Here, we show that GlialCAM, ClC-2 and MLC1 can form a ternary complex in cultured astrocytes, but only under depolarizing conditions. We also provide biochemical evidences that this ternary complex exists in vivo. The formation of this complex changes ClC-2 localization in the membrane and its functional properties. ClC-2 association with GlialCAM/MLC1 depends on calcium flux through L-type calcium channels and activation of calcium-dependent calpain proteases. Based on these studies, we propose that the chloride influx mediated by GlialCAM/MLC1/ClC-2 in astrocytes may be needed to compensate an excess of potassium, as occurs in conditions of high neuronal activity. We suggest that a defect in this compensation may contribute to the pathogenesis of MLC disease.
Subject(s)
Cell Adhesion Molecules, Neuron-Glia/metabolism , Cysts/metabolism , Hereditary Central Nervous System Demyelinating Diseases/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Animals , Astrocytes/metabolism , Brain/metabolism , Brain Diseases/pathology , CLC-2 Chloride Channels , Calcium Channels, L-Type/genetics , Chloride Channels , Cysts/genetics , HEK293 Cells , HeLa Cells , Hereditary Central Nervous System Demyelinating Diseases/genetics , Humans , Membrane Proteins/genetics , Mice , Protein Transport/geneticsABSTRACT
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.
Subject(s)
Anions/metabolism , Membrane Potentials/physiology , Membrane Proteins/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Animals , Anions/chemistry , Carbenoxolone/chemistry , Carbenoxolone/pharmacology , Extracellular Space/chemistry , Extracellular Space/drug effects , Glutamic Acid/genetics , Glutamic Acid/metabolism , Humans , In Vitro Techniques , Kinetics , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Membrane Potentials/drug effects , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/chemistry , Membrane Proteins/genetics , Neurotransmitter Agents/chemistry , Neurotransmitter Agents/pharmacology , Oocytes/chemistry , Oocytes/metabolism , Osmolar Concentration , Permeability , Protein Multimerization , Structure-Activity Relationship , Taurine/chemistry , Taurine/metabolism , Water/chemistry , XenopusABSTRACT
KEY POINTS: The extracellular domain of GlialCAM is necessary for its targeting to cell junctions, as well as for interactions with itself and MLC1 and ClC-2. The C-terminus of GlialCAM is not necessary for interaction but is required for targeting to cell junctions. The first three residues of the transmembrane segment of GlialCAM are required for GlialCAM-mediated ClC-2 activation. ABSTRACT: Mutations in the genes encoding the astrocytic protein MLC1, the cell adhesion molecule GlialCAM or the Cl(-) channel ClC-2 underlie human leukodystrophies. GlialCAM binds to itself, to MLC1 and to ClC-2, and directs these proteins to cell-cell contacts. In addition, GlialCAM dramatically activates ClC-2 mediated currents. In the present study, we used mutagenesis studies combined with functional and biochemical analyses to determine which parts of GlialCAM are required to perform these cellular functions. We found that the extracellular domain of GlialCAM is necessary for cell junction targeting and for mediating interactions with itself or with MLC1 and ClC-2. The C-terminus is also necessary for proper targeting to cell-cell junctions but is not required for the biochemical interaction. Finally, we identified the first three amino acids of the transmembrane segment of GlialCAM as being essential for the activation of ClC-2 currents but not for targeting or biochemical interaction. Our results provide new mechanistic insights concerning the regulation of the cell biology and function of MLC1 and ClC-2 by GlialCAM.
Subject(s)
Brain Diseases/metabolism , Chloride Channels/metabolism , Membrane Proteins/metabolism , Protein Subunits/metabolism , Proteins/metabolism , Astrocytes/metabolism , Brain Diseases/genetics , CLC-2 Chloride Channels , Cell Cycle Proteins , Cell Line , Cell Line, Tumor , Chloride Channels/genetics , HEK293 Cells , HeLa Cells , Humans , Intercellular Junctions/genetics , Intercellular Junctions/metabolism , Membrane Proteins/genetics , Mutation/genetics , Protein Subunits/genetics , Protein Transport/genetics , Proteins/geneticsABSTRACT
The CLC family of chloride channels and transporters is composed by nine members, but only three of them, ClC-Ka/b, ClC-7 and ClC-2, have been found so far associated with auxiliary subunits. These CLC regulatory subunits are small proteins that present few common characteristics among them, both structurally and functionally, and their effects on the corresponding CLC protein are different. Barttin, a protein with two transmembrane domains, is essential for the membrane localization of ClC-K proteins and their activity in the kidney and inner ear. Ostm1 is a protein with a single transmembrane domain and a highly glycosylated N-terminus. Unlike the other two CLC auxiliary subunits, Ostm1 shows a reciprocal relationship with ClC-7 for their stability. The subcellular localization of Ostm1 depends on ClC-7 and not the other way around. ClC-2 is active on its own, but GlialCAM, a transmembrane cell adhesion molecule with two extracellular immunoglobulin (Ig)-like domains, regulates its subcellular localization and activity in glial cells. The common theme for these three proteins is their requirement for a proper homeostasis, since their malfunction leads to distinct diseases. We will review here their properties and their role in normal chloride physiology and the pathological consequences of their improper function.
Subject(s)
Carrier Proteins/metabolism , Chloride Channels/metabolism , Protein Subunits/metabolism , Animals , Humans , Membrane Proteins/metabolismABSTRACT
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.
Subject(s)
Chloride Channels/metabolism , Chlorides/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Amino Acid Sequence , Animals , CLC-2 Chloride Channels , Cell Cycle Proteins , Chloride Channels/chemistry , Chloride Channels/genetics , Databases, Genetic , Gene Expression Regulation, Developmental , Kinetics , Membrane Potentials , Molecular Sequence Data , Oocytes , Phylogeny , Protein Binding , Protein Transport , Proteins/metabolism , RNA, Messenger/metabolism , Xenopus , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/chemistry , Zebrafish Proteins/geneticsABSTRACT
GlialCAM (also named HepaCAM) is a cell adhesion molecule expressed mainly in glial cells from the central nervous system and the liver. GlialCAM plays different roles according to its cellular context. In epithelial cell lines, overexpression of GlialCAM increases cell adhesion and motility but also inhibits cell growth in tumor cell lines, leading to senescence. In glial cells, however, its function is quite different. GlialCAM acts a regulator of subcellular traffic of MLC1, a protein with unknown function involved in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare neurological condition. Moreover, GlialCAM itself has been found to be responsible for some of the cases of this disease. Additionally, GlialCAM also works as an auxiliary subunit of the chloride channel ClC-2, regulating its targeting to cell-cell junctions and modifying its functional properties. In summary, GlialCAM has different functions not only related to its adhesive nature, and defects in these functions lead to neurological disease.
Subject(s)
Central Nervous System/metabolism , Nervous System Diseases/metabolism , Neuroglia/metabolism , Proteins/metabolism , Animals , Cell Cycle Proteins , Central Nervous System/cytology , Humans , Liver/cytology , Liver/metabolism , Nervous System Diseases/geneticsABSTRACT
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.
Subject(s)
Cysts/metabolism , Hereditary Central Nervous System Demyelinating Diseases/metabolism , Membrane Proteins/metabolism , Neuroglia/metabolism , Proteins/metabolism , Animals , Animals, Genetically Modified , Astrocytes/metabolism , Brain/metabolism , Brain/pathology , Cell Cycle Proteins , Cell Line , Cell Membrane/metabolism , Cysts/genetics , Disease Models, Animal , Ependyma/cytology , Ependyma/metabolism , Ependyma/ultrastructure , Gene Expression , Genotype , Hereditary Central Nervous System Demyelinating Diseases/genetics , Humans , Intercellular Junctions/metabolism , Intercellular Junctions/ultrastructure , Membrane Proteins/genetics , Mice , Mice, Knockout , Mutation , Phenotype , Protein Transport , Proteins/genetics , Retina/metabolism , Voltage-Dependent Anion Channels/metabolism , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
During the development of the nervous system the regulation of cell cycle, differentiation, and survival is tightly interlinked. Newly generated neurons must keep cell cycle components under strict control, as cell cycle re-entry leads to neuronal degeneration and death. However, despite their relevance, the mechanisms controlling this process remain largely unexplored. Here we show that Scratch2 is involved in the control of the cell cycle in neurons in the developing spinal cord of the zebrafish embryo. scratch2 knockdown induces postmitotic neurons to re-enter mitosis. Scratch2 prevents cell cycle re-entry by maintaining high levels of the cycle inhibitor p57 through the downregulation of miR-25. Thus, Scratch2 appears to safeguard the homeostasis of postmitotic primary neurons by preventing cell cycle re-entry.
Subject(s)
MicroRNAs/physiology , Mitosis/physiology , Neurons/physiology , Transcription Factors/genetics , Transcription Factors/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Animals , Animals, Genetically Modified , Cell Differentiation/physiology , Cell Survival/physiology , Cyclin-Dependent Kinase Inhibitor p57/genetics , Cyclin-Dependent Kinase Inhibitor p57/metabolism , Gene Expression Regulation, Developmental/physiology , Gene Knockdown Techniques , Green Fluorescent Proteins/genetics , Homeostasis/physiology , Luminescent Proteins/genetics , MicroRNAs/genetics , Neurogenesis/physiology , Neurons/cytology , Spinal Cord/cytology , Spinal Cord/embryology , Spinal Cord/physiology , Zebrafish , Red Fluorescent ProteinABSTRACT
The epithelial-mesenchymal transition (EMT) is required in the embryo for the formation of tissues for which cells originate far from their final destination. Carcinoma cells hijack this program for tumor dissemination. The relevance of the EMT in cancer is still debated because it is unclear how these migratory cells colonize distant tissues to form macrometastases. We show that the homeobox factor Prrx1 is an EMT inducer conferring migratory and invasive properties. The loss of Prrx1 is required for cancer cells to metastasize in vivo, which revert to the epithelial phenotype concomitant with the acquisition of stem cell properties. Thus, unlike the classical EMT transcription factors, Prrx1 uncouples EMT and stemness, and is a biomarker associated with patient survival and lack of metastasis.
Subject(s)
Breast Neoplasms/pathology , Epithelial-Mesenchymal Transition/physiology , Homeodomain Proteins/physiology , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Movement/genetics , Cell Movement/physiology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Epithelial-Mesenchymal Transition/genetics , Female , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , MCF-7 Cells , Middle Aged , Neoplasm Metastasis , Prognosis , Retrospective Studies , Stem Cells/metabolism , Stem Cells/pathologyABSTRACT
Protein transport from endoplasmic reticulum (ER) to Golgi is primarily conducted by coated vesicular carriers such as COPII. Here, we describe zebrafish bulldog mutations that disrupt the function of the cargo adaptor Sec24D, an integral component of the COPII complex. We show that Sec24D is essential for secretion of cartilage matrix proteins, whereas the preceding development of craniofacial primordia and pre-chondrogenic condensations does not depend on this isoform. Bulldog chondrocytes fail to secrete type II collagen and matrilin to extracellular matrix (ECM), but membrane bound receptor beta1-Integrin and Cadherins appear to leave ER in Sec24D-independent fashion. Consequently, Sec24D-deficient cells accumulate proteins in the distended ER, although a subset of ER compartments and Golgi complexes as visualized by electron microscopy and NBD C(6)-ceramide staining appear functional. Consistent with the backlog of proteins in the ER, chondrocytes activate the ER stress response machinery and significantly upregulate BiP transcription. Failure of ECM secretion hinders chondroblast intercalations thus resulting in small and malformed cartilages and severe craniofacial dysmorphology. This defect is specific to Sec24D mutants since knockdown of Sec24C, a close paralog of Sec24D, does not result in craniofacial cartilage dysmorphology. However, craniofacial development in double Sec24C/Sec24D-deficient animals is arrested earlier than in bulldog/sec24d, suggesting that Sec24C can compensate for loss of Sec24D at initial stages of chondrogenesis, but Sec24D is indispensable for chondrocyte maturation. Our study presents the first developmental perspective on Sec24D function and establishes Sec24D as a strong candidate for cartilage maintenance diseases and craniofacial birth defects.