Model animal de malalties associades a canals de clorur en peix zebra

[cat] El clorur compleix un conjunt de funcions essencials perquè la cèl·lula pugui realitzar correctament diferents processos fisiològics, com la regulació de l’excitabilitat en les cèl·lules musculars, participa en el transport transepitelial d’aigua i sals, en la regulació del pH en l’interior dels lisosomes i en la regulació del volum cel·lular, entre d’altres. La família de proteïnes ClC es divideix en dos grups: en canals iònics presents en la membrana plasmàtica i en transportadors Cl-/H+ presents en compartiments intracel·lulars. Fins ara s’ha descrit que ClC-2, els dos canals ClC-K i ClC-7 presenten les subunitats accessòries anomenades GlialCAM, Barttin i Ostm1, respectivament. En aquesta tesi s’han estudiat els canals iònics de membrana plasmàtica ClC-1, ClC-2 i els canals ClC-K. Mutacions en els gens que codifiquen aquestes proteïnes donen lloc a malalties rares en humans i altres espècies animals, com la Miotonia congènita (degut a la pèrdua d’activitat ClC-1, es caracteritza per un retard en la repolarització muscular), un tipus rar de leucodistròfia vacuolitzant (degut a la pèrdua de ClC-2) i síndrome de Bartter tipus III (degut a la pèrdua de ClC-Kb). En aquesta tesi hem identificat i iniciat la caracterització dels gens ClC ortòlegs en el peix zebra. Hem estudiat el patró d’expressió tant en adults com en embrions. En adults, hem descrit que els canals clcn1a i clcn1b són específics de múscul esquelètic, el canal clcn2a és el més abundant en teixits excitables com el cervell, ull i cor; el canal clcn2b és ubic, el canals clcn2c s’expressa en teixits on es dóna intercanvi iònic amb el medi extern com les brànquies i el ronyó i el canals clcnk s’expressa en el ronyó. En embrions, només hem pogut estudiar la localització de clcn2b, el qual s’expressa en el túbul contornejat proximal del pronefros, i de clcn2c, el qual s’expressa en la regió dels arcs branquials. Hem detectat expressió dels gens clcn1a i clcn1b a partir de 1dpf i 2dpf, respectivament. Part important de la tesi ha estat la generació i validació d’anticossos policlonals tant pels canals clc-1a, clc-1b, clc-2a, clc-2b, clc-k com per les subunitats glialcama i barttin. També hem demostrat in vitro que es conserva la interacció dels canals clc-2 amb GlialCAM, modificant el seu tràfic a les unions cel·lulars i les corrents. De la mateixa manera, el paper estabilitzador de Barttin sobre els canals ClC-K també es troba conservat en el peix zebra. Estudis d’immunofluorescència en larves de peix zebra a 3dpf mostren una localització cel·lular diferent en el múscul esquelètic, mentre que clc-1a es localitza al sarcolema, clc-1b es troba envoltant els túbuls T. A més, hem desenvolupat eines de transgènesi amb promotors específics de teixit per tal de realitzar experiments d’expressió en múscul esquelètic (503unc) de proteïnes de fusió fluorescents (tant humanes com de peix zebra). Una part important d’aquest treball ha estat la generació de models de pèrdua de funció en peix zebra mitjançant la tecnologia CRISPR/Cas9. Hem aconseguit generar animals knock out dels gens clcn1a i clcn1b (el doble knock out encara està en procés), els quals han estat validats com a tal gràcies als anticossos generats durant aquesta tesi. Finalment, hem realitzat uns estudis locomotors preliminars i aquests mostren com les línies mutants per clcn1a i per clcn1b no presenten cap alteració en el moviment espontani ni en la resposta a estímuls de vibració. És probable que la manca d’un dels dos canals clc-1 sigui compensada pel seu paràleg, per aquesta raó estem generant una línia nul·la pel canal clc-1. En aquesta línia deficient per clc-1 esperem detectar alteracions motores reminiscents a les observades en pacients de Miotonia congènita.[eng] Chloride is the most abundant anion in body fluids. Chloride transport is involved in many different cellular processes, such as regulating the excitability of muscle cells, transepithelial transport and regulation of pH in lysosomes, among others. The ClC protein family comprises a group of proteins allowing this exchange between the intracellular and extracellular space. They are divided into two subgroups: the plasma membrane chloride channels and the Cl-/H+ co-transporters located in intracellular compartments. The plasma membrane chloride channels are ClC-1 (muscle-specific, is in charge of keeping the resting membrane potential in muscle cells), ClC-2 (ubiquitously expressed) and ClC-Ka and ClC-Kb (located in the distal renal tubule and inner ear, involved in renal salt reabsorption and sound transduction in the ear). Mutations in the genes encoding for these channels lead to human rare diseases. Loss of ClC-1 leads to Myotonia congenital resulting in hyperexcitable muscles, lack of ClC-2 leads to a rare type of vacuolating leukodystrophy and the loss of ClC-Kb leads to Bartter syndrome type III, which is characterized by renal salt loss. In this thesis, we have identified and characterized the zebrafish (Danio rerio) ClC orthologs. To accomplish that, we have generated and validated antibodies against the zebrafish proteins. Furthermore, we have developed transgenesis tools for native and mutant proteins expression experiments and generated clcn1a and clcn1b (human CLCN1 orthologs) loss-of-function alleles in zebrafish with CRISPR/Cas9 technology (the clcn1 null-mutant is in progress). We have validated these lines as knock out and a preliminary locomotor characterization shows these animals display normal spontaneous movement and no difference has been observed between wild-type and knock out animals when applying vibrational stimulus. We hope for the clcn1a-/- clcn1b-/- to show locomotor deficiencies reminiscent to those observed in Myotonia congenita patients

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

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

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

[Abstract] 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.Ministerio de Ciencia e Innovación; SAF2015–70377Ministerio de Ciencia e Innovación; RTI2018–093493-B-I00Generalitat de Catalunya; SGR2014–1178Generalitat de Catalunya; SGR014–2016Instituto de Salud Carlos III; PI16/00267-R-Fede

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

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 phenotyp

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 EGFreceptor. 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

Time to Switch to Second-line Antiretroviral Therapy in Children With Human Immunodeficiency Virus in Europe and Thailand.

Background: Data on durability of first-line antiretroviral therapy (ART) in children with human immunodeficiency virus (HIV) are limited. We assessed time to switch to second-line therapy in 16 European countries and Thailand. Methods: Children aged <18 years initiating combination ART (≥2 nucleoside reverse transcriptase inhibitors [NRTIs] plus nonnucleoside reverse transcriptase inhibitor [NNRTI] or boosted protease inhibitor [PI]) were included. Switch to second-line was defined as (i) change across drug class (PI to NNRTI or vice versa) or within PI class plus change of ≥1 NRTI; (ii) change from single to dual PI; or (iii) addition of a new drug class. Cumulative incidence of switch was calculated with death and loss to follow-up as competing risks. Results: Of 3668 children included, median age at ART initiation was 6.1 (interquartile range (IQR), 1.7-10.5) years. Initial regimens were 32% PI based, 34% nevirapine (NVP) based, and 33% efavirenz based. Median duration of follow-up was 5.4 (IQR, 2.9-8.3) years. Cumulative incidence of switch at 5 years was 21% (95% confidence interval, 20%-23%), with significant regional variations. Median time to switch was 30 (IQR, 16-58) months; two-thirds of switches were related to treatment failure. In multivariable analysis, older age, severe immunosuppression and higher viral load (VL) at ART start, and NVP-based initial regimens were associated with increased risk of switch. Conclusions: One in 5 children switched to a second-line regimen by 5 years of ART, with two-thirds failure related. Advanced HIV, older age, and NVP-based regimens were associated with increased risk of switch

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