RESUMEN
Hyperekplexia is a rare sensorimotor syndrome characterized by pathological startle reflex in response to unexpected trivial stimuli for which there is no specific treatment. Neonates suffer from hypertonia and are at high risk of sudden death due to apnea episodes. Mutations in the human SLC6A5 gene encoding the neuronal glycine transporter GlyT2 may disrupt the inhibitory glycinergic neurotransmission and cause a presynaptic form of the disease. The phenotype of missense mutations giving rise to protein misfolding but maintaining residual activity could be rescued by facilitating folding or intracellular trafficking. In this report, we characterized the trafficking properties of two mutants associated with hyperekplexia (A277T and Y707C, rat numbering). Transporter molecules were partially retained in the endoplasmic reticulum showing increased interaction with the endoplasmic reticulum chaperone calnexin. One transporter variant had export difficulties and increased ubiquitination levels, suggestive of enhanced endoplasmic reticulum-associated degradation. However, the two mutant transporters were amenable to correction by calnexin overexpression. Within the search for compounds capable of rescuing mutant phenotypes, we found that the arachidonic acid derivative N-arachidonoyl glycine can rescue the trafficking defects of the two variants in heterologous cells and rat brain cortical neurons. N-arachidonoyl glycine improves the endoplasmic reticulum output by reducing the interaction transporter/calnexin, increasing membrane expression and improving transport activity in a comparable way as the well-established chemical chaperone 4-phenyl-butyrate. This work identifies N-arachidonoyl glycine as a promising compound with potential for hyperekplexia therapy.
Asunto(s)
Ácidos Araquidónicos/uso terapéutico , Variación Genética/fisiología , Proteínas de Transporte de Glicina en la Membrana Plasmática/genética , Glicina/análogos & derivados , Hiperekplexia/genética , Mutación Missense/fisiología , Neuronas/fisiología , Animales , Ácidos Araquidónicos/farmacología , Células COS , Células Cultivadas , Chlorocebus aethiops , Femenino , Variación Genética/efectos de los fármacos , Glicina/farmacología , Glicina/uso terapéutico , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Hiperekplexia/tratamiento farmacológico , Hiperekplexia/metabolismo , Mutación Missense/efectos de los fármacos , Neuronas/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Ratas , Ratas WistarRESUMEN
Introduction: The inhibitory glycine receptor (GlyR), a mediator of fast synaptic inhibition, is located and held at neuronal synapses through the anchoring proteins gephyrin and collybistin. Stable localization of neurotransmitter receptors is essential for synaptic function. In case of GlyRs, only beta subunits were known until now to mediate synaptic anchoring. Objectives: We identified a poly-proline II helix (PPII) in position 365-373 of the intra-cellular TM3-4 loop of the human GlyRα1 subunit as a novel potential synaptic anchoring site. The potential role of the PPII helix as synaptic anchoring site was tested. Methods: Glycine receptors and collybistin variants were generated and recombinantly expressed in HEK293 cells and cultured neurons. Receptor function was assessed using patch-clamp electrophysiology, protein-protein interaction was studied using co-immuno-precipitation and pulldown experiments. Results: Recombinantly expressed collybistin bound to isolated GlyRα1 TM3-4 loops in GST-pulldown assays. When the five proline residues P365A, P366A, P367A, P369A, P373A (GlyRα1P1-5A) located in the GlyRα1-PPII helix were replaced by alanines, the PPII secondary structure was disrupted. Recombinant GlyRα1P1-5A mutant subunits displayed normal cell surface expression and wildtype-like ion channel function, but binding to collybistin was abolished. The GlyRα1-collybistin interaction was independently confirmed by o-immunoprecipitation assays using full-length GlyRα1 subunits. Surprisingly, the interaction was not mediated by the SH3 domain of collybistin, but by its Pleckstrin homology (PH) domain. The mutation GlyRα1P366L, identified in a hyperekplexia patient, is also disrupting the PPII helix, and caused reduced collybistin binding. Conclusion: Our data suggest a novel interaction between α1 GlyR subunits and collybistin, which is physiologically relevant in vitro and in vivo and may contribute to postsynaptic anchoring of glycine receptors.
Asunto(s)
Prolina/metabolismo , Receptores de Glicina/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Sinapsis/metabolismo , Células HEK293 , Humanos , Hiperekplexia/genética , Hiperekplexia/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Neuronas/metabolismo , Dominios Homólogos a Pleckstrina , Dominios Proteicos Ricos en Prolina , Unión Proteica , Estructura Secundaria de Proteína , Receptores de Glicina/genética , Dominios Homologos srcRESUMEN
The functions of the glycine receptor (GlyR) and GABAA receptor (GABAAR) are both impaired in hyperekplexia, a neurological disorder usually caused by GlyR mutations. Although emerging evidence indicates that cannabinoids can directly restore normal GlyR function, whether they affect GABAAR in hyperekplexia remains unknown. Here we show that dehydroxylcannabidiol (DH-CBD), a synthetic nonpsychoactive cannabinoid, restores the GABA- and glycine-activated currents (IGABA and IGly , respectively) in HEK293 cells coexpressing a major GABAAR isoform (α1ß2γ2) and GlyRα1 carrying a human hyperekplexia-associated mutation (GlyRα1R271Q). Using coimmunoprecipitation and FRET assays, we found that DH-CBD disrupts the protein interaction between GABAAR and GlyRα1R271Q Furthermore, a point mutation of GlyRα1, changing Ser-296 to Ala-296, which is critical for cannabinoid binding on GlyR, significantly blocked DH-CBD-induced restoration of IGABA and IGly currents. This S296A substitution also considerably attenuated DH-CBD-induced disruption of the interaction between GlyRα1R271Q and GABAAR. These findings suggest that, because it restores the functions of both GlyRα1 and GABAAR, DH-CBD may represent a potentially valuable candidate drug to manage hyperekplexia.
Asunto(s)
Cannabinoides/farmacología , Hiperekplexia/genética , Lactonas/farmacología , Receptores de GABA-A/metabolismo , Receptores de Glicina/metabolismo , Potenciales de Acción , Células HEK293 , Humanos , Hiperekplexia/metabolismo , Mutación Missense , Unión Proteica/efectos de los fármacos , Receptores de GABA-A/genética , Receptores de Glicina/genéticaRESUMEN
Startle disease results from mutations in genes encoding inhibitory GlyR α1 and ß subunits or the presynaptic glycine transporter GlyT2. However, the most effective therapies are benzodiazepines that potentiate inhibitory GABAAR function. A recent publication by Zou et al. adds further complexity by suggesting that dominant GlyR α1 mutants assemble into pre- and extrasynaptic GABAARs.
Asunto(s)
Hiperekplexia/genética , Receptores de GABA-A/metabolismo , Receptores de Glicina/genética , Animales , Genes Dominantes , Humanos , Hiperekplexia/metabolismo , Mutación , Receptores de GABA-A/genética , Receptores de Glicina/metabolismoRESUMEN
Hyperekplexia or startle disease is a dysfunction of inhibitory glycinergic neurotransmission characterized by an exaggerated startle in response to trivial tactile or acoustic stimuli. Although rare, this disorder can have serious consequences, including sudden infant death. One of the most frequent causes of hyperekplexia are mutations in the SLC6A5 gene, encoding the neuronal glycine transporter 2 (GlyT2), a key component of inhibitory glycinergic presynapses involved in synaptic glycine recycling though sodium and chloride-dependent co-transport. Most GlyT2 mutations detected so far are recessive, but two dominant missense mutations have been described. The detailed analysis of these mutations has revealed structural cues on the quaternary structure of GlyT2, and opens the possibility that novel selective pharmacochaperones have potential therapeutic effects in hyperekplexia.
Asunto(s)
Proteínas de Transporte de Glicina en la Membrana Plasmática/genética , Hiperekplexia/genética , Mutación/genética , Animales , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Humanos , Hiperekplexia/metabolismo , Neuronas/metabolismo , Receptores de Glicina/genética , Transmisión Sináptica/genéticaRESUMEN
Recently, CLPB deficiency has been shown to cause a genetic syndrome with cataracts, neutropenia, and 3-methylglutaconic aciduria. Surprisingly, the neurological presentation ranges from completely unaffected to patients with virtual absence of development. Muscular hypo- and hypertonia, movement disorder and progressive brain atrophy are frequently reported. We present the foetal, peri- and neonatal features of 31 patients, of which five are previously unreported, using a newly developed clinical severity scoring system rating the clinical, metabolic, imaging and other findings weighted by the age of onset. Our data are illustrated by foetal and neonatal videos. The patients were classified as having a mild (n = 4), moderate (n = 13) or severe (n = 14) disease phenotype. The most striking feature of the severe subtype was the neonatal absence of voluntary movements in combination with ventilator dependency and hyperexcitability. The foetal and neonatal presentation mirrored the course of disease with respect to survival (current median age 17.5 years in the mild group, median age of death 35 days in the severe group), severity and age of onset of all findings evaluated. CLPB deficiency should be considered in neonates with absence of voluntary movements, respiratory insufficiency and swallowing problems, especially if associated with 3-methylglutaconic aciduria, neutropenia and cataracts. Being an important differential diagnosis of hyperekplexia (exaggerated startle responses), we advise performing urinary organic acid analysis, blood cell counts and ophthalmological examination in these patients. The neonatal presentation of CLPB deficiency predicts the course of disease in later life, which is extremely important for counselling.
Asunto(s)
Catarata/metabolismo , Endopeptidasa Clp/deficiencia , Errores Innatos del Metabolismo/metabolismo , Neutropenia/metabolismo , Adolescente , Adulto , Atrofia/metabolismo , Encefalopatías , Niño , Preescolar , Femenino , Feto/metabolismo , Humanos , Hiperekplexia/metabolismo , Lactante , Recién Nacido , Masculino , Trastornos del Movimiento/metabolismo , Fenotipo , Adulto JovenRESUMEN
Glycine receptors (GlyR) belong to the pentameric ligand-gated ion channel (pLGIC) superfamily and mediate fast inhibitory transmission in the vertebrate CNS. Disruption of glycinergic transmission by inherited mutations produces startle disease in man. Many startle mutations are in GlyRs and provide useful clues to the function of the channel domains. E103K is one of few startle mutations found in the extracellular agonist binding site of the channel, in loop A of the principal side of the subunit interface. Homology modeling shows that the side chain of Glu-103 is close to that of Arg-131, in loop E of the complementary side of the binding site, and may form a salt bridge at the back of the binding site, constraining its size. We investigated this hypothesis in recombinant human α1 GlyR by site-directed mutagenesis and functional measurements of agonist efficacy and potency by whole cell patch clamp and single channel recording. Despite its position near the binding site, E103K causes hyperekplexia by impairing the efficacy of glycine, its ability to gate the channel once bound, which is very high in wild type GlyR. Mutating Glu-103 and Arg-131 caused various degrees of loss-of-function in the action of glycine, whereas mutations in Arg-131 enhanced the efficacy of the slightly bigger partial agonist sarcosine (N-methylglycine). The effects of the single charge-swapping mutations of these two residues were largely rescued in the double mutant, supporting the possibility that they interact via a salt bridge that normally constrains the efficacy of larger agonist molecules.
Asunto(s)
Hiperekplexia/genética , Mutación Puntual , Receptores de Glicina/genética , Receptores de Glicina/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Glicina/metabolismo , Células HEK293 , Humanos , Hiperekplexia/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Receptores de Glicina/química , Sarcosina/metabolismo , Alineación de SecuenciaRESUMEN
Hyperekplexia is a rare human neuromotor disorder caused by mutations that impair the efficacy of glycinergic inhibitory neurotransmission. Loss-of-function mutations in the GLRA1 or GLRB genes, which encode the α1 and ß glycine receptor (GlyR) subunits, are the major cause. Paradoxically, gain-of-function GLRA1 mutations also cause hyperekplexia, although the mechanism is unknown. Here we identify two new gain-of-function mutations (I43F and W170S) and characterize these along with known gain-of-function mutations (Q226E, V280M, and R414H) to identify how they cause hyperekplexia. Using artificial synapses, we show that all mutations prolong the decay of inhibitory postsynaptic currents (IPSCs) and induce spontaneous GlyR activation. As these effects may deplete the chloride electrochemical gradient, hyperekplexia could potentially result from reduced glycinergic inhibitory efficacy. However, we consider this unlikely as the depleted chloride gradient should also lead to pain sensitization and to a hyperekplexia phenotype that correlates with mutation severity, neither of which is observed in patients with GLRA1 hyperekplexia mutations. We also rule out small increases in IPSC decay times (as caused by W170S and R414H) as a possible mechanism given that the clinically important drug, tropisetron, significantly increases glycinergic IPSC decay times without causing motor side effects. A recent study on cultured spinal neurons concluded that an elevated intracellular chloride concentration late during development ablates α1ß glycinergic synapses but spares GABAergic synapses. As this mechanism satisfies all our considerations, we propose it is primarily responsible for the hyperekplexia phenotype.