RESUMEN
Muscle-specific kinase myasthenia gravis (MuSK MG) is caused by autoantibodies against MuSK in the neuromuscular junction (NMJ). MuSK MG patients have fluctuating, fatigable skeletal muscle weakness, in particular of bulbar muscles. Severity differs greatly between patients, in spite of comparable autoantibody levels. One explanation for inter-patient and inter-muscle variability in sensitivity might be variations in compensatory muscle responses. Previously, we developed a passive transfer mouse model for MuSK MG. In preliminary ex vivo experiments, we observed that muscle contraction of some mice, in particular those with milder myasthenia, had become partially insensitive to inhibition by µ-Conotoxin-GIIIB, a blocker of skeletal muscle NaV1.4 voltage-gated sodium channels. We hypothesised that changes in NaV channel expression profile, possibly co-expression of (µ-Conotoxin-GIIIB insensitive) NaV1.5 type channels, might lower the muscle fibre's firing threshold and facilitate neuromuscular synaptic transmission. To test this hypothesis, we here performed passive transfer in immuno-compromised mice, using 'high', 'intermediate' and 'low' dosing regimens of purified MuSK MG patient IgG4. We compared myasthenia levels, µ-Conotoxin-GIIIB resistance and muscle fibre action potential characteristics and firing thresholds. High- and intermediate-dosed mice showed clear, progressive myasthenia, not seen in low-dosed animals. However, diaphragm NMJ electrophysiology demonstrated almost equal myasthenic severities amongst all regimens. Nonetheless, low-dosed mouse diaphragms showed a much higher degree of µ-Conotoxin-GIIIB resistance. This was not explained by upregulation of Scn5a (the NaV1.5 gene), lowered muscle fibre firing thresholds or histologically detectable upregulated NaV1.5 channels. It remains to be established which factors are responsible for the observed µ-Conotoxin-GIIIB insensitivity and whether the NaV repertoire change is compensatory beneficial or a bystander effect.
Asunto(s)
Músculo Esquelético , Animales , Ratones , Músculo Esquelético/metabolismo , Músculo Esquelético/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Humanos , Miastenia Gravis/metabolismo , Miastenia Gravis/fisiopatología , Miastenia Gravis/inmunología , Modelos Animales de Enfermedad , Femenino , Receptores Colinérgicos/metabolismo , Receptores Colinérgicos/inmunología , Canales de Sodio Activados por Voltaje/metabolismo , Unión Neuromuscular/metabolismo , Unión Neuromuscular/efectos de los fármacos , Autoanticuerpos , Masculino , Conotoxinas/farmacología , Inmunización PasivaRESUMEN
Human immunoglobulin (Ig) G4 usually displays antiinflammatory activity, and observations of IgG4 autoantibodies causing severe autoimmune disorders are therefore poorly understood. In blood, IgG4 naturally engages in a stochastic process termed "Fab-arm exchange" in which unrelated IgG4s exchange half-molecules continuously. The resulting IgG4 antibodies are composed of two different binding sites, thereby acquiring monovalent binding and inability to cross-link for each antigen recognized. Here, we demonstrate that this process amplifies autoantibody pathogenicity in a classic IgG4-mediated autoimmune disease: muscle-specific kinase (MuSK) myasthenia gravis. In mice, monovalent anti-MuSK IgG4s caused rapid and severe myasthenic muscle weakness, whereas the same antibodies in their parental bivalent form were less potent or did not induce a phenotype. Mechanistically this could be explained by opposing effects on MuSK signaling. Isotype switching to IgG4 in an autoimmune response thereby may be a critical step in the development of disease. Our study establishes functional monovalency as a pathogenic mechanism in IgG4-mediated autoimmune disease and potentially other disorders.
Asunto(s)
Autoanticuerpos/inmunología , Inmunoglobulina G/inmunología , Miastenia Gravis/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores Colinérgicos/inmunología , Animales , Anticuerpos Biespecíficos/administración & dosificación , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/inmunología , Autoanticuerpos/administración & dosificación , Autoanticuerpos/genética , Línea Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/genética , Masculino , Ratones , Miastenia Gravis/patología , Mioblastos , Unión Neuromuscular/inmunología , Unión Neuromuscular/patología , Fosforilación/inmunología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
The C57BL/10ScSn-Dmdmdx/J (BL10-mdx) mouse has been the most commonly used model for Duchenne muscular dystrophy (DMD) for decades. Their muscle dysfunction and pathology is, however, less severe than in patients with DMD, which complicates preclinical studies. Recent discoveries indicate that disease severity is exacerbated when muscular dystrophy mouse models are generated on a DBA2/J genetic background. Knowledge on the natural history of animal models is pivotal for high-quality preclinical testing. However, for BL10-mdx mice on a DBA2/J background (D2-mdx), limited data are available. We addressed this gap in the natural history knowledge. First, we compared histopathological aspects in skeletal muscles of young D2-mdx, BL10-mdx, and wild-type mice. Pathology was more pronounced in D2-mdx mice and differed in severity between muscles within individuals. Secondly, we subjected D2-mdx mice to a functional test regime for 34 weeks and identified that female D2-mdx mice outperform severely impaired males, making females less useful for functional preclinical studies. Direct comparisons between 10- and 34-wk-old D2-mdx mice revealed that disease pathology ameliorates with age. Heart pathology was progressive, with some features already evident at a young age. This natural history study of the D2-mdx mouse will be instrumental for experimental design of future preclinical studies.-Van Putten, M., Putker, K., Overzier, M., Adamzek, W. A., Pasteuning-Vuhman, S., Plomp, J. J., Aartsma-Rus, A. Natural disease history of the D2-mdx mouse model for Duchenne muscular dystrophy.
Asunto(s)
Distrofia Muscular Animal , Distrofia Muscular de Duchenne , Animales , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos mdx , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Distrofia Muscular Animal/fisiopatología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/fisiopatologíaRESUMEN
AIMS: Current treatments of ventricular arrhythmias rely on modulation of cardiac electrical function through drugs, ablation or electroshocks, which are all non-biological and rather unspecific, irreversible or traumatizing interventions. Optogenetics, however, is a novel, biological technique allowing electrical modulation in a specific, reversible and trauma-free manner using light-gated ion channels. The aim of our study was to investigate optogenetic termination of ventricular arrhythmias in the whole heart. METHODS AND RESULTS: Systemic delivery of cardiotropic adeno-associated virus vectors, encoding the light-gated depolarizing ion channel red-activatable channelrhodopsin (ReaChR), resulted in global cardiomyocyte-restricted transgene expression in adult Wistar rat hearts allowing ReaChR-mediated depolarization and pacing. Next, ventricular tachyarrhythmias (VTs) were induced in the optogenetically modified hearts by burst pacing in a Langendorff setup, followed by programmed, local epicardial illumination. A single 470-nm light pulse (1000 ms, 2.97 mW/mm2) terminated 97% of monomorphic and 57% of polymorphic VTs vs. 0% without illumination, as assessed by electrocardiogram recordings. Optical mapping showed significant prolongation of voltage signals just before arrhythmia termination. Pharmacological action potential duration (APD) shortening almost fully inhibited light-induced arrhythmia termination indicating an important role for APD in this process. CONCLUSION: Brief local epicardial illumination of the optogenetically modified adult rat heart allows contact- and shock-free termination of ventricular arrhythmias in an effective and repetitive manner after optogenetic modification. These findings could lay the basis for the development of fundamentally new and biological options for cardiac arrhythmia management.
Asunto(s)
Arritmias Cardíacas/terapia , Channelrhodopsins/farmacología , Optogenética/métodos , Fototerapia/métodos , Adenoviridae , Animales , Channelrhodopsins/administración & dosificación , Terapia Genética/métodos , Vectores Genéticos , Activación del Canal Iónico/efectos de la radiación , Luz , Miocitos Cardíacos/fisiología , Ratas Wistar , Taquicardia Ventricular/terapia , Transgenes/fisiologíaRESUMEN
The ability to administer systemically high doses of manganese as contrast agent while circumventing its toxicity is of particular interest for exploratory MRI studies of the brain. Administering low doses either repeatedly or continuously over time has been shown to enable the acquisition of satisfactory MRI images of the mouse brain without apparent side effects. Here we have systematically compared the obtained MRI contrast and recorded potential systemic side effects such as stress response and muscle strength impairment in relation to the achieved contrast. We show in mice that administering MnCl2 via osmotic infusion pumps allows for a side-effect free delivery of a high cumulative dose of manganese chloride (480mg/kg bodyweight in 8 days). High contrast in MRI was achieved while we did not observe the weight loss or distress seen in other studies where mice received manganese via fractionated intraperitoneal injections of lower doses of manganese. As the normal daily conduct of the mice was not affected, this new manganese delivery method might be of particular use to study brain activity over several days. This may facilitate the phenotyping of new transgenic mouse models, the study of chronic disease models and the monitoring of changes in brain activity in long-term behavioral studies.
Asunto(s)
Cloruros/administración & dosificación , Cloruros/farmacología , Medios de Contraste/administración & dosificación , Medios de Contraste/farmacología , Imagen por Resonancia Magnética/métodos , Compuestos de Manganeso/administración & dosificación , Compuestos de Manganeso/farmacología , Animales , Cloruros/efectos adversos , Medios de Contraste/efectos adversos , Corticosterona/sangre , Aumento de la Imagen , Bombas de Infusión , Infusiones Intravenosas , Inyecciones Intraperitoneales , Masculino , Compuestos de Manganeso/efectos adversos , Ratones , Ratones Endogámicos C57BL , Fuerza Muscular/efectos de los fármacos , Estrés Psicológico/inducido químicamente , Estrés Psicológico/psicología , Pérdida de Peso/efectos de los fármacosRESUMEN
Duchenne muscular dystrophy (DMD) is an X-linked myopathy caused by dystrophin deficiency. Dystrophin is present intracellularly at the sarcolemma, connecting actin to the dystrophin-associated glycoprotein complex. Interestingly, it is enriched postsynaptically at the neuromuscular junction (NMJ), but its synaptic function is largely unknown. Utrophin, a dystrophin homologue, is also concentrated at the NMJ, and upregulated in DMD. It is possible that the absence of dystrophin at NMJs in DMD causes neuromuscular transmission defects that aggravate muscle weakness. We studied NMJ function in mdx mice (lacking dystrophin) and wild type mice. In addition, mdx/utrn(+/-) and mdx/utrn(-/-) mice (lacking utrophin) were used to investigate influences of utrophin levels. The three Duchenne mouse models showed muscle weakness when comparatively tested in vivo, with mdx/utrn(-/-) mice being weakest. Ex vivo muscle contraction and electrophysiological studies showed a reduced safety factor of neuromuscular transmission in all models. NMJs had ~ 40% smaller miniature endplate potential amplitudes compared with wild type, indicating postsynaptic sensitivity loss for the neurotransmitter acetylcholine. However, nerve stimulation-evoked endplate potential amplitudes were unchanged. Consequently, quantal content (i.e. the number of acetylcholine quanta released per nerve impulse) was considerably increased. Such a homeostatic compensatory increase in neurotransmitter release is also found at NMJs in myasthenia gravis, where autoantibodies reduce acetylcholine receptors. However, high-rate nerve stimulation induced exaggerated endplate potential rundown. Study of NMJ morphology showed that fragmentation of acetylcholine receptor clusters occurred in all models, being most severe in mdx/utrn(-/-) mice. Overall, we showed mild 'myasthenia-like' neuromuscular synaptic dysfunction in several Duchenne mouse models, which possibly affects muscle weakness and degeneration.
Asunto(s)
Modelos Animales de Enfermedad , Distrofia Muscular de Duchenne/fisiopatología , Unión Neuromuscular/fisiopatología , Transmisión Sináptica , Potenciales de Acción , Animales , Distrofina/genética , Femenino , Masculino , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Potenciales Postsinápticos Miniatura , Fatiga Muscular , Fuerza Muscular , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Unión Neuromuscular/metabolismo , Receptores Colinérgicos/metabolismo , Respiración , Utrofina/genéticaRESUMEN
Myasthenia gravis (MG) is a severely debilitating autoimmune disease that is due to a decrease in the efficiency of synaptic transmission at neuromuscular synapses. MG is caused by antibodies against postsynaptic proteins, including (i) acetylcholine receptors, the neurotransmitter receptor, (ii) muscle-specific kinase (MuSK), a receptor tyrosine kinase essential for the formation and maintenance of neuromuscular synapses, and (iii) low-density lipoprotein receptor-related protein 4 (Lrp4), which responds to neural Agrin by binding and stimulating MuSK. Passive transfer studies in mice have shown that IgG4 antibodies from MuSK MG patients cause disease without requiring complement or other immune components, suggesting that these MuSK antibodies cause disease by directly interfering with MuSK function. Here we show that pathogenic IgG4 antibodies to MuSK bind to a structural epitope in the first Ig-like domain of MuSK, prevent binding between MuSK and Lrp4, and inhibit Agrin-stimulated MuSK phosphorylation. In contrast, these IgG4 antibodies have no direct effect on MuSK dimerization or MuSK internalization. These results provide insight into the unique pathogenesis of MuSK MG and provide clues toward development of specific treatment options.
Asunto(s)
Autoanticuerpos/inmunología , Inmunoglobulina G/inmunología , Proteínas Relacionadas con Receptor de LDL/inmunología , Miastenia Gravis/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores Colinérgicos/inmunología , Receptores de LDL/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Agrina/inmunología , Animales , Autoanticuerpos/farmacología , Línea Celular , Niño , Preescolar , Epítopos/inmunología , Femenino , Humanos , Inmunización Pasiva , Inmunoglobulina G/farmacología , Proteínas Relacionadas con Receptor de LDL/antagonistas & inhibidores , Masculino , Ratones , Persona de Mediana Edad , Miastenia Gravis/inducido químicamente , Miastenia Gravis/patología , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Multimerización de Proteína/efectos de los fármacos , Multimerización de Proteína/inmunología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de LDL/antagonistas & inhibidoresRESUMEN
The pre-synaptic motor nerve terminal is a highly complex and dynamic compartment within the lower motor neuron responsible for converting electrical signals into secreted chemicals. This self-renewing process of synaptic transmission is accomplished by the calcium-triggered fusion of neurotransmitter-containing vesicles with the plasma membrane and the subsequent retrieval and recycling of vesicle components. Besides this conventional physiological role, the highly active process of vesicle fusion and re-uptake into endosomal sorting pathways acts as a conduit for entry of a range of substances into the intracellular compartment of the motor nerve terminal. Whilst this entry portal sub-serves many vital physiological processes, such as those mediated by neurotrophin trafficking, there is also the potential for substantial pathological consequences resulting from uptake of noxious agents, including autoantibodies, viruses and toxins. These may act locally to induce disease within the nerve terminal, or traffic beyond to the motor neuron cell body and central nervous system to exert their pathological effects. This review focuses on the recent evidence that the ganglioside-rich pre-synaptic membrane acts as a binding site for potentially neurotoxic serum autoantibodies that are present in human autoimmune motor neuropathies. Autoantibodies that bind surface antigens induce membrane lytic effects, whereas their uptake attenuates local injury and transfers any potential pathological consequences to the intracellular compartment. Herein the thesis is explored that a balance exists between local injury at the exofacial leaflet of the pre-synaptic membrane and antibody uptake, which dictates the overall level and site of motor nerve injury in this group of disorders.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Neuronas Motoras/inmunología , Enfermedades de la Unión Neuromuscular/inmunología , Síndromes de Neurotoxicidad/inmunología , Terminales Presinápticos/inmunología , Gangliósidos/inmunología , HumanosRESUMEN
Myasthenia gravis is a paralytic disorder with autoantibodies against acetylcholine receptors at the neuromuscular junction. A proportion of patients instead has antibodies against muscle-specific kinase, a protein essential for acetylcholine receptor clustering. These are generally of the immunoglobulin-G4 subclass and correlate with disease severity, suggesting specific myasthenogenic activity. However, immunoglobulin-G4 subclass antibodies are generally considered to be 'benign' and direct proof for their pathogenicity in muscle-specific kinase myasthenia gravis (or other immunoglobulin-G4-associated disorders) is lacking. Furthermore, the exact electrophysiological synaptic defects caused at neuromuscular junctions by human anti-muscle-specific kinase autoantibodies are hitherto unknown. We show that purified immunoglobulin-G4, but not immunoglobulin-G1-3, from patients with muscle-specific kinase myasthenia gravis binds to mouse neuromuscular junctions in vitro, and that injection into immunodeficient mice causes paralysis. Injected immunoglobulin-G4 caused reduced density and fragmented area of neuromuscular junction acetylcholine receptors. Detailed electrophysiological synaptic analyses revealed severe reduction of postsynaptic acetylcholine sensitivity, and exaggerated depression of presynaptic acetylcholine release during high-rate activity, together causing the (fatigable) muscle weakness. Intriguingly, compensatory transmitter release upregulation, which is the normal homeostatic response in acetylcholine receptor myasthenia gravis, was absent. This conveys extra vulnerability to neurotransmission at muscle-specific kinase myasthenia gravis neuromuscular junctions. Thus, we demonstrate that patient anti-muscle-specific kinase immunoglobulin-G4 is myasthenogenic, independent of additional immune system components, and have elucidated the underlying electrophysiological neuromuscular junction abnormalities.
Asunto(s)
Inmunoglobulina G/efectos adversos , Inmunoglobulina G/sangre , Miastenia Gravis/sangre , Enfermedades de la Unión Neuromuscular/complicaciones , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores Colinérgicos/inmunología , Potenciales de Acción/efectos de los fármacos , Adulto , Animales , Autoanticuerpos/sangre , Modelos Animales de Enfermedad , Electromiografía , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Placa Motora/efectos de los fármacos , Placa Motora/fisiopatología , Contracción Muscular/efectos de los fármacos , Fuerza Muscular/efectos de los fármacos , Fuerza Muscular/fisiología , Miastenia Gravis/complicaciones , Miastenia Gravis/inmunología , Miastenia Gravis/terapia , Conducción Nerviosa/efectos de los fármacos , Conducción Nerviosa/fisiología , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/patología , Unión Neuromuscular/fisiopatología , Unión Neuromuscular/ultraestructura , Enfermedades de la Unión Neuromuscular/patología , Plasmaféresis/métodos , Adulto JovenRESUMEN
Muscle-specific kinase (MuSK) is crucial for acetylcholine receptor (AChR) clustering and thereby neuromuscular junction (NMJ) function. NMJ dysfunction is a hallmark of several neuromuscular diseases, including MuSK myasthenia gravis. Aiming to restore NMJ function, we generated several agonist monoclonal antibodies targeting the MuSK Ig-like 1 domain. These activated MuSK and induced AChR clustering in cultured myotubes. The most potent agonists partially rescued myasthenic effects of MuSK myasthenia gravis patient IgG autoantibodies in vitro. In an IgG4 passive transfer MuSK myasthenia model in NOD/SCID mice, MuSK agonists caused accelerated weight loss and no rescue of myasthenic features. The MuSK Ig-like 1 domain agonists unexpectedly caused sudden death in a large proportion of male C57BL/6 mice (but not female or NOD/SCID mice), likely caused by a urologic syndrome. In conclusion, these agonists rescued pathogenic effects in myasthenia models in vitro, but not in vivo. The sudden death in male mice of one of the tested mouse strains revealed an unexpected and unexplained role for MuSK outside skeletal muscle, thereby hampering further (pre-) clinical development of these clones. Future research should investigate whether other Ig-like 1 domain MuSK antibodies, binding different epitopes, do hold a safe therapeutic promise.
Asunto(s)
Miastenia Gravis , Proteínas Tirosina Quinasas Receptoras , Masculino , Animales , Ratones , Ratones SCID , Proteínas Tirosina Quinasas Receptoras/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Miastenia Gravis/metabolismo , Receptores Colinérgicos/metabolismo , Autoanticuerpos , Debilidad Muscular , AcetilcolinaRESUMEN
Muscle weakness and fatigue are the hallmarks of autoimmune neuromuscular junction disorders. Although a plethora of immunosuppressive treatments exist, no cure is available to date and many patients are left with debilitating muscle weakness. Recent advances in the understanding of the structure and function of the neuromuscular junction, and the development of novel in vitro and in vivo models, have been instrumental in unravelling the pathophysiology of these autoimmune diseases. These advances are providing the rationale for the development of new therapeutic strategies. Restoration of the immune imbalance in these diseases, in parallel with symptomatic therapeutic approaches at the neuromuscular junction, will be crucial to obtain long-term remission or even cure.
Asunto(s)
Enfermedades de la Unión Neuromuscular , Humanos , Unión NeuromuscularRESUMEN
BACKGROUND: Myasthenia gravis (MG) is an autoimmune neuromuscular disorder hallmarked by fluctuating fatigable muscle weakness. Most patients have autoantibodies against acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). These are thought to have three possible pathogenic mode-of-actions: 1) cross-linking and endocytosis of AChRs, 2) direct block of AChRs and 3) complement activation. The relative contributions of these mechanisms to synaptic block and muscle weakness of individual patients cannot be determined. It likely varies between patients and perhaps also with disease course, depending on the nature of the circulating AChR antibodies. NEW METHOD: We developed a new bioassay which specifically enables functional characterization and quantification of complement-mediated synaptic damage at NMJs, without interference of the other pathogenic mechanisms. To this end, we pre-incubated mouse hemi-diaphragm muscle-nerve preparations with mAb35-hG1, a humanized rat AChR monoclonal and subsequently exposed the preparation to normal human serum as a complement source. NMJ-restricted effects were studied. RESULTS: Clearly NMJ-restricted damage occurred. With immunohistology we showed complement deposition at NMJs, and synaptic electrophysiological measurements demonstrated transmission block. In whole-muscle contraction experiments we quantified the effect and characterized its onset and progression during the incubation with normal human serum. COMPARISON WITH EXISTING METHODS: With this new assay the complement-mediated component of myasthenic NMJ pathology can be studied separately. CONCLUSIONS: Our assay will be of importance in detailed mechanistic studies of local complement activation at NMJs, investigations of new complement inhibitors, and laboratory pre-screening of therapeutic efficacy for individual MG patients to optimize care with clinically approved complement inhibitors.
Asunto(s)
Miastenia Gravis , Receptores Colinérgicos , Animales , Autoanticuerpos , Bioensayo , Activación de Complemento , Humanos , Ratones , Miastenia Gravis/terapia , Unión Neuromuscular/patología , RatasRESUMEN
OBJECTIVE: The CACNA1A gene encodes the pore-forming subunit of neuronal Ca(V)2.1 Ca2+ channels. In patients, the S218L CACNA1A mutation causes a dramatic hemiplegic migraine syndrome that is associated with ataxia, seizures, and severe, sometimes fatal, brain edema often triggered by only a mild head trauma. METHODS: We introduced the S218L mutation into the mouse Cacna1a gene and studied the mechanisms for the S218L syndrome by analyzing the phenotypic, molecular, and electrophysiological consequences. RESULTS: Cacna1a(S218L) mice faithfully mimic the associated clinical features of the human S218L syndrome. S218L neurons exhibit a gene dosage-dependent negative shift in voltage dependence of Ca(V)2.1 channel activation, resulting in enhanced neurotransmitter release at the neuromuscular junction. Cacna1a(S218L) mice also display an exquisite sensitivity to cortical spreading depression (CSD), with a vastly reduced triggering threshold, an increased propagation velocity, and frequently multiple CSD events after a single stimulus. In contrast, mice bearing the R192Q CACNA1A mutation, which in humans causes a milder form of hemiplegic migraine, typically exhibit only a single CSD event after one triggering stimulus. INTERPRETATION: The particularly low CSD threshold and the strong tendency to respond with multiple CSD events make the S218L cortex highly vulnerable to weak stimuli and may provide a mechanistic basis for the dramatic phenotype seen in S218L mice and patients. Thus, the S218L mouse model may prove a valuable tool to further elucidate mechanisms underlying migraine, seizures, ataxia, and trauma-triggered cerebral edema.
Asunto(s)
Canales de Calcio Tipo P/genética , Canales de Calcio Tipo P/metabolismo , Canales de Calcio Tipo Q/genética , Canales de Calcio Tipo Q/metabolismo , Depresión de Propagación Cortical/genética , Depresión de Propagación Cortical/fisiología , Trastornos Migrañosos/genética , Trastornos Migrañosos/fisiopatología , Animales , Encéfalo/fisiopatología , Canales de Calcio Tipo N , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Masculino , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , Unión Neuromuscular/fisiopatología , Neuronas/fisiología , Neurotransmisores/metabolismo , SíndromeRESUMEN
Duchenne muscular dystrophy is an X-linked, recessive muscular dystrophy in which the absence of the dystrophin protein leads to fibrosis, inflammation and oxidative stress, resulting in loss of muscle tissue. Drug repurposing, i.e. using drugs already approved for other disorders, is attractive as it decreases development time. Recent studies suggested that simvastatin, a cholesterol lowering drug used for cardiovascular diseases, has beneficial effects on several parameters in mdx mice. To validate properly the effectiveness of simvastatin, two independent labs tested the effects of 12-week simvastatin treatment in either young (starting at 4 weeks of age) or adult (starting at 12 weeks of age) mdx mice. In neither study were benefits of simvastatin treatment observed on muscle function, histology or expression of genes involved in fibrosis, regeneration, oxidative stress and autophagy. Unexpectedly, although the treatment protocol was similar, simvastatin plasma levels were found to be much lower than observed in a previous study. In conclusion, in two laboratories, simvastatin did not ameliorate disease pathology in mdx mice, which could either be due to the ineffectiveness of simvastatin itself or due to the low simvastatin plasma levels following oral administration via the food.
Asunto(s)
Distrofia Muscular Animal/fisiopatología , Distrofia Muscular de Duchenne/fisiopatología , Simvastatina/farmacología , Animales , Modelos Animales de Enfermedad , Fibrosis/fisiopatología , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/efectos de los fármacosRESUMEN
Familial hemiplegic migraine type 1 (FHM1) is caused by mutations in the CACNA1A gene, encoding neuronal presynaptic Ca(V)2.1 (P/Q-type) Ca(2+) channels. These channels mediate neurotransmitter release at many central synapses and at the neuromuscular junction (NMJ). Mutation S218L causes a severe neurological phenotype of FHM and, additionally, ataxia and susceptibility to seizures, delayed brain edema, and fatal coma after minor head trauma. Recently, we generated a Cacna1a S218L knock-in mutant mouse, displaying these features and reduced survival. A first electrophysiological study showed high susceptibility for cortical spreading depression, enhanced neuronal soma Ca(2+) influx, and at diaphragm NMJs, a considerable increase of neurotransmitter release. We here assessed the function of S218L knock-in NMJs at several muscle types in great detail. Pharmacological analyses using specific Ca(V) subtype-blocking toxins excluded compensatory contribution of non-Ca(V)2.1 channels. Endplate potentials were considerably broadened at many NMJs. High rate (40 Hz)-evoked acetylcholine release was slightly reduced; however, it was not associated with block of neurotransmission causing weakness, as assessed with grip strength measurements and in vitro muscle contraction experiments. The synaptopathy clearly progressed with age, including development of an increased acetylcholine release at low-rate nerve stimulation at physiological extracellular Ca(2+) concentration and further endplate potential broadening. Our results suggest enhanced Ca(2+) influx into motor nerve terminals through S218L-mutated presynaptic Ca(V)2.1 channels, likely because of the earlier reported negative shift of activation potential and reduced inactivation. Similar severe aberrations at central synapses of S218L mutant mice and humans may underlie or contribute to the drastic neurological phenotype.
Asunto(s)
Acetilcolina/metabolismo , Canales de Calcio Tipo P/genética , Canales de Calcio Tipo Q/genética , Progresión de la Enfermedad , Técnicas de Sustitución del Gen , Migraña con Aura/genética , Índice de Severidad de la Enfermedad , Animales , Canales de Calcio Tipo N , Canales de Calcio Tipo P/biosíntesis , Canales de Calcio Tipo Q/biosíntesis , Femenino , Masculino , Ratones , Ratones Transgénicos , Migraña con Aura/metabolismo , Potenciales Postsinápticos Miniatura/fisiología , Unión Neuromuscular/metabolismo , Neurotransmisores/metabolismo , Transmisión Sináptica/genéticaRESUMEN
Neuronal membrane gangliosides, forming a large family of sialylated glycosphingolipids, have been hypothesized to play important roles in synaptic transmission. We studied the ex vivo electrophysiological function of neuromuscular junctions of GM2/GD2-synthase*GD3-synthase compound null-mutant mice after acute removal of GM3, the only remaining ganglioside in this mouse, by in vitro treatment with neuraminidase. We found 16% enhancement of the acetylcholine release per nerve impulse at low-rate (0.3 Hz) nerve stimulation. Conversely, the treatment reduced the acetylcholine release evoked by high-rate (40 Hz) nerve stimulation. Also, 25 ms paired-pulse facilitation of endplate potentials was reduced by the neuraminidase-treatment. These effects may indicate a modest modulatory influence of the negative electrical charges carried by the sialic acid molecules of gangliosides on the function of presynaptic Ca(v)2.1 channels, affecting the magnitude and kinetics of the Ca(2+) influx that induces neurotransmitter release from the motor nerve terminal. Our results show that gangliosides are to some extent involved in neurotransmission at the neuromuscular junction, but that their presence is not an absolute requirement in this process.
Asunto(s)
Acetilcolina/metabolismo , Gangliósidos/deficiencia , Unión Neuromuscular/metabolismo , Animales , Calcio/metabolismo , Estimulación Eléctrica/métodos , Gangliósidos/genética , Técnicas In Vitro , Ratones , Ratones Noqueados , Neuraminidasa/farmacología , Unión Neuromuscular/efectos de los fármacos , Potenciales Sinápticos/efectos de los fármacos , Potenciales Sinápticos/genéticaRESUMEN
Autoantibodies to muscle-specific kinase (MuSK) can cause myasthenia gravis (MG). The pathophysiological mechanism remains unknown. We report in vitro electrophysiological and histological studies of the neuromuscular junction in a MuSK MG patient. Low levels of presynaptic acetylcholine release and small miniature endplate potentials were found. This combination of pre- and postsynaptic abnormalities was supported by histology, revealing partially denervated postsynaptic areas, and some degeneration of postsynaptic folds. Results suggest that anti-MuSK antibodies reduce the stability of muscle-nerve contact.
Asunto(s)
Miastenia Gravis/fisiopatología , Unión Neuromuscular/fisiopatología , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores Colinérgicos/inmunología , Adulto , Autoanticuerpos/inmunología , Electrofisiología , Femenino , Humanos , Placa Motora/inmunología , Placa Motora/fisiopatología , Debilidad Muscular/inmunología , Debilidad Muscular/fisiopatología , Músculo Esquelético/inmunología , Músculo Esquelético/fisiopatología , Miastenia Gravis/inmunología , Unión Neuromuscular/inmunologíaRESUMEN
The outer leaflet of neuronal membranes is highly enriched in gangliosides. Therefore, specific neuronal roles have been attributed to this family of sialylated glycosphingolipids, e.g. in modulation of ion channels and transporters, neuronal interaction and recognition, temperature adaptation, Ca(2+) homeostasis, axonal growth, (para)node of Ranvier stability and synaptic transmission. Recent developmental, ageing and injury studies on transgenic mice lacking subsets of gangliosides indicate that gangliosides are involved in maintenance rather than development of the nervous system and that ganglioside family members are able to act in a mutually compensatory manner. Besides having physiological functions, gangliosides are the likely antigenic targets of autoantibodies present in Guillain-Barré syndrome (GBS), a group of neuropathies with clinical symptoms of motor- and/or sensory peripheral nerve dysfunction. Antibody binding to peripheral nerves is thought to either interfere with ganglioside function or activate complement, causing axonal damage and thereby disturbed action potential conduction. The presynaptic motor nerve terminal at the neuromuscular junction (NMJ) may be a prominent target because it is highly enriched in gangliosides and lies outside the blood-nerve barrier, allowing antibody access. The ensuing neuromuscular synaptopathy might contribute to the muscle weakness in GBS patients. Several groups, including our own, have studied the effects of anti-ganglioside antibodies in ex vivo and in vivo experimental settings at mouse NMJs. Here, after providing a background overview on ganglioside synthesis, localization and physiology, we will review those studies, which clearly show that anti-ganglioside antibodies are capable of binding to NMJs and thereby can exert a variety of pathophysiological effects. Furthermore, we will discuss the human clinical electrophysiological and histological evidence produced so far of the existence of a neuromuscular synaptopathy contributing to muscle weakness in GBS patients.
Asunto(s)
Autoanticuerpos/inmunología , Gangliósidos/inmunología , Inmunidad Innata/inmunología , Enfermedades de la Unión Neuromuscular/inmunología , Unión Neuromuscular/inmunología , Animales , Humanos , Modelos Inmunológicos , Modelos NeurológicosRESUMEN
Homozygous rolling Nagoya natural mutant mice display a severe ataxic gait and frequently roll over to their side or back. The causative mutation resides in the Cacna1a gene, encoding the pore-forming alpha(1) subunit of Ca(v)2.1 type voltage-gated Ca(2+) channels. These channels are crucially involved in neuronal Ca(2+) signaling and in neurotransmitter release at many central synapses and, in the periphery, at the neuromuscular junction. We here review the behavioral, histological, biochemical, and neurophysiological studies on this mouse mutant and discuss its usefulness as a model of human neurological diseases associated with Ca(v)2.1 dysfunction.
Asunto(s)
Canales de Calcio Tipo P/genética , Canales de Calcio Tipo Q/genética , Ataxia Cerebelosa , Cerebelo , Ratones Mutantes Neurológicos , Animales , Canales de Calcio Tipo N , Ataxia Cerebelosa/genética , Ataxia Cerebelosa/patología , Ataxia Cerebelosa/fisiopatología , Cerebelo/metabolismo , Cerebelo/patología , Cerebelo/fisiopatología , Modelos Animales de Enfermedad , Humanos , Ratones , Modelos Biológicos , Unión Neuromuscular/fisiopatología , Transmisión Sináptica/fisiologíaRESUMEN
Anti-GQ1b ganglioside antibodies are the serological hallmark of the Miller Fisher syndrome (MFS) variant of the paralytic neuropathy, Guillain-Barré syndrome, and are believed to be the principal pathogenic mediators of the disease. In support of this, we previously showed in an in vitro mouse model of MFS that anti-GQ1b antibodies were able to bind and disrupt presynaptic motor nerve terminals at the neuromuscular junction (NMJ) as one of their target sites, thereby causing muscle paralysis. This injury only occurred through activation of complement, culminating in the formation and deposition of membrane attack complex (MAC, C5b-9) in nerve membranes. Since this step is crucial to the neuropathic process and an important convergence point for antibody and complement mediated membrane injury in general, it forms an attractive pharmacotherapeutic target. Here, we assessed the efficacy of the humanized monoclonal antibody eculizumab, which blocks the formation of human C5a and C5b-9, in preventing the immune-mediated motor neuropathy exemplified in this model. Eculizumab completely prevented electrophysiological and structural lesions at anti-GQ1b antibody pre-incubated NMJs in vitro when using normal human serum (NHS) as a complement source. In a novel in vivo mouse model of MFS generated through intraperitoneal injection of anti-GQ1b antibody and NHS, mice developed respiratory paralysis due to transmission block at diaphragm NMJs, resulting from anti-GQ1b antibody binding and complement activation. Intravenous injection of eculizumab effectively prevented respiratory paralysis and associated functional and morphological hallmarks of terminal motor neuropathy. We show that eculizumab protects against complement-mediated damage in murine MFS, providing the rationale for undertaking clinical trials in this disease and other antibody-mediated neuropathies in which complement activation is believed to be involved.