RESUMEN
Cav1.3 voltage-gated L-type calcium channels (LTCCs) are involved in cardiac pacemaking, hearing and hormone secretion, but are also expressed postsynaptically in neurons. So far, homozygous loss of function mutations in CACNA1D encoding the Cav1.3 α1-subunit are described in congenital sinus node dysfunction and deafness. In addition, germline mutations in CACNA1D have been linked to neurodevelopmental syndromes including epileptic seizures, autism, intellectual disability and primary hyperaldosteronism. Here, a three-generation family with a syndromal phenotype of sinus node dysfunction, idiopathic epilepsy and attention deficit hyperactivity disorder (ADHD) is investigated. Whole genome sequencing and functional heterologous expression studies were used to identify the disease-causing mechanisms in this novel syndromal disorder. We identified a heterozygous non-synonymous variant (p.Arg930His) in the CACNA1D gene that cosegregated with the combined clinical phenotype in an autosomal dominant manner. Functional heterologous expression studies showed that the CACNA1D variant induces isoform-specific alterations of Cav1.3 channel gating: a gain of ion channel function was observed in the brain-specific short CACNA1D isoform (Cav1.3S), whereas a loss of ion channel function was seen in the long (Cav1.3L) isoform. The combined gain-of-function (GOF) and loss-of-function (LOF) induced by the R930H variant are likely to be associated with the rare combined clinical and syndromal phenotypes in the family. The GOF in the Cav1.3S variant with high neuronal expression is likely to result in epilepsy, whereas the LOF in the long Cav1.3L variant results in sinus node dysfunction.
Asunto(s)
Canales de Calcio Tipo L , Epilepsia , Síndrome del Seno Enfermo , Humanos , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Isoformas de Proteínas/metabolismo , Síndrome del Seno Enfermo/genética , Síndrome del Seno Enfermo/metabolismo , Secuenciación del ExomaRESUMEN
Parkinson's disease (PD) is clinically defined by the presence of the cardinal motor symptoms, which are associated with a loss of dopaminergic nigrostriatal neurons in the substantia nigra pars compacta (SNpc). While SNpc neurons serve as the prototypical cell-type to study cellular vulnerability in PD, there is an unmet need to extent our efforts to other neurons at risk. The noradrenergic locus coeruleus (LC) represents one of the first brain structures affected in Parkinson's disease (PD) and plays not only a crucial role for the evolving non-motor symptomatology, but it is also believed to contribute to disease progression by efferent noradrenergic deficiency. Therefore, we sought to characterize the electrophysiological properties of LC neurons in two distinct PD models: (1) in an in vivo mouse model of focal α-synuclein overexpression; and (2) in an in vitro rotenone-induced PD model. Despite the fundamental differences of these two PD models, α-synuclein overexpression as well as rotenone exposure led to an accelerated autonomous pacemaker frequency of LC neurons, accompanied by severe alterations of the afterhyperpolarization amplitude. On the mechanistic side, we suggest that Ca2+-activated K+ (SK) channels are mediators of the increased LC neuronal excitability, as pharmacological activation of these channels is sufficient to prevent increased LC pacemaking and subsequent neuronal loss in the LC following in vitro rotenone exposure. These findings suggest a role of SK channels in PD by linking α-synuclein- and rotenone-induced changes in LC firing rate to SK channel dysfunction.
Asunto(s)
Norepinefrina/fisiología , Enfermedad de Parkinson/fisiopatología , Porción Compacta de la Sustancia Negra/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , alfa-Sinucleína/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Locus Coeruleus/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/fisiología , Síntomas Prodrómicos , RotenonaRESUMEN
Evolving concepts on Parkinson's disease (PD) pathology suggest that α-synuclein (aSYN) promote dopaminergic neuron dysfunction and death through accumulating in the mitochondria. However, the consequence of mitochondrial aSYN localisation on mitochondrial structure and bioenergetic functions in neuronal cells are poorly understood. Therefore, we investigated deleterious effects of mitochondria-targeted aSYN in differentiated human dopaminergic neurons in comparison with wild-type (WT) aSYN overexpression and corresponding EGFP (enhanced green fluorescent protein)-expressing controls. Mitochondria-targeted aSYN enhanced mitochondrial reactive oxygen species (ROS) formation, reduced ATP levels and showed severely disrupted structure and function of the dendritic neural network, preceding neuronal death. Transmission electron microscopy illustrated distorted cristae and many fragmented mitochondria in response to WT-aSYN overexpression, and a complete loss of cristae structure and massively swollen mitochondria in neurons expressing mitochondria-targeted aSYN. Further, the analysis of mitochondrial bioenergetics in differentiated dopaminergic neurons, expressing WT or mitochondria-targeted aSYN, elicited a pronounced impairment of mitochondrial respiration. In a pharmacological compound screening, we found that the pan-caspase inhibitors QVD and zVAD-FMK, and a specific caspase-1 inhibitor significantly prevented aSYN-induced cell death. In addition, the caspase inhibitor QVD preserved mitochondrial function and neuronal network activity in the human dopaminergic neurons overexpressing aSYN. Overall, our findings indicated therapeutic effects by caspase-1 inhibition despite aSYN-mediated alterations in mitochondrial morphology and function.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Enfermedad de Parkinson/genética , Serpinas/farmacología , Proteínas Virales/farmacología , alfa-Sinucleína/genética , Adenosina Trifosfato/genética , Caspasa 1/genética , Muerte Celular/genética , Neuronas Dopaminérgicas/patología , Regulación de la Expresión Génica , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Consumo de Oxígeno/genética , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels encode neuronal and cardiac pacemaker currents. The composition of pacemaker channel complexes in different tissues is poorly understood, and the presence of additional HCN modulating subunits was speculated. Here we show that vesicle-associated membrane protein-associated protein B (VAPB), previously associated with a familial form of amyotrophic lateral sclerosis 8, is an essential HCN1 and HCN2 modulator. VAPB significantly increases HCN2 currents and surface expression and has a major influence on the dendritic neuronal distribution of HCN2. Severe cardiac bradycardias in VAPB-deficient zebrafish and VAPB-/- mice highlight that VAPB physiologically serves to increase cardiac pacemaker currents. An altered T-wave morphology observed in the ECGs of VAPB-/- mice supports the recently proposed role of HCN channels for ventricular repolarization. The critical function of VAPB in native pacemaker channel complexes will be relevant for our understanding of cardiac arrhythmias and epilepsies, and provides an unexpected link between these diseases and amyotrophic lateral sclerosis.-Silbernagel, N., Walecki, M., Schäfer, M.-K. H., Kessler, M., Zobeiri, M., Rinné, S., Kiper, A. K., Komadowski, M. A., Vowinkel, K. S., Wemhöner, K., Fortmüller, L., Schewe, M., Dolga, A. M., Scekic-Zahirovic, J., Matschke, L. A., Culmsee, C., Baukrowitz, T., Monassier, L., Ullrich, N. D., Dupuis, L., Just, S., Budde, T., Fabritz, L., Decher, N. The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function.
Asunto(s)
Corazón/fisiología , Activación del Canal Iónico , Proteínas de la Membrana/fisiología , Neuronas/fisiología , Marcapaso Artificial , Animales , Proteínas Portadoras/fisiología , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Femenino , Células HeLa , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Ratones , Ratones Noqueados , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Proteínas de Transporte Vesicular , Xenopus laevis , Pez CebraRESUMEN
The physiological, intrinsic activity of noradrenergic locus coeruleus (LC) neurons is important for the control of sleep/wakefulness, cognition and autonomous body functions. Dysregulations of the LC-noradrenergic network contribute to the pathogenesis of psychiatric disorders and are key findings in early stages of neurodegenerative diseases. Therefore, identifying ion channels mediating the intrinsic pacemaking mechanism of LC neurons, which is in turn directly coupled to Ca2+ homeostasis and cell survival signaling pathways, can help to foster our understanding of the vulnerability of these neurons in neurodegenerative diseases. Small-conductance Ca2+-activated K+ (SK) channels regulate the intrinsic firing patterns in different central neurons and are essential regulators of the intracellular Ca2+ homeostasis. However, the role of SK channels for the intrinsic pacemaking of LC neurons in mice is still unclear. Therefore we performed qPCR expression analysis as well as patch clamp recordings of in vitro brainstem slices, for instance testing SK channel blockers and activators like apamin and NS309, respectively. Although we found a transcriptional expression of SK1, SK2 and SK3 channels, SK2 was the predominantly expressed subunit in mouse LC neurons. Using perforated-patch clamp experiments, we found that SK channels are essential regulators of the intrinsic pacemaking of LC neurons, mediating a large fraction of the afterhyperpolarization (AHP) in these cells. Consistent with a previous observation that a concerted action of L- and T-type Cav channels is essential for the pacemaking of LC neurons, we found that SK channel activation, and the respective AHP amplitude, is primarily coupled to Ca2+ influx via these types of Ca2+ channels. Our study identified SK2 channels as drug targets for the tuning of the pacemaker frequency in disorders involving a dysregulation of the LC.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Locus Coeruleus/efectos de los fármacos , Neuronas/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Ratones Endogámicos C57BL , Neuronas/metabolismo , Marcapaso Artificial , Canales de Potasio/metabolismoRESUMEN
RATIONALE: Familial sinus node and atrioventricular conduction dysfunction is a rare disorder that leads to paroxysmal dizziness, fatigue, and syncope because of a temporarily or permanently reduced heart rate. To date, only a few genes for familial sinus and atrioventricular conduction dysfunction are known, and the majority of cases remain pathogenically unresolved. OBJECTIVE: We aim to identify the disease gene in a large 3-generation family (n=25) with autosomal dominant sinus node dysfunction (SND) and atrioventricular block (AVB) and to characterize the mutation-related pathomechanisms in familial SND+AVB. METHODS AND RESULTS: Genome-wide linkage analysis mapped the SND+AVB disease locus to chromosome 7q21.1-q31.1 (2-point logarithm of the odds score: 4.64; θ=0); in this region, targeted exome sequencing identified a novel heterozygous mutation (p.Arg52Leu) in the GNB2 gene that strictly cosegregated with the SND+AVB phenotype. GNB2 encodes the ß2 subunit (Gß2) of the heterotrimeric G-protein complex that is being released from G-protein-coupled receptors on vagal stimulation. In 2 heterologous expression systems (HEK-293T cells and Xenopus laevis oocytes), an enhanced activation of the G-protein-activated K+ channel (GIRK; Kir3.1/Kir3.4) was shown when mutant Gß2 was coexpressed with Gγ2; this was in contrast to coexpression of mutant Gß2-Gγ2 with other cardiac ion channels (HCN4, HCN2, and Cav1.2). Molecular dynamics simulations suggested a reduced binding property of mutant Gß2 to cardiac GIRK channels when compared with native Gß2. CONCLUSIONS: A GNB2 gene mutation is associated with familial SND+AVB and leads to a sustained activation of cardiac GIRK channels, which is likely to hyperpolarize the myocellular membrane potential and thus reduces their spontaneous activity. Our findings describe for the first time a role of a mutant G-protein in the nonsyndromic pacemaker disease because of GIRK channel activation.
Asunto(s)
Bloqueo Atrioventricular/genética , Bloqueo Atrioventricular/fisiopatología , Proteínas de Unión al GTP/genética , Mutación/genética , Síndrome del Seno Enfermo/genética , Síndrome del Seno Enfermo/fisiopatología , Adulto , Secuencia de Aminoácidos , Bloqueo Atrioventricular/diagnóstico , Femenino , Perfilación de la Expresión Génica/métodos , Estudio de Asociación del Genoma Completo/métodos , Células HEK293 , Sistema de Conducción Cardíaco/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Síndrome del Seno Enfermo/diagnóstico , Nodo Sinoatrial/fisiología , Adulto JovenRESUMEN
Dysfunction of noradrenergic locus coeruleus (LC) neurons is involved in psychiatric and neurodegenerative diseases and is an early hallmark of Parkinson's disease (PD). The analysis of ion channels underlying the autonomous electrical activity of LC neurons, which is ultimately coupled to cell survival signaling pathways, can lead to a better understanding of the vulnerability of these neurons. In LC neurons somatodendritic Ca(2+) oscillations, mediated by L-type Ca(2+) channels, accompany spontaneous spiking and are linked to mitochondrial oxidant stress. However, the expression and functional implication of low-threshold activated T-type Ca(2+) channels in LC neurons were not yet studied. To this end we performed RT-PCR expression analysis in LC neurons. In addition, we utilized slice patch clamp recordings of in vitro brainstem slices in combination with L-type and T-type Ca(2+) channel blockers. We found the expression of a distinct set of L-type and T-type Ca(2+) channel subtypes mediating a pronounced low-threshold activated Ca(2+) current component. Analyzing spike trains, we revealed that neither L-type Ca(2+) channel nor T-type Ca(2+) channel blockade alone leads to a change in firing properties. In contrast, a combined application of antagonists significantly decreased the afterhyperpolarization amplitude, resulting in an increased firing frequency. Hence, we report the functional expression of T-type Ca(2+) channels in LC neurons and demonstrate their role in increasing the robustness of LC pacemaking by working in concert with Cav1 channels.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo T/metabolismo , Locus Coeruleus/citología , Neuronas/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Biofisica , Agonistas de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo T/genética , Estimulación Eléctrica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Sodio/farmacología , Estadísticas no Paramétricas , Tetrodotoxina/farmacologíaRESUMEN
Voltage-gated potassium (Kv) 1.1 channels undergo a specific enzymatic RNA deamination, generating a channel with a single amino acid exchange located in the inner pore cavity (Kv1.1(I400V)). We studied I400V-edited Kv1.1 channels in more detail and found that Kv1.1(I400V) gave rise to much smaller whole-cell currents than Kv1.1. To elucidate the mechanism behind this current reduction, we conducted electrophysiological recordings on single-channel level and did not find any differences. Next we examined channel surface expression in Xenopus oocytes and HeLa cells using a chemiluminescence assay and found the edited channels to be less readily expressed at the surface membrane. This reduction in surface expression was verified by fluorescence imaging experiments. Western blot analysis for comparison of protein abundances and glycosylation patterns did not show any difference between Kv1.1 and Kv1.1(I400V), further indicating that changed trafficking of Kv1.1(I400V) is causing the current reduction. Block of endocytosis by dynasore or AP180C did not abolish the differences in current amplitudes between Kv1.1 and Kv1.1(I400V), suggesting that backward trafficking is not affected. Therefore, our data suggest that I400V RNA editing of Kv1.1 leads to a reduced current size by a decreased forward trafficking of the channel to the surface membrane. This effect is specific for Kv1.1 because coexpression of Kv1.4 channel subunits with Kv1.1(I400V) abolishes these trafficking effects. Taken together, we identified RNA editing as a novel mechanism to regulate homomeric Kv1.1 channel trafficking. Fine-tuning of Kv1.1 surface expression by RNA editing might contribute to the complexity of neuronal Kv channel regulation.