RESUMEN
Intellectual Disability is a common and heterogeneous disorder characterized by limitations in intellectual functioning and adaptive behaviour, whose molecular mechanisms remain largely unknown. Among the numerous genes found to be involved in the pathogenesis of intellectual disability, 10% are located on the X-chromosome. We identified a missense mutation (c.236 C > G; p.S79W) in the SYN1 gene coding for synapsin I in the MRX50 family, affected by non-syndromic X-linked intellectual disability. Synapsin I is a neuronal phosphoprotein involved in the regulation of neurotransmitter release and neuronal development. Several mutations in SYN1 have been identified in patients affected by epilepsy and/or autism. The S79W mutation segregates with the disease in the MRX50 family and all affected members display intellectual disability as sole clinical manifestation. At the protein level, the S79W Synapsin I mutation is located in the region of the B-domain involved in recognition of highly curved membranes. Expression of human S79W Synapsin I in Syn1 knockout hippocampal neurons causes aberrant accumulation of small clear vesicles in the soma, increased clustering of synaptic vesicles at presynaptic terminals and increased frequency of excitatory spontaneous release events. In addition, the presence of S79W Synapsin I strongly reduces the mobility of synaptic vesicles, with possible implications for the regulation of neurotransmitter release and synaptic plasticity. These results implicate SYN1 in the pathogenesis of non-syndromic intellectual disability, showing that alterations of synaptic vesicle trafficking are one possible cause of this disease, and suggest that distinct mutations in SYN1 may lead to distinct brain pathologies.
Asunto(s)
Discapacidad Intelectual Ligada al Cromosoma X/genética , Mutación Missense , Sinapsinas/genética , Vesículas Sinápticas/genética , Animales , Secuencia de Bases , Humanos , Discapacidad Intelectual Ligada al Cromosoma X/metabolismo , Ratones , Ratones Noqueados , Mutación , Neurogénesis/genética , Plasticidad Neuronal/genética , Neuronas/metabolismo , Linaje , Terminales Presinápticos/metabolismo , Cultivo Primario de Células , Transporte de Proteínas , Sinapsinas/metabolismo , Transmisión Sináptica/genética , Vesículas Sinápticas/metabolismoRESUMEN
The classical view of multiple sclerosis (MS) pathogenesis states that inflammation-mediated demyelination is responsible for neuronal damage and loss. However, recent findings show that impairment of neuronal functions and demyelination can be independent events, suggesting the coexistence of other pathogenic mechanisms. Due to the inflammatory milieu, subtle alterations in synaptic function occur, which are probably at the basis of the early cognitive decline that often precedes the neurodegenerative phases in MS patients. In particular, it has been reported that inflammation enhances excitatory synaptic transmission while it decreases GABAergic transmission in vitro and ex vivo. This evidence points to the idea that an excitation/inhibition imbalance occurs in the inflamed MS brain, even though the exact molecular mechanisms leading to this synaptic dysfunction are as yet not completely clear. Along this line, we observed that acute treatment of primary hippocampal neurons in culture with pro-inflammatory cytokines leads to an increased phosphorylation of synapsin I (SynI) by ERK1/2 kinase and to an increase in the frequency of spontaneous synaptic vesicle release events, which is prevented by SynI deletion. In vivo, the ablation of SynI expression is protective in terms of disease progression and neuronal damage in the experimental autoimmune encephalomyelitis mouse model of MS. Our results point to a possible key role in MS pathogenesis of the neuronal protein SynI, a regulator of excitation/inhibition balance in neuronal networks.
Asunto(s)
Encefalomielitis Autoinmune Experimental/metabolismo , Sinapsinas/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Hipocampo/metabolismo , Inflamación/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esclerosis Múltiple/patología , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Fosforilación , Sinapsis/metabolismo , Sinapsinas/genética , Vesículas Sinápticas/metabolismoRESUMEN
Alterations in the balance of inhibitory and excitatory synaptic transmission have been implicated in the pathogenesis of neurological disorders such as epilepsy. Eukaryotic elongation factor 2 kinase (eEF2K) is a highly regulated, ubiquitous kinase involved in the control of protein translation. Here, we show that eEF2K activity negatively regulates GABAergic synaptic transmission. Indeed, loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and α5-containing GABAA receptors and thus interferes with the excitation/inhibition balance. This cellular phenotype is accompanied by an increased resistance to epilepsy and an impairment of only a specific hippocampal-dependent fear conditioning. From a clinical perspective, our results identify eEF2K as a potential novel target for antiepileptic drugs, since pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy.
Asunto(s)
Quinasa del Factor 2 de Elongación/metabolismo , Epilepsia/enzimología , Neuronas/enzimología , Transmisión Sináptica/fisiología , Animales , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/enzimología , Corteza Cerebral/patología , Condicionamiento Psicológico/fisiología , Modelos Animales de Enfermedad , Quinasa del Factor 2 de Elongación/antagonistas & inhibidores , Quinasa del Factor 2 de Elongación/genética , Epilepsia/patología , Miedo/fisiología , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Hipocampo/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismo , Sinapsinas/genética , Sinapsinas/metabolismo , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The precise subcellular organization of synaptic vesicles (SVs) at presynaptic sites allows for rapid and spatially restricted exocytotic release of neurotransmitter. The synapsins (Syns) are a family of presynaptic proteins that control the availability of SVs for exocytosis by reversibly tethering them to each other and to the actin cytoskeleton in a phosphorylation-dependent manner. Syn ablation leads to reduction in the density of SV proteins in nerve terminals and increased synaptic fatigue under high-frequency stimulation, accompanied by the development of an epileptic phenotype. We analyzed cultured neurons from wild-type and Syn I,II,III(-/-) triple knock-out (TKO) mice and found that SVs were severely dispersed in the absence of Syns. Vesicle dispersion did not affect the readily releasable pool of SVs, whereas the total number of SVs was considerably reduced at synapses of TKO mice. Interestingly, dispersion apparently involved exocytosis-competent SVs as well; it was not affected by stimulation but was reversed by chronic neuronal activity blockade. Altogether, these findings indicate that Syns are essential to maintain the dynamic structural organization of synapses and the size of the reserve pool of SVs during intense SV recycling, whereas an additional Syn-independent mechanism, whose molecular substrate remains to be clarified, targets SVs to synaptic boutons at rest and might be outpaced by activity.
Asunto(s)
Sinapsinas/deficiencia , Transmisión Sináptica/fisiología , Vesículas Sinápticas/fisiología , Vesículas Sinápticas/ultraestructura , Animales , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Sinapsinas/genética , Sinapsinas/fisiología , Transmisión Sináptica/genéticaRESUMEN
Synaptic transmission is critically dependent on synaptic vesicle (SV) recycling. Although the precise mechanisms of SV retrieval are still debated, it is widely accepted that a fundamental role is played by clathrin-mediated endocytosis, a form of endocytosis that capitalizes on the clathrin/adaptor protein complex 2 (AP2) coat and several accessory factors. Here, we show that the previously uncharacterized protein KIAA1107, predicted by bioinformatics analysis to be involved in the SV cycle, is an AP2-interacting clathrin-endocytosis protein (APache). We found that APache is highly enriched in the CNS and is associated with clathrin-coated vesicles via interaction with AP2. APache-silenced neurons exhibit a severe impairment of maturation at early developmental stages, reduced SV density, enlarged endosome-like structures, and defects in synaptic transmission, consistent with an impaired clathrin/AP2-mediated SV recycling. Our data implicate APache as an actor in the complex regulation of SV trafficking, neuronal development, and synaptic plasticity.
Asunto(s)
Complejo 2 de Proteína Adaptadora , Endocitosis , Neurogénesis , Vesículas Sinápticas/metabolismo , Complejo 2 de Proteína Adaptadora/metabolismo , Animales , Células Cultivadas , Vesículas Cubiertas por Clatrina/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/metabolismo , Neuronas/fisiología , Unión Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
Heterozygous mutations in proline-rich transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders, including epilepsy, kinesigenic dyskinesia, and migraine. Most of the mutations lead to impaired PRRT2 expression, suggesting that loss of PRRT2 function may contribute to pathogenesis. We show that PRRT2 is enriched in presynaptic terminals and that its silencing decreases the number of synapses and increases the number of docked synaptic vesicles at rest. PRRT2-silenced neurons exhibit a severe impairment of synchronous release, attributable to a sharp decrease in release probability and Ca(2+) sensitivity and associated with a marked increase of the asynchronous/synchronous release ratio. PRRT2 interacts with the synaptic proteins SNAP-25 and synaptotagmin 1/2. The results indicate that PRRT2 is intimately connected with the Ca(2+)-sensing machinery and that it plays an important role in the final steps of neurotransmitter release.
Asunto(s)
Señalización del Calcio , Exocitosis , Proteínas de la Membrana/metabolismo , Neurotransmisores/metabolismo , Terminales Presinápticos/metabolismo , Animales , Células Cultivadas , Hipocampo/citología , Hipocampo/metabolismo , Ratones , Ratones Endogámicos C57BL , Terminales Presinápticos/fisiología , Ratas , Ratas Sprague-Dawley , Potenciales Sinápticos , Vesículas Sinápticas/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinaptotagminas/metabolismoRESUMEN
Synapsins are a family of neuronal phosphoproteins associated with the cytosolic surface of synaptic vesicles. Experimental evidence suggests a role for synapsins in synaptic vesicle clustering and recycling at the presynaptic terminal, as well as in neuronal development and synaptogenesis. Synapsin knock-out (Syn1(-/-) ) mice display an epileptic phenotype and mutations in the SYN1 gene have been identified in individuals affected by epilepsy and/or autism spectrum disorder. We investigated the impact of the c.1067G>A nonsense transition, the first mutation described in a family affected by X-linked syndromic epilepsy, on the expression and functional properties of the synapsin I protein. We found that the presence of a premature termination codon in the human SYN1 transcript renders it susceptible to nonsense-mediated mRNA decay (NMD). Given that the NMD efficiency is highly variable among individuals and cell types, we investigated also the effects of expression of the mutant protein and found that it is expressed at lower levels compared to wild-type synapsin I, forms perinuclear aggregates and is unable to reach presynaptic terminals in mature hippocampal neurons grown in culture. Taken together, these data indicate that in patients carrying the W356× mutation the function of synapsin I is markedly impaired, due to both the strongly decreased translation and the altered function of the NMD-escaped protein, and support the value of Syn1(-/-) mice as an experimental model mimicking the human pathology.