RESUMO
Autoantibodies against leucine-rich glioma-inactivated 1 (LGI1) occur in patients with encephalitis who present with frequent focal seizures and a pattern of amnesia consistent with focal hippocampal damage. To investigate whether the cellular and subcellular distribution of LGI1 may explain the localization of these features, and hence gain broader insights into LGI1's neurobiology, we analysed the detailed localization of LGI1 and the diversity of its protein interactome, in mouse brains using patient-derived recombinant monoclonal LGI1 antibodies. Combined immunofluorescence and mass spectrometry analyses showed that LGI1 is enriched in excitatory and inhibitory synaptic contact sites, most densely within CA3 regions of the hippocampus. LGI1 is secreted in both neuronal somatodendritic and axonal compartments, and occurs in oligodendrocytic, neuro-oligodendrocytic and astro-microglial protein complexes. Proteomic data support the presence of LGI1-Kv1-MAGUK complexes, but did not reveal LGI1 complexes with postsynaptic glutamate receptors. Our results extend our understanding of regional, cellular and subcellular LGI1 expression profiles and reveal novel LGI1-associated complexes, thus providing insights into the complex biology of LGI1 and its relationship to seizures and memory loss.
Assuntos
Glioma , Peptídeos e Proteínas de Sinalização Intracelular , Animais , Camundongos , Leucina , Proteômica , Autoanticorpos , ConvulsõesRESUMO
Botulinum neurotoxin serotype B (BoNT/B) uses two separate protein and polysialoglycolipid-binding pockets to interact with synaptotagmin 1/2 and gangliosides. However, an integrated model of BoNT/B bound to its neuronal receptors in a native membrane topology is still lacking. Using a panel of in silico and experimental approaches, we present here a new model for BoNT/B binding to neuronal membranes, in which the toxin binds to a preassembled synaptotagmin-ganglioside GT1b complex and a free ganglioside allowing a lipid-binding loop of BoNT/B to interact with the glycone part of the synaptotagmin-associated GT1b. Furthermore, our data provide molecular support for the decrease in BoNT/B sensitivity in Felidae that harbor the natural variant synaptotagmin2-N59Q. These results reveal multiple interactions of BoNT/B with gangliosides and support a novel paradigm in which a toxin recognizes a protein/ganglioside complex.
Assuntos
Gangliosídeos , Sinaptotagmina II , Sítios de Ligação , Gangliosídeos/química , Gangliosídeos/metabolismo , Neurônios/metabolismo , Ligação Proteica , Sinaptotagmina II/química , Sinaptotagmina II/genética , Sinaptotagmina II/metabolismo , Sinaptotagminas/genética , Sinaptotagminas/metabolismoRESUMO
Botulinum neurotoxin type B (BoNT/B) recognizes nerve terminals by binding to 2 receptor components: a polysialoganglioside, predominantly GT1b, and synaptotagmin 1/2. It is widely thought that BoNT/B initially binds to GT1b then diffuses in the plane of the membrane to interact with synaptotagmin. We have addressed the hypothesis that a GT1b-synaptotagmin cis complex forms the BoNT/B receptor. We identified a consensus glycosphingolipid-binding motif in the extracellular juxtamembrane domain of synaptotagmins 1/2 and confirmed by Langmuir monolayer, surface plasmon resonance, and circular dichroism that GT1b interacts with synaptotagmin peptides containing this sequence, inducing α-helical structure. Molecular modeling and tryptophan fluorescence spectroscopy were consistent with the intertwining of GT1b and synaptotagmin, involving cis interactions between the oligosaccharide and ceramide moieties of GT1b and the juxtamembrane and transmembrane domains of synaptotagmin, respectively. Furthermore, a point mutation on synaptotagmin, located outside of the BoNT/B-binding segment, inhibited GT1b binding and blocked GT1b-induced potentiation of BoNT/B binding to synaptotagmin-expressing cells. Our findings are consistent with a model in which a preassembled GT1b-synaptotagmin complex constitutes the high-affinity BoNT/B receptor.
Assuntos
Toxinas Botulínicas Tipo A , Gangliosídeos , Sinaptotagmina I , Animais , Sítios de Ligação , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/metabolismo , Gangliosídeos/química , Gangliosídeos/farmacologia , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Ratos , Sinaptotagmina I/química , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Sinaptotagmina II/química , Sinaptotagmina II/genética , Sinaptotagmina II/metabolismoRESUMO
Autosomal dominant epilepsy with auditory features results from mutations in leucine-rich glioma-inactivated 1 (LGI1), a soluble glycoprotein secreted by neurons. Animal models of LGI1 depletion display spontaneous seizures, however, the function of LGI1 and the mechanisms by which deficiency leads to epilepsy are unknown. We investigated the effects of pure recombinant LGI1 and genetic depletion on intrinsic excitability, in the absence of synaptic input, in hippocampal CA3 neurons, a classical focus for epileptogenesis. Our data indicate that LGI1 is expressed at the axonal initial segment and regulates action potential firing by setting the density of the axonal Kv1.1 channels that underlie dendrotoxin-sensitive D-type potassium current. LGI1 deficiency incurs a >50% down-regulation of the expression of Kv1.1 and Kv1.2 via a posttranscriptional mechanism, resulting in a reduction in the capacity of axonal D-type current to limit glutamate release, thus contributing to epileptogenesis.
Assuntos
Axônios/metabolismo , Proteínas/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Potenciais de Ação , Animais , Venenos Elapídicos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Canal de Potássio Kv1.2/metabolismo , Camundongos Mutantes , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Proteínas/genética , Proteínas/farmacologia , Ratos Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologiaRESUMO
V-ATPase is an important factor in synaptic vesicle acidification and is implicated in synaptic transmission. Rotation in the extra-membranous V1 sector drives proton transfer through the membrane-embedded multi-subunit V0 sector of the V-ATPase. Intra-vesicular protons are then used to drive neurotransmitter uptake by synaptic vesicles. V0a and V0c, two membrane subunits of the V0 sector, have been shown to interact with SNARE proteins, and their photo-inactivation rapidly impairs synaptic transmission. V0d, a soluble subunit of the V0 sector strongly interacts with its membrane-embedded subunits and is crucial for the canonic proton transfer activity of the V-ATPase. Our investigations show that the loop 1.2 of V0c interacts with complexin, a major partner of the SNARE machinery and that V0d1 binding to V0c inhibits this interaction, as well as V0c association with SNARE complex. The injection of recombinant V0d1 in rat superior cervical ganglion neurons rapidly reduced neurotransmission. In chromaffin cells, V0d1 overexpression and V0c silencing modified in a comparable manner several parameters of unitary exocytotic events. Our data suggest that V0c subunit promotes exocytosis via interactions with complexin and SNAREs and that this activity can be antagonized by exogenous V0d.
Assuntos
Proteínas SNARE , ATPases Vacuolares Próton-Translocadoras , Ratos , Animais , Proteínas SNARE/metabolismo , Prótons , Vesículas Sinápticas/metabolismo , Fusão de Membrana , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channel expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca(++) depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca(++) could bind the Nav1.1 C-terminal region with micromolar affinity.
Assuntos
Encéfalo/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Sódio/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Encéfalo/citologia , Células HEK293 , Humanos , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.1 , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Ratos , Canais de Sódio/genética , Espectrometria de Fluorescência , Técnicas do Sistema de Duplo-HíbridoRESUMO
Synaptic vesicle proton V-ATPase is an essential component in synaptic vesicle function. Active acidification of synaptic vesicles, triggered by the V-ATPase, is necessary for neurotransmitter storage. Independently from its proton transport activity, an additional important function of the membrane-embedded sector of the V-ATPase has been uncovered over recent years. Subunits a and c of the membrane sector of this multi-molecular complex have been shown to interact with SNARE proteins and to be involved in modulating neurotransmitter release. The c-subunit interacts with the v-SNARE VAMP2 and facilitates neurotransmission. In this study, we used chromophore-assisted light inactivation and monitored the consequences on neurotransmission on line in CA3 pyramidal neurons. We show that V-ATPase c-subunit V0c is a key element in modulating neurotransmission and that its specific inactivation rapidly inhibited neurotransmission.
Assuntos
Ácidos/metabolismo , Inativação Luminosa Assistida por Cromóforo , Neurotransmissores/metabolismo , Subunidades Proteicas/metabolismo , Vesículas Sinápticas/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Regulação para Baixo , Fluorescência , Neurônios/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos Wistar , Transmissão Sináptica , ATPases Vacuolares Próton-Translocadoras/química , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.