RESUMO
The repertoire of modifications to bile acids and related steroidal lipids by host and microbial metabolism remains incompletely characterized. To address this knowledge gap, we created a reusable resource of tandem mass spectrometry (MS/MS) spectra by filtering 1.2 billion publicly available MS/MS spectra for bile-acid-selective ion patterns. Thousands of modifications are distributed throughout animal and human bodies as well as microbial cultures. We employed this MS/MS library to identify polyamine bile amidates, prevalent in carnivores. They are present in humans, and their levels alter with a diet change from a Mediterranean to a typical American diet. This work highlights the existence of many more bile acid modifications than previously recognized and the value of leveraging public large-scale untargeted metabolomics data to discover metabolites. The availability of a modification-centric bile acid MS/MS library will inform future studies investigating bile acid roles in health and disease.
Assuntos
Ácidos e Sais Biliares , Microbioma Gastrointestinal , Metabolômica , Espectrometria de Massas em Tandem , Animais , Humanos , Ácidos e Sais Biliares/química , Metabolômica/métodos , Poliaminas , Espectrometria de Massas em Tandem/métodos , Bases de Dados de Compostos QuímicosRESUMO
Autoantibodies targeting neuronal membrane proteins can cause encephalitis, seizures, and severe behavioral abnormalities. While antibodies for several neuronal targets have been identified, structural details on how they regulate function are unknown. Here we determined cryo-electron microscopy structures of antibodies derived from an encephalitis patient bound to the γ-aminobutyric acid type A (GABAA) receptor. These antibodies induced severe encephalitis by directly inhibiting GABAA function, resulting in nervous-system hyperexcitability. The structures reveal mechanisms of GABAA inhibition and pathology. One antibody directly competes with a neurotransmitter and locks the receptor in a resting-like state. The second antibody targets the subunit interface involved in binding benzodiazepines and antagonizes diazepam potentiation. We identify key residues in these antibodies involved in specificity and affinity and confirm structure-based hypotheses for functional effects using electrophysiology. Together these studies define mechanisms of direct functional antagonism of neurotransmission underlying autoimmune encephalitis in a human patient.
Assuntos
Encefalite , Receptores de GABA-A , Autoanticorpos , Microscopia Crioeletrônica , Doença de Hashimoto , Humanos , Receptores de GABA-A/metabolismo , Ácido gama-AminobutíricoRESUMO
Social impairment is frequently associated with mitochondrial dysfunction and altered neurotransmission. Although mitochondrial function is crucial for brain homeostasis, it remains unknown whether mitochondrial disruption contributes to social behavioral deficits. Here, we show that Drosophila mutants in the homolog of the human CYFIP1, a gene linked to autism and schizophrenia, exhibit mitochondrial hyperactivity and altered group behavior. We identify the regulation of GABA availability by mitochondrial activity as a biologically relevant mechanism and demonstrate its contribution to social behavior. Specifically, increased mitochondrial activity causes gamma aminobutyric acid (GABA) sequestration in the mitochondria, reducing GABAergic signaling and resulting in social deficits. Pharmacological and genetic manipulation of mitochondrial activity or GABA signaling corrects the observed abnormalities. We identify Aralar as the mitochondrial transporter that sequesters GABA upon increased mitochondrial activity. This study increases our understanding of how mitochondria modulate neuronal homeostasis and social behavior under physiopathological conditions.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Mitocôndrias/metabolismo , Ácido gama-Aminobutírico/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Ácido Aspártico/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Glucose/metabolismo , Homeostase , Humanos , Masculino , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Comportamento Social , Transmissão Sináptica , Ácido gama-Aminobutírico/genéticaRESUMO
Somatosensory over-reactivity is common among patients with autism spectrum disorders (ASDs) and is hypothesized to contribute to core ASD behaviors. However, effective treatments for sensory over-reactivity and ASDs are lacking. We found distinct somatosensory neuron pathophysiological mechanisms underlie tactile abnormalities in different ASD mouse models and contribute to some ASD-related behaviors. Developmental loss of ASD-associated genes Shank3 or Mecp2 in peripheral mechanosensory neurons leads to region-specific brain abnormalities, revealing links between developmental somatosensory over-reactivity and the genesis of aberrant behaviors. Moreover, acute treatment with a peripherally restricted GABAA receptor agonist that acts directly on mechanosensory neurons reduced tactile over-reactivity in six distinct ASD models. Chronic treatment of Mecp2 and Shank3 mutant mice improved body condition, some brain abnormalities, anxiety-like behaviors, and some social impairments but not memory impairments, motor deficits, or overgrooming. Our findings reveal a potential therapeutic strategy targeting peripheral mechanosensory neurons to treat tactile over-reactivity and select ASD-related behaviors.
Assuntos
Transtorno do Espectro Autista/metabolismo , Agonistas GABAérgicos/farmacologia , Ácidos Isonicotínicos/farmacologia , Fenótipo , Células Receptoras Sensoriais/efeitos dos fármacos , Tato/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Ansiedade/tratamento farmacológico , Transtorno do Espectro Autista/tratamento farmacológico , Transtorno do Espectro Autista/genética , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Agonistas GABAérgicos/uso terapêutico , Ácidos Isonicotínicos/uso terapêutico , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos , Proteínas do Tecido Nervoso/genética , Inibição Pré-Pulso/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismoRESUMO
The nervous system is critical for intestinal homeostasis and function, but questions remain regarding its impact on gut immune defense. By screening the major neurotransmitters of C. elegans, we found that γ-aminobutyric acid (GABA) deficiency enhanced susceptibility to pathogenic Pseudomonas aeruginosa PA14 infection. GABAergic signaling between enteric neurons and intestinal smooth muscle promoted gut defense in a PMK-1/p38-dependent, but IIS/DAF-16- and DBL-1/TGF-ß-independent, pathway. Transcriptomic profiling revealed that the neuropeptide, FLP-6, acted downstream of enteric GABAergic signaling. Further data determined that FLP-6 was expressed and secreted by intestinal smooth muscle cells and functioned as a paracrine molecule on the intestinal epithelium. FLP-6 suppressed the transcription factors ZIP-10 and KLF-1 that worked in parallel and converged to the PMK-1/p38 pathway in the intestinal epithelia for innate immunity and gut defense. Collectively, these findings uncover an enteric neuron-muscle-epithelium axis that may be evolutionarily conserved in higher organisms.
Assuntos
Caenorhabditis elegans , Neurônios , Animais , Músculo Liso , Transdução de Sinais , Imunidade InataRESUMO
The recent discovery that genetically modified α cells can regenerate and convert into ß-like cells in vivo holds great promise for diabetes research. However, to eventually translate these findings to human, it is crucial to discover compounds with similar activities. Herein, we report the identification of GABA as an inducer of α-to-ß-like cell conversion in vivo. This conversion induces α cell replacement mechanisms through the mobilization of duct-lining precursor cells that adopt an α cell identity prior to being converted into ß-like cells, solely upon sustained GABA exposure. Importantly, these neo-generated ß-like cells are functional and can repeatedly reverse chemically induced diabetes in vivo. Similarly, the treatment of transplanted human islets with GABA results in a loss of α cells and a concomitant increase in ß-like cell counts, suggestive of α-to-ß-like cell conversion processes also in humans. This newly discovered GABA-induced α cell-mediated ß-like cell neogenesis could therefore represent an unprecedented hope toward improved therapies for diabetes.
Assuntos
Diabetes Mellitus/tratamento farmacológico , Células Secretoras de Glucagon/citologia , Células Secretoras de Insulina/citologia , Ácido gama-Aminobutírico/administração & dosagem , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular/efeitos dos fármacos , Diabetes Mellitus/induzido quimicamente , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Células Secretoras de Glucagon/efeitos dos fármacos , Humanos , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Proteínas do Tecido Nervoso , Ratos , Ratos Wistar , Ácido gama-Aminobutírico/farmacologiaRESUMO
The medial entorhinal cortex (MEC) contains several discrete classes of GABAergic interneurons, but their specific contributions to spatial pattern formation in this area remain elusive. We employed a pharmacogenetic approach to silence either parvalbumin (PV)- or somatostatin (SOM)-expressing interneurons while MEC cells were recorded in freely moving mice. PV-cell silencing antagonized the hexagonally patterned spatial selectivity of grid cells, especially in layer II of MEC. The impairment was accompanied by reduced speed modulation in colocalized speed cells. Silencing SOM cells, in contrast, had no impact on grid cells or speed cells but instead decreased the spatial selectivity of cells with discrete aperiodic firing fields. Border cells and head direction cells were not affected by either intervention. The findings point to distinct roles for PV and SOM interneurons in the local dynamics underlying periodic and aperiodic firing in spatially modulated cells of the MEC. VIDEO ABSTRACT.
Assuntos
Córtex Entorrinal/citologia , Interneurônios/metabolismo , Parvalbuminas/metabolismo , Somatostatina/metabolismo , Processamento Espacial , Animais , Neurônios GABAérgicos/metabolismo , Células de Grade/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vias NeuraisRESUMO
Type 1 diabetes is characterized by the destruction of pancreatic ß cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional ß-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic ß cell mass from α cells.
Assuntos
Artemisininas/farmacologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Modelos Animais de Doenças , Receptores de GABA-A/metabolismo , Transdução de Sinais , Animais , Artemeter , Artemisininas/administração & dosagem , Proteínas de Transporte/metabolismo , Transdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus Tipo 1/patologia , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos , Estabilidade Proteica/efeitos dos fármacos , Ratos , Análise de Célula Única , Fatores de Transcrição/metabolismo , Peixe-Zebra , Ácido gama-Aminobutírico/metabolismoRESUMO
Rapid advances in the neural control of social behavior highlight the role of interconnected nodes engaged in differential information processing to generate behavior. Many innate social behaviors are essential to reproductive fitness and therefore fundamentally different in males and females. Programming these differences occurs early in development in mammals, following gonadal differentiation and copious androgen production by the fetal testis during a critical period. Early-life programming of social behavior and its adult manifestation are separate but yoked processes, yet how they are linked is unknown. This review seeks to highlight that gap by identifying four core mechanisms (epigenetics, cell death, circuit formation, and adult hormonal modulation) that could connect developmental changes to the adult behaviors of mating and aggression. We further propose that a unique social behavior, adolescent play, bridges the preweaning to the postpubertal brain by engaging the same neural networks underpinning adult reproductive and aggressive behaviors.
Assuntos
Agressão , Comportamento Social , Masculino , Animais , Feminino , Encéfalo , Comportamento Sexual Animal , Cognição , MamíferosRESUMO
Heterozygous variants in SLC6A1, encoding the GAT-1 GABA transporter, are associated with seizures, developmental delay, and autism. The majority of affected individuals carry missense variants, many of which are recurrent germline de novo mutations, raising the possibility of gain-of-function or dominant-negative effects. To understand the functional consequences, we performed an in vitro GABA uptake assay for 213 unique variants, including 24 control variants. De novo variants consistently resulted in a decrease in GABA uptake, in keeping with haploinsufficiency underlying all neurodevelopmental phenotypes. Where present, ClinVar pathogenicity reports correlated well with GABA uptake data; the functional data can inform future reports for the remaining 72% of unscored variants. Surface localization was assessed for 86 variants; two-thirds of loss-of-function missense variants prevented GAT-1 from being present on the membrane while GAT-1 was on the surface but with reduced activity for the remaining third. Surprisingly, recurrent de novo missense variants showed moderate loss-of-function effects that reduced GABA uptake with no evidence for dominant-negative or gain-of-function effects. Using linear regression across multiple missense severity scores to extrapolate the functional data to all potential SLC6A1 missense variants, we observe an abundance of GAT-1 residues that are sensitive to substitution. The extent of this missense vulnerability accounts for the clinically observed missense enrichment; overlap with hypermutable CpG sites accounts for the recurrent missense variants. Strategies to increase the expression of the wild-type SLC6A1 allele are likely to be beneficial across neurodevelopmental disorders, though the developmental stage and extent of required rescue remain unknown.
Assuntos
Proteínas da Membrana Plasmática de Transporte de GABA , Haploinsuficiência , Mutação de Sentido Incorreto , Humanos , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Haploinsuficiência/genética , Ácido gama-Aminobutírico/metabolismo , Transtornos do Neurodesenvolvimento/genética , Deficiências do Desenvolvimento/genética , Transtorno Autístico/genética , Células HEK293RESUMO
Ionotropic glutamate receptors (iGluRs) mediate excitatory signals between cells by binding neurotransmitters and conducting cations across the cell membrane. In the mammalian brain, most of these signals are mediated by two types of iGluRs: AMPA and NMDA (i.e. iGluRs sensitive to 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid and N-methyl-D-aspartic acid, respectively). Delta-type iGluRs of mammals also form neurotransmitter-binding channels in the cell membrane, but in contrast, their channel is not activated by neurotransmitter binding, raising biophysical questions about iGluR activation and biological questions about the role of delta iGluRs. We therefore investigated the divergence of delta iGluRs from their iGluR cousins using molecular phylogenetics, electrophysiology, and site-directed mutagenesis. We find that delta iGluRs are found in numerous bilaterian animals (e.g., worms, starfish, and vertebrates) and are closely related to AMPA receptors, both genetically and functionally. Surprisingly, we observe that many iGluRs of the delta family are activated by the classical inhibitory neurotransmitter, γ-aminobutyric acid (GABA). Finally, we identify nine amino acid substitutions that likely gave rise to the inactivity of today's mammalian delta iGluRs, and these mutations abolish activity when engineered into active invertebrate delta iGluRs, partly by inducing receptor desensitization. These results offer biophysical insight into iGluR activity and point to a role for GABA in excitatory signaling in invertebrates.
Assuntos
Receptores Ionotrópicos de Glutamato , Vertebrados , Animais , Receptores Ionotrópicos de Glutamato/metabolismo , Vertebrados/metabolismo , Receptores de AMPA/genética , Invertebrados , Mamíferos/metabolismo , N-Metilaspartato , Neurotransmissores , Ácido gama-AminobutíricoRESUMO
Epilepsies have numerous specific mechanisms. The understanding of neural dynamics leading to seizures is important for disclosing pathological mechanisms and developing therapeutic approaches. We investigated electrographic activities and neural dynamics leading to convulsive seizures in patients and mouse models of Dravet syndrome (DS), a developmental and epileptic encephalopathy in which hypoexcitability of GABAergic neurons is considered to be the main dysfunction. We analyzed EEGs from DS patients carrying a SCN1A pathogenic variant, as well as epidural electrocorticograms, hippocampal local field potentials, and hippocampal single-unit neuronal activities in Scn1a+/- and Scn1aRH/+ DS mice. Strikingly, most seizures had low-voltage-fast onset in both patients and mice, which is thought to be generated by hyperactivity of GABAergic interneurons, the opposite of the main pathological mechanism of DS. Analyzing single-unit recordings, we observed that temporal disorganization of the firing of putative interneurons in the period immediately before the seizure (preictal) precedes the increase of their activity at seizure onset, together with the entire neuronal network. Moreover, we found early signatures of the preictal period in the spectral features of hippocampal and cortical field potential of Scn1a mice and of patients' EEG, which are consistent with the dysfunctions that we observed in single neurons and that allowed seizure prediction. Therefore, the perturbed preictal activity of interneurons leads to their hyperactivity at the onset of generalized seizures, which have low-voltage-fast features that are similar to those observed in other epilepsies and are triggered by hyperactivity of GABAergic neurons. Preictal spectral features may be used as predictive seizure biomarkers.
Assuntos
Epilepsias Mioclônicas , Neurônios GABAérgicos , Hipocampo , Interneurônios , Canal de Sódio Disparado por Voltagem NAV1.1 , Convulsões , Animais , Epilepsias Mioclônicas/fisiopatologia , Epilepsias Mioclônicas/genética , Interneurônios/fisiologia , Interneurônios/metabolismo , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Convulsões/fisiopatologia , Humanos , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/fisiologia , Masculino , Hipocampo/fisiopatologia , Hipocampo/metabolismo , Feminino , Modelos Animais de Doenças , Eletroencefalografia , CriançaRESUMO
Here, we describe a group of basal forebrain (BF) neurons expressing neuronal Per-Arnt-Sim (PAS) domain 1 (Npas1), a developmental transcription factor linked to neuropsychiatric disorders. Immunohistochemical staining in Npas1-cre-2A-TdTomato mice revealed BF Npas1+ neurons are distinct from well-studied parvalbumin or cholinergic neurons. Npas1 staining in GAD67-GFP knock-in mice confirmed that the vast majority of Npas1+ neurons are GABAergic, with minimal colocalization with glutamatergic neurons in vGlut1-cre-tdTomato or vGlut2-cre-tdTomato mice. The density of Npas1+ neurons was high, five to six times that of neighboring cholinergic, parvalbumin, or glutamatergic neurons. Anterograde tracing identified prominent projections of BF Npas1+ neurons to brain regions involved in sleep-wake control, motivated behaviors, and olfaction such as the lateral hypothalamus, lateral habenula, nucleus accumbens shell, ventral tegmental area, and olfactory bulb. Chemogenetic activation of BF Npas1+ neurons in the light period increased the amount of wakefulness and the latency to sleep for 2 to 3 h, due to an increase in long wake bouts and short NREM sleep bouts. NREM slow-wave and sigma power, as well as sleep spindle density, amplitude, and duration, were reduced, reminiscent of findings in several neuropsychiatric disorders. Together with previous findings implicating BF Npas1+ neurons in stress responsiveness, the anatomical projections of BF Npas1+ neurons and the effect of activating them suggest a possible role for BF Npas1+ neurons in motivationally driven wakefulness and stress-induced insomnia. Identification of this major subpopulation of BF GABAergic neurons will facilitate studies of their role in sleep disorders, dementia, and other neuropsychiatric conditions involving BF.
Assuntos
Prosencéfalo Basal , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Neurônios GABAérgicos , Vigília , Animais , Masculino , Prosencéfalo Basal/metabolismo , Prosencéfalo Basal/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/fisiologia , Camundongos Transgênicos , Sono/fisiologia , Vigília/fisiologiaRESUMO
Brain rhythms provide the timing for recruitment of brain activity required for linking together neuronal ensembles engaged in specific tasks. The γ-oscillations (30 to 120 Hz) orchestrate neuronal circuits underlying cognitive processes and working memory. These oscillations are reduced in numerous neurological and psychiatric disorders, including early cognitive decline in Alzheimer's disease (AD). Here, we report on a potent brain-permeable small molecule, DDL-920 that increases γ-oscillations and improves cognition/memory in a mouse model of AD, thus showing promise as a class of therapeutics for AD. We employed anatomical, in vitro and in vivo electrophysiological, and behavioral methods to examine the effects of our lead therapeutic candidate small molecule. As a novel in central nervous system pharmacotherapy, our lead molecule acts as a potent, efficacious, and selective negative allosteric modulator of the γ-aminobutyric acid type A receptors most likely assembled from α1ß2δ subunits. These receptors, identified through anatomical and pharmacological means, underlie the tonic inhibition of parvalbumin (PV) expressing interneurons (PV+INs) critically involved in the generation of γ-oscillations. When orally administered twice daily for 2 wk, DDL-920 restored the cognitive/memory impairments of 3- to 4-mo-old AD model mice as measured by their performance in the Barnes maze. Our approach is unique as it is meant to enhance cognitive performance and working memory in a state-dependent manner by engaging and amplifying the brain's endogenous γ-oscillations through enhancing the function of PV+INs.
Assuntos
Doença de Alzheimer , Cognição , Modelos Animais de Doenças , Ritmo Gama , Animais , Doença de Alzheimer/tratamento farmacológico , Camundongos , Cognição/efeitos dos fármacos , Ritmo Gama/efeitos dos fármacos , Memória/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Camundongos Transgênicos , Humanos , Masculino , Memória de Curto Prazo/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Alanina/análogos & derivados , AzepinasRESUMO
γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter, and its levels in the synaptic space are controlled by the GABA transporter isoforms (GATs). GATs are structurally related to biogenic amine transporters but display interactions with distinct inhibitors used as anti-epileptics. In this study, we engineer the binding pocket of Drosophila melanogaster dopamine transporter to resemble GAT1 and determine high-resolution X-ray structures of the modified transporter in the substrate-free state and in complex with GAT1 inhibitors NO711 and SKF89976a that are analogs of tiagabine, a medication prescribed for the treatment of partial seizures. We observe that the primary binding site undergoes substantial shifts in subsite architecture in the modified transporter to accommodate the two GAT1 inhibitors. We also observe that SKF89976a additionally interacts at an allosteric site in the extracellular vestibule, yielding an occluded conformation. Interchanging SKF89976a interacting residue in the extracellular loop 4 between GAT1 and dDAT suggests a role for this motif in the selective control of neurotransmitter uptake. Our findings, therefore, provide vital insights into the organizational principles dictating GAT1 activity and inhibition.
Assuntos
Drosophila melanogaster , Ácido gama-Aminobutírico , Animais , Transporte Biológico , Drosophila melanogaster/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/química , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Neurotransmissores , Ácido gama-Aminobutírico/metabolismoRESUMO
GABAB receptors (GBRs), the G protein-coupled receptors for GABA, regulate synaptic transmission throughout the brain. A main synaptic function of GBRs is the gating of Cav2.2-type Ca2+ channels. However, the cellular compartment where stable GBR/Cav2.2 signaling complexes form remains unknown. In this study, we demonstrate that the vesicular protein synaptotagmin-11 (Syt11) binds to both the auxiliary GBR subunit KCTD16 and Cav2.2 channels. Through these dual interactions, Syt11 recruits GBRs and Cav2.2 channels to post-Golgi vesicles, thus facilitating assembly of GBR/Cav2.2 signaling complexes. In addition, Syt11 stabilizes GBRs and Cav2.2 channels at the neuronal plasma membrane by inhibiting constitutive internalization. Neurons of Syt11 knockout mice exhibit deficits in presynaptic GBRs and Cav2.2 channels, reduced neurotransmitter release, and decreased GBR-mediated presynaptic inhibition, highlighting the critical role of Syt11 in the assembly and stable expression of GBR/Cav2.2 complexes. These findings support that Syt11 acts as a vesicular scaffold protein, aiding in the assembly of signaling complexes from low-abundance components within transport vesicles. This mechanism enables insertion of pre-assembled functional signaling units into the synaptic membrane.
Assuntos
Camundongos Knockout , Transdução de Sinais , Sinaptotagminas , Animais , Sinaptotagminas/metabolismo , Sinaptotagminas/genética , Camundongos , Humanos , Neurônios/metabolismo , Transmissão Sináptica , Receptores de GABA-B/metabolismo , Receptores de GABA-B/genética , Terminações Pré-Sinápticas/metabolismo , Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo N/genética , Complexo de Golgi/metabolismo , Ligação Proteica , Células HEK293RESUMO
The way the central nervous system (CNS) responds to diverse stimuli is contingent upon the specific brain state of the individual, including sleep and wakefulness. Despite the wealth of readout parameters and data delineating the brain states, the primary mechanisms are yet to be identified. Here we highlight the role of astrocytes, with a specific emphasis on chloride (Cl-) homeostasis as a modulator of brain states. Neuronal activity is regulated by the concentration of ions that determine excitability. Astrocytes, as the CNS homeostatic cells, are recognised for their proficiency in maintaining dynamic homeostasis of ions, known as ionostasis. Nevertheless, the contribution of astrocyte-driven ionostasis to the genesis of brain states or their response to sleep-inducing pharmacological agents has been overlooked. Our objective is to underscore the significance of astrocytic Cl- homeostasis, elucidating how it may underlie the modulation of brain states. We endeavour to contribute to a comprehensive understanding of the interplay between astrocytes and brain states.
Assuntos
Astrócitos , Encéfalo , Cloretos , Homeostase , Astrócitos/metabolismo , Cloretos/metabolismo , Encéfalo/metabolismo , Humanos , Animais , Neurônios/metabolismo , Neurônios/fisiologia , Sono/fisiologia , Vigília/fisiologiaRESUMO
The hypothalamic suprachiasmatic nucleus (SCN) is the master mammalian circadian clock. Its cell-autonomous timing mechanism, a transcriptional/translational feedback loop (TTFL), drives daily peaks of neuronal electrical activity, which in turn control circadian behavior. Intercellular signals, mediated by neuropeptides, synchronize and amplify TTFL and electrical rhythms across the circuit. SCN neurons are GABAergic, but the role of GABA in circuit-level timekeeping is unclear. How can a GABAergic circuit sustain circadian cycles of electrical activity, when such increased neuronal firing should become inhibitory to the network? To explore this paradox, we show that SCN slices expressing the GABA sensor iGABASnFR demonstrate a circadian oscillation of extracellular GABA ([GABA]e) that, counterintuitively, runs in antiphase to neuronal activity, with a prolonged peak in circadian night and a pronounced trough in circadian day. Resolving this unexpected relationship, we found that [GABA]e is regulated by GABA transporters (GATs), with uptake peaking during circadian day, hence the daytime trough and nighttime peak. This uptake is mediated by the astrocytically expressed transporter GAT3 (Slc6a11), expression of which is circadian-regulated, being elevated in daytime. Clearance of [GABA]e in circadian day facilitates neuronal firing and is necessary for circadian release of the neuropeptide vasoactive intestinal peptide, a critical regulator of TTFL and circuit-level rhythmicity. Finally, we show that genetic complementation of the astrocytic TTFL alone, in otherwise clockless SCN, is sufficient to drive [GABA]e rhythms and control network timekeeping. Thus, astrocytic clocks maintain the SCN circadian clockwork by temporally controlling GABAergic inhibition of SCN neurons.
Assuntos
Relógios Circadianos , Ritmo Circadiano , Animais , Ritmo Circadiano/genética , Relógios Circadianos/genética , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Núcleo Supraquiasmático/metabolismo , Ácido gama-Aminobutírico/metabolismo , Mamíferos/metabolismoRESUMO
The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of a central circadian clock that orchestrates overt rhythms of physiology and behavior. Circadian timekeeping requires intercellular communication among SCN neurons, and multiple signaling pathways contribute to SCN network coupling. Gamma-aminobutyric acid (GABA) is produced by virtually all SCN neurons, and previous work demonstrates that this transmitter regulates coupling in the adult SCN but is not essential for the nucleus to sustain overt circadian rhythms. Here, we show that the deletion of the gene that codes for the GABA vesicular transporter Vgat from neuromedin-S (NMS)+ neurons-a subset of neurons critical for SCN function-causes arrhythmia of locomotor activity and sleep. Further, NMS-Vgat deletion impairs intrinsic clock gene rhythms in SCN explants cultured ex vivo. Although vasoactive intestinal polypeptide (VIP) is critical for SCN function, Vgat deletion from VIP-expressing neurons did not lead to circadian arrhythmia in locomotor activity rhythms. Likewise, adult SCN-specific deletion of Vgat led to mild impairment of behavioral rhythms. Our results suggest that while the removal of GABA release from the adult SCN does not affect the pacemaker's ability to sustain overt circadian rhythms, its removal from a critical subset of neurons within the SCN throughout development removes the nucleus ability to sustain circadian rhythms. Our findings support a model in which SCN GABA release is critical for the developmental establishment of intercellular network properties that define the SCN as a central pacemaker.
Assuntos
Relógios Circadianos , Ritmo Circadiano , Humanos , Ritmo Circadiano/fisiologia , Neurônios/metabolismo , Relógios Circadianos/fisiologia , Peptídeo Intestinal Vasoativo/genética , Peptídeo Intestinal Vasoativo/metabolismo , Núcleo Supraquiasmático/metabolismo , Ácido gama-Aminobutírico/metabolismo , Arritmias Cardíacas/metabolismoRESUMO
The brain employs distinct circuitries to encode positive and negative valence stimuli, and dysfunctions of these neuronal circuits have a key role in the etiopathogenesis of many psychiatric disorders. The Dorsal Raphè Nucleus (DRN) is involved in various behaviors and drives the emotional response to rewarding and aversive experiences. Whether specific subpopulations of neurons within the DRN encode these behaviors with different valence is still unknown. Notably, microRNA expression in the mammalian brain is characterized by tissue and neuronal specificity, suggesting that it might play a role in cell and circuit functionality. However, this specificity has not been fully exploited. Here, we demonstrate that microRNA-34a (miR-34a) is selectively expressed in a subpopulation of GABAergic neurons of the ventrolateral DRN. Moreover, we report that acute exposure to both aversive (restraint stress) and rewarding (chocolate) stimuli reduces GABA release in the DRN, an effect prevented by the inactivation of DRN miR-34a or its genetic deletion in GABAergic neurons in aversive but not rewarding conditions. Finally, miR-34a inhibition selectively reduced passive coping with severe stressors. These data support a role of miR-34a in regulating GABAergic neurotransmitter activity and behavior in a context-dependent manner and suggest that microRNAs could represent a functional signature of specific neuronal subpopulations with valence-specific activity in the brain.