RESUMO
RTN4-binding proteins were widely studied as "NoGo" receptors, but their physiological interactors and roles remain elusive. Similarly, BAI adhesion-GPCRs were associated with numerous activities, but their ligands and functions remain unclear. Using unbiased approaches, we observed an unexpected convergence: RTN4 receptors are high-affinity ligands for BAI adhesion-GPCRs. A single thrombospondin type 1-repeat (TSR) domain of BAIs binds to the leucine-rich repeat domain of all three RTN4-receptor isoforms with nanomolar affinity. In the 1.65 Å crystal structure of the BAI1/RTN4-receptor complex, C-mannosylation of tryptophan and O-fucosylation of threonine in the BAI TSR-domains creates a RTN4-receptor/BAI interface shaped by unusual glycoconjugates that enables high-affinity interactions. In human neurons, RTN4 receptors regulate dendritic arborization, axonal elongation, and synapse formation by differential binding to glial versus neuronal BAIs, thereby controlling neural network activity. Thus, BAI binding to RTN4/NoGo receptors represents a receptor-ligand axis that, enabled by rare post-translational modifications, controls development of synaptic circuits.
Assuntos
Inibidores da Angiogênese/metabolismo , Encéfalo/metabolismo , Neurogênese , Neurônios/metabolismo , Proteínas Nogo/metabolismo , Receptores Nogo/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adipocinas/metabolismo , Sequência de Aminoácidos , Animais , Axônios/metabolismo , Adesão Celular , Moléculas de Adesão Celular Neuronais/metabolismo , Complemento C1q/metabolismo , Dendritos/metabolismo , Glicosilação , Células HEK293 , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Ligantes , Camundongos Endogâmicos C57BL , Rede Nervosa/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Domínios Proteicos , Deleção de Sequência , Sinapses/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Synapses are semi-membraneless, protein-dense, sub-micron chemical reaction compartments responsible for signal processing in each and every neuron. Proper formation and dynamic responses to stimulations of synapses, both during development and in adult, are fundamental to functions of mammalian brains, although the molecular basis governing formation and modulation of compartmentalized synaptic assemblies is unclear. Here, we used a biochemical reconstitution approach to show that, both in solution and on supported membrane bilayers, multivalent interaction networks formed by major excitatory postsynaptic density (PSD) scaffold proteins led to formation of PSD-like assemblies via phase separation. The reconstituted PSD-like assemblies can cluster receptors, selectively concentrate enzymes, promote actin bundle formation, and expel inhibitory postsynaptic proteins. Additionally, the condensed phase PSD assemblies have features that are distinct from those in homogeneous solutions and fit for synaptic functions. Thus, we have built a molecular platform for understanding how neuronal synapses are formed and dynamically regulated.
Assuntos
Neurogênese , Plasticidade Neuronal , Densidade Pós-Sináptica , Sinapses/fisiologia , Animais , Encéfalo/fisiologia , Proteína 4 Homóloga a Disks-Large/fisiologia , Hipocampo/fisiologia , Luz , Camundongos , Microscopia Confocal , Neurônios/fisiologia , Espalhamento de Radiação , Transdução de Sinais , Transmissão SinápticaRESUMO
The nervous system is populated by numerous types of neurons, each bearing a dendritic arbor with a characteristic morphology. These type-specific features influence many aspects of a neuron's function, including the number and identity of presynaptic inputs and how inputs are integrated to determine firing properties. Here, we review the mechanisms that regulate the construction of cell type-specific dendrite patterns during development. We focus on four aspects of dendrite patterning that are particularly important in determining the function of the mature neuron: (a) dendrite shape, including branching pattern and geometry of the arbor; (b) dendritic arbor size;
Assuntos
Dendritos/fisiologia , Animais , Pareamento Cromossômico/fisiologia , HumanosRESUMO
Tethering of synaptic vesicles (SVs) to the active zone determines synaptic strength, although the molecular basis governing SV tethering is elusive. Here, we discover that small unilamellar vesicles (SUVs) and SVs from rat brains coat on the surface of condensed liquid droplets formed by active zone proteins RIM, RIM-BP, and ELKS via phase separation. Remarkably, SUV-coated RIM/RIM-BP condensates are encapsulated by synapsin/SUV condensates, forming two distinct SUV pools reminiscent of the reserve and tethered SV pools that exist in presynaptic boutons. The SUV-coated RIM/RIM-BP condensates can further cluster Ca2+ channels anchored on membranes. Thus, we reconstitute a presynaptic bouton-like structure mimicking the SV-tethered active zone with its one side attached to the presynaptic membrane and the other side connected to the synapsin-clustered SV condensates. The distinct interaction modes between membraneless protein condensates and membrane-based organelles revealed here have general implications in cellular processes, including vesicular formation and trafficking, organelle biogenesis, and autophagy.
Assuntos
Encéfalo/metabolismo , Canais de Cálcio/metabolismo , Terminações Pré-Sinápticas/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Canais de Cálcio/genética , Humanos , Camundongos , Ratos , Sinapsinas/genética , Vesículas Sinápticas/genéticaRESUMO
Enzymes or enzyme complexes can be concentrated in different cellular loci to modulate distinct functional processes in response to specific signals. How cells condense and compartmentalize enzyme complexes for spatiotemporally distinct cellular events is not well understood. Here we discover that specific and tight association of GIT1 and ß-Pix, a pair of GTPase regulatory enzymes, leads to phase separation of the complex without additional scaffolding molecules. GIT1/ß-Pix condensates are modular in nature and can be positioned at distinct cellular compartments, such as neuronal synapses, focal adhesions, and cell-cell junctions, by upstream adaptors. Guided by the structure of the GIT/PIX complex, we specifically probed the role of phase separation of the enzyme complex in cell migration and synapse formation. Our study suggests that formation of modular enzyme complex condensates via phase separation can dynamically concentrate limited quantities of enzymes to distinct cellular compartments for specific and optimal signaling.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Animais , Proteínas de Ciclo Celular/química , Proteínas Ativadoras de GTPase/química , Células HEK293 , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Paxilina/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismoRESUMO
Synapses containing γ-aminobutyric acid (GABA) constitute the primary centers for inhibitory neurotransmission in our nervous system. It is unclear how these synaptic structures form and align their postsynaptic machineries with presynaptic terminals. Here, we monitored the cellular distribution of several GABAergic postsynaptic proteins in a purely glutamatergic neuronal culture derived from human stem cells, which virtually lacks any vesicular GABA release. We found that several GABAA receptor (GABAAR) subunits, postsynaptic scaffolds, and major cell-adhesion molecules can reliably coaggregate and colocalize at even GABA-deficient subsynaptic domains, but remain physically segregated from glutamatergic counterparts. Genetic deletions of both Gephyrin and a Gephyrin-associated guanosine di- or triphosphate (GDP/GTP) exchange factor Collybistin severely disrupted the coassembly of these postsynaptic compositions and their proper apposition with presynaptic inputs. Gephyrin-GABAAR clusters, developed in the absence of GABA transmission, could be subsequently activated and even potentiated by delayed supply of vesicular GABA. Thus, molecular organization of GABAergic postsynapses can initiate via a GABA-independent but Gephyrin-dependent intrinsic mechanism.
Assuntos
Proteínas de Transporte , Proteínas de Membrana , Terminações Pré-Sinápticas , Receptores de GABA-A , Sinapses , Ácido gama-Aminobutírico , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Ácido gama-Aminobutírico/metabolismo , Receptores de GABA-A/metabolismo , Receptores de GABA-A/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Terminações Pré-Sinápticas/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genéticaRESUMO
MDGA (MAM domain containing glycosylphosphatidylinositol anchor) family proteins were previously identified as synaptic suppressive factors. However, various genetic manipulations have yielded often irreconcilable results, precluding precise evaluation of MDGA functions. Here, we found that, in cultured hippocampal neurons, conditional deletion of MDGA1 and MDGA2 causes specific alterations in synapse numbers, basal synaptic transmission, and synaptic strength at GABAergic and glutamatergic synapses, respectively. Moreover, MDGA2 deletion enhanced both N-methyl-D-aspartate (NMDA) receptor- and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated postsynaptic responses. Strikingly, ablation of both MDGA1 and MDGA2 abolished the effect of deleting individual MDGAs that is abrogated by chronic blockade of synaptic activity. Molecular replacement experiments further showed that MDGA1 requires the meprin/A5 protein/PTPmu (MAM) domain, whereas MDGA2 acts via neuroligin-dependent and/or MAM domain-dependent pathways to regulate distinct postsynaptic properties. Together, our data demonstrate that MDGA paralogs act as unique negative regulators of activity-dependent postsynaptic organization at distinct synapse types, and cooperatively contribute to adjustment of excitation-inhibition balance.
Assuntos
Hipocampo , Sinapses , Transmissão Sináptica , Animais , Sinapses/metabolismo , Camundongos , Hipocampo/metabolismo , Hipocampo/citologia , Transmissão Sináptica/fisiologia , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Camundongos Knockout , Receptores de AMPA/metabolismo , Receptores de AMPA/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Células CultivadasRESUMO
Synapse formation is mediated by a surprisingly large number and wide variety of genes encoding many different protein classes. One of the families increasingly implicated in synapse wiring is the immunoglobulin superfamily (IgSF). IgSF molecules are by definition any protein containing at least one Ig-like domain, making this family one of the most common protein classes encoded by the genome. Here, we review the emerging roles for IgSF molecules in synapse formation specifically in the vertebrate brain, focusing on examples from three classes of IgSF members: ( a) cell adhesion molecules, ( b) signaling molecules, and ( c) immune molecules expressed in the brain. The critical roles for IgSF members in regulating synapse formation may explain their extensive involvement in neuropsychiatric and neurodevelopmental disorders. Solving the IgSF code for synapse formation may reveal multiple new targets for rescuing IgSF-mediated deficits in synapse formation and, eventually, new treatments for psychiatric disorders caused by altered IgSF-induced synapse wiring.
Assuntos
Encéfalo/metabolismo , Imunoglobulinas/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Sinapses/genética , Animais , Encéfalo/crescimento & desenvolvimento , Moléculas de Adesão Celular/genética , Humanos , Imunoglobulinas/classificação , Imunoglobulinas/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/imunologiaRESUMO
In multicellular organisms, cell-adhesion molecules connect cells into tissues and mediate intercellular signaling between these cells. In vertebrate brains, synaptic cell-adhesion molecules (SAMs) guide the formation, specification, and plasticity of synapses. Some SAMs, when overexpressed in cultured neurons or in heterologous cells co-cultured with neurons, drive formation of synaptic specializations onto the overexpressing cells. However, genetic deletion of the same SAMs from neurons often has no effect on synapse numbers, but frequently severely impairs synaptic transmission, suggesting that most SAMs control the function and plasticity of synapses (i.e., organize synapses) instead of driving their initial establishment (i.e., make synapses). Since few SAMs were identified that mediate initial synapse formation, it is difficult to develop methods that enable experimental control of synaptic connections by targeted expression of these SAMs. To overcome this difficulty, we engineered novel SAMs from bacterial proteins with no eukaryotic homologues that drive synapse formation. We named these engineered adhesion proteins "Barnoligin" and "Starexin" because they were assembled from parts of Barnase and Neuroligin-1 or of Barstar and Neurexin3ß, respectively. Barnoligin and Starexin robustly induce the formation of synaptic specializations in a specific and directional manner in cultured neurons. Synapse formation by Barnoligin and Starexin requires both their extracellular Barnase- and Barstar-derived interaction domains and their Neuroligin- and Neurexin-derived intracellular signaling domains. Our findings support a model of synapse formation whereby trans-synaptic interactions by SAMs drive synapse organization via adhesive interactions that activate signaling cascades.
Assuntos
Moléculas de Adesão Celular Neuronais , Sinapses , Células Cultivadas , Moléculas de Adesão Celular Neuronais/metabolismo , Sinapses/metabolismo , Transmissão Sináptica , Neurônios/metabolismo , Técnicas de Cocultura , Hipocampo/metabolismoRESUMO
α- and ß-neurexins are extensively alternatively spliced, presynaptic cell-adhesion molecules that are thought to organize synapse assembly. However, recent data revealed that, in the hippocampus in vivo, the deletion of one neurexin isoform, Nrxn2, surprisingly increased excitatory synapse numbers and enhanced their presynaptic release probability, suggesting that Nrxn2 restricts, instead of enabling, synapse assembly. To delineate the synaptic function and mechanism of action of Nrxn2, we examined cultured hippocampal neurons as a reduced system. In heterologous synapse formation assays, different alternatively spliced Nrxn2ß isoforms robustly promoted synapse assembly similar to Nrxn1ß and Nrxn3ß, consistent with a general synaptogenic function of neurexins. Deletion of Nrxn2 from cultured hippocampal neurons, however, caused a significant increase in synapse density and release probability, replicating the in vivo data that suggested a synapse-restricting function. Rescue experiments revealed that two of the four Nrxn2ß splice variants (Nrxn2ß-SS4+/SS5- and Nrxn2ß-SS4+/SS5+) reversed the increase in synapse density in Nrxn2-deficient neurons, whereas only one of the four Nrxn2ß splice variants (Nrxn2ß-SS4+/SS5+) normalized the increase in release probability in Nrxn2-deficient neurons. Thus, a subset of Nrxn2 splice variants restricts synapse numbers and restrains their release probability in cultured neurons.
Assuntos
Processamento Alternativo , Sinapses , Sinapses/metabolismo , Hipocampo/metabolismo , Moléculas de Adesão Celular/metabolismo , Neurônios/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/metabolismoRESUMO
Effects of micronutrients on brain connectivity are incompletely understood. Analyzing human milk samples across global populations, we identified the carbocyclic sugar myo-inositol as a component that promotes brain development. We determined that it is most abundant in human milk during early lactation when neuronal connections rapidly form in the infant brain. Myo-inositol promoted synapse abundance in human excitatory neurons as well as cultured rat neurons and acted in a dose-dependent manner. Mechanistically, myo-inositol enhanced the ability of neurons to respond to transsynaptic interactions that induce synapses. Effects of myo-inositol in the developing brain were tested in mice, and its dietary supplementation enlarged excitatory postsynaptic sites in the maturing cortex. Utilizing an organotypic slice culture system, we additionally determined that myo-inositol is bioactive in mature brain tissue, and treatment of organotypic slices with this carbocyclic sugar increased the number and size of postsynaptic specializations and excitatory synapse density. This study advances our understanding of the impact of human milk on the infant brain and identifies myo-inositol as a breast milk component that promotes the formation of neuronal connections.
Assuntos
Aleitamento Materno , Leite Humano , Feminino , Lactente , Humanos , Animais , Camundongos , Ratos , Neurônios , Inositol/farmacologia , AçúcaresRESUMO
Latrophilin-1 (Lphn1, aka CIRL1 and CL1; gene symbol Adgrl1) is an adhesion GPCR that has been implicated in excitatory synaptic transmission as a candidate receptor for α-latrotoxin. Here we analyzed conditional knock-in/knock-out mice for Lphn1 that contain an extracellular myc epitope tag. Mice of both sexes were used in all experiments. Surprisingly, we found that Lphn1 is localized in cultured neurons to synaptic nanoclusters that are present in both excitatory and inhibitory synapses. Conditional deletion of Lphn1 in cultured neurons failed to elicit a detectable impairment in excitatory synapses but produced a decrease in inhibitory synapse numbers and synaptic transmission that was most pronounced for synapses close to the neuronal soma. No changes in axonal or dendritic outgrowth or branching were observed. Our data indicate that Lphn1 is among the few postsynaptic adhesion molecules that are present in both excitatory and inhibitory synapses and that Lphn1 by itself is not essential for excitatory synaptic transmission but is required for some inhibitory synaptic connections.
Assuntos
Camundongos Knockout , Receptores de Peptídeos , Sinapses , Animais , Feminino , Masculino , Camundongos , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/metabolismo , Hipocampo/citologia , Potenciais Pós-Sinápticos Inibidores/fisiologia , Camundongos Endogâmicos C57BL , Inibição Neural/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Sinapses/metabolismo , Sinapses/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
Brain development relies on dynamic morphogenesis and interactions of neurons. Filopodia are thin and highly dynamic membrane protrusions that are critically required for neuronal development and neuronal interactions with the environment. Filopodial interactions are typically characterized by non-deterministic dynamics, yet their involvement in developmental processes leads to stereotypic and robust outcomes. Here, we discuss recent advances in our understanding of how filopodial dynamics contribute to neuronal differentiation, migration, axonal and dendritic growth and synapse formation. Many of these advances are brought about by improved methods of live observation in intact developing brains. Recent findings integrate known and novel roles ranging from exploratory sensors and decision-making agents to pools for selection and mechanical functions. Different types of filopodial dynamics thereby reveal non-deterministic subcellular decision-making processes as part of genetically encoded brain development.
Assuntos
Neurogênese , Pseudópodes , Neurogênese/fisiologia , Neurônios , Morfogênese , EncéfaloRESUMO
The morphology of dendritic spines, the postsynaptic compartment of most excitatory synapses, decisively modulates the function of neuronal circuits as also evident from human brain disorders associated with altered spine density or morphology. Actin filaments (F-actin) form the backbone of spines, and a number of actin-binding proteins (ABP) have been implicated in shaping the cytoskeleton in mature spines. Instead, only little is known about the mechanisms that control the reorganization from unbranched F-actin of immature spines to the complex, highly branched cytoskeleton of mature spines. Here, we demonstrate impaired spine maturation in hippocampal neurons upon genetic inactivation of cyclase-associated protein 1 (CAP1) and CAP2, but not of CAP1 or CAP2 alone. We found a similar spine maturation defect upon overactivation of inverted formin 2 (INF2), a nucleator of unbranched F-actin with hitherto unknown synaptic function. While INF2 overactivation failed in altering spine density or morphology in CAP-deficient neurons, INF2 inactivation largely rescued their spine defects. From our data we conclude that CAPs inhibit INF2 to induce spine maturation. Since we previously showed that CAPs promote cofilin1-mediated cytoskeletal remodeling in mature spines, we identified them as a molecular switch that control transition from filopodia-like to mature spines.
Assuntos
Proteínas do Citoesqueleto , Espinhas Dendríticas , Forminas , Hipocampo , Proteínas dos Microfilamentos , Espinhas Dendríticas/metabolismo , Animais , Camundongos , Forminas/metabolismo , Forminas/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Hipocampo/metabolismo , Hipocampo/citologia , Células Cultivadas , Neurônios/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Camundongos Knockout , Humanos , Proteínas de TransporteRESUMO
Synapse formation in the mammalian brain is a complex and dynamic process requiring coordinated function of dozens of molecular families such as cell adhesion molecules (CAMs) and ligand-receptor pairs (Ephs/Ephrins, Neuroligins/Neurexins, Semaphorins/Plexins). Due to the large number of molecular players and possible functional redundancies within gene families, it is challenging to determine the precise synaptogenic roles of individual molecules, which is key to understanding the consequences of mutations in these genes for brain function. Furthermore, few molecules are known to exclusively regulate either GABAergic or glutamatergic synapses, and cell and molecular mechanisms underlying GABAergic synapse formation in particular are not thoroughly understood. We previously demonstrated that Semaphorin-4D (Sema4D) regulates GABAergic synapse development in the mammalian hippocampus while having no effect on glutamatergic synapse development, and this effect occurs through binding to its high affinity receptor, Plexin-B1. In addition, we demonstrated that RNAi-mediated Plexin-B2 knock-down decreases GABAergic synapse density suggesting that both receptors function in this process. Here, we perform a structure-function study of the Plexin-B1 and Plexin-B2 receptors to identify the protein domains in each receptor which are required for its synaptogenic function. Further, we examine whether Plexin-B2 is required in the presynaptic neuron, the postsynaptic neuron, or both to regulate GABAergic synapse formation. Our data reveal that Plexin-B1 and Plexin-B2 function non-redundantly to regulate GABAergic synapse formation and suggest that the transmembrane domain may underlie functional distinctions. We also provide evidence that Plexin-B2 expression in presynaptic GABAergic interneurons, as well as postsynaptic pyramidal cells, regulates GABAergic synapse formation in hippocampus. These findings lay the groundwork for future investigations into the precise signaling pathways required for synapse formation downstream of Plexin-B receptor signaling.
Assuntos
Moléculas de Adesão Celular , Receptores de Superfície Celular , Semaforinas , Animais , Receptores de Superfície Celular/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , MamíferosRESUMO
During the first two postnatal weeks, intraneuronal chloride concentrations in rodents gradually decrease, causing a shift from depolarizing to hyperpolarizing GABA responses. The postnatal GABA shift is delayed in rodent models for neurodevelopmental disorders and in human patients, but the impact of a delayed GABA shift on the developing brain remains obscure. Here we examine the direct and indirect consequences of a delayed postnatal GABA shift on network development in organotypic hippocampal cultures made from 6- to 7-d-old mice by treating the cultures for 1 week with VU0463271, a specific inhibitor of the chloride exporter KCC2. We verified that VU treatment delayed the GABA shift and kept GABA signaling depolarizing until DIV9. We found that the structural and functional development of excitatory and inhibitory synapses at DIV9 was not affected after VU treatment. In line with previous studies, we observed that GABA signaling was already inhibitory in control and VU-treated postnatal slices. Surprisingly, 14 d after the VU treatment had ended (DIV21), we observed an increased frequency of spontaneous inhibitory postsynaptic currents in CA1 pyramidal cells, while excitatory currents were not changed. Synapse numbers and release probability were unaffected. We found that dendrite-targeting interneurons in the stratum radiatum had an elevated resting membrane potential, while pyramidal cells were less excitable compared with control slices. Our results show that depolarizing GABA signaling does not promote synapse formation after P7, and suggest that postnatal intracellular chloride levels indirectly affect membrane properties in a cell-specific manner.SIGNIFICANCE STATEMENT During brain development, the action of neurotransmitter GABA shifts from depolarizing to hyperpolarizing. This shift is a thought to play a critical role in synapse formation. A delayed shift is common in rodent models for neurodevelopmental disorders and in human patients, but its consequences for synaptic development remain obscure. Here, we delayed the GABA shift by 1 week in organotypic hippocampal cultures and carefully examined the consequences for circuit development. We find that delaying the shift has no direct effects on synaptic development, but instead leads to indirect, cell type-specific changes in membrane properties. Our data call for careful assessment of alterations in cellular excitability in neurodevelopmental disorders.
Assuntos
Cloretos , Hipocampo , Animais , Camundongos , Humanos , Cloretos/metabolismo , Hipocampo/fisiologia , Interneurônios/fisiologia , Sinapses/fisiologia , Ácido gama-Aminobutírico/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
To achieve ultrafast neurotransmission, neurons assemble synapses with highly organized presynaptic and postsynaptic nanomachines that are aligned by synaptic adhesion molecules. How functional assembly of presynaptic active zones is controlled via trans-synaptic interactions remains unknown. Here, we conditionally deleted all three neurexin adhesion molecules from presynaptic neurons of the calyx of Held in the mouse auditory system, a model synapse that allows precise biophysical analyses of synaptic properties. The pan-neurexin deletion had no effect on synapse development or the basic release machinery, but dramatically impaired fast neurotransmitter release. The overall properties of presynaptic calcium ion channels appeared normal, as reflected by the similar characteristics of calcium currents recorded at the nerve terminals. However, the pan-neurexin deletion significantly impaired the tight coupling of calcium influx to exocytosis, thereby suppressing neurotransmitter release. Furthermore, the pan-neurexin deletion reduced the function of calcium-activated BK potassium channels, whose activation depends on their tight association with presynaptic calcium channels. Together, these results suggest that neurexins perform a major function at the calyx synapse in coupling presynaptic calcium channels to release sites.
Assuntos
Canais de Cálcio/metabolismo , Proteínas do Tecido Nervoso/genética , Sinapses/metabolismo , Animais , Exocitose , Deleção de Genes , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Transmissão SinápticaRESUMO
Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation associated with schizophrenia, and additionally predispose to multiple other neurodevelopmental disorders. Engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multicenter effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. Using neurons transdifferentiated from induced pluripotent stem cells that were derived from schizophrenia patients carrying heterozygous NRXN1 deletions, we observed the same synaptic impairment as in engineered NRXN1-deficient neurons. This impairment manifested as a large decrease in spontaneous synaptic events, in evoked synaptic responses, and in synaptic paired-pulse depression. Nrxn1-deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. Human NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1-binding protein, and were associated with characteristic gene-expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Mutação , Moléculas de Adesão de Célula Nervosa/genética , Neurônios/metabolismo , Neurotransmissores/metabolismo , Esquizofrenia/metabolismo , Estudos de Casos e Controles , Transdiferenciação Celular , Células Cultivadas , Estudos de Coortes , Células-Tronco Embrionárias/citologia , Expressão Gênica , Guanilato Quinases/metabolismo , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/citologiaRESUMO
In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.
Assuntos
Canais de Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Canais de Cálcio/genética , Células Cultivadas , Hipocampo/citologia , Camundongos Knockout , Terminações Pré-Sinápticas/ultraestrutura , Isoformas de Proteínas/metabolismoRESUMO
Little is known about the cellular signals that organize synapse formation. To explore what signaling pathways may be involved, we employed heterologous synapse formation assays in which a synaptic adhesion molecule expressed in a nonneuronal cell induces pre- or postsynaptic specializations in cocultured neurons. We found that interfering pharmacologically with microtubules or actin filaments impaired heterologous synapse formation, whereas blocking protein synthesis had no effect. Unexpectedly, pharmacological inhibition of c-jun N-terminal kinases (JNKs), protein kinase-A (PKA), or AKT kinases also suppressed heterologous synapse formation, while inhibition of other tested signaling pathways-such as MAP kinases or protein kinase C-did not alter heterologous synapse formation. JNK and PKA inhibitors suppressed formation of both pre- and postsynaptic specializations, whereas AKT inhibitors impaired formation of post- but not presynaptic specializations. To independently test whether heterologous synapse formation depends on AKT signaling, we targeted PTEN, an enzyme that hydrolyzes phosphatidylinositol 3-phosphate and thereby prevents AKT kinase activation, to postsynaptic sites by fusing PTEN to Homer1. Targeting PTEN to postsynaptic specializations impaired heterologous postsynaptic synapse formation induced by presynaptic adhesion molecules, such as neurexins and additionally decreased excitatory synapse function in cultured neurons. Taken together, our results suggest that heterologous synapse formation is driven via a multifaceted and multistage kinase network, with diverse signals organizing pre- and postsynaptic specializations.