Nils Brose

Synaptotagmin I functions as a calcium regulator of release probability

In all synapses, Ca2+ triggers neurotransmitter release to initiate signal transmission. Ca2+ presumably acts by activating synaptic Ca2+ sensors, but the nature of these sensors—which are the gatekeepers to neurotransmission—remains unclear. One of the candidate Ca2+ sensors in release is the synaptic Ca2+-binding protein synaptotagmin I. Here we have studied a point mutation in synaptotagmin I that causes a twofold decrease in overall Ca2+ affinity without inducing structural or conformational changes. When introduced by homologous recombination into the endogenous synaptotagmin I gene in mice, this point mutation decreases the Ca2+ sensitivity of neurotransmitter release twofold, but does not alter spontaneous release or the size of the readily releasable pool of neurotransmitters. Therefore, Ca2+ binding to synaptotagmin I participates in triggering neurotransmitter release at the synapse.

Neuroligins Determine Synapse Maturation and Function

Synaptogenesis, the generation and maturation of functional synapses between nerve cells, is an essential step in the development of neuronal networks in the brain. It is thought to be triggered by members of the neuroligin family of postsynaptic cell adhesion proteins, which may form transsynaptic contacts with presynaptic alpha- and beta-neurexins and have been implicated in the etiology of autism. We show that deletion mutant mice lacking neuroligin expression die shortly after birth due to respiratory failure. This respiratory failure is a consequence of reduced GABAergic/glycinergic and glutamatergic synaptic transmission and network activity in brainstem centers that control respiration. However, the density of synaptic contacts is not altered in neuroligin-deficient brains and cultured neurons. Our data show that neuroligins are required for proper synapse maturation and brain function, but not for the initial formation of synaptic contacts.

Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles

Neurotransmitter release at synapses between nerve cells is mediated by calcium-triggered exocytotic fusion of synaptic vesicles. Before fusion, vesicles dock at the presynaptic release site where they mature to a fusion-competent state,. Here we identify Munc13-1, a brain-specific presynaptic phorbol ester receptor,, as an essential protein for synaptic vesicle maturation. We show that glutamatergic hippocampal neurons from mice lacking Munc13-1 form ultrastructurally normal synapses whose synaptic-vesicle cycle is arrested at the maturation step. Transmitter release from mutant synapses cannot be triggered by action potentials, calcium-ionophores or hypertonic sucrose solution. In contrast, release evoked by α-latrotoxin is indistinguishable from wild-type controls, indicating that the toxin can bypass Munc13-1-mediated vesicle maturation. A small subpopulation of synapses of any given glutamatergic neuron as well as all synapses of GABA (γ-aminobutyric acid)-containing neurons are unaffected by Munc13-1 loss, demonstrating the existence of multiple and transmitter-specific synaptic vesicle maturation processes in synapses.

Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2

Neuroligins enhance synapse formation in vitro, but surprisingly are not required for the generation of synapses in vivo. We now show that in cultured neurons, neuroligin-1 overexpression increases excitatory, but not inhibitory, synaptic responses, and potentiates synaptic NMDAR/AMPAR ratios. In contrast, neuroligin-2 overexpression increases inhibitory, but not excitatory, synaptic responses. Accordingly, deletion of neuroligin-1 in knockout mice selectively decreases the NMDAR/AMPAR ratio, whereas deletion of neuroligin-2 selectively decreases inhibitory synaptic responses. Strikingly, chronic inhibition of NMDARs or CaM-Kinase II, which signals downstream of NMDARs, suppresses the synapse-boosting activity of neuroligin-1, whereas chronic inhibition of general synaptic activity suppresses the synapse-boosting activity of neuroligin-2. Taken together, these data indicate that neuroligins do not establish, but specify and validate, synapses via an activity-dependent mechanism, with different neuroligins acting on distinct types of synapses. This hypothesis reconciles the overexpression and knockout phenotypes and suggests that neuroligins contribute to the use-dependent formation of neural circuits.

β Phorbol Ester- and Diacylglycerol-Induced Augmentation of Transmitter Release Is Mediated by Munc13s and Not by PKCs

Munc13-1 is a presynaptic protein with an essential role in synaptic vesicle priming. It contains a diacylglycerol (DAG)/beta phorbol ester binding C(1) domain and is a potential target of the DAG second messenger pathway that may act in parallel with PKCs. Using genetically modified mice that express a DAG/beta phorbol ester binding-deficient Munc13-1(H567K) variant instead of the wild-type protein, we determined the relative contribution of PKCs and Munc13-1 to DAG/beta phorbol ester-dependent regulation of neurotransmitter release. We show that Munc13s are the main presynaptic DAG/beta phorbol ester receptors in hippocampal neurons. Modulation of Munc13-1 activity by second messengers via the DAG/beta phorbol ester binding C(1) domain is essential for use-dependent alterations of synaptic efficacy and survival.

Total arrest of spontaneous and evoked synaptic transmission but normal synaptogenesis in the absence of Munc13-mediated vesicle priming

Synaptic vesicles must be primed to fusion competence before they can fuse with the plasma membrane in response to increased intracellular Ca2+ levels. The presynaptic active zone protein Munc13-1 is essential for priming of glutamatergic synaptic vesicles in hippocampal neurons. However, a small subpopulation of synapses in any given glutamatergic nerve cell as well as all γ-aminobutyratergic (GABAergic) synapses are largely independent of Munc13-1. We show here that Munc13-2, the only Munc13 isoform coexpressed with Munc13-1 in hippocampus, is responsible for vesicle priming in Munc13-1 independent hippocampal synapses. Neurons lacking both Munc13-1 and Munc13-2 show neither evoked nor spontaneous release events, yet form normal numbers of synapses with typical ultrastructural features. Thus, the two Munc13 isoforms are completely redundant in GABAergic cells whereas glutamatergic neurons form two types of synapses, one of which is solely Munc13-1 dependent and lacks Munc13-2 whereas the other type employs Munc13-2 as priming factor. We conclude that Munc13-mediated vesicle priming is not a transmitter specific phenomenon but rather a general and essential feature of multiple fast neurotransmitter systems, and that synaptogenesis during development is not dependent on synaptic secretory activity.

Reduced social interaction and ultrasonic communication in a mouse model of monogenic heritable autism.

Autism spectrum conditions (ASCs) are heritable conditions characterized by impaired reciprocal social interactions, deficits in language acquisition, and repetitive and restricted behaviors and interests. In addition to more complex genetic susceptibilities, even mutation of a single gene can lead to ASC. Several such monogenic heritable ASC forms are caused by loss-of-function mutations in genes encoding regulators of synapse function in neurons, including NLGN4. We report that mice with a loss-of-function mutation in the murine NLGN4 ortholog Nlgn4, which encodes the synaptic cell adhesion protein Neuroligin-4, exhibit highly selective deficits in reciprocal social interactions and communication that are reminiscent of ASCs in humans. Our findings indicate that a protein network that regulates the maturation and function of synapses in the brain is at the core of a major ASC susceptibility pathway, and establish Neuroligin-4-deficient mice as genetic models for the exploration of the complex neurobiological disorders in ASCs.

Complexins Regulate a Late Step in Ca2+-Dependent Neurotransmitter Release

Synaptic vesicle fusion at synapses is triggered by increases in cytosolic Ca2+ levels. However, the identity of the Ca2+ sensor and the transduction mechanism of the Ca2+ trigger are unknown. We show that Complexins, stoichiometric components of the exocytotic core complex, are important regulators of transmitter release at a step immediately preceding vesicle fusion. Neurons lacking Complexins show a dramatically reduced transmitter release efficiency due to decreased Ca2+ sensitivity of the synaptic secretion process. Analyses of mutant neurons demonstrate that Complexins are acting at or following the Ca2+-triggering step of fast synchronous transmitter release by regulating the exocytotic Ca2+ sensor, its interaction with the core complex fusion machinery, or the efficiency of the fusion apparatus itself.

Neuroligin 2 is exclusively localized to inhibitory synapses

Neuroligins are cell adhesion proteins that are thought to instruct the formation and alignment of synaptic specializations. The three known rodent neuroligin isoforms share homologous extracellular acetylcholinesterase-like domains that bridge the synaptic cleft and bind beta-neurexins. All neuroligins have identical intracellular C-terminal motifs that bind to PDZ domains of various target proteins. Neuroligin 1 is specifically localized to glutamatergic postsynaptic specializations. We show here that neuroligin 2 is exclusively localized to inhibitory synapses in rat brain and dissociated neurons. In immature neurons, neuroligin 2 is found at synapses and also at GABAA receptor aggregates that are not facing presynaptic termini, indicating that postsynaptic mechanisms lead to synaptic recruitment of neuroligin 2. Our findings identify neuroligin 2 as a new cell adhesion protein specific for inhibitory synapses and open new avenues for identifiying the constituents of this unique type of postsynaptic specialization.

Munc13-1 Is a Presynaptic Phorbol Ester Receptor that Enhances Neurotransmitter Release

Munc13-1, a mammalian homolog of C. elegans unc-13p, is thought to be involved in the regulation of synaptic transmission. We now demonstrate that Munc13-1 is a presynaptic high-affinity phorbol ester and diacylglycerol receptor with ligand affinities similar to those of protein kinase C. Munc13-1 associates with the plasma membrane in response to phorbol ester binding and acts as a phorbol ester-dependent enhancer of transmitter release when overexpressed presynaptically in the Xenopus neuromuscular junction. These observations establish Munc13-1 as a novel presynaptic target of the diacylglycerol second messenger pathway that acts in parallel with protein kinase C to regulate neurotransmitter secretion.