GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles.

GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca2+-dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the "Flash and Freeze-fracture" method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals.

Pre- and postsynaptic nanostructures increase in size and complexity after induction of long-term potentiation.

Synapses, specialized contact sites between neurons, are the fundamental elements of neuronal information transfer. Synaptic plasticity involves changes in synaptic morphology and the number of neurotransmitter receptors, and is thought to underlie learning and memory. However, it is not clear how these structural and functional changes are connected. We utilized time-lapse super-resolution STED microscopy of organotypic hippocampal brain slices and cultured neurons to visualize structural changes of the synaptic nano-organization of the postsynaptic scaffolding protein PSD95, the presynaptic scaffolding protein Bassoon, and the GluA2 subunit of AMPA receptors by chemically induced long-term potentiation (cLTP) at the level of single synapses. We found that the nano-organization of all three proteins increased in complexity and size after cLTP induction. The increase was largely synchronous, peaking at ∼60 min after stimulation. Therefore, both the size and complexity of individual pre- and post-synaptic nanostructures serve as substrates for tuning and determining synaptic strength.

Nedd4-2-dependent regulation of astrocytic Kir4.1 and Connexin43 controls neuronal network activity.

Nedd4-2 is an E3 ubiquitin ligase in which missense mutation is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of γ-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of the Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy.

S-SCAM is essential for synapse formation.

Synapse formation is critical for the wiring of neural circuits in the developing brain. The synaptic scaffolding protein S-SCAM/MAGI-2 has important roles in the assembly of signaling complexes at post-synaptic densities. However, the role of S-SCAM in establishing the entire synapse is not known. Here, we report significant effects of RNAi-induced S-SCAM knockdown on the number of synapses in early stages of network development in vitro. In vivo knockdown during the first three postnatal weeks reduced the number of dendritic spines in the rat brain neocortex. Knockdown of S-SCAM in cultured hippocampal neurons severely reduced the clustering of both pre- and post-synaptic components. This included synaptic vesicle proteins, pre- and post-synaptic scaffolding proteins, and cell adhesion molecules, suggesting that entire synapses fail to form. Correspondingly, functional and morphological characteristics of developing neurons were affected by reducing S-SCAM protein levels; neurons displayed severely impaired synaptic transmission and reduced dendritic arborization. A next-generation sequencing approach showed normal expression of housekeeping genes but changes in expression levels in 39 synaptic signaling molecules in cultured neurons. These results indicate that S-SCAM mediates the recruitment of all key classes of synaptic molecules during synapse assembly and is critical for the development of neural circuits in the developing brain.

An intellectual-disability-associated mutation of the transcriptional regulator NACC1 impairs glutamatergic neurotransmission.

Advances in genome sequencing technologies have favored the identification of rare de novo mutations linked to neurological disorders in humans. Recently, a de novo autosomal dominant mutation in NACC1 was identified (NM_052876.3: c.892C > T, NP_443108.1; p.Arg298Trp), associated with severe neurological symptoms including intellectual disability, microcephaly, and epilepsy. As NACC1 had never before been associated with neurological diseases, we investigated how this mutation might lead to altered brain function. We examined neurotransmission in autaptic glutamatergic mouse neurons expressing the murine homolog of the human mutant NACC1, i.e., Nacc1-R284W. We observed that expression of Nacc1-R284W impaired glutamatergic neurotransmission in a cell-autonomous manner, likely through a dominant negative mechanism. Furthermore, by screening for Nacc1 interaction targets in the brain, we identified SynGAP1, GluK2A, and several SUMO E3 ligases as novel Nacc1 interaction partners. At a biochemical level, Nacc1-R284W exhibited reduced binding to SynGAP1 and GluK2A, and also showed greatly increased SUMOylation. Ablating the SUMOylation of Nacc1-R284W partially restored its interaction with SynGAP1 but did not restore binding to GluK2A. Overall, these data indicate a role for Nacc1 in regulating glutamatergic neurotransmission, which is substantially impaired by the expression of a disease-associated Nacc1 mutant. This study provides the first functional insights into potential deficits in neuronal function in patients expressing the de novo mutant NACC1 protein.

CADPS functional mutations in patients with bipolar disorder increase the sensitivity to stress.

Bipolar disorder is a severe and chronic psychiatric disease resulting from a combination of genetic and environmental risk factors. Here, we identified a significant higher mutation rate in a gene encoding the calcium-dependent activator protein for secretion (CADPS) in 132 individuals with bipolar disorder, when compared to 184 unaffected controls or to 21,070 non-psychiatric and non-Finnish European subjects from the Exome Aggregation Consortium. We found that most of these variants resulted either in a lower abundance or a partial impairment in one of the basic functions of CADPS in regulating neuronal exocytosis, synaptic plasticity and vesicular transporter-dependent uptake of catecholamines. Heterozygous mutant mice for Cadps+/- revealed that a decreased level of CADPS leads to manic-like behaviours, changes in BDNF level and a hypersensitivity to stress. This was consistent with more childhood trauma reported in families with mutation in CADPS, and more specifically in mutated individuals. Furthermore, hyperactivity observed in mutant animals was rescued by the mood-stabilizing drug lithium. Overall, our results suggest that dysfunction in calcium-dependent vesicular exocytosis may increase the sensitivity to environmental stressors enhancing the risk of developing bipolar disorder.

Snapin Specifically Up-Regulates Cav1.3 Ca2+ Channel Variant with a Long Carboxyl Terminus.

Ca2+ entry through Cav1.3 Ca2+ channels plays essential roles in diverse physiological events. We employed yeast-two-hybrid (Y2H) assays to mine novel proteins interacting with Cav1.3 and found Snapin2, a synaptic protein, as a partner interacting with the long carboxyl terminus (CTL) of rat Cav1.3L variant. Co-expression of Snapin with Cav1.3L/Cavß3/α2δ2 subunits increased the peak current density or amplitude by about 2-fold in HEK-293 cells and Xenopus oocytes, without affecting voltage-dependent gating properties and calcium-dependent inactivation. However, the Snapin up-regulation effect was not found for rat Cav1.3S containing a short CT (CTS) in which a Snapin interaction site in the CTL was deficient. Luminometry and electrophysiology studies uncovered that Snapin co-expression did not alter the membrane expression of HA tagged Cav1.3L but increased the slope of tail current amplitudes plotted against ON-gating currents, indicating that Snapin increases the opening probability of Cav1.3L. Taken together, our results strongly suggest that Snapin directly interacts with the CTL of Cav1.3L, leading to up-regulation of Cav1.3L channel activity via facilitating channel opening probability.

The α3 subunit of GABAA receptors promotes formation of inhibitory synapses in the absence of collybistin.

Signaling at nerve cell synapses is a key determinant of proper brain function, and synaptic defects-or synaptopathies-are at the basis of many neurological and psychiatric disorders. Collybistin (CB), a brain-specific guanine nucleotide exchange factor, is essential for the formation of γ-aminobutyric acidergic (GABAergic) postsynapses in defined regions of the mammalian forebrain, including the hippocampus and basolateral amygdala. This process depends on a direct interaction of CB with the scaffolding protein gephyrin, which leads to the redistribution of gephyrin into submembranous clusters at nascent inhibitory synapses. Strikingly, synaptic clustering of gephyrin and GABAA type A receptors (GABAARs) in several brain regions, including the cerebral cortex and certain thalamic areas, is unperturbed in CB-deficient mice, indicating that the formation of a substantial subset of inhibitory postsynapses must be controlled by gephyrin-interacting proteins other than CB. Previous studies indicated that the α3 subunit of GABAARs (GABAAR-α3) binds directly and with high affinity to gephyrin. Here, we provide evidence (i) that a homooligomeric GABAAR-α3A343W mutant induces the formation of submembranous gephyrin clusters independently of CB in COS-7 cells, (ii) that gephyrin clustering is unaltered in the neuronal subpopulations endogenously expressing the GABAAR-α3 in CB-deficient brains, and (iii) that exogenous expression of GABAAR-α3 partially rescues impaired gephyrin clustering in CB-deficient hippocampal neurons. Our results identify an important role of GABAAR-α3 in promoting gephyrin-mediated and CB-independent formation of inhibitory postsynapses.

The murine ortholog of Kaufman oculocerebrofacial syndrome protein Ube3b regulates synapse number by ubiquitinating Ppp3cc.

Kaufman oculocerebrofacial syndrome (KOS) is a severe autosomal recessive disorder characterized by intellectual disability, developmental delays, microcephaly, and characteristic dysmorphisms. Biallelic mutations of UBE3B, encoding for a ubiquitin ligase E3B are causative for KOS. In this report, we characterize neuronal functions of its murine ortholog Ube3b and show that Ube3b regulates dendritic branching in a cell-autonomous manner. Moreover, Ube3b knockout (KO) neurons exhibit increased density and aberrant morphology of dendritic spines, altered synaptic physiology, and changes in hippocampal circuit activity. Dorsal forebrain-specific Ube3b KO animals show impaired spatial learning, altered social interactions, and repetitive behaviors. We further demonstrate that Ube3b ubiquitinates the catalytic γ-subunit of calcineurin, Ppp3cc, the overexpression of which phenocopies Ube3b loss with regard to dendritic spine density. This work provides insights into the molecular pathologies underlying intellectual disability-like phenotypes in a genetically engineered mouse model.

PTPσ Controls Presynaptic Organization of Neurotransmitter Release Machinery at Excitatory Synapses.

Leukocyte common antigen-related receptor tyrosine phosphatases (LAR-RPTPs) are evolutionarily conserved presynaptic organizers. The synaptic role of vertebrate LAR-RPTPs in vivo, however, remains unclear. In the current study, we analyzed the synaptic role of PTPσ using newly generated, single conditional knockout (cKO) mice targeting PTPσ. We found that the number of synapses was reduced in PTPσ cKO cultured neurons in association with impaired excitatory synaptic transmission, abnormal vesicle localization, and abnormal synaptic ultrastructure. Strikingly, loss of presynaptic PTPσ reduced neurotransmitter release prominently at excitatory synapses, concomitant with drastic reductions in excitatory innervations onto postsynaptic target areas in vivo. Furthermore, loss of presynaptic PTPσ in hippocampal CA1 pyramidal neurons had no impact on postsynaptic glutamate receptor responses in subicular pyramidal neurons. Postsynaptic PTPσ deletion had no effect on excitatory synaptic strength. Taken together, these results demonstrate that PTPσ is a bona fide presynaptic adhesion molecule that controls neurotransmitter release and excitatory inputs.

The Synaptic Vesicle Priming Protein CAPS-1 Shapes the Adaptation of Sensory Evoked Responses in Mouse Visual Cortex.

Short-term plasticity gates information transfer across neuronal synapses and is thought to be involved in fundamental brain processes, such as cortical gain control and sensory adaptation. Neurons employ synaptic vesicle priming proteins of the CAPS and Munc13 families to shape short-term plasticity in vitro, but the relevance of this phenomenon for information processing in the intact brain is unknown. By combining sensory stimulation with in vivo patch-clamp recordings in anesthetized mice, we show that genetic deletion of CAPS-1 in thalamic neurons results in more rapid adaptation of sensory-evoked subthreshold responses in layer 4 neurons of the primary visual cortex. Optogenetic experiments in acute brain slices further reveal that the enhanced adaptation is caused by more pronounced short-term synaptic depression. Our data indicate that neurons engage CAPS-family priming proteins to shape short-term plasticity for optimal sensory information transfer between thalamic and cortical neurons in the intact brain in vivo.

Ultrastructural Correlates of Presynaptic Functional Heterogeneity in Hippocampal Synapses.

Although similar in molecular composition, synapses can exhibit strikingly distinct functional transmitter release and plasticity characteristics. To determine whether ultrastructural differences co-define this functional heterogeneity, we combine hippocampal organotypic slice cultures, high-pressure freezing, freeze substitution, and 3D-electron tomography to compare two functionally distinct synapses: hippocampal Schaffer collateral and mossy fiber synapses. We find that mossy fiber synapses, which exhibit a lower release probability and stronger short-term facilitation than Schaffer collateral synapses, harbor lower numbers of docked synaptic vesicles at active zones and a second pool of possibly tethered vesicles in their vicinity. Our data indicate that differences in the ratio of docked versus tethered vesicles at active zones contribute to distinct functional characteristics of synapses.

The Calmodulin Binding Region of the Synaptic Vesicle Protein Mover Is Required for Homomeric Interaction and Presynaptic Targeting.

Neurotransmitter release is mediated by an evolutionarily conserved machinery. The synaptic vesicle (SV) associated protein Mover/TPRGL/SVAP30 does not occur in all species and all synapses. Little is known about its molecular properties and how it may interact with the conserved components of the presynaptic machinery. Here, we show by deletion analysis that regions required for homomeric interaction of Mover are distributed across the entire molecule, including N-terminal, central and C-terminal regions. The same regions are also required for the accumulation of Mover in presynaptic terminals of cultured neurons. Mutating two phosphorylation sites in N-terminal regions did not affect these properties. In contrast, a point mutation in the predicted Calmodulin (CaM) binding sequence of Mover abolished both homomeric interaction and presynaptic targeting. We show that this sequence indeed binds Calmodulin, and that recombinant Mover increases Calmodulin signaling upon heterologous expression. Our data suggest that presynaptic accumulation of Mover requires homomeric interaction mediated by regions distributed across large areas of the protein, and corroborate the hypothesis that Mover functionally interacts with Calmodulin signaling.

Molecular Dissection of Neurobeachin Function at Excitatory Synapses.

Spines are small protrusions from dendrites where most excitatory synapses reside. Changes in number, shape, and size of dendritic spines often reflect changes of neural activity in entire circuits or at individual synapses, making spines key structures of synaptic plasticity. Neurobeachin is a multidomain protein with roles in spine formation, postsynaptic neurotransmitter receptor targeting and actin distribution. However, the contributions of individual domains of Neurobeachin to these functions is poorly understood. Here, we used mostly live cell imaging and patch-clamp electrophysiology to monitor morphology and function of spinous synapses in primary hippocampal neurons. We demonstrate that a recombinant full-length Neurobeachin from humans can restore mushroom spine density and excitatory postsynaptic currents in neurons of Neurobeachin-deficient mice. We then probed the role of individual domains of Neurobeachin by comparing them to the full-length molecule in rescue experiments of knockout neurons. We show that the combined PH-BEACH domain complex is highly localized in spine heads, and that it is sufficient to restore normal spine density and surface targeting of postsynaptic AMPA receptors. In addition, we report that the Armadillo domain facilitates the formation of filopodia, long dendritic protrusions which often precede the development of mature spines, whereas the PKA-binding site appears as a negative regulator of filopodial extension. Thus, our results indicate that individual domains of Neurobeachin sustain important and specific roles in the regulation of spinous synapses. Since heterozygous mutations in Neurobeachin occur in autistic patients, the results will also improve our understanding of pathomechanism in neuropsychiatric disorders associated with impairments of spine function.

Molecular Mechanisms of Synaptic Vesicle Priming by Munc13 and Munc18.

Munc13 catalyzes the transit of syntaxin from a closed complex with Munc18 into the ternary SNARE complex. Here we report a new function of Munc13, independent of Munc18: it promotes the proper syntaxin/synaptobrevin subconfiguration during assembly of the ternary SNARE complex. In cooperation with Munc18, Munc13 additionally ensures the proper syntaxin/SNAP-25 subconfiguration. In a reconstituted fusion assay with SNAREs, complexin, and synaptotagmin, inclusion of both Munc13 and Munc18 quadruples the Ca2+-triggered amplitude and achieves Ca2+ sensitivity at near-physiological concentrations. In Munc13-1/2 double-knockout neurons, expression of a constitutively open mutant of syntaxin could only minimally restore neurotransmitter release relative to Munc13-1 rescue. Together, the physiological functions of Munc13 may be related to regulation of proper SNARE complex assembly.

Increased Excitatory Synaptic Transmission of Dentate Granule Neurons in Mice Lacking PSD-95-Interacting Adhesion Molecule Neph2/Kirrel3 during the Early Postnatal Period.

Copy number variants and point mutations of NEPH2 (also called KIRREL3) gene encoding an immunoglobulin (Ig) superfamily adhesion molecule have been linked to autism spectrum disorders, intellectual disability and neurocognitive delay associated with Jacobsen syndrome, but the physiological roles of Neph2 in the mammalian brain remain largely unknown. Neph2 is highly expressed in the dentate granule (DG) neurons of the hippocampus and is localized in both dendrites and axons. It was recently shown that Neph2 is required for the formation of mossy fiber filopodia, the axon terminal structure of DG neurons forming synapses with GABAergic neurons of CA3. In contrast, however, it is unknown whether Neph2 also has any roles in the postsynaptic compartments of DG neurons. We here report that, through its C-terminal PDZ domain-binding motif, Neph2 directly interacts with postsynaptic density (PSD)-95, an abundant excitatory postsynaptic scaffolding protein. Moreover, Neph2 protein is detected in the brain PSD fraction and interacts with PSD-95 in synaptosomal lysates. Functionally, loss of Neph2 in mice leads to age-specific defects in the synaptic connectivity of DG neurons. Specifically, Neph2-/- mice show significantly increased spontaneous excitatory synaptic events in DG neurons at postnatal week 2 when the endogenous Neph2 protein expression peaks, but show normal excitatory synaptic transmission at postnatal week 3. The evoked excitatory synaptic transmission and synaptic plasticity of medial perforant pathway (MPP)-DG synapses are also normal in Neph2-/- mice at postnatal week 3, further confirming the age-specific synaptic defects. Together, our results provide some evidence for the postsynaptic function of Neph2 in DG neurons during the early postnatal period, which might be implicated in neurodevelopmental and cognitive disorders caused by NEPH2 mutations.

Formation and Maintenance of Functional Spines in the Absence of Presynaptic Glutamate Release.

Dendritic spines are the major transmitter reception compartments of glutamatergic synapses in most principal neurons of the mammalian brain and play a key role in the function of nerve cell circuits. The formation of functional spine synapses is thought to be critically dependent on presynaptic glutamatergic signaling. By analyzing CA1 pyramidal neurons in mutant hippocampal slice cultures that are essentially devoid of presynaptic transmitter release, we demonstrate that the formation and maintenance of dendrites and functional spines are independent of synaptic glutamate release.

ELKS1 localizes the synaptic vesicle priming protein bMunc13-2 to a specific subset of active zones.

Presynaptic active zones (AZs) are unique subcellular structures at neuronal synapses, which contain a network of specific proteins that control synaptic vesicle (SV) tethering, priming, and fusion. Munc13s are core AZ proteins with an essential function in SV priming. In hippocampal neurons, two different Munc13s-Munc13-1 and bMunc13-2-mediate opposite forms of presynaptic short-term plasticity and thus differentially affect neuronal network characteristics. We found that most presynapses of cortical and hippocampal neurons contain only Munc13-1, whereas ∼10% contain both Munc13-1 and bMunc13-2. Whereas the presynaptic recruitment and activation of Munc13-1 depends on Rab3-interacting proteins (RIMs), we demonstrate here that bMunc13-2 is recruited to synapses by the AZ protein ELKS1, but not ELKS2, and that this recruitment determines basal SV priming and short-term plasticity. Thus, synapse-specific interactions of different Munc13 isoforms with ELKS1 or RIMs are key determinants of the molecular and functional heterogeneity of presynaptic AZs.

Synaptic UNC13A protein variant causes increased neurotransmission and dyskinetic movement disorder.

Munc13 proteins are essential regulators of neurotransmitter release at nerve cell synapses. They mediate the priming step that renders synaptic vesicles fusion-competent, and their genetic elimination causes a complete block of synaptic transmission. Here we have described a patient displaying a disorder characterized by a dyskinetic movement disorder, developmental delay, and autism. Using whole-exome sequencing, we have shown that this condition is associated with a rare, de novo Pro814Leu variant in the major human Munc13 paralog UNC13A (also known as Munc13-1). Electrophysiological studies in murine neuronal cultures and functional analyses in Caenorhabditis elegans revealed that the UNC13A variant causes a distinct dominant gain of function that is characterized by increased fusion propensity of synaptic vesicles, which leads to increased initial synaptic vesicle release probability and abnormal short-term synaptic plasticity. Our study underscores the critical importance of fine-tuned presynaptic control in normal brain function. Further, it adds the neuronal Munc13 proteins and the synaptic vesicle priming process that they control to the known etiological mechanisms of psychiatric and neurological synaptopathies.