G protein ß subunits regulate Cav3.3 T-type channel activity and current kinetics via interaction with the Cav3.3 C-terminus.

Ca2+ influx through Cav3.3 T-type channel plays crucial roles in neuronal excitability and is subject to regulation by various signaling molecules. However, our understanding of the partners of Cav3.3 and the related regulatory pathways remains largely limited. To address this quest, we employed the rat Cav3.3 C-terminus as bait in yeast-two-hybrid screenings of a cDNA library, identifying rat Gß2 as an interaction partner. Subsequent assays revealed that the interaction of Gß2 subunit was specific to the Cav3.3 C-terminus. Through systematic dissection of the C-terminus, we pinpointed a 22 amino acid sequence (amino acids 1789-1810) as the Gß2 interaction site. Coexpression studies of rat Cav3.3 with various Gßγ compositions were conducted in HEK-293 cells. Patch clamp recordings revealed that coexpression of Gß2γ2 reduced Cav3.3 current density and accelerated inactivation kinetics. Interestingly, the effects were not unique to Gß2γ2, but were mimicked by Gß2 alone as well as other Gßγ dimers, with similar potencies. Deletion of the Gß2 interaction site abolished the effects of Gß2γ2. Importantly, these Gß2 effects were reproduced in human Cav3.3. Overall, our findings provide evidence that Gß(γ) complexes inhibit Cav3.3 channel activity and accelerate the inactivation kinetics through the Gß interaction with the Cav3.3 C-terminus.

Skewed distribution of spines is independent of presynaptic transmitter release and synaptic plasticity, and emerges early during adult neurogenesis.

Dendritic spines are crucial for excitatory synaptic transmission as the size of a spine head correlates with the strength of its synapse. The distribution of spine head sizes follows a lognormal-like distribution with more small spines than large ones. We analysed the impact of synaptic activity and plasticity on the spine size distribution in adult-born hippocampal granule cells from rats with induced homo- and heterosynaptic long-term plasticity in vivo and CA1 pyramidal cells from Munc13-1/Munc13-2 knockout mice with completely blocked synaptic transmission. Neither the induction of extrinsic synaptic plasticity nor the blockage of presynaptic activity degrades the lognormal-like distribution but changes its mean, variance and skewness. The skewed distribution develops early in the life of the neuron. Our findings and their computational modelling support the idea that intrinsic synaptic plasticity is sufficient for the generation, while a combination of intrinsic and extrinsic synaptic plasticity maintains lognormal-like distribution of spines.

Molecular and functional architecture of striatal dopamine release sites.

Despite the importance of dopamine for striatal circuit function, mechanistic understanding of dopamine transmission remains incomplete. We recently showed that dopamine secretion relies on the presynaptic scaffolding protein RIM, indicating that it occurs at active zone-like sites similar to classical synaptic vesicle exocytosis. Here, we establish using a systematic gene knockout approach that Munc13 and Liprin-α, active zone proteins for vesicle priming and release site organization, are important for dopamine secretion. Furthermore, RIM zinc finger and C2B domains, which bind to Munc13 and Liprin-α, respectively, are needed to restore dopamine release after RIM ablation. In contrast, and different from typical synapses, the active zone scaffolds RIM-BP and ELKS, and RIM domains that bind to them, are expendable. Hence, dopamine release necessitates priming and release site scaffolding by RIM, Munc13, and Liprin-α, but other active zone proteins are dispensable. Our work establishes that efficient release site architecture mediates fast dopamine exocytosis.

Anesthesia triggers drug delivery to experimental glioma in mice by hijacking caveolar transport.

BACKGROUND: Pharmaceutical intervention in the CNS is hampered by the shielding function of the blood-brain barrier (BBB). To induce clinical anesthesia, general anesthetics such as isoflurane readily penetrate the BBB. Here, we investigated whether isoflurane can be utilized for therapeutic drug delivery. METHODS: Barrier function in primary endothelial cells was evaluated by transepithelial/transendothelial electrical resistance, and nanoscale STED and SRRF microscopy. In mice, BBB permeability was quantified by extravasation of several fluorescent tracers. Mouse models including the GL261 glioma model were evaluated by MRI, immunohistochemistry, electron microscopy, western blot, and expression analysis. RESULTS: Isoflurane enhances BBB permeability in a time- and concentration-dependent manner. We demonstrate that, mechanistically, isoflurane disturbs the organization of membrane lipid nanodomains and triggers caveolar transport in brain endothelial cells. BBB tightness re-establishes directly after termination of anesthesia, providing a defined window for drug delivery. In a therapeutic glioblastoma trial in mice, simultaneous exposure to isoflurane and cytotoxic agent improves efficacy of chemotherapy. CONCLUSIONS: Combination therapy, involving isoflurane-mediated BBB permeation with drug administration has far-reaching therapeutic implications for CNS malignancies.

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.

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.

SUMOylation controls the neurodevelopmental function of the transcription factor Zbtb20.

SUMOylation is a dynamic post-translational protein modification that primarily takes place in cell nuclei, where it plays a key role in multiple DNA-related processes. In neurons, the SUMOylation-dependent control of a subset of neuronal transcription factors is known to regulate various aspects of nerve cell differentiation, development, and function. In an unbiased screen for endogenous SUMOylation targets in the developing mouse brain, based on a His6 -HA-SUMO1 knock-in mouse line, we previously identified the transcription factor Zinc finger and BTB domain-containing 20 (Zbtb20) as a new SUMO1-conjugate. We show here that the three key SUMO paralogues SUMO1, SUMO2, and SUMO3 can all be conjugated to Zbtb20 in vitro in HEK293FT cells, and we confirm the SUMOylation of Zbtb20 in vivo in mouse brain. Using primary hippocampal neurons from wild-type and Zbtb20 knock-out (KO) mice as a model system, we then demonstrate that the expression of Zbtb20 is required for proper nerve cell development and neurite growth and branching. Furthermore, we show that the SUMOylation of Zbtb20 is essential for its function in this context, and provide evidence indicating that SUMOylation affects the Zbtb20-dependent transcriptional profile of neurons. Our data highlight the role of SUMOylation in the regulation of neuronal transcription factors that determine nerve cell development, and they demonstrate that key functions of the transcription factor Zbtb20 in neuronal development and neurite growth are under obligatory SUMOylation control.

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.

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.

An Autaptic Culture System for Standardized Analyses of iPSC-Derived Human Neurons.

iPSC-derived human neurons are expected to revolutionize studies on brain diseases, but their functional heterogeneity still poses a problem. Key sources of heterogeneity are the different cell culture systems used. We show that an optimized autaptic culture system, with single neurons on astrocyte feeder islands, is well suited to culture, and we analyze human iPSC-derived neurons in a standardized, systematic, and reproducible manner. Using classically differentiated and transcription factor-induced human glutamatergic and GABAergic neurons, we demonstrate that key features of neuronal morphology and function, including dendrite structure, synapse number, membrane properties, synaptic transmission, and short-term plasticity, can be assessed with substantial throughput and reproducibility. We propose our optimized autaptic culture system as a tool to study functional features of human neurons, particularly in the context of disease phenotypes and experimental therapy.

α-synuclein interacts with PrPC to induce cognitive impairment through mGluR5 and NMDAR2B.

Synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies, are neurodegenerative disorders that are characterized by the accumulation of α-synuclein (aSyn) in intracellular inclusions known as Lewy bodies. Prefibrillar soluble aSyn oligomers, rather than larger inclusions, are currently considered to be crucial species underlying synaptic dysfunction. We identified the cellular prion protein (PrPC) as a key mediator in aSyn-induced synaptic impairment. The aSyn-associated impairment of long-term potentiation was blocked in Prnp null mice and rescued following PrPC blockade. We found that extracellular aSyn oligomers formed a complex with PrPC that induced the phosphorylation of Fyn kinase via metabotropic glutamate receptors 5 (mGluR5). aSyn engagement of PrPC and Fyn activated NMDA receptor (NMDAR) and altered calcium homeostasis. Blockade of mGluR5-evoked phosphorylation of NMDAR in aSyn transgenic mice rescued synaptic and cognitive deficits, supporting the hypothesis that a receptor-mediated mechanism, independent of pore formation and membrane leakage, is sufficient to trigger early synaptic damage induced by extracellular aSyn.

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.

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.

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.

Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses.

The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the proper assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic postsynapses requires the scaffold protein gephyrin and the guanine nucleotide exchange factor collybistin (Cb). In vitro, the pleckstrin homology domain of Cb binds phosphoinositides, specifically phosphatidylinositol 3-phosphate (PI3P). However, whether PI3P is required for inhibitory postsynapse formation is currently unknown. Here, we investigated the role of PI3P at developing GABAergic postsynapses by using a membrane-permeant PI3P derivative, time-lapse confocal imaging, electrophysiology, as well as knockdown and overexpression of PI3P-metabolizing enzymes. Our results provide the first in cellula evidence that PI3P located at early/sorting endosomes regulates the postsynaptic clustering of gephyrin and GABAA receptors and the strength of inhibitory, but not excitatory, postsynapses in cultured hippocampal neurons. In human embryonic kidney 293 cells, stimulation of gephyrin cluster formation by PI3P depends on Cb. We therefore conclude that the endosomal pool of PI3P, generated by the class III phosphatidylinositol 3-kinase, is important for the Cb-mediated recruitment of gephyrin and GABAA receptors to developing inhibitory postsynapses and thus the formation of postsynaptic membrane specializations.

Photoswitchable diacylglycerols enable optical control of protein kinase C.

Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling.

Complexin 3 Increases the Fidelity of Signaling in a Retinal Circuit by Regulating Exocytosis at Ribbon Synapses.

Complexin (Cplx) proteins modulate the core SNARE complex to regulate exocytosis. To understand the contributions of Cplx to signaling in a well-characterized neural circuit, we investigated how Cplx3, a retina-specific paralog, shapes transmission at rod bipolar (RB)→AII amacrine cell synapses in the mouse retina. Knockout of Cplx3 strongly attenuated fast, phasic Ca(2+)-dependent transmission, dependent on local [Ca(2+)] nanodomains, but enhanced slower Ca(2+)-dependent transmission, dependent on global intraterminal [Ca(2+)] ([Ca(2+)]I). Surprisingly, coordinated multivesicular release persisted at Cplx3(-/-) synapses, although its onset was slowed. Light-dependent signaling at Cplx3(-/-) RB→AII synapses was sluggish, owing largely to increased asynchronous release at light offset. Consequently, propagation of RB output to retinal ganglion cells was suppressed dramatically. Our study links Cplx3 expression with synapse and circuit function in a specific retinal pathway and reveals a role for asynchronous release in circuit gain control.

Synaptic adhesion molecule IgSF11 regulates synaptic transmission and plasticity.

Synaptic adhesion molecules regulate synapse development and plasticity through mechanisms that include trans-synaptic adhesion and recruitment of diverse synaptic proteins. We found that the immunoglobulin superfamily member 11 (IgSF11), a homophilic adhesion molecule that preferentially expressed in the brain, is a dual-binding partner of the postsynaptic scaffolding protein PSD-95 and AMPA glutamate receptors (AMPARs). IgSF11 required PSD-95 binding for its excitatory synaptic localization. In addition, IgSF11 stabilized synaptic AMPARs, as determined by IgSF11 knockdown-induced suppression of AMPAR-mediated synaptic transmission and increased surface mobility of AMPARs, measured by high-throughput, single-molecule tracking. IgSF11 deletion in mice led to the suppression of AMPAR-mediated synaptic transmission in the dentate gyrus and long-term potentiation in the CA1 region of the hippocampus. IgSF11 did not regulate the functional characteristics of AMPARs, including desensitization, deactivation or recovery. These results suggest that IgSF11 regulates excitatory synaptic transmission and plasticity through its tripartite interactions with PSD-95 and AMPARs.