Biology subjects

Brose, N.

Publications and source records attributed to Brose, N..

29 records · Page 2Linked to original sources

bioRxiv2022

A two-pool mechanism of vesicle release in medial habenula terminals underlies GABAB receptor-mediated potentiation

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 induces a transition from tonic to phasic release accompanied by a 4-fold increase in readily releasable pool (RRP) size in MHb terminals, mirrored by a similar increase in the docked vesicle number at the presynaptic active zone (AZ). The tonic and phasic release vesicles have distinct coupling distances. We identified two vesicle-associated molecules, synaptoporin and CAPS2, selectively involved in tonic and phasic release, respectively. Synaptoporin mediates augmentation of tonic release and CAPS2 stabilizes readily releasable vesicles during phasic release. A newly developed "Flash and Freeze-fracture" method revealed selective recruitment of CAPS2 to the AZ during phasic release. Thus, we propose a novel two-pool mechanism underlying the GBR-mediated potentiation of release from MHb terminals.

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bioRxiv2022

Characterizing the differential distribution and targets of Sumo paralogs in the mouse brain

SUMOylation is an evolutionarily conserved and essential mechanism whereby Small Ubiquitin Like Modifiers, or SUMO proteins (Sumo in mice), are covalently bound to protein substrates in a highly dynamic and reversible manner. SUMOylation is involved in a variety of basic neurological processes including learning and memory, and central nervous system development, but is also linked with neurological disorders. However, studying SUMOylation in vivo remains challenging due to limited tools to study Sumo proteins and their targets in their native context. More complexity arises from the fact that Sumo1 and Sumo2 are [~]50% homologous, whereas Sumo2 and Sumo3 are nearly identical and indistinguishable with antibodies. While Sumo paralogues can compensate for one anothers loss, Sumo2 is highest expressed and only paralog essential for embryonic development making it critical to uncover roles specific to Sumo2 in vivo. To further examine the roles of Sumo2, and to begin to tease apart the redundancy and similarity between key Sumo paralogs, we generated (His6-)HA epitope-tagged Sumo2 knock-in mouse alleles, expanding the current Sumo knock-in mouse tool-kit comprising of the previously generated His6-HA-Sumo1 knock-in model. Using these HA-Sumo mouse lines, we performed whole brain imaging and mapping to the Allen Brain Atlas to analyze the relative distribution of the Sumo1 and Sumo2 paralogues in the adult mouse brain. We observed differential staining patterns between Sumo1 and Sumo2, including a partial localization of Sumo2 in nerve cell synapses of the hippocampus. Combining immunoprecipitation with mass spectrometry, we identified native substrates targeted by Sumo1 or Sumo2 in the mouse brain. We validated select hits using proximity ligation assays, further providing insight into the subcellular distribution of neuronal Sumo2-conjugates. These mouse models thus serve as valuable tools to study the cellular and biochemical roles of SUMOylation in the central nervous system.

neuroscience↗

bioRxiv2022

Functional Neuroligin-2-MDGA1 interactions differentially regulate synaptic GABAARs and cytosolic gephyrin aggregation

AO_SCPLOWBSTRACTC_SCPLOWThe function of GABAergic synapses is critically shaped by cell adhesion proteins that recruit GABAARs to synapses and mediate transsynaptic signalling, but the synapse-type-specific function of such synaptic adhesion proteins and their mutual interaction remain incompletely understood. A ubiquitous cell adhesion protein at GABAergic synapses is Neuroligin-2 (Nlgn2), which recruits synaptic GABAARs by promoting the assembly of the postsynaptic gephyrin scaffold. While Nlgn2 is present at virtually all GABAergic synapses throughout the forebrain, its loss affects different GABAergic synapse subtypes with different severity, indicating that synapse-specific interactors and synapse-organizer-redundancies define the function of Nlgn2 for a given synapse type. Here we investigated how Nlgn2 function at GABAergic synapses in mouse hippocampal area CA1 is modulated by two recently identified interaction partners, MDGA1 and MDGA2. We show that Nlgn2 and MDGA1 colocalize most prominently in the stratum radiatum (S.R.) of area CA1, and that combined Nlgn2 and MDGA1 deletion causes a layer-specific exacerbation of the loss of gephyrin puncta in layer S.R. seen following Nlgn2 deletion. Intriguingly, combined Nlgn2 and MDGA1 deletion concurrently ameliorates the abnormal cytosolic gephyrin aggregation, the reduction in inhibitory synaptic transmission and the exacerbated anxiety-related behavior seen in Nlgn2 knockout (KO) mice. In contrast, heterozygous deletion of MDGA2 in Nlgn2 KO mice has only minor effects on gephyrin and GABAAR puncta and does not normalize cytosolic gephyrin aggregates, inhibitory synaptic transmission or anxiety-related behavior. Our data indicate that MDGA1, but not MDGA2, modulates Nlgn2 function, primarily by regulating the formation of cytosolic gephyrin aggregates. Given that both Nlgn2 and the MDGA family of proteins have been linked to psychiatric disorders, such as autism and schizophrenia, our data lead to the notion that abnormal gephyrin aggregation may contribute to the pathophysiology of these disorders, and that intervention with gephyrin aggregation could present a novel therapeutic strategy.

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bioRxiv2022

Phosphorylation Determines Whether Neuroligin-3 is at Excitatory or Inhibitory Synapses in Different Regions of the Brain

Neuroligin-3 is a postsynaptic adhesion molecule involved in development, function, and pathologies of synapses in the brain. It is a genetic cause of autism and a potent component of the tumor microenvironment in gliomas. There are four Neuroligins that operate at distinct synapse types, selectively interacting with presynaptic adhesion and postsynaptic scaffold proteins. We investigated the subcellular localization and scaffold specificities of synaptic Neuroligin-3 and demonstrate an unexpected pattern of localization to excitatory synapses in cortical areas, and inhibitory synapses in subcortical areas. Using phosphoproteomics, we identify Neuroligin-3-specific serine phosphorylation in cortex and hippocampus that obstructs a key binding site for inhibitory synapse scaffolds. Using in utero CRISPR/Cas9 knockout and replacement with phosphomimetic mutants, we demonstrate that phosphorylation at this site determines excitatory versus inhibitory synapse localization of Neuroligin-3 in vivo. Our data reveal a mechanism that differentially regulates the balance of Neuroligin-3 between excitatory and inhibitory synapses, adding to our emerging understanding of their role in the development of brain connectivity and associated pathologies.

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bioRxiv2022

Synaptic vesicle-bound molecular bridges organize sequential vesicle states along parallel pathways

Synaptic vesicle tethering, priming, and neurotransmitter release require a coordinated action of multiple protein complexes. While physiological experiments, interaction data, and structural studies of purified systems were essential for our understanding of the function of the individual complexes involved, they cannot combine high structural detail with the unperturbed organization of complexes within cells to resolve how the actions of individual complexes integrate. We employed cryo-electron tomography to simultaneously image multiple presynaptic protein complexes and lipids at molecular resolution in their native composition, conformation and environment. Our results argue that tethers comprising proteins Munc13 and SNAP25 differentially and spatially confine vesicles with single nanometer precision, define vesicle tethering states, and provide molecular mechanisms that guide vesicles towards fusion, which includes molecular priming by conversion to SNARE complex-dependent tethers. These findings present an example of a cellular function performed by an extended molecular assembly comprising multiple, molecularly diverse complexes.

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bioRxiv2022

Munc13 supports vesicle fusogenicity after disrupting active zone scaffolds and synaptic vesicle docking

Active zones consist of protein scaffolds that are tightly attached to the presynaptic plasma membrane. They dock and prime synaptic vesicles, couple them to Ca2+ entry, and target neurotransmitter release to postsynaptic receptor domains. Simultaneous RIM+ELKS ablation disrupts these scaffolds, abolishes vesicle docking and removes active zone-targeted Munc13, but some vesicles remain releasable. This enduring vesicular fusogenicity may be Munc13-independent or be mediated by non-active zone-anchored Munc13. We tested its Munc13-dependence by ablating Munc13-1 and Munc13-2 on top of RIM+ELKS in cultured hippocampal neurons. The hextuple knockout synapses lacked docked vesicles, but other ultrastructural features were near-normal despite the strong genetic manipulation. Removing Munc13 in addition to RIM+ELKS further impaired action potential-evoked release by decreasing the remaining pool of releasable vesicles. We conclude that Munc13 can support some fusogenicity without RIM and ELKS, and that presynaptic recruitment of Munc13, even without active zone-anchoring, suffices to generate some fusion-competent vesicles.

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bioRxiv2021

A fluorescent nanosensor paint reveals the heterogeneity of dopamine release from neurons at individual release sites

The neurotransmitter dopamine is released from discrete axonal structures called varicosities. Its release is essential in behaviour and is critically implicated in prevalent neuropsychiatric diseases. Existing dopamine detection methods are not able to detect and distinguish discrete dopamine release events from multiple varicosities. This prevents an understanding of how dopamine release is regulated across populations of discrete varicosities. Using a near infrared fluorescent (980 nm) dopamine nanosensor paint (AndromeDA), we show that action potential-evoked dopamine release is highly heterogeneous across release sites and also requires molecular priming. Using AndromeDA, we visualize dopamine release at up to 100 dopaminergic varicosities simultaneously within a single imaging field with high temporal resolution (15 images/s). We find that hotspots of dopamine release are highly heterogeneous and are detected at only ~17% of all varicosities. In neurons lacking Munc13 proteins, which prime synaptic vesicles, dopamine release is abolished during electrical stimulation, demonstrating that dopamine release requires vesicle priming. In summary, AndromeDA reveals the spatiotemporal organization of dopamine release.

neuroscience↗

bioRxiv2020

Molecular and functional architecture of striatal dopamine release sites

Dopamine controls striatal circuit function, but its transmission mechanisms are not well understood. We recently showed that dopamine secretion requires RIM, suggesting that it occurs at active zone-like sites similar to conventional synapses. Here, we establish using a systematic conditional gene knockout approach that Munc13 and Liprin-, active zone proteins for vesicle priming and release site organization, are important for dopamine secretion. Correspondingly, RIM zinc finger and C2B domains, which bind to Munc13 and Liprin-, respectively, are needed to restore dopamine release in RIM knockout mice. In contrast, and different from conventional synapses, the active zone scaffolds RIM-BP and ELKS, and the RIM domains that bind to them, are expendable. Hence, dopamine release necessitates priming and release site scaffolding by RIM, Munc13, and Liprin-, but other active zone proteins are dispensable. Our work establishes that molecularly simple but efficient release site architecture mediates fast dopamine exocytosis.

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bioRxiv2020

Synaptotagmin-7 places dense-core vesicles at the cell membrane to promote Munc13-2- and Ca2+-dependent priming

The functional consequences of the co-expression of synaptotagmin-1 and synaptotagmin-7 are unclear. We show that when present separately, synaptotagmin-1 and synaptotagmin-7 act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming. The priming effect of Synaptotagmin-7 extends to the Readily Releasable Pool, whose fusion is executed by synaptotagmin-1, indicating synergistic action of the two Ca2+-sensors, although they are only partially colocalized. Synaptotagmin-7 promotes ubMunc13-2-dependent priming and the absence of synaptotagmin-7 renders phorbolesters less effective in stimulating priming, although synaptotagmin-7 independent priming is also observed. Morphologically, synaptotagmin-7 places vesicles in close membrane apposition (< 6 nm); in its absence vesicles accumulate out of reach of the fusion complex (20-40 nm). We suggest that a synaptotagmin-7-dependent movement toward the membrane is involved in Munc13-2/phorbolester/Ca2+-dependent priming and sets the stage for fast and slow exocytosis triggering.

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bioRxiv2019

Correlating Synaptic Ultrastructure and Function at the Nanoscale

Despite similarities in the composition of the molecular release machinery, synapses can exhibit strikingly different functional transmitter release properties and short- and long-term plasticity characteristics. To address the question whether ultrastructural differences could contribute to this functional synaptic heterogeneity, we employed a combination of hippocampal organotypic slice cultures, high-pressure freezing, freeze substitution, and 3D-electron tomography to resolve the spatial organization of vesicle pools at individual active zones (AZ) in two functionally distinct synapses, namely Schaffer collateral (SC) and mossy fiber (MF) synapses. We found that mature MF and SC synapses harbor equal numbers of docked vesicles at their AZs, MF synapses at rest exhibit a second pool of possibly tethered vesicles in the AZ vicinity, and MF synapses contain at least three morphological types of docked vesicles, indicating that differences in the ultrastructural organization of MF and SC synapses may contribute to their respective functional properties and corresponding plasticity characteristics.

neuroscience↗

bioRxiv2018

Complexin cooperates with Bruchpilot to tether synaptic vesicles to the active zone cytomatrix

Information processing by the nervous system depends on the release of neurotransmitter from synaptic vesicles (SVs) at the presynaptic active zone. Molecular components of the cytomatrix at the active zone (CAZ) regulate the final stages of the SV cycle preceding exocytosis and thereby shape the efficacy and plasticity of synaptic transmission. Part of this regulation is reflected by a physical association of SVs with filamentous CAZ structures. However, our understanding of the protein interactions underlying SV tethering by the CAZ is far from complete. The very C-terminal region of Bruchpilot (Brp), a key component of the Drosophila CAZ, participates in SV tethering. Yet so far, no vesicular or cytoplasmic molecules have been reported to engage in an interaction with Brps C-terminus. Here, we carried out an in vivo screen for molecules that link the Brp C-terminus to SVs. This strategy identified the conserved SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor) regulator Complexin (Cpx) as a vesicular interaction partner of Brp. We show that Brp and Cpx interact genetically and functionally. Interfering with Cpx targeting to SVs mirrored distinctive features of a C-terminal Brp truncation: impaired SV recruitment to the CAZ and enhanced short-term synaptic depression. Extending the study beyond Drosophila synapses, we interrogated active zones of mouse rod bipolar cells. Here, too, we collected evidence for an evolutionarily conserved role of Cpx upstream of SNARE complex assembly where it participates in SV tethering to the CAZ.

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