Biology subjects

Trimbuch, T.

Publications and source records attributed to Trimbuch, T..

16 recordsLinked to original sources

bioRxiv2024

The sodium leak channel NALCN is regulated by neuronal SNARE complex proteins

The sodium leak channel NALCN is vital for the regulation of electrical activity in neurons and other excitable cells, and mutations in the channel or its auxiliary proteins lead to severe neurodevelopmental disorders. Here we show that the neuronal SNARE complex proteins syntaxin and SNAP25, which enable synaptic transmission in the nervous system, inhibit the activity of the NALCN channel complex in both heterologous systems and primary neurons. The existence of this interaction suggests that the neurotransmitter release machinery can regulate electrical signalling directly, and therefore modulate the threshold for its own activity. We further find that reduction of NALCN currents is sufficient to promote cell survival in syntaxin-depleted cells. This suggests that disinhibited NALCN may cause the puzzling phenomenon of rapid neuronal cell death in the absence of syntaxin. This interaction may offer opportunities for future drug development against genetic diseases linked to both NALCN- and SNARE protein-containing complexes.

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bioRxiv2024

Neurotransmitter release is triggered by a calcium-induced rearrangement in the Synaptotagmin-1/SNARE complex primary interface

The Ca2+ sensor synaptotagmin-1 triggers neurotransmitter release together with the neuronal SNARE complex formed by syntaxin-1, SNAP25 and synaptobrevin. Moreover, synaptotagmin-1 increases synaptic vesicle priming and impairs spontaneous vesicle release. The synaptotagmin-1 C2B domain binds to the SNARE complex through a primary interface via two regions (I and II), but how exactly this interface mediates distinct functions of synaptotagmin-1, and the mechanism underlying Ca2+-triggering of release is unknown. Using mutagenesis and electrophysiological experiments, we show that region II is functionally and spatially subdivided: binding of C2B domain arginines to SNAP-25 acidic residues at one face of region II is crucial for Ca2+-evoked release but not for vesicle priming or clamping of spontaneous release, whereas other SNAP-25 and syntaxin-1 acidic residues at the other face mediate priming and clamping of spontaneous release but not evoked release. Mutations that disrupt region I impair the priming and clamping functions of synaptotagmin-1 while, strikingly, mutations that enhance binding through this region increase vesicle priming and clamping of spontaneous release, but strongly inhibit evoked release and vesicle fusogenicity. These results support previous findings that the primary interface mediates the functions of synaptotagmin-1 in vesicle priming and clamping of spontaneous release, and, importantly, show that Ca2+-triggering of release requires a rearrangement of the primary interface involving dissociation of region I, while region II remains bound. Together with modeling and biophysical studies presented in the accompanying paper, our data suggest a model whereby this rearrangement pulls the SNARE complex to facilitate fast synaptic vesicle fusion. Significance statementThe synaptic SNARE complex and synaptotagmin-1 are required for fast neurotransmitter release. The functions of synaptotagmin-1 in preparing synaptic vesicles for fusion and executing the triggering step have been proposed to be regulated through interactions with the SNARE complex via the so-called primary interface. Using site-directed mutagenesis and functional analysis in neurons, we now show that synaptotagmin-1 mediates its release preparatory functions via two contact sites with the SNARE complex at this interface. During Ca2+ triggering, synaptotagmin-1 continues to contact the SNAREs at one site but disconnects the other site. We propose that this switch generates a pulling force on the SNARE complex that in turn triggers release. Biochemical and modeling studies described in the accompanying paper support this hypothesis.

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bioRxiv2024

Brain-wide monosynaptic connectivity mapping with ROInet-seq

Viral projection tracing strategies help establish regional connectomes of mammalian brains. Monosynaptic connectivity tracing with G-deleted rabies virus (RV) establishes synaptic input connectivity, but cannot distinguish networks at cell resolution. We implemented a barcoded {Delta}G rabies virus to introduce unique molecular tags - and make network tracing amenable to readout by RNA-sequencing. First, we optimized and characterized library complexity and uniformity, such that detection of specific barcodes can reliably distinguish the individual monosynaptic input networks of multiple infected neurons in parallel. To deploy the method at scale; to hundreds of cells and full-brain volume per experiment, we developed regions-of-interest network sequencing (ROInet-seq); an accessible, scalable and low-cost spatial assay. ROInet-seq combines routine fluorescent imaging and processing of fixed tissue sections with a simple molecular biology workflow to capture barcode sequences in relevant regions-of-interest, and ultimately describes single-neuron networks brain-wide. In cortical brain areas the assay revealed preserved regional network motives, including co-inputs to single cortical neurons from distant and local sites. Towards improved spatial resolution and simultaneous detection of transcriptomes and networks we finally sampled barcoded {Delta}G rabies virus-infected hippocampus on commercial spatial transcriptomics assays and reveal details of the regions neurons local network architecture.

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bioRxiv2024

Single residues in the complexin N-terminus exhibit distinct phenotypes in synaptic vesicle fusion

The release of neurotransmitters at central synapses is dependent on a cascade of protein interactions, specific to the presynaptic compartment. Amongst those dedicated molecules the cytosolic complexins play an incompletely defined role as synaptic transmission regulators. Complexins are multidomain SNARE complex binding proteins which confer both inhibitory and stimulatory functions. Using systematic mutagenesis and combining reconstituted in vitro membrane fusion assays with electrophysiology in neurons, we deciphered the function of the N-terminus of complexin II (Cpx). The N-terminus (amino acid 1 - 27) starts with a region enriched in hydrophobic amino acids (1-12), which can lead to lipid binding. In contrast to mutants which maintain the hydrophobic character and the stimulatory function of Cpx, non-conservative exchanges largely perturbed spontaneous and evoked exocytosis. Mutants in the downstream region (amino acid 11-18) show differential effects. Cpx-A12W increased spontaneous release without affecting evoked release whereas replacing D15 with amino acids of different shapes or hydrophobic properties (but not charge) not only increased spontaneous release, but also impaired evoked release and surprisingly reduced the size of the readily releasable pool, a novel Cpx function, unanticipated from previous studies. Thus, the exact amino acid composition of the Cpx N-terminus fine tunes the degree of spontaneous and evoked neurotransmitter release. Significance StatementWe describe in this work the importance of the N-terminal domain of the small regulatory cytosolic protein complexin in spontaneous and evoked glutamatergic neurotransmitter release at hippocampal mouse neurons. We show using a combination of biochemical, imaging and electrophysiological techniques that the binding of the proximal region of complexin (amino acids 1-10) to lipids is crucial for spontaneous synaptic vesicular release. Furthermore, we identify a single amino acid at position D15 which is structurally important since it not only is involved in spontaneous release but, when mutated, also decreases drastically the readily releasable pool, a function that was never attributed to complexin.

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bioRxiv2023

The stability of the primed pool of synaptic vesicles and the clamping of spontaneous neurotransmitter release relies on the integrity of the C-terminal half of the SNARE domain of Syntaxin-1A

The SNARE proteins are central in membrane fusion and, at the synapse, neurotransmitter release. However, their involvement in the dual regulation of the synchronous release while maintaining a pool of readily releasable vesicles remains unclear. Using a chimeric approach, we performed a systematic analysis of the SNARE domain of STX1A by exchanging the whole SNARE domain or its N- or C-terminus subdomains with those of STX2. We expressed these chimeric constructs in STX1-null hippocampal mouse neurons. Exchanging the C-terminal half of STX1s SNARE domain with that of STX2 resulted in a reduced RRP accompanied by an increased release rate, while inserting the C-terminal half of STX1s SNARE domain into STX2 lead to an enhanced priming and decreased release rate. Additionally, we found that the mechanisms for clamping spontaneous, but not for Ca2+-evoked release, are particularly susceptible to changes in specific residues on the outer surface of the C-terminus of the SNARE domain of STX1A. Particularly, mutations of D231 and R232 affected the fusogenicity of the vesicles. We propose that the C-terminal half of the SNARE domain of STX1A plays a crucial role in the stabilization of the RRP as well as in the clamping of spontaneous synaptic vesicle fusion through the regulation of the energetic landscape for fusion, while it also plays a covert role in the speed and efficacy of Ca2+-evoked release.

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bioRxiv2022

Syntaxin-1A modulates vesicle fusion in mammalian neurons via juxtamembrane domain dependent palmitoylation of its transmembrane domain

SNAREs are undoubtedly one of the core elements of synaptic transmission. On the contrary to the well characterized function of their SNARE domains bringing the plasma and vesicular membranes together, the level of contribution of their juxtamembrane domain (JMD) and the transmembrane domain (TMD) to the vesicle fusion is still under debate. To elucidate this issue, we analyzed three groups of STX1A mutations: 1) elongation of STX1As JMD by three amino acid insertions in the junction of SNARE-JMD or JMD-TMD; 2) charge reversal mutations in STX1As JMD; and 3) palmitoylation deficiency mutations in STX1As TMD. We found that both JMD elongations and charge reversal mutations have position dependent differential effects on Ca2+-evoked and spontaneous neurotransmitter release in cultured murine hippocampal neurons. Importantly, we show that STX1As JMD regulates the palmitoylation of STX1As TMD and loss of STX1A palmitoylation either through charge reversal mutation K260E or by loss of TMD cysteines particularly inhibits spontaneous vesicle fusion. Interestingly, the retinal ribbon specific STX3B has a glutamate in the position corresponding to the K260E mutation in STX1A and mutating it with E259K acts as a molecular on-switch. Furthermore, palmitoylation of post-synaptic STX3A can be induced by the exchange of its JMD with STX1As JMD together with the incorporation of two cysteines into its TMD. Forced palmitoylation of STX3A dramatically enhances spontaneous vesicle fusion suggesting that STX1A regulates spontaneous release through two distinct mechanisms: one through the C-terminal half of its SNARE domain and the other through the palmitoylation of its TMD.

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bioRxiv2021

Input to layer 1 of somatosensory cortex: Local input outweighs long-range and can modulate the activity of layer 1 interneurons

Neocortical Layer (L) 1 has been proposed to be at the center for top-down and bottom-up integration. It is a locus for interactions between long-range inputs, L1 interneurons and apical tuft dendrites of pyramidal neurons. While input to L1 has been studied intensively, the level and effect of input to this layer has still not been completely characterized. Here we examined the input to L1 of mouse somatosensory cortex with retrograde tracing and optogenetics. Our assays reveal that local input to L1 is predominantly from L2/3 and L5 pyramidal neurons and interneurons, and that subtypes of local L5 and L6b neurons project to L1 with different probabilities. Long-range input from sensory-motor cortices to L1 of S1 arose predominantly from L2/3 neurons. Our optogenetic experiments showed that intra-telencephalic L5 pyramidal neurons drive L1 interneurons but have no effect locally on L5 apical tuft dendrites. Dual retrograde tracing revealed that a fraction of local and long-range neurons were both presynaptic to L5 neurons and projected to L1. Our work highlights the prominent role of local inputs to L1 and shows the potential for complex interactions between long-range and local inputs which are both in position to modify the output of somatosensory cortex.

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bioRxiv2021

Control of neurotransmitter release and presynaptic plasticity by re-orientation of membrane-bound Munc13-1

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C1 domain, a Ca2+/PIP2-binding C2B domain, a MUN domain and a C2C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane in two different orientations mediated by distinct interactions of the C1C2B region with the plasma membrane: i) one involving a polybasic face that yields a perpendicular orientation of Munc13-1 and hinders release; and ii) another involving the DAG-Ca2+-PIP2-binding face that induces a slanted orientation and facilitates release. Here we have tested this model and investigated the role of the C1C2B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair synaptic vesicle priming in primary murine hippocampal cultures, and Ca2+-independent liposome bridging and fusion in in vitro reconstitution assays. A K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca2+-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca2+-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C1-C2B region of Munc13-1 plays a central role in vesicle priming and support the notion that re-orientation of Munc13-1 controls neurotransmitter release and short-term presynaptic plasticity.

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bioRxiv2021

Reexamination of N-terminal domains of Syntaxin-1 in vesicle fusion from central murine synapses

Syntaxin-1 (STX1) and Munc18-1 are two requisite components of synaptic vesicular release machinery, so much so synaptic transmission cannot proceed in their absence. They form a tight binary complex through two major binding modes: one through STX1s N-peptide, the other through STX1s closed conformation driven by its Habc-domain. However, physiological roles of these two reportedly different binding modes in synapses are still controversial. Here we characterized the roles of STX1s N-peptide, Habc-domain, and open conformation with and without N-peptide deletion using our STX1-null mouse model system and exogenous reintroduction of STX1A mutants. We show, on the contrary to the general view, that the Habc-domain is absolutely required and N-peptide is dispensable for synaptic transmission. However, STX1s N-peptide plays a regulatory role, particularly in the Ca2+-sensitivity and the short-term plasticity of vesicular release, whereas STX1s open conformation governs the vesicle fusogenicity. Strikingly, we also show that neurotransmitter release still proceeds when both the interaction modes between STX1 and Munc18-1 are presumably intervened together, necessitating a refinement of the conceptualization of STX1-Munc18-1 interaction.

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bioRxiv2021

Dynamin is primed at endocytic sites for ultrafast endocytosis

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane when the proline-rich domain of this variant is dephosphorylated. When this domain is mutated to include phosphomimetic residues or Syndapin 1s dynamin-interacting domain is mutated, Dynamin 1xA becomes diffuse, and consequently, ultrafast endocytosis slows down by [~]100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

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bioRxiv2020

Functional architecture of the synaptic transducers at a central glutamatergic synapse

Neuronal synapses transduce information via the consecutive action of three transducers: voltage-gated Ca2+-channels, fusion-competent synaptic vesicles, and postsynaptic receptors. Their physical distance is thought to influence the speed and efficiency of neurotransmission. However, technical limitations have hampered resolving their nanoscale arrangement. Here, we developed a new method for live-labeling proteins for electron microscopy (EM), revealing that release-competent vesicles preferentially align with Ca2+-channels and postsynaptic AMPA receptors within 20-30 nm and thereby forming a transsynaptic tripartite nanocomplex. Using functional EM, we show that single action potentials cause vesicles within the nanocomplex to fuse with a 50% probability. The loss of the presynaptic scaffold disrupts the formation of the tripartite transducers. Strikingly, the forced transsynaptic alignment of the Ca2+-channel subunit 2{delta}1 and AMPA receptors suffice to restore neurotransmission in a scaffold lacking synapse. Our results demonstrate a synaptic transducer nanocomplex that actively contributes to the organization of central synapses.

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bioRxiv2019

Vglut2 expression in dopamine neurons contributes to post-lesional striatal reinnervation

In Parkinsons disease, the most vulnerable neurons are found in the ventral tier of the substantia nigra (SN), while the adjacent dopamine (DA) neurons of the ventral tegmental area (VTA) are mostly spared. Although a significant subset of adult VTA DA neurons expresses Vglut2, a vesicular glutamate transporter, and release glutamate as a second neurotransmitter in the striatum, only very few adult SN DA neurons have this capacity. Previous work has demonstrated that lesions created by neurotoxins such as MPTP and 6-hydroxydopamine (6-OHDA) can upregulate the expression of Vglut2 in surviving DA neurons. Currently, the molecular mechanisms explaining the plasticity of Vglut2 expression in DA neurons are unknown, as are the physiological consequences for DA neuron function and survival. Here we aimed to characterize the developmental expression pattern of Vglut2 in DA neurons and the role of this transporter in post-lesional plasticity in these neurons. Using an intersectional genetic lineage-mapping approach, based on Vglut2-Cre and TH-Flpo drivers, we first found that more than 98% of DA neurons expressed Vglut2 at some point in their embryonic development. Expression of this transporter was detectable in most DA neurons until E11.5 and was found to be localized in developing axons. Moderate enhancement of VGLUT2 expression in primary DA neurons caused an increase in axonal arborization length. Compatible with a developmental role, constitutive deletion of Vglut2 caused a regional defect in TH-innervation of the dorsal striatum in E18.5 embryos. Moreover, using an in vitro neurotoxin model, we demonstrate that Vglut2 expression can be upregulated in post-lesional DA neurons by 2.5-fold, arguing that the developmental expression of Vglut2 in DA neurons can be reactivated at postnatal stages and contribute to post-lesional plasticity of dopaminergic axons. In support of this hypothesis, we find fewer mesostriatial dopaminergic projections in the striatum of conditional Vglut2 KO mice 7 weeks after a neurotoxic lesion, compared to control animals. Thus, we propose here that one of the functions of Vglut2 in adult DA neurons is to promote post-lesional recovery of meso-striatal axons.

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bioRxiv2019

Complexin suppresses spontaneous exocytosis by capturing the membrane-proximal regions of VAMP2 and SNAP25

The neuronal protein complexin contains multiple domains that exert both clamping and facilitatory functions to tune spontaneous and action potential triggered synaptic release. We address the clamping mechanism and show that the accessory helix of complexin arrests the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that forms the core machinery of intracellular membrane fusion. In a reconstituted fusion assay, site- and stage-specific photo-cross-linking reveals that prior to fusion the complexin accessory helix laterally binds the membrane-proximal C-terminal ends of SNAP25 and VAMP2. Corresponding complexin interface mutants selectively increase spontaneous release of neurotransmitter in living neurons, implying that the accessory helix suppresses final zippering/assembly of the SNARE four-helix bundle by restraining VAMP2 and SNAP25.

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bioRxiv2019

Human endogenous retrovirus HERV-K(HML-2) RNA causes neurodegeneration through Toll-like receptors

Although human endogenous retroviruses (HERVs) represent a substantial proportion of the human genome and some HERVs have been suggested to be involved in neurological disorders, little is known about their biological function and pathophysiological relevance. HERV-K(HML-2) comprises evolutionarily young proviruses transcribed in the brain. We report that RNA derived from an HERV-K(HML-2) env gene region binds to the human RNA-sensing Toll-like receptor (TLR) 8, activates human TLR8, as well as murine Tlr7, and causes neurodegeneration through TLR8 and Tlr7 in neurons and microglia. HERV-K(HML-2) RNA introduced extracellularly into the cerebrospinal fluid (CSF) of either C57BL/6 wild-type mice or APPPS1 mice, a mouse model for Alzheimers disease (AD), resulted in neurodegeneration. Tlr7-deficient mice were protected against neurodegenerative effects, but were re-sensitized towards HERV-K(HML-2) RNA when neurons ectopically expressed murine Tlr7 or human TLR8. Accordingly, transcriptome datasets of human brain samples from AD patients revealed a specific correlation of upregulated HERV-K(HML-2) and TLR8 RNA expression. HERV-K(HML-2) RNA was detectable more frequently in CSF from AD individuals compared to controls. Our data establish HERV-K(HML-2) RNA as an endogenous ligand for human TLR8 and murine Tlr7 and imply a functional contribution of specific human endogenous retroviral transcripts to neurodegenerative processes such as AD.

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bioRxiv2018

Light induced synaptic vesicle autophagy

The regulated turnover of synaptic vesicle (SV) proteins is thought to involve the ubiquitin dependent tagging and degradation through endo-lysosomal and autophagy pathways. Yet, it remains unclear which of these pathways are used, when they become activated and whether SVs are cleared en-mass together with SV proteins or whether both are degraded selectively. Equally puzzling is how quickly these systems can be activated and whether they function in real time to support synaptic health. To address these questions, we have developed an imaging based system that simultaneously tags presynaptic proteins while monitoring autophagy. Moreover, by tagging SV proteins with a light activated reactive oxygen species (ROS) generator, Supernova, it was possible to temporally control the damage to specific SV proteins and assess their consequence to autophagy mediated clearance mechanisms and synaptic function. Our results show that, in mouse hippocampal neurons, presynaptic autophagy can be induced in as little as 5-10 minutes and eliminates primarily the damaged protein rather than the SV en-mass. Importantly, we also find that autophagy is essential for synaptic function, as light-induced damage to e.g. Synaptophysin only compromises synaptic function when autophagy is simultaneously blocked. These data support the concept that presynaptic boutons have a robust highly regulated clearance system to maintain not only synapse integrity, but also synaptic function.

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bioRxiv2018

Mutant Plasticity Related Gene 1 (PRG1) acts as a potential modifier in SCN1A related epilepsy

Plasticity related gene 1 encodes a cerebral neuron-specific synaptic transmembrane protein that modulates hippocampal excitatory transmission on glutamatergic neurons. In mice, homozygous Prg1-deficiency results in juvenile epilepsy. Screening a cohort of 18 patients with infantile spasms (West syndrome), we identified one patient with a heterozygous mutation in the highly conserved third extracellular phosphatase domain (p.T299S). The functional relevance of this mutation was verified by in-utero electroporation of a mutant Prg1 construct into neurons of Prg1-knockout embryos, and the subsequent inability of hippocampal neurons to rescue the knockout phenotype on the single cell level. Whole exome sequencing revealed the index patient to additionally harbor a novel heterozygous SCN1A variant (p.N541S) that was inherited from her healthy mother. Only the affected child carried both heterozygous PRG1 and SCN1A mutations. The aggravating effect of Prg1-haploinsufficiency on the epileptic phenotype was verified using the kainate-model of epilepsy. Double heterozygous Prg1-/+|Scn1awt/p.R1648Hmice exhibited higher seizure susceptibility than either wildtype, Prg1-/+, or Scn1awt/p.R1648H littermates. Our study provides evidence that PRG1-mutations have a potential modifying influence on SCN1A-related epilepsy in humans.

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