Biology subjects

Rosenmund, C.

Publications and source records attributed to Rosenmund, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Nup153 regulates neuronal responsiveness through HDAC1-mediated epigenetic modulation

Neural activity-dependent gene regulation is central to the development of neural networks and neuronal plasticity. Induction of activity-dependent gene programs is equally important as repression of these programs, and both need to be balanced carefully. However, little is known about how repressive mechanisms modulate neuronal responsiveness across the genome before stimulation. Here, we identify nucleoporin-dependent regulation of neuronal responsiveness, in which Nup153 represses neuronal genes including activity-regulated genes (ARGs), in the basal state. By characterizing the genome-wide landscape of chromatin accessibility, histone modifications and Nup153 chromatin binding, we show that Nup153 bidirectionally regulates chromatin states through both basal activity-dependent and -independent mechanisms and influences associated genes. Mechanistically, Nup153 associates with HDAC1 to modulate histone acetylation and chromatin accessibility at target regulatory regions. Our data suggests Nup153 organizes chromatin states that regulate neuronal gene programs involved in maintaining neuronal responsiveness. TeaserNup153 primes neuronal responsiveness through multi-layered epigenetic regulation.

neuroscience↗

bioRxiv2025

mTORC1-Dependent Signaling in Layer 5b Neurons Is Required for Memory Consolidation

Memory consolidation relies on activity-dependent neuronal plasticity, particularly in deep-layer cortical neurons that integrate long-range inputs from medial temporal lobe (MTL) structures. In the neocortex, apical tuft dendrites of layer 5b pyramidal neurons receive direct input from the MTL within cortical layer 1, and their activation is critical for associative learning. However, the molecular mechanisms that enable learning-induced plasticity in these neurons remain poorly understood. Here, we demonstrate that activity-dependent, mTORC1-regulated translation in layer 5b neurons contributes to learning and memory. Using in vivo paradigms such as exposure to an enriched environment (EE) and in vitro models of network activation by chemical long-term potentiation (cLTP), combined with cell type-specific translational profiling (RiboTag), we identify candidate mRNAs associated with experience-dependent plasticity. We further show that pharmacological inhibition of canonical mTORC1 signaling during memory-relevant tasks abrogated memory consolidation. Our findings identify the mTORC1 pathway as a central regulator of learning-induced protein translation and cortical plasticity.

neuroscience↗

bioRxiv2025

Piccolo Regulates Secretion of the Extracellular Matrix Components Brevican and Tenascin R from Astrocytes to Drive Synapse Formation: Implications for Pontocerebellar Hypoplasia Type 3 (PCH3)

BackgroundAstrocytes are crucial for CNS health, for instance via the secretion of extracellular matrix (ECM) components that are vital for synapse formation and maturation. While the scaffolding protein Piccolo is known for its role at synapses, its function in astrocytes and contribution to neurodegenerative disorders like Pontocerebellar Hypoplasia Type 3 (PCH3) are largely unknown. Understanding these mechanisms is key to elucidating PCH3 pathology. MethodsWe used a multi-faceted approach with a Pclogt/gtrat model. Methods included RNA-sequencing for gene expression and GO analysis. Immunohistochemistry and immunocytochemistry assessed Piccolo localization, ECM component (Brevican [Bcan], Tenascin-R [TNR]) levels, distribution, and secretion in brain sections and primary astrocytes. Golgi morphology was evaluated via GM130 staining. Neuronal network formation and function were investigated by co-culturing wild-type neurons with Pclowt/wt or Pclogt/gt astrocytes in a Banker setup, assessing synapse density (immunostaining, RRP electrophysiology) and spontaneous activity (mEPSCs, mIPSCs). Astrocyte-conditioned media (ACM) experiments determined secreted factor roles. ResultsRNA-seq showed significant DEG increases in older versus young Pclogt/gt rats (P25 vs. P5), which were strongly enriched in cell communication, signaling, and ECM GO terms. We identified a novel astrocyte-specific Piccolo isoform that partially localizes at the Golgi. Piccolo gene trap transposon mutation led to impaired ECM secretion (reduced extracellular Bcan, altered TNR) from astrocytes, correlating with a fragmented Golgi in Pclogt/gt astrocytes. Functionally, Pclogt/gt astrocytes significantly reduced synapse density and altered intrinsic activity (increased mEPSC frequency) in neuronal networks. This synaptic deficit was substantially rescued by Pclowt/wtastrocyte conditioned media (ACM). ConclusionOur findings reveal a critical, unrecognized role for Piccolo in regulating astrocytic ECM secretion, essential for proper neuronal network formation and activity. Astrocytic Piccolo dysfunction disrupts this process, causing impaired synaptogenesis and altered intrinsic network activity, providing a novel cellular and molecular mechanism for neurodegenerative diseases like PCH3 and highlights astrocytes as potential therapeutic targets.

neuroscience↗

bioRxiv2025

Dynamic nanoscale architecture of synaptic vesicle fusion in mouse hippocampal neurons

During neurotransmission, presynaptic action potentials trigger synaptic vesicle fusion with the plasma membrane within milliseconds. To visualize membrane dynamics before, during, and right after vesicle fusion at central synapses under near-native conditions, we developed an experimental strategy for time-resolved in situ cryo-electron tomography with millisecond temporal resolution. We coupled optogenetic stimulation with cryofixation and confirmed the stimulation-induced release of neurotransmitters via cryo-confocal microscopy of a fluorescent glutamate sensor. Our morphometric analysis of tomograms from stimulated and control synapses allowed us to characterize five states of vesicle fusion intermediates ranging from stalk formation to the formation, opening, and collapsing of a fusion pore. Based on these measurements, we generated a coarse-grained simulation of a synaptic vesicle approaching the active zone membrane. Both, our morphofunctional and computational analyses, support a model in which calcium-triggered fusion is initiated from synaptic vesicles in close proximity to the active zone membrane, whereby neither tight docking nor an induction of membrane curvature at the active zone are favorable. Numbers of filamentous tethers closely correlated to the distance between vesicle and membrane, but not to their respective fusion readiness, indicating that the formation of multiple tethers is required for synaptic vesicle recruitment preceding fusion.

neuroscience↗

bioRxiv2024

Presynaptic filopodia form kinapses and modulate membrane mechanics for synchronous neurotransmission and seizure generation.

The structural stability of synapses directly contrasts with their functional plasticity. This conceptual dichotomy is explained by the assumption that all synaptic plasticity is generated via either electrical and/or biochemical signaling. Here, we challenge this dogma by revealing an activity-dependent presynaptic response that is physical in nature. We show that dynamic filopodia emerge during action potential discharge and transiently deform synaptic boutons to enhance connectivity. Filopodia generation requires neuronal activity, calcium and actin, and occurs in intact brain circuits and human brain. Mechanistically, their extension preserves synchronous neurotransmitter release by increasing presynaptic membrane tension. However, filopodia generation becomes maladaptive during dysregulated brain activity, exacerbating seizures in vivo. Therefore, we provide direct evidence that presynaptic mechanical forces determine the extent and timing of synaptic signals.

neuroscience↗

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.

neuroscience↗

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.

neuroscience↗

bioRxiv2024

A lever hypothesis for Synaptotagmin-1 action in neurotransmitter release

Neurotransmiter release is triggered in microseconds by Ca2+-binding to the Synaptotagmin-1 C2 domains and by SNARE complexes that form four-helix bundles between synaptic vesicles and plasma membranes, but the coupling mechanism between Ca2+-sensing and membrane fusion is unknown. Release requires extension of SNARE helices into juxtamembrane linkers that precede transmembrane regions (linker zippering) and binding of the Synaptotagmin-1 C2B domain to SNARE complexes through a primary interface comprising two regions (I and II). The Synaptotagmin-1 Ca2+-binding loops were believed to accelerate membrane fusion by inducing membrane curvature, perturbing lipid bilayers or helping bridge the membranes, but SNARE complex binding orients the Ca2+-binding loops away from the fusion site, hindering these putative activities. Molecular dynamics simulations now suggest that Synaptotagmin-1 C2 domains near the site of fusion hinder SNARE action, providing an explanation for this paradox and arguing against previous models of Sytnaptotagmin-1 action. NMR experiments reveal that binding of C2B domain arginines to SNARE acidic residues at region II remains after disruption of region I. These results and fluorescence resonance energy transfer assays, together with previous data, suggest that Ca2+ causes reorientation of the C2B domain on the membrane and dissociation from the SNAREs at region I but not region II. Based on these results and molecular modeling, we propose that Synaptotagmin-1 acts as a lever that pulls the SNARE complex when Ca2+ causes reorientation of the C2B domain, facilitating linker zippering and fast membrane fusion. This hypothesis is supported by the electrophysiological data described in the accompanying paper. Significance statementNeurotransmiter release requires SNARE complexes that fuse synaptic vesicles with the plasma membrane and the Ca2+-sensor synaptotagmin-1, which was thought to facilitate membrane fusion directly through its Ca2+-binding loops. However, binding of Synaptotagmin-1 to SNARE complexes orients these loops away from the fusion site. Using molecular dynamics simulations, we show that placing Synaptotagmin-1 at the fusion site hinders the action of SNARE complexes. Spectroscopic studies show that Ca2+ binding to Synaptotagmin-1 can change its interactions with SNARE complexes and, together with molecular modeling, suggest that Synaptotagmin-1 acts as a lever, pulling SNARE complexes and thus facilitating their action on the membranes to induce fusion. Functional studies described in the accompanying paper support this hypothesis.

biophysics↗

bioRxiv2024

Molecular architecture of synaptic vesicles.

Synaptic vesicles (SVs) store and transport neurotransmitters to the presynaptic active zone for release by exocytosis. After release, SV proteins and excess membrane are recycled via endocytosis, and new SVs are formed in a clathrin-dependent manner. This process maintains the morphology and complex molecular composition of SVs through multiple recycling rounds. Previous studies explored the molecular composition of SVs through proteomic analysis and fluorescent microscopy, proposing a model for an average SV1,2. However, the structural heterogeneity and molecular architecture of individual SVs are not well described. Here we used cryo-electron tomography to visualize morphological and molecular details of SVs isolated from mouse brains and inside cultured neurons. We describe several classes of small proteins on the SV surface and long proteinaceous densities inside SVs. We identified V-ATPases, determined a structure using subtomogram average, and showed them forming a complex with the membrane-embedded protein synaptophysin. Our bioluminescence assay revealed pairwise interactions between VAMP2 and synaptophysin and V-ATPase Voe1 domains. Interestingly, V-ATPases were randomly distributed on the surface of SVs irrespective of vesicle sizes. A subpopulation of isolated vesicles and vesicles inside neurons contained a partially assembled clathrin coat with a soccer-ball symmetry. We observed a V-ATPase under clathrin cage in several isolated clathrin-coated vesicles. Additionally, from isolated SV preparations and within hippocampal neurons we identified clathrin baskets without vesicles. We determined their preferential location in proximity to the cell membrane. Our analysis advances the understanding of individual SVs diversity and their molecular architecture.

biophysics↗

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.

neuroscience↗

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.

neuroscience↗

bioRxiv2023

Molecular mechanism underlying SNARE-mediated membrane fusion enlightened by all-atom molecular dynamics simulations

The SNARE proteins syntaxin-1, SNAP-25 and synaptobrevin mediate neurotransmiter release by forming tight SNARE complexes that fuse synaptic vesicles with the plasma membranes in microseconds. Membrane fusion is generally explained by the action of proteins on overall membrane properties such as curvature, elastic modulus and tension, and a widespread model envisions that the SNARE motifs, juxtamembrane linkers and C-terminal transmembrane regions of synaptobrevin and syntaxin-1 form continuous helices that act mechanically as semi-rigid rods, squeezing the membranes together as they assemble ( zipper) from the N- to the C-termini. However, the mechanism underlying fast SNARE-induced membrane fusion remains unknown. We have used all-atom molecular dynamics simulations to investigate this mechanism. Our results need to be interpreted with caution because of the limited number and length of the simulations, but they suggest a model of membrane fusion that has a natural physicochemical basis, emphasizes local molecular events over general membrane properties, and explains extensive experimental data. In this model, the central event that initiates fast (microsecond scale) membrane fusion occurs when the SNARE helices zipper into the juxtamembrane linkers which, together with the adjacent transmembrane regions, promote encounters of acyl chains from both bilayers at the polar interface. The resulting hydrophobic nucleus rapidly expands into stalk-like structures that gradually progress to form a fusion pore, aided by the SNARE transmembrane regions and without clearly discernible intermediates. The propensity of polyunsaturated lipids to participate in encounters that initiate fusion suggests that these lipids may be important for the high speed of neurotransmiter release.

biophysics↗

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.

neuroscience↗

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.

neuroscience↗

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.

neuroscience↗

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.

neuroscience↗

bioRxiv2021

Ire1α-Regulated Rate of mRNA Translation is Required for Acquisition of Identity and Polarity in Upper Layer Cortical Neurons

Evolutionary expansion of the neocortex is associated with the increase in upper layer neurons. Here, we present Inositol-Requiring Enzyme 1, Ire1, as an essential determinant of upper layer fate, neuronal polarization and cortical lamination. We demonstrate a non-canonical function of Ire1 in the regulation of global translation rates in the developing neocortex through its dynamic interaction with the ribosome and regulation of eIF4A1 and eEF-2 expression. Inactivation of Ire1 engenders lower protein synthesis rates associated with stalled ribosomes and decreased number of translation start sites. We show unique sensitivity of upper layer fate to translation rates. Whereas eEF-2 is required for cortical lamination, eIF4A1 regulates acquisition of upper layer fate downstream of Ire1 in a mechanism of translational control dependent on 5UTR-embedded structural elements in fate determinant genes. Our data unveil developmental regulation of ribosome dynamics as post-transcriptional mechanisms orchestrating neuronal diversity establishment and assembly of cortical layers. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/449563v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@115a09aorg.highwire.dtl.DTLVardef@e30d2dorg.highwire.dtl.DTLVardef@763b27org.highwire.dtl.DTLVardef@2586e9_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LISmall molecule screening reveals Ire1 upstream of upper layer neuronal identity C_LIO_LIPolarization and proper lamination of layer II/III neurons require Ire1 C_LIO_LIDevelopment of upper layers requires high translation rates driven by eIF4A1 and eEF-2 downstream of Ire1 C_LIO_LIeIF4A1-dependent Satb2 mRNA translation initiation is a mechanism of upper layer fate acquisition C_LI

neuroscience↗

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.

neuroscience↗