Biology subjects

Brose, N.

Publications and source records attributed to Brose, N..

At least 19 recordsLinked to original sources

bioRxiv2026

UFMylation anchors splicing factors at the ER to reprogram nuclear splicing

How organelles communicate stress to the nucleus to coordinate adaptive responses remains a fundamental question in cell biology. Here, we identify a non-canonical retrograde signaling pathway in which stalling-induced UFMylation of ER-associated ribosomes anchors splicing regulators at the ER, directly coupling translational stress to nuclear RNA processing. Phylogenetic profiling linked the UFMylation machinery to a network of nuclear mRNA processing factors. Fractionation-based quantitative proteomics further supported this link and revealed that translational stress triggers UFM1-dependent retention of serine/arginine-rich (SR) splicing factors at the ER, depleting their nuclear pools. Mechanistically, UFMylated ribosomes physically tether SR proteins at the ER surface, driving widespread intron retention that preferentially targets transcripts encoding membrane lipid metabolism and endomembrane-associated processes--a response conserved from plants to mammals. These findings reframe UFMylation from a local ribosome repair signal to a systems-level coordinator of ER-nucleus communication that reprograms nuclear splicing and reshapes membrane-associated gene expression with implications for diverse human diseases linked to UFMylation defects.

cell biology↗

bioRxiv2025

Adaptor protein complex 2 (AP2) participates in biogenesis and homeostasis of myelin sheaths in the central nervous system

Myelination of CNS axons requires oligodendrocytes to undergo extensive morphological changes by producing large amounts of myelin membrane with defined protein composition and structure. The formation of myelin sheaths thus involves efficient trafficking and sorting of future myelin constituents via vesicles that fuse with prospective myelin membranes by exocytotic mechanisms. However, the functional relevance of other trafficking steps in oligodendocytes for myelin biogenesis is largely unknown. Here, we followed the hypothesis that developmental myelination involves endocytic mechanisms. In this model, Golgi-derived vesicles fuse with the oligodendroglial plasma membrane, from which myelin constituents are retrieved by endocytosis into endosomal/lysosomal organelles before their final integration into the growing sheath. Considering that adaptor protein complex-2 subunit-{micro} (AP2M) facilitates AP2-dependent endocytosis, we recombined the Ap2m-gene in myelin-forming oligodendrocytes, causing both hypomyelination and specific changes in the myelin proteome. Most strikingly, lysosomal membrane proteins accumulate in the abaxonal (outermost) myelin layer, identifying this membrane as an active site for retrieving constituents from myelin sheaths. These data demonstrate that the AP2 complex serves a critical function in developmental myelination in vivo. Unexpectedly, we also observed pathological myelin outfoldings indicative of focal hypermyelination. Consistent with the hypothesis that this phenotype reflects impaired maintenance rather than biogenesis of myelin sheaths, recombination of the Ap2m-gene in oligodendrocytes of adult mice caused late-onset progressive focal hypermyelination. These results indicate that, in addition to astrocytic and microglial phagocytosis, oligodendrocytes cell-autonomously contribute to maintaining the structure of healthy myelin sheaths via AP2-dependent mechanisms.

neuroscience↗

bioRxiv2025

Ca2+-phospholipid-dependent regulation of Munc13-1 is essential for post-tetanic potentiation at mossy fiber synapses and supports working memory

Hippocampal mossy fiber (hMF) to CA3 pyramidal cell synapses are thought to support the formation of working memory through presynaptic short-term facilitation (STF) and post-tetanic potentiation (PTP). However, the molecular mechanisms underlying these transient forms of synaptic enhancement remain poorly understood. We show here that Munc13-1-mediated priming of synaptic vesicles (SVs) at active zones controls hMF STF and PTP in response to Ca2+-phospholipid and Ca2+-calmodulin (CaM) signaling. Knock-in mice expressing Munc13-1 variants that are insensitive to Ca2+-phospholipid and Ca2+-CaM signaling exhibit severely impaired STF and PTP at hMF synapses. Moreover, the PTP-induction threshold is strongly increased upon the loss of Ca2+-phospholipid-Munc13-1 signaling. Since these synaptic defects are accompanied by working memory deficits, especially in mice expressing the Ca2+-phospholipid-insensitive Munc13-1 variant, we conclude that Ca2+-dependent regulation of Munc13-1-mediated SV priming co-determines hMF short-term plasticity and working memory formation.

neuroscience↗

bioRxiv2025

Proximity labelling reveals the compartmental proteome of murine sensory neurons

IntroductionUnderstanding the molecular architecture of peripheral sensory neurons is critical as we pursue novel drug targets against pain and neuropathy. Sensory neurons in the dorsal root ganglion (DRG) show extensive compartmentalization, thus understanding each compartment - from the peripheral to central terminals - is key to this effort. MethodsTo systematically profile this spatial complexity, we generated a TurboIDfl/fl transgenic mouse line (ROSA26em1(TurboID)Bros), enabling targeted proximity labelling and deep proteomic profiling of DRG neuron compartments via Tg(Advillin-Cre)+. ResultsOur data reveal distinct proteomic signatures across neuronal compartments that reflect specialized neuronal functions. We provide proteomic insights into previously inaccessible nerve terminals both in the periphery (innervating the skin) and in the spinal cord. Further, using a DRG explant model of chemotherapy-induced peripheral neuropathy, we uncover novel and discrete proteome changes, highlighting neuronal vulnerability. ConclusionTogether, our findings provide a unique proteome atlas of the sensory neuron proteome across anatomical domains and demonstrate the utility of proximity labelling proteomics for detecting compartment-specific molecular alterations in a disease model.

neuroscience↗

bioRxiv2025

Structure and function of otoferlin, a synaptic protein of sensory hair cells essential for hearing

Our sense of hearing relies upon speedy synaptic transmission of sound information from cochlear inner hair cells (IHCs) to spiral ganglion neurons (SGNs). To accomplish this, IHCs employ a sophisticated presynaptic machinery including the multi-C2-domain protein otoferlin which is affected by human deafness mutations. Otoferlin is essential for IHC-exocytosis but how it binds Ca2+ and the target membrane to serve synaptic vesicle (SV) tethering, docking and fusion remained unclear. Here, we obtained cryo-electron-microscopy structures of Ca2+-bound otoferlin and employed molecular dynamics simulations of membrane binding. We show that membrane binding involves C2B-C2G-domains and repositions C2F- and C2G-domains. Progressive disruption of Ca2+-binding by the C2D-domain in mice increasingly altered synaptic sound encoding and eliminated the Ca2+-cooperativity of SV-exocytosis, indicating that this Ca2+-cooperativity reflects binding of several Ca2+-ions to otoferlin. Together, our findings elucidate molecular mechanisms underlying otoferlin-mediated SV-docking and support a role of otoferlin as Ca2+-sensor of SV-fusion in IHCs.

neuroscience↗

bioRxiv2025

Nanobinders for Synaptotagmin 1 enable the analysis of synapticvesicle dynamics in rodent and human models.

Synaptic neurotransmission is a critical hallmark of brain activity and one of the first processes to be affected in neural diseases. Monitoring this process, and in particular synaptic vesicle recycling, in living cells has been instrumental in unraveling mechanisms responsible for neurotransmitter release. However, currently available reporters suffer from major limitations such large probe size or lack of suitability for human neurons, hampering the understanding of human synaptic pathophysiology. Here we describe the NbLumSyt1 toolkit, a panel of nanobody-based affinity probes targeting the luminal domain of the synaptic vesicle protein Synaptotagmin 1 (Syt1). These new tools enable quantitative, non-invasive imaging and functional interrogation of synaptic transmission in human neurons, with unprecedented precision, versatility and cost efficiency, in technologies ranging from fixed-and live-cell super-resolution imaging to electron microscopy and mass spectrometry. Overall, NbLumSyt1 nanobinders provide a valuable platform for human synaptic physiology and pathophysiology, benefiting fundamental neuroscience and translational efforts to study and develop treatments for brain-related disorders.

neuroscience↗

bioRxiv2025

Epiproteomic control of the epitranscriptome drives cortical neurogenesis

Neurological conditions are the leading cause of ill health worldwide. Here, we show that the neurodevelopmental disorder-associated ubiquitin ligase UBE3C regulates the cellular composition of the murine cerebral cortex and human brain organoids, with its loss favoring neurogenesis and suppressing glial fate. Using genetic complementation, we demonstrate that disease-associated UBE3C mutations alter its autoubiquitination activity and disrupt cortical lamination. Proteomic profiling of UBE3C-deficient forebrains and organoids identifies Cbll1 as a UBE3C substrate, and we show that the UBE3C-Cbll1 duo drives N6-methyladenosine (m6A) mRNA methylation. Hyperactivation of m6A writers in UBE3C-deficient neural progenitors impairs cell cycle exit, a defect reversible in vivo by the METTL3 inhibitor STM2457. Our findings uncover an epiproteomic mechanism controlling m6A-mediated gene expression and define a regulatory axis linking ubiquitin signaling to epitranscriptomic control of neural fate. This work provides a mechanistic framework for understanding neurodevelopmental disorders and highlights potential therapeutic strategies.

cell biology↗

bioRxiv2025

Protocol for a minigene splice assay using the pET01 vector

Aberrant splicing is recognized as a key contributor of hereditary disorders, yet characterizing the molecular effects of splice variants is an important task that poses several challenges. Here, we present a protocol for an in vitro splice assay using a minigene approach, which is especially useful when patient samples are not available for RNA analysis or when target genes or isoforms are not expressed in accessible tissues for direct analysis. We describe steps for assay design, including the cloning of minigene plasmids and subsequent transfection, followed by RNA isolation and cDNA synthesis. We also provide details on quantitative fragment analysis using capillary electrophoresis and optional subcloning to facilitate optimal sequencing of multiple splicing products.

genetics↗

bioRxiv2024

Neddylation regulates the development and function of excitatory neurons

The development and function of neurons is orchestrated by a plethora of regulatory mechanisms that control the abundance, localization, interactions, and function of proteins. A key role in this regard is assumed by post-translational protein modifications (PTMs). While some PTM types, such as phosphorylation or ubiquitination, have been explored comprehensively, PTMs involving ubiquitin-like modifiers (Ubls) have remained comparably enigmatic (Ubls). This is particularly true for the Ubl Nedd8 and its conjugation to proteins, i.e. neddylation, in nerve cells. In the present study, we generated a conditional Nedd8 knock-out mouse line and examined the consequences of Nedd8-deletion in cultured postmitotic glutamatergic neurons. Our findings reveal that Nedd8-ablation in young glutamatergic neurons causes alterations in the expression of developmental transcription factors that control neuronal differentiation, ultimately leading to defects in the development of a mature glutamatergic neuronal phenotype. Apparent manifestations of these defects include increased vGlut2 expression levels, reduced vGlut1 and endophilin 1 expression levels, reduced dendrite complexity, and increased transmitter release probability. Collectively, our results highlight a pivotal role for neddylation in controlling the fate of glutamatergic neurons and excitatory synaptic transmission. HighlightsO_LIReduced dendrite complexity in Nedd8-deficient neurons C_LIO_LIIncreased synaptic vesicle fusion probability in Nedd8-deficient neurons C_LIO_LIAltered synaptic vesicle cycling in Nedd8-deficient neurons C_LIO_LINeddylation controls the maturation of glutamatergic neurons C_LI Graphical AbstractResearch Topic

neuroscience↗

bioRxiv2024

Direct cryo-ET detection of native SNARE and Munc13 protein bridges using AI classification and preprocessing

Synaptic transmission requires Munc13 and SNARE proteins for synaptic vesicle priming and fusion. Cryo-electron tomography detected multiple types of Munc13- or SNARE-dependent dependent molecular bridges that tether synaptic vesicles to the presynaptic active zone plasma membrane. To integrate the molecular scenario with structural observations, we obtained de novo, in situ cryo-electron tomography averages of native, mammalian SNARE- and Munc13-dependent tethers. These provide direct evidence that Munc13 and a complex comprising SNARE proteins link synaptic vesicles to the active zone membrane. Furthermore, we determined the plausibility of different molecular compositions of tethers, placed constraints on their conformations and positioning, and proposed the existence of a complex downstream of Munc13 and upstream of SNARE complex formation. Because the detection and subtomogram averaging of membrane-bridging complexes is complicated by the presence of two lipid membranes and multiple protein species and conformations, we developed preprocessing methods and feature-based AI classifiers that outperformed standard methods.

neuroscience↗

bioRxiv2024

SynapseNet: Deep Learning for Automatic Synapse Reconstruction

Electron microscopy is an important technique for the study of synaptic morphology and its relation to synaptic function. The data analysis for this task requires the segmentation of the relevant synaptic structures, such as synaptic vesicles, active zones, mitochondria, presynaptic densities, synaptic ribbons, and synaptic compartments. Previous studies were predominantly based on manual segmentation, which is very time-consuming and prevented the systematic analysis of large datasets. Here, we introduce SynapseNet, a tool for the automatic segmentation and analysis of synapses in electron micrographs. It can reliably segment synaptic vesicles and other synaptic structures in a wide range of electron microscopy approaches, thanks to a large annotated dataset, which we assembled, and domain adaptation functionality we developed. We demonstrated its capability for (semi-)automatic biological analysis in two applications and made it available as an easy-to-use tool to enable novel data-driven insights into synapse organization and function.

bioinformatics↗

bioRxiv2024

Revealing Acute Consequences of Rapid Protein Elimination at Individual Synapses using Auxin-Inducible Degron 2 Technology

A powerful approach to assess a protein of interest (POI) function is its specific elimination. Common knock-out and knock-down strategies, however, are protracted and often irreversible, challenging the assessment of acute or temporary consequences in the same cells and tissues. Here we describe the use of Auxin-Inducible Degron 2 (AID2) technology to study the real-time consequences of acute POI elimination in nerve cell synapses. We demonstrate its capacity in cultured neurons and in vivo to rapidly eliminate postsynaptic scaffold proteins fused at N-terminal, C-terminal, or nested sites to GFP derivatives or HaloTag. We show that acute PSD-95 or gephyrin elimination leads to the concomitant loss of AMPA or GABAA receptors at the same synapses, and that, surprisingly, acute GKAP, but not PSD-95 elimination reduces postsynaptic scaffold size. Our findings highlight the utility of AID2 technology for rapidly eliminating synaptic POIs and studying real-time consequences in the same neurons and synapses.

neuroscience↗

bioRxiv2024

Endogenous tagging of Munc13-1 with a SNAP tag as a tool for monitoring synapse nanoarchitecture

ABSTRACTSynaptic function is governed by highly regulated protein machineries, whose abundance and spatial localization change continually. Studies to determine the dynamic changes in synaptic proteins nanoarchitecture typically rely on immunolabeling, or on the expression of fluorescent proteins. The former employs chemical fluorophores and signal amplification, but requires fixation. The latter enables monitoring of proteins in live microscopy, but uses suboptimal fluorophores. Self-labeling tags have been introduced to combine the advantages of these two approaches, and here we introduce a knock-in mouse line where the essential presynaptic protein Munc13-1 is endogenously fused to the self-labeling SNAP tag. We demonstrate efficient Munc13-1-SNAP labeling in fixed neurons and brain sections by various SNAP dyes, as well as by a novel far-red and cell impermeable compound, SBG-SiR-d12. We introduce and characterize SBG-SiR-d12 here as a highly-efficient dye for SNAP-tag labeling of extracellular epitopes, and of intracellular proteins in fixed and permeabilized tissue. Finally, we show that Munc13-1-SNAP can be labeled in living neurons and monitored through live-cell imaging using confocal- and super resolution microscopy. We conclude that the Unc13aSNAP mouse line is a useful tool for the analysis of presynaptic nanoarchitectural dynamics, with a potential for wide adoption.

neuroscience↗

bioRxiv2023

Presynaptic cAMP-PKA-mediated potentiation induces reconfiguration of synaptic vesicle pools and channel-vesicle coupling at hippocampal mossy fiber boutons

It is widely believed that information storage in neuronal circuits involves nanoscopic structural changes at synapses, resulting in the formation of synaptic engrams. However, direct evidence for this hypothesis is lacking. To test this conjecture, we combined chemical potentiation, functional analysis by paired pre-postsynaptic recordings, and structural analysis by electron microscopy (EM) and freeze-fracture replica labeling (FRL) at the hippocampal mossy fiber synapse, a key synapse in the trisynaptic circuit of the hippocampus. Biophysical analysis of synaptic transmission revealed that forskolin-induced chemical potentiation increased the readily releasable vesicle pool size (RRP) and vesicular release probability (Pr) by 146% and 49%, respectively. Structural analysis of mossy fiber synapses by EM and FRL demonstrated an increase in the number of vesicles close to the plasma membrane and the number of clusters of the priming protein Munc13-1, indicating an increase in the number of both docked and primed vesicles. Furthermore, FRL analysis revealed a significant reduction of the nearest neighbor distance (NND) between Munc13-1 and CaV2.1 Ca2+ channels, suggesting reconfiguration of the channel-vesicle coupling nanotopography. Our results indicate that presynaptic plasticity is associated with structural reorganization of active zones (AZs). We propose that changes in potential nanoscopic organization at synaptic vesicle release sites may be correlates of learning and memory at a plastic central synapse.

neuroscience↗

bioRxiv2023

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 Munc-13-1-Munc13-2 knockout mice with completely blocked synaptic transmission. Neither 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.

neuroscience↗

bioRxiv2023

Pre- and postsynaptic nanostructures increase in size and complexity after LTP induction

Synapses, specialized contact sites between neurons, are the fundamental elements of neuronal information transfer. Synaptic plasticity is related to changes in synaptic morphology and the number of neurotransmitter receptors, and thought to underlie learning and memory. However, it is not clear how these structural and functional changes are connected. We utilized time-lapse super-resolution STED microscopy to visualize structural changes of the synaptic nano-organization of the postsynaptic scaffolding protein PSD95, the presynaptic scaffolding protein Bassoon, and the GluA2 subunit of AMPA receptors by chemically induced long-term potentiation (cLTP) at the level of single synapses. We found that the nano-organization of all three proteins undergoes an increase in complexity and size after cLTP induction. The increase was largely synchronous, peaking at [~]60 min after stimulation. Therefore, both the size and complexity of single pre- and post-synaptic nanostructures serve as substrates for adjusting and determining synaptic strength. Highlights- Time-lapse super-resolution images the structural changes of the PSD95 nano-organization after Cltp - cLTP-induced growth of the PSD95 nano-organization is less than spine head growth and peaks at 60 min, i.e. much slower than the increase in spine volume. - Most PSD95 nanostructures increase in complexity upon cLTP. - Synchronous growth - Nanostructures of pre- and postsynaptic scaffolding proteins and AMPA receptors increase simultaneously and equally strong upon cLTP. - GluA2-containing synaptic AMPA receptors form nanoclusters that increase in size and slightly in number upon cLTP and form subdomains on PSD95. - Bassoon forms complex structures similar to the PSD95 nano-organization.

neuroscience↗

bioRxiv2023

Transcriptional networks predating cognition-associated pyramidal lineages are restructured by erythropoietin

Recombinant human erythropoietin (rhEPO) has potent procognitive effects, hematopoiesis-independent, but underlying mechanisms and physiological role of brain-expressed EPO have remained obscure. Here, we provide encyclopedic transcriptional hippocampal profiling of mice treated with rhEPO. Based on [~]108,000 single nuclei, we unmask multiple pyramidal lineages with their comprehensive molecular signatures. By temporal profiling and gene regulatory analysis, we build a developmental trajectory of CA1 pyramidal neurons derived from multiple predecessor lineages and elucidate gene regulatory networks underlying their fate determination. With EPO as tool, we discover novel populations of newly differentiating pyramidal neurons, overpopulating to [~]200% upon rhEPO with upregulation of genes crucial for neurodifferentiation, dendrite growth, synaptogenesis, memory formation, and cognition. Using a Cre-based approach to visually distinguish pre-existing from newly formed pyramidal neurons for patch-clamp recordings, we learn that rhEPO treatment differentially affects excitatory and inhibitory inputs. Our findings provide mechanistic insight into how EPO modulates neuronal functions and networks.

neuroscience↗

bioRxiv2022

Regulation of excitatory presynaptic activity by Ambra1 protein determines neuronal networks in sex-dimorphic manner

Heterozygous mutation of Ambra1, known as a positive autophagy regulator, produces autismlike behavior in mice and autistic phenotypes in humans in a female-specific manner. However, the substantial roles of the Ambra1 mutation in neurons are still unknown. We find that Ambra1 heterozygotes display a moderate decrease in excitatory synaptic release in-vitro and ex-vivo exclusively in females without autophagy activity, resulting in significant alterations in {gamma}-oscillation power and seizure susceptibility by excitatory/inhibitory (E/I) imbalance. Specifically, Ambra1 deficiency has no effect on neurogenesis and morphogenesis, but selectively decreases excitatory synaptic activity without changes in synapse number, quantal size, synaptic release probability, and synaptic plasticity. Therefore, the limited excitatory synaptopathy by Ambra1 expression levels ultimately determines E/I imbalance in global neural networks leading to the female-specific ASD.

neuroscience↗