Biology subjects

Benseler, F.

Publications and source records attributed to Benseler, F..

8 recordsLinked to original sources

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

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

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

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↗

bioRxiv2021

Dissecting Functional, Structural, and Molecular Requirements for Serotonin Release from Mouse Enterochromaffin Cells

Serotonergic enterochromaffin (EC) cells of the gut epithelium are secretory sensory cells that communicate with vagal neurons. EC cells exhibit many features of neurons in the brain, raising the hypothesis that synapse-like contacts may mediate fast and directed signalling. To dissect functional, structural, and molecular properties underlying serotonin release from genetically identified EC cells, we employed a multidisciplinary in vitro approach combining intestinal epithelial cell and organoid cultures, electrochemistry, correlated light- and electron microscopy, and gene expression and biochemical analyses. Despite the presence of key molecules of the synaptic neurotransmitter release machinery, we found that the majority of serotonin is released with slow kinetics from large dense-core rather than small synaptic-like vesicles. While we cannot exclude synapse-like transmission between EC cells and neurons in vivo, our data support the notion that the predominant mode of serotonin secretion is similar to that of other endocrine cell types.

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.

neuroscience↗