Biology subjects

de Wit, J.

Publications and source records attributed to de Wit, J..

At least 19 recordsLinked to original sources

bioRxiv2026

A systematic cross-modal approach identifies astrocytic VCAM1 as a regulator of hippocampal synapse development

Astrocyte-mediated cell-cell interactions at synapses are essential for circuit formation, yet the molecular mechanisms underlying astrocyte-mediated regulation of hippocampal synapse development remain incompletely characterized. Here, we take a systematic cross-modal approach to identify novel astrocytic cell surface proteins (CSPs) at the tripartite synapse. By using single-cell spatial transcriptomics (scST) targeting CSPs identified in a previous hippocampal synaptic dataset, we find 10 potential candidate astrocytic CSPs. Subsequent systematic protein-level profiling using multiple antibody-based assays establishes GPR37L1, HepaCAM, and VCAM1 as astrocytic peri-synaptic CSPs. To gain insight into the molecular context in which these proteins operate at synapses, we map their synaptic interaction partners using affinity purification-mass spectrometry (AP-MS), revealing distinct hippocampal synaptic interactomes for GPR37L1 and VCAM1. We then develop a custom multi-metric image-based synapse analysis pipeline to assess the roles of GPR37L1 and VCAM1 in synaptic development, using CRISPR/Cas9-mediated gene knockout (KO) in the mouse hippocampus. While loss of GPR37L1 does not substantially affect excitatory and inhibitory synapses, VCAM1 loss impairs excitatory hippocampal synapse development. Conversely, addition of recombinant VCAM1 to cultured hippocampal neurons increases the density of excitatory synapses. Together, these results identify astrocytic VCAM1 as a regulator of hippocampal synapse development.

neuroscience↗

bioRxiv2026

Extracellular neuroligin-ICAM5 coupling drives dendritic growth via actin remodeling

Neuroligins (NLGNs) organize neuronal connectivity by engaging a diverse set of interaction partners, yet how extracellular recognition couples to intracellular growth programs remains unclear. Using affinity proteomics, we identify intercellular adhesion molecule-5 (ICAM5), a cell-surface protein localized to dendritic filopodia, as a novel neuroligin interactor. Surface plasmon resonance and cell-based assays demonstrate direct binding between the ICAM5 and NLGN3 extracellular domains and reveal that ICAM5 engages all neuroligin isoforms. ICAM5 is required for NLGN-induced dendritic outgrowth, but the NLGN3-ICAM5 complex does not contribute to synaptogenesis. Mechanistically, ICAM5 sustains PAK-Cofilin signaling and F-actin organization in growth cones, positioning it as a downstream effector that links neuroligin engagement to actin remodeling. Together, these findings define a neuroligin-ICAM5 axis that couples extracellular recognition to intracellular actin remodeling to control neuronal structural development.

neuroscience↗

bioRxiv2025

Intrabody-guided synapse proteomics defines pyramidal neuron input architecture and uncovers early remodeling in a mouse model of Alzheimer's disease

Across their proximal and distal dendritic domains, pyramidal neurons (PNs) integrate inputs that differ in morphology and function. Hippocampal CA1 PNs are among the earliest affected neurons in Alzheimers disease (AD), but the molecular composition of their inputs and selective vulnerability remain poorly defined. We develop an intrabody-guided proximity-labeling strategy that targets the biotin ligase TurboID to endogenous postsynaptic scaffolds for cell-autonomous mapping of postsynaptic proteomes. Targeting PSD95 or Homer1 enables selective labeling of excitatory postsynaptic proteins in mouse CA1 PNs and resolves subsynaptic organization by comparing the two probes. Mapping the proteomes of major CA1 inputs uncovers a proximal-distal molecular logic that underlies their distinct properties. Applying this approach in the AppNL-G-F AD mouse model reveals an early signaling-driven phase of synaptic remodeling followed by a later translation-linked phase, with persistent downregulation of glutamatergic components. These results provide a molecular atlas of CA1 PN inputs and identify stage-specific mechanisms of synaptic vulnerability in early AD.

neuroscience↗

bioRxiv2025

Enhanced human antigen-specific B cell responses using in vitro 3D tonsil cultures containing stromal cells

The structural complexity of secondary lymphoid organs (SLOs) and their role in shaping antigen-specific B cell responses, pose significant challenges in modeling human germinal center (GC) response in vitro. A human 3D lymphoid model incorporating lymphoid and stromal cell types recapitulates key immune and structural features, enabling the study of antigen-specific B and T cell interactions beyond current 2D culture limitations. In this study, human tonsil cells were cultured with and without tonsil-derived fibroblastic reticular cells (FRCs) either in 2D or within a 3D PEG-4MAL hydrogel culture. Antigen-specific B cell responses in co-cultures were studied by comparing unstimulated cultures to stimulation with antigen (SARS-CoV-2 spike (S) or Influenza hemagglutinin (HA), both with or without adjuvant R848), S-nanoparticles and influenza vaccines. Combination of FRCs with the 3D matrix significantly improved B and T cell survival and facilitated reaggregation into follicle-like structures. Antigen-specific responses were most pronounced in 3D FRC-supported co-cultures, with increasing S- or HA-specific B cell frequencies, antibody-secreting cell differentiation, and secretion of antigen-specific antibodies. Importantly, cell death and unspecific bystander activation was lowest in 3D FRC-supported cultures. Additionally, GC-associated chemokine receptors CXCR4 and CXCR5 showed distinct expression patterns on CD27CD38 B cells, reflecting GC-like dark and light zone organization typically observed in SLOs in vivo. Autologous and allogeneic FRC-supported cultures yielded comparable results, demonstrating the platforms potential for high-throughput applications. The 3D FRC-supported lymphoid cultures offer a physiologically relevant platform for studying human GC responses in vitro, supporting mechanistic research into adaptive immunity and enabling the screening of vaccine immunogens and adjuvants in a controlled setting.

immunology↗

bioRxiv2025

The trimeric structures of the extracellular domains of FAM171A1 and FAM171A2 neuronal proteins belong to a novel structural superfamily

Cell surface molecules play fundamental roles in cell-cell communication, attraction, or repulsion, and when expressed in neurons they are often implicated in neurological disorders. FAM171 is a family of three type-I transmembrane domain cell surface proteins (FAM171A1, FAM171A2, and FAM171B) expressed in several human tissues and especially enriched in the brain. Recent findings suggest that FAM171A1 transduces signals between the cell surface and the cytoskeleton. Genetic evidence links FAM171A1 to multiple cancers and FAM171A2 to neurodegenerative diseases, including Alzheimers and Parkinsons diseases. Despite multiple connections with severe human diseases, no information is currently available on their monomeric structure or oligomerization. Here we show that, structurally, the monomeric ectodomains of human FAM171A1 and FAM171A2 have a new architecture with a novel combination of two domains. Furthermore, their ectodomains oligomerize to form an equilateral trimer. In addition, the ectodomain of FAM171A1 has the propensity to form larger trimer-trimer assemblies at high concentrations. Together, these results provide novel insights into the structure and oligomerization of the extracellular domain of FAM171A1 and FAM171A2, suggesting important roles in ligand binding and signaling.

neuroscience↗

bioRxiv2025

A dynamic gene regulatory code drives synaptic development of hippocampal granule cells

Connecting neurons into functional circuits requires the formation, maturation, and plasticity of synapses. While advances have been made in identifying individual genes regulating synapse development, the molecular programs orchestrating their action during circuit integration of neurons remain poorly understood. Here, we take a multiomic approach to reconstruct gene regulatory networks (GRNs), comprising transcription factors (TFs), regulatory regions, and predicted target genes, in hippocampal granule cells (GCs). We find a dynamic gene regulatory code, with early and late postnatal GRNs regulating cell morphogenesis and synapse organization and plasticity, respectively. Our results predict sequential regulations, with early-active TFs delaying the activation of later GRNs and their putative synaptic targets. Using a loss-of-function approach, we identify Bcl6 as a regulator of pre- and postsynaptic structural maturation, and Smad3 as a modulator of inhibitory synaptic transmission, in GCs. Together, these findings highlight the networks of key TFs and target genes orchestrating GC synapse development.

neuroscience↗

bioRxiv2024

Cell-surface receptor-mediated regulation of synaptic organelle distribution controls dendritic spine maturation

The spine apparatus (SA), an endoplasmic reticulum-related organelle present in a subset of mature dendritic spines, plays a key role in postsynaptic development and has been implicated in various neurological disorders. However, the molecular mechanisms that dictate SA localization at selected synapses remain elusive. Here, we identify a postsynaptic signaling complex comprising the GPCR-like receptor GPR158 and a largely uncharacterized phospholipase C (PLC), PLCXD2, that controls SA abundance. Sparse genetic manipulations in vivo demonstrate that in the absence of GPR158, unrestrained PLCXD2 activity impedes postsynaptic SA incorporation and hampers dendritic spine maturation. Finally, we show that extracellular heparan sulfate proteoglycan (HSPG) binding modulates the GPR158-PLCXD2 interaction. Together, our findings reveal how a postsynaptic receptor signaling complex regulates the local lipid microenvironment to control SA abundance required for the proper maturation of dendritic spines.

neuroscience↗

bioRxiv2024

Decoding Cortical Circuits: Synaptic Signatures and Disease Vulnerabilities of Layer 5 Pyramidal Neuron Types

Cortical layer 5 (L5) intratelencephalic (IT) and pyramidal tract (PT) neurons are embedded in distinct information processing pathways. The morphology, connectivity, electrophysiological properties, and role in behavior of these neurons have been extensively analyzed. However, the molecular composition of their synapses remains largely uncharacterized. Here, we dissect the protein composition of the excitatory postsynaptic compartment of L5 neurons in intact somatosensory circuits, using an optimized proximity biotinylation workflow with subsynaptic resolution. We find distinct synaptic signatures of L5 IT and PT neurons that are defined by proteins regulating synaptic organization and transmission, including cell-surface proteins (CSPs), neurotransmitter receptors and ion channels. In addition, we find a differential vulnerability to disease, with a marked enrichment of autism risk genes in the synaptic signature of L5 IT neurons compared to PT neurons. Our results align with human studies and suggest that the excitatory postsynaptic compartment of L5 IT neurons is notably susceptible in autism. Together, our analysis sheds light on the proteins that regulate synaptic organization and function of L5 neuron types and contribute to their susceptibility in disease. Our approach is versatile and can be broadly applied to other neuron types to create a protein-based, synaptic atlas of cortical circuits.

neuroscience↗

bioRxiv2023

Ecological interactions between host, commensal and pathogenic bacteria in models for the intestinal epithelium.

Gut host physiology and the microbiome intricately interact in the complex ecosystem of the human digestive tract, playing a crucial role in maintaining overall health. In recent decades, the role of the gut microbiota in the defence against pathogens and modulating local and distal immunity has been well-established. The interactions between commensal and potential pathogenic bacteria with the intestinal epithelium can initiate immune responses in the epithelial cells, which, in turn, activate downstream immune responses in other immune cells. These intricate processes involved, especially when multiple microorganisms are present as seen in the intestinal microbiome, remain only partially understood. Previously, it was observed that in adults aged 60 years or older, the commensal Ruminococcus torques (Rt) and Escherichia coli were associated with influenza-like illness and a heightened pro-inflammatory immune profile. In this study, we used a CaCo-2 cell-based model and a human intestinal enteroid (HIE) model to explore epithelial responses to Rt and an adherent invasive E. coli (AIEC) both individually and in co-cultures under anaerobic conditions. Additionally, CaCo-2 cells were co-cultured with peripheral blood mononuclear cells, revealing downstream activation of immune cells. While both systems showed comparable cytokine profiles, they differed in their responses to the different bacteria, with the organoid system being more representative for intestinal epithelial cells in humans. We provide mechanistic evidence of the pro-inflammatory responses associated with these bacteria in the intestinal ecosystem. These models, particularly in the context of combined infections, represent a valuable and promising avenue for future research. They contribute to a deeper understanding of the complex interactions between the gut microbiota, epithelial intestinal cells and immune cells in the gut ecosystem, thereby promoting advances in the field of gut health and host response.

microbiology↗

bioRxiv2022

LRRC37B is a species-specific regulator of voltage-gated channels and excitability in human cortical neurons

The enhanced cognitive abilities characterizing the human species result from specialized features of neurons and circuits, but the underlying molecular mechanisms remain largely unknown. Here we report that the hominid-specific gene LRRC37B encodes a novel receptor expressed in a subset of human cortical pyramidal neurons (CPNs). LRRC37B protein localizes at the axon initial segment (AIS), the specialized domain triggering action potentials. LRRC37B ectopic expression in mouse CPNs in vivo leads to reduced intrinsic excitability, a distinctive feature of some classes of human CPNs. At the molecular level, LRRC37B acts as a receptor for the secreted ligand FGF13A and interacts with the voltage gated sodium channel (VGSC) beta subunit SCN1B, thereby inhibiting the channel function of VGSC, specifically at the AIS. Electrophysiological recordings in adult human cortical slices reveals that endogenous expression of LRRC37B in human CPNs reduces neuronal excitability. LRRC37B thus acts as a species-specific modifier of human cortical neuron function, with important implications for human brain evolution and diseases.

neuroscience↗

bioRxiv2022

TAOK2β represses translation via phosphorylation of eEF2 and ameliorates exaggerated protein synthesis in a mouse model of 16p11.2 microdeletion-driven autism

Microdeletions in the 16p11.2 region of the human genome are frequently associated with autism spectrum disorders (ASDs), but how these genomic rearrangements cause ASD remains unclear. Here, we reveal that TAOK2{beta}, a protein isoform encoded by the human TAOK2 gene located in the 16p11.2 locus, regulates mRNA translation. To identify key functional interaction partners of TAOK2{beta}, we performed proteomic screening from Neuro-2a (N2a) cells, mouse cortices, and cultured neurons. This revealed translation factors as a major class of enriched interacting proteins. Consistently, TAOK2{beta} is present in mouse cortical polyribosomes and cortices from Taok2 knockout mice show increased ribosome density on mRNAs and enhanced protein synthesis. Several lines of evidence support an effect of TAOK2{beta} on translation elongation via phosphorylation of eukaryotic elongation factor (eEF2). TAOK2 can directly phosphorylate eEF2 on Threonine 56 and this phosphorylation is reduced in cortices from Taok2 knockout mice. TAOK2{beta} WT overexpression increased eEF2 phosphorylation levels and reduced protein synthesis, whereas a kinase-dead allele of TAOK2{beta} showed opposite effects. Finally, we show that cortices from the mouse model of the human 16p11.2 microdeletion have increased polysome/monosome (P/M) ratios and protein synthesis, phenocopying Taok2 loss of function. Importantly, defective translation phenotypes observed in the mouse 16p11.2 microdeletion model of ASD could be normalized either by reintroducing Taok2 in vivo or by delivering TAOK2{beta} to cortical neurons derived from 16p11.2 microdeletion mice. Our results uncover a critical role of TAOK2{beta} as a regulator of protein synthesis and support the idea that translational control is a common endpoint of ASD-associated signaling pathways.

neuroscience↗

bioRxiv2021

HyDrop: droplet-based scATAC-seq and scRNA-seq using dissolvable hydrogel beads

Single-cell RNA-seq and single-cell ATAC-seq technologies are being used extensively to create cell type atlases for a wide range of organisms, tissues, and disease processes. To increase the scale of these atlases, lower the cost, and allow for more specialized multi-ome assays, custom droplet microfluidics may provide complementary solutions to commercial setups. We developed HyDrop, a flexible and generic droplet microfluidic platform encompassing three protocols. The first protocol involves creating dissolvable hydrogel beads with custom oligos that can be released in the droplets. In the second protocol, we demonstrate the use of these beads for HyDrop-ATAC, a low-cost non-commercial scATAC-seq protocol in droplets. After validating HyDrop-ATAC, we applied it to flash-frozen mouse cortex and generated 8,502 high-quality single-cell chromatin accessibility profiles in a single run. In the third protocol, we adapt both the reaction chemistry and the capture sequence of the barcoded hydrogel bead to capture mRNA, and demonstrate a significant improvement in throughput and sensitivity compared to previous open-source droplet-based scRNA-seq assays (Drop-seq and inDrop). Similarly, we applied HyDrop-RNA to flash-frozen mouse cortex and generated 9,508 single-cell transcriptomes closely matching reference single-cell gene expression data. Finally, we leveraged HyDrop-RNAs high capture rate to analyse a small population of FAC-sorted neurons from the Drosophila brain, confirming the protocols applicability to low-input samples and small cells. HyDrop is currently capable of generating single-cell data in high throughput and at a reduced cost compared to commercial methods, and we envision that HyDrop can be further developed to be compatible with novel (multi-) omics protocols.

genomics↗

bioRxiv2021

Melanin Concentrating Hormone- and sleep-dependent synaptic downscaling is impaired in Alzheimer's Disease

In Alzheimers disease (AD), pathophysiological changes in the hippocampus cause deficits in episodic memory formation, leading to cognitive impairment 1,2. Neuronal hyperactivity is observed early in AD 3,4. Here, we find that homeostatic mechanisms transiently counteract increased neuronal activity in the hippocampal CA1 region of the AppNL-G-F humanized knock-in mouse model for AD 5, but ultimately fail to maintain neuronal activity at set-point. Spatial transcriptomic analysis in CA1 during the homeostatic response identifies the Melanin-Concentrating Hormone (MCH)-encoding gene. MCH is expressed in sleep-active lateral hypothalamic neurons that project to CA1 and modulate memory 6. We show that MCH regulates synaptic plasticity genes and synaptic downscaling in hippocampal neurons. Furthermore, MCH-neuron activity is impaired in AppNL-G-F mice, disrupting sleep-dependent homeostatic plasticity and stability of neuronal activity in CA1. Finally, we find perturbed MCH-axon morphology in CA1 early in AppNL-G-F mice and in AD patients. Our work identifies dysregulation of the MCH-system as a key player in aberrant neuronal activity in the early stages of AD.

neuroscience↗

bioRxiv2021

Heterotypic Aβ interactions facilitate amyloid assembly and modify amyloid structure

It is still unclear why pathological amyloid deposition initiates in specific brain regions, nor why specific cells or tissues are more susceptible than others. Amyloid deposition is determined by the self-assembly of short protein segments called aggregation-prone regions (APRs) that favour cross-{beta} structure. Here we investigated whether A{beta} amyloid assembly can be modified by heterotypic interactions between A{beta} APRs and short homologous segments in otherwise unrelated human proteins. We identified heterotypic interactions that accelerate A{beta} assembly, modify fibril morphology and affect its pattern of deposition in vitro. Moreover, we found that co-expression of these proteins in an A{beta} reporter cell line promotes A{beta} amyloid aggregation. Importantly, reanalysis of proteomics data of A{beta} plaques from AD patients revealed an enrichment in proteins that share homologous sequences to the A{beta} APRs, suggesting heterotypic amyloid interactions may occur in patients. Strikingly, we did not find such a bias in plaques from overexpression models in mouse. Based on these data, we propose that heterotypic APR interactions may play a hitherto unrealised role in amyloid-deposition diseases.

neuroscience↗

bioRxiv2021

MDGAs are fast-diffusing molecules that delay excitatory synapse development by altering neuroligin behavior

MDGAs are molecules that can bind neuroligins in cis and interfere with trans-synaptic neurexin-neuroligin interactions, thereby impairing synapse development. However, the sub-cellular localization and dynamics of MDGAs, as well as their specific mode of action in neurons are still unclear. Here, using both surface immunostaining of endogenous MDGAs and single molecule tracking of recombinant MDGAs in dissociated hippocampal neurons, we show that MDGA1 and MDGA2 molecules are homogeneously distributed and exhibit fast membrane diffusion, with a small reduction in mobility across neuronal maturation in culture Using shRNAs and CRISPR/Cas9 strategies to knock-down/out MDGA1 or MDGA2, we demonstrate an increase in the density of excitatory synapses accompanied by enhanced membrane immobilization and an increase in the phosphotyrosine level of neuroligins associated with excitatory post-synaptic differentiation. Finally, we show that decreasing MDGA expression level reduces the mobility of AMPA receptors and increases the frequency of AMPA receptor mediated mEPSCs. Overall, our results support a mechanism by which interactions between MDGAs and neuroligin-1 delays the assembly of functional excitatory synapses containing AMPA receptors.

neuroscience↗

bioRxiv2019

Mapping the cell-surface proteome underlying hippocampal mossy fiber synapse identity

Synaptic diversity is a key feature of neural circuits. The structural and functional diversity of closely spaced inputs converging on the same neuron suggests that cell-surface interactions are essential in organizing input properties. Here, we analyzed the cell-surface protein (CSP) composition of hippocampal mossy fiber (MF) inputs on CA3 pyramidal neurons to identify regulators of MF-CA3 synapse properties. We uncover a rich cell-surface repertoire that includes adhesion proteins, guidance cue receptors, extracellular matrix (ECM) proteins, and uncharacterized CSPs. Interactome screening reveals multiple ligand-receptor modules and identifies ECM protein Tenascin-R (TenR) as a ligand of the uncharacterized neuronal receptor IgSF8. Presynaptic Igsf8 deletion impairs MF-CA3 synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition of CA3 neurons. Consequently, loss of IgSF8 increases CA3 neuron excitability. Our findings identify IgSF8 as a regulator of CA3 microcircuit development and suggest that combinations of CSP modules define input identity.

neuroscience↗

bioRxiv2019

Synaptogenic activity of the axon guidance molecule Robo2 is critical for hippocampal circuit function

The developmental transition between axon guidance and synapse formation is critical for circuit assembly but still poorly understood at the molecular level. We hypothesized that this key transition could be regulated by axon guidance cues switching their function to regulate synaptogenesis with subcellular specificity. Here, we report evidence for such a functional switch, describing a novel role for the axon guidance molecule Robo2 in excitatory synapse formation onto dendrites of CA1 pyramidal neurons (PNs) in the mouse hippocampus. Cell-autonomous deletion of Robo2 from CA1 PNs leads to a drastic reduction of the number of excitatory synapses specifically in proximal dendritic compartments. At the molecular level, we show that this novel postsynaptic function of Robo2 depends on both its canonical ligand Slit and a novel interaction with presynaptic Neurexins. Biophysical analysis reveals that Robo2 binds directly to Neurexins via its Ig4-5 domains. In vivo 2-photon Ca2+ imaging of CA1 PNs during spatial navigation in mice shows that sparse deletion of Robo2 during development drastically reduces the likelihood of place cell emergence and alters spatial coding properties of the remaining place cells. Our results identify Robo2 as a novel molecular effector linking synaptic specificity to the acquisition of spatial coding properties characterizing hippocampal circuits.

neuroscience↗

bioRxiv2019

SorCS1-mediated Sorting of Neurexin in Dendrites Maintains Presynaptic Function

The pre- and postsynaptic membranes comprising the synaptic junction differ in protein composition. The mechanisms that maintain the polarized distribution of synaptic membrane proteins are poorly understood. The sorting receptor SorCS1 is a critical trafficking regulator of neuronal receptors, including neurexin (Nrxn), a presynaptic adhesion molecule essential for synaptic transmission. We find that SorCS1 controls a balance between axonal and dendritic Nrxn1 surface levels. Newly synthesized Nrxn1 traffics to the somatodendritic surface, followed by endocytosis. SorCS1 interacts with the Rab11 effector protein Rab11FIP5/Rip11 to facilitate the transition of internalized Nrxn1 from early to recycling endosomes and bias Nrxn1 surface polarization toward the axon. In the absence of SorCS1, Nrxn1 accumulates in early endosomes and mis-polarizes to the dendritic surface, impairing presynaptic function. The axonal/dendritic balance of Nrxn1 surface distribution is activity-dependent, indicating that SorCS1-mediated sorting in somatodendritic endosomes dynamically controls Nrxn1 axonal surface polarization required for proper presynaptic function.

neuroscience↗