Biology subjects

Hoffmann, C.

Publications and source records attributed to Hoffmann, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Reverse engineering Lewy bodies suggests alpha-synuclein pathology as condensatopathy

Understanding how soluble monomeric alpha-synuclein transitions into pathological alpha-synuclein (Lewy bodies) has remained a central challenge in neurodegeneration. Existing models, largely based on treating cells and animals with pre-formed alpha-synuclein fibrils cannot account for the emergence of the first inclusion without pre-existing seeds. Here, we combine in vitro reconstitutions, computational modeling and a minimal cellular system to show that phase separation of alpha-synuclein with an interacting scaffold can recapitulate Lewy body-like structures. An unbiased proteomics screen, live-cell imaging, correlative light-electron microscopy, and soft X-ray tomography show that Lewy body-like structures are associated with redistribution of actin and membrane-bound organelles, with mitochondria accumulating at their periphery in an actin-dependent manner. Structural analyses further indicate that alpha-synuclein condensates provide local environments in which the insoluble beta-sheets can form. Together, these findings support a seed-independent trajectory from soluble alpha-synuclein through condensation and progressive immobilization to Lewy body-like assemblies and, subsequently, insoluble beta-sheet fibrils. This framework offers a new mechanistic route for understanding the disease progression and promises to guide the design of innovative therapeutic interventions.

neuroscience↗

bioRxiv2026

Chemokine and opioid peptide scavenging through constitutive and ligand-induced release of ACKR3-bearing extracellular vesicles

Atypical chemokine receptors (ACKRs) are non-signaling GPCRs that regulate ligand availability, with ACKR3 functioning as a dual scavenger of chemokines and opioid peptides. Here, we demonstrate that following ligand stimulation, besides the canonical internalization, ACKR3 is released on extracellular vesicles (EVs). ACKR3 was also found on EVs released under basal conditions, although to a lesser extent. These observations were confirmed across multiple cellular contexts, including endogenous systems. Mechanistically, basal and ligand-induced EV release are independent of GRKs and {beta}-arrestin but each relies on distinct trafficking routes and C-terminal determinants. Ligand-induced EV release is associated with plasma membrane localization and receptor recycling pathways. In contrast, basal EV release is governed by intracellular sorting processes and influenced by receptor ubiquitination and RAMP3. Functionally, EV-associated ACKR3 retains high-affinity ligand binding, enabling sequestration of CXCL12 and opioid peptides and thereby attenuating their signaling through CXCR4 and MOR. We also show that the release on EVs, in particular under basal conditions, is observed for other receptors such as KOR, CXCR4 and several ACKRs. Collectively, these findings establish EVs as regulators in chemokine and opioid systems and as a previously underappreciated dimension of ACKR3 and more broadly GPCR biology.

Cell Biology↗

bioRxiv2026

Biomolecular condensates provide a unique environment for redox-mediated protein crosslinking

Biomolecular condensates, often formed through liquid-liquid phase separation, are dynamic cellular compartments. Here, we demonstrate that a wide range of fluorescently tagged proteins undergo inadvertent, condensate-mediated crosslinking, resulting in rapid solidification of condensates under common fluorescence imaging conditions. The process is driven by excitation-induced, short-lived reactive oxygen species (ROS), whose otherwise limited crosslinking potential becomes uniquely enabled in the dense phase. In live cells, excitation-induced ROS potently trigger stress granule formation, while the ROS-driven solidification of condensates is modulated by compartment-dependent antioxidant buffering. Our findings demonstrate that condensates create a distinct environment that enables ROS chemistry unlikely to occur in the bulk cytosol. Furthermore, the cellular redox level can be a general regulator of condensate rheology. Beyond biological insights, our findings underscore the need for scrutiny when examining fluorophore-labeled condensates.

biophysics↗

bioRxiv2026

Small aberrant viral genomes induce the innate immune response to arenaviruses

Arenaviruses are a family of negative-sense RNA viruses mainly found in rodents in which they cause chronic asymptomatic infections. Some arenaviruses, however, can occasionally infect humans and cause pathogenic viral haemorrhagic fevers. These viral haemorrhagic fevers are associated with a massively dysregulated immune response characterized by excessive release of pro-inflammatory cytokines resulting in hyperinflammatory immunopathology. Here, we systematically characterize the immune response of human cells to a wide panel of mammalian arenaviruses and identify a clear distinction between New World viruses, which uniformly induce a robust interferon response, and Old World viruses, which do not induce any significant host response. This innate immune response is primarily driven by RIG-I-mediated RNA sensing and might be aided by differences in viral interferon antagonist expression patterns. Furthermore, we identified differences in quantity and type of non-standard viral genomes produced in unpassaged Old World and New World virus infections. Finally, we demonstrate that small non-standard viral RNA expression by New World viruses are key drivers of the innate immune response and might help explain how the host response can shape viral disease phenotypes and inform triage of patients.

microbiology↗

bioRxiv2026

Strength in Unity: a Dual Strategy to Restore NK Cell Cytotoxicity against Pancreatic Ductal Adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC), a condition representing 90% of pancreatic cancers, shows one of the lowest 5-year survival rates across all cancer types. Current therapeutic approaches remain largely inefficient, in part due to the presence of a hostile tumor microenvironment (TME), impeding immune cells infiltration and function. Specifically, Natural Killer (NK) cells from PDAC patients exhibit impaired phenotype and cytotoxic functions. NK cell immunotherapy represents a safe and promising approach to restore NK cell cytotoxicity against PDAC. MethodsWe developed a dual strategy based on i) the re-activation of NK cells through Natural Killer activating multimeric immunotherapeutic complexes (NaMiX) composed of IL-15/IL-15R dimers coupled to anti-NKp46 single-chain variable fragments (scFvs) and ii) the crosslinking of activated NK cells to PDAC cells with a Trispecific Killer Engager (TriKE) targeting NKG2D, NKp30 and the tumor-associated antigen CEA. We evaluated the ability of these constructs to stimulate NK cell functions across BxPC-3 PDAC cell line and patient-derived organoid models and in humanized NSG mice bearing PDAC xenografts. ResultsNaMiX stimulated the activation and cytotoxic functions of NK cells towards pancreatic BxPC-3 cells in vitro while TriKE cross-linked NK cells to BxPC-3 cells. The cytotoxic effects of NaMiX were further enhanced when combined with the crosslinking abilities of TriKE for the killing of NK cell-mediated BxPC-3 spheroid and PDAC patient-derived organoids. In humanized mice bearing BxPC-3 xenografts, NaMiX induced cytotoxic lymphocyte expansion, and increased tumor infiltration of NK cells, while TriKE tended to slow tumor progression. ConclusionsThis proof-of-concept study reports for the first time that activating and engaging NK cells with immunoconjugates are a promising therapeutic avenue for PDAC treatment. Efforts should now focus on the optimization of NK cell therapeutic modalities to favor the infiltration of a high number of NK cells into the tumor.

immunology↗

bioRxiv2026

OKN4395, a first-in-class EP2/EP4/DP1 triple antagonist reprograms prostanoid-driven immunosuppression to restore antitumor immunity

Immune checkpoint inhibitors, particularly T cell targeting anti-PD(L)1 therapies, have revolutionized the treatment landscape for solid malignancies, but challenges related to non-responsiveness and the development of treatment resistance continue to be observed. An additional immunosuppressive axis relates to prostaglandin signaling downstream of cyclooxygenase-2 (COX2), where COX2 inhibitors have shown clinical promise in re-engaging both T and non-T cell immune compartments, yet have suffered from toxicity concerns. We report here the preclinical characterization of OKN4395, a highly potent and specific first-in-class triple antagonist of EP2, EP4, and DP1, major tumor immunosuppressive receptors downstream of COX2. OKN4395 restores immune function on both T cells and NK cells in vitro. Additionally, OKN4395 acts synergistically with anti-PD1 to increase speed and depth of antitumor activity. Overall, these findings robustly support the clinical investigation of OKN4395 in an ongoing Phase 1 trial (NCT06789172) as an innovative cancer immunotherapy for solid tumors, as a single agent and in combination with anti-PD1 therapy. Statement of significanceOKN4395, a first-in-class oral EP2/EP4/DP1 antagonist, reverses prostanoid-driven immunosuppression to restore antitumor immunity. Integrated pharmacology defines mechanism, translational biomarkers as well as both monotherapy and anti-PD1 combination strategies. These data position prostanoid tri-receptor antagonism as a translatable strategy in solid tumors. A global Phase 1 study is underway (NCT06789172).

pharmacology and toxicology↗

bioRxiv2026

Kidney disease reprograms microbiome-host signaling to promote heart failure

BackgroundHeart failure is prevalent in chronic kidney disease (CKD) and linked to chronic inflammation. CKD-typical gut microbiome dysbiosis may stimulate inflammation, as bacterial aromatic metabolites are highly abundant and engage transcriptional programs through the aryl hydrocarbon receptor (AhR). Whether this axis drives cardiac remodeling and is therapeutically targetable remains unknown. MethodsWe used the subtotal nephrectomy model (STNx) and microbiome depletion by oral antibiotics. We investigated cardiac and renal function, AhR activity, metabolite profiles, and immunophenotypes by flow cytometry and transcriptomics. Candidate metabolite indoxyl sulfate (IxS) was tested in experimental HFpEF. In vivo and in vitro AhR inhibition (AhRi) was performed using a clinically tested compound. Mechanistic studies were performed in primary human and murine cardiac fibroblasts and T cells, as well as translational validation using UK Biobank data. ResultsMicrobiome depletion lowered bacterial metabolites and attenuated cardiac fibrosis and diastolic dysfunction in STNx, identifying AhR-driven expansion of interleukin-17A (IL-17A)-producing T helper cells (TH17) as key effector. Plasma IL-17A was stage-dependently elevated in CKD patients, particularly in HFpEF, and associated with all-cause mortality. Bacterial metabolite IxS promoted TH17 polarization and exacerbated cardiac dysfunction in HFpEF. AhRi using a small molecule inhibitor reduced TH17 abundance and attenuated cardiac fibrosis in STNx. Mechanistically, AhR and IL-17A signaling synergistically induced a conserved pro-fibrotic phenotype in human and murine cardiac fibroblasts, and AhR inhibition blocked ECM production in response to CKD patient serum. ConclusionA microbiome-AhR-IL-17A axis drives CKD-associated cardiac fibrosis. AhRi prevents remodeling, highlighting a potential therapeutic avenue to prevent cardiorenal multimorbidity.

immunology↗

bioRxiv2025

Contralesional activity reflects compensation, while brainstem detour pathways support skilled motor recovery after stroke

Large strokes frequently result in lasting motor deficits and trigger extensive reorganization within the brain and spinal cord. Altered neuronal activity in the contralesional hemisphere has been documented in both humans and rodent models, yet its role in functional recovery versus maladaptation remains unresolved. Here, we used chronic wide-field calcium imaging to monitor bilateral cortical activity in mice performing a skilled reach-to-grasp task before and days to weeks after stroke. Strokes produced persistent fine motor impairments, which were only partly alleviated by intensive rehabilitative training. While cortical activity was in particular suppressed in the ipsilesional cortex after stroke, training promoted sustained increases in contralesional sensorimotor activity. However, ridge regression analysis of neural and behavioral data indicated that this activity largely reflected compensatory use of the intact paw rather than recovery of the impaired forelimb. Axonal tracing nevertheless revealed enhanced projections from contralesional motor cortex to ipsilesional brainstem nuclei - including the midbrain and pontine reticular nucleus - specifically in trained animals. These findings identify a rehabilitation-induced corticofugal pathway supporting motor recovery, highlighting a target for neuromodulation strategies in chronic post-stroke impairment.

neuroscience↗

bioRxiv2025

Deciphering Cellular Ecosystems Driving Tumor Progression and Immune Escape from Spatial Transcriptomics and Single-Cell with COMPOTES

Cell-cell communication is central to understanding the complex interactions within the tumor microenvironment. However, current methods fail to identify recurrent communication patterns across patient cohorts from spatial transcriptomics, as they are often limited to single samples or lack essential spatial context. Yet this is essential for understanding how local environments influence cell phenotype and states, and shape the entire cellular ecosystem. We introduce a machine-learning approach that models local, spatially aware ligand-receptor interactions and uses matrix factorization to extract global multicellular programs from large cohorts representing the complex biology of cancer. Applied to a multimodal muscle-invasive bladder cancer cohort of 146 patients, it uncovered 45 communication programs defined by distinct ligand-receptor pairs and cellular niches. In particular, we identified a conserved immune program linked to stalled anti-tumor immunity and a program linking KMT2D loss-of-function mutations with early-stage (T2) tumors, intense proliferation and a favorable response to neoadjuvant chemotherapy.

bioinformatics↗

bioRxiv2025

Multiple instance learning with spatial transcriptomics for interpretable patient-level predictions: application in glioblastoma

Accurate prediction of patient outcomes remains a major challenge in oncology. While recent machine learning (ML) approaches often rely on bulk omics lacking spatial resolution or histology-based multiple instance learning (MIL), spatial transcriptomics (SpT) provides a unique opportunity to capture both molecular content and tissue architecture. However, no generalizable ML framework has yet been established to exploit SpT for patient-level outcomes. We present SpaMIL, a flexible and interpretable MIL framework designed for SpT, with a distillation strategy that enables deployment for hematoxylin and eosin (H&E) slides alone. We evaluate the framework by predicting survival from glioblastoma (GBM) patients, a clinically compelling setting given its aggressiveness with a median survival of only 15 months and the lack of prognostic clinical variables. We analyzed 76 GBM cases from the MOSAIC dataset: 43 with matched SpT, H&E, single-nucleus RNA-seq (scRNA-seq), bulk RNA-seq, and clinical variables, and 33 with H&E for external validation. We developed two main architectures: abMIL, tailored to SpTs spatial molecular structure, and MabMIL, which distills SpT-derived representations into H&E. Model interpretability was achieved through a Shapley-based framework linking prognostic predictions to cell-type compositions via SpT deconvolution. In benchmarking across the five GBM MOSAIC modalities, SpT-based abMIL achieved unprecedented prognostic accuracy (median C-index: 0.72, standard deviation: 0.04), outperforming all other modalities, including established clinical predictors. PCA and deconvolution-based SpT representations surpassed recent foundation models, suggesting the need for further research on SpT foundation models. Our interpretability analysis highlighted malignant and non-malignant cell subpopulations associated with favorable or poor prognosis, consistent with recent reports. Finally, MabMIL maintained strong performance while enabling H&E-only deployment, with improved condorance index over H&E-only baselines in both internal (0.59 vs. 0.57) and external (0.62 vs. 0.55) cohorts.

cancer biology↗

bioRxiv2025

Histology and spatial transcriptomic integration revealed infiltration zone with specific cell composition as a prognostic hotspot in glioblastoma

BackgroundGlioblastoma (GBM), the most aggressive primary brain tumor, has a median survival of approximately 15 months. Twenty percent of patients survive beyond three years, but known clinical factors like age, performance status, resection extent, and MGMT promoter methylation status do not fully explain the observed outcomes. ObjectiveOur objective was to identify novel histology derived biomarkers associated with end-of-spectrum overall survival (OS) to provide novel biological insight with a translational potential. MethodsWe analyzed a total of 748 GBM patients from 3 different cohorts, uniquely enriched in long survivors (n=98 with overall survival (OS) > 5y including n=196 with OS[≥]3y), with clinical data and H&E slides obtained from the primary tumor at baseline. We propose an interpretable machine learning (ML) methodology for the discovery of histological biomarkers. Our method learned to segment each H&E slide into three distinct regions associated with long-term survival, short-term survival, and non-informative tissue. We characterized these regions by integrating unsupervised learning, nuclei segmentation, blood vessels detection, pathologist annotations, and multimodal data including spatial transcriptomics from n=31 patients of the GBM MOSAIC dataset to discover fully interpretable biomarkers. ResultsOur OS prediction model using histology and clinical data as input achieved an area under the curve (AUC) of 0.85 for the classification of patients between OS<2 and OS[≥]3y in external cohort validation, outperforming significantly models trained on clinical data or on histology alone (AUC of 0.76; 0.73, respectively). Two novel biomarkers were predicting poor survival: the presence of regions of lowly infiltrated white matter enriched in malignant cells with a mesenchymal-like phenotype, and lower levels of angiogenesis associated with higher hypoxia response in the main tumor regions. We also found that a subtype of immunosuppressive tumor macrophages - defined by high PLIN2 expression and lipid accumulation- is consistently enriched in histological areas predictive of poor prognosis. ConclusionOur interpretable ML methodology identified a novel prognostic impact of biological processes and cell types according to distinct tumor regions of GBM. These results pave the way for spatially-informed biomarkers to improve risk stratification and for personalized spatially-targeted therapeutic strategies. Key highlightsO_LIOur ML model identified histological biomarkers predicting prognosis independently from known clinical factors C_LIO_LIThe region of lowly infiltrated white matter enriched in malignant cells including a mesenchymal-like phenotype is predictive of poor prognosis C_LIO_LIAngiogenesis is increased in areas predictive of long survival in main non-necrotic tumor regions. C_LIO_LIThe subtype of macrophages expressing PLIN2 and associated with increased lipid metabolism was associated with poor prognosis in all GBM regions. C_LI Highlights O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/681087v4_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@45e619org.highwire.dtl.DTLVardef@105a4b0org.highwire.dtl.DTLVardef@17f257dorg.highwire.dtl.DTLVardef@7669b4_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Dynamic pharmacophores unveil binding mode ensembles for classical partial agonists at the M2 receptor

For the prototypic M2 receptor it has been previously demonstrated that dualsteric partial agonists can stabilize both, active and inactive receptor states, but it remains unclear whether orthosteric partial agonists have a similar mechanism. Here, we apply dynamic pharmacophores to unveil binding mode ensembles for classical M2 partial agonists. We report correlations between the spatial distribution of lipophilic contacts and ligand efficacy and demonstrate the applicability of dynamic pharmacophores to analyze subtle binding mode changes.

bioinformatics↗

bioRxiv2025

Up-to-date MALDI-TOF MS based identification of the complete Corynebacterium diphtheriae species complex for improved diagnostics

The Corynebacterium (C.) diphtheriae species complex (CdSC) comprises the closely related pathogenic species C. diphtheriae, C. belfantii, C. rouxii, C. ulcerans, C. silvaticum, C. ramonii and C. pseudotuberculosis according to the taxonomic revisions after 2018. Due to their close relationship, reliable species identification within the CdSC is also challenging using MALDI-TOF mass spectrometry (MS) for fast and reliable identification of bacteria. However, the most commonly used commercial databases for MALDI-TOF MS do not reflect the current CdSC taxonomy. Therefore, the objective of the present study is to expand the Bruker MALDI-Biotyper database version K (MBTK) in order to achieve reliable identification for all currently known Corynebacterium species within the CdSC. Furthermore, the database extensions were verified with a systematic formal validation procedure in accordance with the German guideline for species identification by MALDI- TOF MS. For this, 321 strains including all seven valid species of the CdSC were identified with a set of 328 spectra using the commercial MBTK database and a customer-supplemented modified MBTK. This custom database version included 33 reference spectra of the CdSC and representatives of all four recently described species. The results of the formal validations for both, the commercial and the extended database, combined partly with a single signal analysis, provide a substantiated basis for the identification of bacteria of the CdSC. A key element for a fast up-to-date identification has been the exchange of user-generated sets of MSPs on the open access MALDI-UP catalogue that includes reference spectra for all members of the CdSC. HighlightsO_LIUsing MALDI-TOF MS, all current species of the C. diphtheriae species complex are accurately identified according to the current taxonomy C_LIO_LIValidation was performed using spectra from different institutions and an approved gold standard protocol C_LIO_LIThe extension and exchange of user created reference spectra via the MALDI User Platform proved to be a key tool for precise identification of bacteria C_LI Importance StatementBacteria belonging to the Corynebacterium (C.) diphtheriae complex are important pathogens for humans and animals. Identification of the species of this complex described since 2018, i.e. C. belfantii, C. rouxii, C. ramonii, and C. silvaticum, using the widely approved MALDI-TOF mass spectrometry is possible. This has been achieved by improved identification using supplemented MALDI reference databases. Comprehensive validation meets the requirements for accurate pathogen identification in accordance with recognised guidelines. This approach of identification is certainly a model for other bacterial species of interest to react on current taxonomic revisions in a short time frame.

microbiology↗

bioRxiv2025

Cytoplasmic tail composition modulates the G protein and arrestin-3 signaling bias of the adhesion GPCR LPHN2

The class B2 Adhesion GPCRs (aGPCRs) combine cell adhesion with GPCR signaling to control diverse developmental and physiological processes. How aGPCRs interact with and integrate distinct groups of effectors including G proteins, arrestins, and G protein receptor kinases (GRKs) remains unclear. Here, we find that diversity in the aGPCR C-terminal intracellular tail modulates G protein activation, arrestin-3 recruitment, and utilization of selective GRKs in LPHN2, a postsynaptic aGPCR essential for synapse formation. The C-terminal tail of LPHN2 is required for G protein activation and arrestin-3 recruitment. LPHN2 with an intact tail recruits arrestin-3 in the absence of G protein activation, suggesting constitutive arrestin-3-biased signaling. Alternative splicing of the LPHN2 tail modulates G protein activation and arrestin-3 binding independently, supporting that it controls G protein vs arrestin-3 bias. GRKs are important but not essential for arrestin-3 recruitment to LPHN2. Moreover, GRK2 increases arrestin-3 recruitment only in a subset of LPHN2 variants. Collectively, these results show that the mechanisms of the interactions of class B2 aGPCRs and arrestin are distinct from those of class A GPCRs and that splicing of the LPHN2 C-terminal tail determines G protein vs arrestin bias.

molecular biology↗

bioRxiv2025

MOSAIC: Intra-tumoral heterogeneity characterization through large-scale spatial and cell-resolved multi-omics profiling.

Precision oncology remains challenging due to gaps in understanding tumor biology and immunology, as well as the scarcity of consistent data across large cohorts. Here, we introduce MOSAIC (Multi-Omics Spatial Atlas in Cancer), a multi-center clinical Omics study designed to systematically profile thousands of cancer samples, with a focus on spatial and single-cell data across multiple tumor types. MOSAIC exploits recent technological advances to integrate spatial and single-cell data with complementary data modalities, including hematoxylin-eosin histology scans, bulk RNA and exome sequences, to generate comprehensive representations of cancer histology, genomics, and transcriptomics. MOSAIC also collects extensive and curated clinical information to ensure that patients meet the precise inclusion criteria for each cohort. The consortium aims to integrate all data modalities using artificial intelligence and other computational approaches in order to identify clinically-relevant biomarkers and cancer subtypes. This paper outlines the core objectives of MOSAIC, its potential impact, early proofs-of-concept, design, and experimental considerations. Additionally, we introduce the MOSAIC Window initiative, featuring the first released dataset from 60 patients, offering a glimpse into the projects groundbreaking potential. Using four selected patients, we demonstrate the power of multi-omic approaches to analyze and interpret intra-tumoral variability within and across patients, providing insights on specific drug sensitivity of cancer subpopulations and revealing potential impacts on therapeutic recommendations.

molecular biology↗

bioRxiv2025

GRKs phosphorylate GPCR C-terminal peptides in a hierarchical manner

Responses from G protein-coupled receptors (GPCRs) are downregulated in a precisely orchestrated process called desensitization. This process consists of two major steps: phosphorylation of the receptor by GPCR kinases (GRKs), predominantly on its C-terminus, and recruitment of arrestin, resulting in different signaling outcomes. We carried out an NMR-based study of the phosphorylation patterns generated by GRK1 and GRK2 on C-terminal peptides of selected receptors (rhodopsin for GRK1, and {beta}1- and {beta}2-adrenergic receptors (ARs) for GRK2). Our data reveal that the kinases are promiscuous with respect to the substrate peptide, but produce clearly defined phosphorylation patterns on each substrate. We found pronounced differences in the rates at which certain residues are phosphorylated, in particular in the PXPP motifs in rhodopsin and {beta}1AR. These results show, that GRKs produce well-defined phosphorylation patterns in absence of further modulators like the full receptor or G{beta}{gamma}, and that the time profile of the phosphorylation barcode seems to be largely encoded in the minimal pair of C-terminal peptide and GRK. The data further suggest that arrestin might encounter different phosphorylation barcodes over time, potentially inducing different responses at different time points in the desensitization process.

biochemistry↗

bioRxiv2025

SynGAP forms biocondensates at sub-micromolar concentrations and recruits PSD95 and receptor oligomers, functioning as a key initiator of PSD formation

A key issue in neuronal circuit regulation is how synapse formation is initiated. Synapse formation could start when one or more synaptic scaffold proteins that can initiate synapse formation reach certain threshold concentrations in the dendritic shaft, which might lead to their oligomerization or even liquid-liquid phase separation (LLPS). By combining in vitro reconstitution of purified proteins with live-cell single-molecule and confocal imaging, we demonstrated that SynGAP alone forms assemblies of nanoscale clusters containing several to several tens of molecules at 10-nM order concentrations and micron-scale LLPS hydrogel-like condensates at submicromolar concentrations. The trimers of SynGAP's intrinsically disordered region (IDR) induced by its coiled-coil domain are responsible for SynGAP condensation. CaMKII-mediated phosphorylation moderately suppresses SynGAP condensation, and also increases condensate liquidity. While PSD95 fails to form assemblies under these conditions, it is recruited to SynGAP condensates by specifically binding to the PDZ-binding motif of SynGAP. SynGAP[PSD95] condensates selectively immobilize postsynaptic transmembrane proteins, Neuroligin1 and AMPAR-TARP2 complexes, in a manner dependent on their oligomerization state, indicating cooperative recruitment dynamics among SynGAP, PSD95, and transmembrane components, which might mimic initial PSD assembly. These findings suggest that SynGAP may act as a primary nucleator of postsynaptic density assembly, challenging the PSD95-centered models.

neuroscience↗

bioRxiv2025

Non-canonical internalization mechanisms of mGlu receptors

Cell surface density of G protein-coupled receptors (GPCRs) is tightly regulated through constitutive and agonist-induced internalization. Whereas the mechanisms of constitutive internalization remain elusive, agonist-induced internalization is accepted to involve receptor phosphorylation by GPCR kinases (GRKs), {beta}-arrestin binding and AP2 recruitment, targeting receptors to clathrin-coated pits. Dimeric class C metabotropic glutamate (mGlu1 to 8) receptors regulate synaptic transmission but their internalization process is ambiguous. Here, we used diffusion-enhanced energy transfer (DERET) to decipher their internalization kinetics. We showed that all mGlu receptors are constitutively internalized. However, only mGlu1, 5 and 3 homodimers are agonist-induced internalized, that require neither GRKs, nor {beta}-arrestins. In contrast, the constitutive internalization involves only {beta}-arrestins. This systematic study further illustrates how different class C receptors are relative to most other GPCRs, revealing non-canonical internalization mechanisms. These insights in mGlu receptor dynamics will help promoting the therapeutic action of drugs targeting mGlu receptors.

neuroscience↗