Biology subjects

Bennett, J. L.

Publications and source records attributed to Bennett, J. L..

12 recordsLinked to original sources

bioRxiv2026

An integrated mass spectrometry strategy for quantifying the proteoform diversity of the extensively modified O-glycoprotein Osteopontin

O-glycosylation is among the most abundant and structurally diverse post-translational modifications in eukaryotes, yet its heterogeneity renders O-glycoproteins exceptionally difficult to characterize. To overcome these challenges, we have developed an integrated mass spectrometry (MS) strategy to define the proteoform landscape of O-glycoproteins and applied it to human osteopontin (OPN). OPN is a disease-associated extracellular matrix protein subject to extensive modification. By combining native MS with serial exoglycosidase digestions, we directly resolved truncation, phosphorylation, sulfation, and O-glycosylation of OPN. Matched glycoproteomic analyses, using tailored (glyco)protease combinations, allowed us to quantify glycan heterogeneity inaccessible to conventional trypsin-based approaches or protein-centric methods. We integrated these datasets using forward compositional simulations to infer the intact OPN proteoform distribution and benchmarked the resulting models against an experimental intact-mass distribution obtained by proton-transfer charge-reduction MS. This comparison revealed that bottom-up O-glycoproteomics systematically underestimates the true extent of glycan sialylation, whereas assuming (near-)complete sialylation accurately reproduced the experimental intact OPN mass distribution. Together, these results provide a comprehensive, quantitative view of OPN compositional diversity and demonstrate how intact-protein and peptide-level measurements can be reconciled to resolve highly heterogeneous glycoform populations. The workflow establishes a broadly applicable framework for characterizing extensively O-glycosylated and multiply modified proteins.

biophysics↗

bioRxiv2026

Microglia drive demyelination via multiple sclerosis antibodies and BTK signaling

Microglia are the predominant immune cells in multiple sclerosis (MS) demyelinating lesions, where they phagocytose myelin, but whether they destroy myelin or merely scavenge its debris is unknown. Here, we explore whether pathogenic autoantibodies found in MS may induce the phagocytic destruction of myelin by microglia. Applying patient-derived, myelin-targeting antibodies to the mouse cortex, we developed an in vivo model of MS with focal demyelination that depended on epitope specificity and Fc gamma receptor and complement binding. Longitudinal monitoring of microglia-myelin interactions using in vivo two-photon microscopy revealed rapid microglial envelopment of intact myelin driving myelin loss, while single-cell RNA sequencing identified a demyelination-associated microglial signature. Parallel changes were observed in human MS lesions, where microglia enveloped intact myelin and similar genes were upregulated. Inhibition of Brutons tyrosine kinase (BTK) limited microglial transcriptional changes and prevented myelin loss following microglial envelopment. These findings directly implicate microglia in pathological myelin loss and support BTK inhibition as a therapeutic strategy to prevent demyelination by modulating microglia behavior.

neuroscience↗

bioRxiv2026

A longitudinal two-photon imaging platform for focal astrocyte ablation in vivo

Investigating the consequences of astrocyte loss in the intact brain is both important and challenging. As integral components of the neuro-glia-vascular unit, astrocytes are involved in a variety of brain processes including water homeostasis, metabolic supply, regulation of cerebral blood flow, and coordination of neuronal circuit activity. Astrocyte impairment has been associated with numerous neurological disorders. However, experimental models combining focal astrocyte ablation with longitudinal in vivo imaging in the intact adult brain have been lacking, limiting efforts to define the causal contribution of astrocyte loss to central nervous system (CNS) pathology and repair. Here, we present an in vivo model of antibody-mediated astrocyte ablation that enables longitudinal imaging and detailed investigation of ensuing cellular responses. It integrates focal induction of aquaporin-4 antibody-mediated astrocyte loss, chronic in vivo two-photon imaging, genetically encoded sensors, and reporter mouse lines. This advancement allows visualization and quantification of cellular and subcellular events in living organisms during lesion progression and recovery. It overcomes many longstanding limitations of previous models that are either constrained by non-specific hypoxic or mechanical tissue damage or require sacrificing animals at discrete time points, hindering the ability to monitor dynamic biological processes over time. In contrast, the selective targeting of astrocytes prevents the formation of the glial border, enabling the investigation of CNS response in a scar-free environment. Overall, this new approach represents a significant technical advancement, enabling comprehensive longitudinal studies of CNS responses to astrocyte loss, thus opening new avenues for understanding astrocytopathy-driven pathology, evaluating therapeutic interventions, and promoting translational research.

neuroscience↗

bioRxiv2026

Language Model Embedding Classifiers Enable Identification of Multiple Sclerosis-Associated BCRs and Repertoires

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease. It affects over 2 million people worldwide but has historically been challenging to diagnose, categorize, and treat. MS has an autoimmune component that involves the production of B-cell receptors (BCRs) and immunoglobulins (Igs) that are associated with disease pathophysiology. We reanalyzed all publicly available RNA sequencing data from MS patients and extracted over 11 million BCR immunoglobulin heavy chain (IGH) sequences obtained from a variety of tissue sources. We developed a new decoder-only BCR DNA embedding model that outperforms state-of-the-art BCR and general-purpose protein language models on sequence embedding tasks. We then trained a language model classifier capable of identifying BCR sequences associated with MS. Using low-dimensional representations of whole repertoire embeddings combined with sequence-disease predictions, we can distinguish MS patient repertoires from healthy, infectious disease, or other autoimmune disease repertoires. Our models also successfully rank known MS-associated myelin-binding IgG sequences relative to controls. These findings provide a methodological foundation for BCR-based MS detection and could facilitate identification and study of disease-associated antibodies from blood. SignificanceAutoimmune diseases are difficult to diagnose and treat. Circulating adaptive immune cells contain accessible information about a patients present and past immune experiences. To better use this information in the context of multiple sclerosis, we extracted a comprehensive dataset of B-cell receptor sequences from archival data, developed a new DNA foundation model to embed these sequences, and applied machine learning methods to classify sequence disease association and patient disease state. These findings will advance our ability to model and predict multiple sclerosis and other diseases.

bioinformatics↗

bioRxiv2026

Molecular dissection of protein complexes isolated from sections of human brain

Molecular studies of brain receptors and transporters have typically relied on recombinant systems, limiting insight into their organization in native tissue. Here, we develop nanobody-based immunoprecipitation coupled with native mass spectrometry to interrogate endogenous protein assemblies from post-mortem mouse and human brain sections. We exemplify our approach by characterizing the synaptic proteins VGluT1 and mGluR2. From a single mouse brain, we discover mGluR2/3 heterodimers, alongside mGluR2 homodimers. Considering regions of human brain heterodimeric mGluR2/3 is highly abundant in the OFC and sgACC ([~]70% and 50%, respectively) and forms regional-specific interactions with additional synaptic proteins. In a modest cohort of biobanked human tissue, associated with depression and suicide, we find increased mGluR2/3 in the OFC. Consistent with this, mice exhibit similar associations between heterodimer levels and stress-susceptibility. Overall, our approach provides a direct means for establishing molecular-behavioural links at the level of receptor organization in brain.

biochemistry↗

bioRxiv2026

Early demyelination by off-target complement injury in a mouse model of neuromyelitis optica

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune CNS disease characterized by serum antibodies targeting astrocytes for complement-mediated lysis. NMOSD lesions show not only astrocyte loss but also early demyelination and oligodendrocyte injury - histological hallmarks that converge with those of other primary demyelinating conditions. How pathology spreads to cause demyelination, particularly in early lesions, remains unclear. Using spinal cord imaging in an acute mouse NMOSD model, we directly observe the spread of pathology from astrocytes to oligodendrocytes in evolving lesions. This spread is characterized by initial calcium dyshomeostasis in oligodendrocytes followed by delayed, non-lytic cell death. Oligodendrocyte death is driven not by astrocyte loss per se but by spill-over of soluble complement proteins, as oligodendrocytes can be cell-autonomously preserved by the cell-type-specific expression of the complement inhibitor CD59. Our findings explain the convergence of glial pathology in antibody-mediated CNS autoimmunity and point towards new approaches to prevent secondary glial injury.

neuroscience↗

bioRxiv2025

Cellular basis for cortical network aging in primates

Large-scale brain networks are vulnerable to change with aging and become dysregulated. How these networks are altered at the cellular level remains unclear owing to challenges of bridging data across scales. Here, we integrate in vivo cortical similarity networks with whole brain spatial transcriptomics to characterize the aging brain in a lifespan cohort of macaques (N=64, ages 1-26 years). Deep-layer excitatory neurons and oligodendrocytes emerged as dominant correlates of cortical similarity, linking infragranular cell type composition to macroscopic network structure. Age-related declines in network strength were most pronounced in transmodal networks, including default mode and limbic, and aligned with regions enriched in inhibitory and glial cell types. Parvalbumin-enriched chandelier cells showed the strongest association with regional vulnerability, suggesting a role in network disconnection. Cell-type enrichment was conserved across species, with both human and macaque transcriptomic data aligning with the cortical functional hierarchy. These findings uncover a cellular basis for cortical network aging and highlight the value of imaging-transcriptomic integration across scales.

neuroscience↗

bioRxiv2024

Astrocytes adopt a progenitor-like migratory strategy for regeneration in adult brain

Mature astrocytes become activated upon non-specific tissue damage and contribute to glial scar formation. Proliferation and migration of adult reactive astrocytes after injury is considered very limited. However, the regenerative behavior of individual astrocytes following selective astroglial loss, as seen in astrocytopathies, such as neuromyelitis optica spectrum disorder, remains unexplored. Here, we performed longitudinal in vivo imaging of cortical astrocytes after focal astrocyte ablation in mice. We discovered that perilesional astrocytes develop a remarkable plasticity for efficient lesion repopulation. A subset of mature astrocytes transforms into reactive progenitor-like (REPL) astrocytes that not only undergo multiple asymmetric divisions but also remain in a multinucleated interstage. This regenerative response facilitates efficient migration of newly formed daughter cell nuclei towards unoccupied astrocyte territories. Our findings define the cellular principles of astrocyte plasticity upon focal lesion, unravelling the REPL phenotype as a fundamental regenerative strategy of mature astrocytes to restore astrocytic networks in the adult mammalian brain. Promoting this regenerative phenotype bears therapeutic potential for neurological conditions involving glial dysfunction.

neuroscience↗

bioRxiv2024

Structural basis of autoantibody binding in Neuromyelitis Optica

Neuromyelitis Optica (NMO) is an autoimmune disease of the central nervous system where pathogenic autoantibodies target the human astrocyte water channel aquaporin-4 causing neurological impairment. Autoantibody binding leads to complement dependent and complement independent cytotoxicity, ultimately resulting in astrocyte death, demyelination, and neuronal loss. Aquaporin-4 assembles in astrocyte plasma membranes as symmetric tetramers or as arrays of tetramers. We report molecular structures of aquaporin-4 alone and bound to Fab fragments from patient-derived NMO autoantibodies using cryogenic electron microscopy. Each antibody binds to epitopes comprised of three extracellular loops of aquaporin-4 with contributions from multiple molecules in the assembly. The structures distinguish between antibodies that bind to the tetrameric form of aquaporin-4, and those targeting higher order orthogonal arrays of tetramers that provide more diverse bridging epitopes. One-Sentence SummaryPathogenic autoantibodies in Neuromyelitis Optica distinguish multiply presented epitopes on arrays of human aquaporin-4.

immunology↗

bioRxiv2024

Lipopeptide antibiotics disrupt interactions of undecaprenyl phosphate with UptA

The peptidoglycan pathway represents one of the most successful antibacterial targets with the last critical step being the flipping of carrier lipid, undecaprenyl phosphate (C55-P), across the membrane to re-enter the pathway. This translocation of C55-P is facilitated by DedA and DUF368 domain-containing family membrane proteins via unknown mechanisms. Here we employ native mass spectrometry to investigate the interactions of UptA, a member of the DedA family of membrane protein from Bacillus subtilis, with C55-P, membrane phospholipids and cell wall-targeting antibiotics. Our results show that UptA, expressed and purified in E. coli, forms monomer-dimer equilibria, and binds to C55-P in a pH-dependent fashion. Specifically, we show that UptA interacts more favourably with C55-P over shorter-chain analogues and membrane phospholipids. Moreover, we demonstrate that lipopeptide antibiotics, amphomycin and aspartocin D, can directly inhibit UptA function by out-competing the substrate for the protein binding, in addition to their propensity to form complex with free C55-P. Overall, this study shows that UptA-mediated translocation of C55-P is potentially mediated by pH and anionic phospholipids and provides insights for future development of antibiotics targeting carrier lipid recycling.

molecular biology↗

bioRxiv2023

Protein interaction kinetics delimit the performance of phosphorylation-driven protein switches.

Post-translational modifications (PTMs) such as phosphorylation and dephosphorylation can rapidly alter protein surface chemistry and structural conformation which can, in turn, switch protein-protein interactions (PPIs) within signaling networks. Recently, de novo designed phosphorylation-responsive protein switches have been created that harness kinase- and phosphatase-mediated phosphorylation that modulate PPIs. PTM-driven protein switches could be useful for investigating PTM dynamics in living cells, developing biocompatible nanodevices, and engineering signaling pathways to program cell behavior. However, little is known about the physical and kinetic constraints of PTM-driven protein switches, which limits their practical application. In this study, we present a theoretical framework to evaluate two-component PTM-driven protein switches based on four performance metrics: effective concentration, dynamic range, response time, and reversibility. Our computational models reveal an intricate relationship between the binding kinetics, phosphorylation kinetics, and switch concentration that governs the sensitivity and reversibility of PTM-driven protein switches. Building upon the insights of our theoretical investigation, we built and evaluated two novel phosphorylation-driven protein switches consisting of phosphorylation-sensitive coiled coils as sensor domains fused to fluorescent proteins as actuator domains. By modulating the phosphorylation state of the switches with a specific protein kinase and phosphatase, we demonstrate fast, reversible transitions between easily differentiated "on" and "off" states. The response of the switches linearly correlated to the concentration of the kinase, demonstrating its potential as a biosensor for kinase measurements in real time. As intended, both switches responded to specific kinase activity with an increase in fluorescence signal and our model could be used to distinguish between two mechanisms of switch activation: dimerization or a structural rearrangement. In summary, the protein switch kinetics model presented here should be useful to guide the design of PTM-driven switches and tune their performance towards concrete applications.

synthetic biology↗

bioRxiv2020

Specific induction of double negative B cells during protective and pathogenic immune responses

1Double negative (DN) (CD19+CD20lowCD27-IgD-) B cells are expanded in patients with autoimmune and infectious diseases; however their role in the humoral immune response remains unclear. Using systematic flow cytometric analyses of peripheral blood B cell subsets, we observed an inflated DN B cell population in patients with variety of active inflammatory conditions: myasthenia gravis, Guillain-Barre syndrome, neuromyelitis optica spectrum disorder, meningitis/encephalitis, and rheumatic disorders. Furthermore, we were able to induce DN B cells in healthy subjects following vaccination against influenza and tick borne encephalitis virus. Transcriptome analysis revealed a gene expression profile in DN B cells that clustered with naive B cells, memory B cells, and plasmablasts. Immunoglobulin VH transcriptome sequencing and analysis of recombinant antibodies revealed clonal expansion of DN B cells, that were targeted against the vaccine antigen. Our study suggests that DN B cells are expanded in multiple inflammatory neurologic diseases and represent an inducible B cell population that responds to antigenic stimulation, possibly through an extra-follicular maturation pathway.

immunology↗