Biology subjects

Yates, J. R.

Publications and source records attributed to Yates, J. R..

At least 19 recordsLinked to original sources

bioRxiv2026

dnoise: Fast Native Data Reduction for Bruker timsTOF

Bruker timsTOF acquisitions produce dense native .d files whose storage, transfer, and archival become substantial at high throughput. We present dnoise, an open-source Rust tool that removes points directly from timsTOF frames and writes a native-compatible .d directory. dnoise retains ions that form coherent streaks across the ion-mobility dimension and applies acquisition-aware gates to signal that cannot be selected for fragmentation. On a three-species benchmark spanning ddaPASEF and diaPASEF at 5- and 15-minute gradients, default MS1-only denoising reduced the frame binary by 35 to 53%. Label-free quantification accuracy was preserved in both modes. ddaPASEF peptide-spectrum-match, peptide, and protein-group counts were unchanged, as expected with the searched MS/MS spectra untouched, and diaPASEF precursor and protein-group counts changed only slightly. Every tested processing run completed in 69 seconds or less on the benchmark workstation. Optional MS/MS denoising produced greater reduction but sacrificed several percent of identifications. Thus, a substantial fraction of native timsTOF frame data can be removed with little analytical change.

bioinformatics↗

bioRxiv2026

Quantitation of Spatial Proteoforms in Alzheimer's Disease

Alzheimers disease (AD) pathogenesis involves complex, multifactorial changes to the brain proteome that conventional unfractionated analyses may obscure. Proteins frequently occupy multiple subcellular compartments as spatial proteoforms, yet the contribution of aberrant protein localization to AD pathogenesis remains poorly understood. To address this, we fractionated post-mortem human hippocampi from 13 AD and 14 non-AD individuals into four subcellular fractions and quantified 6,123 proteins by TMT-LC-MS. Although 75% of proteins were detected in more than one fraction, 78% of significant AD-associated alterations were restricted to a single fraction, demonstrating that subcellular localization is a primary determinant of disease vulnerability. Discordant abundance patterns between fractions revealed retromer complex mislocalization, nuclear transport dysfunction, and insoluble protein accumulation, with the endosomal-lysosomal and protein folding pathways most consistently perturbed. To examine how these perturbations evolve with disease progression, we applied the QUAD strategy to measure protein degradation in two fractions of APPswePS1delta9 mouse cortex at 2, 5, and 12 months. Degradation rates diverged between fractions and genotypes in an age-dependent manner, and cross-dataset comparison identified six proteins altered at the earliest pre-pathological timepoint, implicating vesicle transport and proteostasis disruption as initiating features of AD. These findings establish spatial proteoforms as essential units of pathogenic analysis and reveal disease-relevant signals invisible to bulk tissue approaches.

neuroscience↗

bioRxiv2026

ESCRT-0 regulates AMPA receptor currents and Ca2+- dependent signaling

Membrane protein trafficking is essential for synaptic growth, maintenance, function, and plasticity, and involves the regulated exocytosis and endocytosis of proteins to and from the pre-and post-synaptic membranes. Defects in the clearance of membrane proteins can lead to the accumulation of ubiquitinated membrane proteins and contribute to neurodegenerative disease. The ESCRT (endosomal sorting complexes required for transport) machinery binds and sorts ubiquitinated membrane proteins into lysosomes for degradation, yet the presence and function of ESCRTs in sorting ubiquitinated AMPA and other receptors at the post-synapse remain unclear. Here we show that the ubiquitin-binding ESCRT-0 protein, Hrs, localizes to both pre- and post-synapses, and levels are modulated by neuronal activity, increasing and decreasing with higher and lower neuronal activity, respectively. Phosphoproteomic profiling of Hrs-depleted post-synaptic membranes revealed a role for Hrs in glutamatergic synaptic transmission, including long-term potentiation. In addition, Hrs-depleted neurons showed faster AMPAR current kinetics and reduced amplitude in whole-cell patch-clamp recordings. Genetic deletion of neuronal Hgs in mice led to reductions in phosphorylated CaMKII- and -{beta} (T286/T287) and structural proteins, PSD-95 and gephyrin, suggestive of LTD (long-term depression)-like synaptic depression. In contrast, Hrs overexpression led to increases in Ca2+-dependent signaling, including protein kinase C (PKC) and PKC substrate, AMPAR subunit GluA1-S831, a site which increases conductance. Together, these findings identify a dynamic, bidirectional role for Hrs at the post-synapse as it both senses and is modulated by neuronal activity, ultimately impacting excitatory synaptic strength. Significance StatementSynaptic plasticity relies on dynamic trafficking and turnover of membrane proteins, including AMPA-type glutamate receptors (AMPARs), yet how receptor trafficking intersects with ubiquitin-mediated sorting pathways at synapses remains unclear. We show that the ubiquitin-binding ESCRT-0 protein, Hrs, localizes to both pre- and post-synapses, and its abundance is bidirectionally regulated by neuronal activity. Genetic depletion of Hrs in mice reduces CaMKII phosphorylation and impacts AMPAR channel surface localization. In contrast, neuronal-specific Hrs overexpression led to enhanced GluA1 and protein kinase C substrate phosphorylation, suggesting altered AMPAR trafficking, subunit composition, and/or function. Thus, Hrs emerges as a modulator of glutamatergic signaling, coupling ubiquitin-mediated receptor sorting to the fine-tuning of synaptic transmission, with direct implications for learning and memory in health and disease.

neuroscience↗

bioRxiv2026

Translating Innovation to Clinic: End-to-End Bioprocess Development and cGMP Manufacturing of N332-GT5 HIV Vaccine Candidate for First-in-Human Trials HVTN144

The successful translation of rationally designed HIV-1 immunogens into effective vaccines requires manufacturing platforms that maintain structural conformity while meeting clinical-grade quality standards. We developed and scaled a robust, cGMP-compliant process for N332-GT5 gp140, a germline-targeting envelope trimer designed to initiate broadly neutralizing antibody responses, which is now undergoing first-in-human evaluation in HVTN144. Starting with a stable CHO cell line developed using Leap-In(R) transposon technology, we established a production clone exhibiting high-titer expression (>200 mg/L) and genetic stability through 60 population doublings. The manufacturing process scaled efficiently from Ambr(R) 250 miniature bioreactors to 200-L single-use systems, delivering consistent product quality across multiple cGMP batches. A streamlined three-step purification strategy--affinity capture, multimodal polishing, and viral clearance- yielded >99% trimeric purity with preserved quaternary structure and native-like antigenicity. Orthogonal LC-MS analyses confirmed site-specific glycan occupancy matching design specifications, while robust viral clearance exceeded 18-log and 11-log reductions for model retroviruses. Clinical material manufactured through this platform has been successfully administered in HVTN144. This work establishes a scalable, reproducible manufacturing paradigm for structurally complex HIV-1 envelope immunogens, advancing the field toward rational vaccine design based on germline-targeting principles.

bioengineering↗

bioRxiv2026

De novo design of transmembrane accessory subunits for fold stabilization and expansion

Transmembrane (TM) proteins play essential roles in biology as transporters, ion channels, chaperones, enzymes, and mediators of signal transduction. However, membrane proteins often suffer from inefficient folding and intrinsic instability. Misfolding in cells can cause numerous loss-of-function pathologies. Likewise, denaturation upon purification in the laboratory is a critical barrier to structure determination and characterization of key biochemical mechanisms. Generalizable strategies to stabilize membrane proteins remain limited. Here, we developed an informatics-based de novo design strategy to create synthetic auxiliary subunits that interact with the TM helices of a model pentameric ion channel, thereby bolstering folding while maintaining channel function. Biochemical and structural characterization reveal the synthetic TM subunits can also be used to create larger multi-spanning designer proteins of custom topology. This proof-of-concept motivates the feasibility of computationally designed accessory TM helices as potential pharmacological chaperone "folding correctors" of membrane proteins in disease and as tools in structural biology.

biochemistry↗

bioRxiv2026

Molecular and Structural Characterization Reveals Divergent Extracellular Vesicle Profiles Between Wild Type and Alzheimer's Disease Cerebrocortical Organoids

Alzheimers disease (AD) is a neurodegenerative disorder affecting millions of patients globally. Despite significant efforts from researchers in recent decades, there are still many unanswered questions about AD pathogenesis. AD patient brains manifest changes in extracellular vesicles (EVs) secreted from diseased neurons, and the effect of this phenomenon remains poorly understood. EVs contain a variety of biomolecules and play a critical role in cell-to-cell communication in all eukaryotic organisms. Here, we report a thorough characterization of small EVs purified from cultures of human cerebrocortical organoids. These organoids are differentiated from human patient-derived stem cells that bear a familial AD mutation in the presenilin 1 (PSEN1) gene, or from an isogenic wildtype (WT) control. The organoid conditioned media was aspirated from cultures and processed for EV enrichment using a non-invasive technique that requires no cellular disruption. EVs purified from AD organoid conditioned media have a wider size distribution and show differential expression of tetraspanins CD63, CD9, and CD81 when compared to WT organoid-derived EVs. AD organoid-derived EVs can have single, double, and even triple membranes and display luminal fibrillar material. A deep proteomic profiling of the EVs reveals several statistically significant differences, including evidence for modifications in secretory autophagy. EV isolates from both WT and AD organoids show strong binding to amyloid detecting dyes, both in bulk fluorescence and fluorescence microscopy assays. After a 1-week co-culture of AD organoids with WT organoids, there is evidence of endosomal membrane transfer between the isogenic cultures with an increase in amyloid-{beta} peptides in the WT organoids. These observations support the notion that non-cell-autonomous spread of amyloid-containing EVs in human AD brains can be modeled in a cerebral organoid system.

neuroscience↗

bioRxiv2026

Capturing Cardiomyocyte Cell-to-Cell Heterogeneity via Shotgun Single Cell Top-Down Proteomics

Individual cells exhibit distinct molecular landscapes shaped by proteins and the diverse functional repertoire of their corresponding proteoforms. These structurally diverse variants (e.g., post-translationally modified including truncated proteolyzed forms) collectively orchestrate cellular functions. However, resolving proteoform heterogeneity at single-cell (SC) resolution remains a significant analytical challenge. Here, we present a shotgun SC top-down proteomics (SC-TDP) strategy that enables direct, unbiased proteoform profiling from single cardiomyocytes. Across 13 individual cardiomyocytes isolated from mouse heart, we identified a total of 57 proteins represented by 165 distinct proteoforms, including phosphorylated, succinylated, trimethylated, truncated, amongst others. Notably, proteoform composition varied substantially among cells, revealing a previously unrecognized level of molecular heterogeneity among cardiomyocytes. Together, these findings establish SC-TDP as a powerful tool for uncovering the proteoform diversity at the SC level. Our strategy paves the way for defining functional heterogeneity in cardiac tissue with unprecedented molecular resolution, enabling direct examination of the proteoform landscape that underlies cellular identity and physiology.

cell biology↗

bioRxiv2026

Spt5's central KOW domains and the Pol II Stalk Collaborate to Regulate Chromatin and 3'-End Processing

Spt5 is a universally conserved multidomain transcription elongation factor that acts as a component of all Pol II elongation complexes. Structural studies indicate that several of Spt5s central KOW domains lie adjacent to the Pol II stalk, composed of subunits Rpb4 and Rpb7. However, their in vivo functions are unknown. Here we show that Spt5 and Rpb4/7 jointly modulate 3-end formation and co-transcriptional chromatin integrity in Saccharomyces cerevisiae. We identify mutations in the SPT5 KOW2-3 domains and RPB7 that cause cryptic initiation of transcription and alter 3-end formation of RNA transcripts. Molecular readthrough assays reveal allele-specific changes at both GAL10 and SNR13, consistent with impacts on CPF/CF- and NNS-dependent termination. Proteomic experiments with isolated KOW2-3 domain enrich factors from both pathways as well as chromatin regulators, overlapping known Rpb7 interactors. Together, these findings support a model in which Spt5 KOW2-3/Pol II stalk region acts as a recruitment platform that coordinates pre-mRNA processing and chromatin dynamics during elongation, revealing new roles for the central KOW domains of Spt5. SummaryThis work describes a cooperative in vivo function for Spt5s central KOW domains and the Pol II stalk in Saccharomyces cerevisiae. Allele-specific genetics and reporter assays show cooperative effects of SPT5 and RPB4/7 on cryptic initiation and 3'-end formation; double-mutant analyses reveal synthetic interactions. RT-qPCR at GAL10 and SNR13 demonstrates regulation of both poly(A) and non-coding transcript termination. Spt5 KOW pull-down proteomics enrich poly(A) and non-coding termination factors, as well as chromatin regulators that overlap with known Rpb7 interactors. Together, the data support a model in which Spt5 and the Pol II stalk coordinate chromatin integrity and termination during elongation.

genetics↗

bioRxiv2026

Rapid Histone Post-Translational Modification Analysis Using Alternative Proteases and Tandem Mass Tags

Histone post-translational modifications (PTMs) alter chromatin dynamics and contribute to the regulation of gene expression in health and disease, yet mass spectrometry-based histone PTM analysis remains constrained by inefficient sample preparation workflows. Here, we develop RIPUP (Rapid Identification of histone PTMs in Underivatized Peptides), a streamlined multi-protease workflow that reduces sample preparation to hours while improving PTM coverage and quantitative accuracy. Systematic evaluation of Arg-C Ultra and a recombinant (r)-Chymotrypsin protease under varied conditions, including standard derivatization with propionic anhydride and tandem mass tag (TMT) labeling, demonstrated that Arg-C Ultra with TMT labeling achieves a detection of total PTM that exceeds Trypsin-based approaches. Using the HiP-Frag computational framework for unrestrictive PTM identification, we discovered that TMTs tertiary amine provides charge compensation that rescues the ionization of negatively charged acylations revealing 58 succinylation and 31 glutarylation sites - a dark epigenome largely undetected by propionylation-based methods. Complementary digestion with Arg-C Ultra and r-Chymotrypsin provides orthogonal sequence coverage, enabling detection of PTMs in H2A variants, linker histones, and regions poorly represented by arginine-specific cleavage alone. In HEK293T cells treated with the pan-sirtuin inhibitor nicotinamide, RIPUP quantified 112 statistically significant peptidoforms (adj p < 0.05), predominantly increasing with NAM dose (88 up, 24 down). Application of RIPUP to frozen-thawed rat hippocampal sections within a 3-hour workflow identified >200 PTMs including H3 K27/K36/K37 methylation, H4 N-terminal acetylation patterns, and H2A K118/K119 ubiquitination. This rapid, high-efficiency platform enables timely discovery of epigenetic mechanisms and accelerates the path from PTM identification to therapeutic target validation.

biochemistry↗

bioRxiv2025

The effects of Alcohol Dependence on the CSF Proteome in Mice: Evidence for Blood-Brain Barrier Dysfunction and Neuroinflammation

Alcohol use disorder (AUD) represents a significant neurological health burden, yet the biological mechanisms underlying alcohol-induced brain pathology remain incompletely understood. Moreover, the molecular underpinnings of the transition from alcohol exposure to alcohol dependence are not well-characterized. We used mass spectrometry (MS)-based proteomics in a preliminary discovery study to compare cerebrospinal fluid (CSF) of alcohol-exposed Non-dependent (Non-dep) versus alcohol-dependent (Dep) mice that underwent the chronic intermittent ethanol (alcohol) - two-bottle choice (CIE-2BC) procedure and systemic anti-IL-6 Receptor antibody administration. CSF samples from individual mice were processed for proteomic analysis and digested with trypsin overnight. Peptides were analyzed via data-independent acquisition (DIA)-MS and data were processed in DIA-NN at 1% FDR. We identified 611 unique proteins across both groups, with 140 proteins differentially detected in CSF from Dep mice and 67 proteins specific to alcohol-exposed but Non-dep controls. The Dep-specific proteins revealed signatures of blood-brain barrier (BBB) disruption, neuroinflammation, cellular stress responses, and complement system activation. In contrast, Non-dep-specific proteins indicated preserved protective mechanisms including complement regulation, anti-inflammatory signaling, and neuronal calcium homeostasis. Ethanol-dependent-specific findings include MMP2, BIP, and to a lesser extent VE-cadherin (CDH5) and VCAM1, indicative of the beginnings of endothelial damage and BBB disruption, alongside established neuroinflammation markers GFAP, CHI3L1, and CX3CL1. This work provides novel preliminary protein-level evidence that alcohol exposure and alcohol dependence are dichotomous; despite the small sample size and limited power for moderate effect sizes, there appears to be a clear molecular transition from maintained protective mechanisms to vascular damage, BBB breakdown, and sustained neuroinflammation.

neuroscience↗

bioRxiv2025

Dormant viral pathways underlie space-induced neural senescence: a neuroprotective strategy for spaceflight and neurological diseases

Long-duration spaceflight is associated with neurological symptoms in astronauts, yet the underlying molecular mechanisms remain unclear. Using human brain organoids cultured aboard the International Space Station, we analyzed three independent spaceflights to demonstrate that exposure to the space environment triggers Space-Induced Neural Senescence (SINS), characterized by chromatin remodeling, mitochondrial dysfunction, and activation of viral-like transcriptional programs in the absence of infection. Multi-omics analyses identified upregulation of endogenous LINE-1 (L1) retroelements, whose activity was markedly enhanced in organoids lacking MECP2, a known L1 repressor implicated in Rett syndrome. The resulting accumulation of cytoplasmic L1 DNA elicited an IL-6-mediated inflammatory and neurotoxic response, which was reversed by reverse transcriptase inhibitors (RTi) such as lamivudine or stavudine. Parallel preclinical experiments in Mecp2-deficient mice confirmed that RTi treatment restored neuronal morphology, synaptogenesis, function, cognition, and survival. These findings reveal that the space environment reactivates dormant genomic retroelements, providing an unexpected mechanistic insight into astronaut neurobiology and identifying a potential therapeutic strategy for both space-induced and terrestrial neurological conditions. Our pioneering study demonstrates the value of space-enabling research in accelerating drug discovery and disease treatment on Earth.

neuroscience↗

bioRxiv2025

Lysosomal permeabilization by Group A Streptococcus releases proteins into the macrophage cytosol

The human-specific bacterial pathogen Group A Streptococcus (GAS) is a significant cause of morbidity and mortality due to its ability to cause severe invasive infection. Although macrophages are important for controlling GAS infection, we and others have demonstrated that GAS can persist in macrophages by perforating the phagolysosome using the pore-forming toxin streptolysin O (SLO). In this study, we examined how phagosomal perforation releases lysosomal and bacterial proteins into the cytosol and alters cytosolic protein content in the macrophage. Using IL-1{beta} as a measure of intracellular pathogen detection, we confirmed that cytosolic preparations from macrophages infected with either wild-type (WT) or SLO-deficient ({Delta}SLO) bacteria contained new proteins that are absent in uninfected cytosol controls. Proteomic analysis revealed distinct cytosolic protein profiles in both WT- and {Delta}SLO-infected macrophages. M1 protein was detected only in the cytosol of WT-infected macrophages and corresponded with the IL-1{beta} response, indicating SLO-mediated release of M1 protein from the phagosome, and providing a mechanism for cytosolic recognition of this virulence factor. Unexpectedly, cytosolic extracts of both WT- and {Delta}SLO-infected macrophages contained all histone proteins, suggesting that nucleosomal complexes are released into the cytosol during GAS infection. Our work reveals both a mechanism for the activation of the inflammatory response on a cellular level, and the surprising consequences of phagosomal perforation in GAS infections. These responses may collectively contribute to the pathologies observed during severe invasive GAS infection, and can help inform therapies aimed at improving macrophage function and patient outcomes. ImportanceThe post-pandemic increase in cases of invasive Group A Streptococcus (GAS) infection, along with its sudden onset and severity of symptoms, underscores the need to understand GAS survival in host cells. Macrophages are key first responder immune cells essential for limiting the spread of GAS infection. As professional phagocytes, macrophages typically engulf bacteria in phagosomes and digest them using proteolytic enzymes. However, the human-specific pathogen GAS has acquired several evasion tactics through co-evolution with our immune system. In previous work, we have shown that GAS survives digestion by perforating the phagosome. In this study, we determined that this perforation allows both host and bacterial proteins to leak into the cytosol. These leaked proteins can dictate the subsequent response of the macrophage and may contribute to the pathology observed in invasive infection. Understanding this response can lead to improved therapies for GAS infections.

microbiology↗

bioRxiv2025

Patch-Clamp Single-Cell Proteomics in Acute Brain Slices: A Framework for Recording, Retrieval, and Interpretation

Single-cell proteomics (SCP) is a powerful method for interrogating the molecular composition of neurons, yet its application to acute brain slices has remained limited. Patch-clamp electrophysiology provides direct information on neuronal excitability, synaptic inputs, and ion channel function, making it a natural partner for SCP. However, combining these techniques introduces unique challenges. For instance, after patch-clamping a neuron, its soma must be physically retrieved, and variability during extraction from the brain slice may influence how well proteomic measurements reflect in situ physiology. Here, we introduce a framework for contextualizing patch-SCP outcomes, with an emphasis on retrieval quality (material yield and soma-enriched synaptic content). We used an indiscriminate shotgun strategy in which all patched neurons were collected regardless of electrophysiological outcome to assess soma retrieval in an exploratory dataset of rat medial prefrontal cortex pyramidal neurons. Capacitance during gigaseal-preserved retrieval correlated with protein identifications, suggesting that proteome yield could be linked to soma size. Preservation of neuronal spiking during relocation tended to be associated with broader synaptic enrichment and recovery of transmembrane proteins. By comparison, torn or aspirated neurons produced small proteomes with poor synaptic representation and neurons with little to no characterization displayed more variable outcomes. These results demonstrate that patch-SCP can be used to assess soma retrieval and they provide a framework for interpreting how electrophysiological context and soma retrieval quality shape single-neuron proteomic measurements in semi-intact circuits.

neuroscience↗

bioRxiv2025

Redox regulation of neuroinflammatory pathways contributes to damage in Alzheimers disease brain

The mechanism(s) whereby redox stress mediates aberrant immune signaling in age-related neurological disorders remains largely unknown. Normally, the innate immune system mounts a robust response to infectious stimuli. However, unintentional activation by host-derived factors, such as aggregated proteins associated with neurodegenerative disorders or by cytoplasmic genomic or mitochondrial DNA, can elicit aberrant immune responses. One such immune response is represented by the cytosolic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. Using redox chemical biology and mass spectrometry approaches, we identified S-nitrosylation of STING cysteine 148 as a novel posttranslational redox modification underlying aberrant type 1 interferon signaling in Alzheimers disease (AD). Critically, we observed S-nitrosylated STING (SNO-STING) in postmortem human AD brains, in hiPSC-derived microglia (hiMG) exposed to amyloid-{beta} (A{beta})/-synuclein (Syn) aggregates, and in 5xFAD transgenic mice. Mechanistically, our findings reveal that STING S-nitrosylation is critical in initiating signaling cascades by promoting the formation of disulfide-bonded STING oligomers. This leads to neuroinflammation early in the course of disease in vivo in 5xFAD mice with consequent synaptic loss. Collectively, our research supports the role of SNO-STING in neuroinflammation associated with AD, and points to a novel druggable cysteine residue in STING to prevent this S-nitrosylation reaction with its inherent inflammatory response. One Sentence SummaryS-Nitrosylation of STING triggers activation of cGAS-STING signaling in Alzheimers disease brain and subserves a novel link between excessive nitrosative stress and dysregulated innate immunity, thus contributing to disease progression.

neuroscience↗

bioRxiv2025

Virus glycoprotein nanodisc platform for vaccine design

Transmembrane glycoproteins of enveloped viruses are the targets of neutralizing antibodies and essential vaccine antigens. mRNA-LNP technology allows in situ production of transmembrane glycoproteins upon immunization, but biophysical characterization of transmembrane antigens and in vitro analysis of post-immunization antibody responses typically rely on soluble proteins. Here, we present a methodological platform for assembling transmembrane glycoprotein vaccine candidates into lipid nanodiscs. We demonstrate the utility of the nanodiscs in HIV membrane proximal external region (MPER)-targeting vaccine development by binding assays using surface plasmon resonance (SPR), ex vivo B cell sorting with fluorescence-activated cell sorting (FACS), and by determining the structure of a prototypical HIV MPER-targeting immunogen nanodisc in complex with three broadly neutralizing antibodies (bnAbs), including the MPER bnAb 10E8, to 3.5 [A] by cryogenic electron microscopy (cryo-EM), providing a template for structure-based immunogen design for MPER. Overall, the platform offers a tool for accelerating the development of next-generation viral vaccines.

immunology↗

bioRxiv2025

Proteomic profiling of primary cilia in the developing brain uncovers newregulators of cortical development

In the developing brain, the primary cilia of radial glial cells extend from the surface of the lateral ventricle, serving as the signaling hub to integrate environmental cues critical for brain formation. Dysfunctions in ciliary proteins contribute to a wide range of brain structural abnormalities in ciliopathies, yet identifying the bona fide ciliary protein components in the brain remains a significant challenge. Here, using proximity labeling and quantitative proteomics, we systematically charted proteins localized to the cilia of radial glial cells in the dorsal and ventral regions of the embryonic brain. From this dataset, we identified cohorts of new molecules intrinsic to the cilia at distinct regions of the developing brain. We validated the cilium localization of several components of the translation machinery and studied the mechanistic roles of ciliary candidates previously linked to brain malformation, including Marcks, a key regulator of radial glial polarity, and Ckap2l, a protein associated with Filippi syndrome. These results revealed previously unrecognized ciliary mechanisms that regulate brain development. Thus, our brain proteomic dataset provides a unique resource for understanding ciliary functions in brain development and the molecular etiology of developmental disorders.

developmental biology↗

bioRxiv2025

PrPC-induced signaling in human neurons activates phospholipase Cγ1 and an Arc/Arg3.1 response

Synaptic dysfunction and loss correlate with cognitive decline in neurodegenerative diseases, including Alzheimers disease (AD) and prion disease. Neuronal hyperexcitability occurs in the early stages of AD and experimental prion disease, prior to the onset of dementia, yet the underlying drivers are unclear. Here we identify an increase in the immediate early gene, Arc/Arg3.1, in the human prion disease-affected frontal cortex, suggestive of neuronal hyperactivity. To investigate early signaling events initiated by prion aggregates (PrPSc) in human neurons, we stimulated PrPC in human iPSC-derived excitatory neurons (iNs) with a known PrPSc-mimetic antibody (POM1), which recapitulated the Arc/Arg3.1 response within two hours. Proteomics, RNAseq, and a phosphokinase array in iNs revealed alterations in the EGF receptor and increased phosphorylated phospholipase C (PLC)-{gamma}1 (Y783), which was also observed in the cerebral cortex of prion-infected mice. Thus, PrPC ligands can induce a PLC-{gamma}1 intracellular signaling cascade together with an Arc response, suggestive of a neuronal activity response.

neuroscience↗

bioRxiv2025

Vaccination with mRNA-encoded membrane-bound HIV Envelope trimer induces neutralizing antibodies in animal models

A protective vaccine against HIV will likely need to induce broadly neutralizing antibodies (bnAbs) that engage relatively conserved epitopes on the HIV envelope glycoprotein (Env) trimer. Nearly all vaccine strategies to induce bnAbs require the use of relatively complex immunization regimens involving a series of different immunogens, most of which are Env trimers. Producing protein-based clinical material to evaluate such relatively complex regimens in humans presents major challenges in cost and time. Furthermore, immunization with HIV trimers as soluble proteins induces strong non-neutralizing responses to the trimer base, which is normally occluded on the virion. These base responses could potentially detract from the induction of nAbs and the eventual induction of bnAbs. mRNA vaccine platforms offer potential advantages over protein delivery for HIV vaccine development, including increased production speed, reduced cost, and the ability to deliver membrane-bound trimers that might facilitate improved immuno-focusing to non-base epitopes. We report the design of mRNA-delivered soluble and membrane-bound forms of a stabilized native-like Env trimer (BG505 MD39.3), initial immunogenicity evaluation in rabbits that triggered clinical evaluation, and more comprehensive evaluation of B cell, T cell, and antibody responses in non-human primates. mRNA-encoded membrane-bound Env immunization elicited reduced off-target base-directed Env responses and stronger neutralizing antibody responses, compared with mRNA-encoded soluble Env. Overall, mRNA delivery of membrane-bound Env appears promising for enhancing B cell responses to subdominant epitopes and facilitating rapid translation to clinical testing, which should assist HIV vaccine development. One Sentence SummaryHIV envelope trimer mRNA enables membrane-bound expression and represents a functional immunogen in pre-clinical mammalian models.

immunology↗