Biology subjects

Arosio, P.

Publications and source records attributed to Arosio, P..

18 recordsLinked to original sources

bioRxiv2026

Molecular Disease Stages of Oligodendrocytic and Neuronal Tau Burden in Progressive Supranuclear Palsy

BackgroundProgressive supranuclear palsy (PSP) is a primary tauopathy defined by the accumulation of 4R tau isoforms in neurons, oligodendrocytes and astrocytes. Despite evidence of genetic susceptibility operating through glial cell types, it remains poorly understood how cell type-specific epigenetic-transcriptional programs evolve with progression of tau pathology. MethodsWe conducted single-nucleus chromatin accessibility (snATACseq) and RNA sequencing (snRNAseq) on postmortem frontal cortex samples from PSP patients (n = 8) and matched controls (n = 8), yielding over 144,000 nuclei passing quality control. Tau pathology burden, including neurofibrillary tangles, coiled bodies, and tufted astrocytes, was quantified on AT8-immunostained sections from the same individuals. We integrated differential gene expression analysis, transcription factor motif enrichment, weighted gene co-expression network analysis, and pseudotime modeling anchored to cell type-specific tau pathology burden to delineate molecular pseudo-progression trajectories. ResultsIn eight cell types, 20 subclasses, and 70 subclusters, PSP brains displayed a selective depletion of certain excitatory deep-layer neurons and oligodendrocyte subclusters, with relative preservation of inhibitory neurons and vascular cells. Genetic risk enrichment was localized to astrocytes and oligodendrocytes, whereas excitatory neurons exhibited the greatest transcriptional dysregulation. Oligodendrocyte pseudo-progression indicated a transition from homeostatic myelination programs (MBP, MOBP) through glucocorticoid-responsive stress (FKBP5, ZBTB16), to compensatory myelination (PLP1, CNP) and proteostasis stress (UCHL1, CYRAB, CLU). Neuronal pseudo- progression revealed early dysregulation of synaptic (RORB2, NRG3, NPTX1), microtubule dynamics (KIF2C, RAB27B, TUBA/B), and survival (MEG3, FTX) pathways, alongside a transient increase in neuron-glia interactions (GRIP, CNTNAP4, ERBB4), converging late on ribosomal translation and vesicular trafficking modules across all neuronal subtypes. Cross-modal integration with independent cerebrospinal fluid proteomics identified a concordant subset of glial reactivity, axonal injury, and synaptic markers jointly dysregulated in inhibitory neurons, oligodendrocytes, and excitatory deep-layer neurons. ConclusionPSP pathogenesis reflects a combination of glial genetic susceptibility and staged, cell type-specific transcriptional dysfunction. Oligodendrocytes transition from myelination-competent states to FKBP5-mediated stress states, while neurons show variably timed loss of synaptic excitability and survival programs, preceded by neuron-glia interactions and followed by convergent ribosomal-proteostatic failure. These cytopathology-anchored trajectories outline a potential pathophysiological sequence and may inform candidate selection for stage-specific therapeutic interventions in PSP.

neuroscience↗

bioRxiv2026

Enhanced Workflow for Urinary Extracellular Vesicle Isolation Using Membrane-Sensing Peptides

Urinary extracellular vesicles (uEVs) represent a promising source of non-invasive biomarkers; however, their clinical translation is still limited by suboptimal isolation strategies, which often suffer from poor scalability, co-isolation of abundant urinary proteins, and bias toward specific EV subpopulations. Here, we employ a membrane-sensing peptide (MSP)-based affinity approach for uEVs isolation, that exploits the highly lipid membranes curvature of EV as universal target, enabling pan-specific capture independent of surface marker expression. MSP-functionalized beads were applied to minimally processed urine samples and benchmarked against differential ultracentrifugation (dUC) and size-exclusion chromatography (SEC). Comprehensive characterization by nanoparticle tracking analysis, transmission electron microscopy, high-sensitivity flow cytometry, single-molecule array (SiMoA), and fluorescence nanoparticle tracking analysis, demonstrated that MSP-based isolation preserves vesicle integrity and maintains the native distribution of canonical tetraspanins (CD9, CD63, CD81), without evidence of subpopulation bias. Notably, MSP-based isolation significantly reduced co-isolated contaminants, such as uromodulin, resulting in improved sample purity. By combining high recovery, improved purity, and operational simplicity, MSP workflow offers practical advantages, including reduced processing time, scalability, and compatibility with standard laboratory equipment, without the need for extensive pre-processing. These properties characterize MSP-based affinity capture as a robust and versatile alternative to conventional uEVs isolation approaches, with strong potential for translational and clinical applications.

biochemistry↗

bioRxiv2026

Single-particle light scattering reveals the dynamic heterogeneity of biomolecular condensates

Biomolecular condensates can be heterogeneous in composition, morphology, and material properties, but resolving this heterogeneity at the level of individual submicrometer condensates remains challenging. Here, we combine off-axis holographic imaging with quantitative light-scattering analysis and particle tracking to simultaneously measure the size, optical properties, interfacial structure, and hydrodynamic mobility of hundreds of individual condensates per minute. Applied to condensates formed by the N-terminal domain of Ddx4 (Ddx4N1), the method reveals two populations with distinct scattering signatures: compact particles with well-defined interfaces and structurally heterogeneous, non-compact assemblies. Their relative abundance varies systematically with ionic strength. Synthetic polymer controls show that pronounced structural heterogeneity is not a generic consequence of phase separation and suggest a role for interaction heterogeneity in promoting competing mesoscale structures. Mass-size scaling and Brownian aggregation simulations further show that aggregation with incomplete fusion of smaller protein-rich units provides a physical model for the non-compact population. Simultaneous optical and diffusion measurements further show that the two populations have distinct hydrodynamic properties. These results demonstrate that chemically homogeneous condensate-forming systems can populate distinct mesoscale structures that are obscured by ensemble-averaged measurements.

biophysics↗

bioRxiv2026

Interplay between Local Diffusion, Concentration, and Inter-Protein Alignment Promotes Cross-β-Sheet Transitions at Condensate Interfaces

The interface separating a biomolecular condensate from its surroundings is increasingly recognised as a unique physical and chemical environment that actively regulates condensate behaviour. It controls surface tension, molecular organisation and chemical reactivity, and is also where pathological ageing often begins, with several proteins nucleating cross-{beta}-sheet fibrils preferentially at condensate surfaces. But does ageing emerge there because of protein-specific chemistry, or a more general physical mechanism? Using coarse-grained molecular dynamics simulations, we show that cross-{beta}-sheet nucleation generically occurs preferentially at condensate interfaces across systems of increasing chemical complexity, from simple homopolymers to patterned amphiphilic sequences. In all systems studied, interface-driven ageing has a common physical origin and can be further modulated, but not driven, by protein sequence. Specifically, terminal domains at the condensate interface gain mobility while remaining densely packed and mutually aligned, creating favourable conditions for {beta}-sheet nucleation. Ageing is amplified in amphiphilic sequences, where hydrophilic regions act as surfactants facing the dilute phase, while aggregation-prone hydrophobic domains cluster just beneath the surface, concentrating the nucleation there. This surfactant-like organisation lowers the interfacial free energy, which falls systematically as sequence asymmetry grows. By linking molecular interactions to mesoscale behaviour, these results cast the interface as a central regulator of ageing and reveal a sequence-independent physical mechanism for the liquid-to-solid transition underlying pathological aggregation in biomolecular condensates.

biophysics↗

bioRxiv2026

Engineering nanocondensate formation through sequence composition and patterning

Many biological proteins can assemble into dynamic, non-stoichiometric structures known as biomolecular condensates. Although typically observed at micrometer scales in vitro, recent evidence shows that these condensates often appear as nanoscale assemblies both in vitro and in cells. Moreover, biochemical reactions can be more efficiently promoted in nanoscale condensates than in micron-sized droplets, due to mass-transfer limitations and interfacial effects. Therefore, in analogy with colloids, the function of condensate materials can be engineered by tuning their size distribution. However, controlling the size of condensates remains challenging, as the molecular mechanisms that prevent small condensates from coarsening into larger ones are still poorly understood. Here, we developed and applied a computational pipeline that combines high-throughput molecular simulations, machine learning, and mixed-integer linear programming to design phase-separating peptides that form metastable nanocondensates across a broad range of experimental conditions. In addition to experimentally validating these peptides, we elucidate the underlying molecular mechanisms and derive initial design rules. In particular, we show that scaffold net charge combined with sequence blockiness can lead to high phase separation propensity while simultaneously yielding low interfacial tension, thereby slowing ripening. Moreover, these combined properties induce an electrostatics-driven alignment of molecules at the interface, which generates an additional size-dependent coalescence barrier. We further show that these features are shared by biological proteins, providing a possible mechanistic basis for the widespread occurrence of nanocondensates in biological systems. Altogether, our findings shed light on the molecular mechanisms behind nanocondensate formation, and provide a platform to design nanocondensates for several potential applications in bioengineering and biotechnology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/706365v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@17c379aorg.highwire.dtl.DTLVardef@26686dorg.highwire.dtl.DTLVardef@19206a7org.highwire.dtl.DTLVardef@1f2da71_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Nuclear mechanostability emerges from satellite DNA condensation into chromocenters

As the largest organelle, the nucleus endures significant mechanical stresses over the cellular lifespan, and mechanostability, i.e. the ability to resist deformation, is critical for genome integrity and function. Here, we reveal that nuclear mechanostability is an emergent property arising from the clustering of satellite DNA repeats into nuclear condensates known as chromocenters. Targeted chromocenter disruption in Drosophila testes subjected to natural and artificial mechanical stress compromises nuclear mechanostability, leading to deformed nuclei, DNA damage, and chromosome breaks. Conversely, enhancing chromocenter coalescence through genetic means improves mechanostability. Molecular dynamics simulations suggest that chromocenters enable physically linked chromosomes to respond collectively, rather than individually, to mechanical challenge, and dissipate external forces over a larger nuclear surface. We propose that the satellite DNA-dependent mechanostability framework described here likely extends to other cells and tissues facing mechanical stress, and offers an explanation for the evolutionary success of these non-coding repeats across eukaryotes.

cell biology↗

bioRxiv2025

Competition between protein-RNA clustering and phase separation drives re-entrant phase behavior of hnRNPA1

Phase separation of RNA-binding proteins plays crucial roles in the cell and is modulated by RNA, including promotion and suppression at low and high RNA concentrations, respectively. In complex coacervates, suppression of phase separation is rationalized by charge inversion when increasing the concentration of one component. Here, we show that suppression of biomolecular condensates of the RNA-binding protein hnRNPA1 at high RNA concentration is driven by a different mechanism, namely the competition with formation of nano-sized protein-RNA clusters in the dilute phase. We show that the competition is modulated not only by RNA concentration, but also by the type of RNA, with specific RNA being more effective in promoting cluster formation than unspecific RNA. We further show that protein-RNA clusters convert into amyloid fibrils over a longer time-scale compared to condensates, therefore providing higher kinetic stability. The competition between clustering and phase separation reported in this study provides a unifying framework to understand the distinct assemblies of hnRNPA1 in the nucleus and the cytoplasm, where the protein is exposed to different types and concentrations of RNA.

biophysics↗

bioRxiv2025

De novo design of peptides localizing at the interface of biomolecular condensates

The interface of biomolecular condensates has been shown to play an important role in processes such as protein aggregation and biochemical reactions. Targeted modulation of these interfaces could, therefore, serve as an effective strategy for engineering condensates and modifying aberrant behaviors. However, the molecular grammar driving the preferential localization of molecules at condensate interfaces remains largely unknown. In this study, we developed a computational pipeline that combines highthroughput coarse-grained simulations, machine learning, and mixed-integer linear programming to design peptides that selectively partition at the interfaces of specific condensate targets. Using this workflow, we designed and synthesized peptides that localize at the interface of three distinct condensates formed by different intrinsically disordered protein regions (IDRs). These peptides exhibit surfactant-like architectures, with one tail incorporated into the condensate and the other excluded from the dense phase. In all cases, the tail entering the condensates is enriched in aromatic residues, while the sequence of the excluded tail varies among the IDRs. For hnRNPA1- and LAF1-IDRs, the excluded tail is enriched in lysines and matches the net charge of the condensate-forming protein, promoting electrostatic repulsion. In the case of DDX4-IDR, which exhibits the lowest charge density, the excluded tail mainly consists of uncharged valine residues, which exhibit negligible interactions with the scaffold protein. These results highlight the importance of the net charge of the scaffold as a key physicochemical parameter for designing peptides with preferential interfacial localization. Overall, our pipeline represents a promising strategy for the rational design of interface-localizing peptides and the identification of the corresponding molecular grammar. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/653111v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@eab174org.highwire.dtl.DTLVardef@2852d6org.highwire.dtl.DTLVardef@156960corg.highwire.dtl.DTLVardef@1952e1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Structural defects in amyloid-β fibrils drive secondary nucleation

The nucleation of amyloid fibrils from monomeric protein, catalyzed by the surface of existing fibrils, is an important driver of many disorders such as Alzheimers and Parkinsons diseases. The structural basis of this secondary nucleation process, however, is poorly understood. Here, we ask whether secondary nucleation sites are found predominantly at rare growth defects: defects in the fibril core structure generated during their original assembly. We first demonstrate using the specific inhibitor of secondary nucleation, Brichos, that secondary nucleation sites on Alzheimers disease-associated fibrils composed of A{beta}40 and A{beta}42 peptides are rare compared to the number of protein molecules they contain. We then grow A{beta}40 fibrils under conditions designed to eliminate most growth defects while leaving the regular fibril morphology unchanged, and confirm the latter using cryo-electron microscopy. We measure both the ability of these annealed fibrils to promote secondary nucleation and the stoichiometry of their secondary nucleation sites, finding that both are greatly reduced as predicted. Re-analysis of published data for other proteins suggests that fibril growth defects that expose monomer planes or other structural units may also drive secondary nucleation generally, across most or all amyloids. These findings could unlock structure-based drug design of therapeutics that aim to halt amyloid disorders by inhibiting secondary nucleation sites.

biophysics↗

bioRxiv2024

Enhancement of Enzymatic Activity by Biomolecular Condensates through pH Buffering

Biomolecular condensates can affect enzymatic reactions by locally changing not only concentrations of molecules but also their environment. Since protein conformations can differ between the dense and dilute phase, phase separation can particularly modulate enzymes characterized by a conformation-dependent activity. Here, we generate enzymatic condensates based on a lipase from Bacillus thermocatenulatus, which exhibits an equilibrium between a closed, inactive state, and an open, active conformation. We show that the activity of the enzyme increases inside the dense phase, leading to an enhancement of the overall reaction rate in the phase-separated system. Moreover, we demonstrate that these condensates can generate a more basic environment compared to the surrounding solution. As a consequence, the phase-separated system maintains a high enzymatic activity even in acidic conditions that would be otherwise less favourable for the lipase. We further show that the formation of two phases with distinct pH values optimizes a cascade reaction involving two enzymes with different optimal pH conditions. Our results demonstrate that biomolecular condensates can also affect the dependence of enzymatic rates on solution pH, thereby expanding the accessible pH interval and enabling network reactions with enzymes that require distinct pH values. These findings have crucial implications in biology and biotechnology for biocatalytic engineering.

biochemistry↗

bioRxiv2022

The interface of condensates of the hnRNPA1 low complexity domain promotes formation of amyloid fibrils

The maturation of liquid-like protein condensates into amyloid fibrils has been associated with neurodegenerative diseases. Here, we analyze the amyloid formation mediated by condensation of the low-complexity domain of hnRNPA1, a protein involved in Amyotrophic Lateral Sclerosis (ALS). We show that phase separation and fibrillation are connected but distinct processes which are independently mediated by different regions of the protein sequence. By monitoring the spatial and temporal evolution of amyloid formation we demonstrate that the formation of fibrils does not occur homogeneously inside the droplets but is promoted at the interface of the condensates. Consistently, we further show that coating the interface of the droplets with surfactant molecules inhibits fibril formation. Our results indicate that the interface of biomolecular condensates can act as an important catalyst for fibril formation, and therefore could represent a possible therapeutic target against the formation of aberrant amyloids mediated by condensation.

biophysics↗

bioRxiv2022

A conserved mechanism regulates reversible amyloids via pH-sensing regions

Amyloids were long viewed as irreversible, pathological aggregates, often associated with neurodegenerative diseases1. However, recent insights challenge this view, providing evidence that reversible amyloids can form upon stress conditions and fulfil crucial cellular functions2. Yet, the molecular mechanisms regulating functional amyloids and the differences to their pathological counterparts remain poorly understood. Here we investigate the conserved principles of amyloid reversibility by studying the essential metabolic enzyme pyruvate kinase (PK) in yeast and human cells. We demonstrate that PK forms stress-dependent reversible amyloids through a pH-sensitive amyloid core. Stress- induced cytosolic acidification promotes aggregate formation via protonation of specific glutamate (in yeast) or histidine (in human) residues within the amyloid core. Our work thus unravels a conserved and potentially widespread mechanism underlying amyloid functionality and reversibility, fine-tuned to the respective physiological cellular pH range.

cell biology↗

bioRxiv2021

Dynamic arrest and aging of biomolecular condensates are regulated by low-complexity domains, RNA and biochemical activity

Biomolecular condensates require suitable material properties to properly carry out their function. Yet, modulators that affect the material properties of condensates have remained largely unexplored. Here we apply Differential Dynamic Microscopy (DDM) to probe the material properties of an in vitro model of processing bodies (P-bodies) consisting of condensates formed by the DEAD-box ATPase Dhh1 in the presence of ATP and RNA. DDM allows us to measure the viscosity of liquid droplets and to distinguish between liquid-like and gel- or glass-like condensates. By applying this single-droplet technique we show that condensates within the same population exhibit a distribution of material properties, which can be drastically affected by several modulators such as the low-complexity domains (LCDs) of the protein, the protein/RNA ratio, the type of RNA as well as the enzymatic activity. In particular, we show that structured RNA leads to a large fraction of dynamically arrested condensates with respect to unstructured polyuridylic acid (polyU), emphasizing the role of RNA structure in condensate dynamics. We further demonstrate that the ageing of the condensates and the formation of gel or glass-like structures is reduced by promoting the enzymatic ATPase activity of Dhh1 and the rate of droplet formation and dissolution. Our study shows that not only the reversible formation and dissolution of condensates but also their material properties are regulated on several levels, and that biochemical activity and material turnover can be important to maintain fluid-like properties over time.

biophysics↗

bioRxiv2020

In-vitro and in-vivo characterization of CRANAD-2 for multi-spectral optoacoustic tomography and fluorescence imaging of amyloid-beta deposits in Alzheimer mice

The abnormal deposition of fibrillar beta-amyloid (A{beta}) deposits in the brain is one of the major histopathological hallmarks of Alzheimers disease (AD). Here we characterize curcumin-derivative CRANAD-2 for multi-spectral optoacoustic tomography (MSOT) and fluorescence imaging of brain A{beta} deposits in the arcA{beta} mouse model of AD cerebral amyloidosis. CRANAD-2 shows a specific and quantitative detection of A fibrils in vitro, even in complex mixtures, and it is capable to distinguish between monomeric and fibrillar forms of A. In vivo epifluorescence and MSOT after intravenous CRANAD-2 administration demonstrated higher retention in arcA{beta} compared to non-transgenic littermate mice. Immunohistochemistry showed co-localization of CRANAD-2 and A{beta} deposits in arcA{beta} mouse brain sections, thus verifying the specificity of the probe. In conclusion, we demonstrate suitability of CRANAD-2 for fluorescence- and MSOT-based detection of A{beta} deposits in animal models of AD pathology, which facilitates mechanistic studies and the monitoring of putative treatments targeting A{beta} deposits.

neuroscience↗

bioRxiv2020

Thermodynamic and kinetic design principles for protein aggregation inhibitors

Understanding the mechanism of action of compounds capable of inhibiting protein aggregation is critical to the development of potential ther-apeutics against protein misfolding diseases. A fundamental challenge for progress is the range of possible target species and the disparate timescales involved, since the aggregating proteins are simultaneously the reactants, products, intermediates and catalysts of the reaction. It is a complex problem, therefore, to choose the states of the aggregating proteins that should be bound by the compounds to achieve the most potent inhibition. We present here a comprehensive kinetic theory of protein aggregation inhibition which reveals the fundamental thermodynamic and kinetic signatures characterising effective inhibitors by identifying quantitative relationships between the aggregation and binding rate constants. These results provide general physical laws to guide the design and optimisation of protein aggregation inhibitors.

biophysics↗

bioRxiv2020

Transcranial in vivo detection of Aβ at single plaque resolution with large-field multifocal illumination fluorescence microscopy

The abnormal deposition of beta-amyloid proteins in the brain is one of the major histopathological hallmarks of Alzheimers disease. Currently available intravital microscopy techniques for high-resolution plaque visualization commonly involve highly invasive procedures and are limited to a small field-of-view within the rodent brain. Here, we report the transcranial detection of amyloid-beta deposits at the whole brain scale with 20 m resolution in APP/PS1 and arcA{beta} mouse models of Alzheimers disease amyloidosis using a large-field multifocal (LMI) fluorescence microscopy technique. Highly sensitive and specific detection of amyloid-beta deposits at a single plaque level in APP/PS1 and arcA{beta} mice was facilitated using luminescent conjugated oligothiophene HS-169. Immunohistochemical staining with HS-169, anti-A{beta} antibody 6E10, and conformation antibodies OC (fibrillar) of brain tissue sections further showed that HS-169 resolved compact parenchymal and vessel-associated amyloid deposits. The novel imaging platform offers new prospects for in vivo studies into Alzheimers disease mechanisms in animal models as well as longitudinal monitoring of therapeutic responses at a single plaque level.

neuroscience↗

bioRxiv2020

Dynamics of oligomer populations formed during the aggregation of Alzheimer's Aβ42 peptide

Oligomeric aggregates populated during the aggregation of the A{beta}42 peptide have been identified as potent cytotoxins linked to Alzheimers disease, but the fundamental molecular pathways that control their dynamics have yet to be elucidated. By developing a general approach combining theory, experiment, and simulation, we reveal in molecular detail the mechanisms of A{beta}42 oligomer dynamics during amyloid fibril formation. Even though all mature amyloid fibrils must originate as oligomers, we find that most A{beta}42 oligomers dissociate to their monomeric precursors without forming new fibrils. Only a minority of oligomers converts into fibrillar species. Moreover, the heterogeneous ensemble of oligomeric species interconverts on timescales comparable to aggregation. Our results identify fundamentally new steps that could be targeted by therapeutic interventions designed to combat protein misfolding diseases.

biophysics↗

bioRxiv2018

Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

Processing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid-liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. We recently identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo [Mugler et al., 2016]. Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

biochemistry↗