Biology subjects

Rousseau, F.

Publications and source records attributed to Rousseau, F..

At least 19 recordsLinked to original sources

bioRxiv2026

Two polymorphs of an independent amyloid core in medin's C-terminal region.

Medin, a 50-residue fragment of lactadherin (MFG-E8), forms aortic medial amyloid, the most common localized amyloid in humans, and co-deposits with vascular amyloid-{beta} (A{beta}) in Alzheimer's disease. In the full-length medin fibril the N-terminal half forms the entire protofilament interface, yet the strongest predicted amyloidogenic determinants lie in the C-terminal region. Here we show that this region aggregates on its own. The isolated C-terminal aggregation-prone region (medin C-APR, medin 31-53) begins to aggregate with the same lag time as full-length medin and assembles into two coexisting polymorphs, determined here by cryo-EM at 2.71 and 2.91 [A]. Both are built from the same two-stranded {beta}-arch and a pseudo-21 protofilament pair closed by an aromatic-aliphatic zipper, and differ only in how many accessory chains the exposed hydrophobic face recruits. The medin C-terminus therefore carries an amyloid core of its own, unrelated to that of the full-length fibril: medin has more than one aggregation-prone region, and the structural contribution of each region depends on sequence and assembly context.

biochemistry↗

bioRxiv2026

Characterization of Fetal Cortical Development Using Spectral Analysis of Gyrification (SPANGY)

The prenatal period of human brain development is critical for mental health and cognition across the entire lifespan. During this period, the cortex undergoes a dramatic transformation from a smooth lissencephalic surface into an elaborately folded structure, a process whose precise characterization is essential for understanding neurodevelopmental trajectories. This study represents the first application of Spectral Analysis of Gyrification (SPANGY) to a large multi-centric fetal brain MRI dataset (635 subjects, 20-38 weeks gestational age). SPANGY characterizes geometric variations on a surface based on the wavelength of folds, hence, providing a quantitative local description of gyrification at the individual level. Using rigorous normative modeling (GAMLSS) and statistical harmonization (ComBat-GAM), we established age-specific reference trajectories for multi-scale gyrification features (spectral frequency bands). We provide the first ever quantification of the temporally-ordered emergence of cortical folding in successive waves: the earliest-emerging low frequency, deep fissures are progressively superseded by the accelerating expansion of higher frequency folds. The normative curves provide the first step in taking prenatal neurodevelopmental assessment from qualitative inspection into a rigorous statistical inference, creating an objective reference against which deviations from healthy brain growth can be caught earlier, and with greater precision.

neuroscience↗

bioRxiv2026

Unraveling the potential of short and long read sequencing for human genome profiling

BackgroundShort read sequencing technologies have dominated the field of human whole genome sequencing in the past years in terms of cost, throughput, and accuracy. However, thanks to recent technological evolution, long read approaches have become increasingly competitive and complementary to short reads. With the gap in the cost per genome closing slowly between both approaches, long reads might replace short read sequencing in future research and clinical applications. Still, comprehensive evaluation is necessary to conclude on the performance and general advantages of each technology. ResultsIn this study, we compared the latest chemistries of major suppliers of short and long read technologies: Illumina short reads, Illumina Complete Long Reads (ICLR), Pacific Biosciences HiFi reads (PacBio), and Oxford Nanopore Technologies long reads (ONT). Using the HG002 human reference sample and established bioinformatics guidelines, we assessed their variant calling performance against the latest available truth sets at different levels of coverage. For single nucleotide variant detection, all technologies were equivalent. Despite the latest improvements in chemistry, indel calling with ONT continues to lag in accuracy behind other technologies. In contrast, long reads delivered a clear advantage in structural variant detection, surpassing short reads in both accuracy and sensitivity. The hybrid ICLR approach achieved intermediate performance, narrowing the gap between short and long read sequencing. Furthermore, long reads enhanced haplotype-phasing resolution, enabling the phasing of over 80% of the genome. ConclusionsThese findings highlight the specific strengths and limitations of recent sequencing technologies, aiding the decision-making in future research projects, technological platforms development, and clinical applications.

genomics↗

bioRxiv2026

Proteomic profiling of whole tissue sections in cardiac ATTR amyloidosis reveals increased extracellular matrix remodeling

Cardiac transthyretin amyloidosis (ATTR-CA) is caused by myocardial deposition of misfolded transthyretin, leading to progressive heart failure. Disease pathology, however, extends beyond passive amyloid deposition and also involves active processes such as extracellular matrix (ECM) remodeling and immune activation. Mass spectrometry (MS) is the gold standard for amyloid typing in diagnostics. Here, we applied quantitative MS-driven proteomics on formalin-fixed paraffin-embedded whole cardiac tissue sections from six ATTR-CA cases, ten unaffected controls and four AL-CA controls to investigate protein expression changes. In addition to transthyretin, over 500 proteins were upregulated in ATTR-CA biopsies, including complement and coagulation factors as well as extracellular matrix (ECM) remodeling proteins. Among these, members of the A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) family, metalloproteinases (MMPs), and Tissue Inhibitor of Metalloproteinases (TIMP3) showed significant upregulation. These proteins are key regulators of ECM turnover and structural integrity. Immunohistochemistry confirmed ADAMTS4 enrichment in amyloid deposits, while TIMP3 showed strong expression in cardiomyocytes and weaker staining within amyloid deposits. Together, these findings indicate that ECM remodeling, alongside complement and coagulation activation, represents a reproducible feature of cardiac ATTR amyloidosis. Whole-tissue proteomics provides biological insights that extend beyond amyloid typing, with potential implications for biomarker discovery and therapeutic targeting in ATTR-CA.

pathology↗

bioRxiv2025

Amyloid Explorer: a global atlas of amyloid fibril structures and thermodynamic principles

Amyloid fibrils underpin both functional and pathological protein assemblies and exhibit extensive structural polymorphism. More than 600 high-resolution fibril structures are now available across over 50 protein sequences, yet comparative analysis has been hindered by fragmented resources and inconsistent annotation. Here we present Amyloid Explorer, an open-access platform that integrates the complete fibril archive into a standardized, quality-controlled, and thermodynamically annotated framework. Each entry includes residue- and segment-level stability maps, structural quality metrics, and interactive visualization tools. Global analysis across the dataset reveals conserved rules of amyloid assembly, including cooperative cores, framework polymorphism through reuse of conserved segments in distinct folds, polymorph-specific energetic anchors, and short frustration zones. By combining scale with accessibility, Amyloid Explorer transforms a static archive into a discovery platform for mechanistic insight, classification, and the design of aggregation modulators.

biophysics↗

bioRxiv2025

Phagocytes as Plaque Catalysts: Human Macrophages Actively Generate Pathogenic Aβ42 Fibrils with Seeding and Cross-Seeding Potency

The prevailing view frames microglia and macrophages as guardians against amyloid beta (A{beta}) accumulation in Alzheimers disease (AD). Here, we overturn this paradigm by demonstrating that human phagocytic cells--including differentiated THP-1 macrophages and iPSC-derived microglia--are not merely passive responders but active producers of extracellular, seeding-competent A{beta}42 fibrils, the amyloid species most strongly linked to parenchymal plaque formation and neurodegeneration. These cell-generated aggregates differ structurally and functionally from synthetic fibrils, exhibiting heightened seeding activity and the ability to cross-seed tau aggregation, a key driver of AD progression. Notably, A{beta}42 fibril formation in this system requires active cellular processes and is exacerbated by loss of TREM2, a major AD risk gene. Transcriptomic profiling reveals an early inflammatory response resembling microglial states observed in human AD models, positioning this system as a tractable, human-relevant platform to dissect the interplay between A{beta} aggregation, innate immunity, and genetic susceptibility. Our findings suggest that macrophages and microglia play a dual role in AD, acting both as responders and inadvertent catalysts of pathogenic amyloid formation, with implications for early therapeutic intervention. Significance StatementHow amyloid plaques emerge and spread in Alzheimers disease remains a critical unanswered question. Here, we show that human immune cells--including brain-resident microglia--can actively generate A{beta}42 fibrils, the form of amyloid most strongly linked to neurodegeneration. These cell-produced fibrils not only seed further amyloid buildup but also trigger tau aggregation, a key event in disease progression. We further demonstrate that genetic risk factors like TREM2 amplify this process. Our findings reveal a direct link between immune cell activity, genetic susceptibility, and the earliest stages of Alzheimers pathology, offering new insights into disease mechanisms and potential intervention points.

biochemistry↗

bioRxiv2025

Medin drives Aβ40 to adopt Aβ42-like fibril polymorphs in vitro

Medin, a vascular amyloid derived from MFG-E8, is the most prevalent form of localized human amyloid and co-localizes with A{beta} in Alzheimers disease and, in particular, cerebral amyloid angiopathy (CAA). While it was shown that medin can promote A{beta} aggregation, it remains unclear whether this amyloid-amyloid interaction affects the structure of the resulting fibrils. Here, we investigate how medin modulates A{beta}40 fibril assembly in vitro using cryo-electron microscopy, aggregation kinetics, and immunogold electron microscopy. We show that medin accelerates A{beta}40 aggregation, co-assembles into hybrid fibrils, and modulates fibril morphology. Cryo-EM analysis reveals two fibril populations: one corresponding to a previously described in vitro A{beta}40 morphology, and a second, previously unobserved polymorph with A{beta}42-like features, including a structured N-terminus and a compact hydrophobic C-terminal core. The presence of a peripheral, unresolved cryo-EM density near the fibril surface suggests that the new polymorph is stabilised through heterotypic interactions, yet the atomic details remain unresolved, likely due to substantial structural heterogeneity. Rather than representing a limitation, this highlights how not all determinants critical for fibril assembly are necessarily ordered or resolvable in the final fibril structure, reflecting the inherent dynamic and heterogeneous nature of amyloid interactions. Our findings provide structural evidence that heterotypic co-aggregation can redirect A{beta}40 into distinct conformational states and suggest that dynamic or transient interactions contribute to fibril polymorphism beyond what can be fully captured in static structural models.

biochemistry↗

bioRxiv2025

Energetic profiling reveals thermodynamic principles underlying amyloid fibril maturation

Amyloid fibrils adopt diverse structural polymorphs that underlie disease-specific phenotypes, but the thermodynamic principles guiding their formation and maturation remain poorly understood. Here, we apply energetic profiling to structural time series from cryo-EM datasets of IAPP, tau, and -synuclein to decode the principles governing fibril maturation, stability and polymorphic divergence. By mapping residue-level free energy contributions across experimentally resolved assembly pathways, we reconstruct the complex maturation trajectories of distinct amyloid systems. We find that amyloid assembly is driven by aggregation-prone regions that act as sequence-encoded stabilizing motifs. As assembly progresses, these motifs are reorganized and expanded, while additional regions introduce structural frustration that enables conformational flexibility. In several cases, environmental cofactors such as metal ions or polyanions are observed in association with regions of structural remodeling, where they may act to compensate for otherwise energetically strained conformations. Notably, structurally distinct polymorphs can exhibit similar energetic profiles, while polymorphs with similar folds may differ thermodynamically depending on their assembly environment. This framework connects polymorph structure to both encoded sequence features and extrinsic modifiers, offering mechanistic insight into how amyloid strains mature and diversify over time.

biophysics↗

bioRxiv2025

Targeted fusion of Antibody-Secreting Cells: unlocking monoclonal antibody production with hybridoma technology

Monoclonal antibodies (mAbs) produced by hybridoma technology have extensively proved their value for therapeutic, diagnostic, and biomedical research applications, despite the reported low fusion yields between short-lived B cells and immortal myeloma cells. To improve the efficiency of this process and accelerate the development of new mAbs, we characterized and isolated antibody-secreting cells (ASCs) from the spleen of immunized mice before cell fusion. This approach resulted in a high yield of hybridoma generation by increasing the probability of successive pairing between the most suitable cell fusion partners. Specifically, we developed an optimized workflow combining Fluorescence-Activated Cell Sorting (FACS) with antibody secretion assays, using a panel of five cell-surface markers (CD3, TACI, CD138, MHC-II, and B220) that allowed us to identify a particular ASC subset with key characteristics. Such ASCs exhibited a plasmablast phenotype with high MHC-II expression and secreted high levels of Ag-specific antibodies in immunized mice. These features were also found in hybridomas, suggesting a preferential fusion of myeloma cells with this ASCs subset. Finally, the targeted electrofusion of TACIhighCD138high sorted ASCs led to a 100% fusion yield compared to a non-targeted approach. In particular, over 60% of these generated hybridomas secreted Ag-specific mAbs. Collectively, these results pave the way for a highly efficient method to produce new mAbs by cell fusion, which could facilitate hybridoma generation and expand therapeutic applications of mAbs.

immunology↗

bioRxiv2024

Directed disruption of IL2 aggregation sites produces designer biologics with enhanced specificity coupled to improved production capacity

The pleotropic nature of interleukin-2 (IL2) has allowed it to be used as both a pro-inflammatory and anti-inflammatory therapeutic agent, through promotion of regulatory T cell (Treg) responses via the IL2RA receptor or promotion of CD8 T cell responses via the IL2RB receptor, respectively. However, the utility of IL2 as a treatment is limited by this same pleiotropy, and protein engineering to bias specificity towards either the regulatory T cell (Treg) or CD8 T cell lineage often requires a trade-off in protein production or total bioactivity. Here we use SolubiS, a computational algorithm-based method, to predict mutations within the IL2 structure to improve protein production yield while altering cellular selectivity, to generate a mutein with elevated therapeutic potential. The design and testing process identified the V126R (murine) / V111R (human) mutation as a Treg-enhancing mutein, creating a cation repulsion to inhibit primary binding to IL2RB, with a post-IL2RA confirmational shift enabling secondary IL2RB binding, and hence allowing the trimeric receptor complex to form. In human IL2, additional N110R T151R aggregation-protecting mutations could improve protein yield of the V111R mutation. The approach also generated novel CD8 T cell-promoting mutations. Y79K created a cation-cation repulsion with IL2RA, while Q50W enhanced CD8 T cell activity through potential {pi}-stacking enhancing binding to IL2RB, with the combination highly stimulatory for CD8 T cells. For human IL2, Y65K (homolog to murine Y79K) coupled with E82K prevented IL2RA binding, however it required the aggregation-protecting mutations of N110R T151R to rescue production. These muteins, designed with both cellular specificity and protein production features, have potential as both biological tools and therapeutics.

immunology↗

bioRxiv2024

De novo design of high-affinity single-domain antibodies

Antibody-based therapeutics have become indispensable in modern medicine, but traditional methods of antibody discovery often present with limitations in developability, cross-reactivity, and ethical concerns. While deep learning and generative approaches have shown promise in the design of high affinity protein binders, de novo antibody design remains challenging. Here, we present EvolveX, a structure-based computational pipeline for designing antibody fragments. EvolveX utilizes ModelX and empirical force field FoldX to optimize complementarity- determining regions (CDRs) and TANGO for aggregation analysis. We demonstrate the ability of EvolveX to redesign a single-domain VHH antibody fragment targeting mouse Vsig4 to address two challenges: enhancing stability and affinity for the original target and redesigning it for high affinity to the human ortholog. The redesigned variants of VHH fragments specific to mouse Vsig4 showed improved physicochemical properties, while retaining binding affinities comparable to the original version. Notably, EvolveX improved the binding affinity of VHHs to human Vsig4 by over 1000-fold, transforming low-affinity binders into nanomolar-affinity molecules. Structural analyses by X-ray crystallography and NMR confirmed the accuracy of the designs, which display optimized interactions with the antigen. NGS and re-modelling analysis further demonstrated the efficiency of FoldX-based design pipeline. Collectively, our study highlights EvolveXs potential to overcome current limitations in antibody design, offering a powerful tool for the development of next-generation therapeutics with enhanced specificity, stability, and efficacy.

bioinformatics↗

bioRxiv2024

Normative models combining fetal and postnatal MRI data to characterize neurodevelopmental trajectories during the transition from in- to ex-utero

The perinatal period involves transitioning from an intra- to an extrauterine environment, which requires a complex adaptation of the brain. This period is marked with dynamic and multifaceted cortical changes in both structure and function. Most studies to date have focused either on the fetal or postnatal period, independently. To the best of our knowledge, this is the first neurodevelopmental study targeting the cortical trajectory of typically developing perinatal subjects, combining MRIs from both fetal and postnatal participants. Prior to analysis, preprocessing and segmentation parameters were harmonized across all subjects in order to overcome methodological limitations that arise when studying such different populations. We conducted a normative modeling analysis on a sample of 607 subjects, age ranged 24 to 45 weeks post-conception, to observe changes that arise as participants traverse the birth barrier. We observed that the trajectories of global surface area and several volumetric features, including total gray matter, white matter, brainstem, cerebellum and hippocampi, follow distinct but continuous patterns during this transition. We further report three features presenting a discontinuity in their neurodevelopmental trajectories as participants traverse from a fetal to a postnatal environment: the extra-cerebrospinal fluid volume, the ventricular volume and global gyrification. The current study demonstrates the presence of unique neurodevelopmental patterns for several structural features during the perinatal period, and confirms that not all features are affected in the same way as they cross the birth barrier. SIGNIFICANCE STATEMENTThe perinatal phase comprises the fetal and immediate postnatal period, and is generally described as the time surrounding birth. Comprehensively understanding this period is crucial due to the presence of dynamic and multifaceted brain changes. What makes this investigation unique is that it is the first neurodevelopmental study, to the best of our knowledge, focused on the cortical trajectory of typically developing perinatal subjects through the combination of both fetal and postnatal participants into one analysis. We report that certain brain feature trajectories change drastically as fetuses become newborns, while other features remain continuous. These observations are relevant in both the isolation of biomarkers for later cognitive and physiological disorders and in the understanding of typical cerebral development.

neuroscience↗

bioRxiv2024

Analyzing the implications of protein folding delay caused by translation

Because of vectorial protein production, residues that interact in the native protein structure but are distantly separated in the primary sequence are unavailable simultaneously. Instead, there is a temporal delay during which the N-terminal interaction partner is vulnerable to off-pathway, non-native interactions. In this analysis, we introduce "FoldDelay" (FD), a metric that integrates the topological pattern of atomic interactions of the native structure with translation kinetics to quantify such time delays. The FD metric reveals that many proteins, particularly at eukaryotic translation rates, exhibit residues with FDs in the range of tens of seconds. These residues, predominantly in well-structured, buried regions, often coincide with predicted aggregation-prone regions. We show a correlation between FD and co-translational engagement by the yeast Hsp70 chaperone Ssb, suggesting that fold-delayed regions have a propensity to misfold. In support of this, we show that proteins with high FDs are more frequently co-translationally ubiquitinated and prone to aggregate upon Ssb deletion. Finally, we find that FD cannot be adequately reduced through codon optimization, highlighting the importance of co-translational chaperones to shield these vulnerable regions. This work offers insights into co-translational proteostasis and the delicate balance between efficient folding and potential misfolding and aggregation during translation.

molecular biology↗

bioRxiv2023

N-glycosylation as a eukaryotic protective mechanism against protein aggregation

The tendency for proteins to form aggregates is an inherent part of every proteome and arises from the self-assembly of short protein segments called aggregation-prone regions (APRs). While post-translational modifications (PTMs) have been implicated in modulating protein aggregation, their direct role in APRs remains poorly understood. In this study, we used a combination of proteome-wide computational analyses and biochemical techniques to investigate the potential involvement of PTMs in aggregation regulation. Our findings reveal that while most PTM types are disfavored near APRs, N-glycosylation is enriched and evolutionarily selected, especially in proteins prone to misfolding. Experimentally, we show that N-glycosylation inhibits the aggregation of peptides in vitro through steric hindrance. Moreover, mining existing proteomics data, we find that the loss of N-glycans at the flanks of APRs leads to specific protein aggregation in Neuro2a cells. Our results point towards a novel intrinsic role for N-glycosylation, directly preventing protein aggregation in eukaryotes.

molecular biology↗

bioRxiv2023

Integrating physics in deep learning algorithms: A force field as a PyTorch module

Deep learning algorithms applied to structural biology often struggle to converge to meaningful solutions when limited data is available, since they are required to learn complex physical rules from examples. State-of-the-art force-fields, however, cannot interface with deep learning algorithms due to their implementation. We present MadraX, a forcefield implemented as a differentiable PyTorch module, able to interact with deep learning algorithms in an end-to-end fashion. MadraX is available at madrax.readthedocs.io

bioinformatics↗

bioRxiv2022

Tau amyloid polymorphism is shaped by local structural propensities of its protein sequence

Different tauopathies are characterized by specific amyloid filament folds that are conserved between patients. Disease-specific tau filament folds probably reflect the specific pathological contexts leading to their formation including isoforms or post-translational modifications. Little is known, however, as to whether and how intrinsic conformational tendencies of the tau sequence itself contribute to its polymorphism. Using cryo-EM structure determination we find that a short amyloidogenic C-terminal peptide consisting of residues 350-362 of the tau repeat domain adopts the same polymorphic conformations in isolation as it does in the context of major disease-associated protofilament folds. Biophysical characterisation and molecular modelling show that the amyloid conformations adopted by this peptide constitute core structural motifs stabilizing distinct disease-associated tau filament folds. In accordance this segment also contributes to the efficient propagation of human AD tau seeds in tau reporter cells while it is irrelevant to heparin-induced recombinant seeds. Our findings suggest that tau 350-362 is key to the propagation of disease-associated tau polymorphs and that the conformational preferences of this segment predispose to the topological diversity observed in tau filament folds.

biophysics↗

bioRxiv2022

EndophilinA-dependent coupling between activity-dependent calcium influx and synaptic autophagy is disrupted by a Parkinson-risk mutation

Neuronal activity and neurotransmitter release cause use-dependent decline in protein function. However, it is unclear how this is coupled to local protein turnover and quality control mechanisms. Here we show that the endocytic protein Endophilin-A (EndoA/ENDOA1) couples activity-induced calcium influx to synaptic autophagy and neuronal survival. We identify single mutations in the EndoA flexible region that either increases EndoA diffusion and promotes autophagosome formation in the absence of calcium, or immobilizes EndoA and blocks autophagy, even in the presence of calcium. Hence, the EndoA flexible region is a switch that responds to calcium, regulating EndoA nanoscale synaptic organization and association with autophagosomes driving their formation. Interestingly, a pathogenic variant in the human ENDOA1 variable region that confers risk to Parkinsons disease (PD), also confines ENDOA1 to the synaptic plasma membrane and equally blocks autophagy in flies in vivo and in induced human neurons. Thus, our work reveals a mechanism neurons use to connect neuronal activity to local protein turnover by autophagy, which is critical for neuronal survival.

neuroscience↗

bioRxiv2021

Mapping the sequence specificity of heterotypic amyloid interactions enables the identification of aggregation modifiers

Heterotypic amyloid interactions between related protein sequences have been observed in functional and disease amyloids. While sequence homology seems to favour heterotypic amyloid interactions, we have no systematic understanding of the structural rules determining such interactions nor whether they inhibit or facilitate amyloid assembly. Using structure-based thermodynamic calculations and extensive experimental validation, we performed a comprehensive exploration of the defining role of sequence promiscuity in amyloid interactions. Using this knowledge, we demonstrate, using tau as a model system, that predicted cross-interactions driven by sequence homology indeed can modify nucleation, fibril morphology, kinetic assembly and cellular spreading of aggregates. We also find that these heterotypic amyloid interactions can result in the mis-localisation of brain-expressed protein sequences with prevalent activities in neurodegenerative disorders. Our findings suggest a structural mechanism by which the proteomic background can modulate the aggregation propensity of amyloidogenic proteins and discuss how such sequence-specific proteostatic perturbations could contribute to the selective cellular susceptibility of amyloid disease progression.

biophysics↗