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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2024

Correction of RBFOX1 deficit rescues Huntington's disease mis-splicing and pathology

RNA mis-splicing correction therapies have been developed for neurological disorders like spinal muscular atrophy and neuronal ceroid lipofuscinosis. In Huntingtons disease (HD), pathogenic mis-splicing was initially observed in genes linked to neurodegeneration, such as HTT itself, MAPT, and TAF1. Later, genome-wide analyses identified a broader mis-splicing signature in HD brains, involving additional neurodegeneration-related genes. Correcting each mis-spliced gene individually would be unfeasible, highlighting the need to target upstream splicing factors altered in HD. Our previous motif-enrichment analyses of intronic sequences flanking the exons mis-spliced in HD identified RBFOX and U2AF2 as candidate splicing factors, both of which are reduced in HD brains. In this study, we tested their pathogenic relevance generating conditional transgenic mouse models that overexpress RBFOX1 or U2AF2 in forebrain neurons and combining them with HD mice. Our results show that moderate overexpression of RBFOX1, but not U2AF2, corrects multiple HD-associated mis-splicing events and alleviates HD mice neuropathology and motor symptoms. These findings demonstrate that RBFOX1 downregulation contributes to HD pathology and underscore the therapeutic potential of strategies aimed at increasing RBFOX1 levels.

neuroscience↗

bioRxiv2024

Human cartilage model of the precocious osteoarthritis-inducing COL2A1 p.Arg719Cys reveals pathology-driving matrix defects and a failure of the ER proteostasis network to recognize the defective procollagen-II

Mutations in the COL2A1 gene, encoding procollagen-II, cause various chondrodysplasias, including precocious osteoarthritis with mild spondyloepiphyseal dysplasia engendered by the p.Arg719Cys substitution. The molecular mechanisms underlying these disorders remain incompletely understood, largely owing to the absence of models faithfully recapitulating the human disease. Here, we developed an in vitro human cartilage model using isogenic induced pluripotent stem cell (iPSC) lines carrying either wild-type or Arg719Cys COL2A1. Directed differentiation into chondrocytes yielded cartilage tissues that were analyzed by immunohistochemistry, electron microscopy, SDS-PAGE, and RNA-sequencing. Tissues derived from Arg719Cys heterozygotes displayed a deficient matrix, closely reflecting the human disease phenotype. Arg719Cys procollagen-II was excessively post-translationally modified and partially retained within the endoplasmic reticulum (ER), leading to ER distention. Notably, despite introduction of an aberrant cysteine residue--expected to engage redox-sensitive folding and quality control pathways--Arg719Cys procollagen-II was not detectably recognized by the ER proteostasis network. The resulting inability to mount a quality control response, including activation of the unfolded protein response, indicates a failure in cellular surveillance. As a result, malformed procollagen-II both accumulates intracellularly and is secreted, contributing to the deposition of a structurally compromised extracellular matrix that drives disease pathology. The iPSC-derived cartilage model presented here provides a genetically defined and expandable, human-based system for dissecting the mechanisms of failed proteostasis in collagenopathies. These findings shed light on the types of substitutions in procollagen that cells can or cannot recognize, and underscore the therapeutic potential of targeting cellular surveillance and collagen quality control pathways in COL2A1-related disorders and beyond. SIGNIFICANCEThe p.Arg719Cys substitution in COL2A1 is known to cause dominantly inherited precocious osteoarthritis with mild spondyloepiphyseal dysplasia, but its molecular pathogenesis remains poorly understood. Prior work suggests disrupted fibrillogenesis as the molecular etiology, yet phenotypically relevant human models are lacking. Here, we use human iPSC-derived cartilage to uncover how the Arg719Cys substitution alters procollagen-II folding, modification, trafficking, and matrix deposition. We observe hallmark disease phenotypes, including a sparse collagen-II matrix and distended endoplasmic reticulum (ER), consistent with intracellular accumulation of procollagen-II. Surprisingly, the mutant procollagen-II does not appear to be differentially engaged by the ER proteostasis network, despite the incorporation of an aberrant and apparently non-disulfide bond-forming Cys residue, suggesting a fundamental failure of cellular surveillance. This unrecognized misfolding leads to secretion of defective collagen culminating in matrix dysfunction. These findings establish a mechanistic basis for disease etiology and highlight collagen quality control as a potentially tractable therapeutic target. The iPSC-based model provides a scalable, human-relevant platform for dissecting disease mechanisms and developing therapies for COL2A1-related disorders and other collagenopathies.

cell biology↗

bioRxiv2024

Fructose-2,6-bisphosphate restores TDP-43 pathology-driven genome repair deficiency in motor neuron diseases

TAR DNA-binding protein 43 (TDP-43) proteinopathy plays a critical role in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We recently reported that TDP-43 plays an essential role in DNA double-strand break (DSB) repair via the non-homologous end-joining (NHEJ) pathway. Here, we provide evidence that the brain of patients with ALS exhibit persistent DNA damage in the transcribed regions of the genome. While investigating the mechanistic basis, we found that the activity of polynucleotide kinase 3-phosphatase (PNKP) was severely impaired in the nuclear extracts of patient brains and TDP-43-depleted cells. PNKP is a key player in DSB repair within the transcribed genome, where its 3-phosphate termini processing activity is crucial for preventing persistent DNA strand breaks and neuronal death. The inactivation of PNKP was due to the reduced level of its interacting partner, phosphofructo-2-kinase fructose 2,6 bisphosphatase (PFKFB3), and its biosynthetic product, fructose-2,6-bisphosphate (F2,6BP), an allosteric modulator of glycolysis. Recently, we have demonstrated that F2,6BP acts as a positive modulator of PNKP activity in vivo. Furthermore, F2,6BP supplementation in cultured ALS patient-derived neural progenitor stem cells (NPSCs) reduced the toxic aggregation of polyubiquitinated TDP-43 and cytosolic pTDP-43 (S409/410). Notably, F2,6BP supplementation restored the PNKP activity in the nuclear extracts from autopsied ALS/FTD brain tissues and patient iPSC-derived NPSCs harboring TDP-43 mutations. Importantly, F2,6BP administration significantly restored the genome integrity and motor phenotypes in a Drosophila model of ALS-TDP-43. Collectively, these findings underscore the therapeutic potential of F2,6BP in TDP-43 pathology-associated motor neuron diseases.

neuroscience↗

bioRxiv2024

Fibrotic-like collagen matrices as innovative 3D in vitro models for investigating the impact of pathological ECM on muscle regeneration in muscular dystrophies

Muscular dystrophies are characterized by impaired skeletal muscle contraction due to genetic mutations. Beyond the disruption of muscle function, the extracellular matrix (ECM) surrounding muscle fibers becomes fibrotic, further impeding tissue repair and function. In this study, we designed both healthy and fibrotic matrices to examine the impact of persistent fibrosis on muscle cell behavior. Fibrotic matrices were synthesized by 3D printing of dense collagen solutions in air, followed by slow gelation to yield non-porous, isotropic hydrogels. These matrices were subsequently crosslinked using EDC/NHS chemistry, resulting in a Youngs modulus of approximately 50 kPa. The behavior of C2C12 myoblasts cultured within the fibrotic matrices was compared to cells grown in healthy matrices. Our results showed that the fibrotic matrix had a detrimental effect on cell behavior. Myoblasts were unable to differentiate into mature myotubes, exhibited poor alignment, and suffered from hypoxia. Furthermore, these cells failed to proliferate, secreted inflammatory cytokines, and were unable to remodel their ECM. Using matrices possessing a single characteristic of the fibrotic matrix (i.e stiff or non porous), the predominant factor for each feature of the cell phenotype was determined. These findings underscore the detrimental effects of a fibrotic persistent ECM on muscle homeostasis. The development of these two distinct 3D muscle models, one representing healthy muscle and the other fibrotic, offers a valuable tool for investigating the pathophysiology of muscular dystrophy. HighlightsO_LIStiff and non-porous dense collagen matrices synthesized by 3D printing recapitulate the key characteristics of fibrosis observed in muscular dystrophies C_LIO_LIFibrotic matrices negatively impact myoblast proliferation and differentiation into mature myocytes C_LIO_LIFibrotic matrices impede the 3D organotypic organization of myocytes C_LIO_LIThis 3D pathological model of skeletal muscle offers a valuable tool for investigating how persistent fibrosis influences the progression of muscular dystrophy C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/630059v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@c2403corg.highwire.dtl.DTLVardef@1074147org.highwire.dtl.DTLVardef@14eb055org.highwire.dtl.DTLVardef@11ac419_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Structure-based mechanistic principles for the paradoxical effects of pathological RET mutations

The RET receptor is a transmembrane protein that belongs to the receptor tyrosine kinase family. RET activates signaling pathways regulating cell growth, differentiation, and survival in diverse tissues that include the thyroid and enteric nervous system. Mutations that reduce RET levels or disrupt function can cause Hirschsprungs disease (HSCR), characterized by abnormal distal colon innervation in the developing embryo. In contrast, mutations that constitutively activate RET can lead to tumorigenesis, most notably in multiple endocrine neoplasia type 2 (MEN2) syndromes usually involving the thyroid. Paradoxically, some RET mutations can both reduce RET activity in enteric ganglia, and increase signaling in other tissues, resulting in patients suffering from both HSCR and MEN2A. Although extensive research has been conducted on RET mutations, the structural and mechanistic bases underlying these paradoxical effects remain unclear. Here, we curated data on 70 positions in RET extracellular domains where point-mutations were associated with HSCR, MEN2A, or both. Taking a structure-based approach, we predict the potential effects of mutations in these positions on RET structure. Our analysis suggests that approximately 90% of positions associated with HSCR can, upon mutation, disrupt intramolecular interactions that stabilize RET tertiary structure: residues buried in the protein core, calcium-binding sites, or residues participating in stabilizing intramolecular electrostatic/covalent bonds. A smaller subset of mutations involves substitutions to/from glycines or prolines in key positions. Only a small minority of HSCR-associated positions affect protein-protein interactions needed for signal activation. On the other hand, our analysis showed that [~]75% of mutations in positions that cause MEN2A lead to an unpaired cysteine that can form an intermolecular disulfide bond between two RET monomers. Other mutations that cause MEN2A are also predicted to enhance RET homodimerization via extracellular domains that are proximal to the membrane. Importantly, substitutions that concurrently destabilizes RET tertiary structure and lead to an unpaired cysteine are predicted to cause the paradoxical co-occurrence of HSCR and MEN2A. Our findings suggest a mechanistic basis for almost all identified pathological mutations in RET and imply that therapeutic strategies for targeting RET activity in HSCR and MEN2A may need to be orthogonal.

bioinformatics↗

bioRxiv2025

Large extracellular vesicles containing mitochondria (EVMs) derived from Alzheimer disease cells harbor pathologic functional and molecular profiles and spread mitochondrial dysfunctions

In addition to small extracellular vesicles known as exosomes, cells release large extracellular vesicles containing mitochondria (EVMs). However, the molecular and functional characteristics of EVMs, as well as the impact of EVM secretion on the spreading of mitochondrial dysfunction between cells, remain unknown in the context of Alzheimers Disease (AD). Here, we provide an ultrastructural, biochemical, and functional characterization of EVMs isolated from cells expressing the amyloid precursor protein (APP) with the familial Swedish mutation (APPswe). We identified differential proteomic and lipidomic signatures in APPswe-derived EVMs compared to control EVMs and revealed a specific proteomic profile in EVMs isolated from conditioned media of fibroblasts from AD patients at the prodromal stage of the disease. Our findings show that APP-C terminal fragments (APP-CTFs) pathogenic accumulation in cells potentiates EVM secretion through the budding of the plasma membrane. We lastly demonstrated that APP-CTFs loaded EVMs are active carriers of dysfunctional mitochondria that transfer mitochondrial pathology to naive control recipient cells.

neuroscience↗

bioRxiv2025

Individualized Phenotyping of Functional ALS Pathology in Sensorimotor Cortex

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by the loss of motor neurons in primary motor cortex (MI), leading to muscle weakness, atrophy, and death within a median of three years. Even though ALS is characterized by different disease subtypes affecting different body parts, individiualized phenotyping of functional ALS pathology has so far not been achieved. We recorded 7 Tesla functional MRI (7T-fMRI) data while ALS patients and matched controls moved affected and non-affected body parts in the MR scanner. We applied robust Shared Response Modeling (rSRM) for capturing ALS-specific shared responses for group classification, and Partial Least Squares (PLS) regression for relating the latent variables to clinical subtypes and the degree of disease progression. We show that both functional connectivity and functional activation in MI are a predictor for disease onset site. However, disease severity could best be predicted by functional connectivity rather than pure activation changes. Critically, we show that functionally disease-defining information in MI is not strongest in the area that is behaviorally first-affected, deviating from the behavioral phenotype of the patients. When computing the models weight distribution of the King stage classification and projecting them back into voxel space, the highest mean weights are present in the foot and tongue/face regions that seem to drive disease progress. Our data highlight the importance of 7T-fMRI task-based functional connectivity measures for classifying ALS-patients, and provide evidence that a single 7T-MRI scan can be used for identifying a disease signature of each individual ALS patient.

neuroscience↗

bioRxiv2025

Integrative Gene Co-expression Network Analysis Reveals Protein-Coding and lncRNA Genes Associated with Alzheimer's Disease Pathology

Alzheimers disease (AD) is a complex neurodegenerative disorder marked by widespread molecular changes, many of which remain poorly understood. While AD pathology progresses through specific brain regions, it is unclear whether these regions are affected similarly. Long non-coding RNAs (lncRNAs), emerging as key cellular regulators, remain largely uncharacterized in AD. Understanding how lncRNAs interact with protein-coding genes across brain regions could shed light on AD mechanisms and progression. To investigate this, we performed consensus weighted gene co-expression network analysis on 396 postmortem brain RNA-seq samples using a meta-analytic approach. Our analysis revealed substantial network rewiring in AD, particularly in the temporal cortex compared to the frontal cortex. The temporal cortex exhibited adaptive changes in gene interactions, while the frontal cortex showed a breakdown of healthy correlations--possibly reflecting regional differences in disease progression. We identified 46 protein-coding genes and 27 lncRNAs as key components in the AD network of the temporal cortex. Using known functions of protein-coding genes as reference points, we inferred potential functions for over 100 lncRNAs across both regions. These findings highlight novel lncRNA candidates potentially involved in AD and provide insights into their roles in both healthy and diseased brain states.

neuroscience↗

bioRxiv2025

Allosteric Modulation of Pathological Ataxin-3 Aggregation: A Path to Spinocerebellar Ataxia Type-3 Therapies

Spinocerebellar ataxia type 3 (SCA3) is a rare inherited neurodegenerative disease caused by the expansion of a polyglutamine repeat in the protease ataxin-3 (Atx3). Despite extensive knowledge of the downstream pathophysiology, no disease-modifying therapies are currently available to halt disease progression. The accumulation of protein inclusions enriched in the polyQ-expanded Atx3 in neurons suggests that inhibiting its self-assembly may yield targeted therapeutic approaches. Here it is shown that a supramolecular tweezer, CLR01, binds to a lysine residue on a positively charged surface patch of the Atx3 catalytic Josephin domain. At this site, the binding of CLR01 decreases the conformational fluctuations of the distal flexible hairpin. This results in reduced exposure of the nearby aggregation-prone region, which overlaps with the substrate ubiquitin binding site and primes Atx3 self-assembly, ultimately delaying Atx3 amyloid fibril formation and reducing the secondary nucleation rate, a process linked to fibril proliferation and toxicity. These effects translate into the reversal of synapse loss in a SCA3 cultured cortical neuron model, an improved locomotor function in a C. elegans SCA3 model, and a delay in disease onset, accompanied by reduced severity of motor symptoms in a SCA3 mouse model. This study provides critical insights into Atx3 self-assembly, revealing a novel allosteric site for designing CLR01-inspired therapies targeting pathological aggregation pathways while sparing essential functional sites. These findings emphasize that targeting allosteric sites in amyloid-forming proteins may offer unique opportunities to develop safe therapeutic strategies for various protein misfolding disorders.

biophysics↗

bioRxiv2025

APOE-ϵ4-induced Fibronectin at the blood-brain barrier is a conserved pathological mediator of disrupted astrocyte-endothelia interaction in Alzheimer's disease

Blood-brain barrier (BBB) dysfunction is a key feature of Alzheimers disease (AD), particularly in individuals carrying the APOE-{varepsilon}4 allele. This dysfunction worsens neuroinflammation and hinders the removal of toxic proteins, such as amyloid-beta (A{beta}42), from the brain. In post-mortem brain tissues and in animal models, we previously reported that fibronectin accumulates at the BBB predominantly in APOE-{varepsilon}4 carriers. Furthermore, we found a loss-of-function variant in the fibronectin 1 (FN1) gene significantly reduces aggregated fibronectin levels and decreases AD risk among APOE-{varepsilon}4 carriers. Yet, the molecular mechanisms downstream of fibronectin at the BBB remain unclear. The extracellular matrix (ECM) plays a crucial role in maintaining BBB homeostasis and orchestrating the interactions between BBB cell types, including endothelia and astrocytes. Understanding the mechanisms affecting the ECM and BBB cell types will be critical for developing effective therapies against AD, especially among APOE-{varepsilon}4 carriers. Here, we demonstrate that APOE-{varepsilon}4, A{beta}42, and inflammation drive the induction of FN1 expression in several models including zebrafish, mice, iPSC-derived human 3D astrocyte and 3D cerebrovascular cell cultures, and in human brains. Fibronectin accumulation disrupts astroglial-endothelial interactions and the signalling cascade between vascular endothelial growth factor (VEGF), heparin-binding epidermal growth factor (HBEGF) and Insulin-like growth factor 1 (IGF1). This accumulation of fibronectin in APOE-{varepsilon}4- associated AD potentiates BBB dysfunction, which strongly implicates reducing fibronectin deposition as a potential therapeutic target for AD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/634732v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@1efff03org.highwire.dtl.DTLVardef@14f40cdorg.highwire.dtl.DTLVardef@27743aorg.highwire.dtl.DTLVardef@3fa9a9_HPS_FORMAT_FIGEXP M_FIG C_FIG Accessibility textThis image illustrates the effects of different APOE isoforms (ApoE-{varepsilon}3 and ApoE-{varepsilon}4) on blood-brain barrier (BBB) integrity, focusing on the molecular interactions between astrocytes and endothelial cells. This figure emphasizes the detrimental effects of ApoE-{varepsilon}4 on BBB integrity via fibronectin accumulation and altered signaling pathways. The top section provides a schematic overview of the blood-brain barrier, highlighting astrocytes, endothelial cells, and their interface. The left panel represents the ApoE-{varepsilon}3 condition: Normal fibronectin (FN1) levels support healthy interactions between astrocytes and endothelial cells. Growth factors, including VEGFA, HBEGF, and IGF1, maintain BBB integrity through their respective receptors (VEGFR and EGFR). Green arrows indicate activation of these signaling pathways. The right panel depicts the ApoE-{varepsilon}4 condition: Elevated fibronectin (FN1) disrupts astrocyte-endothelium interactions. FN1 binds integrins and activates focal adhesion kinase (FAK), inhibiting VEGFA, which is required for endothelial HBEGF that in turn activates IGF1 signaling. Red symbols indicate inhibition of HBEGF, VEGFA, and IGF1 pathways, leading to BBB dysfunction. HighlightsAPOE-{varepsilon}4 drives fibronectin deposition in Alzheimers, disrupting astrocyte-endothelia interactions. APOE-{varepsilon}4 and fibronectin co-localize, forming aggregates at blood-brain barrier (BBB). Fibronectin alters the signaling between VEGF, IGF1, and HBEGF impairing BBB function. Reducing fibronectin restores BBB integrity and offsets APOE-{varepsilon}4 pathology.

cell biology↗

bioRxiv2025

Axonal pathology differentially affects human Purkinje cell subtypes in the essential tremor cerebellum

The cerebellar cortex is organized into discrete regions populated by molecularly distinct Purkinje cells (PCs), the sole cortical output neurons. While studies in animal models have shown that PC subtypes differ in their vulnerability to disease, our understanding of human PC subtype and vulnerability remains limited. Here, we demonstrate that human cerebellar regions specialized for motor vs cognitive functions (lobule HV vs Crus I) contain distinct PC populations characterized by specific molecular and anatomical features, which show selective vulnerability in essential tremor (ET), a cerebellar degenerative disorder. Using a known PC subtype marker, neurofilament heavy chain (NEFH), we found that motor lobule HV contains PCs with high NEFH expression, while cognitive lobule Crus I contains PCs with low NEFH expression in post-mortem samples from healthy controls. In the same cerebella, PC axons in lobule HV were 2.2-fold thicker than those in Crus I. Across lobules, axon caliber positively correlated with NEFH expression. In ET cerebella, we identified motor lobule-specific PC axon pathology with a 1.5-fold reduction in caliber and increased axon variability in lobule HV, while Crus I axons were unaffected. Tremor severity and duration in ET correlated with axon diameter variability selectively in lobule HV PCs. Given that axonal caliber is a major determinant of neural signaling capacity, our results (1) suggest that disrupted cerebellar corticonuclear signaling is occurring in ET, (2) provide evidence of region-specific PC subtypes in the human cerebellum and offer insight into how selective PC vulnerability may contribute to the pathophysiology of cerebellar degeneration. Significance StatementThe cerebellar cortex has a uniform laminar architecture but contains heterogeneous cell types. Purkinje cells (PCs), the sole output cells of the cerebellar cortex, include subtypes whose lobular distribution is thought to underlie functional segmentation and patterned degeneration of the cerebellum in animal models. However, human PC subtypes and their disease vulnerability remain unknown. Here, we establish the existence of human PC subtypes that appear conserved across mammalian species using both marker expression and axonal thickness. Consistent with its phenotype, we demonstrate differential PC vulnerability to degeneration in essential tremor cerebella, where motor function- mediating PC subtypes display significant axonal thinning, while axons of cognitive function-mediating PC subtypes are spared. These findings advance Purkinje cell type- oriented research in cerebellar disorders.

neuroscience↗

bioRxiv2025

Dicer loss in Muller glia leads to a defined sequence of pathological events beginning with cone dysfunction

PurposeThe loss of Dicer in Muller glia (MG) results in severe photoreceptor degeneration as it occurs in retinitis pigmentosa or AMD. However, the sequence of events leading to this severe degenerative state is unknown. The aim of this study was to conduct a chronological functional and structural characterization of the pathological events in MG-specific Dicer-cKO mice in vivo and histologically. MethodsTo delete Dicer and mature microRNAs (miRNAs) in MG, two conditional Dicer1 knock-out mouse strains namely RlbpCre:Dicer-cKOMG and GlastCre:Dicer-cKOMG, were created. Optical coherence tomography (OCT), electroretinograms (ERGs) as well as histological analyses were conducted to investigate structural and functional changes up to six months after Dicer deletion. ResultsDicer/miRNA loss in MG leads to 1) impairments of the external limiting membrane (ELM) - retinal pigment epithelium (RPE), 2) cone photoreceptor dysfunction and 3) retinal remodeling and functional loss of the inner retina, 1, 3 and 6 months after Dicer loss, respectively, in both strains. Furthermore, in the Rlbp:Dicer-cKOMG strain, rod photoreceptor impairment was found 4 months after Dicer depletion (4) accompanied by alteration of RPE integrity (5). ConclusionsMG Dicer loss in the adult mouse retina impacts cone function prior to any measurable changes in rod function, suggesting a pivotal role for MG Dicer and miRNAs in supporting cone health. A partially impaired RPE however seems to accelerate rod degeneration and overall degenerative events.

neuroscience↗

bioRxiv2025

Pathological mutation in SMN impairs modulation of GAR1 phase separation linking condensate dysfunction to Spinal Muscular Atrophy

Pathological mutations in liquid-liquid phase separation (LLPS) scaffold proteins have been linked to biomolecular condensate dysfunction in neurodegenerative diseases, while the possible impact of client protein mutations remains unclear. In spinal muscular atrophy (SMA), the disease-associated E134K mutation in the Tudor domain (TD) of the survival motor neuron (SMN) protein disrupts its interaction with GAR1, an RGG-rich protein within the H/ACA small nucleolar ribonucleoprotein complex. The consequences of this impaired interaction have not been elucidated. Here, we identified GAR1 as an LLPS scaffold protein that is phase separated in nuclear compartments and forms gel-like condensates via complex coacervation with RNA in vitro. In cells, we reveal that SMN co-localizes with GAR1 in Cajal bodies and modulates its dynamics. Using confocal microscopy and NMR spectroscopy, we further show that SMN TD is a client protein that regulates the architecture and dynamics of GAR1 condensates through competitive RNA interactions, implicating GAR1 phase separation in RNA accumulation and release processes regulated by SMN. Notably, the SMA-associated E134K variant of SMN exhibits a reduced affinity for GAR1, impairing the modulation of GAR1 condensates and displacement RNA. Our findings suggest a mechanistic link between phase separation dysregulation and SMA, driven by disrupted scaffold-client interactions, that highlights the therapeutic potential of targeting SMN-dependent condensate regulation in SMA.

biochemistry↗

bioRxiv2025

Biomaterial-based 3D human lung models replicate pathological characteristics of early pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and incurable lung disease characterized by tissue scarring that disrupts gas exchange. Epithelial cell dysfunction, fibroblast activation, and excessive extracellular matrix deposition drive this pathology that ultimately leads to respiratory failure. Mechanistic studies have shown that repeated injury to alveolar epithelial cells initiates an aberrant wound-healing response in surrounding fibroblasts through secretion of mediators like transforming growth factor-{beta}, yet the precise biological pathways contributing to disease progression are not fully understood. To better study these interactions there is a critical need for lung models that replicate the cellular heterogeneity, geometry, and biomechanics of the distal lung microenvironment. In this study, induced pluripotent stem cell-derived alveolar epithelial type II (iATII) cells and human pulmonary fibroblasts were arranged to replicate human lung micro-architecture and embedded in soft or stiff poly(ethylene glycol) norbornene (PEG-NB) hydrogels that recapitulated the mechanical properties of healthy and fibrotic lung tissue, respectively. The co-cultured cells were then exposed to pro-fibrotic biochemical cues, including inflammatory cytokines and growth factors. iATIIs and fibroblasts exhibited differentiation pathways and gene expression patterns consistent with trends observed during IPF progression in vivo. A design of experiments statistical analysis identified stiff hydrogels combined with pro-fibrotic biochemical cue exposure as the most effective condition for modeling fibrosis in vitro. Finally, treatment with Nintedanib, one of only two Food and Drug Administration (FDA)-approved drugs for IPF, was assessed. Treatment reduced fibroblast activation, as indicated by downregulation of key activation genes, and upregulated several epithelial genes. These findings demonstrate that human 3D co-culture models hold tremendous potential for advancing our understanding of IPF and identifying novel therapeutic targets. Statement of significanceThis study leverages advanced biomaterials and biofabrication techniques to engineer physiologically relevant, patient-specific, and sex-matched models of pulmonary fibrosis, addressing the critical need for pre-clinical therapeutic drug screening platforms. These human 3D lung models successfully replicated key features of fibrotic lung tissue. Tuning microenvironmental stiffness of 3D PEG-NB hydrogels to match fibrotic lung values and exposing human iATII cells and fibroblasts to pro-inflammatory biochemical cues recreated hallmark characteristics of in vivo fibrosis pathogenesis, including epithelial differentiation and loss, as well as fibroblast activation. The utility of these models was further validated by demonstrating responsiveness to Nintedanib, a clinically available treatment for IPF. These findings highlight the transformative potential of well-defined biomaterial-based 3D models for elucidating complex disease mechanisms and accelerating therapeutic drug discovery for chronic pulmonary diseases like idiopathic pulmonary fibrosis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/637970v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@bd8ed0org.highwire.dtl.DTLVardef@eb3489org.highwire.dtl.DTLVardef@1c64d6aorg.highwire.dtl.DTLVardef@49f60_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

A Novel Mouse Model of Parkinson's Disease for Investigating Progressive Pathology and Neuroprotection

Developing animal models that successfully recapitulate the features of progressive Parkinsons disease (PD) is crucial for understanding disease progression mechanisms and creating effective therapeutic interventions. In this study, we created a mouse model of PD by overexpressing -synuclein through a combined injection of AAV6--synuclein and preformed fibrils (PFFs) into the medial and lateral substantia nigra (SN). We also demonstrated that chronic administration of the c-Abl inhibitor PD180970 provides neuroprotection in this model. Mice injected with the AAV6--synuclein and PFF combination showed a progressive loss of dopaminergic (DA) neurons in the SN and their projections in the striatum over 24 weeks. This neuronal loss coincided with a time-dependent accumulation of phosphorylated -synuclein (p-syn) in the SN. The p-syn aggregates spread to synaptically connected DARPP-32-positive neurons in the striatum and further extended to the cortex. We also observed a contralateral spread of p-syn aggregates. Additionally, -synuclein overexpression led to a significant increase in activated microglia and astrocytes at all timepoints, with the strongest activation occurring early and gradually diminishing over time. Daily administration of PD180970 significantly reduced the loss of DA neurons caused by -synuclein injection and decreased the accumulation of p-syn in the SN. PD180970 treatment also reduced the neuroinflammation significantly. Overall, the combined injection of AAV6--synuclein and preformed fibrils into the mouse brain establishes a robust PD model, enabling detailed mechanistic studies of the disease. We further demonstrate the models utility for chronic neuroprotection studies using the potential drug PD180970, highlighting its broad applicability. Significance StatementThis study establishes a robust mouse model of Parkinsons disease (PD) by combining AAV6-mediated -synuclein overexpression and preformed fibrils (PFFs) to replicate key features of PD, such as progressive dopaminergic neuron loss, phosphorylated -synuclein accumulation, and neuroinflammation. The model captures the spread of pathological aggregates to synaptically connected brain regions, closely mimicking the human disease. By testing the c-Abl inhibitor PD180970, we demonstrate its neuroprotective effects, including reduced neuronal loss, decreased -synuclein accumulation, and neuroinflammation highlighting its therapeutic potential. This model offers a valuable platform for investigating PD mechanisms and evaluating novel interventions, bridging the gap between preclinical and clinical applications.

neuroscience↗

bioRxiv2025

Astrocytic activation of EMMPRIN contributes to their pathological phenotype in ALS.

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterised by the degeneration of upper and lower motoneurons. Onset and progression of the disease are determined by both cell-autonomous neuronal dysfunctions and non-cell-autonomous factors, mainly due to activation of glial cells such as astrocytes and microglia. The Extracellular Matrix Metalloproteinases INducer (EMMPRIN), a glycoprotein expressed by various cell types including neurons, is the major activator of matrix metalloproteinases (MMPs) synthesis and release. EMMPRIN activation can be induced by peptidyl-prolyl isomerase A (PPIA), a chaperone protein with cis/trans isomerase activity, that exhibits cytokine- and chemokine-like behaviour. Previous studies showed that PPIA is highly released in the cerebrospinal fluid (CSF) of ALS patients and animal models where, by activating EMMPRIN on motoneurons, induces neuronal death. Here, we show that EMMPRIN is expressed also by astrocytes, suggesting this cell type as sensitive as motoneurons to PPIA-mediated EMMPRIN activation. We observed that that PPIA-mediated EMMPRIN activation prompt astrocytes toward a pro-inflammatory profile. Interestingly, we found that this pathogenic profile can be reverted by an anti-EMMPRIN antibody. Finally, we provide evidence that the activation of EMMPRIN is relevant for mutant SOD1 and TDP-43 conditions. In conclusion, we demonstrate that EMMPRIN activation in ALS occurs also in astrocytes where it exacerbates their pathological phenotype possibly contributing to the progression of the disease. Furthermore, we suggest the potential use of an anti-EMMPRIN antibody to reduce astrocytic activation during the disease.

neuroscience↗

bioRxiv2025

Pathological tau activates inflammatory nuclear factor-kappa B (NF-κB) and pT181-Qβ vaccine attenuates NF-κB in PS19 tauopathy mice

Tau regulates neuronal integrity. In tauopathy, phosphorylated tau detaches from microtubules and aggregates, and is released into the extracellular space. Microglia are the first responders to the extracellular tau, a danger/damage-associated molecular pattern (DAMP), which can be cleared by proteostasis and activate innate immune response gene expression by nuclear factor-kappa B (NF-{kappa}B). However, longitudinal NF-{kappa}B activation in tauopathies and whether pathological tau (pTau) contributes to NF-{kappa}B activity is unknown. Here, we tau oligomers from human Alzheimers disease brain (AD-TO) activate NF-{kappa}B in mouse microglia and macrophages reducing the I{kappa}B via promoting its secretion in the extracellular space. NF-{kappa}B activity peaks at 9- and 11-months age in PS19Luc+ and hTauLuc+ mice, respectively. Reducing pTau via pharmacological (DOX), genetic (Mapt-/-) or antibody-mediated neutralization (immunization with pT181-Q{beta} vaccine) reduces NF-{kappa}B activity, and together suggest pTau is a driver of NF-{kappa}B and chronic neuroinflammation tauopathies. SummaryNeuronal tau activates microglial NF-{kappa}B constitutively by secreting its inhibitor I{kappa}B. NF-{kappa}B activation in PS19Luc+ and hTauLuc+ mice peaks at 9- and 11-months of age, respectively. Neutralizing pTau with pT181-Q{beta} vaccine (targeting phosphorylated threonine 181 tau) alleviates NF-{kappa}B activity in tauopathy mice.

neuroscience↗

bioRxiv2025

Establishment of a second-generation transgenic marmoset model of polyglutamine disease recapitulating neurological symptoms and pathology

Neurodegenerative diseases, including polyglutamine diseases, remain a major clinical challenge, partly because of limited animal models that recapitulate human disease. Here, we describe a second-generation transgenic marmoset model of spinocerebellar ataxia 3 (SCA3), a polyglutamine disease, which stably expresses expanded CAG repeats in ATXN3. All five offspring of the founder marmoset harbored the transgene with reduced transgene integration sites and without repeat instability or genetic mosaicism, offering improved construct validity. Three of the five marmosets developed progressive motor impairments that segregated into two distinct phenotypes: early onset with rapid progression and late onset with mild progression, accompanied by corresponding patterns in body weight gain and grip strength. Pathological analysis revealed cerebellar Purkinje cell loss, spinal cord neurodegeneration, and widespread intranuclear inclusions. The severity of motor phenotypes correlated with transgene expression levels in disease-relevant brain regions, including the cerebellum, spinal cord, and striatum. By overcoming the common translational limitations of rodent systems, our second-generation model offers a powerful platform for investigating disease mechanisms and testing potential therapeutic interventions. Our results advance the utility of transgenic marmosets as clinically relevant models of neurodegenerative diseases. Summary StatementSecond-generation transgenic marmoset models of spinocerebellar ataxia 3 replicated the progressive motor deficits and neuropathology of the founder marmoset, providing a powerful platform for studying disease mechanisms and developing therapies.

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