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bioRxiv2025

SNCA-targeted epigenome therapy for Parkinsons disease alleviates pathological and behavioral perturbations in a mouse model

Alpha-synuclein (SNCA) overexpression is implicated in Parkinsons disease (PD) pathogenesis, making SNCA downregulation a promising therapeutic strategy. We developed a SNCA-targeted epigenome therapy using an all-in-one lentiviral vector (LV) carrying deactivated CRISPR/(d)Cas9, gRNA targeted at SNCA-intron1, and either the catalytic domain of DNA-methyltransferase3A (DNMT3A), or a synthetic repressor molecule of Kruppel-associated box (KRAB)/ methyl CpG binding protein 2 transcription repression domain (MeCp2-TRD). Therapeutic efficacy was evaluated in a new PD mouse model, generated with an adeno-associated viral vector carrying an engineered minigene comprised of the human (h)A53T-SNCA expressed via the human native regulatory region. Both therapeutic vectors reduced expression of -synuclein in the substantia nigra (SN), with LV/dSaCas9-KRAB-MeCP2(TRD) demonstrating greater repression. LV/dSaCas9-KRAB-MeCP2(TRD) also significantly reduced pathological -synuclein aggregation and phosphorylation (Ser129), and preserved tyrosine hydroxylase expression in the SN and the striatum. Behavioral analysis following LV/dSaCas9-KRAB-MeCP2(TRD) injection, showed significant improvement in motor deficits characteristic of our PD-mouse model. Safety assessments found normal blood counts, serum chemistry, and weights. Collectively, we provide in vivo proof-of-concept for our SNCA-targeted epigenome therapy in a PD-mouse model. Our results support the systems therapeutic potential for PD and related synucleinopathies and establish the foundation for further preclinical studies toward investigational new drug enablement.

neuroscience↗

bioRxiv2025

Dysregulated MEG3 in Myotonic Dystrophy 1: nuclear retention, pathological role, and therapeutic correction by antisense conjugates.

MEG3, a long non-coding RNA (lncRNA), has been shown to play a critical role in regulating apoptosis. Its downregulation inhibits apoptosis in cancer cells, whereas its upregulation has been associated with cell death in both cardiovascular disease and, more recently, Alzheimers Disease. Here we show that MEG3 is upregulated in Myotonic Dystrophy 1 (DM1). Specifically, we show MEG3 upregulation by several-fold in DM1 human muscle cells and in two DM1 mouse models, HSA-LR and LC15. In human DM1 muscle cells we observe nuclear retention of MEG3 and an increase in its transcript diversity. Furthermore, we observe a general trend of nuclear retention in DM1 affecting lncRNAs and microRNAs (miRNAs), in contrast to mRNAs, when compared to healthy cells. This altered nuclear retention may contribute to the pathological effects of non-coding RNA dysregulation in DM1. Importantly, we demonstrate that treatment with antisense conjugates targeting the repeat expansion causing DM1, an approach currently being tested in Clinical Trials, corrects MEG3 levels in HSA-LR mice, without additional therapeutic interventions targeting MEG3.

molecular biology↗

bioRxiv2025

Integrating Artificial Intelligence-Driven Digital Pathology and Genomics to Establish Patient-Derived Organoids as a Novel Alternative Model for Drug Response in Head and Neck Cancer

Patient-derived organoids (PDOs) are emerging as advanced 3D ex vivo novel alternative method (NAM) preclinical models, offering significant advantages over traditional cell lines and monolayer cultures for therapeutic development. In this study, we established PDOs from surgically resected fresh tissues of human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) across anatomical sites, tumor T-categories, and sample types. These PDOs faithfully recapitulate the tumors pathology, mutational profile, and drug response. To enable rapid classification of PDO identity, we developed a new convolutional neural network (CNN) model, TransferNet-PDO, which accurately distinguished tumor versus normal PDOs in culture using digital histopathology images (AUC[≥]0.88). PDOs maintained stable cultures and were cryopreserved between passages 5 and 12. Immunohistochemistry (IHC) staining (PanCK, p63, Cytokeratin 13, Ki67) confirmed squamous phenotype and histologic aggression of the original tumor. For tumors harboring TP53 mutations by whole-exome sequencing (WES), PDOs retained the corresponding p53 functional status as confirmed by IHC (enhanced or loss of expression). Somatic mutational landscape revealed that PDOs preserved driver somatic mutations, copy number variations (CNVs), and clonal architecture including low-prevalence subclones. Drug sensitivity assessment of PDOs showed that cisplatin reduced cell viability, whereas cetuximab and lenvatinib had minimal effects. Chemoradiation led to greater tumor organoid killing compared to radiation or chemotherapy alone. This study presents an integrated HNSCC PDO platform combining tissue biobanking, organoid establishment, multi-omics characterization, functional drug screening, and AI-driven histopathologic classification, providing a comprehensive and scalable system for translational cancer research.

cancer biology↗

bioRxiv2025

Longitudinal Imaging of Experimental Intracerebral Hemorrhage Pathology with Iodine-enhanced Micro-CT

BackgroundIodine-enhanced micro-computed tomography (Micro-CT) enables high-resolution three-dimensional imaging of brain architecture. This study aimed to characterize both acute and chronic pathological changes following intracerebral hemorrhage (ICH) using iodine-enhanced micro-CT. MethodExperimental ICH was induced in 8- to 10-week-old C57BL/6 mice (n = 76) via stereotaxic injection of either 0.075 U collagenase IV or 30 l autologous blood. Iodine-enhanced micro-CT imaging was performed at 4 hours, 1, 3, and 7 days after intracerebral hemorrhage post-ICH to evaluate hematoma formation and erythrolysis. Chronic alterations, including ventriculomegaly and ipsilateral lesion, were assessed at 28 days post-ICH. In parallel, MRI was conducted at 1, 7, and 28 days following autologous blood injection, followed by micro-CT, to facilitate cross-modality quantitative analysis. Lesion volumes were compared between imaging modalities over time. ResultsMicro-CT enabled quantification of hematoma volume and erythrolysis in ICH models. Hematomas extended along perivascular pathways toward the cerebral surface in both collagenase- and autologous blood-induced ICH models. At 28 days post-ICH, micro-CT detected ventriculomegaly and hypodense lesions without concurrent expansion of the choroid plexus. Lesion volume measurements derived from micro-CT correlated with those from MRI, enabling quantitatively assessment of iron deposition and hematoma size alterations after ICH. ConclusionIodine-enhanced micro-CT provides a robust and high-resolution imaging platform for evaluating hematoma evolution, hemolysis, ventricular enlargement, and chronic brain lesions in experimental ICH. When integrated with MRI, these multimodal imaging approaches enhance the characterization of both hematoma volume and iron deposition following ICH.

neuroscience↗

bioRxiv2025

Influenza A Virus infection is associated with TDP-43 pathology and neuronal damage in the brain

Viral pandemics such as COVID-19 have demonstrated long-term neurological consequences, including memory impairment and depression, emphasizing the importance of understanding virus-brain interactions [1]. Similar concerns have been raised for Influenza A virus (IAV), which has been implicated in neurodegenerative disorders [2, 3]. In this study, we investigated the neuropathological effects of highly pathogenic avian influenza (HPAI) H5N1 and H5N8 strains in a mouse model. Although viral RNA was detected in the brain post-infection, no viral proteins were found, suggesting limited or transient brain replication. Despite this, infected brains showed significant neuronal damage, including axonal loss and nuclear condensation, as evidenced by immunofluorescence and Nissl staining. We also observed pathological changes in TDP-43, including conformational alterations and increased phosphorylation, which required antigen retrieval for detection--features reminiscent of those found in frontotemporal dementia and amyotrophic lateral sclerosis [4, 5]. Transcriptomic analysis further revealed strain-specific host responses, including activation of interferon-related genes and downregulation of microtubule-associated pathways. These findings suggest that IAV infection can trigger hallmarks of neurodegeneration in the absence of persistent viral protein expression, possibly through host-driven mechanisms. Our results underscore the need for further investigation into virus-induced molecular pathways contributing to neurodegenerative disease.

neuroscience↗

bioRxiv2025

White adipose tissue undergoes pathological dysfunction in the TDP-43A315T mouse model of amyotrophic lateral sclerosis (ALS)

White adipose tissue (WAT) has a crucial role in maintaining systemic energy homeostasis. Numerous biological pathway studies have highlighted the importance of adipokines in regulating metabolic pathways and contributing to metabolic dysfunction in animal models and patients with ALS. Despite these associations, the specific molecular mechanisms remain poorly understood. Moreover, the direct contribution of WAT to the energy metabolism abnormalities observed in ALS has yet to be clearly defined. The current study sought to identify perturbances in WAT, main source of leptin, during the clinical course of the disease in TDP-43A315T mice using histological, proteomic, and molecular biological techniques. We present the first evidence of a significant histological alteration in WAT prior to the symptomatic stage of the disease in TDP-43A315T mice, providing novel insights into pathological features earlier in the onset of symptoms, and showing WAT as a target organ for ALS. In human ALS cases, we found that circulating leptin levels at the time of diagnosis were lower in the plasma of men with ALS who were overweight or obese and had rapidly progressive ALS, emphasizing the importance of considering sex-specific approaches when analysing adipokines essential for body weight control.

neuroscience↗

bioRxiv2025

Semaphorin 3A and 3F overexpression in TIE2 hyperactive endothelial cells contribute to the pathological lumen expansion in venous malformation

Abstract (formatted)O_ST_ABSBackgroundC_ST_ABSVenous malformation (VM) are developmental defects of the vasculature characterized by tremendously enlarged and dysfunctional veins. Gain-of-function somatic mutations in the endothelial-specific tyrosine kinase receptor TIE2 have been identified as leading driver of VM pathogenesis. The aim of this study was to determine whether the aberrant venous lumen expansion is caused by recruitment of wild-type endothelial cells (EC) to the lesion or by TIE2-mutant EC clonal expansion. MethodsTo investigate the contribution of TIE2-mutant EC and wild-type EC to the aberrant venous lumen expansion, we used a xenograft murine model of VM generated with a combination of TIE2-mutant EC and wild-type EC. To perform longitudinal studies, we employed a three-dimensional (3D) fibrin gel lumen formation assay and a migration assay, both using wild-type EC in competition or confrontation with TIE2-mutant EC. To investigate the mechanisms implicated in VM lumen expansion we used RNA-sequencing and short interference (sh)RNA in the TIE2-mutant EC. ResultsWe demonstrate here that in the VM xenograft model, the aberrant blood vessels were lined almost exclusively by TIE2-mutant EC, and wild-type EC were rarely found. Functionally, the TIE2-mutant EC exerted a competitive advantage over wild-type EC by inhibiting wild-type EC sprouting. In line with these findings, TIE2-mutant EC promoted repulsion of wild-type EC. ShRNA-mediated silencing of Sema3A or Sema3F in TIE2-mutant EC rescued this chemorepellent phenotype and restored the ability of wild-type EC to migrate, sprout and form lumens. Furthermore, knock-down of Sema3A or 3F in TIE2-mutant EC normalized the blood vessel size in vivo. ConclusionsOur results demonstrate that wild-type EC are not recruited to the aberrant veins suggesting VM pathogenesis is fueled by clonal expansion of TIE2-mutant EC. Mechanistically, we show that Sema3A and 3F are overexpressed in TIE2-mutant EC and play a crucial role in the pathological vascular lumen expansion in VM. Abstract (not formatted)Venous malformations (VM) are developmental defects of the vasculature characterized by tremendously enlarged and dysfunctional veins. Gain-of function somatic mutations in the endothelial-specific tyrosine kinase receptor TIE2 have been identified as one of the leading drivers of VM pathogenesis. The aim of this study was to determine whether the aberrant venous lumen expansion is caused by the recruitment of wild-type endothelial cells (EC) to the lesion or instead by TIE2-mutant EC clonal expansion. In a xenograft murine model of VM generated with a combination of TIE2-mutant EC and wild-type EC, we demonstrated that the aberrant blood vessels were lined almost exclusively by TIE2-mutant EC, suggesting lesions form by clonal expansion, while wild-type EC were not recruited to the aberrant veins. Functionally, in a three-dimensional cell competition assay, we showed that TIE2-mutant EC exerted a competitive advantage over wild-type EC by inhibiting wild-type EC sprouting and ability to form vascular lumens. In line with these findings, TIE2-mutant EC repelled wild-type EC by reversing their migration direction in a cell confrontation assay. In seeking to define the mechanism driving this repulsion phenotype, we detected elevated levels of the chemorepellent Semaphorin 3A (Sema3A) and Sema3F in the TIE2-mutant EC. ShRNA-mediated silencing of Sema3A or Sema3F rescued the chemorepellent phenotype and restored the ability of wild-type EC to migrate, sprout and form lumens. Furthermore, Sema3A or 3F knock-down in TIE2-mutant EC normalized the blood vessel morphology and size in vivo. Taken together, these data strongly indicates that Sema3A and 3F are important players in VM pathogenesis.

molecular biology↗

bioRxiv2025

Experimental inoculation of pigs with porcine respirovirus type 1 revealed pathological manifestations in the upper respiratory tract

Several pathogens are known to affect the respiratory tract of pigs resulting in decreased health and welfare. Virological investigations and the use of metagenomic sequencing of samples allowed the identification of new viruses in pigs, such as porcine respirovirus type 1 (PRV1) and swine orthopneumovirus (SOV). The pathogenicity of PRV1 has been investigated experimentally by one research group, whereas SOV is yet to be studied. In this experimental trial, groups of weaners were inoculated with a European isolate of PRV1 (n = 8), a pool of SOV RNA-positive clinical nasal swab material (n = 8) or culture medium serving as controls (n = 4). Four days post inoculation (DPI), two controls and four pigs from each of the PRV1 and SOV groups were euthanized and necropsied. All SOV inoculated pigs tested negative for SOV at DPI 4 and therefore four of these pigs were transferred to the stable with the PRV1- infected pigs to assess direct-contact transmission. Nasal swab samples were collected at regular intervals after challenge and blood samples were collected at DPI 0, 4, and 14. At 14 DPI, the remaining pigs (n = 10) were euthanized and necropsied. Nasal swab samples and tissues from the respiratory tract were examined for PRV1 RNA by reverse transcriptase real- time PCR and blood samples were investigated for PRV1 antibodies by ELISA. Respiratory tissues were also evaluated macro- and microscopically and selected tissues were investigated for in situ detection of PRV1 mRNA by RNAscope. No clinical signs, except for nasal discharge, were observed in any of the pigs. PRV1-shedding was observed from DPI 2 to 11 with peaks between DPI 4 and 7, and PRV1 was transmitted horizontally to all direct-contact pigs. The highest viral load was detected in the upper respiratory tract, i.e. nose, upper and lower trachea compared to the lower respiratory tract, i.e., bronchioles, and alveoli. Generally, a chronic tracheitis at 4 DPI, developing into chronic, erosive tracheitis at 14 DPI was observed in the PRV1 groups and was supported by in situ detection of PRV1 by RNAscope. Three pigs also developed mild, bronchointerstitial pneumonia at 14 DPI. In conclusion, these results showed that PRV1 is a primary porcine respiratory pathogen. Author summaryRespiratory diseases in pigs impair porcine health and welfare and are often caused by a complex interaction between pathogens. We investigated the pathogenesis of two recently discovered viruses: porcine respirovirus type 1 (PRV1) and swine orthopneumovirus (SOV). An experimental model in weaner pigs showed that PRV1 is capable of efficient replication, horizontal transmission and caused pathological manifestations mainly in the upper respiratory tract. The lesions consisted of chronic, erosive tracheitis in all PRV1 infected pigs, resulting in a breakage of the tracheal epithelial barrier, and a few pigs developed bronchointerstitial pneumonia. These findings confirm that PRV1 should be considered a primary porcine respiratory pathogen, but in contrast, SOV did not result in a successful infection, and the reasons why remain to be elucidated. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/666573v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@d93a11org.highwire.dtl.DTLVardef@17341e9org.highwire.dtl.DTLVardef@1ec4ca4org.highwire.dtl.DTLVardef@9bd42f_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstract created with Biorender.com

microbiology↗

bioRxiv2025

Antisense oligonucleotides targeting Valosin-containing protein improve muscle pathology and molecular defects in cell and mouse models of multisystem proteinopathy.

Valosin-containing protein (VCP) related disease, also known as multisystem proteinopathy 1 (MSP1), is an autosomal dominant disease caused by gain-of-function pathogenic variants of the VCP gene. The disease is associated with inclusion body myopathy, early-onset Pagets disease of bone, frontotemporal dementia, and familial amyotrophic lateral sclerosis. There is currently no treatment for this progressive disease associated with early demise resulting from proximal limb girdle and respiratory muscle weakness. We hypothesize that regulating VCP hyperactivity to normal levels can reduce the disease pathology. In this study, we assessed the effect of antisense oligonucleotides (ASOs) specifically targeting the human VCP gene in the patient (R155H) iPSC-derived skeletal muscle progenitor cells (SMPCs). ASOs were well tolerated up to a concentration of 5 {micro}M, and significantly reduced VCP protein expression in the SMPCs by 48% (95% CI [39-56]). We also treated the transgenic mouse model of VCP disease with the overexpressed humanized VCP gene severe A232E pathogenic variant with weekly subcutaneous ASO injections starting from 6 months of age for 3 months. In the skeletal muscle of transgenic mice, ASOs resulted in 30% (95%CI [27-32]) knockdown of VCP protein compared to control ASO. The ASO-mediated reduction of VCP expression in muscle tissue was associated with improvement in autophagy flux and reduction in TDP-43 expression, hallmarks of VCP disease. In addition, ASO-treated VCP A232E mice showed improvements in functional tests of muscle strength, such as rotarod and inverted screen test compared to mice treated with control ASO. These results suggest that targeting VCP could be beneficial in preventing the progression of the VCP myopathy and hold promise for the treatment of patients with VCP MSP1.

molecular biology↗

bioRxiv2025

Manual lymph drainage massage of the head and neck improves cognition and reduces pathological biomarkers in the 5x-FAD mouse model of Alzheimers disease

Alzheimers disease (AD) affects 6.9 million people over the age of 65 in the US and is expected to double by 2060. While FDA approved immunotherapies slow cognitive decline in some individuals with AD, they do not improve cognition, are costly, and have significant side-effects. Therefore, new targets, approaches, and treatments for AD are a necessity. There are no FDA approved therapies for AD that target the brains lymphatic system. It is well established that the toxic protein, amyloid-beta (A{beta}), accumulates in the AD brain. Recent studies have shown that A{beta} is cleared via interstitial fluid and cerebrospinal fluid through a pathway involving the glymphatic system--meningeal lymphatic vessels--leading to deep and superficial cervical lymphatic vessels and nodes. Therefore, any blockage along this route can cause inefficient drainage and result in pathological buildup of A{beta}, which can lead to AD. Here, we propose a new approach to treating AD by manual lymph drainage (MLD), which is a light skin massage traditionally used to reduce fluid accumulation in lymphedema. This therapy has also been demonstrated to be safe in individuals with AD, but its effects on cognition and biomarkers of AD has never been investigated. In this study we demonstrate that repeated MLD of the head and neck, including the superficial cervical lymphatic vessels (scLVs), improves cognitive function in AD as measured in both the Y-maze and nest-building tests. We also show that this coincides with a reduction in plasma levels of neurofilament light chain (NfL), a non-specific biomarker for neuronal cell death and axonal damage. MLD was also shown to reduce A{beta} in the hippocampus of these mice. Combined, this data provides compelling proof-of-principle evidence for the potential of MLD as a standalone or adjunct therapy in the treatment of AD.

neuroscience↗

bioRxiv2025

Amyloid β interaction with membranes: Removal of cholesterol from the membranes to catalyze aggregation and amyloid pathology

The interplay between the cholesterol metabolism and assembly of A{beta}42 (the 42- residue form of the amyloid-{beta} peptide) peptides in pathological aggregates is considered as one of the major molecular mechanisms in development of Alzheimers disease (AD). Numerous in vitro studies led to the finding that the high cholesterol levels in membranes accelerate the production of A{beta} aggregates. The molecular mechanisms explaining how cholesterol localized inside the membrane bilayer catalyzes the assembly of A{beta} aggregates above the membrane remain unknown. We addressed this problem by combining different AFM modalities, including imaging and force spectroscopy, with fluorescence spectroscopy. Our combined studies revealed that A{beta}42 was capable of removing cholesterol from the membrane. Importantly, physiologically low concentrations of A{beta}42 demonstrate such ability of monomeric A{beta}42. Extracted cholesterol interacts with A{beta}42 and accelerates its on- membrane aggregation. We propose a model of interaction of A{beta}42 with membranes based on the ability of A{beta}42 to extract cholesterol, which explains several AD associated observations related to cholesterol interplay with A{beta}42 aggregation resulting in the AD onset and progression.

biophysics↗

bioRxiv2025

Combining Selenocystine Based Fluorescence Assay with Structural Prediction for Enhanced Detection and Analysis of Cystine Stone Pathology

BackgroundCystine stones, a rare but recurrent type of kidney stones, primarily result from cystinuria, an inherited disorder caused by mutations in the genes SLC3A1 and SLC7A9, which encode renal cystine transporters. These mutations impair cystine reabsorption, resulting in elevated urinary cystine concentrations and stone formation. Current diagnostic methods are limited, particularly for detecting molecular-level dysfunctions. We aimed to develop a nonradioactive method for functional assessment of cystine transporters and mutation-specific pathologies. MethodsWe designed an innovative diagnostic approach combining a selenocystine-based fluorescence assay with structural predictions using AlphaFold to rapidly and accurately assess cystine transporter function and the molecular impacts of genetic mutations. ResultsOur assay demonstrated comparable transport efficiencies of cystine and selenocystine by the SLC3A1/SLC7A9 complex, and effectively differentiated mild, moderate, and severe functional impairments associated with known clinical mutations, including A354T and P482L. Structural modeling further provided mechanistic insights into mutation-induced dysfunctions. ConclusionThis integrated approach--combining a sensitive selenocystine fluorescence assay with AI-powered structural analysis--enables rapid, precise diagnosis of cystinuria variants and delivers mechanistic insights for personalized therapeutic strategies.

genetics↗

bioRxiv2025

Aldehyde dehydrogenase protein: a placental glycogen trophoblast marker suitable to monitor placental pathologies in mouse models

This study focuses on developing new approaches to identify and quantify glycogen trophoblast cells in the placenta. Due to limitations with currently available methods, alternative markers for these cells are being explored. Here we report on one promising candidate, Aldh1a3, a gene that was recently shown to be expressed in the mouse placenta only in glycogen trophoblast cells and their progenitors. Our study validates ALDH1A3 protein as glycogen trophoblast marker in the mouse placenta with high specificity when compared to CDKN1C (p57KIP2). This marker will be useful for the isolation of the glycogen trophoblast subpopulation and facilitate examining its function in placental programming and understanding its role in pathological pregnancy models. Furthermore, trophoblast giant cells with high expression of ALDH1A3 were detected in the decidua of human term placentas. These were interspersed with extravillous interstitial trophoblast cells or were located distal of the interstitial trophoblast layer of the decidua basalis.

developmental biology↗

bioRxiv2025

Morphogen-guided neocortical organoids recapitulate regional areal identity and model neurodevelopmental disorder pathology

The human neocortex exhibits characteristic regional patterning (arealization) critical for higher-order cognitive function. Disrupted arealization is strongly implicated in neurodevelopmental disorders (NDDs), but current neocortical organoid models largely fail to recapitulate this patterning, limiting mechanistic understanding. Here, we establish a straightforward method for generating arealized organoids through short-term early exposure to anterior (FGF8) or posterior (BMP4/CHIR-99021) morphogens. These treatments created distinct anterior and posterior signaling centers, supporting long-lasting polarization, which we validated with single-cell RNA sequencing that revealed area-specific molecular signatures matching prenatal human cortex. To demonstrate the utility of this platform, we modeled Fragile X Syndrome (FXS) in organoids with distinct anterior and posterior regional identities. FXS organoids showed highly disrupted SOX4/SOX11 expression gradients along the anterior-posterior axis, consistent with alterations found in autism spectrum disorder (ASD) and demonstrate how regional patterning defects may contribute to NDD pathology. Together, our study provides a robust platform for generating neocortical organoids with anterior-posterior molecular signatures and highlights the importance of modeling NDDs using experimental platforms with neuroanatomic specificity.

developmental biology↗

bioRxiv2025

Impact of Membrane Fluidity on α-syn Fibril Structures and Neuronal Pathology

Conformational variations in -syn fibrils are thought to underlie the distinct clinical features of synucleinopathies, including Lewy body dementia (LBD), Parkinsonss disease (PD), and multiple system atrophy (MSA), suggesting that distinct fibril structures act as molecular fingerprints linked to disease phenotype. While the origins of these conformational variations remain unclear, increasing evidence points to membranes as key modulators of fibrils conformations. In this study, we investigated how age-related alterations in membrane composition and fluidity influence -syn fibril formation and cellular outcomes. Using complex mixture membranes that mimic normal neuronal membranes and their age-related modifications in fatty acid chains, we found that -syn fibrils grown with these membranes displayed distinct 2D ssNMR spectral patterns compared to lipid-free -syn fibrils, reflecting differences in rigid fibril cores. Moreover, fibrils grown with age-related membranes exhibited weaker membrane association than those grown with normal neuronal membranes. These membrane-associated fibrils induce stronger neuronal pathologies than lipid-free fibrils, though the severity differed in intraneuronal aggregation and inflammation responses. Overall, our findings provide new insights into how age-related changes in membrane composition shape -syn fibril structure and pathogenicity, strengthening the link between membrane dynamics and amyloid-driven neurodegeneration.

molecular biology↗

bioRxiv2025

Transforming Hyperspectral Data into Insight: The DREAM Approach for Pathology

Quantitative pathology remains limited due to the need for chemical staining and subjective interpretation of tissue features. Autofluorescence imaging offers a label-free alternative: however, high-dimensional excitation-emission datasets pose challenges for visualization and reproducible analysis. Here, we present Dimensionality Reduction for Enhanced Autofluorescence Microscopy (DREAM), a method that condenses multi-excitation emission spectra into a compact, information-rich format using phasor-based tools. Applied to unstained esophageal tissue samples, DREAM enables high-contrast visualizations that distinguish key histological structures without the need for exogenous labeling. Quantitative assessments across multiple datasets show DREAM improves colorfulness, sharpness, and consistency over single-laser acquisitions, supporting its potential to advance objective, label-free diagnostics through enhanced spectral visualization.

biophysics↗

bioRxiv2025

TDP-43 pathology triggers SRRM4-dependent cryptic splicing of G3BP1 in ALS/FTD

Loss of nuclear TDP-43 is a defining feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet how this leads to selective neuronal vulnerability is poorly understood. Here, using human iPSC-derived neurons and a large multi-omics dataset of ALS/FTD patients, we demonstrate that TDP-43 pathology induces the inclusion of an in-frame cryptic exon in human G3BP1. The resulting CRYPTIC G3BP1 protein contains an additional 10-amino acids within the highly conserved NTF2L domain, which acts as a dominant negative and disrupts stress granule dynamics. We further show that cryptic exon inclusion in G3BP1 upon TDP-43 loss is enriched in neurons. Mechanistically, the loss of TDP-43 unmasks a binding site for the neuron-specific splicing regulator SRRM4 within intron 2 of G3BP1, enabling the inclusion of the cryptic exon. Collectively, our findings reveal that neuron-specific regulatory mechanisms intersect with TDP-43 -mediated splicing and suggest a mechanistic basis for the increased neuronal vulnerability observed in ALS/FTD.

neuroscience↗

bioRxiv2025

Dengue NS1 Antibodies drive Immune Complex Formation, Hyperglycaemia and systemic pathology in a murine NS1 plasmid challenge model

Dengue virus (DV) NS1, a secreted virotoxin and key pathogenic factor, can trigger immune responses with poorly understood long-term effects. This study assessed immunopathology in mice administered with DV NS1 plasmid DNA via intraperitoneal (IP), intramuscular (IM), or intravenous (IV) route for DV serotypes 1-4. IP delivery caused the most pronounced effects, including elevated AST/ALT and GRP78 levels, hyperglycemia, and altered organ weights, with DV4 NS1 showing the strongest hepatic damage. Despite serum NS1 antigen being undetectable, mice developed strong NS1-specific antibodies (Abs) and immune complexes. Liver histology revealed degeneration and immune cell depletion. DV NS1 plasmid DNA was detected in liver tissue, but not RNA. DV could infect and replicate in murine pancreatic beta cells. In liver cells, DV increased GAPDH expression, while NS1-Ab-positive serums reduced it. Findings indicated that NS1-specific Abs, not the antigen, drove immune-metabolic dysfunctions, emphasizing the need to evaluate Ab-mediated effects in dengue pathogenesis. Significance StatementDengue virus (DV) remains a leading tropical pathogen, yet the long-term effects of its secreted virotoxin NS1 are incompletely understood. Using a murine model, we demonstrated that NS1 plasmid DNA administration drove systemic and organ-specific pathology. Serotype-specific differences in immune responses, biochemical alterations, and histopathological changes underscored NS1s complex role in disease severity. Elevated immune complexes and liver enzyme profiles highlighted mechanisms of immune modulation and hepatic injury, key features of dengue, while increased serum glucose and GRP78 levels pointed to early markers of diabetes onset. These findings provide foundational evidence that NS1- and NS1 antibody-mediated pathways link dengue pathogenesis with metabolic dysfunction, offering critical insights into host-pathogen interactions and comorbidity development.

microbiology↗