Biology subjects

Varga, A.

Publications and source records attributed to Varga, A..

9 recordsLinked to original sources

bioRxiv2026

Rapid direct neuronal reprogramming of human dental pulp stem cells

Direct neuronal reprogramming offers an alternative to induced pluripotent stem cell-based differentiation by converting somatic cells directly into neurons without passage through pluripotency. However, commonly used fibroblast-based protocols are often slow and inefficient. Here, we evaluated human dental pulp stem cells (DPSCs), which originate from the cranial neural crest and possess intrinsic neurogenic potential, as a developmentally relevant source of induced neurons (iNs). Using an all-in-one lentiviral vector, we converted DPSCs into iNs within 17 days, compared with 28 days for fibroblasts reprogrammed with the same vector, and achieved significantly higher neuronal purity under the respective established protocols. Multi-omic profiling revealed coordinated suppression of mesenchymal and cell-cycle programs and induction of neuronal, synaptic, and metabolic pathways. Single-nucleus RNA sequencing resolved fibroblast-like, transitional, maturing neuronal, GABAergic-like, and alternative fates, while trajectory inference suggested divergent neuronal and non-neuronal conversion paths. Whole-cell recordings showed that a subset of DPSC-iNs developed early neuronal excitability and voltage-gated inward and outward currents. Together, our findings establish DPSCs as an accessible and developmentally relevant source for rapid direct neuronal conversion. This integrated molecular, single-nucleus, and electrophysiological characterization defines the cellular heterogeneity of DPSC-to-neuron reprogramming and provides a framework for protocol refinement and future patient-specific disease modeling.

neuroscience↗

bioRxiv2026

ViTAMIn-O: Democratizing computer vision-based machine learning for stem cell research

Deep Learning (DL) holds exciting potential in automating the prediction of organoid differentiation results. Nevertheless, current models lack adaptability, openness, and robustness in performance. Additionally, broad employments of predictive models in wet-lab settings necessitate machine learning expertise, often not readily available in biologically oriented laboratories. To offer an intuitive solution, we present ColabViTAMIn-O, a code-free platform together with ViTAMIn-O. ViTAMIn-O is a fully open organoid-specific DL model trained and tested on a total of 34 organoid categories, incorporating annotated images across transmitted light microscopy (TLM) modalities at single-organoid resolution. It is adaptable to downstream prediction tasks of varying dataset sizes and outperforms established models even with linear-probing. It performs reliably within a few-shot framework and is even extensible to human embryo TLM imaging data at single specimen level. By releasing our platform, centralized model hub, and datasets, we hope to encourage broader deployments of specialized DL models in stem-cell laboratories.

bioinformatics↗

bioRxiv2026

Non-muscle actinopathy-associated loss-of-function actin variants modulate cytoskeletal reorganization

Variants in ACTB gene encoding for cytoplasmic {beta}-actin result in a group of rare disorders called non-muscle actinopathies (NMA). We investigated the cellular effects of a missense variant, G302A, and a four-amino-acid deletion, S338-I341, associated with the subgroup of NMA - ACTB pLoF (predicted loss-of-function) disorder in patient-derived fibroblast cells. We found that neither of the mutations affected the organization of actin or the width of the actin-filament bundles, while the mutation G302A reduced the stiffness of the cells as measured by using atomic force microscopy. The latter effect might be associated with the misorganization of tubulin and with the increased size and number of focal adhesions. When we challenged the cells by monolayer stretching and followed the mechanically-induced reorganization of the actin cytoskeleton, we found that G302A mutant cells showed more dense actin filament bundles within the cells compared to wild type cells. At the same time, the extent of cofilin reorganization from the cell periphery was increased upon stretch, and this correlated with an increased cofilin phosphorylation. In the case of the deletion, while the extent of cofilin phosphorylation increased, the extent of reorganization was unaltered; rather, the phosphorylation of myosin light chain, important in counteracting external force, was drastically reduced. We could partially rescue this fascinating effect by overexpressing the active form of the formin mDia. Our findings open the possibility to validate the cellular phenotype in the most affected patients cells, in neurons.

biophysics↗

bioRxiv2026

Pathogenic tau in the mouse locus coeruleus induces noradrenergic hyperactivity and neuropsychiatric phenotypes reminiscent of early Alzheimers disease

Alzheimers disease (AD), though defined as a cognitive disorder, often presents neuropsychiatric symptoms such as anxiety, depression, agitation and sleep disruptions years before the onset of frank memory impairment. An early pathological feature is the accumulation of hyperphosphorylated "pretangle" tau (pTau) in the locus coeruleus (LC), the brains primary source of norepinephrine (NE). While clinical studies link LC pTau burden to behavioral abnormalities, causal mechanisms remain unclear. We developed a translationally-relevant mouse model that recapitulates the LC-first phenomenon using cell type-specific viral expression of pathogenic P364S mutant human tau in LC neurons. Three months post-infusion, pTau accumulation induced anxiety-and compulsive-like behaviors and reduced sleep spindles without altering overall sleep architecture. Consistent with the behavioral phenotypes, electrophysiological recordings revealed significant increases in spontaneous and evoked firing of LC neurons, accompanied by robust astrocytic reactivity with no apparent cell death. Transcriptomic analysis identified upregulation of Hcn2 and downregulation of Clic6, suggesting changes in neuronal excitability. To further define molecular mechanisms, we developed a cell type-specific proteomics approach, which showed synaptic and metabolic alterations associated with LC-specific tau pathology. Early anxiety-like behaviors observed at 3 months diminished at later timepoints (6-9 months) and were replaced by anxiolytic characteristics. These findings demonstrate that pTau triggers phenotypes reflective of LC-NE hyperactivity in the early stages of AD pathogenesis, laying the foundation for the development of LC-based disease-modifying therapies to address neuropsychiatric manifestations.

neuroscience↗

bioRxiv2025

Phosphoproteomic profiling reveals post-translational dysregulation in Huntington's disease patient-derived neurons

Huntingtons disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin (HTT) gene. Although transcriptomic and proteomic changes have been characterized in patient-derived neurons, the contribution of post-translational modifications (PTMs), such as phosphorylation, remains poorly understood. Here, we present the first phosphoproteomic analysis by mass spectrometry (P-MS) of human induced neurons (iNs) directly reprogrammed from HD patient fibroblasts. We identified 177 phosphopeptides with significantly altered abundance in HD-iNs, mapping to phosphoproteins associated with key signaling pathways known to be affected in HD, such as splicing and autophagy. By integrating P-MS data with previously published proteomic and transcriptomic data from the same donors, we identified distinct subsets of ON-OFF phosphopeptides that exhibited a complete loss of phosphorylation in either HD- or Ctrl-iNs, without corresponding changes at the RNA or protein level. An exception was MXRA8, previously described in glial cells as a mediator of blood-brain barrier integrity and astrocyte-mediated neuroinflammation. This protein showed increased protein abundance despite the absence of phosphorylation in HD- iNs, suggesting a compensatory mechanism - a pattern also observed in human post mortem cortical HD tissue. Additionally, MXRA8 showed altered protein-protein interactions with lysosomal and metabolic regulators in HD-iNs, highlighting its potential role in autophagy impairment as well as in neurovascular dysfunction. These findings uncover a distinct layer of post-translational dysregulation in HD, suggesting that phospho-switch proteins such as MXRA8 may be candidate effectors of pathology and thus site-specific phosphorylation loss may contribute to impaired signaling and proteostasis in human HD neurons.

neuroscience↗

bioRxiv2025

The Baraitser-Winter Cerebrofrontofacial Syndrome recurrent R196H variant in cytoplasmic β-actin impairs its cellular polymerization and stability

Variants in cytoskeletal actin encoding genes are associated with a broad spectrum of disorders, called non-muscle actinopathies. Among them, Baraitser-Winter cerebrofrontofacial syndrome (BWCFF) displays the most severe symptoms, such as intellectual disability and epilepsy. The exact consequences of the mutation on actins properties, however, are not fully understood. Here we explored the cellular effects of the R196H mutation in patient-derived fibroblasts. We show that the heterozygous mutation causes an actin polymerization defect in cells, leading to a fifty percent decrease in filamentous (F-) actin content. This effect can be rescued by the addition of the actin-polymerizing and stabilizing drug, jasplakinolide. We observed no significant defects either in the organization of the cellular actin cytoskeleton, analyzed by superresolution (STED) microscopy, or in the structure of purified filaments stabilized with phalloidin, explored with atomic force microscopy (AFM). The reduced F-actin content correlated with an approximately fourfold reduction in the stiffness of patient-derived cells probed with AFM. Manipulating the cells by mechanical forces through the application of the Dual Laser Optical Tweezers (DLOT) technique suggests that the mutation weakens the attachment of cytoskeletal actin to the plasma membrane. Inducing dynamical reorganization of actin by uniaxial stretching revealed that the interaction of cofilin with actin is also weakened by the mutation. Based on the existing cofilin-actin structures, the binding of cofilin may weaken the interaction of the neighboring residue E195 with K113, one of the lateral contacts stabilizing the filament. Thus, the mutation possibly exerts its effect through the destabilization of the interfilament interactions, potentially interfering allosterically with cofilin binding during actin depolymerization.

biophysics↗

bioRxiv2025

Cholesteryl esters and high protein-to-lipid ratios distinguish Non-Vesicular Extracellular Particles from Extracellular Vesicles

Extracellular vesicles (EVs) are central to intercellular communication, yet the mechanisms underlying their biogenesis and diversity remain incompletely understood. Here, we integrate meta-analysis, advanced lipidomic, protein-to-lipid profiling, and super-resolution imaging to define the fundamental principles governing EV heterogeneity. Our meta-analysis of published transmission electron microcrographs across kingdoms reveals a highly conserved 110 nm average diameter and 200 nm upper size limit for intraluminal vesicles (ILVs), which are secreted as exosomes. Besides classical EV populations, we also characterize a distinct nanoparticle class: 167000 xg pellet of non-vesicular extracellular particles (167k-NVEPs), which exhibit a significantly higher protein-to-lipid ratio than 14000 xg pellet of large EVs (14k-lEVs) and 100000 xg pellet of small EVs (100k-sEVs), as measured by both biochemical assays and Raman spectroscopy. Lipid profiling demonstrates that 167k-NVEPs exhibit significant enrichment in cholesteryl esters and triacylglycerols, lipids typically associated with lipid droplets and the endosome/lysosome system. Analysis of lipid carbon-chain lengths reveals distinct signatures: 167k-NVEPs show pronounced enrichment at 16 and 18 carbons, while 100k-sEVs display enrichment at 32 and 34 carbons. This divergence indicates a potential connection to flexible biogenesis pathways. Marker heterogeneity across EV populations, confirmed by confocal and super-resolution microscopy, further underscores the limitations of relying on canonical tetraspanins for EV classification. Notably, 167k-NVEPs (likely exomeres) exhibit enrichment of Arf6 and CD63. Together, our findings provide compositional, biophysical, and molecular evidence supporting the formal recognition of 167k-NVEPs as a distinct class of extracellular particles and enabling exploring in disease biology and therapeutic delivery. Significance StatementExtracellular vesicles (EVs) are critical mediators of intercellular communication, yet their classification remains clouded by ambiguity in terms of their composition and biogenesis. This study resolves critical uncertainties through a cross-kingdom meta-analysis, establishing a conserved [~]110nm diameter and [~]200 nm upper size limit for intraluminal vesicles (ILVs), the precursors to exosomes. More significantly, we identify non-vesicular extracellular particles (167k-NVEPs) as a distinct class based on their unique sterol-rich lipidome, enrichment in lipids of 16 and 18 carbon chain length, elevated protein-to-lipid ratio, and functional cargo delivery. These features, alongside evidence of non-canonical origin and functional cargo delivery, establish NVEPs as a discrete class of extracellular particles.

cell biology↗

bioRxiv2020

Decreased calmodulin recruitment triggers PMCA4 dysfunction and pancreatic injury in cystic fibrosis

Exocrine pancreatic damage is a common complication of cystic fibrosis (CF), which can significantly debilitate the quality of life and life expectancy of CF patients. The cystic fibrosis transmembrane conductance regulator (CFTR) has a major role in pancreatic ductal ion secretion, however, it presumably has an influence on intracellular signaling as well. Here we describe in multiple model systems, including iPSC-derived human pancreatic organoids from CF patients, that the activity of PMCA4 is impaired by the decreased expression of CFTR in ductal cells. The regulation of PMCA4, which colocalizes and physically interacts with CFTR on the apical membrane of the ductal cells, is dependent on the calmodulin binding ability of CFTR. Moreover, CFTR seems to be involved in the process of the apical recruitment of calmodulin, which enhances its role in calcium signaling and homeostasis. Sustained intracellular Ca2+ elevation in CFTR KO cells undermined the mitochondrial function and increased apoptosis. Based on these, the prevention of sustained intracellular Ca2+ overload may improve the exocrine pancreatic function and may have a potential therapeutic aspect in CF.

cell biology↗

bioRxiv2018

Purkinje cell number-correlated cerebrocerebellar circuit anomaly in the valproate model of autism

While cerebellar alterations may play a crucial role in the development of core autism spectrum disorder (ASD) symptoms, their pathophysiology on the function of cerebrocerebellar circuit loops is largely unknown. We combined multimodal MRI (9.4 T) brain assessment of the prenatal rat valproate (VPA) model and correlated immunohistological analysis of the cerebellar Purkinje cell number to address this question. We hypothesized that a suitable functional MRI (fMRI) paradigm might show some altered activity related to disrupted cerebrocerebellar information processing. Two doses of maternal VPA (400 and 600 mg/kg, s.c.) were used, and while the higher VPA dose induced a global decrease in whole brain volume, the lower dose induced a focal gray matter density decrease in the cerebellum and brainstem. Increased cortical BOLD responses to whisker stimulation were detected in both VPA groups, but it was more pronounced and extended to cerebellar regions in the 400 mg/kg VPA group. Immunohistological analysis revealed a decreased number of Purkinje cells in both VPA groups. In a detailed analysis, we revealed that the Purkinje cell number interacts with the cerebral BOLD response distinctively in the two VPA groups that highlights atypical function of the cerebrocerebellar circuit loops with potential translational value as an ASD biomarker.

neuroscience↗