Biology subjects

Han, C.

Publications and source records attributed to Han, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Glycosaminoglycan presentation-dependent regulation of glial responses by Orion during Drosophila neuronal pruning

Neuronal pruning refines neural circuits by eliminating unnecessary connections to ensure proper nervous system function. In Drosophila mushroom body gamma-neuron remodeling, the chemokine-like protein Orion is required for astrocyte infiltration and phagocytosis. This study investigated whether glial responses depend on how Orion is presented. We found that Orion interacts with glycosaminoglycans (GAG) associated to proteoglycans, which are essential for proper axon pruning. Tethering Orion to the gamma-neuron plasma membrane, mimicking glycosaminoglycan binding, was sufficient to induce astrocyte infiltration into axon bundles. However, membrane-bound Orion alone could not efficiently support phagocytosis, which additionally requires a soluble and mobile Orion pool. We also identified exosomes carrying GAG-bound Orion as potential mediators of neuronal remodeling. These findings provide the first evidence that glial infiltration can be uncoupled from phagocytosis, demonstrating that the mode of Orion presentation regulates distinct glial mechanisms during neuronal remodeling.

neuroscience↗

bioRxiv2026

Deciphering the gene-centric spatial dynamics across tissue and time with GeneSPOT

Spatial omics technologies provide unprecedented opportunities to investigate how molecular programs are organized within tissues. However, existing analytical frameworks primarily focus on identifying cellular states and characterizing differentially expressed molecular features across these states. The relational spatial organization of molecular features and its dynamics across biological contexts therefore remain largely unexplored. Here, we present GeneSPOT, a gene-centric computational framework for characterizing the spatial relationships among genes and quantifying their reorganization across biological conditions. Although gene-centric in design, GeneSPOT generalizes to other molecular features. Across diverse applications, GeneSPOT resolved tissue structures, integrated paired and unpaired spatial multi-omics data, unveiled the spatial convergence of gene programs during embryonic development, and identified aging-associated spatial reorganization among genes whose expression did not change significantly with age. GeneSPOT thus complements conventional cell-centric analysis, providing a framework for deciphering how spatial relationships among molecular features are reorganized across biological conditions and over time.

bioinformatics↗

bioRxiv2026

Association of object mnemonic discrimination deficit with age and AD biomarkers in older adults

Background Blood-based Alzheimers disease (AD) biomarkers provide scalable detection of AD pathology, yet their relationships with hippocampal-dependent mnemonic discrimination remain unclear. This study examined how object mnemonic discrimination relates to age, hippocampal structure, and plasma AD biomarkers in cognitively normal (CN) older adults and individuals with amnestic mild cognitive impairment (aMCI). Methods Forty-six CN and forty-one aMCI participants completed an object mnemonic similarity task (MST). Mnemonic discrimination was quantified using the overall lure discrimination index (LDI) and similarity-specific LDIs. Plasma pTau181, pTau217, pTau231, and GFAP were assayed, and hippocampal volumes were derived from 7T MR scanner. Multiple linear regression models assessed associations between LDI and biomarkers, controlling for age, sex, education, APOE4 status, and diagnosis. Repeated-measures ANCOVA and linear mixed-effects models evaluated similarity-dependent age effects. ROC analyses and stepwise logistic regression quantified diagnostic classification performance. Results LDI was significantly reduced in aMCI compared to CN. Total hippocampal volume and plasma pTau217 showed the strongest associations with LDI, exceeding those of pTau181, pTau231, and GFAP. LDI discriminated aMCI from CN with high accuracy (AUC = 0.836), comparable to hippocampal volume and plasma biomarkers. A combined model including LDI, pTau181, pTau217, and hippocampal volume achieved the highest diagnostic accuracy (AUC = 0.931). Age effects were similarity-dependent: discrimination declined with age only for low-similarity lures, independent of diagnosis. Sex differences emerged within aMCI, with females showing lower LDI than males. Conclusions Object mnemonic discrimination provides a sensitive behavioral marker linked to hippocampal volume and plasma pTau217, showing discriminative power comparable to established AD biomarkers in separating aMCI from CN participants. Similarity-dependent age effects highlight age-related deficits are not universal for lures with varying similarity levels.

neuroscience↗

bioRxiv2026

A role for CREB5 in skin wound healing

Compared with skin wounds, oral mucosal wounds heal more quickly, with minimal scarring, faster re-epithelialization, and reduced inflammation. One differentiating factor may be the differential transcription factor-associated gene networks involved in tissue regeneration. One such transcription factor, BATF3, was recently shown by us to promote wound-healing responses in vitro and in vivo. Our prior analyses also suggest that CREB5 is a differentially regulated transcription factor in oral wounds and may be involved in early wound-healing gene expression programs. CREB5 expression was induced in immortalized skin keratinocytes (HaCaT) to examine its effect on in vitro wound healing relative to immortalized gingival keratinocytes (TIGK). CREB5 overexpression let to differential expression of predicted downstream genes and improved skin keratinocyte migration in vitro. This work suggests that examining transcription factors and gene networks that regulate wound-healing responses in the oral mucosa may lead to the discovery of novel targets to improve skin wound healing.

cell biology↗

bioRxiv2026

LARP4 is a B cell-specific metabolic checkpoint for plasma cell differentiation and a therapeutic target in systemic lupus erythematosus

RNA-binding protein LARP4 plays an important role in T cell activation and differentiation, but its role in B cell biology and the pathogenesis of systemic lupus erythematosus (SLE) remains unclear. This study found that LARP4 was specifically highly expressed in B cells of SLE patients and was positively correlated with disease activity. By constructing T cell-specific and B cell-specific conditional knockout mice, we found that deletion of LARP4 in B cells, but not in T cells, significantly alleviated pristane-induced and Bm12-induced lupus nephritis. Further analysis showed that LARP4 deletion selectively inhibited B cell differentiation into plasma cells, but did not affect germinal center B cell formation. Integrated transcriptomic and metabolomics analyses revealed that this effect is due to reduced phosphatidic acid synthesis and decreased mTORC1 activity caused by mitochondrial oxidative phosphorylation dysfunction. Furthermore, we used LIPEP, a LARP4 inhibitory peptide that effectively mimicked the therapeutic effects of LARP4 gene knockout in the MRL/lpr spontaneous lupus model and outperformed cyclophosphamide in reducing glomerular immune complex deposition and improving extrarenal dermatitis. These results indicates that LARP4 is a key metabolic checkpoint regulating B cell differentiation into Plasma cells and suggest that it may be a potential therapeutic target for SLE.

immunology↗

bioRxiv2026

AI-enabled reconstruction of 3D spatial multi-omics at single-cell resolution

Three-dimensional (3D) spatial multi-omics provides unparalleled insights into biological activities, yet remains technically prohibitive. Here, we introduce Histo3D-MO, a hybrid experimental-computational pipeline for reconstructing single-cell-resolution 3D spatial multi-omics maps. Notably, Histo3D-MO integrates sparse, omics-disjoint spatial measurements with dense Hematoxylin and Eosin (H&E) histology through SPatial multi-Omics from h&E imaGEs (SPONGE), achieving cell-level 3D mapping across multiple omics layers. Validated using held-out slices, SPONGE substantially outperforms existing omics prediction methods. We further developed an algorithmic suite for 3D cell-type propagation and tissue-domain annotation, enabling whole-volume characterization of the tumor microenvironment. Applied to the in-house hepatocellular carcinoma data, Histo3D-MO revealed spatially organized patterns of translation efficiency, volumetric decoupling between malignant cells and monocytes, and depth-associated monocyte differentiation trajectories. Together, these results establish Histo3D-MO as a scalable framework for reconstructing single-cell-resolution 3D spatial multi-omics and interrogating tissue organization across complex biological systems.

bioinformatics↗

bioRxiv2026

A post-mitotic bistable switch actively safeguards mature neurons against reciprocal transdifferentiation in vivo

How mature cells actively defend their identity against alternative fates is poorly understood. Here we show that two types of fully differentiated Drosophila somatosensory neurons, derived from distinct lineages, are continuously prevented from transdifferentiating into one another by a post-mitotic bistable switch. Two homeodomain transcription factors, Hmx and Cut, are co-expressed in both neuronal types but adopt opposite stoichiometric dominances through mutual repression, each defining one identity. Disrupting this balance in mature neurons triggers deterministic, lineage-crossing reprogramming of transcriptome, dendritic and axonal architecture, and sensory behavior without reversion to a progenitor state. This regulatory logic is evolutionarily conserved: Drosophila sensory identities share transcriptomic signatures with distinct human A{delta} mechanoreceptors, and human CUX and HMX proteins functionally replace their fly counterparts. Mature neuronal identity is thus not a fixed endpoint but a poised equilibrium actively maintained by ongoing competition between opposing selectors. TEASERMature somatosensory identity is a poised equilibrium actively maintained by continuous competition between opposing terminal selectors.

Developmental Biology↗

bioRxiv2026

Cardiomyocytes Undergo a Mesenchymal-Like Fate Transition in Myocardial Fibrosis

BACKGROUNDMyocardial fibrosis, a pathological hallmark of adverse cardiac remodeling and heart failure, has been conventionally attributed to the activation of resident fibroblasts. Although recent studies suggest contributions from non-fibroblast lineages, direct in vivo genetic evidence that cardiomyocytes can undergo a mesenchymal-like fate transition during myocardial fibrosis remains absent. This study aims to investigate whether such a transition occurs and to elucidate the underlying regulatory mechanisms. METHODSHuman myocardial infarction (MI) tissues were analyzed by immunohistochemistry and integrated with public single-nucleus RNA sequencing (snRNA-seq) data to detect mesenchymal-like signatures in cardiomyocytes. Genetic lineage-tracing was performed in MI mice, and in cardiomyocyte-specific Hgs (hepatocyte growth factor-regulated tyrosine kinase substrate) gene knockout mice, to map the fate of cardiomyocyte-derived cells. Mechanistic insights were obtained through proteomic and snRNA-seq analysis of Hgs knockout hearts and validated through gain- and loss-of-function experiments targeting Aldh1a2 (aldehyde dehydrogenase 1 family member A2). RESULTSIn human MI samples, a subset of cardiomyocytes showed reduced expression of cardiomyocyte markers concurrent with acquisition of mesenchymal-associated markers. Genetic lineage tracing demonstrated that adult cardiomyocytes can adopt a mesenchymal-like cell fate during post-MI remodeling. We identify HGS as a factor constraining this transition. Hgs knockout in adult cardiomyocytes upregulated Aldh1a2, triggered the mesenchymal-like fate transition, and gave rise to cells expressing markers of activated fibroblasts or osteoblasts, accompanied by pronounced myocardial fibrosis and calcification. Forced Aldh1a2 overexpression in cardiomyocytes drove the mesenchymal-like fate transition in vitro and in vivo, whereas Aldh1a2 deletion in cardiomyocytes mitigated MI-induced myocardial fibrosis. CONCLUSIONSThis study provides in vivo genetic evidence that adult cardiomyocytes possess the capacity to undergo a mesenchymal-like fate transition under pathological conditions. Our data suggest that HGS and ALDH1A2 serve as regulators of the transition, offering a new basis for understanding cellular and molecular mechanisms of myocardial fibrosis. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIMyocardial fibrosis is primarily driven by resident fibroblast activation, with additional contributions from cardiac CD34+ cells, pericytes, and macrophages. C_LIO_LIAdult cardiomyocytes exhibit phenotypic plasticity and transdifferentiate into epicardial-like or pacemaker cells under specific conditions. C_LI What New Information Does This Article Contribute?O_LIA subset of cardiomyocytes adopts a mesenchymal-like cell fate during myocardial fibrosis, marked by downregulation of cardiomyocyte identity markers and loss of aligned cell-cell contacts. C_LIO_LIThese cells acquire mesenchymal morphology and markers, ECM components, migratory gene signatures, and proliferative capacity. C_LIO_LIHGS and ALDH1A2 act as regulators of this mesenchymal-like fate transition. C_LI Myocardial fibrosis drives heart failure progression, yet the cellular sources of pathological fibroblasts remain incompletely defined. Here, we demonstrate that a subset of cardiomyocytes adopts a mesenchymal-like cell fate during myocardial fibrosis by using an integrated approach combining human MI samples, murine genetic lineage tracing, and snRNA-seq. Mechanistically, we identify HGS and ALDH1A2 as regulators of this transition. Cardiomyocyte-specific Hgs deletion upregulates Aldh1a2, triggering the mesenchymal-like fate transition. Furthermore, Aldh1a2 overexpression drives this transition, while its deletion attenuates MI-induced fibrosis. These findings reveal a previously unrecognized plasticity of adult cardiomyocytes and identify potential therapeutic targets for fibrotic heart disease.

genetics↗

bioRxiv2026

FBXL21 regulates diurnal proteostasis and stress response by targeting DNAJB6 and client proteins

Circadian regulation of proteostasis, a key determinant of muscle health, remains poorly understood. Here, we identified DNAJB6, an Hsp40 (DnaJ) co-chaperone, as a substrate of the circadian E3 ligase FBXL21. FBXL21 mediated the ubiquitination-dependent proteasomal degradation of both DNAJB6 and its client proteins including Desmin; causative mutations of DNAJB6 in myopathies, however, rendered resistance to FBXL21-directed degradation. Fbxl21 KO C2C12 cells displayed aberrant accumulation of Desmin, and showed aggravated cytoplasmic accumulation of TDP-43, another DNAJB6 client protein, in heat shock response. Under timed exercise as a physiological stressor, WT mice displayed robust diurnal rhythms in the levels of stress granule markers (G3BP1 and FUS) and TDP-43 as a function of exercise timing. In contrast, the Fbxl21 hypomorph Psttm mutant mice showed elevated expression of these proteins without exercise, which was exacerbated under exercise-induced stress conditions; importantly, these abnormalities were rescued by skeletal muscle-specific FBXL21 expression. Our study elucidates a novel diurnal regulatory mechanism of skeletal muscle proteostasis via FBXL21 as a chaperone-linked E3 ligase, highlighting the FBXL21-DNAJB6 axis as a potential therapeutic target for myopathies.

cell biology↗

bioRxiv2026

DHHC3-dependent S-Acylation of CRY1 regulates its subcellular localization and repressor function in the circadian clock

The circadian clock is essential for maintaining cellular homeostasis and physiological fitness. At the molecular level, core clock proteins function via transcriptional-translational feedback loops in the cellular oscillator, and are highly regulated by post-translational modifications. Our unbiased screening of core clock proteins revealed that Cryptochrome 1 (CRY1), the central transcriptional repressor in the circadian clock, undergoes a novel post-translational modification known as S-acylation. We show that this reversible lipidation of CRY1 is required for its nuclear import and interaction with key clock components. Further, we mapped four cysteine residues as CRY1 S-acylation sites and identified DHHC3 as the primary protein acyltransferase for CRY1. Importantly, loss of CRY1 S-acylation, either via cysteine mutagenesis or genetic deletion of DHHC3, impaired CRY1 repressor function and consequently cellular circadian rhythms, suggesting that dynamic S-acylation couples cytoplasmic regulation of CRY1 and its transcriptional repressor function in the nucleus. Together, our findings identify S-acylation as a previously unknown post-translational modification of CRY1 critical for circadian clock function and establish DHHC3 as a pivotal circadian regulatory enzyme. Targeting CRY1 S-acylation or its regulatory enzymes may constitute an innovative therapeutic approach against clock-associated diseases.

biochemistry↗

bioRxiv2026

Cryptochrome Loss Drives COPD-like Lung Pathology through Disrupted Alveolar Epithelial Proliferation and Immune Homeostasis

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by alveolar destruction, impaired epithelial regeneration and chronic inflammation. While circadian disruption has been linked to COPD pathogenesis, the cellular mechanisms remain unclear. Here, we identify the core clock components Cryptochrome 1 and 2 (Cry1/2) as essential regulators of alveolar epithelial cell proliferation and pulmonary immune homeostasis. Cry1/2 double knockout (dKO) mice exhibit spontaneous emphysema-like pathology, including airspace enlargement, increased lung compliance, and inflammatory cell infiltration without environmental insults. Mechanistically, Cry1/2-deficient alveolar epithelial cells display reduced proliferative capacity, and transcriptomic profiling revealed a pronounced shift toward a more proximal airway-like phenotype. Notably, these cells share a gene signature with human COPD lungs in pathways involving immune regulation. Furthermore, Cry1/2-deficient macrophages show elevated responsiveness to LPS. Bone marrow (BM) transplantation revealed that mice receiving Cry1/2 dKO BM suffer from enhanced lung inflammation without airspace enlargement, supporting a critical role of epithelial Cry1/2 in maintaining alveolar integrity. Importantly, treatment with Nobiletin, a circadian rhythm-modulating compound, mitigates NF-{kappa}B activation and ameliorates lung inflammation and structural damage in Cry1/2 dKO mice. These findings establish CRY1/2 as critical circadian regulators of epithelial cell proliferation and immune homeostasis, and highlight the therapeutic potential of targeting circadian pathways in COPD.

genetics↗

bioRxiv2026

miR-128 Regulates Hypertensive Vascular Remodeling via PPAR-γ

OBJECTIVEThis study aimed to explore the role and underlying mechanism of microRNA-128 (miR-128) in regulating vascular remodeling in spontaneously hypertensive rats (SHRs), focusing on its targeting of peroxisome proliferator-activated receptor {gamma} (PPAR-{gamma}) and modulation of the Toll-like receptor 4/nuclear factor-{kappa}B (TLR4/NF-{kappa}B) inflammatory pathway. METHODSAll experimental procedures were approved by the Animal Care and Use Committee of Fujian Medical University. In vivo, ten-week-old male SHRs were randomly assigned to three groups: renal denervation (RDN, n=6), sacubitril/valsartan (Sac/Val, n=6), and Sham (n=6). Age-matched Wistar-Kyoto (WKY) rats served as normotensive controls (n=6).Eight weeks after intervention, mesenteric arteries were harvested for histological, functional, and molecular analyses. Serum miR-128 levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The expression levels of key proteins in the vascular wall were assessed via immunofluorescence (IF), immunohistochemistry (IHC), and Western blotting (WB). Bioinformatics analysis and RNA sequencing (RNA-seq) were employed to identify core genes and signaling pathways associated with hypertension-induced pathological inflammation. RESULTSIn vivo, in the SHR sham-operated group, elevated blood pressure, severe vascular remodeling, and impaired vasodilatory function were observed, accompanied by downregulated miR-128 expression and upregulated TLR4/NF-{kappa}B signaling activity (all p < 0.0001).RDN postoperative, miR-128 expression was significantly restored, which in turn inhibited the TLR4/NF-{kappa}B pathway, reduced the production of pro-inflammatory cytokines (including IL-1{beta}, IL-6, and TNF-), and ameliorated vascular dilation dysfunction in SHRs (all p < 0.0001). Mechanistically, miR-128 negatively regulated the TLR4/NF-{kappa}B signaling pathway while upregulating the expression of PPAR-{gamma} (p < 0.05). CONCLUSIONRDN not only exerts a hypotensive effect but also improves hypertensive vascular remodeling. miR-128 inhibits excessive inflammation in vascular smooth muscle cells and alleviates vascular remodeling in SHRs via the PPAR-{gamma}/TLR4/NF-{kappa}B axis. These findings identify miR-128 as a potential therapeutic target for RDN in the treatment of hypertension, providing a novel regulatory strategy for the precision management of cardiovascular diseases.

bioinformatics↗

bioRxiv2026

Serine Stabilizes SLC7A11 and Enhances Cystine Influx to Protect Against Acute Pancreatitis

Lethal sterile inflammatory diseases are linked to amino acid metabolism, but the role of serine remains unclear. Here, we show that dysregulated serine metabolism and reduced plasma serine levels correlate with disease severity of acute pancreatitis (AP) in patients and mouse models. Elevating serine levels via exogenous serine supplementation or pancreatic phosphoglycerate dehydrogenase (PHGDH) overexpression mitigates pancreatic injury, whereas a serine deprivation diet or pancreatic PHGDH knockdown exacerbates AP. Serine prevents cell death and oxidative stress in pancreatic acinar cells, human induced pluripotent stem cells-derived pancreatic organoids and mouse pancreatic tissue. Serine enhances cysteine and glutathione biosynthesis primarily by promoting solute carrier family 7 member 11 (SLC7A11)-dependent cystine uptake rather than by serving as a direct substrate. Mechanistically, the E3 ubiquitin ligase NEDD4 mediates ubiquitination and degradation of SLC7A11, whereas serine binds to NEDD4 and thereby inhibits SLC7A11 degradation. Similarly to serine, pharmacological inhibition of NEDD4 alleviates lipid peroxidation and pancreatic injury. These findings identify serine as a critical signaling regulator of SLC7A11 stability and oxidative stress, and provides a new therapeutic strategy for AP and associated sterile inflammatory disorders. HighlightsAcute pancreatitis (AP) is linked to abnormal serine metabolism and serine depletion. Serine prevents cell death in AP acinar cells, human pancreatic organoids and mice. Serine promotes SLC7A11-dependent cystine uptake and glutathione levels in acinar cells. Serine reduces NEDD4-mediated ubiquitination of SLC7A11. In briefSerine protects against cell death and pancreatic injury in acute pancreatitis by stabilizing SLC7A11 through disruption of NEDD4-mediated ubiquitination in acinar cells.

pharmacology and toxicology↗

bioRxiv2026

A Conserved Metabolic Oxidative Axis Underlies Immune Cell Cryo-vulnerability

Immunotherapy has emerged as a transformative approach for treating cancer and other diseases, yet its widespread deployment requires effective cryopreservation strategies to enable scalable global distribution. However, many immune cell types remain acutely vulnerable to freeze-thaw stress, and the underlying mechanisms of this cryo-vulnerability are not well defined. In this study, we combined metabolic flux analysis, ROS quantification, lipidomics, and preclinical xenograft models to investigate how metabolic state influences cryopreservation outcomes. We found that immune cell activation induces a metabolic shift characterized by elevated glucose utilization and excessive ROS production, leading to profound post-thaw loss of viability and function, as demonstrated by a [~]25% survival rate in natural killer (NK) cells. Targeted pretreatments--including inhibitors of glucose metabolism, antioxidants, and suppression of lipid peroxidation--restored post-thaw recovery to nearly 90% while preserving effector activity and antitumor efficacy. Similar protective effects were observed across {beta} T cells, {gamma}{delta} T cells, and macrophages, defining a conserved metabolic-oxidative pathway of cryo-vulnerability and offering applicable strategies to enhance immune cell preservation.

immunology↗

bioRxiv2026

A role for ETV1 and endothelial cell-derived extracellular vesicle microRNAs in priming fibroblast response to vesicle-bound FGF2

Communication between various cell types following wounding is paramount for proper healing and regeneration of injured tissue. Endothelial cells and fibroblasts are critical cellular players involved in cutaneous wound repair, yet their communication mechanisms are not well understood. It has previously been shown that extracellular vesicles derived from endothelial cells (ECEVs) induce dermal fibroblasts to express a gene signature correlated with FGF2-mediated cancer associated fibroblast (CAF) activation, under the control of transcription factor ETV1. In this report, we utilize loss-of-function studies to define the mechanistic role of ETV1 in conferring this ECEV-induced transcriptomic shift and functional change in fibroblasts. Additionally, we identify highly expressed ECEV microRNAs and examine their potential contribution to the ECEV mechanism through downstream gene modulation. In summary, we describe a plausible mechanism by which both ETV1 and top ECEV microRNAs promote a genotypic and phenotypic shift in dermal fibroblasts that have taken up ECEVs.

cell biology↗

bioRxiv2026

Hidden stop codons orchestrate mRNA fate by ambushing ribosomal frameshifting associated with codon usage

In eukaryotes, the mRNA stability is generally shaped by codon usage bias, the uneven preferences for synonymous codons, in a translation-dependent manner. However, the conserved mechanism linking codon to mRNA decay remains elusive. Hidden stop codons (HSCs), defined as stop codons located in the +1 or -1 frame relative to canonical ORF, are widespread across all genomes but largely uncharacterized. Here, we demonstrate that in both fungi and human cells, HSCs play an conserved and mechanistic role in promoting mRNA decay by rapidly terminating out-of-frame translation promoted by nonoptimal codons, primarily through +1 ribosomal frameshifting. In the filamentous fungus Neurospora crassa, partially deleting HSCs via synonymous substitutions in the clock gene frequency increases mRNA stability and disrupts circadian rhythmicity. In human cells, acute depletion of translation termination factor eRF1 impairs recognition of HSCs, leading to global stabilization of mRNAs enriched in nonoptimal codons and HSCs. In both Neurospora and humans, these mRNAs are in part degraded through the NMD pathway via UPF1. Collectively, these findings suggest that in eukaryotes, HSCs serve as surveillance elements to monitor ribosomal frameshifting and out-of-translation promoted by nonoptimal codons, thereby initiating mRNA decay through NMD.

molecular biology↗

bioRxiv2025

A high-content imaging workflow to screen for molecules that reduce cellular uptake of α-synuclein preformed fibrils

A classical pathological hallmark of many neurodegenerative diseases is the formation of protein-rich aggregates and inclusions. In Parkinsons disease (PD), -synuclein (-syn) constitutes a major protein component of pathological inclusions, termed Lewy bodies. These -syn aggregates are hypothesized to spread throughout the nervous system by cell-to-cell transmission acting as templates to amplify aggregate formation. In vitro generated -syn aggregates, commonly called preformed fibrils (PFFs), have been used to investigate a number of aspects related to -syn mediated pathology across different model systems. Here we describe a semi-automated assay to screen for small molecules that interfere with the cellular uptake and accumulation of PFFs. The assay uses dopaminergic progenitor cells (DPCs), derived from human induced pluripotent stem cells (hiPSCs). In an initial screen, we tested 1520 small molecules and identified several molecules that strongly reduce intracellular PFF load in DPCs. From these hits, candidate compounds were validated in dopaminergic neurons (DNs) to demonstrate the utility of the assay. This assay provides a robust, scalable and adaptable tool to screen for molecules that affect PFF uptake in hiPSC-derived cell models. Within the scope of this screen, it led to the identification of a set of compounds with diverse annotated targets that effectively reduce the uptake of synuclein aggregates in DPCs and DNs.

neuroscience↗

bioRxiv2025

An automated workflow for quantifying the formation of synuclein aggregates in human dopaminergic neurons

Parkinsons disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (-syn) aggregates termed Lewy bodies. To model PD pathology in vitro, preformed fibrils of -syn (PFFs), which can be taken up by cells, provide a seed that drives misfolding and aggregation of endogenous -syn, with new aggregates amplifying this process. External application of PFFs to dopaminergic neurons (DNs) increases aggregate formation, marked by -syn phosphorylation at serine 129 (pS129-syn), a pathological PD marker. Building on this, we developed an automated synuclein seeding assay to quantify new -syn aggregates in iPSC-derived DNs. Using pS129-syn as a readout, we show that PFFs elicit a time- and dose-dependent increase in pS129-syn aggregates. Our high-throughput assay further revealed that aggregate formation depends on endogenous -syn levels. Treatment with PFFs produced a greater increase in pS129-syn aggregates in iPSC DNs derived from a PD patient with a triplication in the SNCA gene, which encodes the -syn protein and which elevates total -syn levels, relative to DNs from an isogenic iPSC line from the same individual, in which the SNCA gene mutation had been corrected by CRISPR/Cas9. In contrast, no pS129-syn signal was detected in neurons in which all copies of the SNCA gene had been knocked out (KO). This high-content imaging assay for synuclein seeding offers a platform for assessing compounds and therapeutics that may impede -syn aggregate formation.

neuroscience↗