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AbstractDespite being one of the most prevalent RNA modifications,the role of N6?methyladenosine (m6A) in amyotrophic lateral sclerosis (ALS) remains ambiguous. In this investigation,we explore the contribution of genetic defects of m6A?related genes to ALS pathogenesis. We scrutinized the mutation landscape of m6A genes through a comprehensive analysis of whole?exome sequencing cohorts,encompassing 508 ALS patients and 1660 population?matched controls. Our findings reveal a noteworthy enrichment of RNA binding motif protein X?linked (RBMX) variants among ALS patients,with a significant correlation between pathogenic m6A variants and adverse clinical outcomes. Furthermore,Rbmx knockdown in NSC?34 cells overexpressing mutant TDP43Q331K results in cell death mediated by an augmented p53 response. Similarly,RBMX knockdown in ALS motor neurons derived from induced pluripotent stem cells (iPSCs) manifests morphological defects and activation of the p53 pathway. Transcriptional analysis using publicly available single?cell sequencing data from the primary motor cortex indicates that RBMX?regulated genes selectively influence excitatory neurons and exhibit enrichment in ALS?implicated pathways. Through integrated analyses,our study underscores the emerging roles played by RBMX in ALS,suggesting a potential nexus between the disease and dysregulated m6A?mediated mRNA metabolism. The dysregulation of m6A modification has gained recognition as a crucial factor in the development of amyotrophic lateral sclerosis (ALS). Among the m6A reader proteins,RNA binding motif protein X?linked (RBMX) stands out with a notable enrichment of variants in ALS patients,and the presence of pathogenic RBMX variants is associated with a faster disease progression. In vitro experiments have provided evidence that reducing RBMX levels can result in neuronal defects. Additionally,bioinformatic analyses have supported these findings by revealing that RBMX?associated genes specifically impact excitatory neurons. Furthermore,these genes are involved in the regulation of pathways and genes associated with neurodegeneration and RNA metabolism,underscoring the relevance of RBMX in ALS pathogenesis. View Publication

BackgroundCertain structural variants (SVs) including large-scale genetic copy number variants,as well as copy number-neutral inversions and translocations may not all be resolved by chromosome karyotype studies. The identification of genetic risk factors for Parkinson’s disease (PD) has been primarily focused on the gene-disruptive single nucleotide variants. In contrast,larger SVs,which may significantly influence human phenotypes,have been largely underexplored. Optical genomic mapping (OGM) represents a novel approach that offers greater sensitivity and resolution for detecting SVs. In this study,we used induced pluripotent stem cell (iPSC) lines of patients with PD-linked SNCA and PRKN variants as a proof of concept to (i) show the detection of pathogenic SVs in PD with OGM and (ii) provide a comprehensive screening of genetic abnormalities in iPSCs.ResultsOGM detected SNCA gene triplication and duplication in patient-derived iPSC lines,which were not identified by long-read sequencing. Additionally,various exon deletions were confirmed by OGM in the PRKN gene of iPSCs,of which exon 3–5 and exon 2 deletions were unable to phase with conventional multiplex-ligation-dependent probe amplification. In terms of chromosomal abnormalities in iPSCs,no gene fusions,no aneuploidy but two balanced inter-chromosomal translocations were detected in one line that were absent in the parental fibroblasts and not identified by routine single nucleotide variant karyotyping.ConclusionsIn summary,OGM can detect pathogenic SVs in PD-linked genes as well as reveal genomic abnormalities for iPSCs that were not identified by other techniques,which is supportive for OGM’s future use in gene discovery and iPSC line screening.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-024-10902-1. View Publication

Amyotrophic lateral sclerosis (ALS) is a major neurodegenerative disease for which there is currently no curative treatment. The blood-brain barrier (BBB),multiple physiological functions formed by mainly specialized brain microvascular endothelial cells (BMECs),serves as a gatekeeper to protect the central nervous system (CNS) from harmful molecules in the blood and aberrant immune cell infiltration. The accumulation of evidence indicating that alterations in the peripheral milieu can contribute to neurodegeneration within the CNS suggests that the BBB may be a previously overlooked factor in the pathogenesis of ALS. Animal models suggest BBB breakdown may precede neurodegeneration and link BBB alteration to the disease progression or even onset. However,the lack of a useful patient-derived model hampers understanding the pathomechanisms of BBB dysfunction and the development of BBB-targeted therapies. In this study,we differentiated BMEC-like cells from human induced pluripotent stem cells (hiPSCs) derived from ALS patients to investigate BMEC functions in ALS patients. TARDBP N345K/+ carrying patient-derived BMEC-like cells exhibited increased permeability to small molecules due to loss of tight junction in the absence of neurodegeneration or neuroinflammation,highlighting that BMEC abnormalities in ALS are not merely secondary consequences of disease progression. Furthermore,they exhibited increased expression of cell surface adhesion molecules like ICAM-1 and VCAM-1,leading to enhanced immune cell adhesion. BMEC-like cells derived from hiPSCs with other types of TARDBP gene mutations (TARDBP K263E/K263E and TARDBP G295S/G295S) introduced by genome editing technology did not show such BMEC dysfunction compared to the isogenic control. Interestingly,transactive response DNA-binding protein 43 (TDP-43) was mislocalized to cytoplasm in TARDBP N345K/+ carrying model. Wnt/?-catenin signaling was downregulated in the ALS patient (TARDBP N345K/+)-derived BMEC-like cells and its activation rescued the leaky barrier phenotype and settled down VCAM-1 expressions. These results indicate that TARDBP N345K/+ carrying model recapitulated BMEC abnormalities reported in brain samples of ALS patients. This novel patient-derived BMEC-like cell is useful for the further analysis of the involvement of vascular barrier dysfunctions in the pathogenesis of ALS and for promoting therapeutic drug discovery targeting BMEC. View Publication

Diabetic keratopathy (DK),a significant complication of diabetes,often leads to corneal damage and vision impairment. Effective models are essential for studying DK pathogenesis and evaluating potential therapeutic interventions. This study developed a novel biomimetic full-thickness corneal model for the first time,incorporating corneal epithelial cells,stromal cells,endothelial cells,and nerves to simulate DK conditions in vitro. By exposing the model to a high-glucose (HG) environment,the pathological characteristics of DK,including nerve bundle disintegration,compromised barrier integrity,increased inflammation,and oxidative stress,were successfully replicated. Transcriptomic analysis revealed that HG downregulated genes associated with axon and synapse formation while upregulating immune response and oxidative stress pathways,with C-C Motif Chemokine Ligand 5 (CCL5) identified as a key hub gene in DK pathogenesis. The therapeutic effects of Lycium barbarum glycopeptide (LBGP) were evaluated using this model and validated in db/db diabetic mice. LBGP promoted nerve regeneration,alleviated inflammation and oxidative stress in both in vitro and in vivo models. Notably,LBGP suppressed the expression of CCL5,highlighting its potential mechanism of action. This study establishes a robust biomimetic platform for investigating DK and other corneal diseases,and identifies LBGP as a promising therapeutic candidate for DK. These findings provide valuable insights into corneal disease mechanisms and pave the way for future translational research and clinical applications. Graphical abstractImage 1 Highlights•A full-thickness biomimetic corneal model containing corneal epithelium,nerves,stroma,and endothelium was constructed.•Using this model,the pathological characteristics of diabetic keratopathy were successfully replicated in vitro.•Lycium barbarum glycopeptide (LBGP) alleviated high-glucose-induced damage in vitro and in vivo models.•CCL5 plays an important role in the pathogenesis of diabetic keratopathy. View Publication

BackgroundTrichloroethylene (TCE) is a ubiquitous pollutant with potential capacity to induce congenital heart disease (CHD). However,the mechanisms underlying TCE-induced CHD are largely unraveled.MethodsWe exposed zebrafish embryos to TCE to investigate its cardiac development toxicity and related response factor through bulk RNA sequencing. We constructed transgenic fluorescent fish and employed the CRISPR/dCas9 system along with single-cell RNA sequencing to identify the genetic cause of TCE-induced CHD.ResultsWe found that early-stage exposure to TCE induced significant cardiac defects characterized by elongated SV-BA distance,thinned myocardium,and attenuated contractility. Gremlin1 encoding gene,grem1a,a putative target showing high expression at the beginning of cardiac development,was sharply down-regulated by TCE. Consistently,grem1a knockdown in zebrafish induced cardiac phenotypes generally like those of the TCE-treated group,accompanying the disarrangement of myofibril structure. Single-cell RNA-seq depicted that mitochondrial respiration in grem1a-repressed cardiomyocytes was greatly enhanced,ultimately leading to a branch from the normal trajectory of myocardial development. Accordingly,in vitro results demonstrated that GREM1 repression increased mitochondrial content,ATP production,mitochondrial reactive oxygen species,mitochondrial membrane potential,and disrupted myofibril expansion in hPSC-CMs.ConclusionsThese results suggested that TCE-induced gremlin1 repression could result in mitochondrial hyperfunction,thereby hampering cardiomyocyte development and causing cardiac defects in zebrafish embryos. This study not only provided a novel insight into the etiology for environmental stressor-caused cardiac development defects,but also offered a potential therapeutic and preventive target for TCE-induced CHD.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-025-02314-9. View Publication

Organoids,derived from human induced pluripotent stem cells (hiPSCs),are intricate three-dimensional in vitro structures that mimic many key aspects of the complex morphology and functions of in vivo organs such as the retina and heart. Traditional histological methods,while crucial,often fall short in analyzing these dynamic structures due to their inherently static and destructive nature. In this study,we leveraged the capabilities of optical coherence tomography (OCT) for rapid,non-invasive imaging of both retinal,cerebral,and cardiac organoids. Complementing this,we developed a sophisticated deep learning approach to automatically segment the organoid tissues and their internal structures,such as hollows and chambers. Utilizing this advanced imaging and analysis platform,we quantitatively assessed critical parameters,including size,area,volume,and cardiac beating,offering a comprehensive live characterization and classification of the organoids. These findings provide profound insights into the differentiation and developmental processes of organoids,positioning quantitative OCT imaging as a potentially transformative tool for future organoid research. View Publication

PIEZO1 is critical to numerous physiological processes,transducing diverse mechanical stimuli into electrical and chemical signals. Recent studies underscore the importance of visualizing endogenous PIEZO1 activity and localization to understand its functional roles. To enable physiologically and clinically relevant studies on human PIEZO1,we genetically engineered human induced pluripotent stem cells (hiPSCs) to express a HaloTag fused to endogenous PIEZO1. Combined with advanced imaging,our chemogenetic platform allows precise visualization of PIEZO1 localization dynamics in various cell types. Furthermore,the PIEZO1-HaloTag hiPSC technology facilitates the non-invasive monitoring of channel activity across diverse cell types using Ca2+-sensitive HaloTag ligands,achieving temporal resolution approaching that of patch clamp electrophysiology. Finally,we use lightsheet microscopy on hiPSC-derived neural organoids to achieve molecular scale imaging of PIEZO1 in three-dimensional tissue. Our advances establish a platform for studying PIEZO1 mechanotransduction in human systems,with potential for elucidating disease mechanisms and targeted drug screening. PIEZO1 is critical in numerous physiological processes,but monitoring its activity and localization in cells can be challenging. Here,the authors present a chemogenetic platform to visualize endogenous human PIEZO1 localization and activity in native cellular conditions,expanding the knowledge on mechanotransduction across single cells and tissue organoids. View Publication

PurposeDilated cardiomyopathy (DCM) is a prevalent form of heart failure with limited therapeutic options. This study explores a novel treatment strategy involving the delivery of exosome-derived miRNA-185-5p inhibitors encapsulated in liposomes,aiming to target cardiac tissue and alleviate myocardial apoptosis and cuproptosis in DCM.MethodsThe miRNA-185-5p inhibitor,identified in our previous study and extracted from exosomes,was encapsulated in liposomes functionalized with a cardiac-targeting peptide. This system was used in both in vitro and in vivo models of DCM induced by doxorubicin (DOX). We evaluated the effects of this treatment on cardiac function,apoptosis,cuproptosis,oxidative stress,and fibrosis using echocardiography,histological analysis,Western blotting,and biochemical assays.ResultsIn vitro experiments demonstrated that the Lipo@miR-185-5p inhibitor markedly attenuated apoptosis and cuproptosis in H9C2 cells and iPSC-derived cardiomyocytes,with a 42.6% reduction in apoptotic cell rates and over 50% downregulation of cuproptosis-related markers (both P < 0.01). In vivo,the targeted liposomal formulation significantly improved cardiac function in DOX-induced DCM mice,as evidenced by a 27.3% increase in left ventricular ejection fraction (LVEF) and a 36.5% reduction in myocardial fibrosis area (P < 0.01),along with enhanced survival. These findings underscore the therapeutic potential of this targeted delivery strategy for the treatment of dilated cardiomyopathy.ConclusionLipo@miR-185-5p inhibitor,utilizing exosome-derived miRNA and targeted liposomal delivery,effectively alleviates DCM-induced myocardial dysfunction. This approach represents a promising therapeutic strategy for cardiovascular diseases by targeting specific molecular mechanisms involved in heart failure. View Publication

SummaryApolipoprotein E4 (APOE4) is the leading genetic risk factor for Alzheimer’s disease. While most studies examine the role of APOE4 in aging,APOE4 causes persistent changes in brain structure as early as infancy and is associated with altered functional connectivity that extends beyond adolescence. Here,we used human induced pluripotent stem cell-derived cortical and ganglionic eminence organoids (COs and GEOs) to examine APOE4’s influence during the development of cortical excitatory and inhibitory neurons. We show that APOE4 reduces cortical neurons and increases glia by promoting gliogenic transcriptional programs. In contrast,APOE4 increases proliferation and differentiation of GABAergic progenitors resulting in early and persistent increases in GABAergic neurons. Multi-electrode array recordings in assembloids revealed that APOE4 disrupts neural network function resulting in heightened excitability and synchronicity. Together,our data provide new insights on how APOE4 influences cortical neurodevelopmental processes and the establishment of functional networks. Highlights•APOE4 accelerates differentiation and maturation at developmental time points•APOE4 results in a loss of cortical neurons and increase in astrocytes and outer radial glia•APOE4 enhances proliferation,differentiation,and maturation of GABAergic neurons•APOE4 alters GABA-related genes,linked to increased GABA response and synchronicity Meyer-Acosta et al. reveal that Alzheimer’s disease genetic risk factor APOE4 decreases cortical neurons and increases glia in cortical organoids and enhances GABAergic neuron maturation in ganglionic eminence organoids derived from iPSCs. These cellular changes result in heightened excitability and synchronicity in APOE4-fused organoids,providing insight into the cellular processes that may underlie altered brain structure observed in APOE4 infants. View Publication

Alternative splicing is a major contributor of transcriptomic complexity,but the extent to which transcript isoforms are translated into stable,functional protein isoforms is unclear. Furthermore,detection of relatively scarce isoform-specific peptides is challenging,with many protein isoforms remaining uncharted due to technical limitations. Recently,a family of advanced targeted MS strategies,termed internal standard parallel reaction monitoring (IS-PRM),have demonstrated multiplexed,sensitive detection of pre-defined peptides of interest. Such approaches have not yet been used to confirm existence of novel peptides. Here,we present a targeted proteogenomic approach that leverages sample-matched long-read RNA sequencing (LR RNAseq) data to predict potential protein isoforms with prior transcript evidence. Predicted tryptic isoform-specific peptides,which are specific to individual gene product isoforms,serve as “triggers” and “targets” in the IS-PRM method,Tomahto. Using the model human stem cell line WTC11,LR RNAseq data were generated and used to inform the generation of synthetic standards for 192 isoform-specific peptides (114 isoforms from 55 genes). These synthetic “trigger” peptides were labeled with super heavy tandem mass tags (TMT) and spiked into TMT-labeled WTC11 tryptic digest,predicted to contain corresponding endogenous “target” peptides. Compared to DDA mode,Tomahto increased detectability of isoforms by 3.6-fold,resulting in the identification of five previously unannotated isoforms. Our method detected protein isoform expression for 43 out of 55 genes corresponding to 54 resolved isoforms. This LR RNA seq-informed Tomahto targeted approach,called LRP-IS-PRM,is a new modality for generating protein-level evidence of alternative isoforms – a critical first step in designing functional studies and eventually clinical assays. View Publication

Background: Effective molecular diagnosis of congenital diseases hinges on comprehensive genomic analysis,traditionally reliant on various methodologies specific to each variant type-whole exome or genome sequencing for single nucleotide variants (SNVs),array CGH for copy-number variants (CNVs),and microscopy for structural variants (SVs). Methods: We introduce a novel,integrative approach combining exome sequencing with chromosome conformation capture,termed Exo-C. This method enables the concurrent identification of SNVs in clinically relevant genes and SVs across the genome and allows analysis of heterozygous and mosaic carriers. Enhanced with targeted long-read sequencing,Exo-C evolves into a cost-efficient solution capable of resolving complex SVs at base-pair accuracy. Results: Applied to 66 human samples Exo-C achieved 100% recall and 73% precision in detecting chromosomal translocations and SNVs. We further benchmarked its performance for inversions and CNVs and demonstrated its utility in detecting mosaic SVs and resolving diagnostically challenging cases. Conclusions: Through several case studies,we demonstrate how Exo-C's multifaceted application can effectively uncover diverse causative variants and elucidate disease mechanisms in patients with rare disorders. View Publication

Peptide-based supramolecular nanostructures offer a versatile platform with substantial promise for clinical translation in regenerative medicine. These systems allow for the incorporation of biologically active sequences and can be engineered to modulate tissue-specific parameters such as stiffness,diffusivity,and biodegradability. We developed here a bioactive supramolecular nanostructure containing a peptide designed based on glial cell-derived neurotrophic factor. These nanostructures form scaffolds that mimic important trophic effects provided by this growth factor on iPSC-derived human dopaminergic neurons. Our in vitro data show that the nanostructures promote cell viability,confer neuroprotection against 6-hydroxydopamine toxicity,enhance neuronal morphology,facilitate electrophysiological maturation,and induce genes involved in neuronal survival. We also found that the scaffold promoted axonal extension in midbrain human organoids. These findings suggest that the supramolecular system could be useful to improve outcomes in cell-based therapies for Parkinson’s disease,where progressive dopaminergic degeneration is a hallmark. View Publication