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IntroductionCRISPR/Cas9-edited induced pluripotent stem cells (iPSCs) are valuable research models for mechanistic studies. However,gene conversion between a gene-pseudogene pair that share high sequence identity and form direct repeats in proximity on the same chromosome can interfere with the precision of gene editing. Mutations in the human beta-glucocerebrosidase gene (GBA1) are associated with Gaucher disease,Parkinson’s disease,and Lewy body dementia. During the creation of a GBA1 KO iPSC line,we detected about 70% gene conversion from its pseudogene GBAP1. These events maintained the reading frame and resulted from GBA1-specific cleavage by CRISPR/Cas9,without disrupting the GBA1 gene.MethodTo increase the percentage of alleles with out-of-frame indels for triggering nonsense-mediated decay of the GBA1 mRNA,we supplied the cells with two single-stranded oligodeoxynucleotide (ssODN) donors as homology-directed repair (HDR) templates.ResultsWe demonstrate that HDR using the ssODN templates effectively competes with gene conversion and enabled biallelic KO clone isolation,whereas the nonallelic homologous recombination (NAHR)-based deletion rate remained the same.DiscussionHere,we report a generalizable method to direct cellular DNA repair of double strand breaks at a target gene towards the HDR pathway using exogenous ssODN templates,allowing specific editing of one gene in a gene-pseudogene pair without disturbing the other. View Publication

Prenatal immune challenges pose significant risks to human embryonic brain and eye development. However,our knowledge about the safe usage of anti-inflammatory drugs during pregnancy is still limited. While human induced pluripotent stem cells (hIPSC)-derived brain organoid models have started to explore functional consequences upon viral stimulation,these models commonly lack microglia,which are susceptible to and promote inflammation. Furthermore,microglia are actively involved in neuronal development. Here,we generate hIPSC-derived microglia precursor cells and assemble them into retinal organoids. Once the outer plexiform layer forms,these hIPSC-derived microglia (iMG) fully integrate into the retinal organoids. Since the ganglion cell survival declines by this time in 3D-retinal organoids,we adapted the model into 2D and identify that the improved ganglion cell number significantly decreases only with iMG presence. In parallel,we applied the immunostimulant POLY(I:C) to mimic a fetal viral infection. While POLY(I:C) exposure alters the iMG phenotype,it does not hinder their interaction with ganglion cells. Furthermore,iMG significantly enhance the supernatant’s inflammatory secretome and increase retinal cell proliferation. Simultaneous exposure with the non-steroidal anti-inflammatory drug (NSAID) ibuprofen dampens POLY(I:C)-mediated changes of the iMG phenotype and ameliorates cell proliferation. Remarkably,while POLY(I:C) disrupts neuronal calcium dynamics independent of iMG,ibuprofen rescues this effect only if iMG are present. Mechanistically,ibuprofen targets the enzymes cyclooxygenase 1 and 2 (COX1/PTGS1 and COX2/PTGS2) simultaneously,from which iMG mainly express COX1. Selective COX1 blockage fails to restore the calcium peak amplitude upon POLY(I:C) stimulation,suggesting ibuprofen’s beneficial effect depends on the presence and interplay of COX1 and COX2. These findings underscore the importance of microglia in the context of prenatal immune challenges and provide insight into the mechanisms by which ibuprofen exerts its protective effects during embryonic development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03366-x. View Publication

ObjectivesMutations in Tissue Inhibitor of Metalloproteinases 3 (TIMP3) cause Sorsby's Fundus Dystrophy (SFD),a dominantly inherited,rare form of macular degeneration that results in vision loss. TIMP3 is synthesized primarily by retinal pigment epithelial (RPE) cells,which constitute the outer blood-retinal barrier. One major function of RPE is the synthesis and transport of vital nutrients,such as glucose,to the retina. Recently,metabolic dysfunction in RPE cells has emerged as an important contributing factor in retinal degenerations. We set out to determine if RPE metabolic dysfunction was contributing to SFD pathogenesis.MethodsQuantitative proteomics was conducted on RPE of mice expressing the S179C variant of TIMP3,known to be causative of SFD in humans. Proteins found to be differentially expressed (P < 0.05) were analyzed using statistical overrepresentation analysis to determine enriched pathways,processes,and protein classes using g:profiler and PANTHER Gene Ontology. We examined the effects of mutant TIMP3 on RPE metabolism using human ARPE-19 cells expressing mutant S179C TIMP3 and patient-derived induced pluripotent stem cell-derived RPE (iRPE) carrying the S204C TIMP3 mutation. RPE metabolism was directly probed using isotopic tracing coupled with GC/MS analysis. Steady state [U–13C6] glucose isotopic tracing was preliminarily conducted on S179C ARPE-19 followed by [U–13C6] glucose and [U–13C5] glutamine isotopic tracing in SFD iRPE cells.ResultsQuantitative proteomics and enrichment analysis conducted on RPE of mice expressing mutant S179C TIMP3 identified differentially expressed proteins that were enriched for metabolism-related pathways and processes. Notably these results highlighted dysregulated glycolysis and glucose metabolism. Stable isotope tracing experiments with [U–13C6] glucose demonstrated enhanced glucose utilization and glycolytic activity in S179C TIMP3 APRE-19 cells. Similarly,[U–13C6] glucose tracing in SFD iRPE revealed increased glucose contribution to glycolysis and the TCA cycle. Additionally,[U–13C5] glutamine tracing found evidence of altered malic enzyme activity.ConclusionsThis study provides important information on the dysregulation of RPE glucose metabolism in SFD and implicates a potential commonality with other retinal degenerative diseases,emphasizing RPE cellular metabolism as a therapeutic target. Highlights•SFD mice display alterations in proteins associated with metabolism.•SFD RPE cells have increased glycolytic activity and glucose contribution to the TCA cycle.•Glutamine contribution to energy metabolism is unaltered in SFD RPE cells however there is reduced malic enzyme activity.•SFD RPE cells display metabolic dysfunction potentially implicating metabolism as a viable therapeutic target. View Publication

The reconstruction of damaged neural circuits is critical for neurological repair after brain injury. Classical brain-computer interfaces (BCIs) allow direct communication between the brain and external controllers to compensate for lost functions. Importantly,there is increasing potential for generalized BCIs to input information into the brains to restore damage,but their effectiveness is limited when a large injured cavity is caused. Notably,it might be overcome by transplantation of brain organoids into the damaged region. Here,we construct innovative BCIs mediated by implantable organoids,coined as organoid-brain-computer interfaces (OBCIs). We assess the prolonged safety and feasibility of the OBCIs,and explore neuroregulatory strategies. OBCI stimulation promotes progressive differentiation of grafts and enhances structural-functional connections within organoids and the host brain,promising to repair the damaged brain via regenerating and regulating,potentially directing neurons to preselected targets and recovering functional neural networks in the future. Damaged neural circuits could be improved by generalized BCIs via inputting information into the brains,which is restricted when a large injured cavity caused. Here,the authors construct BCIs mediated by organoid grafts to repair the damaged brain View Publication

Mutations in GBA1 encoding the lysosomal enzyme ?-glucocerebrosidase (GCase) are among the most prevalent genetic susceptibility factors for Parkinson’s disease (PD),with 10–30% of carriers developing the disease. To identify genetic modifiers contributing to the incomplete penetrance,we examined the effect of 1634 human transcription factors (TFs) on GCase activity in lysates of an engineered human glioblastoma line homozygous for the pathogenic GBA1 L444P variant. Using an arrayed CRISPR activation library,we uncovered 11 TFs as regulators of GCase activity. Among these,activation of MITF and TFEC increased lysosomal GCase activity in live cells,while activation of ONECUT2 and USF2 decreased it. While MITF,TFEC,and USF2 affected GBA1 transcription,ONECUT2 might control GCase trafficking. The effects of MITF,TFEC,and USF2 on lysosomal GCase activity were reproducible in iPSC-derived neurons from PD patients. Our study provides a systematic approach to identifying modulators of GCase activity and deepens our understanding of the mechanisms regulating GCase. View Publication

Genetic and experimental evidence suggests that Alzheimer’s disease (AD) risk alleles and genes may influence disease susceptibility by altering the transcriptional and cellular responses of macrophages,including microglia,to damage of lipid-rich tissues like the brain. Recently,sc/nRNA sequencing studies identified similar transcriptional activation states in subpopulations of macrophages in aging and degenerating brains and in other diseased lipid-rich tissues. We collectively refer to these subpopulations of microglia and peripheral macrophages as DLAMs. Using macrophage sc/nRNA-seq data from healthy and diseased human and mouse lipid-rich tissues,we reconstructed gene regulatory networks and identified 11 strong candidate transcriptional regulators of the DLAM response across species. Loss or reduction of two of these transcription factors,BHLHE40/41,in iPSC-derived microglia and human THP-1 macrophages as well as loss of Bhlhe40/41 in mouse microglia,resulted in increased expression of DLAM genes involved in cholesterol clearance and lysosomal processing,increased cholesterol efflux and storage,and increased lysosomal mass and degradative capacity. These findings provide targets for therapeutic modulation of macrophage/microglial function in AD and other disorders affecting lipid-rich tissues. Factors regulating lipid and lysosomal clearance in microglia and peripheral macrophage are not known. Here,authors nominate and validate transcription factors BHLHE40 and BHLHE41 as regulators of these processes in health and disease. View Publication

ObjectiveThyroid hormone (TH) action is mediated by thyroid hormone receptor (THR) isoforms. While THR?1 is likely the main isoform expressed in liver,its role in human hepatocytes is not fully understood.MethodsTo elucidate the role of THR?1 action in human hepatocytes we used CRISPR/Cas9 editing to knock out THR?1 in induced pluripotent stem cells (iPSC). Following directed differentiation to the hepatic lineage,iPSC-derived hepatocytes were then interrogated to determine the role of THR?1 in ligand-independent and -dependent functions.ResultsWe found that the loss of THR?1 promoted alterations in proliferation rate and metabolic pathways regulated by T3,including gluconeogenesis,lipid oxidation,fatty acid synthesis,and fatty acid uptake. We observed that key genes involved in liver metabolism are regulated through both T3 ligand-dependent and -independent THR?1 signaling mechanisms. Finally,we demonstrate that following THR?1 knockout,several key metabolic genes remain T3 responsive suggesting they are THR? targets.ConclusionsThese results highlight that iPSC-derived hepatocytes are an effective platform to study mechanisms regulating TH signaling in human hepatocytes. Graphical abstractImage 1 Highlights•THR?1 is essential for T3 effects in human iPSC-derived hepatocytes (iHEPs).•THR?1 knockout reduces iPSC and progenitor cell proliferative capacity.•T3 regulates key genes involved in lipid and carbohydrate metabolism through THR?1.•THR?1 plays a strong ligand-independent role. View Publication

Backgroundc-Jun is a key regulator of gene expression. Through the formation of homo- or heterodimers,c-JUN binds to DNA and regulates gene transcription. While c-Jun plays a crucial role in embryonic development,its impact on nervous system development in higher mammals,especially for some deep structures,for example,thalamus in diencephalon,remains unclear.MethodsTo investigate the influence of c-JUN on early nervous system development,c-Jun knockout (KO) mice and c-JUN KO H1 embryonic stem cells (ESCs)-derived neural progenitor cells (NPCs),cerebral organoids (COs),and thalamus organoids (ThOs) models were used. We detected the dysplasia via histological examination and immunofluorescence staining,omics analysis,and loss/gain of function analysis.ResultsAt embryonic day 14.5,c-Jun knockout (KO) mice exhibited sparseness of fibers in the brain ventricular parenchyma and malformation of the thalamus in the diencephalon. The absence of c-JUN accelerated the induction of NPCs but impaired the extension of fibers in human neuronal cultures. COs lacking c-JUN displayed a robust PAX6+/NESTIN+ exterior layer but lacked a fibers-connected core. Moreover,the subcortex-like areas exhibited defective thalamus characteristics with transcription factor 7 like 2-positive cells. Notably,in guided ThOs,c-JUN KO led to inadequate thalamus patterning with sparse internal nerve fibers. Chromatin accessibility analysis confirmed a less accessible chromatin state in genes related to the thalamus. Overexpression of c-JUN rescued these defects. RNA-seq identified 18 significantly down-regulated genes including RSPO2,WNT8B,MXRA5,HSPG2 and PLAGL1 while 24 genes including MSX1,CYP1B1,LMX1B,NQO1 and COL2A1 were significantly up-regulated.ConclusionOur findings from in vivo and in vitro experiments indicate that c-JUN depletion impedes the extension of nerve fibers and renders the thalamus susceptible to dysplasia during early mouse embryonic development and human ThO patterning. Our work provides evidence for the first time that c-JUN is a key transcription regulator that play important roles in the thalamus/diencephalon development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-024-01303-8. View Publication

Febrile seizures (FS) are a common childhood neurological condition triggered by fever in children without prior neurological disorders. While generally benign,some individuals,particularly those with complex FS or genetic predispositions,may develop epilepsy or other neurological comorbidities. The mechanisms underlying this transition remain unclear. Mutations in SCN1A,encoding the NaV1.1 sodium channel ?-subunit,have been linked to several epilepsy syndromes associated with FS. This study examines phenotypic variability in individuals carrying the same SCN1A c.434T?>?C mutation,using induced pluripotent stem cell (iPSC)-derived neurons from two siblings with FS. Despite sharing the mutation,only the older sibling developed temporal lobe epilepsy (TLE). Transcriptomic analysis revealed downregulation of GABAergic pathway genes in both siblings’ neurons,aligning with SCN1A-associated epilepsy. However,neurons from the sibling with TLE exhibited additional abnormalities,including altered AMPA receptor subunit composition,changes in GABAA receptor subunits and chloride cotransporters expression,and reduced brain-derived neurotrophic factor (BDNF) levels,indicative of developmental immaturity. Voltage-clamp recordings confirmed impaired GABAergic and AMPA receptor-mediated synaptic activity. These findings suggest that combined GABAergic dysfunction,aberrant AMPA receptor composition,and reduced BDNF signaling contribute to the more severe phenotype and increased epilepsy susceptibility.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-09208-3. View Publication

Genome editing is poised to revolutionize treatment of genetic diseases,but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons,which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this,we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage,compared to just days in isogenic iPSCs. Furthermore,Cas9-treated neurons upregulated unexpected DNA repair genes,including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells,we uncovered unforeseen challenges and opportunities for precise therapeutic editing. View Publication

Peripheral nerve and large-scale muscle injuries result in significant disability,necessitating the development of biomaterials that can restore functional deficits by promoting tissue regrowth in an electroactive environment. Among these materials,graphene is favored for its high conductivity,but its low bioactivity requires enhancement through biomimetic components. In this study,we extrusion printed graphene-poly(lactide-co-glycolide) (graphene) lattice scaffolds,aiming to increase bioactivity by incorporating decellularized extracellular matrix (dECM) derived from mouse pup skeletal muscle. We first evaluated these scaffolds using human-induced pluripotent stem cell (hiPSC)-derived motor neurons co-cultured with supportive glia,observing significant improvements in axon outgrowth. Next,we tested the scaffolds with C2C12 mouse and human primary myoblasts,finding no significant differences in myotube formation between dECM-graphene and graphene scaffolds. Finally,using a more complex hiPSC-derived 3D motor neuron spheroid model co-cultured with human myoblasts,we demonstrated that dECM-graphene scaffolds significantly improved axonal expansion towards peripheral myoblasts and increased axonal network density compared to graphene-only scaffolds. Features of early neuromuscular junction formation were identified near neuromuscular interfaces in both scaffold types. These findings suggest that dECM-graphene scaffolds are promising candidates for enhancing neuromuscular regeneration,offering robust support for the growth and development of diverse neuromuscular tissues. View Publication

Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations are situated in the last exon and we previously demonstrated escape from nonsense-mediated decay by detecting mutant ADNP mRNA in patient blood. In this study,wild-type and ADNP mutants are investigated at the protein level and therefore optimal detection of the protein is required. Detection of ADNP by means of western blotting has been ambiguous with reported antibodies resulting in non-specific bands without unique ADNP signal. Validation of an N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials,resulting in a unique band signal around 150 kDa for ADNP,above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. Our antibody panel was subsequently tested by immunoblotting,comparing parental and homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines and showed disappearance of the 150 kDa signal,indicative for intact ADNP. By means of both a GFPSpark and Flag-tag N-terminally fused to a human ADNP expression vector,we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. Furthermore,we were also able to visualize endogenous ADNP with our C-terminal antibody panel in heterozygous cell lines carrying ADNP patient mutations,while the truncated ADNP mutants could only be detected with epitope-tag-specific antibodies,suggesting that addition of an epitope-tag possibly helps stabilizing the protein. However,western blotting of patient-derived hiPSCs,immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detect a native mutant ADNP protein. In addition,an N-terminal immunoprecipitation-competent ADNP antibody enriched truncating mutants in overexpression lysates,whereas implementation of the same method failed to enrich a possible native mutant protein in immortalized patient-derived lymphoblastoid cell lines. This study aims to shape awareness for critical assessment of mutant ADNP protein analysis in Helsmoortel-Van der Aa syndrome. View Publication