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Central nervous system (CNS) cancers are responsible for high rates of morbidity and mortality worldwide. Malignant CNS tumors such as adult Glioblastoma (GBM) and pediatric embryonal CNS tumors such as medulloblastoma (MED) and atypical teratoid rhabdoid tumors (ATRT) present relevant therapeutic challenges due to the lack of response to classic treatment regimens with radio and chemotherapy. Recent findings on the Zika virus’ (ZIKV) ability to infect and kill CNS neoplastic cells draw attention to the virus’ oncolytic potential. Studies demonstrating the safety of using ZIKV for treating malignant CNS tumors,enabling the translation of this approach to clinical trials,are scarce in the literature. Here we developed a co-culture model of mature human cerebral organoids assembled with GBM,MED or ATRT tumor cells and used these assembloids to test ZIKV oncolytic effect,replication potential and preferential targeting between normal and cancer cells. Our hybrid co-culture models allowed the tracking of tumor cell growth and invasion in cerebral organoids. ZIKV replication and ensuing accumulation in the culture medium was higher in organoids co-cultured with tumor cells than in isolated control organoids without tumor cells. ZIKV infection led to a significant reduction in tumor cell proportion in organoids with GBM and MED cells,but not with ATRT. Tumoroids (3D cultures of tumor cells alone) were efficiently infected by ZIKV. Interestingly,ZIKV rapidly replicated in GBM,MED,and ATRT tumoroids reaching significantly higher viral RNA accumulation levels than co-cultures. Moreover,ZIKV infection reduced viable cells number in MED and ATRT tumoroids but not in GBM tumoroids. Altogether,our findings indicate that ZIKV has greater replication rates in aggressive CNS tumor cells than in normal human cells comprising cerebral organoids. However,such higher ZIKV replication in tumor cells does not necessarily parallels oncolytic effects,suggesting cellular intrinsic and extrinsic factors mediating tumor cell death by ZIKV. 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

BackgroundAlzheimer’s disease (AD) is characterized by progressive amyloid beta (A?) deposition in the brain,with eventual widespread neurodegeneration. While the cell-specific molecular signature of end-stage AD is reasonably well characterized through autopsy material,less is known about the molecular pathways in the human brain involved in the earliest exposure to A?. Human model systems that not only replicate the pathological features of AD but also the transcriptional landscape in neurons,astrocytes and microglia are crucial for understanding disease mechanisms and for identifying novel therapeutic targets.MethodsIn this study,we used a human 3D iPSC-derived neurosphere model to explore how resident neurons,microglia and astrocytes and their interplay are modified by chronic amyloidosis induced over 3–5 weeks by supplementing media with synthetic A?1 -?42 oligomers. Neurospheres under chronic A? exposure were grown with or without microglia to investigate the functional roles of microglia. Neuronal activity and oxidative stress were monitored using genetically encoded indicators,including GCaMP6f and roGFP1,respectively. Single nuclei RNA sequencing (snRNA-seq) was performed to profile A? and microglia driven transcriptional changes in neurons and astrocytes,providing a comprehensive analysis of cellular responses.ResultsMicroglia efficiently phagocytosed A? inside neurospheres and significantly reduced neurotoxicity,mitigating amyloidosis-induced oxidative stress and neurodegeneration following different exposure times to A?. The neuroprotective effects conferred by the presence of microglia was associated with unique gene expression profiles in astrocytes and neurons,including several known AD-associated genes such as APOE. These findings reveal how microglia can directly alter the molecular landscape of AD.ConclusionsOur human 3D neurosphere culture system with chronic A? exposure reveals how microglia may be essential for the cellular and transcriptional responses in AD pathogenesis. Microglia are not only neuroprotective in neurospheres but also act as key drivers of A?-dependent APOE expression suggesting critical roles for microglia in regulating APOE in the AD brain. This novel,well characterized,functional in vitro platform offers unique opportunities to study the roles and responses of microglia to A? modelling key aspects of human AD. This tool will help identify new therapeutic targets,accelerating the transition from discovery to clinical applications.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03433-3. Highlights Well-characterized functional human iPSC-derived 3D neurospheres (hiNS) consisting of neurons and astrocytes can be supplemented with microglia/macrophages (hiMG).Chronic amyloidosis in the presence of hiMG recapitulate key features and gene expression profiles of AD.hiMG within the model phagocytose A? and mitigate A?-induced neurotoxicity,reducing oxidative stress and neuronal damagehiMG are essential for A? to upregulate AD-like gene expression signatures in astrocytes.Immunohistochemical analysis reveals hiMG-dependent colocalization of A? and APOE. Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03433-3. 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

Viruses depend on host metabolic pathways and flaviviruses are specifically linked to lipid metabolism. During dengue virus infection lipid droplets are degraded to fuel replication and Zika virus (ZIKV) infection depends on triglyceride biosynthesis. Here,we systematically investigated the neutral lipid–synthesizing enzymes diacylglycerol O-acyltransferases (DGAT) and the sterol O-acyltransferase (SOAT) 1 in orthoflavivirus infection. Downregulation of DGAT1 and SOAT1 compromises ZIKV infection in hepatoma cells but only SOAT1 and not DGAT inhibitor treatment reduces ZIKV infection. DGAT1 interacts with the ZIKV capsid protein,indicating that protein interaction might be required for ZIKV replication. Importantly,inhibition of SOAT1 severely impairs ZIKV infection in neural cell culture models and cerebral organoids. SOAT1 inhibitor treatment decreases extracellular viral RNA and E protein level and lowers the specific infectivity of virions,indicating that ZIKV morphogenesis is compromised,likely due to accumulation of free cholesterol. Our findings provide insights into the importance of cholesterol and cholesterol ester balance for efficient ZIKV replication and implicate SOAT1 as an antiviral target. Exploring the role of neutral lipid-synthesizing enzymes in Zika virus infection using different cell culture models,inhibition of cholesterol esterification is found to impair ZIKV morphogenesis. View Publication

ABSTRACTExtracellular vesicles (EVs) and secretory factors play crucial roles in intercellular communication,but the molecular mechanisms and dynamics governing their interplay in human pluripotent stem cells (hPSCs) are poorly understood. Here,we demonstrate that hPSC?secreted milk fat globule?EGF factor 8 (MFGE?8) is the principal corona protein at the periphery of EVs,playing an essential role in controlling hPSC stemness. MFGE?8 depletion reduced EV?mediated self?renewal and survival in hPSC cultures. MFGE?8 in the EV corona bound to integrin ?v?5 expressed in the peripheral zone of hPSC colonies. It activated cyclin D1 and dynamin?1 via the AKT/GSK3? axis,promoting the growth of hPSCs and facilitating the endocytosis of EVs. Internalization of EVs alleviated oxidative stress and cell death by transporting redox and stress response proteins that increased GSH levels. Our findings demonstrate the critical role of the extracellular association of MFGE?8 and EVs in modulating the self?renewal and survival of hPSCs. View Publication

Monkeypox virus (MPV) is known to inflict injuries and,in some cases,lead to fatalities in humans. However,the underlying mechanisms responsible for its pathogenicity remain poorly understood. We investigated functions of MPV core proteins,H3L,A35R,A29L,and I1L,and discovered that H3L induced transcriptional perturbations and injuries. We substantiated that H3L upregulated IL1A expression. IL1A,in consequence,caused cellular injuries,and this detrimental effect was mitigated when countered with IL1A blockage. We also observed that H3L significantly perturbed the transcriptions of genes in cardiac system. Mechanistically,H3L occupied the promoters of genes governing cellular injury,leading to alterations in the binding patterns of H3K27me3 and H3K4me3 histone marks,ultimately resulting in expression perturbations. In vivo and in vitro models confirmed that H3L induced transcriptional disturbances and cardiac dysfunction,which were ameliorated when IL1A was blocked or repressed. Our study provides valuable insights into comprehensive understanding of MPV pathogenicity,highlights the significant roles of H3L in inducing injuries,and potentially paves the way for the development of therapeutic strategies targeting IL1A. View Publication

SummaryProtein turnover is a critical component of gene expression regulation and cellular homeostasis,yet methods for measuring turnover rates that are scalable and applicable to different models are still needed. We introduce an improved D2O (heavy water) labeling strategy to investigate the landscape of protein turnover in cell culture,with accurate calibration of per-residue deuterium incorporation in multiple cell types. Applying this method,we mapped the proteome-wide turnover landscape of pluripotent and differentiating human induced pluripotent stem cells (hiPSCs). Our analysis highlights the role of APC/C (anaphase-promoting complex/cyclosome) and SPOP (speckle-type POZ protein) degrons in the fast turnover of cell-cycle-related and DNA-binding hiPSC proteins. Upon pluripotency exit,many short-lived hiPSC proteins are depleted,while RNA-binding and -splicing proteins become hyperdynamic. The ability to identify fast-turnover proteins also facilitates secretome profiling,as exemplified in hiPSC-cardiomyocyte and primary human cardiac fibroblast analysis. This method is broadly applicable to protein turnover studies in primary,pluripotent,and transformed cells. Graphical abstract Highlights•D2O labeling measures protein turnover in primary,pluripotent,and transformed cells•D2O incorporates into multiple amino acids in vitro,including Ala,Glu,Asp,and Pro•Protein turnover analysis shows hiPSC differentiation alters fast-turnover proteins•We show application to secretome analysis in human cardiac myocytes and fibroblasts MotivationDynamic stable isotope labeling by amino acids in cell culture coupled with mass spectrometry is commonly used to measure protein turnover in cell culture but requires altering culture medium composition and may not label some peptides. We describe a simple and convenient alternative for measuring protein turnover kinetics in cultured cells by adding low-volume D2O (heavy water) to standard tissue culture media. Addressing a critical gap,we determined the number of deuterium-accessible atoms on all 20 proteinogenic amino acids across multiple cell types. This allows accurate interpretation of D2O-labeled mass spectra to measure protein turnover kinetics and secretome flux on a proteome scale. Alamillo et al. present a D2O labeling mass spectrometry method to measure protein turnover rates that is compatible with multiple cell cultures and medium formulations. The method reveals a parsimonious protein turnover landscape in human induced pluripotent stem cells and identifies hyperdynamic proteins that are unique to self-renewal states. View Publication

To build in vitro tissues for therapeutic applications,it is essential to replicate the spatial distribution of cells that occurs during morphogenesis in vivo. However,it remains technically challenging to simultaneously regulate the geometric alignment and aggregation of cells during tissue fabrication. Here,we introduce the acoustofluidic bioassembly induced morphogenesis,which is the combination of precise arrangement of cells by the mechanical forces produced by acoustofluidic cues,and the morphological and functional changes of cells in the following in vitro and in vivo cultures. The acoustofluidic bioassembly can be used to create tissues with regulated nano-,micro-,and macro-structures. We demonstrate that the neuromuscular tissue fabricated with the acoustofluidic bioassembly exhibits enhanced contraction dynamics,electrophysiology,and therapeutic efficacy. The potential of the acoustofluidic bioassembly as an in situ application is demonstrated by fabricating artificial tissues at the defect sites of living tissues. The acoustofluidic bioassembly induced morphogenesis can provide a pioneering platform to fabricate tissues for biomedical applications. Tissue engineering is essential for drug screening and regenerative medicine. Here,authors developed an acoustofluidic method that can induce morphogenesis of therapeutic tissues at varied dimensions/scales. View Publication