技术资料

ABSTRACTProneural genes are conserved drivers of neurogenesis across the animal kingdom. How their functions have adapted to guide human-specific neurodevelopmental features is poorly understood. Here,we mined transcriptomic data from human fetal cortices and generated from human embryonic stem cell-derived cortical organoids (COs) to show that NEUROG1 and NEUROG2 are most highly expressed in basal neural progenitor cells,with pseudotime trajectory analyses indicating that NEUROG1-derived lineages predominate early and NEUROG2 lineages later. Using ChIP-qPCR,gene silencing and overexpression studies in COs,we show that NEUROG2 is necessary and sufficient to directly transactivate known target genes (NEUROD1,EOMES,RND2). To identify new targets,we engineered NEUROG2-mCherry knock-in human embryonic stem cells for CO generation. The mCherry-high CO cell transcriptome is enriched in extracellular matrix-associated genes,and two genes associated with human-accelerated regions: PPP1R17 and FZD8. We show that NEUROG2 binds COL1A1,COL3A1 and PPP1R17 regulatory elements,and induces their ectopic expression in COs,although NEUROG2 is not required for this expression. Neurog2 similarly induces Col3a1 and Ppp1r17 in murine P19 cells. These data are consistent with a conservation of NEUROG2 function across mammalian species. Summary: Analysis of human cortical organoids reveals that NEUROG1 lineages prevail early and NEUROG2 lineages later,and that NEUROG2 targets include COL genes and PPP1R17,a human-accelerated region-associated gene. View Publication

Lateral Meningocele Syndrome (LMS),a disorder associated with NOTCH3 pathogenic variants,presents with neurological,craniofacial and skeletal abnormalities. Mouse models of the disease exhibit osteopenia that is ameliorated by the administration of Notch3 antisense oligonucleotides (ASO) targeting either Notch3 or the Notch3 mutation. To determine the consequences of LMS pathogenic variants in human cells and whether they can be targeted by ASOs,induced pluripotent NCRM1 and NCRM5 stem (iPS) cells harboring a NOTCH36692-93insC insertion were created. Parental iPSCs,NOTCH36692-93insC and isogenic controls,free of chromosomal aberrations as determined by human CytoSNP850 array,were cultured under conditions of neural crest,mesenchymal and osteogenic cell differentiation. The expected cell phenotype was confirmed by surface markers and a decline in OCT3/4 and NANOG mRNA. NOTCH36692-93insC cells displayed enhanced expression of Notch target genes HES1,HEY1,2 and L demonstrating a NOTCH3 gain-of-function. There was enhanced osteogenesis in NOTCH36692-93insC cells as evidenced by increased mineralized nodule formation and ALPL,BGLAP and BSP expression. ASOs targeting NOTCH3 decreased both NOTCH3 wild type and NOTCH36692-93insC mutant mRNA by 40% in mesenchymal and 90% in osteogenic cells. ASOs targeting the NOTCH3 insertion decreased NOTCH36692-93insC by 70–80% in mesenchymal cells and by 45–55% in osteogenic cells and NOTCH3 mRNA by 15–30% and 20–40%,respectively. In conclusion,a NOTCH3 pathogenic variant causes a modest increase in osteoblastogenesis in human iPS cells in vitro and NOTCH3 and NOTCH3 mutant specific ASOs downregulate NOTCH3 transcripts associated with LMS. View Publication

Mutations in FUS and TARDBP cause amyotrophic lateral sclerosis (ALS),but the precise mechanisms of selective motor neuron degeneration remain unresolved. To address if pathomechanisms are shared across mutations and related to either gain- or loss-of-function,we performed single-cell RNA sequencing across isogenic induced pluripotent stem cell-derived neuron types,harbouring FUS P525L,FUS R495X,TARDBP M337V mutations or FUS knockout. Transcriptional changes were far more pronounced in motor neurons than interneurons. About 20% of uniquely dysregulated motor neuron transcripts were shared across FUS mutations,half from gain-of-function. Most indicated mitochondrial impairments,with attenuated pathways shared with mutant TARDBP M337V as well as C9orf72-ALS patient motor neurons. Mitochondrial motility was impaired in ALS motor axons,even with nuclear localized FUS mutants,demonstrating shared toxic gain-of-function mechanisms across FUS- and TARDBP-ALS,uncoupled from protein mislocalization. These early mitochondrial dysfunctions unique to motor neurons may affect survival and represent therapeutic targets in ALS. In this study,the authors performed single-cell RNA-sequencing across various isogenic mutant FUS and TDP43 neurons. Mitochondrial dysfunction emerged as pathway unique to motor neurons demonstrating shared toxic gain of-function mechanisms,uncoupled from protein mislocalization. View Publication

SummaryRibosomopathies arise from the disruptions in ribosome biogenesis within the nucleolus,which is organized via liquid-liquid phase separation (LLPS). The roles of LLPS in ribosomopathies remain poorly understood. Here,we generated human induced pluripotent stem cell (hiPSC) models of ribosomopathy caused by mutations in small nucleolar RNA (snoRNA) gene SNORD118. Mutant hiPSC-derived neural progenitor cells (NPCs) or neural crest cells (NCCs) exhibited ribosomopathy hallmark cellular defects resulting in reduced organoid growth,recapitulating developmental delay in patients. SNORD118 mutations in NPCs disrupted nucleolar morphology and LLPS properties coupled with impaired ribosome biogenesis and a translational downregulation of fibrillarin (FBL),the key LLPS effector acting via the intrinsically disordered region (IDR) motif. IDR-depleted FBL failed to rescue NPC defects,whereas a chimeric FBL with swapped IDR motif from an unrelated protein mitigated ribosomopathy and organoid growth defects. Thus,SNORD118 human iPSC models revealed aberrant phase separation and nucleolar functions as potential pathogenic mechanisms in ribosomopathies. Graphical abstract Highlights•SNORD118 mutant hiPSC-derived cells and organoids recapitulate the ribosomopathy defects•Mutations impair ribosome biogenesis and translation of phase separation effector FBL•Phase separation and nucleolar organization are defective in SNORD118 mutant cells•Impaired phase separation causes ribosomopathy and growth defects in hiPSC models Natural sciences; Biological sciences; Cell biology; Stem cell research View Publication

Haloperidol is a typical antipsychotic used to treat schizophrenia and induces dopamine D2 receptor antagonism. Long-term use of haloperidol can reduce brain size in animals and humans; however,the underlying mechanism of this effect remains unclear. Notch1 signaling regulates the development and function of the nervous system by balancing stem cell proliferation and differentiation. Therefore,we investigated the effects of long-term exposure to haloperidol on human-derived brain organoids,which served as sophisticated in vitro models of human brain development. Long-term exposure to haloperidol reduced the size of brain organoids and decreased the ventricular zone and Notch1 signaling. When propionate,which protects against haloperidol-induced toxicity,was combined with haloperidol,it rescued both the overall size of brain organoids and Notch1 expression levels. Additionally,treatment with valproic acid,a Notch1 activator,partially restored the size of brain organoids and the thickness of the ventricular layer. Taken together,these data suggest that long-term exposure to haloperidol impairs neurodevelopment via Notch1 signaling in brain organoids. These findings contribute to our understanding of antipsychotic drug safety and provide information for new neurodevelopmental toxicity assessments.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-08855-w. View Publication

Alternative splicing (AS) is a crucial mechanism contributing to proteomic diversity,which is highly regulated in tissue- and development-specific patterns. Retinal tissue exhibits one of the highest levels of AS. In particular,photoreceptors have a distinctive AS pattern involving the inclusion of microexons not found in other cell types. PROM1 whose encoded protein Prominin-1 is located in photoreceptor outer segments (OSs),undergoes exon 4 inclusion from the 12th post-conception week of human development through adulthood. Exon 4 skipping in PROM1 is associated with late-onset mild maculopathy,however its role in photoreceptor maturation and function is unknown. In this study retinal organoids,a valuable model system,were employed in combination with phosphorodiamidate morpholino oligos (PMOs) to assess the role of exon 4 AS in the development of human retina. Retinal organoids were treated with the PMOs for four weeks after which RT-PCR,western blotting and immunofluorescence analysis were performed to assess exon 4 exclusion and its impact on photoreceptors. The transcriptome of treated ROs was studied by bulk RNA-Seq. Our data demonstrate that 55% skipping of PROM1 exon 4 resulted in decreased Prominin-1 expression by 40%,abnormal accumulation of cones in the basal side of the retinal organoids as well as detectable cone photoreceptor cilium defects. Transcriptomic and western blot analyses revealed decreased expression of cone,inner segment and connecting cilium basal body markers,increased expression of genes associated with stress response and the ubiquitin-proteasome system,and downregulation of autophagy. Importantly,the use of retinal organoids provides a valuable platform to study AS and unravel disease mechanisms in a more physiologically relevant context,opening avenues for further research and potential therapeutic interventions. Together our data indicate that cones may be more sensitive to PROM1 exon 4 skipping and/or reduced Prominin-1 expression,corroborating the pathogenesis of late-onset mild maculopathy. View Publication

Several differentiation protocols have enabled the generation of intermediate mesoderm (IM)-derived cells from human pluripotent stem cells (hPSC). However,the substantial variability between existing protocols for generating IM cells compromises their efficiency,reproducibility,and overall success,potentially hindering the utility of urogenital system organoids. Here,we examined the role of high levels of Nodal signaling and BMP activity,as well as WNT signaling in the specification of IM cells derived from a UCSD167i-99-1 human induced pluripotent stem cells (hiPSC) line. We demonstrate that precise modulation of WNT and BMP signaling significantly enhances IM differentiation efficiency. Treatment of hPSC with 3 ?M CHIR99021 induced TBXT+/MIXL1+ mesoderm progenitor (MP) cells after 48 h of differentiation. Further treatment with a combination of 3 ?M CHIR99021 and 4 ng/mL BMP4 resulted in the generation of OSR1+/GATA3+/PAX2+ IM cells within a subsequent 48 h period. Molecular characterization of differentiated cells was confirmed through immunofluorescence staining and RT-qPCR. Hence,this study establishes a consistent and reproducible protocol for differentiating hiPSC into IM cells that faithfully recapitulates the molecular signatures of IM development. This protocol holds promise for improving the success of protocols designed to generate urogenital system organoids in vitro,with potential applications in regenerative medicine,drug discovery,and disease modeling. View Publication

Sustained smouldering,or low-grade activation,of myeloid cells is a common hallmark of several chronic neurological diseases,including multiple sclerosis1. Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells2. However,how these metabolic features act to perpetuate inflammation of the central nervous system is unclear. Here,using a multiomics approach,we identify a molecular signature that sustains the activation of microglia through mitochondrial complex I activity driving reverse electron transport and the production of reactive oxygen species. Mechanistically,blocking complex I in pro-inflammatory microglia protects the central nervous system against neurotoxic damage and improves functional outcomes in an animal disease model in vivo. Complex I activity in microglia is a potential therapeutic target to foster neuroprotection in chronic inflammatory disorders of the central nervous system3. Blocking mitochondrial complex I in pro-inflammatory microglia protects the central nervous system against neurotoxic damage and improves functional outcomes in vivo in an animal disease model. View Publication

Huntington disease (HD) is a neurodegenerative disease caused by the abnormal expansion of a polyglutamine tract resulting from a mutation in the HTT gene. Oxidative stress has been identified as a significant contributing factor to the development of HD and other neurodegenerative diseases,and targeting anti-oxidative stress has emerged as a potential therapeutic approach. CHCHD2 is a mitochondria-related protein involved in regulating cell migration,anti-oxidative stress,and anti-apoptosis. Although CHCHD2 is highly expressed in HD cells,its specific role in the pathogenesis of HD remains uncertain. We postulate that the up-regulation of CHCHD2 in HD models represents a compensatory protective response against mitochondrial dysfunction and oxidative stress associated with HD. To investigate this hypothesis,we employed HD mouse striatal cells and human induced pluripotent stem cells (hiPSCs) as models to examine the effects of CHCHD2 overexpression (CHCHD2-OE) or knockdown (CHCHD2-KD) on the HD phenotype. Our findings demonstrate that CHCHD2 is crucial for maintaining cell survival in both HD mouse striatal cells and hiPSCs-derived neurons. Our study demonstrates that CHCHD2 up-regulation in HD serves as a compensatory protective response against oxidative stress,suggesting a potential anti-oxidative strategy for the treatment of HD. View Publication

Regulation of gene expression through enhancers is one of the major processes shaping the structure and function of the human brain during development. High-throughput assays have predicted thousands of enhancers involved in neurodevelopment,and confirming their activity through orthogonal functional assays is crucial. Here,we utilized Massively Parallel Reporter Assays (MPRAs) in stem cells and forebrain organoids to evaluate the activity of ~ 7000 gene-linked enhancers previously identified in human fetal tissues and brain organoids. We used a Gaussian mixture model to evaluate the contribution of background noise in the measured activity signal to confirm the activity of ~ 35% of the tested enhancers,with most showing temporal-specific activity,suggesting their evolving role in neurodevelopment. The temporal specificity was further supported by the correlation of activity with gene expression. Our findings provide a valuable gene regulatory resource to the scientific community. View Publication

Klotho,a well-known aging suppressor protein,has been implicated in neuroprotection and the regulation of neuronal senescence. While previous studies have demonstrated its anti-aging properties in human brain organoids,its potential to mitigate neurodegenerative processes triggered by ?-amyloid remains underexplored. In this study,we utilised human induced pluripotent stem cells (iPSCs) engineered with a doxycycline-inducible system to overexpress KLOTHO and generated 2D cortical neuron cultures from these cells. These neurons were next exposed to pre-aggregated ?-amyloid 1–42 oligomers to model the neurotoxicity associated with Alzheimer’s disease. Our data reveal that upregulation of KLOTHO significantly reduced ?-amyloid-induced neuronal degeneration and apoptosis,as evidenced by decreased cleaved caspase-3 expression and preservation of axonal integrity. Additionally,KLOTHO overexpression prevented the loss of dendritic branching and mitigated reductions in axonal diameter,hallmark features of neurodegenerative pathology. These results highlight Klotho’s protective role against ?-amyloid-induced neurotoxicity in human cortical neurons and suggest that its age-related decline may contribute to neurodegenerative diseases such as Alzheimer’s disease. Our findings underscore the therapeutic potential of Klotho-based interventions in mitigating age-associated neurodegenerative processes.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13041-025-01199-6. View Publication

Microglia play a pivotal role in neurodegenerative disease pathogenesis,but the mechanisms underlying microglia dysfunction and toxicity remain to be elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia,we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs),termed iMGs,harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited evidence of lipid dysmetabolism,autophagy dysregulation and deficient phagocytosis,a canonical microglia function. Mutant PFN1 also displayed enhanced binding affinity for PI3P,a critical signaling molecule involved in autophagic and endocytic processing. Our cumulative data implicate a gain-of-toxic function for mutant PFN1 within the autophagic and endo-lysosomal pathways,as administration of rapamycin rescued phagocytic dysfunction in ALS-PFN1 iMGs. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and implicate microglial vesicular degradation pathways in the pathogenesis of these disorders. Mutations in profilin 1 (PFN1),which modulates actin dynamics,are associated with ALS. Here the authors show that expression of ALS-PFN1 is sufficient to induce deficits in human microglia-like cells,including impaired phagocytosis and lipid metabolism,and that gain-of-function interactions between ALS-PFN1 and PI3P may underlie these deficits. View Publication