技术资料

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

CRISPR-Cas9-mediated gene editing has vast applications in basic and clinical research and is a promising tool for several disorders. Our lab previously developed a non-integrating RNA virus,measles virus (MeV),as a single-cycle reprogramming vector by replacing the viral attachment protein with the reprogramming factors for induced pluripotent stem cell generation. Encouraged by the MeV reprogramming vector efficiency,in this study,we develop a single-cycle MeV vector to deliver the gRNA(s) and Cas9 nuclease to human cells for efficient gene editing. We show that the MeV vector achieved on-target gene editing of the reporter (mCherry) and endogenous genes (HBB and FANCD1) in human cells. Additionally,the MeV vector achieved precise knock-in via homology-directed repair using a single-stranded oligonucleotide donor. The MeV vector is a new and flexible platform for gene knock-out and knock-in modifications in human cells,capable of incorporating new technologies as they are developed. Graphical abstract Devaux and colleagues developed a novel single-cycle measles vector allowing gene editing of human cells. They show that Measles can express the CRISPR-Cas9 and gRNA from one genome. Finally,they demonstrate that these vectors can efficiently perform KO and knock-in in human cells without excessive off-target effects. View Publication

There has been a recent drive to replace in vivo studies with in vitro studies in the field of toxicity testing. Therefore,instead of conventional animal or planar cell culture models,there is an urgent need for in vitro systems whose conditions can be strictly controlled,including cell–cell interactions and sensitivity to low doses of chemicals. Neural organoids generated from human-induced pluripotent stem cells (iPSCs) are a promising in vitro platform for modeling human brain development. In this study,we developed a new tool based on various iPSCs to study and predict chemical-induced toxicity in humans. The model displayed several neurodevelopmental features and showed good reproducibility,comparable to that of previously published models. The results revealed that basic fibroblast growth factor plays a key role in the formation of the embryoid body,as well as complex neural networks and higher-order structures such as layered stacking. Using organoid models,pesticide toxicities were assessed. Cells treated with low concentrations of rotenone underwent apoptosis to a greater extent than those treated with high concentrations of rotenone. Morphological changes associated with the development of neural progenitor cells were observed after exposure to low doses of chlorpyrifos. These findings suggest that the neuronal organoids developed in this study mimic the developmental processes occurring in the brain and nerves and are a useful tool for evaluating drug efficacy,safety,and toxicity. View Publication

Genomic instability is the main cause of abnormal embryo development and abortion. NLRP7 dysfunctions affect embryonic development and lead to Hydatidiform Moles,but the underlying mechanisms remain largely elusive. Here,we show that NLRP7 knockout affects the genetic stability,resulting in increased DNA damage in both human embryonic stem cells and blastoids,making embryonic cells in blastoids more susceptible to apoptosis. Mechanistically,NLRP7 can interact with factors related to alternative splicing and DNA damage response,including DDX39B,PRPF8,THRAP3 and PARP1. Moreover,NLRP7 dysfunction leads to abnormal alternative splicing of genes involved in homologous recombination in human embryonic stem cells,Such as Brca1 and Rad51. These results indicate that NLRP7-mediated Alternative splicing is potentially required for the maintenance of genome integrity during early human embryogenesis. Together,this study uncovers that NLRP7 plays an essential role in the maintenance of genetic stability during early human embryonic development by regulating alternative splicing of homologous recombination-related genes. NLRP7 plays an essential role in the maintenance of genetic stability during early human embryonic development by regulating alternative splicing of homologous recombination-related genes. View Publication

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common genetic form of stroke. It is caused by a cysteine-altering variant in one of the 34 epidermal growth factor-like repeat (EGFr) domains of Notch3. NOTCH3 pathogenic variants in EGFr 1–6 are associated with high disease severity,whereas those in EGFr 7–34 are associated with late stroke onset and increased survival. However,whether and how the position of the NOTCH3 variant directly affects the disease severity remains unclear. In this study,we aimed to generate human-induced pluripotent stem cells (hiPSCs) from patients with CADASIL with EGFr 1–6 and 7–34 pathogenic variants to evaluate whether the NOTCH3 position affects the cell phenotype and protein profile of the generated hiPSCs lines. Six hiPSCs lines were generated: two from patients with CADASIL with EGFr 1–6 pathogenic variants,two from patients with EGFr 7–34 variants,and two from controls. Notch3 aggregation and protein profiles were tested in the established six hiPSCs lines. Cell analysis revealed that the NOTCH3 variants did not limit the cell reprogramming efficiency. However,EGFr 1–6 variant position was associated with increased accumulation of Notch3 protein in pluripotent stem cells and proteomic changes related with cytoplasmic reorganization mechanisms. In conclusion,our analysis of hiPSCs derived from patients with CADASIL support the clinical association between the NOTCH3 variant position and severity of CADASIL.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12017-025-08840-6. View Publication

Huntington’s disease (HD) causes selective degeneration of striatal and cortical neurons,resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells,grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids,confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons,while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity,trajectories,synaptic density,and communication pathways upon cell-cell contact,while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells,emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment. Mosaic organoids where pathological and healthy cells are grown together,reveal the rescue of phenotypes in pathological cells due to communication with healthy cells without harming them,as demonstrated by single-cell RNA-sequencing data. View Publication

ObjectiveGene discovery studies in individuals with diabetes diagnosed within 6 months of life (neonatal diabetes,NDM) can provide unique insights into the development and function of human pancreatic beta-cells.MethodsWe performed genome sequencing in a cohort of 43 consanguineous individuals with NDM in whom all the known genetic causes had previously been excluded. We used quantitative PCR and RNA-sequencing in CRISPR-edited human induced pluripotent stem cells (iPSCs),and CUT&RUN-sequencing in EndoC-?H1 cells to investigate the effect of PAX4 loss on human pancreatic development.ResultsWe describe the identification of homozygous PAX4 loss-of-function variants in 2 individuals with transient NDM: a p.(Arg126?) stop-gain variant and a c.-352_104del deletion affecting the first 4 PAX4 exons. We confirmed the p.(Arg126?) variant causes nonsense mediated decay in CRISPR-edited iPSC-derived pancreatic endoderm cells. Integrated analysis of CUT&RUN-sequencing in EndoC-?H1 cells and RNA-sequencing in PAX4-depleted islet stem cell models identified genes directly regulated by PAX4 involved in both pancreatic islet development and glucose-stimulated insulin secretion.ConclusionWe report the first human cases of complete loss of PAX4,establishing it as a novel cause of NDM and highlighting its role in human beta cell development. Both probands had transient NDM which remitted in early infancy but relapsed at the ages of 2.4 and 6.7 years,demonstrating that in contrast to mouse models,PAX4 is not essential for the development of human pancreatic beta-cells. Highlights•Homozygous loss-of-function variants in PAX4 are a novel genetic cause of transient neonatal diabetes.•PAX4 directly regulates genes involved in pancreatic beta cell development and glucose-sensitive insulin secretion.•The role of PAX4 in humans differs to that observed in mouse and is not essential for beta cell development. View Publication

Periventricular heterotopia (PH),a common form of gray matter heterotopia associated with developmental delay and drug-resistant seizures,poses a challenge in understanding its neurophysiological basis. Human cerebral organoids (hCOs) derived from patients with causative mutations in FAT4 or DCHS1 mimic PH features. However,neuronal activity in these 3D models has not yet been investigated. Here we show that silicon probe recordings reveal exaggerated spontaneous spike activity in FAT4 and DCHS1 hCOs,suggesting functional changes in neuronal networks. Transcriptome and proteome analyses identify changes in neuronal morphology and synaptic function. Furthermore,patch-clamp recordings reveal a decreased spike threshold specifically in DCHS1 neurons,likely due to increased somatic voltage-gated sodium channels. Additional analyses reveal increased morphological complexity of PH neurons and synaptic alterations contributing to hyperactivity,with rescue observed in DCHS1 neurons by wild-type DCHS1 expression. Overall,we provide new comprehensive insights into the cellular changes underlying symptoms of gray matter heterotopia. Periventricular heterotopia (PH) is associated with neurodevelopmental delay. Here authors report patient-derived organoids with FAT4 and DCHS1 mutations mimic PH features,showing hyperactivity,synaptic changes and cell morphological alterations. View Publication

Alzheimer’s disease (AD) is a devastating neurodegenerative condition that affects memory and cognition,characterized by neuronal loss and currently lacking a cure. Mutations in PSEN1 (Presenilin 1) are among the most common causes of early-onset familial AD (fAD). While changes in neuronal excitability are believed to be early indicators of AD progression,the link between PSEN1 mutations and neuronal excitability remains to be fully elucidated. This study examined iPSC-derived neurons (iNs) from fAD patients with PSEN1 mutations S290C or A246E,alongside CRISPR-corrected isogenic cell lines,to investigate early changes in excitability. Electrophysiological profiling revealed reduced excitability in both PSEN1 mutant iNs compared to their isogenic controls. Neurons bearing S290C and A246E mutations exhibited divergent passive membrane properties compared to isogenic controls,suggesting distinct effects of PSEN1 mutations on neuronal excitability. Additionally,both PSEN1 backgrounds exhibited higher current density of voltage-gated potassium (Kv) channels relative to their isogenic iNs,while displaying comparable voltage-gated sodium (Nav) channel current density. This suggests that the Nav/Kv imbalance contributes to impaired neuronal firing in fAD iNs. Deciphering these early cellular and molecular changes in AD is crucial for understanding disease pathogenesis. View Publication

Purpose: CLN3 Batten disease (also known as juvenile neuronal ceroid lipofuscinosis) is a lysosomal storage disorder that typically initiates with retinal degeneration but is followed by seizure onset,motor decline and premature death. Patient-derived CLN3 disease induced pluripotent stem cell-RPE cells show defective phagocytosis of photoreceptor outer segment (POS). Because modifier genes are implicated in CLN3 disease,our goal here was to investigate a direct link between CLN3 mutation and POS phagocytosis defect. Methods: Isogenic control and CLN3 mutant stem cell lines were generated by CRISPR-Cas9-mediated biallelic deletion of exons 7 and 8. A transgenic CLN3Δ7-8/Δ7-8 (CLN3) Yucatan miniswine was also used to study the impact of CLN3Δ7-8/Δ7-8 mutation on POS phagocytosis. POS phagocytosis by cultured RPE cells was analyzed by Western blotting and immunohistochemistry. Electroretinogram,optical coherence tomography and histological analysis of CLN3Δ7-8/Δ7-8 and wild-type miniswine eyes were carried out at 6,36,or 48 months of age. Results: CLN3Δ7-8/Δ7-8 RPE (CLN3 RPE) displayed decreased POS binding and consequently decreased uptake of POS compared with isogenic control RPE cells. Furthermore,wild-type miniswine RPE cells phagocytosed CLN3Δ7-8/Δ7-8 POS less efficiently than wild-type POS. Consistent with decreased POS phagocytosis,lipofuscin/autofluorescence was decreased in CLN3 miniswine RPE at 36 months of age and was followed by almost complete loss of photoreceptors at 48 months of age. Conclusions: CLN3Δ7-8/Δ7-8 mutation (which affects ≤85% of patients) affects both RPE and POS and leads to photoreceptor cell loss in CLN3 disease. Furthermore,both primary RPE dysfunction and mutant POS independently contribute to impaired POS phagocytosis in CLN3 disease. View Publication

AbstractA basic assumption underlying induced pluripotent stem cell (iPSC) models of neurodegeneration is that disease-relevant pathologies present in brain tissue are also represented in donor-matched cells differentiated from iPSCs. However,few studies have tested this hypothesis in matched iPSCs and neuropathologically characterized donated brain tissues. To address this,we assessed iPSC-neuron production of ?-amyloid (A?) A?40,A?42,and A?43 in 24 iPSC lines matched to donor brains with primary neuropathologic diagnoses of sporadic AD (sAD),familial AD (fAD),control,and other neurodegenerative disorders. Our results demonstrate a positive correlation between A?43 production by fAD iPSC-neurons and A?43 accumulation in matched brain tissues but do not reveal a substantial correlation in soluble A? species between control or sAD iPSC-neurons and matched brains. However,we found that the ApoE4 genotype is associated with increased A? production by AD iPSC-neurons. Pathologic tau phosphorylation was found to be increased in AD and fAD iPSC-neurons compared to controls and positively correlated with the relative abundance of longer-length A? species produced by these cells. Taken together,our results demonstrate that sAD-predisposing genetic factors influence iPSC-neuron phenotypes and that these cells are capturing disease-relevant and patient-specific components of the amyloid cascade. View Publication

SummaryCytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43 is a pathological hallmark of the motor neuron (MN) disease amyotrophic lateral sclerosis (ALS). Furthermore,while mutations in TARDBP (encoding TDP-43) have been associated with ALS,the pathogenic consequences of these mutations remain poorly understood. Using CRISPR-Cas9,we engineered two homozygous knock-in induced pluripotent stem cell lines carrying mutations in TARDBP encoding TDP-43A382T and TDP-43G348C,two common yet understudied ALS TDP-43 variants. Motor neurons (MNs) differentiated from knock-in iPSCs had normal viability and displayed no significant changes in TDP-43 subcellular localization,phosphorylation,solubility,or aggregation compared with isogenic control MNs. However,our results highlight synaptic impairments in both TDP-43A382T and TDP-43G348C MN cultures,as reflected in synapse abnormalities and alterations in spontaneous neuronal activity. Collectively,our findings suggest that MN dysfunction may precede the occurrence of TDP-43 pathology and neurodegeneration in ALS and further implicate synaptic and excitability defects in the pathobiology of this disease. Graphical abstract Highlights•Mutant MNs maintain viability but are more vulnerable to cellular stress•Mutant MNs do not show TDP-43 pathology•TDP-43 variants lead to a progressive decline in spontaneous neuronal activity•Functional impairments are accompanied by abnormal synaptic marker expression Molecular neuroscience; Cellular neuroscience View Publication