技术资料

BackgroundMicroglia play important roles in maintaining brain homeostasis and neurodegeneration. The discovery of genetic variants in genes predominately or exclusively expressed in myeloid cells,such as Apolipoprotein E (APOE) and triggering receptor expressed on myeloid cells 2 (TREM2),as the strongest risk factors for Alzheimer’s disease (AD) highlights the importance of microglial biology in the brain. The sequence,structure and function of several microglial proteins are poorly conserved across species,which has hampered the development of strategies aiming to modulate the expression of specific microglial genes. One way to target APOE and TREM2 is to modulate their expression using antisense oligonucleotides (ASOs).MethodsIn this study,we identified,produced,and tested novel,selective and potent ASOs for human APOE and TREM2. We used a combination of in vitro iPSC-microglia models,as well as microglial xenotransplanted mice to provide proof of activity in human microglial in vivo.ResultsWe proved their efficacy in human iPSC microglia in vitro,as well as their pharmacological activity in vivo in a xenografted microglia model. We demonstrate ASOs targeting human microglia can modify their transcriptional profile and their response to amyloid-? plaques in vivo in a model of AD.ConclusionsThis study is the first proof-of-concept that human microglial can be modulated using ASOs in a dose-dependent manner to manipulate microglia phenotypes and response to neurodegeneration in vivo.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-024-00725-9. View Publication

Fluorescence microscopy has undergone rapid advancements,offering unprecedented visualization of biological events and shedding light on the intricate mechanisms governing living organisms. However,the exploration of rapid biological dynamics still poses a significant challenge due to the limitations of current digital camera architectures and the inherent compromise between imaging speed and other capabilities. Here,we introduce sHAPR,a high-speed acquisition technique that leverages the operating principles of sCMOS cameras to capture fast cellular and subcellular processes. sHAPR harnesses custom fiber optics to convert microscopy images into one-dimensional recordings,enabling acquisition at the maximum camera readout rate,typically between 25 and 250 kHz. We have demonstrated the utility of sHAPR with a variety of phantom and dynamic systems,including high-throughput flow cytometry,cardiomyocyte contraction,and neuronal calcium waves,using a standard epi-fluorescence microscope. sHAPR is highly adaptable and can be integrated into existing microscopy systems without requiring extensive platform modifications. This method pushes the boundaries of current fluorescence imaging capabilities,opening up new avenues for investigating high-speed biological phenomena. The authors introduce a highspeed acquisition technique,sHAPR,for rapid exploration of biodynamics using fluorescence microscopy. The method leverages sCMOS cameras and custom fibre optics to convert microscopy images into 1D recordings,enabling acquisition at the maximum camera readout rate. View Publication

SummaryThe neurodevelopmental disorders Prader-Willi syndrome (PWS) and Schaaf-Yang syndrome (SYS) both arise from genomic alterations within human chromosome 15q11–q13. A deletion of the SNORD116 cluster,encoding small nucleolar RNAs,or frameshift mutations within MAGEL2 result in closely related phenotypes in individuals with PWS or SYS,respectively. By investigation of their subcellular localization,we observed that in contrast to a predominant cytoplasmic localization of wild-type (WT) MAGEL2,a truncated MAGEL2 mutant was evenly distributed between the cytoplasm and the nucleus. To elucidate regulatory pathways that may underlie both diseases,we identified protein interaction partners for WT or mutant MAGEL2,in particular the survival motor neuron protein (SMN),involved in spinal muscular atrophy,and the fragile-X-messenger ribonucleoprotein (FMRP),involved in autism spectrum disorders. The interactome of the non-coding RNA SNORD116 was also investigated by RNA-CoIP. We show that WT and truncated MAGEL2 were both involved in RNA metabolism,while regulation of transcription was mainly observed for WT MAGEL2. Hence,we investigated the influence of MAGEL2 mutations on the expression of genes from the PWS locus,including the SNORD116 cluster. Thereby,we provide evidence for MAGEL2 mutants decreasing the expression of SNORD116,SNORD115,and SNORD109A,as well as protein-coding genes MKRN3 and SNRPN,thus bridging the gap between PWS and SYS. Graphical abstract Mutations within MAGEL2 from chromosomal region 15q11–q13 cause Schaaf-Yang syndrome,which is phenotypically related to Prader-Willi syndrome,caused by deletion of the SNORD116 cluster within the same locus. We correlate mutations within MAGEL2 to spinal muscular atrophy and autism and also demonstrate its influence on the abundance of SNORD116. View Publication

Propionic acidemia (PA) is a metabolic disorder caused by a deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC) due to mutations in the PCCA or PCCB genes,which encode the two PCC subunits. PA may lead to several types of cardiomyopathy and has been linked to cardiac electrical abnormalities such as QT interval prolongation,life-threatening arrhythmias,and sudden cardiac death. To gain insights into the mechanisms underlying PA-induced proarrhythmia,we recorded action potentials (APs) and ion currents using whole-cell patch-clamp in ventricular-like induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) from a PA patient carrying two pathogenic mutations in the PCCA gene (p.Cys616_Val633del and p.Gly477Glufs*9) (PCCA cells) and from a healthy subject (healthy cells). In cells driven at 1 Hz,PCC deficiency increased the latency and prolonged the AP duration (APD) measured at 20% of repolarization,without modifying resting membrane potential or AP amplitude. Moreover,delayed afterdepolarizations appeared at the end of the repolarization phase in unstimulated and paced PCCA cells. PCC deficiency significantly reduced peak sodium current (INa) but increased the late INa (INaL) component. In addition,L-type Ca2+ current (ICaL) density was reduced,while the inward and outward density of the Na+/Ca2+ exchanger current (INCX) was increased in PCCA cells compared to healthy ones. In conclusion,our results demonstrate that at the cellular level,PCC deficiency can modify the ion currents controlling cardiac excitability,APD,and intracellular Ca2+ handling,increasing the risk of arrhythmias independently of the progressive late-onset cardiomyopathy induced by PA disease. View Publication

ABSTRACTRAD50?interacting protein1 (RINT1) deficiency has been implicated in recurrent acute liver failure (RALF) triggered by fever or infections. RINT1,together with neuroblastoma amplified sequence and Zeste White 10 (forming the NRZ complex),localizes at the interface between the endoplasmic reticulum and Golgi apparatus,where it plays a key role in vesicular trafficking. However,the mechanisms by which RINT1 deficiency leads to RALF remain unclear. This study aimed to describe a woman with RALF harboring a homozygous missense mutation in RINT1. Induced pluripotent stem cells (iPSCs) were generated from the patient's mononuclear cells and differentiated into hepatocyte?like cells (HLCs). Upon exposure to high temperature (40°C),RINT1?deficient HLCs exhibited cellular damage characteristic of RALF. Furthermore,these cells also demonstrated heightened sensitivity to cytokines and viral mimetics while showing comparatively lower responsiveness to bacterial infection?related stimuli. Transcriptome sequencing revealed dysregulated gene expression associated with the extracellular matrix (ECM). Additionally,glycosaminoglycan disaccharide analysis revealed abnormal levels of chondroitin sulfate,heparan sulfate,and hyaluronan in RINT1?deficient HLCs. In conclusion,HLCs derived from RINT1?deficient iPSCs serve as a valuable model for investigating RINT1?related liver pathogenesis. The results suggest that cytokine responses,particularly those triggered by viral infections,play a central role in the development of RALF. Furthermore,ECM alterations provided novel insights into the potential role of RINT1 defects in RALF. RAD50?interacting protein1 (RINT1) deficiency causes recurrent acute liver failure (RALF) during fever or infections. To investigate its underlying mechanism,induced pluripotent stem cells were generated from a patient with RINT1 deficiency and differentiated into hepatocyte?like cells (HLCs). RINT1?deficient HLCs exhibited damage resembling RALF when exposed to high temperatures and were more susceptible to cytokines and viral mimetics than to bacterial infection?related factors. Furthermore,RNA?seq and disaccharide analyses revealed dysregulation of extracellular matrix?related genes and abnormalities in extracellular matrix levels. View Publication

Remarkable advances in protocol development have been achieved to manufacture insulin-secreting islets from human pluripotent stem cells (hPSCs). Distinct from current approaches,we devised a tunable strategy to generate islet spheroids enriched for major islet cell types by incorporating PDX1+ cell budding morphogenesis into staged differentiation. In this process that appears to mimic normal islet morphogenesis,the differentiating islet spheroids organize with endocrine cells that are intermingled or arranged in a core-mantle architecture,accompanied with functional heterogeneity. Through in vitro modelling of human pancreas development,we illustrate the importance of PDX1 and the requirement for EphB3/4 signaling in eliciting cell budding morphogenesis. Using this new approach,we model Mitchell-Riley syndrome with RFX6 knockout hPSCs illustrating unexpected morphogenesis defects in the differentiation towards islet cells. The tunable differentiation system and stem cell-derived islet models described in this work may facilitate addressing fundamental questions in islet biology and probing human pancreas diseases. The ability to differentiate human pluripotent stem cells (hPSCs) into insulin producing cells holds potential for diabetes treatments,but many of these approaches lack the complexity needed for in vitro disease modeling. Here they develop an hPSC-derived islet spheroid system,offering an experimental model to study pancreatic budding and islet morphogenesis with human cells. View Publication

Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the Slc6a8 gene. The impaired creatine uptake in the brain leads to developmental delays with intellectual disability. We hypothesized that deficient creatine uptake in CTD cerebral cells impact methylation balance leading to alterations of genes and proteins expression by epigenetic mechanism. In this study,we determined the status of nucleic acid methylation in both Slc6a8 knockout mouse model and brain organoids derived from CTD patients’ cells. We also investigated the effect of dodecyl creatine ester (DCE),a promising prodrug that increases brain creatine content in the mouse model of CTD. The level of nucleic acid methylation was significantly reduced compared to healthy controls in both in vivo and in vitro CTD models. This hypo-methylation tended to be regulated by DCE treatment in vivo. These results suggest that increased brain creatine after DCE treatment restores normal levels of DNA methylation,unveiling the potential of using DNA methylation as a marker to monitor the drug efficacy. View Publication

AbstractThe hippocampus is a brain area linked to cognition. The mechanisms that maintain cognitive activity in humans are poorly understood. Centenarians display extreme longevity which is generally accompanied by better quality of life,lower cognitive impairment,and reduced incidence of pathologies including neurodegenerative diseases. We performed transcriptomic studies in hippocampus samples from individuals of different ages (centenarians [?97 years],old,and young) and identified a differential gene expression pattern in centenarians compared to the other two groups. In particular,several isoforms of metallothioneins (MTs) were highly expressed in centenarians. Moreover,we identified that MTs were mainly expressed in astrocytes. Functional studies in human primary astrocytes revealed that MT1 and MT3 are necessary for their homeostasis maintenance. Overall,these results indicate that the expression of MTs specifically in astrocytes is a mechanism for protection during aging. Higher levels of MT1 and MT3 are detected in hippocampus of very old individuals (over 90) compared with old and young individuals. MTs colocalize with astrocytic markers. View Publication

Oral facial cleft (OFC) comprises cleft lip with or without cleft palate (CL/P) or cleft palate only. Genome wide association studies (GWAS) of isolated OFC have identified common single nucleotide polymorphisms (SNPs) in many genomic loci where the presumed effector gene (for example,IRF6 in the 1q32 locus) is expressed in embryonic oral epithelium. To identify candidates for functional SNPs at eight such loci we conduct a massively parallel reporter assay in a fetal oral epithelial cell line,revealing SNPs with allele-specific effects on enhancer activity. We filter these SNPs against chromatin-mark evidence of enhancers and test a subset in traditional reporter assays,which support the candidacy of SNPs at loci containing FOXE1, IRF6, MAFB, TFAP2A,and TP63. For two SNPs near IRF6 and one near FOXE1,we engineer the genome of induced pluripotent stem cells,differentiate the cells into embryonic oral epithelium,and discover allele-specific effects on the levels of effector gene expression,and,in two cases,the binding affinity of transcription factors FOXE1 or ETS2. Conditional analyses of GWAS data suggest the two functional SNPs near IRF6 account for the majority of risk for CL/P at this locus. This study connects genetic variation associated with OFC to mechanisms of pathogenesis. Non-syndromic orofacial cleft is a relatively common congenital anomaly. Many non-coding genetic variants are associated with this disorder but only a subset is functional. Here the authors use reporter assays and stem cells to reveal members of this subset. View Publication

Proteomic analysis of human cerebral organoids may reveal how psychedelics regulate biological processes,shedding light on drug-induced changes in the brain. This study elucidates the proteomic alterations induced by lysergic acid diethylamide (LSD) in human cerebral organoids. By employing high-resolution mass spectrometry-based proteomics,we quantitatively analyzed the differential abundance of proteins in cerebral organoids exposed to LSD. Our findings indicate changes in proteostasis,energy metabolism,and neuroplasticity-related pathways. Specifically,LSD exposure led to alterations in protein synthesis,folding,autophagy,and proteasomal degradation,suggesting a complex interplay in the regulation of neural cell function. Additionally,we observed modulation in glycolysis and oxidative phosphorylation,crucial for cellular energy management and synaptic function. In support of the proteomic data,complementary experiments demonstrated LSD’s potential to enhance neurite outgrowth in vitro,confirming its impact on neuroplasticity. Collectively,our results provide a comprehensive insight into the molecular mechanisms through which LSD may affect neuroplasticity and potentially contribute to therapeutic effects for neuropsychiatric disorders. View Publication

A patient with the PSEN1 E280A mutation and homozygous for APOE3 Christchurch (APOE3Ch) displayed extreme resistance to Alzheimer’s disease (AD) cognitive decline and tauopathy,despite having a high amyloid burden. To further investigate the differences in biological processes attributed to APOE3Ch,we generated induced pluripotent stem (iPS) cell-derived cerebral organoids from this resistant case and a non-protected control,using CRISPR/Cas9 gene editing to modulate APOE3Ch expression. In the APOE3Ch cerebral organoids,we observed a protective pattern from early tau phosphorylation. ScRNA sequencing revealed regulation of Cadherin and Wnt signaling pathways by APOE3Ch,with immunostaining indicating elevated ?-catenin protein levels. Further in vitro reporter assays unexpectedly demonstrated that ApoE3Ch functions as a Wnt3a signaling enhancer. This work uncovered a neomorphic molecular mechanism of protection of ApoE3 Christchurch,which may serve as the foundation for the future development of protected case-inspired therapeutics targeting AD and tauopathies. View Publication

Extraembryonic mesoderm (ExM) is crucial for human development,yet its specification is poorly understood. Human embryonic stem cell (hESC)-based models,including embryoids and differentiated derivatives,are emerging as promising tools for studying ExM development. Despite this,the signaling mechanisms and developmental dynamics that underlie ExM specification from hESCs remain challenging to study. Here,we report that the modulation of BMP,WNT,and Nodal signaling pathways can rapidly (4-5 days) and efficiently (?~90%) induce the differentiation of both naive and primed hESCs into ExM-like cells (ExMs). We reveal that ExM specification from hESCs predominantly proceeds through intermediates exhibiting a primitive streak (PS)-like gene expression pattern and delineate the regulatory roles of WNT and Nodal signaling in this process. Furthermore,we find that the initial pluripotent state governs hESC-based ExM specification by influencing signal response,cellular composition,developmental progression,and transcriptional characteristics of the resulting ExMs. Our study provides promising models for dissecting human ExM development and sheds light on the signaling principles,developmental dynamics,and influences of pluripotency states underlying ExM specification from hESCs. Extraembryonic mesoderm (ExM) is crucial but its formation is unclear. Here,authors develop efficient systems to specify ExM from hESCs and dissect the signaling mechanisms,specification dynamics,and impact of pluripotent states in ExM formation. View Publication