技术资料

Recent advances in imaging suggested that spatial organization of hematopoietic cells in their bone marrow microenvironment (niche) regulates cell expansion,governing progression,and leukemic transformation of hematological clonal disorders. However,our ability to interrogate the niche in pre-malignant conditions has been limited,as standard murine models of these diseases rely largely on transplantation of the mutant clones into conditioned mice where the marrow microenvironment is compromised. Here,we leveraged live-animal microscopy and ultralow dose whole body or focal irradiation to capture single cells and early expansion of benign/pre-malignant clones in the functionally preserved microenvironment. 0.5 Gy whole body irradiation (WBI) allowed steady engraftment of cells beyond 30 weeks compared to non-conditioned controls. In-vivo tracking and functional analyses of the microenvironment showed no change in vessel integrity,cell viability,and HSC-supportive functions of the stromal cells,suggesting minimal inflammation after the radiation insult. The approach enabled in vivo imaging of Tet2 + /− and its healthy counterpart,showing preferential localization within a shared microenvironment while forming discrete micro-niches. Notably,stationary association with the niche only occurred in a subset of cells and would not be identified without live imaging. This strategy may be broadly applied to study clonal disorders in a spatial context. View Publication

Streptococcus pneumoniae is a global priority respiratory pathogen that kills over a million people annually. The pore-forming cytotoxin,pneumolysin (PLY) is a major virulence factor. Here,we found that recombinant PLY as well as wild-type pneumococcal strains,but not the isogenic PLY mutant,upregulated the shedding of extracellular vesicles (EVs) harboring membrane-bound toxin from human THP-1 monocytes. PLY-EVs induced cytotoxicity and hemolysis dose-dependently upon internalization by recipient monocyte-derived dendritic cells. Proteomics analysis revealed that PLY-EVs are selectively enriched in key inflammatory host proteins such as IFI16,NLRC4,PTX3,and MMP9. EVs shed from PLY-challenged or infected cells induced dendritic cell maturation and primed them to infection. In vivo,zebrafish administered with PLY-EVs showed pericardial edema and mortality. Adoptive transfer of bronchoalveolar-lavage-derived EVs from infected mice to healthy recipients induced lung damage and inflammation in a PLY-dependent manner. Our findings identify that host EVs released during infection mediate pneumococcal pathogenesis. Subject areas: Microbiology,Bacteriology,Cell biology View Publication

The growth factor Neuregulin-1 (NRG1) has pleiotropic roles in proliferation and differentiation of the stem cell niche in different tissues. It has been implicated in gut,brain and muscle development and repair. Six isoform classes of NRG1 and over 28 protein isoforms have been previously described. Here we report a new class of NRG1,designated NRG1-VII to denote that these NRG1 isoforms arise from a myeloid-specific transcriptional start site (TSS) previously uncharacterized. Long-read sequencing was used to identify eight high-confidence NRG1-VII transcripts. These transcripts presented major structural differences from one another,through the use of cassette exons and alternative stop codons. Expression of NRG1-VII was confirmed in primary human monocytes and tissue resident macrophages and induced pluripotent stem cell-derived macrophages (iPSC-derived macrophages). Isoform switching via cassette exon usage and alternate polyadenylation was apparent during monocyte maturation and macrophage differentiation. NRG1-VII is the major class expressed by the myeloid lineage,including tissue-resident macrophages. Analysis of public gene expression data indicates that monocytes and macrophages are a primary source of NRG1. The size and structure of class VII isoforms suggests that they may be more diffusible through tissues than other NRG1 classes. However,the specific roles of class VII variants in tissue homeostasis and repair have not yet been determined. The online version contains supplementary material available at 10.1186/s12864-024-10723-2. View Publication

Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are the strongest genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level,ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However,whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here,we combine patient-derived and mouse models to dissect the effects of ATXN2 intermediate expansions in an ALS background. iPSC-derived motor neurons from ATXN2-ALS patients show altered stress granules,neurite damage and abnormal electrophysiological properties compared to healthy control and other familial ALS mutations. In TDP-43 Tg -ALS mice,ATXN2-Q33 causes reduced motor function,NMJ alterations,neuron degeneration and altered in vitro stress granule dynamics. Furthermore,gene expression changes related to mitochondrial function and inflammatory response are detected and confirmed at the cellular level in mice and human neuron and organoid models. Together,these results define pathogenic defects underlying ATXN2-ALS and provide a framework for future research into ATXN2-dependent pathogenesis and therapy. Subject terms: Amyotrophic lateral sclerosis,Molecular neuroscience,Cellular neuroscience View Publication

Hypomethylating agents (HMAs) are frontline therapies for Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML). However,acquired resistance and treatment failure are commonplace. To address this,we perform a genome-wide CRISPR-Cas9 screen in a human MDS-derived cell line,MDS-L,and identify TOPORS as a loss-of-function target that synergizes with HMAs,reducing leukemic burden and improving survival in xenograft models. We demonstrate that depletion of TOPORS mediates sensitivity to HMAs by predisposing leukemic blasts to an impaired DNA damage response (DDR) accompanied by an accumulation of SUMOylated DNMT1 in HMA-treated TOPORS-depleted cells. The combination of HMAs with targeting of TOPORS does not impair healthy hematopoiesis. While inhibitors of TOPORS are unavailable,we show that inhibition of protein SUMOylation with TAK-981 partially phenocopies HMA-sensitivity and DDR impairment. Overall,our data suggest that the combination of HMAs with inhibition of SUMOylation or TOPORS is a rational treatment option for High-Risk MDS (HR-MDS) or AML. Subject terms: Myelodysplastic syndrome,Acute myeloid leukaemia,Sumoylation View Publication

DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies,although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein ( TOPORS ),which encodes a ubiquitin/SUMO E3 ligase,augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis,suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks,which undergo SUMOylation,followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain,which mediates ubiquitination,is responsible for HMA resistance. In TOPORS knockout cells,DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination,resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1,thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently,the ubiquitination inhibitor,TAK-243,and the SUMOylation inhibitor,TAK-981,show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks. Subject terms: Myelodysplastic syndrome,Myelodysplastic syndrome View Publication

Three-dimensionality (3D) was proven essential for developing reliable models for different anatomical compartments and many diseases. However,the neuronal compartment still poses a great challenge as we still do not understand precisely how the brain computes information and how the complex chain of neuronal events can generate conscious behavior. Therefore,a comprehensive model of neuronal tissue has not yet been found. The present work was conceived in this framework: we aimed to contribute to what must be a collective effort by filling in some information on possible 3D strategies to pursue. We compared directly different kinds of scaffolds (i.e.,PDMS sponges,thermally crosslinked hydrogels,and glass microbeads) in their effect on neuronal network activity recorded using micro-electrode arrays. While the overall rate of spiking activity remained consistent,the type of scaffold had a notable impact on bursting dynamics. The frequency,density of bursts,and occurrence of random spikes were all affected. The examination of inter-burst intervals revealed distinct burst generation patterns unique to different scaffold types. Network burst propagation unveiled divergent trends among configurations. Notably,it showed the most differences,underlying that functional variations may arise from a different 3D spatial organization. This evidence suggests that not all 3D neuronal constructs can sustain the same level of richness of activity. Furthermore,we commented on the reproducibility,efficacy,and scalability of the methods,where the beads still offer superior performances. By comparing different 3D scaffolds,our results move toward understanding the best strategies to develop functional 3D neuronal units for reliable pre-clinical studies. View Publication

Despite advances in breast cancer screening and treatment,prognosis for metastatic disease remains dismal at 30% five-year survival. This is due,in large,to the failure of current therapeutics to target properties unique to metastatic cells. One of the drivers of metastasis is miR-10b,a small noncoding RNA implicated in cancer cell invasion,migration,viability,and proliferation. We have developed a nanodrug,termed MN-anti-miR10b,that delivers anti-miR-10b antisense oligomers to cancer cells. In mouse models of metastatic triple-negative breast cancer,MN-anti-miR10b has been shown to prevent onset of metastasis and eliminate existing metastases in combination with chemotherapy,even after treatment has been stopped. Recent studies have implicated miR-10b in conferring stem cell-like properties onto cancer cells,such as chemoresistance. In this study,we show transcriptional evidence that inhibition of miR-10b with MN-anti-miR10b activates developmental processes in cancer cells and that stem-like cancer cells have increased miR-10b expression. We then demonstrate that treatment of breast cancer cells with MN-anti-miR10b reduces their stemness,confirming that these properties make metastatic cells susceptible to the nanodrug actions. Collectively,these findings indicate that inhibition of miR-10b functions to impair breast cancer cell stemness,positioning MN-anti-miR10b as an effective treatment option for stem-like breast cancer subtypes. View Publication

CD8 + cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide–MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell–cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell–cell contact,promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux,providing the necessary energy through oxidative phosphorylation,and its own protein translation on 55S ribosomes. However,the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Here,we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton,mitochondria,and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 55S by puromycin. We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs. View Publication

Riboflavin transporter deficiency type 2 (RTD2) is a rare neurodegenerative autosomal recessive disease caused by mutations in the SLC52A2 gene encoding the riboflavin transporters,RFVT2. Riboflavin (Rf) is the precursor of FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide),which are involved in different redox reactions,including the energetic metabolism processes occurring in mitochondria. To date,human induced pluripotent stem cells (iPSCs) have given the opportunity to characterize RTD2 motoneurons,which reflect the most affected cell type. Previous works have demonstrated mitochondrial and peroxisomal altered energy metabolism as well as cytoskeletal derangement in RTD2 iPSCs and iPSC-derived motoneurons. So far,no attention has been dedicated to astrocytes. Here,we demonstrate that in vitro differentiation of astrocytes,which guarantee trophic and metabolic support to neurons,from RTD2 iPSCs is not compromised. These cells do not exhibit evident morphological differences nor significant changes in the survival rate when compared to astrocytes derived from iPSCs of healthy individuals. These findings indicate that differently from what had previously been documented for neurons,RTD2 does not compromise the morpho-functional features of astrocytes. View Publication

Previous studies have shown that aggregated alpha-synuclein (α-s) protein,a key pathological marker of Parkinson’s disease (PD),can propagate between cells,thus participating in disease progression. This prion-like propagation has been widely studied using in vivo and in vitro models,including rodent and human cell cultures. In this study,our focus was on temporal assessment of functional changes during α-s aggregation and propagation in human induced pluripotent stem cell (hiPSC)-derived neuronal cultures and in engineered networks. Here,we report an engineered circular tripartite human neuronal network model in a microfluidic chip integrated with microelectrode arrays (MEAs) as a platform to study functional markers during α-s aggregation and propagation. We observed progressive aggregation of α-s in conventional neuronal cultures and in the exposed (proximal) compartments of circular tripartite networks following exposure to preformed α-s fibrils (PFF). Furthermore,aggregated forms propagated to distal compartments of the circular tripartite networks through axonal transport. We observed impacts of α-s aggregation on both the structure and function of neuronal cells,such as in presynaptic proteins,mitochondrial motility,calcium oscillations and neuronal activity. The model enabled an assessment of the early,middle,and late phases of α-s aggregation and its propagation during a 13-day follow-up period. While our temporal analysis suggested a complex interplay of structural and functional changes during the in vitro propagation of α-s aggregates,further investigation is required to elucidate the underlying mechanisms. Taken together,this study demonstrates the technical potential of our introduced model for conducting in-depth analyses for revealing such mechanisms. Subject terms: Parkinson's disease,Neurological models View Publication

Hematopoietic aging is associated with decreased hematopoietic stem cell (HSC) self-renewal capacity and myeloid skewing. We report that culture of bone marrow (BM) HSCs from aged mice with epidermal growth factor (EGF) suppressed myeloid skewing,increased multipotent colony formation,and increased HSC repopulation in primary and secondary transplantation assays. Mice transplanted with aged,EGF-treated HSCs displayed increased donor cell engraftment within BM HSCs and systemic administration of EGF to aged mice increased HSC self-renewal capacity in primary and secondary transplantation assays. Expression of a dominant negative EGFR in Scl/Tal1 + hematopoietic cells caused increased myeloid skewing and depletion of long term-HSCs in 15-month-old mice. EGF treatment decreased DNA damage in aged HSCs and shifted the transcriptome of aged HSCs from genes regulating cell death to genes involved in HSC self-renewal and DNA repair but had no effect on HSC senescence. These data suggest that EGFR signaling regulates the repopulating capacity of aged HSCs. Subject areas: Human physiology,cellular physiology,molecular medicine,stem cells research,functional aspects of cell biology View Publication