参考文献

Objective Friedreich's ataxia (FRDA) is an autosomal recessive trinucleotide repeat expansion disorder caused by epigenetic silencing of the frataxin gene (FXN). Current research suggests that damage and variation of mitochondrial DNA (mtDNA) contribute to the molecular pathogenesis of FRDA. We sought to establish the extent of the mutation burden across the mitochondrial genome in FRDA cells and investigate the molecular mechanisms connecting FXN downregulation and the acquisition of mtDNA damage. Methods Damage and mutation load in mtDNA of a panel of FRDA and control fibroblasts were determined using qPCR and next-generation MiSeq sequencing,respectively. The capacity of FRDA and control cells to repair oxidative lesions in their mtDNA was measured using a quantitative DNA damage assay. Comprehensive RNA sequencing gene expression analyses were conducted to assess the status of DNA repair and metabolism genes in FRDA cells. Results Acute or prolonged downregulation of FXN expression resulted in a significant increase in mtDNA damage that translated to a significant elevation of mutation load in mtDNA. The predominant mutations identified throughout the mtDNA were CtextgreaterT,GtextgreaterA transitions (P = 0.007). Low FXN expression reduced capacity to repair oxidative damage in mtDNA. Downregulation of FXN expression strongly correlated (r = 0.73) with decreased levels of base excision repair (BER) DNA glycosylase NTHL1. Interpretation Downregulation of FXN expression in FRDA cells elevates mtDNA damage,increases mutation load of the mitochondrial genome,and diminishes DNA repair capacity. Progressive accumulation of mtDNA mutations in vulnerable FRDA patient cells reduces mitochondrial fitness ultimately leading to cell death. View Publication

Rett syndrome (RTT) is a postnatal neurodevelopmental disorder that primarily affects girls. Mutations in the methyl-CpG-binding protein 2 (MECP2) gene account for approximately 95 % of all RTT cases. To model RTT in vitro,we generated induced pluripotent stem cells (iPSCs) from fibroblasts of two RTT patients with different mutations (MECP2 (R306C) and MECP2 (1155$$32)) in their MECP2 gene. We found that these iPSCs were capable of differentiating into functional neurons. Compared to control neurons,the RTT iPSC-derived cells had reduced soma size and a decreased amount of synaptic input,evident both as fewer Synapsin 1-positive puncta and a lower frequency of spontaneous excitatory postsynaptic currents. Supplementation of the culture media with choline rescued all of these defects. Choline supplementation may act through changes in the expression of choline acetyltransferase,an important enzyme in cholinergic signaling,and also through alterations in the lipid metabolite profiles of the RTT neurons. Our study elucidates the possible mechanistic pathways for the effect of choline on human RTT cell models,thereby illustrating the potential for using choline as a nutraceutical to treat RTT. View Publication

The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs,FUS and TDP-43,we integrate CLIP-seq and RNA Bind-N-Seq technologies,and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns,are enriched in 3' untranslated regions and alter genes distinct from TDP-43. However,unlike FUS and TDP-43,TAF15 has a minimal role in alternative splicing. In human neural progenitors,TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons,the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G,but contrasts with late-stage sporadic ALS patients. Taken together,our findings reveal convergent and divergent roles for FUS,TAF15 and TDP-43 in RNA metabolism. View Publication

Autism spectrum disorders (ASD) are common,complex and heterogeneous neurodevelopmental disorders. Cellular and molecular mechanisms responsible for ASD pathogenesis have been proposed based on genetic studies,brain pathology and imaging,but a major impediment to testing ASD hypotheses is the lack of human cell models. Here,we reprogrammed fibroblasts to generate induced pluripotent stem cells,neural progenitor cells (NPCs) and neurons from ASD individuals with early brain overgrowth and non-ASD controls with normal brain size. ASD-derived NPCs display increased cell proliferation because of dysregulation of a β-catenin/BRN2 transcriptional cascade. ASD-derived neurons display abnormal neurogenesis and reduced synaptogenesis leading to functional defects in neuronal networks. Interestingly,defects in neuronal networks could be rescued by insulin growth factor 1 (IGF-1),a drug that is currently in clinical trials for ASD. This work demonstrates that selection of ASD subjects based on endophenotypes unraveled biologically relevant pathway disruption and revealed a potential cellular mechanism for the therapeutic effect of IGF-1 View Publication

Bat immunity has received increasing attention because some bat species are being decimated by the fungal disease,White Nose Syndrome,while other species are potential reservoirs of zoonotic viruses. Identifying specific immune processes requires new specific tools and reagents. In this study,we describe a new mouse monoclonal antibody (mAb) reactive with Eptesicus fuscus immunoglobulins. The epitope recognized by mAb BT1-4F10 was localized to immunoglobulin light (lambda) chains; hence,the mAb recognized serum immunoglobulins and B lymphocytes. The BT1-4F10 epitope appeared to be restricted to Microchiropteran immunoglobulins and absent from Megachiropteran immunoglobulins. Analyses of sera and other E. fuscus fluids showed that most,if not all,secreted immunoglobulins utilized lambda light chains. Finally,mAb BT1-4F10 permitted the identification of B cell follicles in splenic white pulp. This Microchiropteran-specific mAb has potential utility in seroassays; hence,this reagent may have both basic and practical applications for studying immune process. View Publication

Positive expectations contribute to the clinical benefits of the placebo effect. Such positive expectations are mediated by the brain's reward system; however,it remains unknown whether and how reward system activation affects the body's physiology and,specifically,immunity. Here we show that activation of the ventral tegmental area (VTA),a key component of the reward system,strengthens immunological host defense. We used 'designer receptors exclusively activated by designer drugs' (DREADDs) to directly activate dopaminergic neurons in the mouse VTA and characterized the subsequent immune response after exposure to bacteria (Escherichia coli),using time-of-flight mass cytometry (CyTOF) and functional assays. We found an increase in innate and adaptive immune responses that were manifested by enhanced antibacterial activity of monocytes and macrophages,reduced in vivo bacterial load and a heightened T cell response in the mouse model of delayed-type hypersensitivity. By chemically ablating the sympathetic nervous system (SNS),we showed that the reward system's effects on immunity are,at least partly,mediated by the SNS. Thus,our findings establish a causal relationship between the activity of the VTA and the immune response to bacterial infection. View Publication

A large number of EBV immortalized LCLs have been generated and maintained in genetic/epidemiological studies as a perpetual source of DNA and as a surrogate in vitro cell model. Recent successes in reprograming LCLs into iPSCs have paved the way for generating more relevant in vitro disease models using this existing bioresource. However,the overall reprogramming efficiency and success rate remain poor and very little is known about the mechanistic changes that take place at the transcriptome and cellular functional level during LCL-to-iPSC reprogramming. Here,we report a new optimized LCL-to-iPSC reprogramming protocol using episomal plasmids encoding pluripotency transcription factors and mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate using this optimized protocol. Further,we investigated the transcriptional changes in mRNA and miRNA levels,using FC-abs ≥ 2.0 and FDR ≤ 0.05 cutoffs; 5,228 mRNAs and 77 miRNAs were differentially expressed during LCL-to-iPSC reprogramming. The functional enrichment analysis of the upregulated genes and activation of human pluripotency pathways in the reprogrammed iPSCs showed that the generated iPSCs possess transcriptional and functional profiles very similar to those of human ESCs. View Publication

The developmental outcomes of preimplantation mammalian embryos are regulated directly by the surrounding microenvironment,and inappropriate concentrations of amino acids,or the loss of amino acid-sensing mechanisms,can be detrimental and impact further development. A specific role for l-proline in the differentiation of embryonic stem (ES) cells,a cell population derived from the blastocyst,has been shown in culture. l-proline acts as a signalling molecule,exerting its effects through cell uptake and subsequent metabolism. Uptake in ES cells occurs predominantly through the sodium-coupled neutral amino acid transporter 2,Slc38a2 (SNAT2). Dynamic expression of amino acid transporters has been shown in the early mammalian embryo,reflecting functional roles for amino acids in embryogenesis. The expression of SNAT2 and family member Slc38a1 (SNAT1) was determined in mouse embryos from the 2-cell stage through to the early post-implantation pre-gastrulation embryo. Key changes in expression were validated in cell culture models of development. Both transporters showed temporal dynamic expression patterns and changes in intracellular localisation as differentiation progressed. Changes in transporter expression likely reflect different amino acid requirements during development. Findings include the differential expression of SNAT1 in the inner and outer cells of the compacted morula and nuclear localisation of SNAT2 in the trophectoderm and placental lineages. Furthermore,SNAT2 expression was up-regulated in the epiblast prior to primitive ectoderm formation,an expression pattern consistent with a role for the transporter in later developmental decisions within the pluripotent lineage. We propose that the differential expression of SNAT2 in the epiblast provides evidence for an l-proline-mediated mechanism contributing to the regulation of embryonic development. View Publication

Human pluripotent stem cells (hPSCs) have gained a solid foothold in basic research and drug industry as they can be used in??vitro to study human development and have potential to offer limitless supply of various somatic cell types needed in drug development. Although the hepatic differentiation of hPSCs has been extensively studied,only a little attention has been paid to the role of the extracellular matrix. In this study we used laminin-511,laminin-521,and fibronectin,found in human liver progenitor cells,as culture matrices for hPSC-derived definitive endoderm cells. We observed that laminin-511 and laminin-521 either alone or in combination support the hepatic specification and that fibronectin is not a vital matrix protein for the hPSC-derived definitive endoderm cells. The expression of the laminin-511/521-specific integrins increased during the definitive endoderm induction and hepatic specification. The hepatic cells differentiated on laminin matrices showed the upregulation of liver-specific markers both at mRNA and protein levels,secreted human albumin,stored glycogen,and exhibited cytochrome P450 enzyme activity and inducibility. Altogether,we found that laminin-511 and laminin-521 can be used as stage-specific matrices to guide the hepatic specification of hPSC-derived definitive endoderm cells. View Publication

Induced pluripotent stem cells (iPSCs) have the ability to differentiate in any specialized somatic cell type,which makes them an attractive tool for a wide variety of scientific approaches,including regenerative medicine. However,their pluripotent state and their growth in compact colonies render them difficult to access and,therefore,restrict delivery of specific agents for cell manipulation. Thus,our investigation focus was set on the evaluation of the capability of Layer-by-Layer (LbL) designed microcarriers to serve as a potential drug delivery system to iPSCs,as they offer several appealing advantages. Most notably,these carriers allow for the transport of active agents in a protected environment and for a rather specific delivery through surface modifications. As we could show,charge and mode of LbL carrier application as well as the size of the iPSC colonies determine the interaction with and the uptake rate by iPSCs. None of the examined conditions had an influence on iPSC colony properties such as colony morphology and size or maintenance of pluripotent properties. An overall interaction rate of LbL carriers with iPSCs of up to 20 % was achieved. Those data emphasize the applicability of LbL carriers for stem cell research. Additionally,the potential use of LbL carriers as a promising delivery tool for iPSCs was contrasted to viral particles and liposomes. The identified differences among those delivery tools have substantiated our major conclusion that LbL carrier uptake rate is influenced by characteristic features of the iPSC colonies (most notably colony size) in addition to their surface charges. View Publication

The AMP-activated protein kinase,AMPK,is an energy-sensing,metabolic switch implicated in various metabolic disorders; however,its role in inflammation is not well defined. We have previously shown that loss of AMPK exacerbates experimental autoimmune encephalomyelitis (EAE) disease severity. In this study,we investigated the mechanism through which AMPK modulates inflammatory disease like EAE. AMPKα1 knockout (α1KO) mice with EAE showed severe demyelination and inflammation in the brain and spinal cord compared with wild-type due to higher expression of proinflammatory Th17 cytokines,including IL-17,IL-23,and IL-1β,impaired blood-brain barrier integrity,and increased infiltration of inflammatory cells in the CNS. Infiltrated CD4 cells in the brains and spinal cords of α1KO with EAE were significantly higher compared with wild-type EAE and were characterized as IL-17 (IL-17 and GM-CSF double-positive) CD4 cells. Increased inflammatory response in α1KO mice was due to polarization of macrophages (Mϕ) to proinflammatory M1 type phenotype (IL-10(low)IL-23/IL-1β/IL-6(high)),and these M1 Mϕ showed stronger capacity to induce allogenic as well as Ag-specific (myelin oligodendrocyte glycoprotein [MOG]35-55) T cell response. Mϕ from α1KO mice also enhanced the encephalitogenic property of MOG35-55-primed CD4 T cells in B6 mice. The increased encephalitogenic MOG-restricted CD4(+) T cells were due to an autocrine effect of IL-1β/IL-23-mediated induction of IL-6 production in α1KO Mϕ,which in turn induce IL-17 and GM-CSF production in CD4 cells. Collectively,our data indicate that AMPK controls the inflammatory disease by regulating the M1 phenotype-Th17 axis in an animal model of multiple sclerosis. View Publication

p40,a Lactobacillus rhamnosus GG (LGG)-derived protein,transactivates epidermal growth factor receptor (EGFR) in intestinal epithelial cells,leading to amelioration of intestinal injury and inflammation. To elucidate mechanisms by which p40 regulates mucosal immunity to prevent inflammation,this study aimed to determine the effects and mechanisms of p40 on regulation of a proliferation-inducing ligand (APRIL) expression in intestinal epithelial cells for promoting immunoglobulin A (IgA) production. p40 upregulated April gene expression and protein production in mouse small intestine epithelial (MSIE) cells,which were inhibited by blocking EGFR expression and kinase activity. Enteroids from Egfr(fl/fl),but not Egfr(fl/fl)-Vil-Cre mice with EGFR specifically deleted in intestinal epithelial cells,exhibited increased April gene expression by p40 treatment. p40-conditioned media from MSIE cells increased B-cell class switching to IgA(+) cells and IgA production,which was suppressed by APRIL receptor-neutralizing antibodies. Treatment of B cells with p40 did not show any effects on IgA production. p40 treatment increased April gene expression and protein production in small intestinal epithelial cells,fecal IgA levels,IgA(+)B220(+),IgA(+)CD19(+),and IgA(+) plasma cells in lamina propria of Egfr(fl/fl),but not of Egfr(fl/fl)-Vil-Cre,mice. Thus p40 upregulates EGFR-dependent APRIL production in intestinal epithelial cells,which may contribute to promoting IgA production. View Publication