技术资料

Early cognitive deficits in Alzheimer's disease (AD) seem to be correlated to dysregulation of glutamate receptors evoked by amyloid-beta (Aβ) peptide. Aβ interference with the activity of N-methyl-d-aspartate receptors (NMDARs) may be a relevant factor for Aβ-induced mitochondrial toxicity and neuronal dysfunction. To evaluate the role of mitochondria in NMDARs activation mediated by Aβ,we followed in situ single-cell simultaneous measurement of cytosolic free Ca(2+)(Cai(2+)) and mitochondrial membrane potential in primary cortical neurons. Our results show that direct exposure to Aβ + NMDA largely increased Cai(2+) and induced immediate mitochondrial depolarization,compared with Aβ or NMDA alone. Mitochondrial depolarization induced by rotenone strongly inhibited the rise in Cai(2+) evoked by Aβ or NMDA,suggesting that mitochondria control Ca(2+) entry through NMDARs. However,incubation with rotenone did not preclude mitochondrial Ca(2+) (mitCa(2+)) retention in cells treated with Aβ. Aβ-induced Cai(2+) and mitCa(2+) rise were inhibited by ifenprodil,an antagonist of GluN2B-containing NMDARs. Exposure to Aβ + NMDA further evoked a higher mitCa(2+) retention,which was ameliorated in GluN2B(-/-) cortical neurons,largely implicating the involvement of this NMDAR subunit. Moreover,pharmacologic inhibition of endoplasmic reticulum (ER) inositol-1,4,5-triphosphate receptor (IP3R) and mitCa(2+) uniporter (MCU) evidenced that Aβ + NMDA-induced mitCa(2+) rise involves ER Ca(2+) release through IP3R and mitochondrial entry by the MCU. Altogether,data highlight mitCa(2+) dyshomeostasis and subsequent dysfunction as mechanisms relevant for early neuronal dysfunction in AD linked to Aβ-mediated GluN2B-composed NMDARs activation. View Publication

SummaryMesenchymal stromal cells (MSCs) have been modified via genetic or pharmacological engineering into potent antigen-presenting cells-like capable of priming responding CD8 T cells. In this study,our screening of a variant library of Accum molecule revealed a molecule (A1) capable of eliciting antigen cross-presentation properties in MSCs. A1-reprogrammed MSCs (ARM) exhibited improved soluble antigen uptake and processing. Our comprehensive analysis,encompassing cross-presentation assays and molecular profiling,among other cellular investigations,elucidated A1’s impact on endosomal escape,reactive oxygen species production,and cytokine secretion. By evaluating ARM-based cellular vaccine in mouse models of lymphoma and melanoma,we observe significant therapeutic potency,particularly in allogeneic setting and in combination with anti-PD-1 immune checkpoint inhibitor. Overall,this study introduces a strong target for developing an antigen-adaptable vaccination platform,capable of synergizing with immune checkpoint blockers to trigger tumor regression,supporting further investigation of ARMs as an effective and versatile anti-cancer vaccine. Graphical abstract Highlights•Treatment with A1/antigen mix reprograms MSCs into antigen-presenting cells•The antigen cross-presenting ability of ARM cells require ROS and UPR•ARMs synergize with immune-checkpoint inhibitors in priming potent antitumoral activity Classification Description: Immunology; Pharmaceutical engineering; Cancer View Publication

Amyotrophic lateral sclerosis (ALS) involves motor neuron death due to mislocalized TDP-43. Pathologic TDP-43 associates with stress granules (SGs),and lowering the SG-associated protein ataxin-2 (ATXN2) using Atxn2-targeting antisense oligonucleotides prolongs survival in TAR4/4 sporadic ALS mice but failed in clinical trials likely due to poor target engagement. Here we show that an AAV with potent motor neuron transduction delivering Atxn2-targeting miRNAs reduces Atxn2 throughout the central nervous system at doses 40x lower than published work. In TAR4/4 mice,miAtxn2 increased survival (50%) and strength,and reduced motor neuron death,inflammation,and phosphorylated TDP-43. TAR4/4 transcriptomic dysregulation recapitulated ALS gene signatures that were rescued by miAtxn2,identifying potential therapeutic mechanisms and biomarkers. In slow progressing hemizygous mice,miAtxn2 slowed disease progression,and in ALS patient-derived lower motor neurons,our AAV vector transduced >95% of cells and potently reduced ATXN2 at MOI 4 logs lower than previously reported. These data support AAV-RNAi targeting ATXN2 as a translatable therapy for sporadic ALS. Amado et al. develop a gene therapy for sporadic ALS using motor neuron-targeting AAVs to deliver RNAi targeting ataxin-2. In a mouse model,survival,strength,and disease-related pathology are improved; and human motor neurons are strongly transduced. View Publication

Characterization of molecules with tightly controlled expression patterns during differentiation represents an approach to understanding regulation of hematopoietic stem cell commitment. The multidrug resistance-1 (MDR1) gene product,P-glycoprotein,and the breast cancer resistance protein (BCRP) are expressed differentially during hematopoiesis,with the highest levels in primitive bone marrow stem cell populations that are CD34(low) and CD34(-),respectively. Roles for ATP-binding cassette (ABC) transporter superfamily members in conferring drug resistance have been extensively described. However,recent hematopoietic overexpression studies have begun to reveal previously unknown roles for ABC transporter function in normal and malignant hematopoiesis. Expression of MDR1 and BCRP transporters in the myeloid lineage has been reported in blasts from acute myeloid leukemia,but very low to undetectable in normal myelomonocytic cells. Retroviral-mediated dysregulated expression of the MDR1 transporter resulted in increased hematopoietic repopulating activity and myeloproliferative disease in mice. A distinct functional role for the BCRP transporter as a negative regulator of hematopoietic repopulating activity has recently been demonstrated using the same approach. Additionally,the presence of BCRP expression specifically on hematopoietic side-population stem cells and neural stem/progenitors,makes BCRP an attractive candidate marker for isolation of stem cells with the ability to respond to diverse environmental cues. Regulation of stem cell biology by ABC transporters has emerged as an important new field of investigation. In light of these findings,it will be critical to further characterize this family of proteins in hematopoietic lineage-restricted stem cells and in pluripotent stem cells capable of crossing lineage barriers. View Publication

Acute coronary syndrome (ACS) is the most severe clinical manifestation of coronary heart disease.We performed an epigenome-wide analysis of circulating CD4+ and CD8+ T cells isolated from ACS patients and healthy subjects (HS),enrolled in the DIANA clinical trial,by reduced-representation bisulphite sequencing (RRBS). In CD4+ T cells,we identified 61 differentially methylated regions (DMRs) associated with 57 annotated genes (53% hyper- and 47% hypo-methylated) by comparing ACS patients vs HS. In CD8+ T cells,we identified 613 DMRs associated with 569 annotated genes (28% hyper- and 72% hypo-methylated) in ACS patients as compared to HS. In CD4+vs CD8+ T cells of ACS patients we identified 175 statistically significant DMRs associated with 157 annotated genes (41% hyper- and 59% hypo-methylated). From pathway analyses,we selected six differentially methylated hub genes (NFATC1,TCF7,PDGFA,PRKCB,PRKCZ,ABCA1) and assessed their expression levels by q-RT-PCR. We found an up-regulation of selected genes in ACS patients vs HS (P < 0.001). ABCA1,TCF7,PDGFA,and PRKCZ gene expression was positively associated with CK-MB serum concentrations (r = 0.75,P = 0.03; r = 0.760,P = 0.029; r = 0.72,P = 0.044; r = 0.74,P = 0.035,respectively).This pilot study is the first single-base resolution map of DNA methylome by RRBS in CD4+ and CD8+ T cells and provides specific methylation signatures to clarify the role of aberrant methylation in ACS pathogenesis,thus supporting future research for novel epigenetic-sensitive biomarkers in the prevention and early diagnosis of this pathology. View Publication

B cell-activating factor of the tumor necrosis factor (TNF) family,or BAFF,is mainly produced in monocytes and dendritic cells,and indispensable for proliferation,differentiation and survival of B cells. BAFF is a type II membrane-bound protein and the extracellular C-terminal fragment is released from the cells as soluble BAFF (sBAFF),which binds to specific receptors on B cells. Accumulating evidence suggests that BAFF plays an important role in the pathogenesis of autoimmune diseases,such as systemic lupus erythematosus (SLE). In this study,we developed a sensitive sandwich ELISA system to quantify the amount of sBAFF using our own mAb. Treatment of peripheral T cells of SLE patients with an anti-CD3 antibody triggered robust expression of BAFF and subsequent release of sBAFF from the cells. On the other hand,the stimulus induced only marginal elevation of sBAFF from normal T cells. These data indicate that BAFF is expressed in T cells upon stimulation at least under pathological conditions. Expression of BAFF was also largely induced in a human T cell line,Loucy (American Type Tissue Collection CRL-2629),in response to several stimuli,while other T cell lines so far examined produced the cytokine almost constitutively. These data suggest that Loucy recapitulates some of the characteristics of SLE T cells. Investigation of molecular and cellular mechanisms of production of BAFF in Loucy demonstrated that expression of BAFF was regulated through a signal transduction pathway which involves c-jun NH2-terminal kinase and p38,and that shedding of BAFF was catalyzed by a membrane-bound protease,furin. View Publication

The generation of amyloid-beta peptide (Abeta) and its accumulation in amyloid plaques are generally recognized as key characteristics of Alzheimer's disease. A number of reports have indicated that Abeta can regulate the proliferation of neural precursor cells and adult neurogenesis,suggesting that this may underpin the cognitive decline and compromised olfaction also associated with the condition. Here we report that Abeta(1-42) treatment both in vitro and in vivo,as well as endogenous generation of Abeta in C100 and APP/PS1 transgenic models of Alzheimer's disease,stimulate neurogenesis of young adult subventricular zone precursors. The neurogenic effect of Abeta(1-42) was found to require expression of the p75 neurotrophin receptor (p75(NTR)) by the precursor cells,and activation of p75(NTR) by metalloprotease cleavage. However,precursors from 12-month-old APP/PS1 mice failed to respond to Abeta(1-42). Our results suggest that overstimulation of p75(NTR)-positive progenitors during early life might result in depletion of the stem cell pool and thus a more rapid decline in basal neurogenesis. This,in turn,could lead to impaired neurogenic function in later life. View Publication

Some cases of pre-B cell acute lymphoblastic leukemia (pre-B-ALL) are caused by the Philadelphia (Ph) chromosome-encoded BCR-ABL oncogene,and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL-mediated transformation in vitro; however,the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph+ pre-B-ALL,we found that deletion of both Pik3r1 and Pik3r2,genes encoding class IA PI3K regulatory isoforms,severely impaired transformation. BCR-ABL-dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes,but the cells were smaller,proliferated more slowly,and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However,they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR,PI-103,was more effective than rapamycin at suppressing proliferation of mouse pre-B-ALL and human CD19+CD34+)Ph+ ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K,alone or together with mTOR,in the treatment of Ph+ ALL. View Publication

Familial hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiac disorder linked to arrhythmia and sudden cardiac death. While the causes of HCM have been identified as genetic mutations in the cardiac sarcomere,the pathways by which sarcomeric mutations engender myocyte hypertrophy and electrophysiological abnormalities are not understood. To elucidate the mechanisms underlying HCM development,we generated patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a ten-member family cohort carrying a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Diseased iPSC-CMs recapitulated numerous aspects of the HCM phenotype including cellular enlargement and contractile arrhythmia at the single-cell level. Calcium (Ca2+) imaging indicated dysregulation of Ca2+ cycling and elevation in intracellular Ca2+ ([Ca2+] i) are central mechanisms for disease pathogenesis. Pharmacological restoration of Ca2+ homeostasis prevented development of hypertrophy and electrophysiological irregularities. We anticipate that these findings will help elucidate the mechanisms underlying HCM development and identify novel therapies for the disease. textcopyright 2013 Elsevier Inc. View Publication

HIV-1 replication in simian cells is restricted at an early postentry step because of the presence of an inhibitory cellular factor. This block reduces the usefulness of HIV-1-based lentiviral vectors in primate animal models. Here,we demonstrate that substitution of the cyclophilin A (CyPA) binding region in the capsid of an HIV-1-based lentiviral vector (LV) with that of the macrophage tropic HIV-1 Ba-L resulted in a vector that was resistant to the inhibitory effect and efficiently transduced simian cells. Notably,the chimeric gag LV efficiently transduced primary simian hematopoietic progenitor cells,a critical cellular target in gene therapy. The alterations in the CyPA binding region did not affect CyPA incorporation; however,transduction by the gag chimeric LV seemed to be relatively insensitive to cyclosporin A,indicating that it does not require CyPA for early postentry steps. In dual infection experiments,the gag chimeric LV failed to remove the block to transduction of the WT LV,suggesting that the gag chimeric LV did not saturate the inhibitory simian cellular factor. These data suggest that the CyPA binding region of capsid contains a viral determinant involved in the postentry restriction of HIV-1-based lentiviral vectors. Overall,the findings demonstrate that the host range of HIV-1-based LV can be altered by modifications in the packaging construct. View Publication

Prior experimental strategies to induce transplantation tolerance have focused largely on modifying adaptive immunity. However,less is known concerning the role of innate immune signaling in the induction of transplantation tolerance. Using a highly immunogenic murine skin transplant model that resists transplantation tolerance induction when innate immunity is preserved,we show that absence of MyD88,a key innate Toll like receptor signal adaptor,abrogates this resistance and facilitates inducible allograft acceptance. In our model,absence of MyD88 impairs inflammatory dendritic cell responses that reduce T cell activation. This effect increases T cell susceptibility to suppression mediated by CD4+ CD25+ regulatory T cells. Therefore,this study provides evidence that absence of MyD88 promotes inducible allograft acceptance and implies that inhibiting innate immunity may be a potential,clinically relevant strategy to facilitate transplantation tolerance. View Publication

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