RESUMO
Targeting autophagy in cancer cells and in the tumor microenvironment are current goals of cancer therapy. However, components of canonical autophagy play roles in other biological processes, adding complexity to this goal. One such alternative function of autophagy proteins is LC3-associated phagocytosis (LAP), which functions in phagosome maturation and subsequent signaling events. Here, we show that impairment of LAP in the myeloid compartment, rather than canonical autophagy, induces control of tumor growth by tumor-associated macrophages (TAM) upon phagocytosis of dying tumor cells. Single-cell RNA sequencing (RNA-seq) analysis revealed that defects in LAP induce pro-inflammatory gene expression and trigger STING-mediated type I interferon responses in TAM. We found that the anti-tumor effects of LAP impairment require tumor-infiltrating T cells, dependent upon STING and the type I interferon response. Therefore, autophagy proteins in the myeloid cells of the tumor microenvironment contribute to immune suppression of T lymphocytes by effecting LAP.
Assuntos
Tolerância Imunológica/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Fagocitose/fisiologia , Animais , Autofagia/imunologia , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Tolerância Imunológica/imunologia , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Células Mieloides/metabolismo , Fagossomos/fisiologia , Linfócitos T/metabolismo , Microambiente Tumoral/fisiologiaRESUMO
Spatial transcriptomics technologies have recently emerged as a powerful tool for measuring spatially resolved gene expression directly in tissues sections, revealing cell types and their dysfunction in unprecedented detail. However, spatial transcriptomics technologies are limited in their ability to separate transcriptionally similar cell types and can suffer further difficulties identifying cell types in slide regions where transcript capture is low. Here, we describe a conceptually novel methodology that can computationally integrate spatial transcriptomics data with cell-type-informative paired tissue images, obtained from, for example, the reverse side of the same tissue section, to improve inferences of tissue cell type composition in spatial transcriptomics data. The underlying statistical approach is generalizable to any spatial transcriptomics protocol where informative paired tissue images can be obtained. We demonstrate a use case leveraging cell-type-specific immunofluorescence markers obtained on mouse brain tissue sections and a use case for leveraging the output of AI annotated H&E tissue images, which we used to markedly improve the identification of clinically relevant immune cell infiltration in breast cancer tissue. Thus, combining spatial transcriptomics data with paired tissue images has the potential to improve the identification of cell types and hence to improve the applications of spatial transcriptomics that rely on accurate cell type identification.
Assuntos
Modelos Estatísticos , Transcriptoma , Animais , Teorema de Bayes , Imunofluorescência , CamundongosRESUMO
Improvements in whole genome amplification (WGA) would enable new types of basic and applied biomedical research, including studies of intratissue genetic diversity that require more accurate single-cell genotyping. Here, we present primary template-directed amplification (PTA), an isothermal WGA method that reproducibly captures >95% of the genomes of single cells in a more uniform and accurate manner than existing approaches, resulting in significantly improved variant calling sensitivity and precision. To illustrate the types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a tool for mapping genome-wide interactions of mutagens with single living human cells at base-pair resolution. In addition, we utilized PTA for genome-wide off-target indel and structural variant detection in cells that had undergone CRISPR-mediated genome editing, establishing the feasibility for performing single-cell evaluations of biopsies from edited tissues. The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate WGA, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.
Assuntos
Variação Genética , Genoma Humano , Técnicas de Amplificação de Ácido Nucleico , Análise de Célula Única , Moldes Genéticos , Pareamento de Bases/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Humanos , Mutagênicos/metabolismo , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
The MYC proto-oncogenes (c-MYC, MYCN , MYCL ) are among the most deregulated oncogenic drivers in human malignancies including high-risk neuroblastoma, 50% of which are MYCN -amplified. Genetically engineered mouse models (GEMMs) based on the MYCN transgene have greatly expanded the understanding of neuroblastoma biology and are powerful tools for testing new therapies. However, a lack of c-MYC-driven GEMMs has hampered the ability to better understand mechanisms of neuroblastoma oncogenesis and therapy development given that c-MYC is also an important driver of many high-risk neuroblastomas. In this study, we report two transgenic murine neuroendocrine models driven by conditional c-MYC induction in tyrosine hydroxylase (Th) and dopamine ß-hydroxylase (Dbh)-expressing cells. c-MYC induction in Th-expressing cells leads to a preponderance of Pdx1 + somatostatinomas, a type of pancreatic neuroendocrine tumor (PNET), resembling human somatostatinoma with highly expressed gene signatures of δ cells and potassium channels. In contrast, c-MYC induction in Dbh-expressing cells leads to onset of neuroblastomas, showing a better transforming capacity than MYCN in a comparable C57BL/6 genetic background. The c-MYC murine neuroblastoma tumors recapitulate the pathologic and genetic features of human neuroblastoma, express GD2, and respond to anti-GD2 immunotherapy. This model also responds to DFMO, an FDA-approved inhibitor targeting ODC1, which is a known MYC transcriptional target. Thus, establishing c-MYC-overexpressing GEMMs resulted in different but related tumor types depending on the targeted cell and provide useful tools for testing immunotherapies and targeted therapies for these diseases.
RESUMO
BACKGROUND: Neuroblastoma is a common pediatric cancer, where preclinical studies suggest that a mesenchymal-like gene expression program contributes to chemotherapy resistance. However, clinical outcomes remain poor, implying we need a better understanding of the relationship between patient tumor heterogeneity and preclinical models. RESULTS: Here, we generate single-cell RNA-seq maps of neuroblastoma cell lines, patient-derived xenograft models (PDX), and a genetically engineered mouse model (GEMM). We develop an unsupervised machine learning approach ("automatic consensus nonnegative matrix factorization" (acNMF)) to compare the gene expression programs found in preclinical models to a large cohort of patient tumors. We confirm a weakly expressed, mesenchymal-like program in otherwise adrenergic cancer cells in some pre-treated high-risk patient tumors, but this appears distinct from the presumptive drug-resistance mesenchymal programs evident in cell lines. Surprisingly, however, this weak-mesenchymal-like program is maintained in PDX and could be chemotherapy-induced in our GEMM after only 24 h, suggesting an uncharacterized therapy-escape mechanism. CONCLUSIONS: Collectively, our findings improve the understanding of how neuroblastoma patient tumor heterogeneity is reflected in preclinical models, provides a comprehensive integrated resource, and a generalizable set of computational methodologies for the joint analysis of clinical and pre-clinical single-cell RNA-seq datasets.
Assuntos
Neuroblastoma , RNA-Seq , Análise de Célula Única , Neuroblastoma/genética , Neuroblastoma/patologia , Humanos , Animais , Análise de Célula Única/métodos , Camundongos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Resistencia a Medicamentos Antineoplásicos/genética , Transcriptoma , Análise da Expressão Gênica de Célula ÚnicaRESUMO
Neuroblastoma is a common pediatric cancer, where preclinical studies suggest that a mesenchymal-like gene expression program contributes to chemotherapy resistance. However, clinical outcomes remain poor, implying we need a better understanding of the relationship between patient tumor heterogeneity and preclinical models. Here, we generated single-cell RNA-seq maps of neuroblastoma cell lines, patient-derived xenograft models (PDX), and a genetically engineered mouse model (GEMM). We developed an unsupervised machine learning approach ('automatic consensus nonnegative matrix factorization' (acNMF)) to compare the gene expression programs found in preclinical models to a large cohort of patient tumors. We confirmed a weakly expressed, mesenchymal-like program in otherwise adrenergic cancer cells in some pre-treated high-risk patient tumors, but this appears distinct from the presumptive drug-resistance mesenchymal programs evident in cell lines. Surprisingly however, this weak-mesenchymal-like program was maintained in PDX and could be chemotherapy-induced in our GEMM after only 24 hours, suggesting an uncharacterized therapy-escape mechanism. Collectively, our findings improve the understanding of how neuroblastoma patient tumor heterogeneity is reflected in preclinical models, provides a comprehensive integrated resource, and a generalizable set of computational methodologies for the joint analysis of clinical and pre-clinical single-cell RNA-seq datasets.
RESUMO
We present the first comprehensive investigation of clonal hematopoiesis (CH) in 2,860 long-term survivors of pediatric cancer with a median follow-up time of 23.5 years. Deep sequencing over 39 CH-related genes reveals mutations in 15% of the survivors, significantly higher than the 8.5% in 324 community controls. CH in survivors is associated with exposures to alkylating agents, radiation, and bleomycin. Therapy-related CH shows significant enrichment in STAT3, characterized as a CH gene specific to survivors of Hodgkin lymphoma, and TP53. Single-cell profiling of peripheral blood samples revealed STAT3 mutations predominantly present in T cells and contributed by SBS25, a mutational signature associated with procarbazine exposure. Serial sample tracking reveals that larger clone size is a predictor for future expansion of age-related CH clones, whereas therapy-related CH remains stable decades after treatment. These data depict the distinct dynamics of these CH subtypes and support the need for longitudinal monitoring to determine the potential contribution to late effects. SIGNIFICANCE: This first comprehensive CH analysis in long-term survivors of pediatric cancer presents the elevated prevalence and therapy exposures/diagnostic spectrum associated with CH. Due to the contrasting dynamics of clonal expansion for age-related versus therapy-related CH, longitudinal monitoring is recommended to ascertain the long-term effects of therapy-induced CH in pediatric cancer survivors. See related commentary by Collord and Behjati, p. 811. This article is highlighted in the In This Issue feature, p. 799.
Assuntos
Hematopoiese Clonal , Doença de Hodgkin , Humanos , Criança , Hematopoese/genética , Mutação , Doença de Hodgkin/genética , Doença de Hodgkin/terapia , SobreviventesRESUMO
A lack of relevant genetic models and cell lines hampers our understanding of hepatoblastoma pathogenesis and the development of new therapies for this neoplasm. Here, we report an improved MYC-driven hepatoblastoma-like murine model that recapitulates the pathological features of embryonal type of hepatoblastoma, with transcriptomics resembling the high-risk gene signatures of the human disease. Single-cell RNA-sequencing and spatial transcriptomics identify distinct subpopulations of hepatoblastoma cells. After deriving cell lines from the mouse model, we map cancer dependency genes using CRISPR-Cas9 screening and identify druggable targets shared with human hepatoblastoma (e.g., CDK7, CDK9, PRMT1, PRMT5). Our screen also reveals oncogenes and tumor suppressor genes in hepatoblastoma that engage multiple, druggable cancer signaling pathways. Chemotherapy is critical for human hepatoblastoma treatment. A genetic mapping of doxorubicin response by CRISPR-Cas9 screening identifies modifiers whose loss-of-function synergizes with (e.g., PRKDC) or antagonizes (e.g., apoptosis genes) the effect of chemotherapy. The combination of PRKDC inhibition and doxorubicin-based chemotherapy greatly enhances therapeutic efficacy. These studies provide a set of resources including disease models suitable for identifying and validating potential therapeutic targets in human high-risk hepatoblastoma.
Assuntos
Hepatoblastoma , Neoplasias Hepáticas , Humanos , Animais , Camundongos , Hepatoblastoma/tratamento farmacológico , Hepatoblastoma/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Linhagem Celular , Oncogenes , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genéticaRESUMO
Ribbon synapses continuously transmit graded membrane potential changes into changes of synaptic vesicle exocytosis and rely on intense synaptic membrane trafficking. The synaptic ribbon is considered central to this process. In the present study we asked whether tonically active ribbon synapses are associated with the generation of certain lipids, specifically the highly active signaling phospholipid phosphatidic acid (PA). Using PA-sensor proteins, we demonstrate that PA is enriched at mouse retinal ribbon synapses in close vicinity to the synaptic ribbon in situ. As shown by heterologous expression, RIBEYE, a main component of synaptic ribbons, is responsible for PA binding at synaptic ribbons. Furthermore, RIBEYE is directly involved in the synthesis of PA. Using various independent substrate binding and enzyme assays, we demonstrate that the B domain of RIBEYE possesses lysophosphatidic acid (LPA) acyltransferase (LPAAT) activity, which leads to the generation of PA from LPA. Since an LPAAT-deficient RIBEYE mutant does not recruit PA-binding proteins to artificial synaptic ribbons, whereas wild-type RIBEYE supports PA binding, we conclude that the LPAAT activity of the RIBEYE(B) domain is a physiologically relevant source of PA generation at the synaptic ribbon. We propose that PA generated at synaptic ribbons likely facilitates synaptic vesicle trafficking.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ácidos Fosfatídicos/metabolismo , Sinapses/ultraestrutura , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Aciltransferases/metabolismo , Oxirredutases do Álcool/química , Animais , Células COS , Chlorocebus aethiops , Proteínas Correpressoras , Concanavalina A/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lipossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Proteínas do Tecido Nervoso/química , Fosfolipídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-raf/genética , Retina/citologia , Células Bipolares da Retina/metabolismo , Células Bipolares da Retina/ultraestrutura , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Membranas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Transfecção , Triptofano/genética , Triptofano/metabolismoRESUMO
Treatment of acute lymphoblastic leukemia (ALL) necessitates continuous risk assessment of leukemic disease burden and infections that arise in the setting of immunosuppression. This study was performed to assess the feasibility of a hybrid capture next-generation sequencing panel to longitudinally measure molecular leukemic disease clearance and microbial species abundance in 20 pediatric patients with ALL throughout induction chemotherapy. This proof of concept helps establish a technical and conceptual framework that we anticipate will be expanded and applied to additional patients with leukemia, as well as extended to additional cancer types. Molecular monitoring can help accelerate the attainment of insights into the temporal biology of host-microbe-leukemia interactions, including how those changes correlate with and alter anticancer therapy efficacy. We also anticipate that fewer invasive bone marrow examinations will be required, as these methods improve with standardization and are validated for clinical use.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Criança , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Análise de Sequência de DNARESUMO
Guanylate cyclase activating protein 2 (GCAP2) is a recoverin-like Ca2+-sensor protein known to modulate guanylate cyclase activity in photoreceptor outer segments. GCAP2 is also present in photoreceptor ribbon synapses where its function is unknown. Synaptic ribbons are active zone-associated presynaptic structures in the tonically active photoreceptor ribbon synapses and contain RIBEYE as a unique and major protein component. In the present study, we demonstrate by various independent approaches that GCAP2 specifically interacts with RIBEYE in photoreceptor synapses. We show that the flexible hinge 2 linker region of RIBEYE(B) domain that connects the nicotinamide adenine dinucleotide (NADH)-binding subdomain with the substrate-binding subdomain (SBD) binds to the C terminus of GCAP2. We demonstrate that the RIBEYE-GCAP2 interaction is induced by the binding of NADH to RIBEYE. RIBEYE-GCAP2 interaction is modulated by the SBD. GCAP2 is strongly expressed in synaptic terminals of light-adapted photoreceptors where GCAP2 is found close to synaptic ribbons as judged by confocal microscopy and proximity ligation assays. Virus-mediated overexpression of GCAP2 in photoreceptor synaptic terminals leads to a reduction in the number of synaptic ribbons. Therefore, GCAP2 is a prime candidate for mediating Ca2+-dependent dynamic changes of synaptic ribbons in photoreceptor synapses.
Assuntos
Proteínas Ativadoras de Guanilato Ciclase/metabolismo , NAD/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Sinapses/metabolismo , Oxirredutases do Álcool , Sequência de Aminoácidos , Animais , Bovinos , Células Cultivadas , Proteínas Correpressoras , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/genética , Técnicas In Vitro , Camundongos , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Terminações Pré-Sinápticas/metabolismo , Ligação Proteica , Conformação Proteica , Retina/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Survival in high-risk pediatric neuroblastoma has remained around 50% for the last 20 years, with immunotherapies and targeted therapies having had minimal impact. Here, we identify the small molecule CX-5461 as selectively cytotoxic to high-risk neuroblastoma and synergistic with low picomolar concentrations of topoisomerase I inhibitors in improving survival in vivo in orthotopic patient-derived xenograft neuroblastoma mouse models. CX-5461 recently progressed through phase I clinical trial as a first-in-human inhibitor of RNA-POL I. However, we also use a comprehensive panel of in vitro and in vivo assays to demonstrate that CX-5461 has been mischaracterized and that its primary target at pharmacologically relevant concentrations, is in fact topoisomerase II beta (TOP2B), not RNA-POL I. This is important because existing clinically approved chemotherapeutics have well-documented off-target interactions with TOP2B, which have previously been shown to cause both therapy-induced leukemia and cardiotoxicity-often-fatal adverse events, which can emerge several years after treatment. Thus, while we show that combination therapies involving CX-5461 have promising anti-tumor activity in vivo in neuroblastoma, our identification of TOP2B as the primary target of CX-5461 indicates unexpected safety concerns that should be examined in ongoing phase II clinical trials in adult patients before pursuing clinical studies in children.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , Indóis/uso terapêutico , Morfolinas/uso terapêutico , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Pirimidinas/uso terapêutico , Sulfonamidas/uso terapêutico , Animais , Benzotiazóis , Western Blotting , Linhagem Celular Tumoral , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Imunofluorescência , Camundongos , Camundongos Nus , Simulação de Dinâmica Molecular , Naftiridinas , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Importance: Bloodstream infection (BSI) is a common, life-threatening complication of treatment for cancer. Predicting BSI before onset of clinical symptoms would enable preemptive therapy, but there is no reliable screening test. Objective: To estimate sensitivity and specificity of plasma microbial cell-free DNA sequencing (mcfDNA-seq) for predicting BSI in patients at high risk of life-threatening infection. Design, Setting, and Participants: A prospective pilot cohort study of mcfDNA-seq for predicting BSI in pediatric patients (<25 years of age) with relapsed or refractory cancers at St Jude Children's Research Hospital, a specialist quaternary pediatric hematology-oncology referral center. Remnant clinical blood samples were collected during chemotherapy and hematopoietic cell transplantation. Samples collected during the 7 days before and at onset of BSI episodes, along with negative control samples from study participants, underwent blinded testing using a mcfDNA-seq test in a Clinical Laboratory Improvement Amendments/College of American Pathologists-approved laboratory. Main Outcomes and Measures: The primary outcomes were sensitivity of mcfDNA-seq for detecting a BSI pathogen during the 3 days before BSI onset and specificity of mcfDNA-seq in the absence of fever or infection in the preceding or subsequent 7 days. Results: Between August 9, 2017, and June 4, 2018, 47 participants (27 [57%] male; median age [IQR], 10 [5-14] years) were enrolled; 19 BSI episodes occurred in 12 participants, and predictive samples were available for 16 episodes, including 15 bacterial BSI episodes. In the 3 days before the onset of infection, predictive sensitivity of mcfDNA-seq was 75% for all BSIs (12 of 16; 95% CI, 51%-90%) and 80% (12 of 15; 95% CI, 55%-93%) for bacterial BSIs. The specificity of mcfDNA-seq, evaluated on 33 negative control samples from enrolled participants, was 82% (27 of 33; 95% CI, 66%-91%) for any bacterial or fungal organism and 91% (30 of 33; 95% CI, 76%-97%) for any common BSI pathogen, and the concentration of pathogen DNA was lower in control than predictive samples. Conclusions and Relevance: A clinically relevant pathogen can be identified by mcfDNA-seq days before the onset of BSI in a majority of episodes, potentially enabling preemptive treatment. Clinical application appears feasible pending further study. Trial Registration: ClinicalTrials.gov identifier: NCT03226158.
Assuntos
Infecções Relacionadas a Cateter/sangue , Ácidos Nucleicos Livres/sangue , Neoplasias/sangue , Sepse/sangue , Adolescente , Infecções Relacionadas a Cateter/complicações , Infecções Relacionadas a Cateter/microbiologia , Infecções Relacionadas a Cateter/patologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Neoplasias/complicações , Neoplasias/microbiologia , Neoplasias/patologia , Sepse/complicações , Sepse/microbiologia , Sepse/patologia , Análise de Sequência de DNARESUMO
Identification of genomic and epigenomic determinants of drug resistance provides important insights for improving cancer treatment. Using agnostic genome-wide interrogation of mRNA and miRNA expression, DNA methylation, SNPs, CNAs and SNVs/Indels in primary human acute lymphoblastic leukemia cells, we identified 463 genomic features associated with glucocorticoid resistance. Gene-level aggregation identified 118 overlapping genes, 15 of which were confirmed by genome-wide CRISPR screen. Collectively, this identified 30 of 38 (79%) known glucocorticoid-resistance genes/miRNAs and all 38 known resistance pathways, while revealing 14 genes not previously associated with glucocorticoid-resistance. Single cell RNAseq and network-based transcriptomic modelling corroborated the top previously undiscovered gene, CELSR2. Manipulation of CELSR2 recapitulated glucocorticoid resistance in human leukemia cell lines and revealed a synergistic drug combination (prednisolone and venetoclax) that mitigated resistance in mouse xenograft models. These findings illustrate the power of an integrative genomic strategy for elucidating genes and pathways conferring drug resistance in cancer cells.
Assuntos
MicroRNAs , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animais , Resistencia a Medicamentos Antineoplásicos/genética , Genômica , Glucocorticoides/farmacologia , Humanos , Camundongos , MicroRNAs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológicoRESUMO
Synaptic ribbons are large, dynamic structures in the active zone complex of ribbon synapses and important for the physiological properties of these tonically active synapses. RIBEYE is a unique and major protein component of synaptic ribbons. The aim of the present study was to understand how the synaptic ribbon is built and how the construction of the ribbon could contribute to its ultrastructural plasticity. In the present study, we demonstrate that RIBEYE self-associates using different independent approaches (yeast two-hybrid analyses, protein pull downs, synaptic ribbon-RIBEYE interaction assays, coaggregation experiments, transmission electron microscopy and immunogold electron microscopy). The A-domain [RIBEYE(A)] and B-domain [RIBEYE(B)] of RIBEYE contain five distinct sites for RIBEYE-RIBEYE interactions. Three interaction sites are present in the A-domain of RIBEYE and mediate RIBEYE(A)-RIBEYE(A) homodimerization and heterodimerization with the B-domain. The docking site for RIBEYE(A) on RIBEYE(B) is topographically and functionally different from the RIBEYE(B) homodimerization interface and is negatively regulated by nicotinamide adenine dinucleotide. The identified multiple RIBEYE-RIBEYE interactions have the potential to build the synaptic ribbon: heterologously expressed RIBEYE forms large electron-dense aggregates that are in part physically associated with surrounding vesicles and membrane compartments. These structures resemble spherical synaptic ribbons. These ribbon-like structures coassemble with the active zone protein bassoon, an interaction partner of RIBEYE at the active zone of ribbon synapses, emphasizing the physiological relevance of these RIBEYE-containing aggregates. Based on the identified multiple RIBEYE-RIBEYE interactions, we provide a molecular mechanism for the dynamic assembly of synaptic ribbons from individual RIBEYE subunits.
Assuntos
Oxirredutases do Álcool/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Sinapses/fisiologia , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Animais , Bactérias/metabolismo , Células COS , Linhagem Celular , Chlorocebus aethiops , Proteínas Correpressoras , Dimerização , Imuno-Histoquímica , Microscopia Eletrônica , NAD/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Pichia/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/fisiologia , Estrutura Terciária de Proteína , Retina/metabolismo , Células-Tronco/metabolismo , Sinapses/ultraestrutura , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Intra-tumor heterogeneity stems from genetic, epigenetic, functional, and environmental differences among tumor cells. A major source of genetic heterogeneity comes from DNA sequence differences and/or whole chromosome and focal copy number variations (CNVs). Whole chromosome CNVs are caused by chromosomal instability (CIN) that is defined by a persistently high rate of chromosome mis-segregation. Accordingly, CIN causes constantly changing karyotypes that result in extensive cell-to-cell genetic heterogeneity. How the genetic heterogeneity caused by CIN influences gene expression in individual cells remains unknown. METHODS: We performed single-cell RNA sequencing on a chromosomally unstable glioblastoma cancer stem cell (CSC) line and a control normal, diploid neural stem cell (NSC) line to investigate the impact of CNV due to CIN on gene expression. From the gene expression data, we computationally inferred large-scale CNVs in single cells. Also, we performed copy number adjusted differential gene expression analysis between NSCs and glioblastoma CSCs to identify copy number dependent and independent differentially expressed genes. RESULTS: Here, we demonstrate that gene expression across large genomic regions scales proportionally to whole chromosome copy number in chromosomally unstable CSCs. Also, we show that the differential expression of most genes between normal NSCs and glioblastoma CSCs is largely accounted for by copy number alterations. However, we identify 269 genes whose differential expression in glioblastoma CSCs relative to normal NSCs is independent of copy number. Moreover, a gene signature derived from the subset of genes that are differential expressed independent of copy number in glioblastoma CSCs correlates with tumor grade and is prognostic for patient survival. CONCLUSIONS: These results demonstrate that CIN is directly responsible for gene expression changes and contributes to both genetic and transcriptional heterogeneity among glioblastoma CSCs. These results also demonstrate that the expression of some genes is buffered against changes in copy number, thus preserving some consistency in gene expression levels from cell-to-cell despite the continuous change in karyotype driven by CIN. Importantly, a gene signature derived from the subset of genes whose expression is buffered against copy number alterations correlates with tumor grade and is prognostic for patient survival that could facilitate patient diagnosis and treatment.
Assuntos
Instabilidade Cromossômica , Glioblastoma/genética , Glioblastoma/patologia , Células-Tronco Neoplásicas/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Gradação de Tumores , Células-Tronco Neoplásicas/patologia , Células-Tronco Neurais/metabolismo , Análise de SobrevidaRESUMO
The cerebellum develops from a restricted number of cell types that precisely organize to form the circuitry that controls sensory-motor coordination and some higher-order cognitive processes. To acquire an enhanced understanding of the molecular processes that mediate cerebellar development, we performed single-cell RNA-sequencing of 39,245 murine cerebellar cells at twelve critical developmental time points. Using recognized lineage markers, we confirmed that the single-cell data accurately recapitulate cerebellar development. We then followed distinct populations from emergence through migration and differentiation, and determined the associated transcriptional cascades. After identifying key lineage commitment decisions, focused analyses uncovered waves of transcription factor expression at those branching points. Finally, we created Cell Seek, a flexible online interface that facilitates exploration of the dataset. Our study provides a transcriptional summarization of cerebellar development at single-cell resolution that will serve as a valuable resource for future investigations of cerebellar development, neurobiology, and disease.
Assuntos
Diferenciação Celular , Movimento Celular , Neurogênese , Transcriptoma , Animais , Cerebelo/citologia , Cerebelo/embriologia , Embrião de Mamíferos , Desenvolvimento Embrionário , Feminino , Camundongos , Camundongos Endogâmicos ICR , Análise de Célula ÚnicaRESUMO
Optic neuritis is one of the first manifestations of multiple sclerosis. Its pathogenesis is incompletely understood, but considered to be initiated by an auto-immune response directed against myelin sheaths of the optic nerve. Here, we demonstrate in two frequently used and well-validated mouse models of optic neuritis that ribbon synapses in the myelin-free retina are targeted by an auto-reactive immune system even before alterations in the optic nerve have developed. The auto-immune response is directed against two adhesion proteins (CASPR1/CNTN1) that are present both in the paranodal region of myelinated nerves as well as at retinal ribbon synapses. This occurs in parallel with altered synaptic vesicle cycling in retinal ribbon synapses and altered visual behavior before the onset of optic nerve demyelination. These findings indicate that early synaptic dysfunctions in the retina contribute to the pathology of optic neuritis in multiple sclerosis.
Assuntos
Autoimunidade , Esclerose Múltipla/patologia , Células Fotorreceptoras de Vertebrados/metabolismo , Sinapses/metabolismo , Animais , Anticorpos/metabolismo , Bovinos , Ativação do Complemento , Contactina 1/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/líquido cefalorraquidiano , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/sangue , Esclerose Múltipla/líquido cefalorraquidiano , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Neurite Óptica/metabolismo , Neurite Óptica/patologia , Células Fotorreceptoras de Vertebrados/ultraestrutura , Retina/metabolismo , Sinapses/ultraestrutura , Vesículas Sinápticas/metabolismoRESUMO
The embryonic site of definitive hematopoietic stem cell (dHSC) origination has been debated for decades. Although an intra-embryonic origin is well supported, the yolk sac (YS) contribution to adult hematopoiesis remains controversial. The same developmental origin makes it difficult to identify specific markers that discern between an intraembryonic versus YS-origin using a lineage trace approach. Additionally, the highly migratory nature of blood cells and the inability of pre-circulatory embryonic cells (i.e., 5-7 somite pairs (sp)) to robustly engraft in transplantation, even after culture, has precluded scientists from properly answering these questions. Here we report robust, multi-lineage and serially transplantable dHSC activity from cultured 2-7sp murine embryonic explants (Em-Ex). dHSC are undetectable in 2-7sp YS explants. Additionally, the engraftment from Em-Ex is confined to an emerging CD31+CD45+c-Kit+CD41- population. In sum, our work supports a model in which the embryo, not the YS, is the major source of lifelong definitive hematopoiesis.
Assuntos
Linhagem da Célula , Embrião de Mamíferos/citologia , Hematopoese , Células-Tronco Hematopoéticas/fisiologia , Saco Vitelino/citologia , Animais , Movimento Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Circular RNAs comprise a poorly understood new class of noncoding RNA. In this study, we used a combination of targeted deletion, high-resolution splicing detection, and single-cell sequencing to deeply probe ASXL1 circular splicing. We found that efficient circular splicing required the canonical transcriptional start site and inverted AluSx elements. Sequencing-based interrogation of isoforms after ASXL1 overexpression identified promiscuous linear splicing between all exons, with the two most abundant non-canonical linear products skipping the exons that produced the circular isoforms. Single-cell sequencing revealed a strong preference for either the linear or circular ASXL1 isoforms in each cell, and found the predominant exon skipping product is frequently co-expressed with its reciprocal circular isoform. Finally, absolute quantification of ASXL1 isoforms confirmed our findings and suggests that standard methods overestimate circRNA abundance. Taken together, these data reveal a dynamic new view of circRNA genesis, providing additional framework for studying their roles in cellular biology.