scholarly journals Aicardi-Goutières syndrome associated mutation at ADAR1 gene locus activates innate immune response in mouse brain

2021 ◽  
Author(s):  
Xinfeng Guo ◽  
Clayton A. Wiley ◽  
Richard A. Steinman ◽  
Yi Sheng ◽  
Beihong Ji ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Rna Editing ◽  
Mutant Mouse ◽  
Cell Types ◽  
Blue Staining ◽  
Sequencing Analysis ◽  
Protein Coding ◽  
Brain Neurons ◽  
Elevated Type ◽  
The Brain

Abstract BackgroundAicardi-Goutières syndrome (AGS) is a severe infant or juvenile-onset autoimmune disease characterized by inflammatory encephalopathy with an elevated Type 1 interferon-stimulated gene (ISG) expression signature in the brain. Mutations in seven different protein-coding genes, all linked to DNA/RNA metabolism or sensing, have been identified in AGS patients, but none of them has been demonstrated to activate IFN pathway in the brain of an animal. The molecular mechanism of inflammatory encephalopathy in AGS has not been well defined. Adenosine Deaminase Acting on RNA 1 (ADAR1) is one of the AGS associated genes. It carries out A-to-I RNA editing that converts adenosine to inosine at double stranded RNA regions. Whether an AGS associated mutation in ADAR1 activates IFN pathway and causes autoimmune pathogenesis in the brain is yet to be determined.MethodsMutations in the ADAR1 gene found in AGS patients were introduced into mouse genome via CRISPR/Case9 technology. Molecular activities of the specific p.K999N mutation were investigated by measuring the RNA editing levels in brain mRNA substrates of ADAR1 through RNA sequencing analysis. IFN pathway activation in the brain was assessed by measuring ISG expression at the mRNA and protein level through real-time RT-PCR and Luminex assays respectively. The locations in the brain and neural cell types that express ISGs were determined by RNA in situ hybridization (ISH). Potential AGS-related brain morphologic changes were assessed with immunohistological analysis. Von Kossa and Luxol Fast Blue staining was performed on brain tissue to assess calcification and myelin, respectively. ResultsMice bearing the ADAR1 p.K999N were viable though smaller than wild type sibs. RNA sequencing analysis of neuron-specific RNA substrates revealed altered RNA editing activities of the mutant ADAR1 protein. Mutant mice exhibited dramatically elevated levels of multiple ISGs within the brain. RNA ISH of brain sections showed selective activation of ISG expression in neurons and microglia in a patchy pattern. ISG-15 mRNA was upregulated in ADAR1 mutant brain neurons whereas CXCL10 mRNA was elevated in adjacent astroglia. No calcification or gliosis was detected in mutant brain.Conclusions We demonstrated that an AGS-associated mutation in ADAR1 was sufficient to activate the IFN pathway in the brain. Neurons and microglia expressed different ISGs. The ADAR1 p.K999N mutant mouse replicated aspects of the brain interferonopathy of AGS. Other brain changes seen in AGS (gliosis, calcification, death) did not occur, indicating that clinical AGS mutations may be necessary but not sufficient for development of the full phenotype. This mutant mouse presents a robust tool for investigation of AGS and neuroinflammatory diseases including the modeling of potential “second hits” that enable severe phenotypes of clinically variable diseases.

scholarly journals Aicardi-Goutières syndrome-associated mutation at ADAR1 gene locus activates innate immune response in mouse brain

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Xinfeng Guo ◽  
Clayton A. Wiley ◽  
Richard A. Steinman ◽  
Yi Sheng ◽  
Beihong Ji ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Rna Editing ◽  
Mouse Brain ◽  
Mutant Mouse ◽  
Cell Types ◽  
Mutant Mice ◽  
Blue Staining ◽  
Sequencing Analysis ◽  
Elevated Type ◽  
The Brain

Abstract Background Aicardi-Goutières syndrome (AGS) is a severe infant or juvenile-onset autoimmune disease characterized by inflammatory encephalopathy with an elevated type 1 interferon-stimulated gene (ISG) expression signature in the brain. Mutations in seven different protein-coding genes, all linked to DNA/RNA metabolism or sensing, have been identified in AGS patients, but none of them has been demonstrated to activate the IFN pathway in the brain of an animal. The molecular mechanism of inflammatory encephalopathy in AGS has not been well defined. Adenosine Deaminase Acting on RNA 1 (ADAR1) is one of the AGS-associated genes. It carries out A-to-I RNA editing that converts adenosine to inosine at double-stranded RNA regions. Whether an AGS-associated mutation in ADAR1 activates the IFN pathway and causes autoimmune pathogenesis in the brain is yet to be determined. Methods Mutations in the ADAR1 gene found in AGS patients were introduced into the mouse genome via CRISPR/Cas9 technology. Molecular activities of the specific p.K999N mutation were investigated by measuring the RNA editing levels in brain mRNA substrates of ADAR1 through RNA sequencing analysis. IFN pathway activation in the brain was assessed by measuring ISG expression at the mRNA and protein level through real-time RT-PCR and Luminex assays, respectively. The locations in the brain and neural cell types that express ISGs were determined by RNA in situ hybridization (ISH). Potential AGS-related brain morphologic changes were assessed with immunohistological analysis. Von Kossa and Luxol Fast Blue staining was performed on brain tissue to assess calcification and myelin, respectively. Results Mice bearing the ADAR1 p.K999N were viable though smaller than wild type sibs. RNA sequencing analysis of neuron-specific RNA substrates revealed altered RNA editing activities of the mutant ADAR1 protein. Mutant mice exhibited dramatically elevated levels of multiple ISGs within the brain. RNA ISH of brain sections showed selective activation of ISG expression in neurons and microglia in a patchy pattern. ISG-15 mRNA was upregulated in ADAR1 mutant brain neurons whereas CXCL10 mRNA was elevated in adjacent astroglia. No calcification or gliosis was detected in the mutant brain. Conclusions We demonstrated that an AGS-associated mutation in ADAR1, specifically the p.K999N mutation, activates the IFN pathway in the mouse brain. The ADAR1 p.K999N mutant mouse replicates aspects of the brain interferonopathy of AGS. Neurons and microglia express different ISGs. Basal ganglia calcification and leukodystrophy seen in AGS patients were not observed in K999N mutant mice, indicating that development of the full clinical phenotype may need an additional stimulus besides AGS mutations. This mutant mouse presents a robust tool for the investigation of AGS and neuroinflammatory diseases including the modeling of potential “second hits” that enable severe phenotypes of clinically variable diseases.

Noncoding RNAs and RNA Editing in Brain Development, Functional Diversification, and Neurological Disease

2007 ◽  
Vol 87 (3) ◽  
pp. 799-823 ◽  
Author(s):  
Mark F. Mehler ◽  
John S. Mattick
Keyword(s):  
Nervous System ◽  
Rna Editing ◽  
Neurological Diseases ◽  
Cell Types ◽  
State Transitions ◽  
Long Term Memory ◽  
Protein Coding ◽  
Continuous State ◽  
Wide Range

The progressive maturation and functional plasticity of the nervous system in health and disease involve a dynamic interplay between the transcriptome and the environment. There is a growing awareness that the previously unexplored molecular and functional interface mediating these complex gene-environmental interactions, particularly in brain, may encompass a sophisticated RNA regulatory network involving the twin processes of RNA editing and multifaceted actions of numerous subclasses of non-protein-coding RNAs. The mature nervous system encompasses a wide range of cell types and interconnections. Long-term changes in the strength of synaptic connections are thought to underlie memory retrieval, formation, stabilization, and effector functions. The evolving nervous system involves numerous developmental transitions, such as neurulation, neural tube patterning, neural stem cell expansion and maintenance, lineage elaboration, differentiation, axonal path finding, and synaptogenesis. Although the molecular bases for these processes are largely unknown, RNA-based epigenetic mechanisms appear to be essential for orchestrating these precise and versatile biological phenomena and in defining the etiology of a spectrum of neurological diseases. The concerted modulation of RNA editing and the selective expression of non-protein-coding RNAs during seminal as well as continuous state transitions may comprise the plastic molecular code needed to couple the intrinsic malleability of neural network connections to evolving environmental influences to establish diverse forms of short- and long-term memory, context-specific behavioral responses, and sophisticated cognitive capacities.

scholarly journals Development of a Personalized Intestinal Fibrosis Model Using Human Intestinal Organoids Derived From Induced Pluripotent Stem Cells

2021 ◽  
Author(s):  
Hannah Q Estrada ◽  
Shachi Patel ◽  
Shervin Rabizadeh ◽  
David Casero ◽  
Stephan R Targan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Rna Sequencing ◽  
Pluripotent Stem Cells ◽  
Cell Types ◽  
Mesenchymal Cells ◽  
Sequencing Analysis ◽  
Intestinal Fibrosis ◽  
Induced Pluripotent

Abstract Background Intestinal fibrosis is a serious complication of Crohn’s disease. Numerous cell types including intestinal epithelial and mesenchymal cells are implicated in this process, yet studies are hampered by the lack of personalized in vitro models. Human intestinal organoids (HIOs) derived from induced pluripotent stem cells (iPSCs) contain these cell types, and our goal was to determine the feasibility of utilizing these to develop a personalized intestinal fibrosis model. Methods iPSCs from 2 control individuals and 2 very early onset inflammatory bowel disease patients with stricturing complications were obtained and directed to form HIOs. Purified populations of epithelial and mesenchymal cells were derived from HIOs, and both types were treated with the profibrogenic cytokine transforming growth factor β (TGFβ). Quantitative polymerase chain reaction and RNA sequencing analysis were used to assay their responses. Results In iPSC-derived mesenchymal cells, there was a significant increase in the expression of profibrotic genes (Col1a1, Col5a1, and TIMP1) in response to TGFβ. RNA sequencing analysis identified further profibrotic genes and demonstrated differential responses to this cytokine in each of the 4 lines. Increases in profibrotic gene expression (Col1a1, FN, TIMP1) along with genes associated with epithelial-mesenchymal transition (vimentin and N-cadherin) were observed in TGFβ -treated epithelial cells. Conclusions We demonstrate the feasibility of utilizing iPSC-HIO technology to model intestinal fibrotic responses in vitro. This now permits the generation of near unlimited quantities of patient-specific cells that could be used to reveal cell- and environmental-specific mechanisms underpinning intestinal fibrosis.

scholarly journals IKAP—Identifying K mAjor cell Population groups in single-cell RNA-sequencing analysis

GigaScience ◽  
2019 ◽  
Vol 8 (10) ◽  
Author(s):  
Yun-Ching Chen ◽  
Abhilash Suresh ◽  
Chingiz Underbayev ◽  
Clare Sun ◽  
Komudi Singh ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Cell Types ◽  
Sequencing Analysis ◽  
Sequencing Data ◽  
Peripheral Blood Mononuclear ◽  
Biologically Relevant ◽  
Single Cell Rna Sequencing ◽  
Cell Groups ◽  
Cell Ontology

AbstractBackgroundIn single-cell RNA-sequencing analysis, clustering cells into groups and differentiating cell groups by differentially expressed (DE) genes are 2 separate steps for investigating cell identity. However, the ability to differentiate between cell groups could be affected by clustering. This interdependency often creates a bottleneck in the analysis pipeline, requiring researchers to repeat these 2 steps multiple times by setting different clustering parameters to identify a set of cell groups that are more differentiated and biologically relevant.FindingsTo accelerate this process, we have developed IKAP—an algorithm to identify major cell groups and improve differentiating cell groups by systematically tuning parameters for clustering. We demonstrate that, with default parameters, IKAP successfully identifies major cell types such as T cells, B cells, natural killer cells, and monocytes in 2 peripheral blood mononuclear cell datasets and recovers major cell types in a previously published mouse cortex dataset. These major cell groups identified by IKAP present more distinguishing DE genes compared with cell groups generated by different combinations of clustering parameters. We further show that cell subtypes can be identified by recursively applying IKAP within identified major cell types, thereby delineating cell identities in a multi-layered ontology.ConclusionsBy tuning the clustering parameters to identify major cell groups, IKAP greatly improves the automation of single-cell RNA-sequencing analysis to produce distinguishing DE genes and refine cell ontology using single-cell RNA-sequencing data.

Abstract MP227: Single Cell RNA Sequencing Analysis Reveals Insulin Signaling Defects In Cardiomyocytes Derived From A Novel Mouse Model Of Heart Failure With Preserved Ejection Fraction

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Jijun Huang ◽  
Zhaojun Xiong ◽  
Shuxun Ren ◽  
Nancy Cao ◽  
Jianfang Liu ◽  
...  
Keyword(s):  
Heart Failure ◽  
Insulin Resistance ◽  
Ejection Fraction ◽  
Mouse Model ◽  
Rna Sequencing ◽  
Insulin Signaling ◽  
Mitochondrial Gene ◽  
Cell Types ◽  
Sequencing Analysis ◽  
Preserved Ejection Fraction

Heart failure with preserved ejection fraction (HFpEF) is an emerging form of heart failure worldwide with no effective therapies in contrast with heart failure with reserved ejection fraction (HFrEF). To simulate multiple risk-factors associated with HFpEF in clinic, we developed a HFpEF mouse model by introducing cardiac hypertrophy with transverse aortic constriction (TAC) in ObOb ( Lep ob/ob ) mice, which has intrinsic systemic metabolic dysfunctions including obesity and insulin resistance. We first validated pathological changes in diastolic but not systolic parameters in the Ob-TAC vs. Ob-sham mice up to 10 weeks post-TAC by echocardiography. To evaluate the global transcriptome change in difference cell types, we conducted single nuclei RNA sequencing (snRNA-seq) from whole hearts of lean mice (c57), ObOb, and Ob-TAC mice (male only). 10x genomic 3’ GEM kit was used to generate the cDNA library and sequencing was done by Novaseq SP platform. A total of 13k nuclei were recovered from QC, nFeature RNA (&lt 2500) and mitochondrial gene (&lt 5%) filtering. By UMAP dimension reduction analysis, we annotated major cardiac cell types in the integrated snRNA-seq dataset, including 3 clusters of Cardiomyocytes (CMs). By pathway analysis of the differentially expressed genes in each CM clusters, we found that insulin resistance and glucagon pathway were enriched among the up regulated genes in CMs in HFpEF vs. lean control, while cell migration, signal transduction including insulin substrates were down regulated. Thus, we hypothesized that the altered crosstalk between glucagon and insulin signaling might contribute to the development of HFpEF in this mouse modal. This hypothesis was validated in a proof-of-concept study showing significant improvement of HFpEF features by inhibiting the glucagon receptors post-TAC with injection of a glucagon receptor antagonist.

scholarly journals RNA Sequencing Analysis Reveals Interactions between Breast Cancer or Melanoma Cells and the Tissue Microenvironment during Brain Metastasis

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Ryo Sato ◽  
Teppei Nakano ◽  
Mari Hosonaga ◽  
Oltea Sampetrean ◽  
Ritsuko Harigai ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Brain Metastasis ◽  
Cancer Cells ◽  
Cancer Patients ◽  
Rna Sequencing ◽  
Brain Tissue ◽  
Molecular Mechanisms ◽  
Sequencing Analysis ◽  
Primary Tumors ◽  
The Brain

Metastasis is the main cause of treatment failure and death in cancer patients. Metastasis of tumor cells to the brain occurs frequently in individuals with breast cancer, non–small cell lung cancer, or melanoma. Despite recent advances in our understanding of the causes and in the treatment of primary tumors, the biological and molecular mechanisms underlying the metastasis of cancer cells to the brain have remained unclear. Metastasizing cancer cells interact with their microenvironment in the brain to establish metastases. We have now developed mouse models of brain metastasis based on intracardiac injection of human breast cancer or melanoma cell lines, and we have performed RNA sequencing analysis to identify genes in mouse brain tissue and the human cancer cells whose expression is associated specifically with metastasis. We found that the expressions of the mouse genes Tph2, Sspo, Ptprq, and Pole as well as those of the human genes CXCR4, PLLP, TNFSF4, VCAM1, SLC8A2, and SLC7A11 were upregulated in brain tissue harboring metastases. Further characterization of such genes that contribute to the establishment of brain metastases may provide a basis for the development of new therapeutic strategies and consequent improvement in the prognosis of cancer patients.

scholarly journals Cellular and genetic drivers of RNA editing variation in the human brain

2021 ◽  
Author(s):  
Ryn Cuddleston ◽  
Junhao Li ◽  
Xuanjia Fan ◽  
Alexey Kozenkov ◽  
Matthew Lalli ◽  
...  
Keyword(s):  
Human Brain ◽  
Rna Sequencing ◽  
Rna Editing ◽  
Genetic Regulation ◽  
Cell Types ◽  
Brain Regions ◽  
Brain Cell ◽  
Cell Type ◽  
Sequencing Data ◽  
Regulatory Effects

Posttranscriptional adenosine-to-inosine modifications amplify the functionality of RNA molecules in the brain, yet the cellular and genetic regulation of RNA editing is poorly described. We quantified base-specific RNA editing across three major cell populations from the human prefrontal cortex: glutamatergic neurons, medial ganglionic eminence GABAergic neurons, and oligodendrocytes. We found more selective editing and RNA hyper-editing in neurons relative to oligodendrocytes. The pattern of RNA editing was highly cell type-specific, with 189,229 cell type-associated sites. The cellular specificity for thousands of sites was confirmed by single nucleus RNA-sequencing. Importantly, cell type-associated sites were enriched in GTEx RNA-sequencing data, edited ~twentyfold higher than all other sites, and variation in RNA editing was predominantly explained by neuronal proportions in bulk brain tissue. Finally, we discovered 661,791 cis-editing quantitative trait loci across thirteen brain regions, including hundreds with cell type-associated features. These data reveal an expansive repertoire of highly regulated RNA editing sites across human brain cell types and provide a resolved atlas linking cell types to editing variation and genetic regulatory effects.

scholarly journals Multiscale Co-Expression in the Brain

2020 ◽  
Author(s):  
Benjamin D. Harris ◽  
Megan Crow ◽  
Stephan Fischer ◽  
Jesse Gillis
Keyword(s):  
Motor Cortex ◽  
Single Cell ◽  
Rna Sequencing ◽  
Primary Motor Cortex ◽  
Cell Types ◽  
Cell Type ◽  
Single Cell Rna Sequencing ◽  
Cell Type Specific ◽  
The Brain ◽  
Regulatory Landscape

ABSTRACTSingle-cell RNA-sequencing (scRNAseq) data can reveal co-regulatory relationships between genes that may be hidden in bulk RNAseq due to cell type confounding. Using the primary motor cortex data from the Brain Initiative Cell Census Network (BICCN), we study cell type specific co-expression across 500,000 cells. Surprisingly, we find that the same gene-gene relationships that differentiate cell types are evident at finer and broader scales, suggesting a consistent multiscale regulatory landscape.

scholarly journals Telomere-to-telomere genome assembly of asparaginase-producing Trichoderma simmonsii

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Dawoon Chung ◽  
Yong Min Kwon ◽  
Youngik Yang
Keyword(s):  
Rna Sequencing ◽  
Draft Genome ◽  
Sequencing Analysis ◽  
Sequencing Data ◽  
Terrestrial Plants ◽  
Protein Coding ◽  
Oxford Nanopore ◽  
Wide Range ◽  
The Family ◽  
Encoding Genes

Abstract Background Trichoderma is a genus of fungi in the family Hypocreaceae and includes species known to produce enzymes with commercial use. They are largely found in soil and terrestrial plants. Recently, Trichoderma simmonsii isolated from decaying bark and decorticated wood was newly identified in the Harzianum clade of Trichoderma. Due to a wide range of applications in agriculture and other industries, genomes of at least 12 Trichoderma spp. have been studied. Moreover, antifungal and enzymatic activities have been extensively characterized in Trichoderma spp. However, the genomic information and bioactivities of T. simmonsii from a particular marine-derived isolate remain largely unknown. While we screened for asparaginase-producing fungi, we observed that T. simmonsii GH-Sj1 strain isolated from edible kelp produced asparaginase. In this study, we report a draft genome of T. simmonsii GH-Sj1 using Illumina and Oxford Nanopore technologies. Furthermore, to facilitate biotechnological applications of this species, RNA-sequencing was performed to elucidate the transcriptional profile of T. simmonsii GH-Sj1 in response to asparaginase-rich conditions. Results We generated ~ 14 Gb of sequencing data assembled in a ~ 40 Mb genome. The T. simmonsii GH-Sj1 genome consisted of seven telomere-to-telomere scaffolds with no sequencing gaps, where the N50 length was 6.4 Mb. The total number of protein-coding genes was 13,120, constituting ~ 99% of the genome. The genome harbored 176 tRNAs, which encode a full set of 20 amino acids. In addition, it had an rRNA repeat region consisting of seven repeats of the 18S-ITS1–5.8S-ITS2–26S cluster. The T. simmonsii genome also harbored 7 putative asparaginase-encoding genes with potential medical applications. Using RNA-sequencing analysis, we found that 3 genes among the 7 putative genes were significantly upregulated under asparaginase-rich conditions. Conclusions The genome and transcriptome of T. simmonsii GH-Sj1 established in the current work represent valuable resources for future comparative studies on fungal genomes and asparaginase production.

scholarly journals Self-renewing endometrial epithelial organoids of the human uterus

2019 ◽  
Vol 116 (46) ◽  
pp. 23132-23142 ◽  
Author(s):  
Harriet C. Fitzgerald ◽  
Pramod Dhakal ◽  
Susanta K. Behura ◽  
Danny J. Schust ◽  
Thomas E. Spencer
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Hormonal Regulation ◽  
Hormone Treatment ◽  
Cell Types ◽  
First Trimester ◽  
Human Endometrium ◽  
Receptor Expression ◽  
Sequencing Analysis ◽  
Growth And Survival

The human endometrium is essential in providing the site for implantation and maintaining the growth and survival of the conceptus. An unreceptive endometrium and disrupted maternal−conceptus interactions can cause infertility due to pregnancy loss or later pregnancy complications. Despite this, the role of uterine glands in first trimester human pregnancy is little understood. An established organoid protocol was used to generate and comprehensively analyze 3-dimensional endometrial epithelial organoid (EEO) cultures from human endometrial biopsies. The derived EEO expand long-term, are genetically stable, and can be cryopreserved. Using endometrium from 2 different donors, EEO were derived and then treated with estrogen (E2) for 2 d or E2 and medroxyprogesterone acetate (MPA) for 6 d. EEO cells were positive for the gland marker, FOXA2, and exhibited appropriate hormonal regulation of steroid hormone receptor expression. Real-time qPCR and bulk RNA-sequencing analysis revealed effects of hormone treatment on gene expression that recapitulated changes in proliferative and secretory phase endometrium. Single-cell RNA sequencing analysis revealed that several different epithelial cell types are present in the EEO whose proportion and gene expression changed with hormone treatment. The EEO model serves as an important platform for studying the physiology and pathology of the human endometrium.

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