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.

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 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 Reconstruction of 1,000 projection neurons reveals new cell types and organization of long-range connectivity in the mouse brain

2019 ◽  
Author(s):  
Johan Winnubst ◽  
Erhan Bas ◽  
Tiago A. Ferreira ◽  
Zhuhao Wu ◽  
Michael N. Economo ◽  
...  
Keyword(s):  
Long Range ◽  
Mouse Brain ◽  
Neuronal Cell ◽  
Cell Types ◽  
Neuronal Morphology ◽  
Projection Neurons ◽  
Axonal Arbor ◽  
Flow Of Information ◽  
Organizational Principles ◽  
The Brain

SummaryNeuronal cell types are the nodes of neural circuits that determine the flow of information within the brain. Neuronal morphology, especially the shape of the axonal arbor, provides an essential descriptor of cell type and reveals how individual neurons route their output across the brain. Despite the importance of morphology, few projection neurons in the mouse brain have been reconstructed in their entirety. Here we present a robust and efficient platform for imaging and reconstructing complete neuronal morphologies, including axonal arbors that span substantial portions of the brain. We used this platform to reconstruct more than 1,000 projection neurons in the motor cortex, thalamus, subiculum, and hypothalamus. Together, the reconstructed neurons comprise more than 75 meters of axonal length and are available in a searchable online database. Axonal shapes revealed previously unknown subtypes of projection neurons and suggest organizational principles of long-range connectivity.

scholarly journals Cell type-specific biotin labeling in vivo resolves regional neuronal proteomic differences in mouse brain

2021 ◽  
Author(s):  
Sruti Rayaprolu ◽  
Sara Bitarafan ◽  
Ranjita Betarbet ◽  
Sydney N Sunna ◽  
Lihong Cheng ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Neurological Diseases ◽  
Neuronal Cell ◽  
Cell Types ◽  
Proteomic Profiling ◽  
Cell Type ◽  
Specific Expression ◽  
Cell Type Specific ◽  
The Brain

Isolation and proteomic profiling of brain cell types, particularly neurons, pose several technical challenges which limit our ability to resolve distinct cellular phenotypes in neurological diseases. Therefore, we generated a novel mouse line that enables cell type-specific expression of a biotin ligase, TurboID, via Cre-lox strategy for in vivo proximity-dependent biotinylation of proteins. Using adenoviral-based and transgenic approaches, we show striking protein biotinylation in neuronal cell bodies and axons throughout the mouse brain. We quantified more than 2,000 neuron-derived proteins following enrichment that mapped to numerous subcellular compartments. Synaptic, transmembrane transporters, ion channel subunits, and disease-relevant druggable targets were among the most significantly enriched proteins. Remarkably, we resolved brain region-specific proteomic profiles of Camk2a neurons with distinct functional molecular signatures and disease associations that may underlie regional neuronal vulnerability. Leveraging the neuronal specificity of this in vivo biotinylation strategy, we used an antibody-based approach to uncover regionally unique patterns of neuron-derived signaling phospho-proteins and cytokines, particularly in the cortex and cerebellum. Our work provides a proteomic framework to investigate cell type-specific mechanisms driving physiological and pathological states of the brain as well as complex tissues beyond the brain.

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 Transposon-mediated, cell type-specific transcription factor recording in the mouse brain

2019 ◽  
Author(s):  
Alexander J. Cammack ◽  
Arnav Moudgil ◽  
Tomas Lagunas ◽  
Michael J. Vasek ◽  
Mark Shabsovich ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Brain ◽  
Regulation Of Gene Expression ◽  
Cell Types ◽  
Mouse Tissue ◽  
Specific Cell ◽  
Cell Type ◽  
Cell Fate Decisions ◽  
Cell Type Specific ◽  
The Brain

AbstractTranscription factors (TFs) play a central role in the regulation of gene expression, controlling everything from cell fate decisions to activity dependent gene expression. However, widely-used methods for TF profiling in vivo (e.g. ChIP-seq) yield only an aggregated picture of TF binding across all cell types present within the harvested tissue; thus, it is challenging or impossible to determine how the same TF might bind different portions of the genome in different cell types, or even to identify its binding events at all in rare cell types in a complex tissue such as the brain. Here we present a versatile methodology, FLEX Calling Cards, for the mapping of TF occupancy in specific cell types from heterogenous tissues. In this method, the TF of interest is fused to a hyperactive piggyBac transposase (hypPB), and this bipartite gene is delivered, along with donor transposons, to mouse tissue via a Cre-dependent adeno-associated virus (AAV). The fusion protein is expressed in Cre-expressing cells where it inserts transposon “Calling Cards” near to TF binding sites. These transposons permanently mark TF binding events and can be mapped using high-throughput sequencing. Alternatively, unfused hypPB interacts with and records the binding of the super enhancer (SE)-associated bromodomain protein, Brd4. To demonstrate the FLEX Calling Card method, we first show that donor transposon and transposase constructs can be efficiently delivered to the postnatal day 1 (P1) mouse brain with AAV and that insertion profiles report TF occupancy. Then, using a Cre-dependent hypPB virus, we show utility of this tool in defining cell type-specific TF profiles in multiple cell types of the brain. This approach will enable important cell type-specific studies of TF-mediated gene regulation in the brain and will provide valuable insights into brain development, homeostasis, and disease.

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.

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