CLIPseq of hnRNP A1 from the spinal cords of mice with experimental autoimmune encephalomyelitis
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ABSTRACT: Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis (MS) a chronic autoimmune disease of the central nervous system. We have observed dysfunction of the RNA binding protein hnRNP A1 in neurons from the brains of patients with MS, and the spinal cords of mice with EAE. Here, we sought to characterize the consequences of EAE-induced dysfunction of hnRNP A1 on the RNAs it binds by using CLIPseq to establish both the normal central nervous system RNA binding profile of hnRNP A1 in the spinal cords of naive mice, and any alterations to the binding profile of hnRNP A1 in the spinal cords of mice with EAE.
Project description:Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS); its cause is unknown. To understand the pathogenesis of MS, researchers often use the experimental autoimmune encephalomyelitis (EAE) mouse model. Here, our aim was to build a proteome map of the biological changes that occur during MS at the major onset sites—the brain and the spinal cord. We performed quantitative proteome profiling in five specific brain regions and the spinal cord of EAE and healthy mice with high-resolution mass spectrometry based on tandem mass tags.
Project description:Myelin Basic Protein (MBP) induced experimental autoimmune encephalomyelitis (EAE) in the Lewis rat, produces an an acute weakness, or paralysis of the tail and hind limb ataxia ,weakness or paralysis associated with increased permiability of the blood brain barrier, inflammation and demyelination in central nervous system (CNS). Clinical symptoms , ascending weakness or paralysis of the tail followed by the hind limbs and in rare cases the fore limbs occurs 8 and 14 days post immunisation (dpi) and is generally resolved completely by day 20 dpi. We have carried out transcriptome analysis of total RNA from the spinal cords of female Lewis rats at the peak of disease (EAE) and age matched healthy controls to identify exon expression changes associated with the disease. In these data sets we include the exon expression data obtained from total RNA preparations from the spinal cords of female Lewis rats sacrificed at the clinical peak of MBP induced EAE and age matched , untreated, healthy controls. 8 total RNA samples were prepared. A two way ANOVA comparison carried out in Partek Genomics Suite was used to detect differences in exon expression in the spinal cord of female lewis rats with MBP induced EAE and age matched healthy controls.
Project description:Disruption of blood-brain barrier (BBB) integrity is a hallmark of several neurological disorders. Here we show that the BBB is dynamically and differentially affected during the preclinical, progression and remission phase of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We isolated vascular fragments, representing the BBB, from spinal cords of imatinib and PBS treated mice at the preclinical, progression and remission phase of MOG-EAE as well as from non-immunized, naive mice.
Project description:Experimental autoimmune encephalomyelitis (EAE) is a murine model of multiple sclerosis, a chronic neurodegenerative and inflammatory autoimmune condition of the central nervous system (CNS). Pathology is driven by the infiltration of autoreactive CD4+ lymphocytes into the CNS where they attack neuronal sheaths causing ascending paralysis. We used an isotope-coded protein labelling approach to investigate the proteome of CD4+ cells isolated from the spinal cord and brain of mice at various stages of EAE progression in two EAE disease models; PLP139-151-induced relapsing-remitting EAE and MOG35-55-induced chronic EAE, which emulate the two forms of human multiple sclerosis. A total of 1120 proteins were quantified across disease onset, peak-disease and remission phases of disease and of these, 13 up-regulated proteins of interest were identified with functions relating to the regulation of inflammation, leukocyte adhesion and migration, tissue repair and the regulation of transcription/translation. Proteins implicated in processes such as inflammation (S100A4 and S100A9) and tissue repair (Annexin A1), which represent key events during EAE progression were validated by quantitative PCR. This is the first targeted analysis of autoreactive cells purified from the CNS during EAE, highlighting fundamental CD4+ cell-driven processes that occur during the initiation of relapse and remission stages of disease.
Project description:We sought to identify alterations in RNA expression and splicing in human grey matter from the brains of people with Multiple Sclerosis (MS) where we observe mislocalization of the RNA binding protein hnRNP A1. Thus, RNA was extracted from fresh-frozen post-mortem brain samples of three people with MS and compared to RNA extracted from fresh-frozen post-mortem brain samples of three healthy controls by RNAseq. Regions of interest in human grey matter were defined by immunostaining for hnRNP A1 to confirm grey matter regions with hnRNP A1 mislocalization in MS tissue, and grey matter regions with normal hnRNP A1 localization in control tissue, and the same region from an adjacent tissue section was dissected with a razor blade and used for RNA extraction and sequencing.
Project description:Myelin Basic Protein (MBP) induced experimental autoimmune encephalomyelitis (EAE) in the Lewis rat, produces an an acute weakness, or paralysis of the tail and hind limb ataxia ,weakness or paralysis associated with increased permiability of the blood brain barrier, inflammation and demyelination in central nervous system (CNS). Clinical symptoms , ascending weakness or paralysis of the tail followed by the hind limbs and in rare cases the fore limbs occurs 8 and 14 days post immunisation (dpi) and is generally resolved completely by day 20 dpi. We have carried out transcriptome analysis of total RNA from the spinal cords of female Lewis rats at the peak of disease (EAE) and age matched healthy controls to identify gene expression changes associated with the disease. In these data sets we include the exon and gene expression data obtained from total RNA preparations from the spinal cords of female Lewis rats sacrificed at the clinical peak of MBP induced EAE and age matched , untreated, healthy controls. This data was used to obtain 2265 mapped IDS wich identified 1190 known genes which were differentially expressed in the spinal cord in EAE compared to healthy animals. 8 total RNA samples were prepared. A two way ANOVA comparison carried out in Partek Genomics Suite was used to detect gene transcripts for which the expression levels varied significantly (un-adjusted p-values M-bM-^IM-$ 0.05) from the healthy controls. 2265 mapped IDs were uploaded to the Ingenuity pathway analysis suite (IPA) where 1190 known genes were identified as being differentially regulated between groups. An FDR M-bM-^IM-$ 5% and fold change limit of +/- 4.0 further refined the data set to identify the 72 most highly and significantly differentially regulated genes in the spinal cord at the clinical peak of disease in MBP induced EAE in the Lewis rat.
Project description:Myelin Basic Protein (MBP) induced experimental autoimmune encephalomyelitis (EAE) in the Lewis rat, produces an an acute weakness, or paralysis of the tail and hind limb ataxia ,weakness or paralysis associated with increased permiability of the blood brain barrier, inflammation and demyelination in central nervous system (CNS). Clinical symptoms , ascending weakness or paralysis of the tail followed by the hind limbs and in rare cases the fore limbs occurs 8 and 14 days post immunisation (dpi) and is generally resolved completely by day 20 dpi. We have carried out transcriptome analysis of total RNA from the spinal cords of female Lewis rats at the peak of disease (EAE) and age matched healthy controls to identify exon expression changes associated with the disease. In these data sets we include the exon expression data obtained from total RNA preparations from the spinal cords of female Lewis rats sacrificed at the clinical peak of MBP induced EAE and age matched , untreated, healthy controls.
Project description:Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, cross-linking and immunoprecipitation coupled with high-throughput sequencing, and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins, and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and auto-regulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells. CLIPseq for hnRNP A1, hnRNP A2/B1, hnRNP F, hnRNP M, and hnRNP U in human 293T cells
Project description:Myelin Basic Protein (MBP) induced experimental autoimmune encephalomyelitis (EAE) in the Lewis rat, produces an an acute weakness, or paralysis of the tail and hind limb ataxia ,weakness or paralysis associated with increased permiability of the blood brain barrier, inflammation and demyelination in central nervous system (CNS). Clinical symptoms , ascending weakness or paralysis of the tail followed by the hind limbs and in rare cases the fore limbs occurs 8 and 14 days post immunisation (dpi) and is generally resolved completely by day 20 dpi. We have carried out transcriptome analysis of total RNA from the spinal cords of female Lewis rats at the peak of disease (EAE) and age matched healthy controls to identify gene expression changes associated with the disease. In these data sets we include the exon and gene expression data obtained from total RNA preparations from the spinal cords of female Lewis rats sacrificed at the clinical peak of MBP induced EAE and age matched , untreated, healthy controls. This data was used to obtain 2265 mapped IDS wich identified 1190 known genes which were differentially expressed in the spinal cord in EAE compared to healthy animals.