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  1. Article: Loss of Endothelial TDP-43 Leads to Blood Brain Barrier Defects in Mouse Models of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

    Cheemala, Ashok / Kimble, Amy L / Tyburski, Jordan D / Leclair, Nathan K / Zuberi, Aamir R / Murphy, Melissa / Jellison, Evan R / Reese, Bo / Hu, Xiangyou / Lutz, Cathleen M / Yan, Riqiang / Murphy, Patrick A

    bioRxiv : the preprint server for biology

    2023  

    Abstract: Loss of nuclear TDP-43 occurs in a wide range of neurodegenerative diseases, and specific mutations in ... ...

    Abstract Loss of nuclear TDP-43 occurs in a wide range of neurodegenerative diseases, and specific mutations in the
    Language English
    Publishing date 2023-12-14
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2023.12.13.571184
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article: Targeted inCITE-Seq Analysis Identifies the Loss of Nuclear TDP-43 in Endothelium as a Mediator of Blood Brain Barrier Signaling Pathway Dysfunction in Neurodegeneration.

    Omar, Omar M F / Kimble, Amy L / Cheemala, Ashok / Tyburski, Jordan D / Pandey, Swati / Wu, Qian / Reese, Bo / Jellison, Evan R / Li, Yunfeng / Hao, Bing / Yan, Riqiang / Murphy, Patrick A

    bioRxiv : the preprint server for biology

    2023  

    Abstract: Despite the importance of the endothelium in the regulation of the blood brain barrier (BBB) in aging and neurodegenerative disease, difficulties in extracting endothelial cell (EC) nuclei have limited analysis of these cells. In addition, nearly all ... ...

    Abstract Despite the importance of the endothelium in the regulation of the blood brain barrier (BBB) in aging and neurodegenerative disease, difficulties in extracting endothelial cell (EC) nuclei have limited analysis of these cells. In addition, nearly all Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Degeneration (FTD), and a large portion of Alzheimer's Disease (AD) exhibit neuronal TDP-43 aggregation, leading to loss of nuclear function, but whether TDP-43 is similarly altered in human BBB ECs is unknown. Here we utilize a novel technique for the enrichment of endothelial and microglial nuclei from human cortical brain tissues, combined with inCITE-seq, to analyze nuclear proteins and RNA transcripts in a large cohort of healthy and diseased donors. Our findings reveal a unique transcriptional signature in nearly half of the capillary endothelial cells across neurodegenerative states, characterized by reduced levels of nuclear β-Catenin and canonical downstream genes, and an increase in TNF/NF-kB target genes. We demonstrate that this does not correlate with increased nuclear p65/NF-kB, but rather a specific loss of nuclear TDP-43 in these disease associated ECs. Comparative analysis in animal models with targeted disruption of TDP-43 shows that this is sufficient to drive these transcriptional alterations. This work reveals that TDP-43 is a critical governor of the transcriptional output from nuclear p65/NF-kB, which has paradoxical roles in barrier maintenance and also barrier compromising inflammatory responses, and suggests that disease specific loss in ECs contributes to BBB defects observed in the progression of AD, ALS and FTD.
    Language English
    Publishing date 2023-12-14
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2023.12.13.571178
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Identification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen.

    Hensel, Jessica A / Heineman, Brent D / Kimble, Amy L / Jellison, Evan R / Reese, Bo / Murphy, Patrick A

    Scientific reports

    2021  Volume 11, Issue 1, Page(s) 19835

    Abstract: The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and ... ...

    Abstract The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and -EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.
    MeSH term(s) Alternative Splicing ; Animals ; Carrier Proteins ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endothelial Cells/metabolism ; Exons ; Extracellular Matrix/metabolism ; Fibronectins/chemistry ; Fibronectins/metabolism ; Flow Cytometry ; Gene Expression Regulation ; Gene Knockout Techniques ; Genes, Reporter ; Mice ; Protein Binding ; Protein Interaction Domains and Motifs ; RNA, Messenger/genetics
    Chemical Substances Carrier Proteins ; Fibronectins ; RNA, Messenger
    Language English
    Publishing date 2021-10-06
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2615211-3
    ISSN 2045-2322 ; 2045-2322
    ISSN (online) 2045-2322
    ISSN 2045-2322
    DOI 10.1038/s41598-021-99079-1
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Splice factor polypyrimidine tract-binding protein 1 (Ptbp1) primes endothelial inflammation in atherogenic disturbed flow conditions.

    Hensel, Jessica A / Nicholas, Sarah-Anne E / Kimble, Amy L / Nagpal, Arjun S / Omar, Omar M F / Tyburski, Jordan D / Jellison, Evan R / Ménoret, Antoine / Ozawa, Manabu / Rodriguez-Oquendo, Annabelle / Vella, Anthony T / Murphy, Patrick A

    Proceedings of the National Academy of Sciences of the United States of America

    2022  Volume 119, Issue 30, Page(s) e2122227119

    Abstract: NF-κB-mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through adhesion molecules Icam1 and Vcam1. The endothelium is primed for cytokine activation of NF-κB by exposure to low and disturbed blood flow (LDF)but ... ...

    Abstract NF-κB-mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through adhesion molecules Icam1 and Vcam1. The endothelium is primed for cytokine activation of NF-κB by exposure to low and disturbed blood flow (LDF)but the molecular underpinnings are not fully understood. In an experimental in vivo model of LDF, platelets were required for the increased expression of several RNA-binding splice factors, including polypyrimidine tract binding protein (Ptbp1). This was coordinated with changes in RNA splicing in the NF-κB pathway in primed cells, leading us to examine splice factors as mediators of priming. Using Icam1 and Vcam1 induction by tumor necrosis factor (TNF)-α stimulation as a readout, we performed a CRISPR Cas9 knockout screen and identified a requirement for Ptbp1 in priming. Deletion of Ptbp1 had no effect on cell growth or response to apoptotic stimuli, but reversed LDF splicing patterns and inhibited NF-κB nuclear translocation and transcriptional activation of downstream targets, including Icam1 and Vcam1. In human coronary arteries, elevated PTBP1 correlates with expression of TNF pathway genes and plaque. In vivo, endothelial-specific deletion of Ptbp1 reduced Icam1 expression and myeloid cell infiltration at regions of LDF in atherosclerotic mice, limiting atherosclerosis. This may be mediated, in part, by allowing inclusion of a conserved alternative exon in Ripk1 leading to a reduction in Ripk1 protein. Our data show that Ptbp1, which is induced in a subset of the endothelium by platelet recruitment at regions of LDF, is required for priming of the endothelium for subsequent NF-κB activation, myeloid cell recruitment and atherosclerosis.
    MeSH term(s) Alternative Splicing ; Animals ; Atherosclerosis/genetics ; Atherosclerosis/metabolism ; Endothelium/metabolism ; Heterogeneous-Nuclear Ribonucleoproteins/genetics ; Humans ; Inflammation/genetics ; Inflammation/metabolism ; Mice ; NF-kappa B/genetics ; NF-kappa B/metabolism ; Polypyrimidine Tract-Binding Protein/genetics ; Polypyrimidine Tract-Binding Protein/metabolism
    Chemical Substances Heterogeneous-Nuclear Ribonucleoproteins ; NF-kappa B ; PTBP1 protein, human ; Ptbp1 protein, mouse ; Polypyrimidine Tract-Binding Protein (139076-35-0)
    Language English
    Publishing date 2022-07-18
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
    ZDB-ID 209104-5
    ISSN 1091-6490 ; 0027-8424
    ISSN (online) 1091-6490
    ISSN 0027-8424
    DOI 10.1073/pnas.2122227119
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 1148: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (1.OG)
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  5. Article ; Online: A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue.

    Kimble, Amy L / Silva, Jordan / Omar, Omar M / Murphy, Melissa / Hensel, Jessica A / Nicholas, Sarah-Anne E / Jellison, Evan R / Reese, Bo / Murphy, Patrick A

    Laboratory investigation; a journal of technical methods and pathology

    2021  Volume 102, Issue 2, Page(s) 204–211

    Abstract: Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh ... ...

    Abstract Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.
    MeSH term(s) Animals ; Brain/cytology ; Brain/metabolism ; Cell Fractionation/methods ; Cell Nucleus/metabolism ; Cells, Cultured ; Cryopreservation/methods ; Flow Cytometry/methods ; HEK293 Cells ; Human Umbilical Vein Endothelial Cells/metabolism ; Humans ; Mice, Inbred C57BL ; RNA/isolation & purification ; RNA/metabolism ; Reproducibility of Results ; Sequence Analysis, RNA/methods ; Single-Cell Analysis/methods ; Tissue Banks ; Transcriptional Regulator ERG/metabolism ; Mice
    Chemical Substances ERG protein, human ; Transcriptional Regulator ERG ; RNA (63231-63-0)
    Language English
    Publishing date 2021-11-13
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 80178-1
    ISSN 1530-0307 ; 0023-6837
    ISSN (online) 1530-0307
    ISSN 0023-6837
    DOI 10.1038/s41374-021-00698-z
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Ud II Zs.164: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 2021: Bestellungen von Artikeln über das Online-Bestellformular
    ab Jg. 2022: Lesesaal (EG)

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  6. Article ; Online: Alternative RNA splicing in the endothelium mediated in part by Rbfox2 regulates the arterial response to low flow.

    Murphy, Patrick A / Butty, Vincent L / Boutz, Paul L / Begum, Shahinoor / Kimble, Amy L / Sharp, Phillip A / Burge, Christopher B / Hynes, Richard O

    eLife

    2018  Volume 7

    Abstract: Low and disturbed blood flow drives the progression of arterial diseases including atherosclerosis and aneurysms. The endothelial response to flow and its interactions with recruited platelets and leukocytes determine disease progression. Here, we report ...

    Abstract Low and disturbed blood flow drives the progression of arterial diseases including atherosclerosis and aneurysms. The endothelial response to flow and its interactions with recruited platelets and leukocytes determine disease progression. Here, we report widespread changes in alternative splicing of pre-mRNA in the flow-activated murine arterial endothelium in vivo. Alternative splicing was suppressed by depletion of platelets and macrophages recruited to the arterial endothelium under low and disturbed flow. Binding motifs for the Rbfox-family are enriched adjacent to many of the regulated exons. Endothelial deletion of
    MeSH term(s) Adaptation, Physiological ; Alternative Splicing ; Animals ; Endothelium, Vascular/physiology ; Gene Expression Regulation ; Mice ; RNA Splicing Factors/metabolism ; Regional Blood Flow
    Chemical Substances RNA Splicing Factors ; Rbfox2 protein, mouse
    Language English
    Publishing date 2018-01-02
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2687154-3
    ISSN 2050-084X ; 2050-084X
    ISSN (online) 2050-084X
    ISSN 2050-084X
    DOI 10.7554/eLife.29494
    Database MEDical Literature Analysis and Retrieval System OnLINE

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