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  • 1.
    Muthukrishnan, Uma
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    The release of histone proteins from cells via extracellular vesicles2018Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Histones are chromatin-associated proteins localized to the nucleus. However, extracellular histones are present in biofluids from healthy individuals and become elevated under disease conditions, such as neurodegeneration and cancer. Hence, extracellular histones may have important biological functions in healthy and diseased states, which are not understood. Histones have been reported in the proteomes of extracellular vesicles (EVs), including microvesicles and exosomes. The main aim of this thesis was to determine whether or not extracellular histones are secreted via EVs/exosomes.

    In an initial study (Paper I), I optimized methods for human embryonic kidney (HEK293) cell culture, transfection and protein detection using western blotting.

    In the main study (Paper II), I used oligodendrocyte cell lines (rat OLN-93 and mouse Oli-neu) to investigate the localization of histones to EVs. Western blotting of EVs purified from OLN-93 cell-conditioned media confirmed the presence of linker and core histones in them. Immunolocalization and transmission electron microscopy confirmed that histones are localized to EVs, as well as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). This suggests that histones are secreted via the MVB/exosome pathway.

    Localization of histones in EVs was investigated by biochemical/proteolytic degradation and purification followed by western blotting. Surprisingly, histones were associated with the membrane but not the luminal fraction. Overexpression of tagged histones in HEK293 cells confirmed their conserved, membrane localization. OLN-93 cell EVs contained both double stranded and single stranded DNA but nuclease and protease digestion showed that the association of histones and DNA with EVs was not interdependent.

    The abundance of histones in EVs was not affected by differentiation in Oli-neu cells. However, histone release was upregulated as an early response to cellular stress in OLN-93 cells and occurred before the release of markers of stress including heat shock proteins. Interestingly, a notable upregulation in secretion of small diameter (50-100 nm) EVs was observed following heat stress, suggesting that a sub-population of vesicles may be involved specifically in histone secretion in response to stress. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) as a possible mechanism underlying increased histone secretion.

    In Paper III, I developed methods to quantify extracellular histone proteins in human ascites samples from ovarian cancer patients.

     

    In summary, we show for the first time that membrane-associated histones are secreted via the MVB/exosome pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease. 

  • 2.
    Muthukrishnan, Uma
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Natarajan, Balasubramanian
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Mäger, Imre
    Levén May, Hanna
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Jones, Iwan
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Corso, Giulia
    Nordin, Joel Z.
    Wiklander, Oscar
    Johansson, Henrik J.
    Lehtiö, Janne
    Hällbrink, Mattias
    Wood, Matthew J.
    Sandblad, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nagaev, Ivan
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Baranov, Vladimir
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Mincheva-Nilsson, Lucia
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Rome, Sophie
    Pini, Adrian
    Andaloussi, Samir EL
    Gilthorpe, Jonathan D.
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    The exosome membrane localization of histones is independent of DNA and upregulated in response to stressManuscript (preprint) (Other academic)
    Abstract [en]

    Extracellular histones contribute to many acute and chronic diseases but also populate the secretomes of healthy cells and biofluids. However, a secretory pathway for histones has not been described. Here we report that core and linker histones localize to multivesicular bodies and are secreted via exosomes. Histones are tightly associated with the exosome membrane, with N-terminal domains exposed, in a DNA-independent manner. Furthermore, rapid upregulation of exosomal histones occurs following heat stress, accompanied by enhanced vesicle secretion and a shift towards a population of smaller vesicles. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) complex as a possible mechanism underlying increased histone secretion.We show for the first time that membrane-associated histones are actively secreted from intact cells via the multivesicular body/exosomal pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease.

  • 3. Singel, Kelly L.
    et al.
    Grzankowski, Kassondra S.
    Khan, A. N. M. Nazmul H.
    Grimm, Melissa J.
    D'Auria, Anthony C.
    Morrell, Kayla
    Eng, Kevin H.
    Hylander, Bonnie
    Mayor, Paul C.
    Emmons, Tiffany R.
    Lénárt, Nikolett
    Fekete, Rebeka
    Környei, Zsuzsanna
    Muthukrishnan, Uma
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Gilthorpe, Jonathan D.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Itagaki, Kiyoshi
    Hauser, Carl J.
    Leifer, Cynthia
    Moysich, Kirsten B.
    Odunsi, Kunle
    Dénes, Ádám
    Segal, Brahm H.
    Mitochondrial DNA in the tumour microenvironment activates neutrophils and is associated with worse outcomes in patients with advanced epithelial ovarian cancer2019In: British Journal of Cancer, ISSN 0007-0920, E-ISSN 1532-1827, Vol. 120, no 2, p. 207-217Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesised that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worse outcomes.

    METHODS: Banked ascites supernatants from patients with newly diagnosed advanced EOC were analysed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELP and ELANE.

    RESULTS: The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n = 68, log-rank, p = 0.0178). NETs were detected in resected tumours. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivo stimulated T cell proliferation. Increased SELP mRNA expression correlated with worse overall survival (n = 302, Cox model, p = 0.02).

    CONCLUSION: In this single-centre retrospective analysis, ascites mtDNA correlated with worse PFS in advanced EOC. Mitochondrial and other DAMPs in ascites may activate neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumour immunity. These pathways are potential prognostic markers and therapeutic targets.

  • 4. Singel, Kelly L.
    et al.
    Grzankowski, Kassondra S.
    Khan, ANM Nazmul H.
    Grimm, Melissa J.
    D’Auria, Anthony C.
    Morrell, Kayla
    Eng, Kevin H.
    Hylander, Bonnie
    Mayor, Paul C.
    Emmons, Tiffany R.
    Lénárt, Nikolett
    Fekete, Rebeka
    Környei, Zsuzsanna
    Muthukrishnan, Uma
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Gilthorpe, Jonathan D.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Itagaki, Kiyoshi
    Hauser, Carl J.
    Leifer, Cynthia
    Moysich, Kirsten B.
    Odunsi, Kunle
    Dénes, Ádám
    Segal, Brahm H.
    Mitochondrial DNA in the tumor microenvironment activates neutrophils and is associated with worsened outcomes in patients with advanced epithelial ovarian cancerManuscript (preprint) (Other academic)
    Abstract [en]

    Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesized that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worsened outcomes.Banked ascites supernatants from patients with newly diagnosed advanced EOC were analyzed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELPand ELANE. The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n=68, log-rank, p=0.0178). NETs were detected in resected tumors. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivostimulated T cell proliferation. Increased SELPmRNA expression correlated with worsened overall survival (n=302, Cox model, p=0.02).In this single-center retrospective analysis, ascites mtDNA correlated with worsened PFS in advanced EOC. Our results support mitochondrial and other DAMPs activating neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumor immunity. These pathways are potential prognostic markers and therapeutic targets.

  • 5. Tossell, Kyoko
    et al.
    Andreae, Laura C
    Cudmore, Chloe
    Lang, Emily
    Muthukrishnan, Uma
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Lumsden, Andrew
    Gilthorpe, Jonathan D
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Irving, Carol
    Lrrn1 is required for formation of the midbrain-hindbrain boundary and organiser through regulation of affinity differences between midbrain and hindbrain cells in chick2011In: Developmental Biology, ISSN 0012-1606, E-ISSN 1095-564X, Vol. 352, no 2, p. 341-352Article in journal (Refereed)
    Abstract [en]

    The midbrain-hindbrain boundary (MHB) acts as an organiser/signalling centre to pattern tectal and cerebellar compartments. Cells in adjacent compartments must be distinct from each other for boundary formation to occur at the interface. Here we have identified the leucine-rich repeat (LRR) neuronal 1 (Lrrn1) protein as a key regulator of this process in chick. The Lrrn family is orthologous to the Drosophila tartan/capricious (trn/caps) family. Differential expression of trn/caps promotes an affinity difference and boundary formation between adjacent compartments in a number of contexts; for example, in the wing, leg and eye imaginal discs. Here we show that Lrrn1 is expressed in midbrain cells but not in anterior hindbrain cells. Lrrn1 is down-regulated in the anterior hindbrain by the organiser signalling molecule FGF8, thereby creating a differential affinity between these two compartments. Lrrn1 is required for the formation of MHB--loss of function leads to a loss of the morphological constriction and loss of Fgf8. Cells overexpressing Lrrn1 violate the boundary and result in a loss of cell restriction between midbrain and hindbrain compartments. Lrrn1 also regulates the glycosyltransferase Lunatic Fringe, a modulator of Notch signalling, maintaining its expression in midbrain cells which is instrumental in MHB boundary formation. Thus, Lrrn1 provides a link between cell affinity/compartment segregation, and cell signalling to specify boundary cell fate.

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