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  • 1.
    Christoffersson, Gustaf
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Lomei, Jalal
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Engblom, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computational Science.
    Phillipson, Mia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Vascular sprouts induce local attraction of proangiogenic neutrophils2017In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 102, p. 741-751Article in journal (Refereed)
  • 2.
    Fatsis-Kavalopoulos, Nikos
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Gradientech AB, Uppsala, Sweden.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Xie, Beichen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Hernández Vera, Rodrigo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Idevall Hagren, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution2019In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 19, no 6, p. 1071-1081Article in journal (Refereed)
    Abstract [en]

    The function and behaviour of any given cell in a healthy tissue, or in a tumor, is affected by interactions with its neighboring cells. It is therefore important to create methods that allow for reconstruction of tissue niches in vitro for studies of cell-cell signaling and associated cell behaviour. To this end we created the cell assembly generator (CAGE), a microfluidic device which enables the organization of different cell types into precise cell clusters in a flow chamber compatible with high-resolution microscopy. In proof-of-concept paracrine signalling experiments, 4-cell clusters consisting of one pancreatic -cell and three breast cancer cells were formed. It has previously been established that extracellular ATP induces calcium (Ca2+) release from the endoplasmic reticulum (ER) to the cytosol before it is cleared back into the ER via sarcoplasmic/ER Ca2+ ATPase (SERCA) pumps. Here, ATP release from the -cell was stimulated by depolarization, and dynamic changes in Ca2+ levels in the adjacent cancer cells measured using imaging of the calcium indicator Fluo-4. We established that changes in the concentration of cytosolic Ca2+ in the cancer cells were proportional to the distance from the ATP-releasing -cell. Additionally, we established that the relationship between distance and cytosolic calcium changes were dependent on Ca2+-release from the ER using 5-cell clusters composed of one -cell, two untreated cancer cells and two cancer cells pretreated with Thapsigargin (to deplete the ER of Ca2+). These experiments show that the CAGE can be used to create exact cell clusters, which affords precise control for reductionist studies of cell-cell signalling and permits the formation of heterogenous cell models of specific tissue niches.

  • 3.
    Heldin, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Hernández Vera, Rodrigo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Fredlund Fuchs, Peder
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gerwins, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    FGD5 sustains vascular endothelial growth factor A (VEGFA) signaling through inhibition of proteasome-mediated VEGF receptor 2 degradation2017In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 40, p. 125-132Article in journal (Refereed)
    Abstract [en]

    The complete repertoire of endothelial functions elicited by FGD5, a guanine nucleotide exchange factor activating the Rho GTPase Cdc42, has yet to be elucidated. Here we explore FGD5's importance during vascular endothelial growth factor A (VEGFA) signaling via VEGF receptor 2 (VEGFR2) in human endothelial cells. In microvascular endothelial cells, FGD5 is located at the inner surface of the cell membrane as well as at the outer surface of EEAl-positive endosomes carrying VEGFR2. The latter finding prompted us to explore if FGD5 regulates VEGFR2 dynamics. We found that depletion of FGD5 in microvascular cells inhibited their migration towards a stable VEGFA gradient. Furthermore, depletion of FGD5 resulted in accelerated VEGFR2 degradation, which was reverted by lactacystin-mediated proteasomal inhibition. Our results thus suggest a mechanism whereby FGD5 sustains VEGFA signaling and endothelial cell chemotaxis via inhibition of proteasome-dependent VEGFR2 degradation.

  • 4.
    Hernández Vera, Rodrigo
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Fatsis-Kavalopoulos, Nikos
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Gradientech AB, Uppsala Science Park, Uppsala, Sweden.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Modular microfluidic systems cast from 3D-printed molds for imaging leukocyte adherence to differentially treated endothelial cultures2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 11321Article in journal (Refereed)
    Abstract [en]

    Microfluidic systems are very useful for in vitro studies of interactions between blood cells and vascular endothelial cells under flow, and several commercial solutions exist. However, the availability of customizable, user-designed devices is largely restricted to researchers with expertise in photolithography and access to clean room facilities. Here we describe a strategy for producing tailor-made modular microfluidic systems, cast in PDMS from 3D-printed molds, to facilitate studies of leukocyte adherence to endothelial cells. A dual-chamber barrier module was optimized for culturing two endothelial cell populations, separated by a 250 μm wide dividing wall, on a glass slide. In proof-of-principle experiments one endothelial population was activated by TNFα, while the other served as an internal control. The barrier module was thereafter replaced with a microfluidic flow module, enclosing both endothelial populations in a common channel. A suspension of fluorescently-labeled leukocytes was then perfused through the flow module and leukocyte interactions with control and tnfα-treated endothelial populations were monitored in the same field of view. Time-lapse microscopy analysis confirmed the preferential attachment of leukocytes to the TNFα-activated endothelial cells. We conclude that the functionality of these modular microfluidic systems makes it possible to seed and differentially activate adherent cell types, and conduct controlled side-by-side analysis of their capacity to interact with cells in suspension under flow. Furthermore, we outline a number of practical considerations and solutions associated with connecting and switching between the microfluidic modules, and the advantages of simultaneously and symmetrically analyzing control and experimental conditions in such a microfluidic system.

  • 5. Kasza, Zsolt
    et al.
    Fredlund Fuchs, Peder
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tamm, Christoffer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eriksson, Anna S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heindryckx, Femke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Larsson, Erik
    Le Jan, Sebastien
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gerwins, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Radiology.
    Kjellen, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    MicroRNA-24 Suppression of N-Deacetylase/N-Sulfotransferase-1 (NDST1) Reduces Endothelial Cell Responsiveness to Vascular Endothelial Growth Factor A (VEGFA)2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 36, p. 25956-25963Article in journal (Refereed)
    Abstract [en]

    Heparan sulfate (HS) proteoglycans, present at the plasma membrane of vascular endothelial cells, bind to the angiogenic growth factor VEGFA to modulate its signaling through VEGFR2. The interactions between VEGFA and proteoglycan co-receptors require sulfated domains in the HS chains. To date, it is essentially unknown how the formation of sulfated protein-binding domains in HS can be regulated by microRNAs. In the present study, we show that microRNA-24 (miR-24) targets NDST1 to reduce HS sulfation and thereby the binding affinity of HS for VEGFA. Elevated levels of miR-24 also resulted in reduced levels of VEGFR2 and blunted VEGFA signaling. Similarly, suppression of NDST1 using siRNA led to a reduction in VEGFR2 expression. Consequently, not only VEGFA binding, but also VEGFR2 protein expression is dependent on NDST1 function. Furthermore, overexpression of miR-24, or siRNA-mediated reduction of NDST1, reduced endothelial cell chemotaxis in response to VEGFA. These findings establish NDST1 as a target of miR-24 and demonstrate how such NDST1 suppression in endothelial cells results in reduced responsiveness to VEGFA.

  • 6.
    Kreuger, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Failure to Genotype: A Cautionary Note on an Elusive loxP Sequence2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 10, article id e0165012Article in journal (Refereed)
    Abstract [en]

    Here we report on a technical difficulty we encountered while optimizing genotyping strategies to identify mice derived from Exoc3l2(tm1a( KOMP)Wtsi) embryonic stem cells obtained from the Knockout Mouse Project Repository. The Exoc3l2(tm1a(KOMP)Wtsi) construct encodes a "knockout-first" design with loxP sites that confer conditional potential (KO1st). We designed primers that targeted wild-type sequences flanking the most downstream element of the construct, an 80 base pair synthetic loxP region, which BLAST alignment analysis reveals is an element common to over 10,000 conditional gene-targeting mouse models. As PCR products amplified from KO1st and wild-type templates would have different lengths (and different mobility in an agarose gel) this strategy was designed to determine the zygosity of individual mice from a single PCR. In parallel we performed PCR with a primer specifically targeting the synthetic loxP sequence. Unexpectedly, while the latter strategy detected the synthetic loxP region and correctly genotyped KO1st chimeric mice, the same individuals were genotyped as wild-type when using the primers that flanked the synthetic loxP region. We discuss the possibility that secondary DNA structures, formed due to the palindromic nature of the synthetic loxP region, may have caused the KO1st template to elude the PCR when using primers that flanked this region. This brief report aims to raise awareness regarding this potential source of false-negative genotype results, particularly for those who are devising genotyping strategies for similarly engineered animal models.

  • 7.
    Noborn, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hermansson, Erik
    Zhang, Xiao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Ancsin, John
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Damas, Ana
    Dacklin, Ingrid
    Presto, Jenny
    Johansson, Jan
    Saraiva, Maria
    Lundgren, Erik
    Kisilevsky, Robert
    Westermark, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Li, Jin-ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparan sulfate/heparin promotes transthyretin fibrillization through selective binding to a basic motif in the protein2011In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 14, p. 5584-5589Article in journal (Refereed)
    Abstract [en]

    Transthyretin (TTR) is a homotetrameric protein that transports thyroxine and retinol. Tetramer destabilization and misfolding of the released monomers result in TTR aggregation, leading to its deposition as amyloid primarily in the heart and peripheral nervous system. Over 100 mutations of TTR have been linked to familial forms of TTR amyloidosis. Considerable effort has been devoted to the study of TTR aggregation of these mutants, although the majority of TTR-related amyloidosis is represented by sporadic cases due to the aggregation and deposition of the otherwise stable wild-type (WT) protein. Heparan sulfate (HS) has been found as a pertinent component in a number of amyloid deposits, suggesting its participation in amyloidogenesis. This study aimed to investigate possible roles of HS in TTR aggregation. Examination of heart tissue from an elderly cardiomyopathic patient revealed substantial accumulation of HS associated with the TTR amyloid deposits. Studies demonstrated that heparin/HS promoted TTR fibrillization through selective interaction with a basic motif of TTR. The importance of HS for TTR fibrillization was illustrated in a cell model; TTR incubated with WT Chinese hamster ovary cells resulted in fibrillization of the protein, but not with HS-deficient cells (pgsD-677). The effect of heparin on TTR fibril formation was further demonstrated in a Drosophila model that overexpresses TTR. Heparin was colocalized with TTR deposits in the head of the flies reared on heparin-supplemented medium, whereas no heparin was detected in the nontreated flies. Heparin of low molecular weight (Klexane) did not demonstrate this effect.

  • 8.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Heparan Sulfate in the Amyloidosis and Inflammation of Alzheimer’s Disease2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Alzheimer’s disease (AD) is a neurodegenerative disorder, with extensive evidence implicating the misfolding, aggregation and deposition of the amyloid-β (Aβ) peptide as central to the pathogenesis. Heparan sulfate (HS) is an interactive glycosaminoglycan, attached to core proteins as HS proteoglycans (HSPGs). HSPGs are present on cell surfaces and in the extracellular matrix where they facilitate multiple signaling functions, but HS is also consistently present in all amyloid deposits, including those of AD. In amyloidosis HS has been studied as an aggregation template, promoting fibril formation and serving a scaffold function in the resulting deposits. The objective of this thesis was to assess how cell surface HS is potentially implicated in Aβ amyloidosis and the associated neuroinflammation of AD.  

    In AD brain we determined that HS predominantly accumulated in Aβ deposits with dense cores and found glial-expressed HSPGs within these deposits. Aβ elevated HSPG levels in primary glial cultures, implicating activated glia as one source of the Aβ-associated HS. Next, we determined that microglial HSPGs are critical for the upregulation of interleukin-1β and tumor necrosis factor-α following exposure to lipopolysaccharide, an established inflammatory insult. Together these results raise the possibility that Aβ-induced expression of microglial HSPGs may promote neuroinflammation.  

    Multiple mechanisms of Aβ toxicity have been proposed and different Aβ assemblies exert their toxicity through alternative routes. We found that three different preparations of Aβ aggregates all exhibited HS-dependent cytotoxicity, which in part correlated with Aβ internalization. Furthermore, heparin treatment attenuated Aβ cytotoxicity and uptake. In Aβ-positive AD microvasculature, HS deposited with Apolipoprotein E (ApoE) and its receptor, the low density lipoprotein receptor-related protein 1 (LRP1). In cell culture, HS and LRP1 co-operated in Aβ interactions and the addition of ApoE increased the levels of cell-associated Aβ in a HS- and LRP1-dependent manner. This ApoE-mediated increase in cell-associated Aβ may promote toxicity and vascular degeneration, but equally HS-mediated internalization of Aβ could represent a clearance route across the blood-brain-barrier.

    The findings presented here illustrate multiple roles for cell-surface HSPGs in interactions relevant to the pathogenesis of AD.

    List of papers
    1. Heparan sulfate accumulation with Abeta deposits in Alzheimer's disease and Tg2576 mice is contributed by glial cells
    Open this publication in new window or tab >>Heparan sulfate accumulation with Abeta deposits in Alzheimer's disease and Tg2576 mice is contributed by glial cells
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    2008 (English)In: Brain Pathology, ISSN 1015-6305, E-ISSN 1750-3639, Vol. 18, no 4, p. 548-561Article in journal (Refereed) Published
    Abstract [en]

    Amyloid beta-peptide (Abeta) plaques, one of the major neuropathological lesions in Alzheimer's disease (AD), can be broadly subdivided into two morphological categories: neuritic and diffuse. Heparan sulfate (HS) and HS proteoglycans (HSPGs) are codeposits of multiple amyloidoses, including AD. Although HS has been considered a limiting factor in the initiation of amyloid deposition, the pathological implications of HS in Abeta deposits of AD remain unclear. In this study, immunohistochemistry combined with fluorescence and confocal microscopy was employed to gain deeper insight into the accumulation of HS with Abeta plaques in sporadic and familial AD. Here we demonstrate that HS preferentially accumulated around the Abeta40 dense cores of neuritic plaques, but was largely absent from diffuse Abeta42 plaques, suggesting that Abeta42 deposition may occur independently of HS. A codeposition pattern of HS with Abeta deposits in Tg2576 mice was also examined. We identified the membrane-bound HSPGs, glypican-1 (GPC1) and syndecan-3 (SDC3), in glial cells associated with Abeta deposits, proximal to sites of HS accumulation. In mouse primary glial cultures, we observed increased levels of GPC1 and SDC3 following Abeta stimulation. These results suggest that HS codeposits with Abeta40 in neuritic plaques and is mainly derived from glial cells.

    Keywords
    b-Amyloid, glial cells, heparan sulfate
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-98662 (URN)10.1111/j.1750-3639.2008.00152.x (DOI)000259146700009 ()18422760 (PubMedID)
    Available from: 2009-03-02 Created: 2009-03-02 Last updated: 2017-12-13Bibliographically approved
    2. Microglial heparan sulfate proteoglycans mediate pro-inflammatory signaling
    Open this publication in new window or tab >>Microglial heparan sulfate proteoglycans mediate pro-inflammatory signaling
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    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Microglia are the central nervous system’s (CNS) first line of defense against pathogenic insults and acute inflammatory responses are necessary for the resolution of infection. However, unregulated and/or chronic activation of microglia is associated with neurodegeneration. Heparan sulfate proteoglycans (HSPGs) have been attributed various roles in inflammation, but the possibility that HSPGs are integral to pro-inflammatory signaling mechanisms has not been fully explored. To analyze the relevance of microglial HSPGs in the pro-inflammatory response we isolated primary microglia from mice overexpressing human heparanase (Hpa-tg), the HS-degrading endoglucuronidase, and challenged them with the pro-inflammatory endotoxin lipopolysaccharide (LPS). The LPS-induced upregulation of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) was inhibited in Hpa-tg microglia, as was upregulation of the LPS-receptor CD14. Analysis of HSPG structures revealed that Hpa-tg microglia produced truncated HS chains. Importantly, co-treatment of microglia with heparin attenuated LPS-induced cytokine upregulation. Together these findings implicate microglial HSPGs as key facilitators of the pro-inflammatory response. Astrocytes constitute a critical support network in the CNS, but are also implicated in inflammation. LPS induced comparable levels of TNF-α in Hpa-tg and Ctrl astrocytes, indicating that the mechanism of HSPG-dependent inflammation is specific to microglia.  We conclude that microglial HSPGs are required for pro-inflammatory signaling events and that heparanase, through its HS-degrading activity, can regulate this mechanism.

    National Category
    Neurosciences Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-159923 (URN)
    Available from: 2011-10-12 Created: 2011-10-11 Last updated: 2018-01-12
    3. Heparan sulfate mediates amyloid-beta internalization and cytotoxicity
    Open this publication in new window or tab >>Heparan sulfate mediates amyloid-beta internalization and cytotoxicity
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    2010 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 20, no 5, p. 533-541Article in journal (Refereed) Published
    Abstract [en]

    Heparan sulfate (HS) has been found associated with amyloid deposits, including the toxic amyloid-beta (Abeta) peptide aggregates in cerebral vasculature and neuronal tissues in patients with Alzheimer's disease. However, the pathophysiological significance of the HS-Abeta interaction has remained unclear. In the present study, we applied cell models to gain insight into the roles of HS in relation to Abeta toxicity. Wild-type Chinese hamster ovary (CHO-WT) cells showed loss of viability following exposure to Abeta40, whereas the HS-deficient cell line, pgsD-677, was essentially resistant. Immunocytochemical analysis showed Abeta internalization by CHO-WT, but not pgsD-677 cells. Abeta40 toxicity was also attenuated in human embryonic kidney cells overexpressing heparanase. Finally, addition of heparin to human umbilical vein endothelial cells prevented internalization of added Abeta40 and protected against Abeta toxicity. Taken together, these findings suggest that cell-surface HS mediates Abeta internalization and toxicity.

    Keywords
    Aβ, cytotoxicity, heparanase, heparan sulfate, heparin
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-122999 (URN)10.1093/glycob/cwp205 (DOI)000276525700004 ()20053627 (PubMedID)
    Available from: 2010-04-22 Created: 2010-04-22 Last updated: 2017-12-12Bibliographically approved
    4. Apolipoprotein-E increases cell-associated amyloid-β through a heparan sulfate-dependent pathway
    Open this publication in new window or tab >>Apolipoprotein-E increases cell-associated amyloid-β through a heparan sulfate-dependent pathway
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    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    The increased risk of Alzheimer’s disease (AD) associated with specific apolipoprotein E (ApoE) isoforms appears to relate to altered amyloid-β (Aβ) homeostasis. The efficiency of Aβ clearance from the brain is reduced in the presence of the AD-associated ApoE4 isoform, which may explain the accumulation of Aβ deposits in the parenchyma and vasculature. The low density lipoprotein receptor-related protein 1 (LRP1) and heparan sulfate proteoglycans (HSPGs) are involved in Aβ uptake, with LRP1 further implicated in Aβ transcytosis across the blood brain barrier. However, both are also established ApoE receptors and function co-operatively to mediate cell interactions with lipoproteins and Aβ. Here we determined that HS, ApoE and LRP1 co-occur in Aβ40-positive microvessels of AD brain, establishing the relevance of studying interactions between these molecules. Using Chinese hamster ovary (CHO) cells deficient in HS or LRP1 we found that ApoE increases the levels of cell-associated Aβ in primarily a HSPG-dependent manner. Furthermore, in this model we found that ApoE is alternatively processed in the absence of cell surface HS, supporting a role for HSPGs in ApoE metabolism. The findings presented here raise the possibility that ApoE can increase Aβ associations with HSPGs and LRP1 in the vasculature. This may facilitate clearance, but if unbalanced could contribute to Aβ accumulation and the pathogenesis of AD.

    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-159926 (URN)
    Available from: 2011-10-12 Created: 2011-10-11 Last updated: 2018-01-12
  • 9.
    O'Callaghan, Paul
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Li, Jin-Ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lindahl, Ulf
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Zhang, Xiao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Microglial Heparan Sulfate Proteoglycans Facilitate the Cluster-of-Differentiation 14 (CD14)/Toll-like Receptor 4 (TLR4)-Dependent Inflammatory Response2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 24, p. 14904-14914Article in journal (Refereed)
    Abstract [en]

    Microglia rapidly mount an inflammatory response to pathogens in the central nervous system (CNS). Heparan sulfate proteoglycans (HSPGs) have been attributed various roles in inflammation. To elucidate the relevance of microglial HSPGs in a pro-inflammatory response we isolated microglia from mice overexpressing heparanase (Hpa-tg), the HS-degrading endoglucuronidase, and challenged them with lipopolysaccharide (LPS), a bacterial endotoxin. Prior to LPS-stimulation, the LPS-receptor cluster-of-differentiation 14 (CD14) and Toll-like receptor 4 (TLR4; essential for the LPS response) were similarly expressed in Ctrl and Hpa-tg microglia. However, compared with Ctrl microglia, Hpa-tg cells released significantly less tumor necrosis factor-α (TNFα), essentially failed to up-regulate interleukin-1β (IL1β) and did not initiate synthesis of proCD14. Isolated primary astroyctes expressed TLR4, but notably lacked CD14 and in contrast to microglia, LPS challenge induced a similar TNFα response in Ctrl and Hpa-tg astrocytes, while neither released IL1β. The astrocyte TNFα-induction was thus attributed to CD14-independent TLR4 activation and was unaffected by the cells HS status. Equally, the suppressed LPS-response in Hpa-tg microglia indicated a loss of CD14-dependent TLR4 activation, suggesting that microglial HSPGs facilitate this process. Indeed, confocal microscopy confirmed interactions between microglial HS and CD14 in LPS-stimulated microglia and a potential HS-binding motif in CD14 was identified. We conclude that microglial HSPGs facilitate CD14-dependent TLR4 activation and that heparanase can modulate this mechanism.

  • 10.
    O'Callaghan, Paul
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Noborn, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Li, Jin-ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Zhang, Xiao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Apolipoprotein E increases cell association of amyloid-β 40 through heparan sulfate and LRP1 dependent pathways2014In: Amyloid: Journal of Protein Folding Disorders, ISSN 1350-6129, E-ISSN 1744-2818, Vol. 21, no 2, p. 76-87Article in journal (Refereed)
  • 11.
    O'Callaghan, Paul
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sandwall, Elina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Li, Jin-Ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Yu, Hong
    Ravid, Rivka
    Guan, Zhi-Zhong
    van Kuppevelt, Toin H
    Nilsson, Lars N G
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hyman, Bradley T
    Kalimo, Hannu
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Zhang, Xiao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Heparan sulfate accumulation with Abeta deposits in Alzheimer's disease and Tg2576 mice is contributed by glial cells2008In: Brain Pathology, ISSN 1015-6305, E-ISSN 1750-3639, Vol. 18, no 4, p. 548-561Article in journal (Refereed)
    Abstract [en]

    Amyloid beta-peptide (Abeta) plaques, one of the major neuropathological lesions in Alzheimer's disease (AD), can be broadly subdivided into two morphological categories: neuritic and diffuse. Heparan sulfate (HS) and HS proteoglycans (HSPGs) are codeposits of multiple amyloidoses, including AD. Although HS has been considered a limiting factor in the initiation of amyloid deposition, the pathological implications of HS in Abeta deposits of AD remain unclear. In this study, immunohistochemistry combined with fluorescence and confocal microscopy was employed to gain deeper insight into the accumulation of HS with Abeta plaques in sporadic and familial AD. Here we demonstrate that HS preferentially accumulated around the Abeta40 dense cores of neuritic plaques, but was largely absent from diffuse Abeta42 plaques, suggesting that Abeta42 deposition may occur independently of HS. A codeposition pattern of HS with Abeta deposits in Tg2576 mice was also examined. We identified the membrane-bound HSPGs, glypican-1 (GPC1) and syndecan-3 (SDC3), in glial cells associated with Abeta deposits, proximal to sites of HS accumulation. In mouse primary glial cultures, we observed increased levels of GPC1 and SDC3 following Abeta stimulation. These results suggest that HS codeposits with Abeta40 in neuritic plaques and is mainly derived from glial cells.

  • 12.
    O'Callaghan, Paul
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Zarb, Yvette
    Zurich Univ, Zurich Univ Hosp, Clin Neurosci Ctr, Dept Neurosurg, Zurich, Switzerland.
    Noborn, Fredrik
    Univ Gothenburg, Inst Biomed, Dept Clin Chem & Transfus Med, Sahlgrenska Acad, Gothenburg, Sweden.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Modeling the structural implications of an alternatively spliced Exoc3l2, a paralog of the tunneling nanotube-forming M-Sec2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 8, article id e0201557Article in journal (Refereed)
    Abstract [en]

    The exocyst is a molecular tether that retains secretory vesicles at the plasma membrane prior to SNARE-mediated docking and fusion. However, individual exocyst complex components (EXOCs) may also function independently of exocyst assembly. Alternative splice variants of EXOC mRNA and paralogs of EXOC genes have been described and several have been attributed functions that may be independent of the exocyst complex. Here we describe a novel splice variant of murine Exoc3l2, which we term Exoc3l2a. We discuss possible functional implications of the resulting domain excision from this isoform of EXOC3L2 based on structural similarities with its paralog M-Sec (EXOC3L3), which is implicated in tunneling nanotube formation. The identification of this Exoc3l2 splice variant expands the potential for subunit diversity within the exocyst and for alternative functionality of this component independently of the exocyst.

  • 13.
    O'Callaghan, Paul
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Zhang, Xiao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Li, Jin-Ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparan Sulfate Proteoglycans as Relays of Neuroinflammation2018In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 66, no 4, p. 305-319Article, review/survey (Refereed)
    Abstract [en]

    Heparan sulfate proteoglycans (HSPGs) are implicated as inflammatory mediators in a variety of settings, including chemokine activation, which is required to recruit circulating leukocytes to infection sites. Heparan sulfate (HS) polysaccharide chains are highly interactive and serve co-receptor roles in multiple ligand:receptor interactions. HS may also serve as a storage depot, sequestering ligands such as cytokines and restricting their access to binding partners. Heparanase, through its ability to fragment HS chains, is a key regulator of HS function and has featured prominently in studies of HS's involvement in inflammatory processes. This review focuses on recent discoveries regarding the role of HSPGs, HS, and heparanase during inflammation, with particular focus on the brain. HS chains emerge as critical go-betweens in multiple aspects of the inflammatory responserelaying signals between receptors and cells. The molecular interactions proposed to occur between HSPGs and the pathogen receptor toll-like receptor 4 (TLR4) are discussed, and we summarize some of the contrasting roles that HS and heparanase have been assigned in diseases associated with chronic inflammatory states, including Alzheimer's disease (AD). We conclude by briefly discussing how current knowledge could potentially be applied to augment HS-mediated events during sustained neuroinflammation, which contributes to neurodegeneration in AD.

  • 14.
    Philipson, Ola
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lord, Anna
    Gumucio, Astrid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Nilsson, Lars N. G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Animal models of amyloid-β-related pathologies in Alzheimer’s disease2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 6, p. 1389-1409Article, review/survey (Refereed)
    Abstract [en]

    In the early 1990s, breakthrough discoveries on the genetics of Alzheimer's disease led to the identification of missense mutations in the amyloid-beta precursor protein gene. Research findings quickly followed, giving insights into molecular pathogenesis and possibilities for the development of new types of animal models. The complete toolbox of transgenic techniques, including pronuclear oocyte injection and homologous recombination, has been applied in the Alzheimer's disease field, to produce overexpressors, knockouts, knockins and regulatable transgenics. Transgenic models have dramatically advanced our understanding of pathogenic mechanisms and allowed therapeutic approaches to be tested. Following a brief introduction to Alzheimer's disease, various nontransgenic and transgenic animal models are described in terms of their values and limitations with respect to pathogenic, therapeutic and functional understandings of the human disease.

  • 15.
    Sandwall, Elina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Zhang, Xiao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Li, Jin-Ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparan sulfate mediates amyloid-beta internalization and cytotoxicity2010In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 20, no 5, p. 533-541Article in journal (Refereed)
    Abstract [en]

    Heparan sulfate (HS) has been found associated with amyloid deposits, including the toxic amyloid-beta (Abeta) peptide aggregates in cerebral vasculature and neuronal tissues in patients with Alzheimer's disease. However, the pathophysiological significance of the HS-Abeta interaction has remained unclear. In the present study, we applied cell models to gain insight into the roles of HS in relation to Abeta toxicity. Wild-type Chinese hamster ovary (CHO-WT) cells showed loss of viability following exposure to Abeta40, whereas the HS-deficient cell line, pgsD-677, was essentially resistant. Immunocytochemical analysis showed Abeta internalization by CHO-WT, but not pgsD-677 cells. Abeta40 toxicity was also attenuated in human embryonic kidney cells overexpressing heparanase. Finally, addition of heparin to human umbilical vein endothelial cells prevented internalization of added Abeta40 and protected against Abeta toxicity. Taken together, these findings suggest that cell-surface HS mediates Abeta internalization and toxicity.

  • 16.
    Zhang, Xiao
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Wang, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hjertstrom, Elina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jia, Juan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gong, Feng
    Zcharia, Eyal
    Nilsson, Lars N. G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Vlodavsky, Israel
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Li, Jin-Ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparanase overexpression impairs inflammatory response and macrophage-mediated clearance of amyloid-beta in murine brain2012In: Acta Neuropathologica, ISSN 0001-6322, E-ISSN 1432-0533, Vol. 124, no 4, p. 465-478Article in journal (Refereed)
    Abstract [en]

    Neuroinflammation is typically observed in neurodegenerative diseases such as Alzheimer's disease, as well as after traumatic injury and pathogen infection. Resident immune cells, microglia and astrocytes, are activated and joined by blood-borne monocytes that traverse the blood-brain barrier and convert into activated macrophages. The activated cells express various cytokines, chemokines and proteolytic enzymes. To study the role of heparan sulfate proteoglycans in neuroinflammation, we employed a transgenic mouse overexpressing heparanase, an endoglucuronidase that specifically degrades heparan sulfate side chains. Neuroinflammation was induced by systemic challenge with lipopolysaccharide, or by localized cerebral microinjection of aggregated amyloid-beta peptide, implicated in Alzheimer's disease. Lipopolysaccharide-treated control mice showed massive activation of resident microglia as well as recruitment of monocyte-derived macrophages into the brain parenchyma. Microinjection of aggregated amyloid-beta elicited a similar inflammatory response, albeit restricted to the injection site, which led to dispersion and clearance of the amyloid. In the heparanase-overexpressing mice, all aspects of immune cell recruitment and activation were significantly attenuated in both inflammation models, as was amyloid dispersion. Accordingly, an in vitro blood-brain barrier model constructed from heparanase-overexpressing cerebral vascular cells showed impaired transmigration of monocytes compared to a corresponding assembly of control cells. Our data indicate that intact heparan sulfate chains are required at multiple sites to mediate neuroinflammatory responses, and further point to heparanase as a modulator of this process, with potential implications for Alzheimer's disease.

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