A J Roebroek

Université de Sherbrooke, Sherbrooke, Quebec, Canada

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Publications (84)403.13 Total impact

  • Anton J M Roebroek, Bart Van Gool
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    ABSTRACT: Molecular genetic strategies applying embryonic stem cell (ES cell) technologies to study the function of a gene in mice or to generate a mouse model for a human disease are continuously under development. Next to (conditional) inactivation of genes the application and importance of approaches to generate knock-in mutations are increasing. In this chapter the principle and application of recombinase-mediated cassette exchange (RMCE) are discussed as being a new emerging knock-in strategy, which enables easy generation of a series of different knock-in mutations within one gene. An RMCE protocol, which was used to generate a series of different knock-in mutations in the Lrp1 gene of ES cells, is described in detail as an example of how RMCE can be used to generate highly efficiently an allelic series of differently modified ES cell clones from a parental modified ES cell clone. Subsequently the differently modified ES cell clones can be used to generate an allelic series of mutant knock-in mice.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1194:63-76. · 1.29 Impact Factor
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    ABSTRACT: The N-methyl-D-aspartate receptors are key mediators of excitatory transmission and are implicated in many forms of synaptic plasticity. These receptors are heterotetrameres consisting of two obligatory NR1 and two regulatory subunits, usually NR2A or NR2B. The NR2B subunits are abundant in the early postnatal brain, while the NR2A/NR2B ratio increases during early postnatal development. This shift is driven by NMDA receptor activity. A functional interplay of the Low Density Lipoprotein Receptor Related Protein 1 (LRP1) NMDA receptor has already been reported. Such abilities as interaction of LRP1 with NMDA receptor subunits or its important role in tPa-mediated NMDA receptor signaling were already demonstrated. Moreover, mice harboring a conditional neuronal knock-out mutation of the entire Lrp1 gene display NMDA-associated behavioral changes. However, the exact role of LRP1 on NMDA receptor function remains still elusive. To provide a mechanistic explanation for such effects we investigated whether an inactivating knock-in mutation into the NPxY2 motif of LRP1 might influence the cell surface expression of LRP1 and NMDA receptors in primary cortical neurons. Here we demonstrate that a knock-in into the NPxY2 motif of LRP1 results in an increased surface expression of LRP1 and NR2B NMDA receptor subunit due to reduced endocytosis rates of LRP1 and the NR2B subunit in primary neurons derived from LRP1DeltaNPxY2 animals. Furthermore, we demonstrate an altered phosphorylation pattern of S1480 and Y1472 in the NR2B subunit at the surface of LRP1DeltaNPxY2 neurons, while the respective kinases Fyn and casein kinase II are not differently regulated compared with wild type controls. Performing co-immunoprecipitation experiments we demonstrate that binding of LRP1 to NR2B might be linked by PSD95, is phosphorylation dependent and this regulation mechanism is impaired in LRP1DeltaNPxY2 neurons. Finally, we demonstrate hyperactivity and changes in spatial and reversal learning in LRP1DeltaNPxY2 mice, confirming the mechanistic interaction in a physiological readout. In summary, our data demonstrate that LRP1 plays a critical role in the regulation of NR2B expression at the cell surface and may provide a mechanistic explanation for the behavioral abnormalities detected in neuronal LRP1 knock-out animals reported earlier.
    Molecular Neurodegeneration 07/2013; 8(1):25. · 4.01 Impact Factor
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    ABSTRACT: ApoM is mainly associated with HDL. Nevertheless, we have consistently observed positive correlations of apoM with plasma LDL cholesterol in humans. Moreover, LDL receptor deficiency is associated with increased plasma apoM in mice. Here, we tested the idea that plasma apoM concentrations are affected by the rate of LDL receptor-mediated clearance of apoB-containing particles. We measured apoM in humans each carrying one of three different LDL receptor mutations (n = 9) or the apoB3500 mutation (n = 12). These carriers had increased plasma apoM (1.34 ± 0.13 µM, P = 0.003, and 1.23 ± 0.10 µM, P = 0.02, respectively) as compared with noncarriers (0.93 ± 0.04 µM). When we injected human apoM-containing HDL into Wt (n = 6) or LDL receptor-deficient mice (n = 6), the removal of HDL-associated human apoM was delayed in the LDL receptor-deficient mice. After 2 h, 54 ± 5% versus 90 ± 8% (P < 0.005) of the initial amounts of human apoM remained in the plasma of Wt and LDL receptor-deficient mice, respectively. Finally, we compared the turnover of radio-iodinated LDL and plasma apoM concentrations in 45 normocholesterolemic humans. There was a negative correlation between plasma apoM and the fractional catabolic rate of LDL (r = -0.38, P = 0.009). These data suggest that the plasma clearance of apoM, despite apoM primarily being associated with HDL, is influenced by LDL receptor-mediated clearance of apoB-containing particles.
    The Journal of Lipid Research 07/2012; 53(10):2198-204. · 4.39 Impact Factor
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    ABSTRACT: In mammals, seven proprotein convertases (PCs) cleave secretory proteins after basic residues, and four of them are called furin-like PCs: furin, PC5, PACE4, and PC7. In vitro, they share many substrates. However, furin is essential during development since deficient embryos die at embryonic day 11 and exhibit multiple developmental defects, particularly defects related to the function of endothelial cells. To define the role of furin in endothelial cells, an endothelial cell-specific knockout (ecKO) of the Furin gene was generated. Newborns die shortly after birth, indicating that furin is essential in these cells. Magnetic resonance imaging revealed that ecKO embryos exhibit ventricular septal defects (VSD) and/or valve malformations. In addition, primary cultures of wild-type and ecKO lung endothelial cells revealed that ecKO cells are unable to grow. Growth was efficiently rescued by extracellular soluble furin. Analysis of the processing of precursors of endothelin-1 (ET-1), adrenomedullin (Adm), transforming growth factor β1 (TGF-β1), and bone morphogenetic protein 4 (BMP4) confirmed that ET-1, Adm, and TGF-β1 are in vivo substrates of endothelial furin. Mature ET-1 and BMP4 forms were reduced by ~90% in ecKO purified endothelial cells from lungs.
    Molecular and cellular biology 06/2012; 32(17):3382-91. · 6.06 Impact Factor
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    ABSTRACT: Determination of the in vivo significance of LDL receptor-related protein 1 (LRP1) dysfunction on lipid metabolism and atherosclerosis development in absence of its main ligand apoE. LRP1 knock-in mice carrying an inactivating mutation in the NPxYxxL motif were crossed with apoE-deficient mice. In the absence of apoE, relative to LRP1 wild-type animals, LRP1 mutated mice showed an increased clearance of postprandial lipids despite a compromised LRP1 endocytosis rate and inefficient insulin-mediated translocation of the receptor to the plasma membrane, likely due to inefficient slow recycling of the mutated receptor. Postprandial lipoprotein improvement was explained by increased hepatic clearance of triglyceride-rich remnant lipoproteins and accompanied by a compensatory 1.6-fold upregulation of LDLR expression in hepatocytes. One year-old apoE-deficient mice having the dysfunctional LRP1 revealed a 3-fold decrease in spontaneous atherosclerosis development and a 2-fold reduction in LDL-cholesterol levels. These findings demonstrate that the NPxYxxL motif in LRP1 is important for insulin-mediated translocation and slow perinuclear endosomal recycling. These LRP1 impairments correlated with reduced atherogenesis and cholesterol levels in apoE-deficient mice, likely via compensatory LDLR upregulation.
    PLoS ONE 01/2012; 7(6):e38330. · 3.73 Impact Factor
  • EMBO J. 01/2012; Jan 17..
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    ABSTRACT: Surface-exposed calreticulin (ecto-CRT) and secreted ATP are crucial damage-associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)-based (reactive oxygen species (ROS)-regulated) pathway for ecto-CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS-mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80(high), CD83(high), CD86(high), MHC-II(high)) and functional stimulation (NO(high), IL-10(absent), IL-1β(high)) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto-CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK-orchestrated pathways that require a functional secretory pathway and phosphoinositide 3-kinase (PI3K)-mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase-8 signalling are dispensable for this ecto-CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto-CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase-8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK-dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS-mediated ER stress.
    The EMBO Journal 01/2012; 31(5):1062-79. · 9.82 Impact Factor
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    ABSTRACT: According to the "amyloid hypothesis", the amyloid-β (Aβ) peptide is the toxic intermediate driving Alzheimer's disease (AD) pathogenesis. Recent evidence suggests that the low density lipoprotein receptor-related protein 1 (LRP1) transcytoses Aβ out of the brain across the blood-brain barrier (BBB). To provide genetic evidence for LRP1-mediated transcytosis of Aβ across the BBB we analyzed Aβ transcytosis across primary mouse brain capillary endothelial cells (pMBCECs) derived from wild-type and LRP1 knock-in mice. Here, we show that pMBCECs in vitro express functionally active LRP1. Moreover, we demonstrate that LRP1 mediates transcytosis of [(125)I]-Aβ(1-40) across pMBCECs in both directions, whereas no role for LRP1-mediated Aβ degradation was detected. Analysis of [(125)I]-Aβ(1-40) transport across pMBCECs generated from mice harboring a knock-in mutation in the NPxYxxL endocytosis/sorting domain of endogenous LRP1 revealed a reduced Aβ clearance from brain-to-blood and blood-to-brain compared with wild-type derived pMBCECs. Therefore, for the first time, we present genetic evidence that LRP1 modulates the pathogenic actions of soluble Aβ in the brain by clearing Aβ across the BBB.
    Neurobiology of aging 12/2011; 32(12):2323.e1-11. · 5.94 Impact Factor
  • Anton J M Roebroek, Philip L S M Gordts, Sara Reekmans
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    ABSTRACT: Molecular genetic strategies to study gene function in mice or to generate a mouse model for a human disease are continuously under development. The application and importance of knock-in approaches are increasing. This chapter elaborates on novel developments for the generation of knock-in mice. Special emphasis is given to recombinase-mediated cassette exchange, a new emerging knock-in strategy that enables easy generation of a series of different knock-in mutations within one gene.
    Methods in molecular biology (Clifton, N.J.) 01/2011; 693:257-75. · 1.29 Impact Factor
  • Anton J M Roebroek, Philip L S M Gordts, Sara Reekmans
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    ABSTRACT: Recombinase-mediated cassette exchange (RMCE) is a powerful tool to generate a series of knock-in mutations into a particular gene of the mouse. It uses standard ES cell technologies to introduce an exchangeable cassette into the gene of interest by homologous recombination. Because the introduced exchangeable cassette is flanked by heterotypic specific recombination target sequences, site-specific recombinases can be used in a subsequent RMCE step to exchange the cassette by other sequences. Here, an experimental procedure for the application of RMCE in E14 ES cells using heterotypic FRT recombination target sequences and the site-specific recombinase Flp is elaborated.
    Methods in molecular biology (Clifton, N.J.) 01/2011; 693:277-81. · 1.29 Impact Factor
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    ABSTRACT: Plasma apolipoprotein (apo)M is mainly associated with high-density lipoprotein (HDL). HDL-bound apoM is antiatherogenic in vitro. However, plasma apoM is not associated with coronary heart disease in humans, perhaps because of a positive correlation with plasma low-density lipoprotein (LDL). We explored putative links between apoM and very-low-density (VLDL)/LDL metabolism and the antiatherogenic potential of apoM in vivo. Plasma apoM was increased approximately 2.1 and approximately 1.5 fold in mice lacking LDL receptors (Ldlr(-/-)) and expressing dysfunctional LDL receptor-related protein 1 (Lrp1(n2/n2)), respectively, but was unaffected in apoE-deficient (ApoE(-/-)) mice. Thus, pathways controlling catabolism of VLDL and LDL affect plasma apoM. Overexpression (approximately 10-fold) of human apoM increased (50% to 70%) and apoM deficiency decreased ( approximately 25%) plasma VLDL/LDL cholesterol in Ldlr(-/-) mice, whereas apoM did not affect plasma VLDL/LDL in mice with intact LDL receptors. Moreover, plasma clearance of apoM-enriched VLDL/LDL was slower than that of control VLDL/LDL in mice lacking functional LDL receptors and LRP1, suggesting that apoM impairs the catabolism of VLDL/LDL that occurs independently of the LDL receptor and LRP1. ApoM overexpression decreased atherosclerosis in ApoE(-/-) (60%) and cholate/cholesterol-fed wild-type mice (70%). However, in Ldlr(-/-) mice the antiatherogenic effect of apoM was attenuated by its VLDL/LDL-raising effect. The data suggest that defect LDL receptor function leads to increased plasma apoM concentrations, which in turn, impairs the removal of VLDL/LDL from plasma. This mechanism opposes the otherwise antiatherogenic effect of apoM.
    Circulation Research 04/2010; 106(10):1624-34. · 11.86 Impact Factor
  • Atherosclerosis Supplements - ATHEROSCLER SUPPL. 01/2010; 11(2):85-86.
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    ABSTRACT: The proximal NPXY and distal NPXYXXL motifs in the intracellular domain of LRP1 play an important role in regulation of the function of the receptor. The impact of single and double inactivating knock-in mutations of these motifs on receptor maturation, cell surface expression, and ligand internalization was analyzed in mutant and control wild-type mice and MEFs. Single inactivation of the proximal NPXY or in combination with inactivation of the distal NPXYXXL motif are both shown to be associated with an impaired maturation and premature proteasomal degradation of full-length LRP1. Therefore, only a small mature LRP1 pool is able to reach the cell surface resulting indirectly in severe impairment of ligand internalization. Single inactivation of the NPXYXXL motif revealed normal maturation, but direct impairment of ligand internalization. In conclusion, the proximal NPXY motif proves to be essential for early steps in the LRP1 biosynthesis, whereas NPXYXXL appears rather relevant for internalization.
    Cellular and Molecular Life Sciences CMLS 10/2009; 67(1):135-45. · 5.62 Impact Factor
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    ABSTRACT: The purpose of this study was to determine the significance of the intracellular NPxYxxL motif of LRP1 for the atheroprotective role of this multifunctional receptor. LRP1 knock-in mice carrying an inactivating mutation in the NPxYxxL motif were crossed with LDLR-deficient mice, a model for atherosclerosis. In this LDLR(-/-) background the mutated mice showed a more atherogenic lipoprotein profile, which was associated with a decreased clearance of postprandial lipids because of a compromised endocytosis rate and reduced lipase activity. On an atherogenic diet LRP1 mutant mice revealed a 50% increased development of atherosclerosis. This aggravation was accompanied by an increase in smooth muscle cell (SMC) and collagen content and apoptotic cells in the lesions. The mutation showed, however, a limited impact on basal PDGFR-beta expression and signaling and the antimigratory property of apoE on PDGF-BB-stimulated SMCs. Additionally, levels of LRP1 atherogenic ligands, like MMP2, t-PA, FVIII, and the inflammatory ligand TNF-alpha showed to be significantly elevated. These findings demonstrate that the NPxYxxL motif is essential for the atheroprotective role of LRP1. This motif is relevant for normal control of lipid metabolism and of atherogenic and inflammatory ligands, but has no pronounced effect on regulating PDGF-BB/PDGFR-beta signaling in SMCs.
    Arteriosclerosis Thrombosis and Vascular Biology 09/2009; 29(9):1258-64. · 6.34 Impact Factor
  • P Gordts, A Bartelt, J Heeren, A Roebroek
    Atherosclerosis Supplements - ATHEROSCLER SUPPL. 01/2009; 10(2).
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    ABSTRACT: Furin is a proprotein convertase which activates a variety of regulatory proteins in the constitutive exocytic and endocytic pathway. The effect of genetic ablation of fur was studied in the endocrine pancreas to define its physiological function in the regulated secretory pathway. Pdx1-Cre/loxP furin KO mice show decreased secretion of insulin and impaired processing of known PC2 substrates like proPC2 and proinsulin II. Both secretion and PC2 activity depend on granule acidification, which was demonstrated to be significantly decreased in furin-deficient beta cells by using the acidotrophic agent 3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine (DAMP). Ac45, an accessory subunit of the proton pump V-ATPase, was investigated as a candidate substrate. Ac45 is highly expressed in islets of Langerhans and furin was able to cleave Ac45 ex vivo. Furthermore, the exact cleavage site was determined. In addition, reduced regulated secretion and proinsulin II processing could be obtained in the insulinoma cell line betaTC3 by downregulation of either furin or Ac45. Together, these data establish an important role for furin in regulated secretion, particularly in intragranular acidification most likely due to impaired processing of Ac45.
    Proceedings of the National Academy of Sciences 09/2008; 105(34):12319-24. · 9.81 Impact Factor
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    ABSTRACT: Furin is one of seven proprotein convertase family members that promote proteolytic maturation of proproteins. It is induced in activated T cells and is reported to process a variety of substrates including the anti-inflammatory cytokine transforming growth factor (TGF)-beta1 (refs 2-4), but the non-redundant functions of furin versus other proprotein convertases in T cells are unclear. Here we show that conditional deletion of furin in T cells allowed for normal T-cell development but impaired the function of regulatory and effector T cells, which produced less TGF-beta1. Furin-deficient T regulatory (Treg) cells were less protective in a T-cell transfer colitis model and failed to induce Foxp3 in normal T cells. Additionally, furin-deficient effector cells were inherently over-active and were resistant to suppressive activity of wild-type Treg cells. Thus, our results indicate that furin is indispensable in maintaining peripheral tolerance, which is due, at least in part, to its non-redundant, essential function in regulating TGF-beta1 production. Targeting furin has emerged as a strategy in malignant and infectious disease. Our results suggest that inhibiting furin might activate immune responses, but may result in a breakdown in peripheral tolerance.
    Nature 09/2008; 455(7210):246-50. · 38.60 Impact Factor
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    ABSTRACT: Proprotein convertases are serine endoproteases implicated in the proteolytic processing of a large variety of regulatory proteins. An important role of proprotein convertases in tumorigenic processes has been suggested by various studies. In this study, the role of the proprotein convertase furin in PLAG1 proto-oncogene-induced salivary gland tumorigenesis was investigated. PLAG1 overexpression in salivary glands has previously been shown to result in salivary gland tumors in 100% of mice within 5 weeks after birth. MMTV-cre-mediated inactivation of fur without over-expression of PLAG1 caused smaller but histologically normal salivary glands. Moreover, the lymph nodes close to the salivary glands were enlarged, and histology showed that they had activated follicles. When genetic ablation of 1 or 2 alleles of fur and overexpression of the PLAG1 transgene were simultaneously achieved, a significant delay in tumorigenesis was observed. Collectively, these results suggest an important role for furin in PLAG1-induced salivary gland tumorigenesis in mice.
    International Journal of Oncology 06/2008; 32(5):1073-83. · 2.66 Impact Factor

Publication Stats

2k Citations
403.13 Total Impact Points

Institutions

  • 2012
    • Université de Sherbrooke
      Sherbrooke, Quebec, Canada
  • 2008–2011
    • Johannes Gutenberg-Universität Mainz
      • Institut für Pathobiochemie
      Mainz, Rhineland-Palatinate, Germany
  • 1991–2011
    • KU Leuven
      • Department of Human Genetics
      Leuven, VLG, Belgium
  • 1998
    • Catholic University of Louvain
      Walloon Region, Belgium
  • 1994–1996
    • Maastricht University
      • Genetica en Celbiologie
      Maastricht, Provincie Limburg, Netherlands
    • University of Tsukuba
      Tsukuba, Ibaraki, Japan
  • 1985–1992
    • Radboud University Nijmegen
      • • Department of Biochemistry
      • • Department of Urology
      Nijmegen, Provincie Gelderland, Netherlands