Ioannis Dafnis

National Center for Scientific Research Demokritos, Athínai, Attica, Greece

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Publications (5)19.72 Total impact

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    ABSTRACT: The K146N/R147W substitutions in apoE3 were described in patients with a dominant form of type III hyperlipoproteinemia. The effects of these mutations on the in vivo functions of apoE were studied by adenovirus mediated gene transfer in different mouse models. Expression of the apoE3[K146N/R147W] mutant in apoE-/- or apoA-I-/-xapoE-/- mice exacerbated the hypercholesterolemia and increased plasma apoE and triglyceride levels. In apoE-/- mice the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and caused the accumulation of discoidal apoE-containing HDL. The WT apoE3 cleared the cholesterol of apoE-/- mice without induction of hypertriglyceridemia and promoted formation of spherical HDL. A unique property of the truncated apoE3[K146N/R147W]-202 mutant, compared to similarly truncated apoE forms, is that it did not correct the hypercholesterolemia. The contribution of LPL and LCAT in the induction of the dyslipidemia was studied. Treatment of apoE-/- mice with apoE3[K146N/R147W] and LPL corrected the hypertriglyceridemia but did not prevent the formation of discoidal HDL. Treatment with LCAT corrected hypertriglyceridemia and generated spherical HDL. The combined data indicate that the K146N/R147W substitutions convert the full length and the truncated apoE3[K146N/R147W] mutant into a dominant negative ligand that prevents receptor-mediated remnant clearance, exacerbates the dyslipidemia and inhibits the biogenesis of HDL.
    The Journal of Lipid Research 04/2014; · 4.39 Impact Factor
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    ABSTRACT: Apolipoprotein (apo) E4 isoform has consistently emerged as a susceptibility factor for late-onset Alzheimers disease (AD) although the exact mechanism is not clear. A rare apoE4 mutant, apoE4[L28P] Pittsburgh, burdens carriers with added risk for late-onset AD and may be a useful tool for gaining insight on the role of apoE4 in disease pathogenesis. Towards this end, we evaluated the effect of the L28P mutation on the structural and functional properties of apoE4. ApoE4[L28P] was found to have significantly perturbed thermodynamic properties, to have reduced helical content and to expose a larger portion of hydrophobic surface to the solvent. Furthermore, this mutant is thermodynamically destabilized and more prone to proteolysis. When interacting with lipids, apoE4[L28P] formed populations of lipoprotein particles with structural defects. The structural perturbations brought about by the mutation were accompanied by aberrant functions associated with the pathogenesis of AD. Specifically, apoE4[L28P] promoted the cellular uptake of extracellular amyloid-beta peptide 42 (Aβ42) by human neuroblastoma SK-N-SH cells as well as by primary mouse neuronal cells and led to increased formation of intracellular reactive oxygen species (ROS) that persisted for at least 24hrs. Furthermore, lipoprotein particles containing apoE4[L28P] induced intracellular ROS formation and reduced SK-N-SH cell viability. Overall, our findings suggest that the L28P mutation leads to significant structural and conformational perturbations in apoE4, and can induce functional defects associated with neuronal Aβ42 accumulation and oxidative stress. We propose that these structural and functional changes underlie the observed added risk for AD development in carriers of apoE4[L28P].
    Journal of Biological Chemistry 03/2014; · 4.65 Impact Factor
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    ABSTRACT: Apolipoprotein (apo) E4 isoform, a major risk factor for Alzheimer disease (AD), is more susceptible to proteolysis than apoE2 and apoE3 isoforms. ApoE4 fragments have been found in AD patients' brain. In the present study, we examined the effect of full-length apoE4 and apoE4 fragments apoE4[Δ(186-299)] and apoE4[Δ(166-299)] on inflammation in human neuroblastoma SK-N-SH and human astrocytoma SW-1783 cells. Western blot and zymography analysis showed that treatment of SK-N-SH cells with apoE4[Δ(186-299)], but not full-length apoE4 or the shorter apoE4[Δ(166-299)] fragment, leads to increased extracellular levels of matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1). Real-time PCR showed that interleukin (IL)-1β gene expression is also increased in SK-N-SH cells treated with apoE4[Δ(186-299)]. Treatment of SK-N-SH cells with IL-1β leads to increased MMP9 and TIMP1 extracellular levels, suggesting that the induction of IL-1β may be the mechanism by which apoE4[Δ(186-299)] regulates MMP9 and TIMP1 levels in these cells. In contrast to SK-N-SH cells, treatment of SW-1783 cells with apoE4[Δ(186-299)], and to a lesser extent with apoE4, leads to increased TIMP1 extracellular levels without affecting MMP9 levels. Additionally, apoE4[Δ(186-299)] leads to decreased IL-10 gene expression in SK-N-SH cells, whereas both apoE4 and apoE4[Δ(186-299)] lead to decreased TNFα gene expression without affecting IL-1β and IL-10 gene expression in SW-1783 cells. Overall, our findings indicate that a specific apoE4 fragment (apoE4[Δ(186-299)]), with molecular mass similar that of apoE4 fragments detected in AD patients' brain, can influence the level of inflammatory molecules in brain cell lines. It is possible that these phenomena contribute to AD pathogenesis.
    Neuroscience 03/2012; 210:21-32. · 3.12 Impact Factor
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    ABSTRACT: Eukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], produce the Ca(2+)-mobilizing agent inositol 1,4,5-trisphosphate, and regulate signaling in multicellular organisms. Bacterial PtdIns-specific PLCs, also present in trypanosomes, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence that Tetrahymena species contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca(2+). One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca(2+), modestly activated by GTPγS in vitro, and inhibited by the compound U73122 [1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLC in vivo resulted in rapid upregulation of PtdIns(4,5)P(2) levels, suggesting its functional importance in regulating phosphoinositide turnover in Tetrahymena. By in silico and molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in three T. thermophila strains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P(3)-Ca(2+) regulatory axis in ciliates.
    Eukaryotic Cell 12/2010; 10(3):412-22. · 3.59 Impact Factor
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    ABSTRACT: Apolipoprotein E (apoE) plays a crucial role in lipid transport in circulation and the brain. The apoE4 isoform is a major risk factor for Alzheimer's disease (AD). ApoE4 is more susceptible to proteolysis than other apoE isoforms and apoE4 fragments have been found in brains of AD patients. These apoE4 fragments have been hypothesized to be involved in the pathogenesis of AD, although the mechanism is not clear. In this study we examined the effect of lipid-free apoE4 on amyloid precursor protein processing and 40-amino-acid Aβ variant and 42-amino-acid Aβ variant levels in human neuroblastoma SK-N-SH cells. We discovered that a specific apoE4 fragment, apoE4[Δ(166-299)], can promote the cellular uptake of extracellular 40-amino-acid Aβ variant and 42-amino-acid Aβ variant either generated after amyloid precursor protein transfection or added exogenously. A longer length fragment, apoE4[Δ(186-299)], or full-length apoE4 failed to elicit this effect. ApoE4[Δ(166-299)] effected a 20% reduction of cellular sphingomyelin levels, as well as changes in cellular membrane micro-fluidity. Following uptake, approximately 50% of 42-amino-acid Aβ variant remained within the cell for at least 24 h, and led to increased formation of reactive oxygen species. Overall, our findings suggest a direct link between two early events in the pathogenesis of AD, apoE4 proteolysis and intraneuronal presence of amyloid beta peptide.
    Journal of Neurochemistry 11/2010; 115(4):873-84. · 3.97 Impact Factor