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2017-05-22 - Colloque/Présentation - poster - Anglais - 1 page(s)

Andre Séverine , Daldal Fatima, Lecomte Marine, Muller Robert , Vander Elst Luce , Laurent Sophie , Burtea Carmen , "The phospholipase A2 signaling as a new therapeutic biomarker of Alzheimer’s disease: modulation through phage display-derived peptides able to cross the blood-brain barrier" in 12th Meeting of the Belgian Neutoscience Society, Ghent, Belgique, 2017

  • Codes CREF : Histologie (DI3212), Sciences biomédicales (DI3200), Biochimie pharmaceutique (DI3491), Biologie moléculaire (DI3111), Neuropathologie (DI332C), Biologie cellulaire (DI311D)
  • Unités de recherche UMONS : Chimie générale, organique et biomédicale (M108)
  • Instituts UMONS : Institut des Sciences et Technologies de la Santé (Santé), Institut des Biosciences (Biosciences)
  • Centres UMONS : Centre de Recherche en Microscopie et Imagerie Médicale (CMMI)

Abstract(s) :

(Anglais) Introduction The treatment of Alzheimer’s disease (AD) is a real challenge up to now. Phospholipase A2 (PLA2) signaling pathway was recently revealed to be involved in this pathology, its inhibition already showing neuron protection against apoptosis induced by amyloid beta (Aβ). Using the phage-display technology, we have identified a PLA2-targeted peptide (PLP25) aiming to limit the activation of PLA2. Furthermore, the blood-brain barrier (BBB) is a restrictive barrier protecting the brain against xenobiotics and limiting the access of most molecules, including potential therapeutic agents. Non-invasive crossing strategies are thus indispensable to deliver therapeutic agents to the central nervous system (CNS) without BBB disruption. Because of its involvement in LDL transcytosis, LDL receptor (LDLR) was targeted by phage display and the peptide LRP2 was identified as a new vector able to facilitate the crossing of this barrier. LRP2 will be thus used as vector of our therapeutic peptide aiming to improve its CNS delivery. Methods The specificity of both peptides PLP25 and LRP2 to their respective target was validated by immunofluorescence. The inhibitory potential of PLP25 targeting PLA2 was evaluated by the dosage of arachidonic acid (AA) released by human astrocytes (1321N1) and mouse neurons (N18) induced by H2O2 and glutamate respectively, known as PLA2 activators. The localization of PLA2 after its activation was evaluated on these same cells by immunofluorescence. The endocytosis mechanism of LDLR-targeted LRP2 was investigated in human brain endothelial cells ACBRI376, human astrocytes 1321N1 and mouse neurons N18(H) by immunofluorescence. The ability of LRP2 to accede the brain was assessed in vivo on NMRI mice by Magnetic Resonance Imaging (MRI) using LRP2-grafted Ultrasmall Superparamagnetic Particles of Iron Oxide (USPIO-LRP2). After brain collection, USPIO derivatives were detected by Perls’-DAB. Results and Prospects The AA released by 1321N1 and N18(H) cells was increased after PLA2 induction with H2O2 or glutamate respectively. On 1321N1, the pre-incubation with PLP25 before PLA2 stimulation allowed to decrease AA levels released by these cells, ranging from 20% to 60% as compared to induced cells, with a more potent effect at low concentration. On differentiated N18(H) cells, PLP25 produced the same effects. Generally, PLP25 restored PLA2 activity in the range of negative control both in astrocytes and neurons. In order to observe the effect of PLP25 on cellular localization of PLA2, this last one was detected in a first stage on 1321N1 by immunofluorescence. H2O2 stimulation resulted in the concentration of PLA2 at the plasma membrane and its processes, showed by a higher fluorescent labeling. A similar experiment performed on glutamate-induced N18(H) revealed the migration of PLA2 in filopodia of neuronal growth cones. These results agree with the known translocation of the induced PLA2 from the cytoplasm to cellular membranes, where it binds via its C2 domain to the enzymatic substrate, the phosphatidylcholine. PLA2 is involved in the dynamics of actin cytoskeleton and filipodia formation in astrocytes and neurons, playing a key role in neuronal plasticity. Being specific to the C2 domain of PLA2, PLP25 prevented the migration of PLA2 to cell membrane and filopodia, as suggested by the decreased fluorescent labeling of these structures. This effect was moreover facilitated when PLP25 was combined to LRP2. Concerning the crossing of the BBB, the peptide LRP2 is endocytosed via a caveolae-mediated non-degradation pathway in ACBRI376 cells, which compose the BBB, whereas the lysosome degradation is bypassed. Interestingly, LRP2 is also able to penetrate 1321N1 and N18(H) cells mostly via the caveolae pathway, even if a small proportion seems to taken by lysosomes. These results confirm that LRP2 could be used as vector through the BBB for the therapeutic agent. In vivo MRI experiments showed that USPIO-LRP2 (a negative contrast agent) induces a darkening of brains as compared to USPIO-NSP (nonspecific). These observations were confirmed by the increased negative contrast (%SNR) obtained by the analysis of signal intensity. The Perls’-DAB staining performed on brain slices of the injected mice, highlighting iron contained in USPIO, revealed an important brown staining for USPIO-LRP2, as opposed to USPIO-NSP or PBS that do not show any staining. The evaluation of peptide LRP2 coupled to the therapeutic peptide PLP25 is currently in progress.