Development of an original phospholipase A2-targeted peptide able to reduce amyloid pathology in a mouse model of Alzheimer’s disease

Abstract

Event Abstract Back to Event Development of an original phospholipase A2-targeted peptide able to reduce amyloid pathology in a mouse model of Alzheimer’s disease Séverine André1, 2, Jean M. Atakana Lukulu1, 2, Marine Lecomte1, 2, Robert N. Muller1, 2, 3, Luce Vander Elst1, 2, Sophie Laurent1, 2, 3 and Carmen Burtea1, 2* 1 University of Mons, Belgium 2 Laboratory of Nuclear Magnetic Resonance and Molecular Imaging, University of Mons, Belgium 3 Centre for Microscopy and Molecular Imaging (CMMI), Belgium Introduction Alzheimer’s disease (AD) is the most common dementia worldwide and, since 1993, no new drug was approved by the FDA for its treatment because more than 95% of them have failed during clinical trials [1]. Moreover, actual therapies are only symptomatic and do not slow the progression of the disease. In this context, identification of new targets is crucial and phospholipase A2 (PLA2) signaling pathway has been proposed to be involved in this pathology [2], its inhibition showing neuron protection against apoptosis induced by amyloid beta (Aβ) [3]. Using the phage-display technology, we have identified a PLA2-targeted peptide (PLP25) aiming to reversibly and specifically inhibit this enzyme, contrariwise to existing inhibitory molecules. In order to improve its brain availability, we combined this peptide to a vector peptide (LRP2) targeting the LDL receptor (LDLR) and able to cross the blood-brain barrier. This complex (PLP25-LRP2), evaluated in vitro and in vivo, is expected to be beneficial for AD therapy. Methods The in vitro evaluation of PLP25-LRP2 aimed to determine: (i) the inhibition of arachidonic acid (AA) release by activated PLA2 in H2O2, glutamate or A stimulated cells; (ii) the effect on cellular localization of PLA2 and of AA-depending enzymes such as COX2 and ALOX5; and (iii) the actin cytoskeleton reorganization in cells treated with this complex. For in vivo validation, the PLP25-LRP2 complex was injected to APP/PS1 mice. Its therapeutic potential was first evaluated by assessing the spatial memory of APP/PS1 mice by the Barnes maze and detecting amyloid burden by Magnetic Resonance Imaging (MRI) using USPIO-PHO (peptide PHO identified by phage display and specific to Aβ, grafted to Ultrasmall Superparamagnetic Particles of Iron Oxide) [4,5]. Secondly, different biomarkers were evaluated ex vivo by immunohistochemistry, such as the PLA2 itself, amyloid plaques (AP), phosphorylated Tau protein (p-Tau) and NMDA receptors (NMDAR). Results H2O2, glutamate and A stimulate PLA2 activity and AA released by the induced cells, whereas our PLP25-LRP2 therapeutic complex reduces this release by 20% to 30%. In neuronal cells, glutamate and Aβ stimulate PLA2 translocation from cytosol to organelle membranes and plasma membrane processes, whereas PLP25-LRP2 is able to prevent this translocation, leading to PLA2 inhibition. Using AA released by PLA2, COX2 and ALOX5 relocated like PLA2 upon activation, while the reduced AA restricted their relocation. Finally, PLA2 inhibition by PLP25-LRP2 has also restrained the filopodial dynamics and reorganization of actin cytoskeleton, phenomena associated to neuronal excitotoxicity, characteristic to AD. Injected to APP/PS1 mice, our therapeutic complex seems to improve the spatial memory as shown by a lower number of errors and a higher percentage of time spent in the correct quadrant of the Barnes maze, compared to the mice injected with a non-specific peptide (NSP). Moreover, the PLP25-LRP2-treated mice exhibit a lower number of AP than mice injected with NSP, shown by both MRI and IHC. They also present a cellular localization of p-Tau similar to healthy mice, revealing the key role of Aβ in Tau pathology; the expression levels of PLA2 and NMDAR tend to be restored to the healthy control levels. Acknowledgements This work is supported by the Rotary Foundation – Hope in Head campaign 2015, the FRMH (Fond pour la Recherche Médicale en Hainaut) and the grant “Actions de Recherche Concertée” financed by the Fédération Wallonie-Bruxelles, the Waloon Region, Feder, Interreg and COST actions.