Blood-brain barrier (BBB) crossing and brain penetration are really challenging for the delivery of therapeutic agents and imaging probes. The development of new crossing strategies is needed, and a wide range of approaches (invasive or not) have been proposed so far. The receptor-mediated transcytosis is an attractive mechanism, allowing the non-invasive penetration of the BBB. Among available targets, the low-density lipoprotein (LDL) receptor (LDLR) shows favorable characteristics mainly because of the lysosome-bypassed pathway of LDL delivery to the brain, allowing an intact discharge of the carried ligand to the brain targets. The phage display technology was employed to identify a dodecapeptide targeted to the extracellular domain of LDLR (ED-LDLR). This peptide was able to bind the ED-LDLR in the presence of natural ligands and dissociated at acidic pH and in the absence of calcium, in a similar manner as the LDL. In vitro, our peptide was endocytosed by endothelial cells through the caveolae-dependent pathway, proper to the LDLR route in BBB, suggesting the prevention of its lysosomal degradation. The in vivo studies performed by magnetic resonance imaging and fluorescent lifetime imaging suggested the brain penetration of this ED-LDLR-targeted peptide.

The diagnosis of Alzheimer’s disease (AD) is a critical step in the management of patients. We have developed a non-invasive diagnosis tool based on magnetic resonance molecular imaging (MRMI) of amyloid-β peptide using ultra-small particles of iron

Introduction Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive and irreversible loss of cognitive functions. Actual therapies are only symptomatic and the AD treatment is a real challenge. Phospholipase A2 (PLA2) signaling pathway plays a critical role in memory impairment and neurodegeneration by modulating the activity of both cholinergic and glutamatergic systems, and contributing to the amyloid beta peptide- and neurofibrillary tangles-induced neurotoxicity [1]. This convinced us to target one of its key enzymes with phage display-derived peptides aiming to modulate its activity and develop in this way a new therapeutic strategy for the AD patients. On the other hand, the treatment of brain pathologies has to deal with the blood-brain barrier (BBB) that protects the brain against xenobiotics. New BBB crossing strategies are thus very important for the treatment of the central nervous system pathologies, and LDL receptor (LDLR) seems to be an optimal shuttling system due to its involvement in LDL transcytosis [2]. We have therefore identified several LDLR-targeted peptides by phage display in order to facilitate the access to the brain of our therapeutic peptides. Methods A randomized linear peptide library fused to the p3 coat proteins of the M13 bacteriophage was used to identify PLA2- and LDLR-specific peptides. The selection of peptides consists to expose the library to the target (i.e., PLA2 or LDLR), collect bound phages, amplify them in Escherichia coli and repeat. The stringency, affinity and specificity of the selected clones was improved during the panning rounds by increasing the selective pressure. Affinity tests were performed by ELISA and the phage clones were selected based on their dissociation constants (Kd) and inhibition constants-50 (IC50). The amino acid sequence of the expressed peptides was deciphered after DNA sequencing, and the LDLR-targeted peptides were synthesized. Their endocytosis and binding to LDLR expressed by mouse brain was assessed by cytochemistry and immunohistochemistry. The characterization of PLA2-targeted clones is still in progress, so the corresponding peptides are not yet synthesized. Results Sequence analysis of the PLA2-targeted peptides showed a redundancy of some amino acids likely able to regulate PLA2 activity by preventing or facilitating it’s binding to membrane phospholipids; the first ability is required during the later stages of the disease, while the second one is mandatory to arrest the disease progression. Their Kd ranges from 100 fM to 100 pM, showing great promise as therapeutic agents (Figure 1). Concerning the BBB crossing strategy, Kd values of the selected clones range from 1 pM to 100 pM. Among them, two clones were selected because they are not destabilized by competitors, the acidic pH allows their dissociation from the receptor and the absence of calcium inhibits their binding. The corresponding peptides were synthesized and their endocytosis was confirmed on endothelial cells, while their colocalisation with LDLR was very good on brain slices (Figure 2). When their transcytosis potential will be confirmed, these peptides will be used to design multi-functional pharmaceutical molecules able to cross the BBB and modulate PLA2 activity, paving the way toward a more effective therapy of AD.

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 [1], its inhibition already showing neuron protection against apoptosis induced by amyloid beta (Aβ) [2]. 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 [3], 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 [4]. PLA2 is involved in the dynamics of actin cytoskeleton and filipodia formation in astrocytes and neurons, playing a key role in neuronal plasticity [5,6]. 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.

Summary This project concerns the development of an original therapeutic strategy for Alzheimer’s disease (AD). We aim to modulate a new pathological biomarker, the phospholipase A2, reported to be involved in memory deficits and neurodegeneration. For this purpose, we identified a peptide specific to this molecule that we coupled to another one able to facilitate the blood-brain barrier crossing through the LDL receptor. This peptide complex, tested in vitro and in vivo, showed interesting results that allow us to expect a more effective therapy of AD. Introduction Alzheimer’s disease (AD), one of the main causes of dementia worldwide, is characterized by a progressive and irreversible loss of cognitive functions. Actual therapies are only symptomatic and the targeting of amyloid beta (Aβ) and Tau protein does not seem to be effective, as shown by a failure percentage of 95% during clinical trials [1]. Phospholipase A2 (PLA2) signaling pathway was recently revealed to be involved in this pathology [2] and the modulation of some isoforms was already attempted in the treatment of neurological disorders [3]. In this context, we aim to target specifically a PLA2 isoform in order to develop a new therapeutic strategy for the AD patients, as an alternative of the existing inhibitory molecules that are non-specific or irreversible. We have developed a therapeutic complex combining a PLA2-targeted peptide (PL-P25) and a LDLR-targeted peptide (LRP2), this last one being used as vector through the blood-brain barrier (BBB). This complex, already evaluated on in vitro and in vivo models, is expected to be beneficial for AD therapy. Methods Peptides PLP25 and LRP2 were identified by phage display and coupled using streptavidin as a scaffold. The inhibitory potential of different combinations of these peptides was evaluated by the dosage of the arachidonic acid (AA) released by cells after PLA2 stimulation (glutamate in neuronal cells and H2O2 in astrocytes) and allowed the selection of the complex St-(PLP25)3-(LRP2)1. The cellular localization of PLA2, COX2 and ALOX5 as well as the reorganisation of actin cytoskeleton were studied by immunofluorescence. After in vitro validation, this complex was synthesized by coupling peptides to a MAP dendrimer (MAP-(PLP25)3-(LRP2)1). After excluding the eventual toxic effects and verified its efficacy in vitro (AA release), the therapeutic potential of MAP-(PLP25)3-(LRP2)1 was evaluated in vivo by assessing the spatial memory of APP/PS1 mice using the Barnes maze. The detection of amyloid plaques (AP) in their brain was assessed 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] and by immunohistochemistry (IHC). The phosphorylated Tau protein (p-Tau) was also detected by IHC. Results and Prospects PLA2 activation results in an increase of AA released by cells, whereas St-(PLP25)3-(LRP2)1 can reduce this release by 20% to 30% as compared to induced cells. Stimulated PLA2 translocates from cytosol to organelle membranes and plasma membrane processes and St-(PLP25)3-(LRP2)1 is able to prevent this translocation, leading to PLA2 inhibition. St-(PLP25)3-(LRP2)1 shows the same results when PLA2 is stimulated by Aβ in neuronal cells, suggesting its ability to protect against deleterious effects of Aβ. AA released by PLA2 from cell membranes is the substrate of COX2 and ALOX5, these enzymes being relocated like PLA2 upon activation. The reduced AA availability impacted COX2 and ALOX5 membrane relocation, which was restricted. Finally, PLA2 is involved in filopodial dynamics and a restructuration of actin cytoskeleton following its activation. St-(PLP25)3-(LRP2)1 blocked this phenomenon involved in excitotoxicity. Injected to APP/PS1 mice, MAP-(PLP25)3-(LRP2)1 seems to improve the spatial memory as shown by a lower number of errors and a higher percentage of time in the correct quadrant in the Barnes maze, compared to mice injected with a non-specific peptide (NSP). Moreover, MAP-(PLP25)3-(LRP2)1 treated mice exhibit a lower number of amyloid plaques than mice injected with NSP, shown by both MRI and IHC, and present a localization of p-Tau similar to healthy mice. This last result reveals the key role of Aβ in Tau pathology.

Introduction Alzheimer’s disease (AD) is one of the main causes of dementia in the elderly, being characterized by a progressive and irreversible loss of cognitive functions. Actual therapies are only symptomatic and the AD treatment is a real challenge. Indeed, AD is a multifactorial pathology triggered by hereditary (familial AD) and environmental (sporadic AD) factors, making its management especially challenging. Phospholipase A2 (PLA2) signaling pathway was recently revealed to be involved in this pathology [1] and the modulation of some isoforms was already attempted in the treatment of neurological disorders [2]. The goal of the present work is to target specifically a PLA2 isoform in order to develop a new therapeutic strategy for the AD patients, as an alternative of the existing molecules that are non-specific or irreversible. We have therefore identified a PLA2-targeted peptide (PL-P25) by phage display able to inhibit the PLA2 in preliminary in vitro tests. On the other hand, the blood-brain barrier (BBB), localized at the interface between the blood and the brain, protects the brain against xenobiotics and limits the access of most molecules, including potential therapeutic agents. The development of non-invasive BBB crossing strategies is thus crucial to accede to the central nervous system (CNS) without BBB disruption. LDL receptor (LDLR) seems to be an interesting target for drug delivery due to its involvement in LDL transcytosis [3]. A LDLR-targeted peptide (LR-P2) was identified in order to facilitate the access to the brain of our therapeutic peptide. Methods A randomized linear peptide library fused to the p3 coat proteins of the M13 bacteriophage was used to identify PLA2- and LDLR-specific peptides. Peptide specificity was evaluated by immunofluorescence on mouse brain slices or human brain endothelial cells (ACBRI376) by colocalization with their respective target. The inhibitory effect of PL-P25 (alone or multivalent by coupling to streptavidin), targeted to PLA2, was evaluated by the dosage of AA release from H2O2-induced human astrocytes (1321N1) and from glutamate-induced mouse neurons (N18TG2). The mechanism of transcytosis of LR-P2, targeting LDLR, was investigated by immunofluorescence allowing to observe its colocalization with caveolae or lysosomes. After LR-P2 coupling to iron oxide nanoparticles (USPIO-LRP2), its crossing over the BBB was evaluated after in vivo injection of NMRI mice by nuclear magnetic resonance (NMR) relaxometry and Perls’-DAB histochemistry staining. Results and Prospects The PLA2-targeted peptide PL-P25, selected for its affinity among two others, showed a good colocalization with the target on mouse brain slices, confirming its specificity. Its inhibitory potential on the PLA2 activity was confirmed on 1321N1 and N18TG2 cells, with an effect that ranged from 20% to 60% compared to positive control (induced but non inhibited cells), and a more potent activity at low concentrations (20µM) and for the multivalent model. Concerning the BBB crossing strategy, LR-P2 has co-localized with LDLR expressed by human brain endothelial cells, while its endocytosis via a caveolae-mediated non-degradation pathway was confirmed on these cells. After in vivo administration, USPIO-LRP2 was found within the brain parenchyma of NMRI mice, around the 3rd ventricle and brain capillaries by iron staining. NMR relaxometry has also confirmed the presence of USPIO-LRP2 in the brain, whereas the control nanoparticles coupled to a non-specific peptide (USPIO-NSP) was not able to cross the BBB as proven by both methods. This peptide will be used as vector in order to facilitate the access to the brain of our therapeutic peptide described above. Resumé en Français: L’objectif de ce projet est le développement d’une nouvelle stratégie thérapeutique de la maladie d’Alzheimer (MA) par modulation d’une molécule connue pour son implication dans les déficits de mémoire et la neurodégénérescence, appelée phospholipase A2. Un peptide spécifique à cette molécule a été identifié et sera couplé à un peptide facilitateur ciblant le récepteur aux LDL dans le but de passer la barrière hémato-encéphalique protégeant le cerveau. Cette nouvelle molécule pharmaceutique multifonctionnelle ainsi développée devra permettre une thérapie plus efficace de la MA. Samenvatting in het Nederlands: Dit project heeft tot doel om een nieuwe behandelingsstrategie voor de ziekte van Alzheimer te ontwikkelen, door modulatie van een molecule, phospholipase A2, waarvan geweten is dat ze te maken heeft met geheugenstoornissen en neurodegeneratie. Een voor deze molecule specifieke peptide werd geïdentificeerd en deze zal gekoppeld worden aan een faciliterende molecule die zich richt op de lipoproteïne receptor met een zwakke dichtheid (LDL) met als oogpunt de hemato-encefalische afscherming die de hersenen beschermt te doorbreken. De aldus ontwikkelde multifunctionele farmaceutische molecule zal een meer efficiënte behandeling van de ziekte van Alzheimer mogelijk maken.