<p>Alzheimer Disease (AD) is the most common neurodegenerative disorder in the world. One of the neuropathological hallmarks of AD is the senile plaques in the brain. The plaques are mainly composed of the amyloid β (Aβ) peptide. Aβ is generated from the amyloid precursor protein, APP, when it is first cleaved by the β-secretase and subsequently the γ-secretase complex. The γ-secretase complex cleaves at different sites, called γ and ε, where the γ-cleavage site generates Aβ peptides of different lengths and ε-cleavage generates the APP intracellular domain (AICD). The two major forms of Aβ is 40 and 42 amino acids long peptides, where the latter is more prone to aggregate and is the main component in senile plaques. The γ-secretase complex is composed of four proteins; Pen-2, Aph-1, nicastrin and presenilin (PS). The PS protein harbours the catalytic site of the complex, where two aspartate residues in position 257 and 385 (Presenilin 1 numbering) are situated. Most Familial AD (FAD) mutations in the PS gene cause a change in the γ-cleavage site, leading to a shift from producing Aβ40 to the longer more toxic variant Aβ42. Frequently, this often leads to impairments of the AICD production. Another substrate for the γ-secretase complex is Notch. It is important to maintain the Notch signaling since an intracellular domain (NICD) is formed after cleavage by the γ-secretase complex in the membrane (S3-site) and this domain is involved in transcription of genes important for cell fate decisions.</p><p>It has been reported that certain APP luminal juxtamembrane mutations could drastically alter Aβ secretion, however their effect on AICD production remains unknown. In this study we want to analyse wether the juxtamembrane region is important for the AICD production. To gain more insight into the luminal juxtamembrane function for γ-secretase-dependent proteolysis, we have made a juxtamembrane chimeric construct. A four-residue sequence preceding the transmembrane domain (TMD) of APP (GSNK), was replaced by its topological counterpart from the human Notch1 receptor (PPAQ). The resulting chimeric vector C99GVP-PPAQ and the wildtype counterpart were expressed in cells lacking PS1 and PS2 (BD8) together with PS1wt. We observed that the chimeric construct did not alter production of AICD when using a cell based luciferase reporter gene assay monitoring AICD production. We also introduced a PS1 variant lacking a big portion of the large hydrophilic loop, PS1∆exon10, since our group has previously observed that this region affect Aβ production¹⁴³. We found that the absence of the large hydrophilic loop in PS1 gave a 2-fold decrease in AICD-GVP formation from C99GVPwt compared to PS1wt.  The activity of PS1wt and PS1Δexon10 using C99GVP-PPAQ as a substrate gave similar result as the C99GVPwt substrate, i.e. a 2-fold decrease in AICD-GVP formation when comparing PS1Δexon10 with PS1wt. From this data we therefore suggest that the four residues in the juxtramembrane domain (JMD) (GSNK) is not altering ε-cleavage of APP when changed to Notch1 counterpart, PPAQ. Furthermore, we also show that the 2-fold decrease in AICD-production by the PS1Δexon10 molecule is not changed between the two substrates C99GVPwt and C99GVP-PPAQ. This indicates that the luminal region of APP is not directly involved in the ε-site processing. If the luminal region is affecting processing in the γ-cleavage sites, remains however to be investigated.</p>

Alzheimer Disease (AD) is the most common neurodegenerative disorder in the world. One of the neuropathological hallmarks of AD is the senile plaques in the brain. The plaques are mainly composed of the amyloid β (Aβ) peptide. Aβ is generated from the amyloid precursor protein, APP, when it is first cleaved by the β-secretase and subsequently the γ-secretase complex. The γ-secretase complex cleaves at different sites, called γ and ε, where the γ-cleavage site generates Aβ peptides of different lengths and ε-cleavage generates the APP intracellular domain (AICD). The two major forms of Aβ is 40 and 42 amino acids long peptides, where the latter is more prone to aggregate and is the main component in senile plaques. The γ-secretase complex is composed of four proteins; Pen-2, Aph-1, nicastrin and presenilin (PS). The PS protein harbours the catalytic site of the complex, where two aspartate residues in position 257 and 385 (Presenilin 1 numbering) are situated. Most Familial AD (FAD) mutations in the PS gene cause a change in the γ-cleavage site, leading to a shift from producing Aβ40 to the longer more toxic variant Aβ42. Frequently, this often leads to impairments of the AICD production. Another substrate for the γ-secretase complex is Notch. It is important to maintain the Notch signaling since an intracellular domain (NICD) is formed after cleavage by the γ-secretase complex in the membrane (S3-site) and this domain is involved in transcription of genes important for cell fate decisions. It has been reported that certain APP luminal juxtamembrane mutations could drastically alter Aβ secretion, however their effect on AICD production remains unknown. In this study we want to analyse wether the juxtamembrane region is important for the AICD production. To gain more insight into the luminal juxtamembrane function for γ-secretase-dependent proteolysis, we have made a juxtamembrane chimeric construct. A four-residue sequence preceding the transmembrane domain (TMD) of APP (GSNK), was replaced by its topological counterpart from the human Notch1 receptor (PPAQ). The resulting chimeric vector C99GVP-PPAQ and the wildtype counterpart were expressed in cells lacking PS1 and PS2 (BD8) together with PS1wt. We observed that the chimeric construct did not alter production of AICD when using a cell based luciferase reporter gene assay monitoring AICD production. We also introduced a PS1 variant lacking a big portion of the large hydrophilic loop, PS1∆exon10, since our group has previously observed that this region affect Aβ production143. We found that the absence of the large hydrophilic loop in PS1 gave a 2-fold decrease in AICD-GVP formation from C99GVPwt compared to PS1wt.  The activity of PS1wt and PS1Δexon10 using C99GVP-PPAQ as a substrate gave similar result as the C99GVPwt substrate, i.e. a 2-fold decrease in AICD-GVP formation when comparing PS1Δexon10 with PS1wt. From this data we therefore suggest that the four residues in the juxtramembrane domain (JMD) (GSNK) is not altering ε-cleavage of APP when changed to Notch1 counterpart, PPAQ. Furthermore, we also show that the 2-fold decrease in AICD-production by the PS1Δexon10 molecule is not changed between the two substrates C99GVPwt and C99GVP-PPAQ. This indicates that the luminal region of APP is not directly involved in the ε-site processing. If the luminal region is affecting processing in the γ-cleavage sites, remains however to be investigated.

La preuve de concept d‟une approche theranostique pour la Maladie de Alzheimer basée sur les nanotechnologies a été explorée. Des nouvelles nanoparticules polymeriques fluorescentes on été conçus, et leur internalisation et aptitude à traverser un nouveau modèle in vitro de barrière hémato-encéphalique humaine on été étudiées en détails. Une petite librairie de nanoparticules polymerique a été préparés, et leur capacité de capturer le peptide β-Amyloïde1-42, considéré comme une des principales causes de la dégénérescence neuronale, a été évaluées et quantifiées en utilisant une méthode expressément conçus. / The proof of concept of an original nanotechnology-based theranostic approach for Alzheimer‟s disease has been explored. Novel fluorescently tagged nanoparticles have been designed and employed for internalization and transcytosis studies across a recently developed human in vitro blood-brain barrier model. A small library of polymeric nanoparticles have been designed and their ability to capture the Amyloid β1-42 peptide, considered one of the causes of the Alzheimer‟s disease, has been investigated and quantified using an on purpose designed method.

The proof of concept of an original nanotechnology-based theranostic approach for Alzheimer‟s disease has been explored. Novel fluorescently tagged nanoparticles have been designed and employed for internalization and transcytosis studies across a recently developed human in vitro blood-brain barrier model. A small library of polymeric nanoparticles have been designed and their ability to capture the Amyloid β1-42 peptide, considered one of the causes of the Alzheimer‟s disease, has been investigated and quantified using an on purpose designed method.

Les canaux potassiques sensibles à l’ATP (KATP) jouent un rôle primordial dans plusieurs processus cellulaires. La modulation de ces canaux par des molécules activatrices constituerait des applications pharmacologiques et médicinales très intéressantes. À cet effet nous avons conçu et synthétisé de nouvelles molécules hybrides cromakalim-diazoxide et diazoxide-amine/aminoacide. Nous avons également, évalué l’activité myorelaxante de ces composés sur l’aorte de rates. Les résultats obtenus ne montrent pas un effet myorelaxant significatif. Des études sur d’autres tissus, notamment les cellules β pancréatiques et le muscle utérin, sont envisagées afin d’explorer une éventuelle sélectivité tissulaire. Par ailleurs, les interactions protéine-protéine jouent un rôle fondamental dans presque tous les processus cellulaires. Elles sont fortement impliquées dans la formation de la structure dimérique de la protéase du VIH-1 et l’agrégation du peptide β amyloïde impliquée dans la maladie d’Alzheimer. L’inhibition de ces interactions serait donc d’un avantage thérapeutique pour le traitement du SIDA et de la maladie d’Alzheimer. Nous avons conçu et synthétisé d’une part, des pinces moléculaires à base de motifs carbonylhydrazides et oligohydrazides (Azatide), et d’autre part, des molécules pentapeptidiques avec un peudoaminoacide central alcoolfluoré. Enfin, nous avons testé la capacité des pinces moléculaires à perturber le feuillet β terminal de la PR du VIH-1 afin d'inhiber sa dimérisation et donc son activité. Nous avons réalisé de même une étude de relation structure-activité et d’après l’ensemble des résultats obtenus, il semblerait que la flexibilité est délétère pour l’activité inhibitrice. Nous avons également évalué la capacité des nouvelles molécules peptidomimétiques alcool fluorées à accélérer ou inhiber l’agrégation du peptide Aβ1-42 dans le but de diminuer la présence de petits oligomères neurotoxiques. Les résultats obtenus sont très prometteurs, nous avons réussi à développer d’une part un pentapeptide capable d’inhiber totalement l’agrégation de Aβ1-42, et d’autre part des pseudopentapeptides capables d’accélérer son agrégation. Nous avons aussi démontré l’influence de l’atome de fluor sur la structuration d’un pentapeptide. Des études par RMN et DC sont en cours. / ATP-sensitive potassium channels (KATP) play an important role in many cellular processes. The modulation of these channels by activating molecules may constitute very interesting pharmacological and medicinal applications. For this purpose, we have designed and synthesized new hybrid molecules cromakalim-diazoxide and diazoxide-amine/aminoacid. We also evaluated the relaxant activity of these compounds on aorta of rats. The obtained results do not show a significant relaxant effect. Studies on other tissues, including pancreatic  cells and uterine muscle, are envisaged to explore the potency of these compounds and their possible tissue selectivity.Otherwise, Protein-protein interactions play a fundamental role in almost all cellular processes. They are strongly involved in the formation of the dimeric structure of HIV-1 protease and β amyloid peptide aggregation involved in Alzheimer's disease. Inhibition of these interactions would be a therapeutic advantage for the treatment of AIDS and Alzheimer's disease. We designed and synthesized on one hand, molecular tongs based on carbonylhydrazide oligohydrazid (Azatide) fragments and in the other hand, pentapeptide molecules with a central fluorinated and hydroxylated aminoacid. Finally, we tested the ability of molecular tongs to disrupt the terminal β sheet of the HIV-1 PR to inhibit its dimerization and thus its activity. We have also conducted a structure-activity relationship study and According to the results it seems that flexibility is detrimental to the inhibitory activity. We evaluated as well the ability of new fluorinated and hydroxylated peptidomimetics to accelerate or inhibit the aggregation of Aβ1-42 peptide in order to reduce the presence of small toxic oligomers. The results are very promising that we succeeded in developing a pentapeptide able to completely inhibit the aggregation of Aβ1-42, and in the other hand pseudopentapeptides able to accelerate its aggregation. We also demonstrated the influence of fluorine on the structure of a pentapeptides. Studies by NMR and DC are in progress.