The processing of amyloid precursor protein (APP) by - and -secretases produces amyloid (A) peptide, the principal component of the neuritic plaques found in Alzheimers disease (AD) pathology. The enzyme -secretase is a multimeric protein consisting of presenilins-1/2 (PS1/PS2), nicastrin, anterior pharynx defective 1 (APH-1) and presenilin enhancer-2 (PEN-2). Recently it was discovered that p23, a transmembrane protein involved in intracellular protein trafficking, negatively regulates -secretase activity. In the present study, I evaluated the levels/expression of the nicastrin and p23 and their possible colocalization with PS1 in normal adult and developing brains. Additionally, I have studied the alterations of p23 levels in both animal model of neurodegeneration and in postmortem AD brains. Nicastrin and p23 were widely distributed throughout the brain and colocalized in all brain regions with PS1. The levels of nicastrin and p23 were relatively high at the early stages of postnatal development and then declined gradually as age increased. Interestingly, p23 level/expression was found to be altered following kainic acid-induced neurodegeneration in the adult rat brain. Additionally, p23 levels were reduced in the brains of individuals with AD. These results, taken together, suggest that both nicastrin and p23 are expressed in neurons throughout the brain and their levels decline gradually during development to reach an adult profile. Additionally, my results indicate that a decreased level of p23 may contribute to AD pathogenesis by increasing the production of A-related peptides.

No description available.

γ-Secretase plays a pivotal role in the production of neurotoxic amyloid β-peptide (Aβ), the principal component of amyloid plaques present in Alzheimer’s disease. It consists of a core complex of presenilin (PS), nicastrin, anterior pharynx-defective 1 (Aph-1), and presenilin enhancer 2 (Pen-2) proteins. PS harbors the catalytic aspartates required for regulated intramembrane proteolysis and the paralogs (PS1 and PS2) contribute to the assembly of distinct subpopulations of γ-secretases that may fulfill distinct roles. To characterize the molecular environments of distinct γ-secretases complexes in-depth quantitative comparisons were performed on 1) wild-type PS1 and its derivative carrying point mutations known to cause heritable early-onset AD in mice, and 2) PS1- or PS2-containing γ-secretase complexes equipped with N-terminal tandem-affinity purification (TAP) tags on PS paralogs in HEK293 cells. Isobaric labeling of co-purifying peptides for quantitative mass spectrometry revealed that γ-secretase complexes interact with other protein networks, including the cellular catenin-cadherin network, the molecular machinery that targets and fuses synaptic vesicles to cellular membranes, and the H+-transporting lysosomal ATPase macro-complex. The study revealed mature γ-secretase complexes containing PS1 or mutant PS1 to be indistinguishable in their protein composition, confirmed several previously proposed γ-secretase interactors, identified many novel interactors and uncovered a subset of proteins which can engage in robust interactions with γ-secretase complexes in individual cell types but may escape detection when whole brains are used as biological source materials. Interestingly, signal peptide peptidase (SPP), a Type II TM cleaving aspartyl protease, was pre-dominantly found to co-purify with PS2-containing γ-secretase complexes and could be shown not to influence their maturation but to affect cleavage or release of cellular Aβ. A model emerged from this work that suggests PS1 and PS2 paralogs may divide up the task of handling a broad range of membrane stubs at least in part by associating with different molecular environments.

γ-Secretase plays a pivotal role in the production of neurotoxic amyloid β-peptide (Aβ), the principal component of amyloid plaques present in Alzheimer’s disease. It consists of a core complex of presenilin (PS), nicastrin, anterior pharynx-defective 1 (Aph-1), and presenilin enhancer 2 (Pen-2) proteins. PS harbors the catalytic aspartates required for regulated intramembrane proteolysis and the paralogs (PS1 and PS2) contribute to the assembly of distinct subpopulations of γ-secretases that may fulfill distinct roles. To characterize the molecular environments of distinct γ-secretases complexes in-depth quantitative comparisons were performed on 1) wild-type PS1 and its derivative carrying point mutations known to cause heritable early-onset AD in mice, and 2) PS1- or PS2-containing γ-secretase complexes equipped with N-terminal tandem-affinity purification (TAP) tags on PS paralogs in HEK293 cells. Isobaric labeling of co-purifying peptides for quantitative mass spectrometry revealed that γ-secretase complexes interact with other protein networks, including the cellular catenin-cadherin network, the molecular machinery that targets and fuses synaptic vesicles to cellular membranes, and the H+-transporting lysosomal ATPase macro-complex. The study revealed mature γ-secretase complexes containing PS1 or mutant PS1 to be indistinguishable in their protein composition, confirmed several previously proposed γ-secretase interactors, identified many novel interactors and uncovered a subset of proteins which can engage in robust interactions with γ-secretase complexes in individual cell types but may escape detection when whole brains are used as biological source materials. Interestingly, signal peptide peptidase (SPP), a Type II TM cleaving aspartyl protease, was pre-dominantly found to co-purify with PS2-containing γ-secretase complexes and could be shown not to influence their maturation but to affect cleavage or release of cellular Aβ. A model emerged from this work that suggests PS1 and PS2 paralogs may divide up the task of handling a broad range of membrane stubs at least in part by associating with different molecular environments.

<p>The cancer stem cell hypothesis claims that only a small subpopulation of cells within a tumor is responsible for tumor growth, recurrence after treatment and metastasis. These cells have been termed tumor-initiating cells or cancer stem cells and are functionally defined by their capacity to elicit the growth of tumors in immune-compromised animals that recapitulate the cellularity of the tumor from which they were isolated. Several reports demonstrate that tumor-initiating cells are resistant to most current treatments. Hence, novel therapies for breast cancer should be developed that specifically target these tumorigenic cells. The Notch signaling pathway is hyperactive in human breast cancer as well as in mouse mammary tumor-initiating cells. In this study, I have found that inhibitors of the pathway target breast tumor-initiating cells from various breast cancer subtypes and may provide a novel therapy for breast cancer. MRK-003, a gamma-secretase inhibitor that blocks Notch signaling, inhibited the self-renewal of breast tumor-initiating cells <em>in vitro</em> and reduced tumor growth in xenograft models. MRK-003 inhibited proliferation of tumor cells within xenografts and induced their apoptosis and differentiation towards the myoepithelial lineage. Expression of the Notch pathway antagonists led to similar outcome in human breast tumor cell lines. Notably, tumors in MRK-003 treated mice were devoid of tumor initiating cells, suggesting that inhibitors of Notch signaling may lead to durable cancer cures. These findings suggest that GSIs target breast tumor-initiating cells and may prove to be effective novel anti breast cancer drugs. <strong> </strong></p> <p><strong> </strong></p> / Doctor of Philosophy (PhD)

<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.

Le développement et la fonction des cellules dendritiques plasmacytoïdes (pDCs) doivent être finement régulés afin d’éviter le développement de maladies auto-immunes ou de leucémies. Il a été montré récemment qu’une fraction des leucémies humaines dérivées des pDCs possède des mutations de type perte de fonction dans le locus IKZF1 qui code pour le facteur de transcription Ikaros. Ainsi l’étude de la fonction d’Ikaros dans les pDCs pourrait aider à comprendre son possible rôle de gène suppresseur de tumeur. Les souris hypomorphiques pour Ikaros (IkL/L) ne possèdent pas de pDCs matures dans la rate et les ganglions lymphatiques mais accumulent des pDCs immatures dans la moelle osseuse (MO). De manièreintéressante, les pDCs de MO IkL/L montrent une activation ectopique de la voie Notch. Nous avons trouvé qu’un inhibiteur de gamma-secrétase (GSI), qui inhibe la voie Notch, permet de restaurer la différenciation de pDCs fonctionnelles dans les cultures de cellules de MO. Les principaux progéniteurs dendritiques affectés par le GSI sont le progéniteur myéloïde commun (CMP) et le progéniteur des macrophages et des cellules dendritiques (MDP). Comme le GSI inhibe la voie Notch, nous avons aussi inactivé RBPJ, le facteur de transcription situé en aval des récepteurs Notch. Contre toute attente, l’inactivation de RBPJ ne récapitule pas les effets observés avec le GSI. De plus, les cellules IkL/L déficientes pour RBPJ continuent de répondre au GSI, ce qui suggère que le GSI possède d’autres cibles en plus de la voie Notch dans ce système. Nos données montrent ainsi qu’Ikaros est requis pour la différenciation terminale des pDCs et qu’il agit en partie en bloquant une voie GSI dépendante Notch indépendante. / The development and function of plasmacytoid dendritic cells (pDCs) must be tightly regulated to prevent autoimmune disease or leukemia. It was recently discovered that a fraction of human pDC-derived neoplasms exhibit loss of function mutations of the IKZF1 locus, which encodes the Ikaros transcription factor. Deciphering the function of Ikaros in pDCs could thus help understand its probable tumor suppressor function. Mice hypomorphic for Ikaros (IkL/L) are devoid of mature pDCs in the spleen and lymph nodes but accumulate immature pDCs in the bone marrow (BM). Interestingly IkL/L BM pDCs exhibit an ectopic activation of the Notch pathway. We found that a gamma secretase inhibitor (GSI), which inhibits Notch signalling,rescues the differentiation of functional pDCs in BM cultures. The main dendritic cell progenitors affected by GSI are the common myeloid progenitors (CMP) and the macrophage and dendritic cell progenitors (MDP). As GSI inhibits the activation of the Notch pathway, we also inactivated RBPJ, the transcriptional effector of the Notch pathway. Surprisingly, RBPJ inactivation did not recapitulate the effect of GSI. Moreover, RBPJdeficient IkL/L cells still respond to GSI, demonstrating that GSI targets additional events besides Notch in this system. Our data thus show that Ikaros is required for terminal differentiation of pDCs, and acts in part by blocking a Notch independent GSI-sensitive pathway.

Une des caractéristiques histopathologiques de la maladie d'Alzheimer (AD) est la présence de plaques amyloïdes formées par les peptides amyloïdes β (Aβ) de 40 et 42 résidus, qui sont les produits de clivage par des protéases de l'APP. Afin de comprendre le rôle des variations structurelles du TM dans le traitement de l'APP, les peptides APP_TM4K ont été étudiés dans la bicouche lipidique en utilisant l’ATR-FTIR et ssNMR. Tandis que la structure secondaire globale du peptide APP_TM4K est hélicoidale, hétérogénéité de conformation et d'orientation a été observée pour le site de clivage γ et , que peuvent avoir des implications dans le mécanisme de clivage et donc dans la production d’Aβ. Les peptides Aβ s'agrègent pour produire des fibrilles et aussi de manière transitoire d'oligomères neurotoxiques. Nous avons constaté qu'en présence de Ca2+, l’Aβ (1-40) forme de préférence des oligomères, tandis qu'en absence de Ca2+ l'Aβ (1-40) s’agrège sous forme de fibrilles. Dans les échantillons sans Ca2+, l’ATR-FTIR révèle la conversion des oligomères en feuillets β antiparallèles en la conformation caractéristique des fibrilles en feuillets β parallèles. Ces résultats nous ont amené à conclure que les Ca2+ stimulent la formation d'oligomères d'Aβ (1-40), qui sont impliqués dans l’AD. Les positions et une orientation précise de deux nouveaux médicaments puissants modulateurs de la γ-sécrétase - le benzyl-carprofen et le sulfonyl-carprofen  dans la bicouche lipidique, ont été obtenus à partir des expériences des ssNMR. Ces résultats indiquent que le mécanisme probable de modulation du clivage par la y-sécrétase est une interaction directe avec le domaine TM de l’APP. / A histopathological characteristic of Alzheimer’s disease (AD) is the presence of amyloid plaques formed by amyloid β(A) peptides of 40 and 42 residues-long, which are the cleavage products of APP by proteases. To understand the role of structural changes in the TM domain of APP, APP_TM4K peptides were studied in the lipid bilayer using ATR-FTIR and ssNMR. While the overall secondary structure of the APP_TM4K peptide is helical, conformational and orientational heterogeneity was observed for the y- and for the -cleavage sites, which may have implications for the cleavage mechanism and therefore the production of Aβ. Starting from its monomeric form, Aβ peptides aggregate into fibrils and / or oligomers, the latter being the most neurotoxic. We found that in the presence of Ca2 +, Aβ (1-40) preferably forms oligomers, whereas in the absence of a2 + Aβ (1-40) aggregates into fibrils. In samples without Ca2 +, ATR-FTIR shows conversion from antiparallel β sheet conformation of oligomers into parallel β sheets, characteristic of fibrils. These results led us to conclude that Ca2 +stimulates the formation of oligomers of Aβ (1-40), that have been implicated in the pathogenesis of AD. Position and precise orientation of two new drugs  powerful modulators of γ-secretase  benzyl-carprofen and carprofen sulfonyl  in the lipid bilayer were obtained from neutron scattering and ssNMR experiments. These results indicate that carprofen-derivatives can directly interact with APP. Such interaction would interfere with proper APP-dimer formation, which is necessary for the sequential cleavage by β -secretase, diminishing or greatly reducing Aβ42 production.