Down syndrome (DS) is a congenital disorder caused by partial or complete triplication of human chromosome 21. By age 40, nearly all individuals with DS develop amyloid beta (Aβ) plaques and tau neurofibrillary tangles, the pathological hallmarks of Alzheimer’s disease (AD). This increased susceptibility to Alzheimer’s Disease in Down syndrome (AD-DS) has primarily been attributed to an over-dosage of the amyloid precursor protein (APP), which generates neurotoxic Aβ fragments when cleaved by β- secretase. However, the complete molecular mechanisms of AD-DS are not completely understood, as trisomy of chromosome 21 can induce AD-like neuropathology independently of APP triplication. In addition to classical AD neuropathology, enlarged, APP-positive early endosomes appear early in AD-DS pathogenesis and are the first site of Aβ accumulation. In AD, these endocytic abnormalities have been linked to dysfunction of the endosomal-sorting system known as the retromer complex. Mechanistically, retromer dysfunction can influence amyloid beta production by increasing interaction of the retromer cargo APP with beta-secretase. However, recent studies have also implicated the retromer complex in the development of tau pathology, both through regulation of tau phosphorylation and degradation via lysosomes. Given that retromer dysfunction is associated with the endosomal phenotype found in AD-DS, and that the retromer system can modulate key aspects of AD-DS neuropathology, the objective of the current study is to investigate the role of the retromer complex in the development of AD-DS. We first examined the retromer system in cortices and hippocampi from human patients with DS. Retromer recognition core proteins were significantly decreased in both the hippocampi and cortices of young and aged DS subjects compared to controls. Correlative analyses showed a significant inverse relationship between recognition core proteins and levels of soluble forms of Ab 1-40 and 1-42 in both hippocampus and cortex tissue, and phosphorylated tau epitopes PHF1 and PHF13 in the cortex of the same patients. While this does not indicate causation, these correlative analyses support the hypothesis that dysregulation of the retromer system and AD-like pathology are closely related. Next, we analyzed the retromer system in euploid and trisomic induced pluripotent stem cell (iPSC) -derived neurons and observed an age-related decrease in retromer proteins and elevation of Ab 1-40, Ab 1-42 and phosphorylated tau proteins in trisomic neurons compared to euploid controls. Additionally, we found that pharmacological stabilization of the retromer complex can reduce Ab and phospho-tau in trisomic iPSC-derived neurons. While total levels of retromer proteins are unaffected, we hypothesize that retromer function improves with TPT-172 treatment, as levels of early endosome proteins decrease while autophagy and lysosomal proteins increase with treatment. Treatment of trisomic neurons with TPT-172 also led to reductions in the neuronal tau kinase CDK5 and its activator p25, providing a potential mechanistic link between tau phosphorylation and pharmacological stabilization of the retromer system. Additionally, we examined the effects of genetic overexpression of VPS35, the backbone of the retromer core, in trisomic neurons. We observed similar decreases in AD pathology measures, however, the magnitude of the effect was smaller with genetic overexpression than with pharmacological stabilization using TPT-172. We hypothesized that trisomy 21 may can some inherent instability of the retromer system, possibly due to overabundance of retromer cargo protein APP, that is better overcome by enhancing stability of the complex rather than increasing VPS35 protein level via genetic overexpression. To further explore the role of the retromer system in the AD-DS phenotype, we performed a full characterization of cognitive function and the retromer complex at 2, 5, 9 and 12 months of age in the Ts65dn mouse model of DS. While we observed accelerated aging related cognitive and pathological changes in DS mice, we did not observe any protein levels changes in the retromer complex. However, because these mice do develop endosomal dysfunction that could be indicative of retromer dysfunction, we treated mice with TPT-172 from 4 to 9 months of age and examined cognitive function, the retromer system and AD pathology measures. We observed improvements in cognitive function tests and synaptic function in Ts65dn mice receiving TPT-172, as well as reductions in tau pathology, early endosome proteins, and early endosome size. Taken together, these data demonstrate that retromer complex deficiency occurs in human DS and may contribute to the development of AD-like pathology and cognitive decline in AD-DS. Because pharmacological stabilization of the retromer system improves AD-like pathology and cognitive function in models of DS, we conclude the retromer represents a potential therapeutic target for AD-DS. / Biomedical Sciences

Chez la levure et les mammifères le complexe rétromère est impliqué dans différentes étapes de trafic intracellulaire qui modulent divers processus cellulaires et développementaux. Chez les mammifères, le rétromère se compose des protéines Vacuolar Protein Sorting (VPS) 35, VPS26, VPS29 et d’un dimere de Sorting Nexins (SNX). Les composants du rétromère sont conservés chez les plantes et sont localisés dans le même endosome de tri. Dans ce travail, nous avons étudié le mécanisme d’assemblage et de recrutement du complexe à la membrane et analysé le rôle du rétromère dans le développement d’Arabidopsis. Nos resultsts montrent que chez les plantes, contrairement aux mammifères, VPS35 recrute le sous-complexe VPS à la membrane, indépendamment des SNXs. Ceci nous a permis de proposer un modèle d’assemblage du rétromère très original. L’analyse de mutants perte de fonction pour le rétromère révèle que VPS26, VPS29 et VPS35 n’ont pas de fonctions indépendantes, mais agissent ensemble dans la régulation de processus développementaux. De plus, par un crible double hybride chez la levure, nous avons isolé un interacteur de VPS35 : Ethylene Insensitive 2 (EIN2). EIN2 est une protéine localisée au Réticulum Endoplasmique et impliquée dans la voie de signalisation d’une hormone végétale : l’éthylène. Nous montrons que des mutants perte de fonction pour le rétromère présentent des défauts dans la réponse à l’éthylène, indiquant un rôle du rétromère dans la perception de cette hormone par les plantes. Ces résultats, combinés avec nos données antérieures montrant que le rétromère est impliqué dans la voie de signalisation de l’auxine, révèlent un lien entre signalisation de l’auxine et de l’éthylènevia le complexe rétromère. / In yeast and mammals, the retromer complex mediates various steps of intracellular trafficking and regulates a variety of cellular and developmental processes. In mammals, it is composed of Vacuolar Protein Sorting (VPS) 35, VPS26, VPS29 and a dimer of Sorting Nexins (SNX). Retromer components are conserved in plants and we showed that they colocalize to the same sorting endosome. In this work, we have investigated the mechanism of assembly and recruitment of the retromer to the endosomal membrane and studied its function in Arabidopsis development. We report that, unlike animals, plant VPS35 recruits the other VPS retromer components to the membrane of endosomes, independently of SNXs. This data allowed us to propose an original model of assembly of the plant retromer complex. By analyzing a series of retromer loss-of-function mutants, we show that VPS26, VPS29 and VPS35 always act together in modulating developmental processes. To identify retromer partners, we carried out a Yeast-2-Hybrid (Y2H) screen using VPS35 as a bait. We found that VPS35 can bind, among other proteins, Ethylene Insensitive 2 (EIN2). EIN2 is an Endoplasmic Reticulum-located protein involved in the signaling pathway of a plant hormone: ethylene. We demonstrate that retromer mutants are affected in ethylene signaling, which indicates that the retromer complex participates in a proper perception of ethylene by plants. Combined with our previous data in which we showed that the retromer is involved in the auxin signalling pathway, our present work reveals a link between auxin and ethylene signaling through the retromer complex.

The parasite Plasmodium falciparum causes the most virulent form of human malaria and is responsible for the vast majority of malaria-related deaths. During the asexual intraerythrocytic stage, the parasite must transport newly synthesized proteins and endocytosed cargo to a variety of organelles, many of which are formed de novo and have no human equivalent. This process in mammalian cells would utilize an endosomal protein trafficking system, but no endosomal structures or proteins have been described in the parasite. Prior work on the parasite genome indicated that several proteins, which could potentially coordinate an endosomal network, were encoded in the genome and expressed during the asexual parasite stages. In this study, we have localized and attempted to further characterize these proteins in the context of the endosomal system. Two well-conserved protein components of the late endosome, the retromer cargo-selective complex and Rab7, were found on a previously un-described inherited structure adjacent to the parasite Golgi apparatus and in close opposition to nascent rhoptries (specialized secretory organelles required for invasion). The retromer cargo-selective complex was also in close proximity to its putative cargo, a P. falciparum homolog of the sortilin family of protein sorting receptors, PfSortilin. Another protein, PfFCP, the sole FYVE domain-containing protein in the P. falciparum genome, was localized to the membrane of a specialized acidic organelle, known as the food vacuole, where the parasite catabolizes the majority of its host cell hemoglobin. We analyzed the effects of a PfFCP dominant negative mutant and found that it altered food vacuole morphology and trafficking. A previous report localized the early endosome phosphoinositide, phosphatidylinositol 3-phosphate, to the food vacuole membrane, and in conjunction with our studies on PfFCP, this has raised doubts about the food vacuole as a lysosome equivalent in the parasite. The combination of both early and late endosome protein homologs in the parasite, and their potential function, has led to a new model of protein trafficking within the parasite that includes the food vacuole as a terminal early endosome and the apical organelles as lysosome equivalents. / Ph. D.

Le rétromère est un complexe multiprotéique qui régule le trafic intracellulaire et qui est conservé chez les eucaryotes. Chez la levure et les animaux, le complexe rétromère est impliqué dans le recyclage des régulateurs de la polarité cellulaire. Chez Arabidopsis thaliana, les travaux de notre équipe ont démontré que ce complexe est requis pour la localisation polaire des transporteurs d’auxine de la famille PIN. Au cours de mon travail de thèse, j’ai approfondi la caractérisation de la fonction du composant AtVPS29 du rétromère et démontré son rôle dans la régulation de la croissance cellulaire polarisée et dans la polarité planaire chez la plante modèle Arabidopsis thaliana. Les mutants perte de fonction vps29 présentent des défauts de croissance polaire des tubes polliniques et des poils absorbants, et également des défauts de morphogenèse des cellules épidermiques des cotylédons. Les petites GTPases ROP (Rho-Of-Plant) sont les régulateurs majeurs de la polarité cellulaire chez les plantes. ROP1 est requis pour la croissance du tube pollinique, et ROP2 pour la morphogenèse des poils absorbants et des cellules épidermiques des cotylédons. Le travail décrit ici est destiné à comprendre les liens fonctionnels et moléculaires entre AtVPS29 et les protéines ROP. Mes résultats suggèrent que l’activité de ROP2 est dépendante de VPS29. J’ai également établi que VPS29 était nécessaire pour assurer la localisation subcellulaire de ROP1 et de ROP2. Chez les mutants vps29, l’analyse de la voie de signalisation ROP2 suggère que l’activité de ROP2 est fortement dérégulée. Ainsi, nos données montrent que le complexe rétromère de plante régule le trafic membranaire des protéines ROP, assurant la localisation de leur activité dans les bons territoires cellulaires, et in fine, la croissance polaire des cellules. Ces données suggèrent que le complexe rétromère de plante pourrait réguler la formation et/ou le maintien de la polarité chez différents types de cellules végétales. Ils soulignent également l’importance du trafic endocytique dans la régulation de la polarité cellulaire chez les plantes. / The retromer complex is a coat pentameric complex, strongly conserved among eukaryotes and involved in intracellular trafficking. In yeast and animals, the retromer complex participates in the polar targeting of regulators of cell polarity. Our group has previously shown that the retromer is required for the polar localization of auxin carrier proteins of the PIN-FORMED (PIN) family in Arabidopsis thaliana. Here, I demonstrate that the plant retromer component AtVPS29 regulates cell polarity, with a specific focus on polar cell growth and planar cell polarity in the model plant A. thaliana. Null vps29 mutants display defects in both pollen tube and root hair tip growth, as well as in the diffuse polar growth of epidermal pavement cells. Rho-of-plant (ROP) small GTPases are master regulators of cell polarity in plants. ROP1 is required for pollen tube growth, while ROP2 coordinates root hair and pavement cell morphogenesis. The aim of my work was focused on the functional and the molecular links between the AtVPS29 and ROP proteins. My results indicate that ROP2 signaling activity is VPS29-dependent in root hairs and in pavement cells. They also suggest that VPS29 is required for the proper localization of ROP1 and ROP2. The analysis of ROP2 signaling pathway in the vps29 mutant, suggest that ROP2 activity is completely deregulated in the absence of VPS29. Altogether, my data indicate that the plant retromer mediates ROP membrane trafficking to allow the proper localization of its activity, a prerequisite for cell polar growth. These findings suggest that the plant retromer might regulate maintenance and/or establishment of cell polarity in various cell types and further emphasize the importance of endocytic recycling in the regulation of polarity in plants.