Neurons connect to their remote targets via axons, which usually survive for the lifetime of an organism. Spatiotemporal regulation of the axonal proteome by local protein synthesis (LPS) plays a critical role in neuronal wiring and axon survival, raising the intriguing possibility that some neurological disorders involve LPS dysfunction. To visualise LPS in situ, I optimised multiple imaging techniques to investigate Netrin-1-induced translation in cultured retinal axons. Total axonal protein synthesis measured by metabolic and puromycin labelling indicates axons experience stage-dependent alterations in translation rate upon Netrin-1 stimulation. Remarkably, Netrin-1 triggers a burst of β-actin synthesis starting within 20 seconds of cue application at multiple non-repetitive sites visualised by single molecule translation imaging, an approach that allows direct visualisation of translation dynamics in response to external stimuli. Further studies have shown that local translation can occur on Rab7a-associated late endosomes, where mRNA recruitment and translation are coordinately regulated. Notably, mRNAs encoding mitochondria-related proteins are found translating on late endosomes docking in the vicinity of mitochondria, suggesting late endosomes act as ‘platforms’ for the localised synthesis of mitochondrial proteins necessary for maintaining mitochondrial integrity. Moreover, this process is affected in axons expressing the Charcot-Marie-Tooth disease type 2B (CMT2B)-related Rab7a mutants, leading to abnormal mitochondrial biogenesis and activity and compromised axon survival. Finally, attenuated de novo protein synthesis is observed in axons expressing amyotrophic lateral sclerosis (ALS)-associated fused in sarcoma (FUS) mutants and hypomethylated wild-type FUS. Live imaging reveals mislocalised mutant or hypomethylated FUS granules are transported along axons and accumulate at growth cones, possibly irreversibly trapping RNA molecules, resulting in reduced distance travelled by RNA granules in axons. Furthermore, mutant FUS expression results in defective retinal projections in vivo, highlighting the importance of RNA metabolism and local translation in axonal homeostatic mechanisms. In conclusion, aberrant translational activity in axons leads to prominent axonopathy, which recapitulates features of early stages of neurological diseases, providing the basis for novel therapeutic strategies.

Le tri et le transport efficace des hydrolases acides vers le lysosome jouent un rôle critique pour la fonction des cellules. Plus de 50 maladies humaines sont dues à des mutations des enzymes lysosomales, des protéines régulant des processus-clés du transport vers le lysosome ou des enzymes effectuant des modifications posttraductionnelles importantes pour la fonction du lysosome. L’objectif de cette thèse est d’identifier des protéines et des mécanismes permettant à la cellule de réguler le transport des enzymes vers le lysosome. Nous avons formulé l’hypothèse que des protéines mutées dans des maladies lysosomales et dont les fonctions étaient inconnues pouvaient jouer un rôle dans le transport vers le lysosome. Les céroïdes-lipofuscinoses neuronales forment une famille de maladies lysosomales rares mais sont aussi les maladies neurodégénératives infantiles les plus fréquentes. Plusieurs gènes impliqués dans les NCL encodent des protéines aux fonctions inconnues. Les travaux présentés dans cette thèse ont identifié la protéine « ceroid lipofuscinosis neuronal-5 » (CLN5) qui est localisée à l’endosome et au lysosome comme élément nécessaire au recrutement et à l’activation de rab7. Rab7 est une protéine Rab-clé qui contrôle le trafic à l’endosome tardif. Cette petite GTPase est impliquée dans le recrutement de retromer, un complexe protéique qui régule le trafic de l’endosome vers l’appareil de Golgi des récepteurs de tri lysosomal comme sortilin et le récepteur du mannose-6-phosphate. Dans les cellules où CLN5 est déplété, les récepteurs de tri lysosomal sont moins recyclés plus rapidement dégradés. En utilisant des expériences de photomarquage nous avons aussi pu démontrer que Rab7 est moins activées en l’absence de CLN5. Pour exécuter leur fonction les protéines rabs doivent être recrutée à la membrane et activées par l’échange d’une molécule de GDP pour une molécule de GTP. Le recrutement des Rabs à la membrane nécessite une modification posttraductionnelle lipidique pour être facilités. En utilisant un modèle de levures nous avons démontré que l’homologue de Rab7, Ypt7 est palmitoylée. Nous avons aussi démontré que la palmitoyltransférase Swif1 est nécessaire au recrutement de Ypt7 à la membrane. Nous avons aussi remarqué que les sous- unités de retromer chez la levure sont moins recrutées lorsque les palmitoyltransférases sont déplétées. Dans les cellules de mammifères nous avons démontré que Rab7 est également palmitoylé et que cette palmitoylation est possiblement effectuée par les palmitoyltransférases DHHC1 et DHHC8. La palmitoylation de Rab7 a lieu sur les cystéines en C-terminal qui sont nécessaires au recrutement membranaire et qui auparavant étaient uniquement décrites comme prénylées. En utilisant la méthode de « click chemistry » nous avons découvert que lorsque la prénylation de Rab7 est bloquée le niveau de palmitoylation augmente. Pour caractériser l’interaction entre CLN5 et Rab7 nous avons performé des expériences afin d’établir définitivement la topologie de cette protéine. Nous avons ainsi démontré que CLN5 est une protéine hautement glycosylée qui est initialement traduite en protéine transmembranaire et subséquemment clivée par un membre de la famille des peptidase de peptide signal (SPP). Cette protéine soluble peut alors possiblement interagir avec CLN3 qui est aussi palmitoylée pour recruter et activer Rab7. Nos études suggèrent pour la première fois que CLN5 pourrait être un recruteur et un activateur de Rab7 qui agirait avec la protéine CLN3 pour séquestrer Rab7 avec les autres récepteurs palmitoylés et permettre leur recyclage vers l’appareil de Golgi. / The proper sorting and trafficking of acid hydrolases plays a critical role in the normal function of cells. Over 50 known human diseases are caused by mutations of lysosomal enzymes, of proteins that regulate key processes of transport to the lysosome or of enzymes that perform posttranslational modifications which are important for the function of the lysosome. The main objective of this thesis is to identify proteins and mechanisms that allow the cell to regulate the transport of enzymes toward the lysosome. We formulated the hypothesis that proteins mutated in lysosomal diseases and that have no known functions could play a role in transport toward the lysosome. Neuronal ceroid-lipofuscinoses form a family of lysosomal storage disorders that are very rare but are also the most frequent infantile neurodegenerative diseases. The work presented in this thesis identified ceroid-lipofuscinosis neuronal-5 (CLN5), which is located at the late-endosomal/lysosomal compartment as a necessary element for the recruitment and activation of Rab7. Rab7 is an important GTPase that controls traffic from the late-endosome to the trans-Golgi network. Rab7 has been implicated in the recruitment of the retromer complex, which regulates retrograde transport of the lysosomal sorting receptor such as sortilin and the mannose-6-phosphate receptor. In the cells where CLN5 is depleted, the lysosomal sorting receptors are less recycled and degraded more rapidly. Using photolabelling assays we were also able to show that Rab7 is less activated in the absence of CLN5. To perform their function, Rab proteins have to be recruited to membranes and activated by the exchange of a GDP nucleotide for GTP. The recruitment of Rabs to membranes necessitates a lipidic posttranslational modification to raise the affinity. Using yeast as a model we demonstrated that the Rab7 homolog, Ypt7 is palmitoylated. We have also showed that the yeast palmitoyltransferase Swif1 is required for Ypt7 membrane recruitment. We have also observed that retromer subunits in yeast are less recruited when palmitoyltranferases are depleted. In mammals we have shown that Rab7 is also palmitoylated and that this palmitoylation may be done by palmitoyltransferases DHHC1 and DHHC8. The palmitoylation of Rab7 occurs on the C-terminal cysteines that are required for membrane recruitment and were previously only shown to be prenylated. By using Click chemistry we have discovered that when Rab7 prenylation is blocked the level of palmitoylation is augmented. To characterize the interaction of Rab7 and CLN5 we performed experiments to definitively establish the topology of this latter protein. Our results show that CLN5 is a heavily glycosylated protein that is initially translated as a type II transmembrane protein and subsequently cleaved by a member of the signal-peptide peptidase (SPP) family. This protein can then possibly interact with another member of the CLN family, CLN3 that is predicted to be palmitoylated to recruit and activate Rab7. Our studies establish for the first time that CLN5 is required for the recruitment and activation of Rab7 and may cooperate with the possibly palmitoylated protein CLN3 to sequester Rab7 in specific membrane domains with sorting receptors to allow their recycling toward the trans-Golgi network.

In Saccharomyces cerevisiae, experiments with temperature-sensitive mutants of the PI4-kinase Pik1p revealed that the PI4P pool generated by this enzyme is essential for Golgi morphology and normal secretory function and that the PI4P pool at the Golgi represents a regulatory signal on its own. In order to function as a spatial and temporal regulator of membrane traffic, PI4P synthesis and turnover must be tightly regulated. It remains elusive which factors are involved in the targeting and regulation of Pik1p. Little is also known about PI4P binding proteins mediating the effects of this phosphoinositide on Golgi function. Since it has been shown that multiple pathways leave the Golgi towards the plasma membrane one can ask the question whether Pik1p and its product PI4P specifically control one pathway? Here we demonstrate an interaction of Pik1p with the 14-3-3 proteins Bmh1p and Bmh2p. Interestingly, overexpression of Bmh1p and Bmh2p results in multiple genetic interactions with genes involved in late steps of exocytosis and it affects the forward transport of the general amino acid permease Gap1p. The detected interaction depends on the phosphorylation state of Pik1p and Pik1p phosphorylation accompanies its shuttling out of the nucleus into the cytoplasm where presumably the binding to Bmh1/2p occurs. Therefore, we reason that these interactions might serve the sequestration of Pik1p away from the Golgi. This study reveals that Pik1p shows a strong effect on the delivery of Gap1p to the surface whereas the transport of exocytosis markers implicated in the direct Golgi-to-plasma membrane pathway are not significantly disturbed. Cells carrying a deletion of gga2 also show a strong defect in delivery of Gap1p to the surface. In addition, pik1-101 gga2[delta]double mutants display synthetic genetic and membrane transport phenotypes and recruitment of Gga2 to the TGN partially depends on functional Pik1p. Therefore, our results suggest a role of Pik1p in the TGN to endosome pathway.

Utilizing various systems such as cell-based assays but also multicellular organisms such as Drosophila melanogaster and C.elegans, for example, the endocytic system has been shown to consist of a network of biochemically and morphologically distinct organelles that carry out specialized tasks in the uptake, recycling and catabolism of growth factors and nutrients, serving a plethora of key biological functions (Mellman, 1996). Different classes of endosomes were found to exhibit a characteristic intracellular steady state distribution. This distribution pattern observed at steady state results from a dynamic interaction of endosomes with the actin and the microtubule cytoskeleton. It remains unclear, however, which microtubule-based motors besides Dynein control the intracellular distribution and motility of early endosomes and how their function is integrated with the sorting and transport of cargo. The first part of this thesis research outlines the search for such motor. I describe the identification of KIF16B which functions as a novel endocytic motor protein. This molecular motor, a kinesin-3, transports early endosomes to the plus end of microtubules, in a process regulated by the small GTPase Rab5 and its effector, the phosphatidylinositol-3-OH kinase hVPS34. In vivo, KIF16B overexpression relocated early endosomes to the cell periphery and inhibited transport to the degradative pathway. Conversely, expression of dominant-negative mutants or ablation of KIF16B by RNAi caused the clustering of early endosomes to the peri-nuclear region, delayed receptor recycling to the plasma membrane and accelerated degradation. These results suggest that KIF16B, by regulating the plus end motility of early endosomes, modulates the intracellular localization of early endosomes and the balance between receptor recycling and degradation. In displaying Rab5 and PI(3)P-containing cargo selectivity, a remarkable property of KIF16B is that it is subjected to the same regulatory principles governing the membrane tethering and fusion machinery (Zerial and McBride, 2001). Since KIF16B can modulate growth factor degradation, we propose that this motor could have also important implications for signaling. Importantly, KIF16B has provided novel insight into how intracellular localization of endosomes governs the transport activity of these organelles. The second part of this thesis describes the proof-of-principle of a genome-wide screening strategy aimed at gaining insights into the next level of understanding: How the spatial distribution of organelles is linked to their function in an experimental system which features cellular polarity, for example, a tissue or organ. The suitability of C. elegans as a model organism to identify genes functioning in endocytosis has been demonstrated by previous genetic screens (Grant and Hirsh 1999; Fares and Greenwald, 2001). Offering excellent morphological resolution and polarization, the nematode intestine represents a good system to study the apical sorting of a transmembrane marker. The steady state localization of such a marker is likely the result of a dynamic process that depends on biosynthetic trafficking to the apical surface, apical endocytosis and recycling occurring through apical recycling endosomes. Therefore, mis-sorting of this marker upon RNA-mediated interference will be indicative of a failure in one of the aforementioned processes. Furthermore, since it is still largely unclear why apical endosomes maintain their polarized localization, this screen will also monitor the morphology of this endocytic compartment using a second marker. Following image acquisition based on an automated confocal microscope, data can be analyzed using custom-built software allowing objective phenotypic analysis. The successful establishment of the proof-of-principle marks the current state-of-the-art of this large-scale screening project.

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Alzheimers sjukdom är den vanligaste orsaken till demens och i samband med att befolkningen blir större och allt äldre ökar även antalet patienter. Vid sjukdomen sker en hjärnatrofi och de mikroskopiska fynd man ser är extracellulära plack av β-amyloid, intracellulära neurofibriller av fosforylerat tau och förlust av nervcellsutskott, axoner, synapser och dendriter. Några av de tidiga patologiska förändringarna man kan se är störningar i nervcellernas lysosomala system som fyller en viktig roll vid nedbrytning av makromolekyler. I en tidigare studie har man påvisat förhöjda nivåer av proteiner från det lysosomala systemet i cerebrospinalvätska. Syftet med den här studien var att mäta nivåer av det lysosomala systemets proteiner i human hjärnvävnad från patienter med Alzheimer och jämföra dessa med kontrollprover. De sex proteiner som analyserades med Western blot var EEA1, PICALM, LAMP-1, LAMP-2, LC3 och TFEB. Resultaten visar på signifikant ökning i temporala cortex av LAMP-1 och LAMP-2 och en signifikant minskning av LC3 och EEA1 hos patienter med Alzheimers sjukdom. För att kunna dra riktiga slutsatser kring hur de ökade nivåerna i cerebrospinalvätska speglar de olika sjukdomsmekanismerna i hjärnan krävs vidare analyser av fler patientprover samt prover från andra områden i hjärnan. / Alzheimer's disease is the most common cause of dementia, and when the population becomes larger and older also the number of patients increase. A cerebral atrophy and microscopic findings of extracellular plaques of β-amyloid, intracellular neurofibrillary of phosphorylated tau and loss of nerve cell protrusions, axons, synapses and dendrites are seen during the disease. One of the early pathological changes is the disruption of the neuronal lysosomal network that plays an important role in the degradation of macromolecules. In a previous study elevated levels of proteins of the lysosomal network in cerebrospinal fluid from Alzheimer’s disease patients was demonstrated. The purpose of this study was to measure levels of the lysosomal network system in the brain. The six proteins EEA1, PICALM, LAMP-1, LAMP -2, LC3 and TFEB were analyzed in human brain tissue from five Alzheimer's disease cases and five control cases by Western blot. The results show a significant increase in the temporal cortex of LAMP-1 and LAMP -2 and a significant decrease of LC3 and EEA1 in patients with Alzheimer's disease. In order to draw proper conclusions about how the increased levels in cerebrospinal fluid reflect the different disease mechanisms in the brain it requires further analysis of more patient samples and from other areas of the brain.