Synucleins represent a conserved family of small proteins that include α-, β-, and γ- isoforms, which are highly expressed in neurons of the vertebrate nervous system. The normal function of these proteins is not well understood. However, in humans α- synuclein dysfunction is causatively linked to Parkinson’s Disease (PD), where it abnormally accumulates in neuronal cell bodies as protein aggregates that are associated with neuronal death. Although the associations between synuclein accumulation and cellular death are established in PD, the extent to which this occurs in other contexts, such as neuronal injury, is unknown. Furthermore, the effects of synuclein aggregation on the function of synapses, where synuclein is normally localized, are not well understood. To address these questions I took advantage of the experimentally accessible nervous system of the sea lamprey (Petromyzon marinus). I used molecular cloning and phylogenetic analyses to characterize three lamprey synuclein orthologues, one of which is highly expressed within a class of neurons called the giant reticulospinal (RS) neurons. Spinal cord injury induces the accumulation of synuclein protein only within a population of poor surviving RS neurons, and this accumulation is correlated with cellular death. Thus, similar to PD, the abundance of synuclein protein is associated with neuronal toxicity. In a related project, I demonstrated that elevating synuclein levels at synapses, such as occurs in PD, is deleterious to synaptic function through an inhibition of synaptic vesicle (SV) recycling. By injecting excess synuclein protein directly into the axons of giant RS neurons, and analyzing the ultrastructural morphology of synapses, I have shown that clathrin-mediated synaptic vesicle endocytosis was greatly inhibited. The conserved N-terminal domain was sufficient to inhibit vesicle recycling, and injecting synuclein mutants with disrupted N-terminal α-helices caused reduced defects in SV recycling. Therefore the α-helical structure of the N-terminus is necessary to inhibit SV recycling at early stages of clathrin-mediated endocytosis. Binding interactions with clathrin-mediated endocytosis components, such as the phosphoinositide lipid PI(4)P support this hypothesis. These studies provide a better understanding of the mechanisms by which synuclein dysfunction leads to neuronal death after injury and synaptic dysfunction in PD and other synuclein-associated diseases. / text

Σα αρνητικά και θετικά κατά Gram βακτήρια E. coli και S. αureus αντίστοιχα αναγνωρίζονται και δεσμεύονται στην επιφάνεια των αιμοκυττάρων της C. capitata. Η πρόσδεση των βακτηρίων ενεργοποιεί τόσο τις β1 ιντεγκρίνες, όσο και σηματοδοτικά μονοπάτια που περιλαμβάνουν τα μόρια μεταγωγής σήματος Ras, Raf, MEK, ERK, FAK, Src, και GRB2. Οι παραπάνω ενεργοποιήσεις, σε συνδυασμό με τη συμμετοχή του κυτταροσκελετού της ακτίνης και της τουμπουλίνης, καταλήγουν στην επαγωγή της έκκρισης μορίων απαραίτητων για την κυτταροφαγία των βακτηρίων που είναι και το τελικό αποτέλεσμα των παραπάνω διαδικασιών. Σα συνθετικά πολυμερή σφαιρίδια, αλλά και πιθανόν και άλλοι αβιοτικοί παράγοντες, παρότι δεν έχουν καμία προηγούμενη εξελικτική σχέση με τα αιμοκύτταρα, ως σύγχρονο προϊόν της ανθρώπινης γνώσης, αναγνωρίζονται και δεσμεύονται στην επιφάνεια των αιμοκυττάρων από άγνωστους μέχρις στιγμής υποδοχείς. Η κυτταροφαγία τους προωθείται μέσω ενεργοποίησης σηματοδοτικών μονοπατιών που περιλαμβάνουν την ενεργοποίηση των μορίων FAK, Src και MAP κινασών καθώς και με τη συμμετοχή του κυτταροσκελετού της ακτίνης και της τουμπουλίνης. Ο LPS αναγνωρίζεται και δεσμεύεται στην επιφάνεια των αιμοκυττάρων, ενεργοποιεί άγνωστους μέχρις στιγμής υποδοχείς και διαμέσου σηματοδοτικών μονοπατιών που περιλαμβάνουν τις Ras, ενεργοποιεί τις MAP κινάσες και το σύστημα της έκκρισης. Αν και ενεργοποιεί και τις τρεις MAP κινάσες, μόνο η ERK και η p38 απαιτούνται τόσο στη διαδικασία της έκκρισης, όσο και στη διαδικασία της ενδοκυττάρωσής του. Η FAK, αν και ενεργοποιείται από τον LPS, δεν εμπλέκεται στην διαδικασία της ενδοκυττάρωσής του. Σα παραπάνω δείχνουν ότι τα αιμοκύτταρα έχουν αναπτύξει διακριτούς μηχανισμούς για την κυτταροφαγία των παθογόνων, των μικρομορίων και των αβιοτικών παραγόντων, γεγονός που δείχνει την ικανότητα εξέλιξης των εντόμων έτσι ώστε να καλύπτουν τις ανάγκες της επιβίωσή τους. / Gram negative and Gram positive bacteria, E. coli and S. αureus respectively, are recognized and they are bound on the C. capitata hemocyte surface. After binding, they activate β1 integrins and intracellular signalling pathways, involving the kinases Ras, Raf, MEK, ERK, FAK, Src, and GRB2. This signal transduction, with the participation of the cytoskeleton of actin and tuboulin filaments, leads to a regulated secretion, that is a prerequisite for phagocytosis. Latex beads and probably other abiotic factors, despite having no previous evolutionary relation to the hemocytes, being a new product of human knowledge, are recognized and they are bound on the hemocyte surface, by hitherto unknown receptors. They activate intracellular signalling pathways that involves FAK, Src and MAP kinases and they promote, with the participation of actin and tuboulin cytoskeleton, their phagocytosis. LPS is recognized and bound on the hemocyte surface and activates so far unknown receptors and through unknown intracellular signalling pathways involving Ras, activates the MAP kinases and the regulated secretion. Although it activates all three MAPKs, only the ΕRΚ and p38 are required not only for the secretion, but also for its internalization. Although FAK is activated by LPS, it does not get involved in the process of its internalization. All of the above mentioned results indicate that the hemocytes have developed distinct mechanisms for phagocytosis of pathogens, micromolecules and abiotic factors, a fact that underlines insects evolutionary adaptations, so that they can survive.

No description available.

Endocytose joue un rôle dans l'activation du récepteur Notch. Des mutations dans le gène drosophilien lethal giant discs (lgd), provoque une prolifération cellulaire en perturbant l'endocytose de Notch. Les orthologues murins mlgd1 et 2 peuvent sauver ce phénotype, démontrant une fonction conservée. Cependant, des publications récentes suggèrent que les orthologs humains de lgd (hgd1/2) sont nucléaires. Dans cette étude, il est démontré que chez la Drosophile, le mutant dlgd(08) provoque l'accumulation de Notch dans des vésicules et une surprolifération de neuroblastes . Ceci suggère que Notch est activé a l'intérieur des endosomes dans les neuroblastes. L'immunohistochimie de cellules Hela indique que hlgd1 et 2 ne sont pas nucléaires, mais associés à des strctures endosomales. Enfin, la baisse d'expression par shRNA des gènes murins mlgd1 et mlgd2 provoque une différenciation accélérée des cellules souches hématopoïétiques dans la lignée lymphopoïèse T et bloque la transition DN3 / CD4+CD8+, suggérant une suractivation de Notch. / Endocytosis plays a role in the activation of the Notch receptor. Mutations in the Drosophila gene lethal giant discs (lgd), causes cellular overgrowth by perturbing Notch endocytosis. This Drosophila phenotype is rescued by the murine orthologs mlgd1 and 2, indicating conserved function. However, recent publications suggest that the human orthologs (hlgd1/2) are nuclear. This study demonstrates that the dlgd(08) mutant in Drosophila causes accumulation of Notch in vesicles and the overproliferation of neuroblasts. This suggests Notch is activated from within endosomes in neuroblasts. Immunohistochemistry of Hela cells indicates that hlgd1 is associated with early endosome while, hlgd2 with later endosome and lysosome, and not with the nucleus. Finally, down regulation of murine mlgd1 and mlgd2 by shRNA caused an accelerated differentiation of hematopoietic stem cell into the T lymphopoiesis lineage and blocked the DN3 to CD4+CD8+ transition, suggesting that Notch is overactivated in these cells.

At the plasma membrane, both NGF receptors have been shown to localized to lipid rafts, specific subdomains that are enriched in cholesterol, sphingolipids and the presence of caveolin proteins (Cav1 and/or Cav2). The focus of this work is on this membrane microenvironment mediated modulation of NGF signaling which via two receptors: p75NTR and TrkA. In the present work we found that overexpression of Cav-1 in mouse dorsal root ganglia neurons significantly impacted neurite extension. Similarly, overexpression of Cav-1 in PC12 cells strongly inhibits their ability to grow neurites in response to NGF. It inhibits NGF signaling without, impairing transient MAPK pathway activation. Rather, it does so by sequestering NGF receptors in lipid rafts, which correlates with the cell surface localization of downstream effectors, and phosphorylated-Rsk2, resulting in the prevention of the phosphorylation of CREB. By contrast, overexpression of Cav-2 potentiates NGF induced differentiation, which is accompanied by sustained activation of downstream effectors, and standard internalization of the receptors. This differential effect could be due to the different localization of Caveolins, that modifies the microenvironment, thereby affecting NGF signaling. Furthermore, PC12 cells expressing the non-phosphorylatable Cav-1 mutant (S80V), neither TrkA trafficking or CREB phosphorylation are inhibited and the response resembles that observed in Cav-2 expressing PC12 cells. These studies underline the interplay between caveolins and NGF signalling, offering insight into the potential impact of Caveolin-1 and mutations thereof in certain cancers where NGF signaling is involved.

No description available.

Biological information processing in living systems like cells, tissues and organs critically depends on the physical interactions of molecular signaling components in time and space. How endocytic transport of signaling molecules contributes to the regulation of developmental signaling in the complex in vivo environment of a developing organism is not well understood. In a previously performed genome-wide screen on endocytosis, several genes have been identified, that selectively regulate transport of signaling molecules to different types of endosomes, without disrupting endocytosis. My PhD thesis work provides the first functional in vivo characterization of one of these candidate genes, the novel, highly conserved Rab5 effector protein P95 (PPP1R21). Cell culture studies suggest that P95 is a novel endocytic protein important to maintain the balance of distinct endosomal sub-populations and potentially regulates the sorting of signaling molecules between them (unpublished work, Zerial lab). The scientific evidence presented in this study demonstrates that zebrafish P95 is essential for early zebrafish embryogenesis. Both, knockdown and overexpression of zebrafish P95 compromise accurate morphogenetic movements and patterning of the zebrafish gastrula, showing that P95 functions during zebrafish gastrulation. P95 is functionally required to maintain signaling activity of signaling pathways that control embryonic patterning, in particular for WNT/β-catenin signaling activity. Knockdown of zebrafish P95 amplifies the recruitment of β-catenin to early endosomes, which correlates with the limitation of β-catenin to translocate to the nucleus and function as transcriptional activator. The obtained results suggest that zebrafish P95 modulates the cytoplasmic pools of β-catenin in vivo, via endosomal transport of β-catenin. In conclusion, the data presented in this thesis work provides evidence that the cytoplasm-to-nucleus shuttling of β-catenin is modulated by endocytic trafficking of β-catenin in vivo. We propose the endocytic modulation of β-catenin cytoplasm-to-nucleus trafficking as potential new mechanism to fine-tune the functional output of WNT/β-catenin signaling during vertebrate gastrulation.

The chloride/proton antiporter ClC-5 is primarily expressed in the kidney where it aids in re-absorption of proteins from the glomerular filtrate. Functional disruption of ClC-5 causes Dent’s Disease – a renal condition characterized by proteinuria and kidney failure in a third of all cases. The majority of disease-causing mutations translate into premature truncations of the carboxy-terminal (Ct) region of ClC-5 and are predicted to disrupt the protein-protein interactions mediated by this domain. In this thesis, direct ATP binding to the two cystathionine β-synthase (CBS) domains of ClC-5 was demonstrated. ATP binding enhanced the global compactness of the ClC-5 Ct region likely through a clamping motion of the CBS domains around the nucleotide. Along with ATP, the sodium proton exchange regulatory factor 2 (NHERF2) also binds ClC-5; however, the molecular mechanism behind this interaction was unknown as ClC-5 lacked the PDZ binding motif traditionally localized at the Ct end of bait proteins. Here, we also identified a class I PDZ binding motif (657-660; TSII) within the internal sequence of ClC-5. Despite the buried position of this motif in the Ct peptide’s X-ray crystal structure (PDB: 2J9L), the high propensity of this region for dynamic flexibility prompted us to test whether it could mediate NHERF2 interactions. In support of this hypothesis, we demonstrated that the motif is transiently available to interact directly with NHERF2 in vivo and to enable an enhancement in receptor-mediated endocytosis in mammalian cells. Collectively, these results gave further evidence that the intracellular Ct region of ClC-5 serves as a hub to mediate interactions essential for its maturation, stability, and trafficking in renal epithelium, as well as providing further insights into the molecular basis of Dent’s Disease.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

<p>Fibroblast growth factor homologous factors (FHFs) are noncanonical members of the fibroblast growth factor family (FGFs, FGF11-FGF14) that bind directly to voltage gated sodium channels (VGSCs), thereby regulating channel activity and consequently neuronal excitability. Mutations in FGF14 cause spinocerebellar ataxia while FGF13 is a candidate for X-linked mental retardation. Since FGF13 and FGF14 are nearly identical within their respective VGSC-interacting domains, those distinct pathological consequences have generally been attributed to regional differences in expression. I have shown that FGF13 and FGF14 have non-overlapping subcellular distributions and biological roles even in hippocampal neurons where both are prominent. While both FHFs are abundant in the axon initial segment (AIS), only FGF13 is observed within the soma and dendrites. shRNA knockdown and rescue strategies showed that FGF14 regulates axonal VGSCs, while FGF13 only affects VGSCs in the somatodendritic compartment. Thus, FGF13 and FGF14 have nonredundant roles in hippocampal neurons, with FGF14 acting as an AIS-dominant positive regulator and FGF13 serving as a somatodendritic negative regulator. Both of these FHFs also perform important non-VGSC regulatory roles. FGF14 is a novel potassium channel regulator, which binds to KCNQ2 and regulates both localization and function. FGF14 is also capable of serving as a “bridge” between VGSCs and KCNQ2 thus implicating it as a broad organizer of the AIS. FGF13, on the other hand is involved in a new form of neuronal plasticity called axon initial segment structural plasticity. Knockdown of FGF13 impairs AIS structural plasticity and reduces L-type CaV current through channels known to be important to this new form of plasticity. Both of these novel non-VGSC roles are specific to the FHF in question because FGF13 does not regulate KCNQ2 whereas FGF14 knockdown does not affect AIS position. These data imply wider roles for FHFs in neuronal regulation that may contribute to differing roles in neural disease.</p> / Dissertation