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Identifier 000351331
Title Μηχανισμοί νευροεκφυλισμού και γήρανσης στο νηματώδη Caenorhabditis elegans : ο ρόλος της ενδοκύττωσης μέσω κλαθρίνης και της ενδοκυτταρικής μεταφοράς κυστιδίων
Alternative Title Molecular mechanisms of neurodegeneration and ageing in the nematode C. elegans
Author Τρουλλινάκη, Κωστούλα
Thesis advisor Οικονόμου, Αναστάσιος
Ταβερναράκης, Νεκτάριος
Abstract Necrotic cell death is a type of death that is activated under extreme stress and is involved in many pathological conditions such as stroke and ischemia, and in neurodegenerative diseases such as Alzheimer, Parkinson, Huntington and ALS (amyotrophic lateral sclerosis). Recent discoveries have shed light into this process and provide evidence that the mechanisms of necrotic cell death are conserved from nematodes to humans and involve normal cellular activities that are destabilized under adverse conditions and become detrimental for the cell. In the nematode Caenorabditis elegans, neurons expressing mutant degenerins (ion channels) die by cell death that exhibit morphological and ultrastructural characteristics reminiscent of necrotic cell death caused by excitotoxicity after ischemia in mammals. Electron microscopy studies of dying neurons in animals expressing a toxic form of MEC-4 degenerin have shown that the first detectable abnormality is an infolding of the plasma membrane and the production of small electrondense whorls (Hall et al., 1997). These whorls are internalized and form large membranous structures. These ultrastructural features suggest that degeneration may involve enhanced endocytosis at the plasma membrane and intracellular trafficking of membranes and vesicles. Interestingly, disrupted trafficking has been implicated in human neurodegenerative diseases, such as Alzheimer {Katayama, 1999 #455; Nixon, 2000 #453; Sherrington, 1995 , Huntington {Davies, 1997 #456; DiFiglia, 1995 #457} #454} and ALS (De Vos et al., 2007; Kieran et al., 2005). In this thesis we utilized C. elegans, a well-characterized animal model of necrotic cell death, to investigate the involvement of clathrin-mediated endocytosis and intracellular trafficking in necrosis. Endocytosis serves various functions in eukaryotic cells, including the uptake of extracellular molecules and ligands, the regulation of cell signaling pathways, the recycling of proteins to the Golgi and plasma membrane, and the degradation of proteins from the secretory and endocytic pathways. In addition it is required for the retrieval of synaptic vesicle components after release of neurotransmitters in response to action potentials (Cremona and De Camilli, 1997; Harris et al., 2001). We show that clathrin-mediated endocytosis is essential for necrotic cell death in the nematode since elimination or dysfunction of proteins that participate in all steps ameliorates neurodegeneration induced by various insults -both genetic and environmental. By studying its interaction with known necrosis effectors we found that it synergizes with calpain and cathepsin proteases whereas it acts in parallel with autophagy and V-ATPase to mediate necrotic cell death. By using genetically encoded fluorescent markers to monitor structures that arise from clathrin-mediated endocytosis (CME) during necrosis in vivo, we found that early and recycling endosomes increase in number during the first steps of cell death, whereas their number decreases at the later stages, compared to normal neurons. Concerning clathrin-coated pits or vesicles their number does not change significantly at the beginning of necrosis but it drops at later stages. Kinesins are motor proteins that transport proteins and organelles such as lysosomes, mitochondria and endosomes across microtubules. Mutations in neuronal kinesin genes have been implicated in many human neurodegenerative diseases. We checked the involvement of two kinesins (monomeric kinesin UNC-104 and UNC-116 which is the heavy chain of kinesin 1) in necrotic death and found they are required. Their dysfunction ameliorates neurodege neration induced by various insults, without affecting the transport of the toxic protein that is used. These kinesins act in the same pathway with calpains and in parallel with autophagy and the V-ATPase concerning their role on cell death. On the other hand, kinesin UNC-104 synergizes with the lysosomal proteases cathepsins, whereas kinesin 1 acts in parallel with cathepsins. Based on our findings we propose that clathrin-mediated endocytosis and trafficking by kinesins are two processes that are destabilized during necrosis and contribute to the destruction of the cell. Thus, interfering with these cellular functions may protect neurons against neurodegeneration in humans. Protein synthesis is a critical cellular process affecting cell growth, reproduction and survival, depending on the signals that a cell receives. Studies in many organisms, including humans, have revealed that ageing is accompanied by marked alterations in both general and specific protein synthesis (Ward, 2000). The activity of many translation factors decline with age (Kimball et al., 1992; Moldave et al., 1979; Takahashi et al., 1985; Vargas and Castaneda, 1981, 1983) and protein synthesis rate is reduced (Lee et al., 1999a; Rattan, 1996). These alterations indicate a link between the ageing process and the regulation of protein synthesis. However, these studies do not clarify whether these alterations are simply a corollary of the ageing process or have a causative role in senescent decline. In this thesis we manipulated mRNA translation by interfering with a protein factor that participates in the initiation of the process, the eukaryotic initiation factor 4E (eIF4E). eIF4E is a key regulator of protein synthesis as it binds the 7-methyl guanosine cap at the 5’ end of all nuclear mRNAs and mediates the formation of the mRNA translation initiation complex (Gingras et al., 1999). The C. elegans genome encodes 5 isoforms of this factor, called IFE-1 to IFE-5 that differ in cap-binding specificity and anatomical expression (Keiper et al., 2000). By systematically knocking down each of these genes, we found that elimination of a specific eIF4E isoform, IFE-2, that functions in somatic tissues, reduces global protein synthesis, protects from oxidative stress and extends lifespan in C. elegans (Syntichaki et al., 2007). Elimination of the three other isoforms expressed in the germline (IFE-1, IFE-3 and IFE-5), or IFE-4 (Dinkova et al., 2005), an additional somaspecific isoform which regulates translation of a small set of mRNAs, did not alter animal lifespan. The somatic origin of lifespan extension was confirmed by the fact that the absence of functional germline does not abolish the effect of IFE-2 elimination. By examining the link between protein synthesis and other mechanisms known to influence longevity, we found that it is independent of the insulin-like signalling, as IFE-2 depletion further extends the lifespan of long-lived daf-2 and age-1 mutants (Friedman and Johnson, 1988; Kenyon et al., 1993; Kimura et al., 1997). Moreover, lifespan extension is independent of the forkhead transcription factor DAF-16 that mediates the effects of the insulin-like signalling pathway on ageing (Kenyon, 2005). Similarly, lack of IFE-2 enhances the long-lived phenotype of clk and dietaryrestricted eat mutant animals (Hekimi et al., 1995; Lakowski and Hekimi, 1996, 1998). Knockdown of the phosphatidyl inositol kinase TOR that controls protein synthesis in response to nutrient cues further increases the longevity of ife-2 mutants (Gingras et al., 2004; Proud, 2007). Thus, this thesis reveals a new link between protein synthesis and ageing suggesting that signalling via eIF4E in the soma is a novel pathway influencing ageing in C. elegans. Protein synthesis is one of the most energy-consuming processes of the cell, utilizing ~50% of the total energy (Proud, 2002). ife-2 mutants have lower protein synthesis rates than wild type animals. We propose that downregulation of protein synthesis in the soma results in notable energy savings that could be diverted to cellular repair and maintenance processes, thus contributing to longevity. In agreement with this scenario, it was found that IFE-2 depletion protects animals from oxidative stress induced by paraquat or NaN3 and significantly improves the lifespan of mev-1 mutants that are hypersensitive to oxidative stress and are short-lived (Ishii et al., 1998).
Language Greek
Subject Cell death
Necrosis
Neuron
Protein synthesis
eIF4E
Κυτταρικός θάνατος
Νέκρωση
Νευρώνας
Πρωτεινοσύνθεση
Issue date 2009-09-24
Collection   School/Department--School of Sciences and Engineering--Department of Biology--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/b/5/5/metadata-dlib-d22f510a18b46b03cf43232f69a76a61_1259130401.tkl Bookmark and Share
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