Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of upper and lower motor neurons, with no effective treatment to date. Only 5-10% of ALS cases are familial, of which 20% are caused by missense mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD I). Although ALS has a multi-factorial aetiology, oxidative stress is hypothesized to be one of the key pathogenic mechanisms. It is thus proposed that manipulation of the expression of anti- oxidant genes may serve as a therapeutic strategy for the protection of motor neurons. It has been previously found that three specific anti-oxidant genes (PTGR 1 /L TB4DH - prostaglandin reductase I / leukotriene B4 12-hydroxydehydrogenase, PRDX3 - peroxiredoxin 3, and NRF2 - nuclear factor erythroid 2-related factor 2) are down- regulated in the presence of mutant SOD 1. The main aim of this project is to alter the expression of the target genes using viral vectors and study the effect on the vulnerability of cells expressing mutant SOD I both in vitro and in vivo. Lentiviral vectors expressing each of the three target genes were tested in the NSC34 cell line (hybrid cell line of mouse motor neurons and neuroblastoma), and the oxidative stress levels as well as cell survival was measured in wild type cells and cells stably expressing the human SOD I gene incorporating the ALS-causing G93A mutation. In the ALS tissue culture model, stable over-expression was achieved, and two of the genes showed cytoprotective properties. Virally delivered PTGR I exhibited no significant effect on oxidation stress levels or cell survival, and as such this gene was not investigated further. However, cells over-expressing either PRDX3 or NRF2 showed a significant decrease in endogenous oxidation stress levels by 40% and 50% respectively compared to controls, whereas cell survival was increased by 30%. These two genes were taken forward to the ALS mouse model, in which intramuscular injections of adeno-associated virus serotype 6 expressing either of the target genes were administered at a pre-symptornatic stage. Behavioural tests and quantitative readouts of motor function allowed the investigation of this therapeutic strategy. However no significant effect was seen in survival, disease onset or disease progression. Quantification of the efficiency of viral delivery showed that only 5% of the lumbar motor neurons were transduced through retrograde transport accounting for the absence of a therapeutic effect. These results suggest that such a delivery is not sufficient to induce a therapeutic effect, and a more widespread CNS delivery is needed.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurological disorder caused by the selective degeneration of upper and lower motor neurons, for which there are currently no effective treatments. 10% of ALS cases are familial, of which 15-20% are caused by mutations in the copper/zinc superoxide dismutase gene (SOD1). The primary triggers for motor neuron degeneration in ALS are unknown, but research in patients and SOD1 models has revealed several mechanisms which may contribute. These include: oxidative stress, mitochondrial abnormalities, inflammation and protein aggregation. This study focused on the pre-clinical testing of two-potential therapeutics for ALS in the SOD1G93A murine model of the disease; metformin, an anti type II diabetes drug which has been shown to have anti-inflammatory and anti-oxidant properties and the ability to bring about mitochondrial biogenesis, and trehalose, a chemical chaperone and enhancer of autophagy, which has been shown to reduce protein misfolding and aggregation. We performed an initial study in which oral metformin administration from 35 days increased the survival of functional motor units in the hindlimbs of male and female SOD1G93A mice at 100 days. Consequently we performed a dose-response survival study in SOD1G93A mice with longitudinal monitoring of weight and neurological score. Surprisingly, metformin had no effect in males and brought about a dose-dependent negative effect on the onset of neurological symptoms and on disease progression in females. We hypothesise this negative effect may have resulted from a metformin-induced reduction in oestrogen production. Oral trehalose administration was tested using the same survival study format. Although trehalose treatment brought about a dose-dependent delay in weight-loss in males, it had no effect on the onset or progression of neurological symptoms or on survival in male or female mice. We conclude that neither metformin nor trehalose represent strong candidates for clinical trial in ALS patients when administered orally.

One of the aims of this study was to explore the psychological experiences of people diagnosed and living with motor neurone disease (MND). As qualitative research within this area is currently limited a literature review of other similar chronic conditions such as Parkinson's disease (PD), multiple sclerosis (MS) and Huntington's disease (HD), as well as MND, were evaluated using an meta-ethnographical approach. This approach was used as it allows for multiple cases, accounts, narratives or studies to be assimilated and interpreted so as to develop new understandings in areas where research may be limited. The review found that people living with one of the four conditions experienced similar difficulties and could be conceptualised under three main concepts. The second aim of this study was to explore the psychological experiences of people with a recent diagnosis of MND and the transitional process they experienced to accept their diagnosis and to make adjustments in their lives. An interpretative phenomenological approach (IPA) was used and three themes were constructed from the analysis of the data collected. The first described the sense of devastation people experienced when they realised they had been diagnosed with a life limiting condition. The second theme describes the processes of accepting and adjusting to the diagnosis. The third theme describes the progressive losses that were experienced as symptoms affected functional abilities and how this impacted on their lives. Ideas for future research were also discussed as well reflections on some of the difficulties that were experienced whilst completing the study.

Recent evidence suggests that the angiogenic factor, vascular endothelial growth factor (VEGF), has a role in determining the survival of motor neurones. VEGF has a pivotal function in the growth of coordinated vascular and neuronal networks in the developing embryo, and promotes the survival of neurones in vitro. Transgenic (VEGFδ/δ) mice which lack a hypoxia-responsive regulatory element in the VEGF gene express low levels of this neurotrophic factor in neural tissue, and develop adult-onset degeneration of motor neurones, which resembles amyotrophic lateral sclerosis (ALS).

Η Πλάγια Αμυοτροφική Σκλήρυνση είναι μια ετερογενής νόσος, που οφείλεται στην εκφύλιση των ανώτερων και κατώτερων κινητικών νευρώνων. Αρχικά η νόσος εκδηλώνεται είτε με μυϊκή αδυναμία και σπαστικότητα των άνω ή κάτω άκρων, είτε με δυσχέρεια στην ομιλία. Η εξέλιξη της νόσου είναι μοιραία και ο αναμενόμενος χρόνος επιβίωσης είναι 3 – 5 χρόνια. Με την εμφάνιση της νόσου έχουν συσχετιστεί πολλοί γενετικοί και περιβαλλοντικοί παράγοντες. Ταυτοχρόνως, έχουν προταθεί και πολλοί μηχανισμοί παθογένειας. Παρόλα αυτά, η ALS παραμένει ανίατη και οι υπάρχουσες θεραπείες αποσκοπούν στην βελτίωση της κλινικής εικόνας ή / και στην παράταση της επιβίωσης. Ιδιαίτερα στον ελληνικό πληθυσμό, η γενετική αιτία της νόσου δεν έχει διερευνηθεί επαρκώς. Μεταλλάξεις στα γονίδια SOD1, TARDBP και FUS στις οποίες οφείλεται το 30% των οικογενών περιπτώσεων ALS σύμφωνα με τη βιβλιογραφία, δεν απαντώνται στους ασθενείς με ελληνική καταγωγή. Για αυτό το λόγο, με τη βοήθεια της αλληλούχησης ολόκληρου του γονιδιώματος και χρησιμοποιώντας διάφορες προσεγγίσεις πάνω στα δεδομένα που εξήχθησαν, εστιάσαμε την έρευνά μας σε συγκεκριμένες παραλλαγές. Στόχος μας ήταν ο έλεγχος για πιθανές συσχετίσεις μεταξύ των παραλλαγών αυτών και της εμφάνισης της νόσου. Η επιλογή των παραλλαγών στηρίχθηκε στους κοινούς μεταξύ των ασθενών πολυμορφισμούς που δεν υπήρχαν στο δείγμα αναφοράς. Από αυτή την προσέγγιση, επιλέχθηκαν οι κοινοί πολυμορφισμοί rs6850200 (TBC1D1), rs1861869 (FTO), rs2892469 (FTO), rs4773203 και rs10581954 (A2LD1), rs34030508 (FAM181B) και rs6676539 (KAZN). Η δεύτερη προσέγγιση, περιλάμβανε τις νέες παραλλαγές που εντοπίστηκαν στο γονιδίωμα τουλάχιστον ενός εκ των ασθενών. Μεταξύ αυτών, επιλέχθηκαν 2 παραλλαγές, στην 5’ αμετάφραστη περιοχή των γονιδίων FGF13 και SLC36A1. Σε δεύτερο χρόνο, αντιπαρατέθηκαν οι παραλλαγές των γονιδιωμάτων, με τη βάση δεδομένων Human Gene Mutation Database (HGMD) και συγκεκριμένα, με τις μεταλλάξεις που έχουν συσχετιστεί με την ALS. Ωστόσο, δεν κρίθηκε σκόπιμη η περαιτέρω διερεύνηση των αποτελεσμάτων αυτών. Το δείγμα των ασθενών επεκτάθηκε για τις επιλεγμένες παραλλαγές και τα αποτελέσματα της γονοτύπησης των ασθενών συγκρίθηκαν με τα αποτελέσματα της γονοτύπησης που πραγματοποιήθηκε σε δείγμα αναφοράς. Οι παραλλαγές που έδωσαν στατιστικά σημαντικό αποτέλεσμα, αναλύθηκαν και σε πληθυσμό Σαρδηνίων ασθενών ALS έναντι υγιών ατόμων. Εξετάσθηκε επίσης και η περίπτωση μιας οικογένειας στην οποία υπήρχαν τέσσερα άτομα τα οποία εμφάνιζαν ιδιαίτερο φαινότυπο και είχαν διαγνωστεί με πιθανή ALS. Συλλέχθηκε το πλήρες ιατρικό ιστορικό των ατόμων της οικογένειας που νοσούσαν, ενώ με τη χρήση της μεθόδου νέας γενιάς αλληλούχησης εντοπίστηκε πιθανή μετάλλαξη. Μεταξύ των συνολικά οχτώ παραλλαγών που μελετήθηκαν (από την πρώτη και δεύτερη προσέγγιση), από την ανάλυση των δύο προέκυψε στατιστικά σημαντική διαφορά μεταξύ υγιών και ασθενών σε επίπεδο γονοτύπων, αλλά όχι σε επίπεδο αλληλομόρφων. Οι συσχετίσεις με την ALS αφορούν τους πολυμορφισμούς, rs6850200 (TBC1D1), rs1861869 και rs2892469 (FTO), στον ελληνικό πληθυσμό. Επιπλέον, οι rs1861869 και rs2892469 συνδέθηκαν με απλότυπο. Οι διαφορές αυτές δεν υπήρχαν στο δείγμα των Σαρδηνίων. Αναφορικά με την οικογένεια, επιβεβαιώθηκε η ύπαρξη μετάλλαξης μεγέθους 25 βάσεων στο γονίδιο SPG7 σε ομόζυγη κατάσταση, στα μέλη της οικογένειας που φέρουν τον φαινότυπο της ALS / HSP (Οικογενούς Σπαστικής Παραπληγίας) και σε ετερόζυγη κατάσταση στα υγιή άτομα της οικογένειας που ελέγχθηκαν. / Amyotrophic Lateral Sclerosis is a heterogeneous disorder, caused by upper and lower motor neuron degeneration. At the first stages of the disease, muscle weakness as well as spasticity of upper or lower limbs or alternatively, impaired speech become obvious. The patients deteriorate rapidly and finally die 3 – 5 years after the initiation of the symptoms. Many genetic and environmental factors have been correlated with ALS. At the same time, a wide range of pathogenesis’ mechanisms have been proposed. Nevertheless, ALS remains a fatal disease and the existing therapies, aim to symptom relief and/ or a slight prolongation of life time expectancy. In particular, the genetic causes of ALS in Greek population, have not been thoroughly investigated. Mutations in SOD1, TARDBP and FUS exons to which 30% of FALS cases are attributed according to the bibliography, are under-represented in patients with a Hellenic ancestry. Herein, we obtained whole genome sequencing data from 10 Greek ALS patients. To interpret our data we used several approaches, which then helped us focus on certain variations. The aim of the project was to investigate those variations and their possible association with the disease. The variations of interest were chosen according to the following criteria; polymorphisms (i) being common to all patients, (ii) not present in healthy individuals and (iii) located near genes. From this approach, we chose the common polymorphisms rs6850200 (TBC1D1), rs2892469 (FTO), rs1861869 (FTO), rs4773203 and rs10581954 (A2LD1), rs34030508 (FAM181B) and rs6676539 (KAZN). The second approach, includes the annotation of all the novel variants found in at least one patient. Among these variants, two were chosen, both located in the 5’ untranslated regions of the genes FGF13 and SLC36A1. Subsequently, the patients’ genomes variants were juxtaposed with the Human Gene Mutation Database (HGMD) and only those associated with ALS were selected. In this case, however, no further investigation has been performed. Our patients’ population was expanded for the overall chosen variations and their genotypes were compared to the genotypes of healthy individuals. Whenever the outcome had a statistical significance, a replication study was attempted in Sardinian ALS patients compared to healthy individuals. In parallel, we came across a case of a family with interesting phenotype features; four individuals in this family were diagnosed with possible ALS / HSP (Hereditary spastic paraparesis) and their full medical history was acquired. Through a next generation sequencing method a new possible mutation was identified. Overall, eight variations were studied (from the first and second approach). The polymorphisms rs6850200 (TBC1D1), rs1861869 and rs2892469 (FTO) showed a statistically significant association with ALS at a genotype level in the Greek population. At the same time, rs1861869 and rs2892469 were correlated with the existence of a haplotype. On the contrary, rs1861869 and rs2892469 showed no statistical significant differences, when Sardinian ALS patients were compared to healthy Sardinian individuals. As far as the family mentioned above is concerned, the existence of a homozygous 25bp deletion in the SPG7 gene was confirmed. All the members of the family, who manifested symptoms, bear the deletion.

TDP-43 est une protéine de liaison aux acides nucléiques qui joue un rôle essentiel dans le métabolisme de l'ARN. À l'état physiologique, un contrôle strict des niveaux d’expression de cette protéine est critique pour la fonction et la survie cellulaire. Une boucle d'autorégulation négative est à la base de ce contrôle du taux intracellulaire de TDP-43. Laquelle a été identifiée comme le constituant principal des inclusions observées chez une majorité des patients atteints de Sclérose Latérale Amyotrophique (SLA) ou de Dégénérescence Lobaire Fronto-Temporale (DLFT). A ce jour, plus de 50 mutations faux-sensdu gène TARDBP/TDP-43 ont été décrites chez des patients DLFT/SLA, démontrant le rôle clé de TDP-43 dans ces pathologies neurodégénératives. Notons cependant que les conséquences fonctionnelles de ces mutations ne sont pas complètement déterminées. Plusieurs études suggèrent qu’une élévation des niveaux d’accumulation de TDP-43 pourraitparticiper aux mécanismes physiopathologiques. La modulation du cycle de production de TDP-43 pourrait donc constituer une nouvelle stratégie thérapeutique. Ce travail de recherche avait donc pour principal objectif d’identifier des modulateurs génétiques de la production de TDP-43 en utilisant un nouveau modèle de drosophile transgénique mimant les principales étapes d’autorégulation de TDP-43. Nous avons ainsi pu montrer que la modulation des niveaux d’expression de la protéine TCERG1 et de plusieurs facteurs d'épissage, parmi lesquels SRSF1, SRSF3 et SF3B1, influe sur les niveaux de production deTDP-43. Nous avons également montré que la présence des mutations DLFT/SLA n’altère pas la capacité de la protéine à s’autoréguler. / TDP-43 is a DNA/RNA binding protein that plays an important role in RNA metabolism. In the physiological state, strict control of its expression levels is critical for cell function and survival. TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. This protein has been identified as the principal component of the inclusions observed in a majority of patients with Amyotrophic Lateral Sclerosis (ALS) or FrontoTemporal Lobar Degeneration (FTLD). To date, more than 50 missense mutations of the TARDBP / TDP-43 gene have been described in FTLD / ALS patients, demonstrating the key role of TDP-43 in these neurodegenerative pathologies. However, the functional consequences of TDP-43 mutations are not completely determined. Several studies suggest that high accumulation levels of TDP-43 may participate in pathophysiological mechanisms. The modulation of the production cycle of TDP-43 may therefore provide a new therapeutic strategy. The main goal of this research project was to identify genetic modulators of TDP-43 production by using a novel transgenic Drosophila model mimicking main steps of TDP-43 the autoregulatory mechanism. We identified several splicing factors, including SF2, Rbp1 and Sf3b1, as genetic modulators of TDP-43 production. We have also shown that modulation of TCERG1 expression levels affect TDP-43 production levels in flies. Finally, we found that FTLD/ALSlinked TDP-43 mutations do not alter TDP-43’s ability to self-regulate its expression and consequently of the homeostasis of TDP-43 protein levels.