Molecular Interventions towards Multiple Sclerosis Treatment

Multiple sclerosis (MS) is an autoimmune life-threatening disease, afflicting millions of people worldwide. Although the disease is non-curable, considerable therapeutic advances have been achieved through molecular immunotherapeutic approaches, such as peptides vaccination, administration of monoclonal antibodies, and immunogenic copolymers. The main aims of these therapeutic strategies are to shift the MS-related autoimmune response towards a non-inflammatory T helper 2 (Th2) cells response, inactivate or ameliorate cytotoxic autoreactive T cells, induce secretion of anti-inflammatory cytokines, and inhibit recruitment of autoreactive lymphocytes to the central nervous system (CNS). These approaches can efficiently treat autoimmune encephalomyelitis (EAE), an essential system to study MS in animals, but they can only partially inhibit disease progress in humans. Nevertheless, modern immunotherapeutic techniques remain the most promising tools for the development of safe MS treatments, specifically targeting the cellular factors that trigger the initiation of the disease

The mobile element Minos as a tool for functional genomic analysis in drosophila

At the present thesis the features of the mobile element Minos as a tool for genomic analysis in Drosophila have been examined. We have developed systems for the in vivo mobilisation of Minos – based transposons through the inducible expression of the transposase protein, which are characterised by high levels of transposition. The analysis of approximately 100 insertions of Minos in Drosophila revealed the ability of the element to insert in genes that have not been targeted by other mobile elements so far. Furthermore, Minos has been found to preferentially target the introns in comparison to the exons of the genes. The computational analysis of the primary structure and some physical properties of the DNA at the insertion sites showed that the primary structure of the DNA that surrounds the dinucleotides - target ΤΑ affects the target choice of the Minos transposase less, compared to other mobile elements that belong to the superfamily Tc1/mariner. The physical properties of the DNA at the insertion sites have an important impact on the target choice of the Minos transposase. Despite the fact that the Minοs insertions in introns have not been found to be mutagenic, we showed that the deletion of adjacent exonic sequences and, as a consequence, the mutation of the relative gene, can be generated through the mobilization of the transposon from the initial insertion site. We developed an enhancer trapping system with the use of the gene Gal4 as a first reporter gene. The genes expression patterns that have been observed with the certain technique and the expression patterns of the endogenous genes, as they were detected with the use of the RNA in situ hybridization technique, were similar. The analysis of approximately 1000 insertions of Minos that have been characterized by the Drosophila Genes Disruption Project group proved the ability of the element to insert with a higher frequency in new genes, than the P element. The use of Minos as the basic tool for the disruption of the Drosophila genes by the Drosophila Genes Disruption Project will greatly advance the completion of the relative project.Στην παρούσα εργασία μελετήθηκαν οι ιδιότητες του μεταθετού στοιχείου Minos ως εργαλείου γονιδιωματικής ανάλυσης στη Δροσόφιλα. Αναπτύχθηκαν συστήματα in vivo κινητοποίησης τρανσποζονίων του στοιχείου μέσω επαγόμενης έκφρασης τρανσποζάσης, που χαρακτηρίζονται από υψηλά ποσοστά κινητοποίησης των τρανσποζονίων. H ανάλυση 100 περίπου ενθέσεων του Minos στη Δροσόφιλα έδειξε τη δυνατότητα του στοιχείου να εντίθεται σε γονίδια στα οποία δεν έχουν επιλεχθεί ενθέσεις άλλων μεταθετών στοιχείων μέχρι τώρα. Επίσης, βρέθηκε ότι τα εσώνια των γονιδίων αποτελούν προτιμούμενους στόχους σε σχέση με τα εξώνια. Η ανάλυση της πρωτοταγούς δομής και των φυσικών ιδιοτήτων τoυ DNA στα σημεία ένθεσης του στοιχείου έδειξε ότι η πρωτοταγής ακολουθία του DNA που περιβάλλει τα δινουκλεοτίδια ΤΑ στα σημεία ένθεσης του στοιχείου επηρεάζει σε μικρότερο βαθμό την επιλογή της θέσης ένθεσης του Minos, σε σχέση με άλλα μεταθετά στοιχεία της υπεροικογένειας Tc1/mariner. Oι φυσικές ιδιότητες του DNA στις θέσεις ένθεσης του στοιχείου φαίνεται ότι επηρεάζουν σε σημαντικό βαθμό την επιλογή της θέσης ένθεσης. Αν και οι ενθέσεις του Minοs σε εσώνια γονιδίων δεν προκαλούν γονιδιακές μεταλλαγές, δείξαμε ότι η απαλειφή γειτονικών περιοχών εσωνίων και επομένως η μεταλλαγή του σχετικού γονιδίου μπορεί να γίνει μετά από την κινητοποποίηση του στοιχείου από την αρχική θέση ένθεσης. Αναπτύχθηκε ένα σύστημα παγίδευσης ενισχυτών με χρήση του γονιδίου Gal4 ως πρώτου γονιδίου αναφοράς. Τα πρότυπα γονιδιακής έκφρασης που ανιχνεύτηκαν με την τεχνική παγίδευσης ενισχυτών και τα πρότυπα έκφρασης των ενδογενών γονιδίων, όπως προέκυψαν μετά από την τεχνική του υβριδισμού με RNA, ήταν παρόμοια. Η ανάλυση 1000 ενθέσεων που χαρακτηρίστηκαν από την ομάδα του Προγράμματος μεταλλαγής των γονιδίων της Δροσόφιλας κατέδειξε το γεγονός ότι το στοιχείο Minοs εντίθεται με μεγαλύτερη συχνότητα σε νέα γονίδια σε σχέση με το στοιχείο Ρ. Η χρησιμοποίηση του Minοs ως βασικού εργαλείου μεταλλαγής των γονιδίων της Δροσόφιλας από το Πρόγραμμα μεταλλαγής των γονιδίων της Δροσόφιλας θα επισπεύσει την ολοκλήρωση του σχετικού Προγράμματο

Neuronal <i>atg1</i> Coordinates Autophagy Induction and Physiological Adaptations to Balance mTORC1 Signalling

The mTORC1 nutrient-sensing pathway integrates metabolic and endocrine signals into the brain to evoke physiological responses to food deprivation, such as autophagy. Nevertheless, the impact of neuronal mTORC1 activity on neuronal circuits and organismal metabolism remains obscure. Here, we show that mTORC1 inhibition acutely perturbs serotonergic neurotransmission via proteostatic alterations evoked by the autophagy inducer atg1. Neuronal ATG1 alters the intracellular localization of the serotonin transporter, which increases the extracellular serotonin and stimulates the 5HTR7 postsynaptic receptor. 5HTR7 enhances food-searching behaviour and ecdysone-induced catabolism in Drosophila. Along similar lines, the pharmacological inhibition of mTORC1 in zebrafish also stimulates food-searching behaviour via serotonergic activity. These effects occur in parallel with neuronal autophagy induction, irrespective of the autophagic activity and the protein synthesis reduction. In addition, ectopic neuronal atg1 expression enhances catabolism via insulin pathway downregulation, impedes peptidergic secretion, and activates non-cell autonomous cAMP/PKA. The above exert diverse systemic effects on organismal metabolism, development, melanisation, and longevity. We conclude that neuronal atg1 aligns neuronal autophagy induction with distinct physiological modulations, to orchestrate a coordinated physiological response against reduced mTORC1 activity

Effect of DR on median and maximum lifespan of <i>Drosophila melanogaster</i> wild-derived strains.

<p>A yeast concentration range of 0.1 to 2.0 captured the DR response of all strains. Both median and maximum lifespans were affected by yeast concentration, in all cases. Maximum lifespan for <i>W^Dah</i>: F = 114.5, R² = 0.9106, <i>p</i><1×10⁻⁴, for <i>FRA</i>: F = 52.76, R² = 0.8242, <i>p</i><1×10⁻⁴, for <i>GRE</i>: F = 58.22, R² = 0.8381, <i>p</i><1×10⁻⁴, for <i>GER</i>: F = 91.41, R² = 0.8904, <i>p</i><1×10⁻⁴, for <i>NETH</i>: F = 48.92, R² = 0.8130, <i>p</i><1×10⁻⁴, one-way ANOVA test, <i>n</i> = 100). Maximum lifespan was calculated as the average lifespan of the most long-lived 10% of flies.</p

Norms of reaction of wild-derived <i>Drosophila</i> populations.

<p>Lifespan and fecundity showed different shapes of response to yeast concentration. The effect of yeast concentration on lifespan best fitted a third-order polynomial equation (non-linear regression) and on fecundity a linear equation (linear regression). Goodness of fit is high in both models, as represented by R² values: For mean lifespans: 0.88, 0.74, 0.80, 0.88, 0.79 for <i>W^Dah</i>, <i>FRA</i>, <i>GRE</i>, <i>GER</i>, <i>NETH</i> strains respectively. For fecundity: 0.90, 0.87, 0.87, 0.89, 0.91 for <i>W^Dah</i>, <i>FRA</i>, <i>GRE</i>, <i>GER</i>, <i>NETH</i> strains respectively. For mean lifespans, best-fit values ranged for B0: 6.6 to 28, for B1: 86.5 to 176, for B2: -81.6 to -151, for B3: 20.9 to 36.8. Linear regression analysis for fecundity: for <i>W^Dah</i>: F = 484.2, DFn = 1, DFd = 48, <i>p</i><1×10⁻⁴, for <i>FRA</i>: F = 335.7, DFn = 1, DFd = 48, <i>p</i><1×10⁻⁴, for <i>GRE</i>: F = 309.9, DFn = 1, DFd = 48, <i>p</i><1×10⁻⁴, for <i>GER</i>: F = 373.6, DFn = 1, DFd = 48, <i>p</i><1×10⁻⁴, for <i>NETH</i>: F = 508.2, DFn = 1, DFd = 48, <i>p</i><1×10⁻⁴. Lifespan curves and fecundity slopes differed significantly among strains (for lifespan curves: F = 63.23, DFn = 16, DFd = 230, <i>p</i><1×10⁻⁴, for fecundity slopes: F = 27.10, DFn = 4, DFd = 240, <i>p</i><1×10⁻⁴).</p

Wild-derived strain <i>NETH</i> had similar greatest mean lifespan/fecundity values to laboratory strain <i>W^Dah.</i>

<p>Under all food conditions tested in this study, the combined greatest mean lifespan and greatest mean fecundity values were seen in the laboratory-adapted <i>W^Dah</i> and wild-derived <i>NETH</i> strains, compared to each of the other strains (for lifespan: <i>p</i><0.001, log rank test, for fecundity: <i>p</i><0.01, one-way ANOVA with Bonferroni's Multiple Comparison test). Greatest mean lifespans for <i>GRE</i>, <i>GER</i> and <i>FRA</i> strains were obtained from 0.5 yeast concentration food, while for <i>W^Dah</i> and <i>NETH</i> strains from 1.0 yeast concentration. For all strains greatest fecundity occurred at 2.0 yeast concentration food. Wild-derived strain <i>NETH</i> had similar greatest mean lifespan/mean fecundity values to the laboratory strain <i>W^Dah</i> (for lifespan: <i>p</i>>0.05, log rank test, for fecundity: <i>p</i>>0.05, one-way ANOVA with Bonferroni's Multiple Comparison test). Data are derived from results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074681#pone-0074681-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074681#pone-0074681-g002" target="_blank">2</a>. Error bars indicate standard deviation (SD).</p

Lifespan and fecundity of wild–derived <i>Drosophila melanogaster</i> strains responded to DR.

<p>The mean lifespans of all wild-derived strains and the laboratory strain exhibited a tent-shaped response to DR with highest mean lifespan values at 0.5 or 1.0 yeast concentration. Female fecundity showed a monotonic increase with yeast concentration in all strains. Both mean lifespan and fecundity were significantly affected by food. Mean lifespan for <i>W^Dah</i>: F = 94.21, R² = 0.8933, <i>p</i><1×10⁻⁴, for <i>FRA</i>: F = 35.42, R² = 0.7589, <i>p</i><1×10⁻⁴, for <i>GRE</i>: F = 81.23, R² = 0.8784, <i>p</i><1×10⁻⁴, for <i>GER</i>: F = 96.66, R² = 0.8957, <i>p</i><1×10⁻⁴, for <i>NETH</i>: F = 43.25, R² = 0.7936, <i>p</i><1×10⁻⁴, one-way ANOVA test. Fecundity for <i>W^Dah</i>: F = 136.5, R² = 0.9239, <i>p</i><1×10⁻⁴, for <i>FRA</i>: F = 158.3, R² = 0.9337, <i>p</i><1×10⁻⁴, for <i>GRE</i>: F = 103.1, R² = 0.9016, <i>p</i><1×10⁻⁴, for <i>GER</i>: F = 107.6, R² = 0.9054, <i>p</i><1×10⁻⁴, for <i>NETH</i>: F = 123.5, R² = 0.9165, <i>p</i><1×10⁻⁴, one-way ANOVA test. Points: mean lifespan. Bars: estimate of mean number of eggs laid/fly/day ± standard error; connected points: mean lifespan in days (<i>n</i>  = 100). Data shown are from a single trial with second generation flies of the wild-derived strains.</p

<i>Drosophila melanogaster</i> wild-derived strains used in this study.

<p><i>Drosophila melanogaster</i> wild-derived strains used in this study.</p

Minos as a Genetic and Genomic Tool in Drosophila melanogaster

Much of the information about the function of D. melanogaster genes has come from P-element mutagenesis. The major drawback of the P element, however, is its strong bias for insertion into some genes (hotspots) and against insertion into others (coldspots). Within genes, 5′-UTRs are preferential targets. For the successful completion of the Drosophila Genome Disruption Project, the use of transposon vectors other than P will be necessary. We examined here the suitability of the Minos element from Drosophila hydei as a tool for Drosophila genomics. Previous work has shown that Minos, a member of the Tc1/mariner family of transposable elements, is active in diverse organisms and cultured cells; it produces stable integrants in the germ line of several insect species, in the mouse, and in human cells. We generated and analyzed 96 Minos integrations into the Drosophila genome and devised an efficient “jump-starting” scheme for production of single insertions. The ratio of insertions into genes vs. intergenic DNA is consistent with a random distribution. Within genes, there is a statistically significant preference for insertion into introns rather than into exons. About 30% of all insertions were in introns and ∼55% of insertions were into or next to genes that have so far not been hit by the P element. The insertion sites exhibit, in contrast to other transposons, little sequence requirement beyond the TA dinucleotide insertion target. We further demonstrate that induced remobilization of Minos insertions can delete nearby sequences. Our results suggest that Minos is a useful tool complementing the P element for insertional mutagenesis and genomic analysis in Drosophila

Lowered Insulin Signalling Ameliorates Age-Related Sleep Fragmentation in <i>Drosophila</i>

<div><p>Sleep fragmentation, particularly reduced and interrupted night sleep, impairs the quality of life of older people. Strikingly similar declines in sleep quality are seen during ageing in laboratory animals, including the fruit fly <i>Drosophila</i>. We investigated whether reduced activity of the nutrient- and stress-sensing insulin/insulin-like growth factor (IIS)/TOR signalling network, which ameliorates ageing in diverse organisms, could rescue the sleep fragmentation of ageing <i>Drosophila</i>. Lowered IIS/TOR network activity improved sleep quality, with increased night sleep and day activity and reduced sleep fragmentation. Reduced TOR activity, even when started for the first time late in life, improved sleep quality. The effects of reduced IIS/TOR network activity on day and night phenotypes were mediated through distinct mechanisms: Day activity was induced by adipokinetic hormone, dFOXO, and enhanced octopaminergic signalling. In contrast, night sleep duration and consolidation were dependent on reduced S6K and dopaminergic signalling. Our findings highlight the importance of different IIS/TOR components as potential therapeutic targets for pharmacological treatment of age-related sleep fragmentation in humans.</p></div