M. J. Martínez-Sebastián

Causative role of oxidative stress in a Drosophila model of Friedreich ataxia

Friedreich ataxia (FA), the most common form of hereditary ataxia, is caused by a deficit in the mitochondrial protein frataxin. While several hypotheses have been suggested, frataxin function is not well understood. Oxidative stress has been suggested to play a role in the pathophysiology of FA, but this view has been recently questioned, and its link to frataxin is unclear. Here, we report the use of RNA interference (RNAi) to suppress the Drosophila frataxin gene (fh) expression. This model system parallels the situation in FA patients, namely a moderate systemic reduction of frataxin levels compatible with normal embryonic development. Under these conditions, fh‐RNAi flies showed a shortened life span, reduced climbing abilities, and enhanced sensitivity to oxidative stress. Under hyperoxia, fh‐RNAi flies also showed a dramatic reduction of aconitase activity that seriously impairs the mitochondrial respiration while the activities of succinate dehydrogenase, respiratory complex I and II, and indirectly complex III and IV are normal. Remarkably, frataxin overexpression also induced the oxidative‐mediated inactivation of mitochondrial aconitase. This work demonstrates, for the first time, the essential function of frataxin in protecting aconitase from oxidative stress‐dependent inactivation in a multicellular organism. Moreover our data support an important role of oxidative stress in the progression of FA and suggest a tissue‐dependent sensitivity to frataxin imbalance. We propose that in FA, the oxidative mediated inactivation of aconitase, which occurs normally during the aging process, is enhanced due to the lack of frataxin.—Llorens, J. V., Navarro, J. A., Martínez‐Sebastián, M. J., Baylies, M. K., Schneuwly, S., Botella, J. A., Moltó, M. D. Causative role of oxidative stress in a Drosophila model of Friedreich ataxia. FASEB J. 21, 333–344 (2007)

Distribution, frequency and diversity of Bacillus thuringiensis in olive tree environments in Spain

Summary Bacillus thuringiensis was isolated from samples collected from olive tree related habitats (olive groves, olive storage facilities and oil mills) of different olive producing regions in Spain. This bacterium was found in a high percentage (92%) of samples. From 72 samples analysed, 2244 sporulating colonies were selected and observed under phase contrast microscopy. From these, 414 colonies (18%) were classified as B. thuringiensis based on the production of parasporal crystals. A great variability of spore, crystal size and morphology was observed. The results indicate that olive groves are as rich as soil olive mills and olive storage facilities regarding the B. thuringiensis population. Analysis by SDS-PAGE of the proteins in the parasporal crystal showed a considerable heterogeneity between B. thuringiensis strains. This was especially relevant in samples obtained from oil mills and storage centres, indicating the high polymorphism in this species. Strains isolated from the same sample showing the same SDS-PAGE profile were not considered different, and just one of them was selected for further tests. A total of 188 out of the 414 B. thuringiensis isolates were shown to be different strains and tested for their toxicity against adult animals of the olive fly Bactrocera oleae . Approximately 40% of the strains caused similar or higher mortality than the dipteran active strain B. thuringiensis israelensis (45% mortality). Most strains (71%) producing a toxicity higher than 65% came from olive mills and olive storage facilities. Three of these strains exhibited a mortality higher than 70% in our test conditions.

The banding pattern of polytene chromosomes of Drosophila guanche compared with that of D. subobscura.

A detailed map of the salivary gland chromosomes of Drosophila guanche is presented and compared to the standard gene arrangements of D. subobscura. Generally, the polytene chromosomc banding patterns of the two species show a high degrce of homology. Only Segment I of the sex chromosome (Chromosome A) shows marked differences. The banding pattern proposed for this segment in D. guanche could have originated from a cluster of overlapping inversions including A1 arrangement.

Overexpression of Human and Fly Frataxins in Drosophila Provokes Deleterious Effects at Biochemical, Physiological and Developmental Levels

Background Friedreichs ataxia (FA), the most frequent form of inherited ataxias in the Caucasian population, is caused by a reduced expression of frataxin, a highly conserved protein. Model organisms have contributed greatly in the efforts to decipher the function of frataxin; however, the precise function of this protein remains elusive. Overexpression studies are a useful approach to investigate the mechanistic actions of frataxin; however, the existing literature reports contradictory results. To further investigate the effect of frataxin overexpression, we analyzed the consequences of overexpressing human (FXN) and fly (FH) frataxins in Drosophila. Methodology/Principal Findings We obtained transgenic flies that overexpressed human or fly frataxins in a general pattern and in different tissues using the UAS-GAL4 system. For both frataxins, we observed deleterious effects at the biochemical, histological and behavioral levels. Oxidative stress is a relevant factor in the frataxin overexpression phenotypes. Systemic frataxin overexpression reduces Drosophila viability and impairs the normal embryonic development of muscle and the peripheral nervous system. A reduction in the level of aconitase activity and a decrease in the level of NDUF3 were also observed in the transgenic flies that overexpressed frataxin. Frataxin overexpression in the nervous system reduces life span, impairs locomotor ability and causes brain degeneration. Frataxin aggregation and a misfolding of this protein have been shown not to be the mechanism that is responsible for the phenotypes that have been observed. Nevertheless, the expression of human frataxin rescues the aconitase activity in the fh knockdown mutant. Conclusion/Significance Our results provide in vivo evidence of a functional equivalence for human and fly frataxins and indicate that the control of frataxin expression is important for treatments that aim to increase frataxin levels.

TORC1 Inhibition by Rapamycin Promotes Antioxidant Defences in a Drosophila Model of Friedreich’s Ataxia

Friedreich’s ataxia (FRDA), the most common inherited ataxia in the Caucasian population, is a multisystemic disease caused by a significant decrease in the frataxin level. To identify genes capable of modifying the severity of the symptoms of frataxin depletion, we performed a candidate genetic screen in a Drosophila RNAi-based model of FRDA. We found that genetic reduction in TOR Complex 1 (TORC1) signalling improves the impaired motor performance phenotype of FRDA model flies. Pharmacologic inhibition of TORC1 signalling by rapamycin also restored this phenotype and increased the lifespan and ATP levels. Furthermore, rapamycin reduced the altered levels of malondialdehyde + 4-hydroxyalkenals and total glutathione of the model flies. The rapamycin-mediated protection against oxidative stress is due in part to an increase in the transcription of antioxidant genes mediated by cap-n-collar (Drosophila ortholog of Nrf2). Our results suggest that autophagy is indeed necessary for the protective effect of rapamycin in hyperoxia. Rapamycin increased the survival and aconitase activity of model flies subjected to high oxidative insult, and this improvement was abolished by the autophagy inhibitor 3-methyladenine. These results point to the TORC1 pathway as a new potential therapeutic target for FRDA and as a guide to finding new promising molecules for disease treatment.

Gypsy endogenous retrovirus maintains potential infectivity in several species of Drosophilids

BackgroundSequences homologous to the gypsy retroelement from Drosophila melanogaster are widely distributed among drosophilids. The structure of gypsy includes an open reading frame resembling the retroviral gene env, which is responsible for the infectious properties of retroviruses.ResultsIn this study we report molecular and phylogeny analysis of the complete env gene from ten species of the obscura group of the genus Drosophila and one species from the genus Scaptomyza.ConclusionThe results indicate that in most cases env sequences could produce a functional Env protein and therefore maintain the infectious capability of gypsy in these species.

Tirant: A New Retrotransposon-Like Element in Drosophila melanogaster

In this paper we report a new retrotransposon-like element ofDrosophila melanogaster calledTirant. This sequence is moderately repeated in the genome of this species and it has been found to be widely dispersed throughout its distribution area. From Southern blot andin situ analyses, this sequence appears to be mobile inD. melanogaster, since its chromosome location and the hybridization patterns vary among the different strains analyzed. In this way, partial sequencing ofTirant ends suggests that it is a retrotransposon, since it is flanked by two LTRs.The presence of sequences homologous toTirant has been also investigated in 28 species of the genusDrosophila by means of Southern analyses. These sequences were only detected in species frommelanogaster andobscura groups. These data suggest that ancestral sequences ofTirant appeared after the Sophophora radiation and before the divergence of those groups.

Metal Homeostasis Regulators Suppress FRDA Phenotypes in a Drosophila Model of the Disease

Friedreich’s ataxia (FRDA), the most commonly inherited ataxia in populations of European origin, is a neurodegenerative disorder caused by a decrease in frataxin levels. One of the hallmarks of the disease is the accumulation of iron in several tissues including the brain, and frataxin has been proposed to play a key role in iron homeostasis. We found that the levels of zinc, copper, manganese and aluminum were also increased in a Drosophila model of FRDA, and that copper and zinc chelation improve their impaired motor performance. By means of a candidate genetic screen, we identified that genes implicated in iron, zinc and copper transport and metal detoxification can restore frataxin deficiency-induced phenotypes. Taken together, these results demonstrate that the metal dysregulation in FRDA includes other metals besides iron, therefore providing a new set of potential therapeutic targets.

A heterochromatic P sequence in the D. subobscura genome.

The study of a heterochromatic P sequence of D. subobscura reveals that it is a degraded element, located at the centromeric region of the A chromosome (X chromosome in this species), and that it is strongly diverged from the euchromatic P sequences previously described in this species. This heterochromatic sequence is composed of some P element fragments embedded in undefined β-heterochromatic sequences. These mosaic P sequences do not show any transcriptional activity and seem to be ancient parasites of the D. subobscura genome. Phylogenetic analyses indicate that both the euchromatic and heterochromatic P sequences of D. subobscura could come from an ancestral element which was present before the divergence of the subobscura species cluster.

Gene activity of polytene chromosomes in Drosophila species of the obscura group

The polytene chromosome puffing patterns of Drosophila guanche were established and compared with those of Drosophila subobscura. A total of 150 loci, active in some of the 17 developmental stages studied, were described and 23 of them were found to form the characteristic puffing pattern of D. guanche. Taking into account the number of puffs as well as the gene activity of each chromosome and the total gene activity, D. guanche seems to be less active than D. subobscura. Although both species show a degree of homology in their puffing patterns lower than that found for sibling species, the degree of homology is stronger than that between species belonging to the same group but to different subgroups. Thus, D. guanche and D. subobscura must be considered as phylogenetically closely related species, belonging to the same subgroup.