Spatial and temporal requirement of Mlp60A isoforms during muscle development and function in Drosophila melanogaster.

Many myofibrillar proteins undergo isoform switching in a spatio-temporal manner during muscle development. The biological significance of the variants of several of these myofibrillar proteins remains elusive. One such myofibrillar protein, the Muscle LIM Protein (MLP), is a vital component of the Z-discs. In this paper, we show that one of the Drosophila MLP encoding genes, Mlp60A, gives rise to two isoforms: a short (279 bp, 10 kDa) and a long (1461 bp, 54 kDa) one. The short isoform is expressed throughout development, but the long isoform is adult-specific, being the dominant of the two isoforms in the indirect flight muscles (IFMs). A concomitant, muscle-specific knockdown of both isoforms leads to partial developmental lethality, with most of the surviving flies being flight defective. A global loss of both isoforms in a Mlp60A-null background also leads to developmental lethality, with muscle defects in the individuals that survive to the third instar larval stage. This lethality could be rescued partially by a muscle-specific overexpression of the short isoform. Genetic perturbation of only the long isoform, through a P-element insertion in the long isoform-specific coding sequence, leads to defective flight, in around 90% of the flies. This phenotype was completely rescued when the P-element insertion was precisely excised from the locus. Hence, our data show that the two Mlp60A isoforms are functionally specialized: the short isoform being essential for normal embryonic muscle development and the long isoform being necessary for normal adult flight muscle function.

Genetic repression of the antioxidant enzymes reduces the lifespan in Drosophila melanogaster.

Aging is a biological process associated with gradual loss of function caused by cellular and molecular damages ultimately leading to mortality. Free radicals are implicated in oxidative damage which affects the longevity of organisms. Natural cellular defenses involving antioxidant enzymes delay or prevent oxidative damage and, therefore, influence the aging process and longevity has been shown in many species including Drosophila. We and others have shown that oxidative resistance is an important mechanism in the aging process in Drosophila. Therefore, we hypothesized that repressing endogenous antioxidant defenses shortens longevity in Drosophila. To study the influence of natural defense mechanisms against oxidative stress in aging, we have investigated the effect of genetic repression of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), on longevity in Drosophila using transgenic RNAi flies and in vivo inhibition of the enzymes with chemical inhibitors. RNAi lines of Drosophila viz., UAS-sod1-IR and UAS-cat-IR, are driven ubiquitously using Act5C-Gal4 and Tubulin-Gal4 to achieve the suppression of SOD1 and CAT activities, respectively. We show that genetic repression of SOD1 and CAT by RNAi in transgenic flies led to drastically reduced longevity (SOD1, 77%; CAT, 83%), presenting the evidence for the role of endogenous antioxidant defenses in lifespan extension in Drosophila. Further, our study shows that the enzyme inhibitors, diethyldithiocarbamate and 3-amino-1,2,4-triazole, although lower the enzyme activities in vivo in flies, but did not affect longevity, which could be attributed to the factors such as bioavailability and metabolism of the inhibitors and adaptive mechanisms involving de novo synthesis of the enzymes. Our study of genetic repression using transgenic RNAi provides experimental evidence that extended longevity is associated with endogenous antioxidant defenses and aging is correlated with oxidative stress resistance.

A cis-regulatory mutation in troponin-I of Drosophila reveals the importance of proper stoichiometry of structural proteins during muscle assembly.

Rapid and high wing-beat frequencies achieved during insect flight are powered by the indirect flight muscles, the largest group of muscles present in the thorax. Any anomaly during the assembly and/or structural impairment of the indirect flight muscles gives rise to a flightless phenotype. Multiple mutagenesis screens in Drosophila melanogaster for defective flight behavior have led to the isolation and characterization of mutations that have been instrumental in the identification of many proteins and residues that are important for muscle assembly, function, and disease. In this article, we present a molecular-genetic characterization of a flightless mutation, flightless-H (fliH), originally designated as heldup-a (hdp-a). We show that fliH is a cis-regulatory mutation of the wings up A (wupA) gene, which codes for the troponin-I protein, one of the troponin complex proteins, involved in regulation of muscle contraction. The mutation leads to reduced levels of troponin-I transcript and protein. In addition to this, there is also coordinated reduction in transcript and protein levels of other structural protein isoforms that are part of the troponin complex. The altered transcript and protein stoichiometry ultimately culminates in unregulated acto-myosin interactions and a hypercontraction muscle phenotype. Our results shed new insights into the importance of maintaining the stoichiometry of structural proteins during muscle assembly for proper function with implications for the identification of mutations and disease phenotypes in other species, including humans.

Attenuation of rotenone-induced mitochondrial oxidative damage and neurotoxicty in Drosophila melanogaster supplemented with creatine.

Creatine (Cr), an ergogenic nutritional supplement is demonstrated to possess bioenergetic, antiexcitotoxic and antioxidant properties. This study investigated the neuroprotective effects of Cr against rotenone induced oxidative stress, mortality and neurotoxicty in Drosophila melanogaster. We found significant diminution in the endogenous levels of oxidative markers in whole body homogenates of flies exposed to Cr (2-10 mM). Cr supplementation resulted in reduced mortality in flies exposed to rotenone (500 microM) and better performance in a negative geotaxis assay. Further Cr (10 mM) markedly offset rotenone induced mitochondrial oxidative stress, completely restored the GSH levels, nitric oxide levels, activity of Mn-SOD and dopamine depletion. In an oxidative stress bioassay, flies given Cr prophylaxis exhibited marked resistance to paraquat exposure. These data allow us to hypothesize that the neuroprotective action of Cr in Drosophila may be related to its direct antioxidant activity and ability to abrogate rotenone induced mitochondrial oxidative stress.