Inhibition of mitochondrial calcium uptake 1 in Drosophila neurons.

The mitochondrial calcium uptake 1 (MICU1) is a regulatory subunit of the mitochondrial calcium uniporter that plays an important role in calcium sensing. It contains two EF-hand domains that are well conserved across diverse species from protozoa to plants and metazoans. The loss of MICU1 function in mammals is attributed to several neurological disorders that involve movement dysfunction. The CG4495 gene in Drosophila melanogaster was identified as a putative homolog of MICU1 in the HomoloGene database of the National Centre for Biotechnology Information (NCBI). In agreement with previous studies that have shown the development of neurological disorders and movement defects in MICU1 loss-of-function organisms, we attempted to identify the function of CG4495/MICU1 in Drosophila neurons. We analyzed survival and locomotor ability of these flies and additionally performed biometric analysis of the Drosophila developing eye. The inducible RNA interference-mediated inhibition of CG4495/MICU1 in the Ddc-Gal4-expressing neurons of Drosophila presented with reduction in survival coupled with a precocious loss of locomotor ability. Since the pro-survival Bcl-2 family genes have been shown to be protective towards mitochondria, and CG4495/MICU1 has a mitochondrial targeting sequence, we attempted to rescue the phenotypes resulting from the inhibition of CG4495/MICU1 by overexpressing Buffy, the sole Bcl-2 homologue in Drosophila. The co-expression of CG4495/MICU1-RNAi along with Buffy resulted in the suppression of the phenotypes induced by the inhibition of CG4495/MICU1. Subsequently, the inhibition of CG4495/MICU1 in the Drosophila developing eye, a neuron-rich organ, resulted in reduced number of ommatidia and a highly fused ommatidial array. These developmental eye defects were rescued by the overexpression of Buffy. Our study suggests an important role for MICU1 in the normal function of neurons in Drosophila.

Manipulation of components that control feeding behavior in Drosophila melanogaster increases sensitivity to amino acid starvation.

Feeding is a complex behavior that is regulated by several internal mechanisms. Neuropeptides are able to survey quantities of stored energy and inform the organism if nutrient intake is required. In addition to this homeostatic regulation, a post-feeding reward system positively reinforces feeding. Slight adjustments to either system can tilt the balance to affect the energy reserves and survivorship in times of nutrient adversity. Neuropeptide F (NPF), a homolog of the mammalian neuropeptide Y, acts to induce feeding within the homeostatic regulation of this behavior. Drosophila and other insects bear a shorter form of NPF known as short NPF (sNPF) that can influence feeding. A neural hormone regulator, the dopamine transporter (DAT), works to clear dopamine from the synapses. This action may manipulate the post-feeding reward circuit in that lowered dopamine levels depress feeding, and excess dopamine levels encourage feeding. Here, we have overexpressed and impaired the activities of NPF, sNPF, and DAT in Drosophila, and we examined their ability to survive during conditions of amino acid starvation. Too much or too little NPF or sNPF, which are key players in homeostatic feeding regulation, leads to increased sensitivity to amino acid starvation and diminished survivorship when compared to controls. When DAT, a member of the post-feeding reward system, is either overexpressed or reduced via mutation, Drosophila has increased sensitivity to amino acid starvation. Taken together, these results indicate that subtle variation in the expression of key components of these systems impacts survivorship during adverse nutrient conditions.

Altered expression of CG5961, a putative Drosophila melanogaster homologue of FBXO9, provides a new model of Parkinson disease.

F-box proteins act as the protein recognition component of the Skp-Cul-F-box class of ubiquitin ligases. Two members of a gene sub-family encoding these proteins, FBXO7 and FBXO32, have been implicated in the onset and progression of degenerative disease. FBXO7 is responsible for rare genetic forms of Parkinson disease, while FBXO32 has been implicated in muscle wasting. The third gene in this family, FBXO9, is related to growth signaling, but the role of this gene in degenerative disease pathways has not been thoroughly investigated. Characterizing the putative Drosophila melanogaster homologue of this gene, CG5961, enables modeling and analysis of the consequence of targeted alteration of gene function and the effects on the overall health of the organism. Comparison of the protein domains of Homo sapiens FBXO9 and the putative D. melanogaster homologue CG5961 revealed a high degree of conservation between the protein domains. Directed expression of CG5961 (via CG5961(EP)) and inhibition of CG5961 (through a stable RNAi transgene) in the developing D. melanogaster eye caused abnormalities in adult structures (ommatidia and inter-ommatidial bristles). Directed expression of either CG5961 or CG5961-RNAi in the dopaminergic neurons led to a reduced lifespan compared to that in lacZ controls. We showed that protein structures of CG5961 and FBXO9 are highly similar and studied the effects of altered expression of CG5961 in neuron-rich tissues. Our results suggest that CG5961 activity is necessary for the proper formation of neuronal tissue and that targeted alteration of gene expression in dopaminergic neurons leads to a reduced lifespan.

Identifying potential PARIS homologs in D. melanogaster.

Mitochondrial destruction leads to the formation of reactive oxygen species, increases cellular stress, causes apoptotic cell death, and involves a cascade of proteins including PARKIN, PINK1, and Mitofusin2. Mitochondrial biogenesis pathways depend upon the activity of the protein PGC-1α. These two processes are coordinated by the activity of a transcriptional repressor, Parkin interacting substrate (PARIS). The PARIS protein is degraded through the activity of the PARKIN protein, which in turn eliminates the transcriptional repression that PARIS imposes upon a downstream target, PGC-1α. Genes in this pathway have been implicated in Parkinson's disease, and there is a strong relationship between mitochondrial dysfunction and pre-mature neuron death. The identification of a PARIS homolog in Drosophila melanogaster would increase our understanding of the roles that PARIS and interacting genes play in higher organisms. We identified three potential PARIS homologs in D. melanogaster, one of which encodes a protein with similar domains to the Homo sapiens PARIS protein, CG15436. The Drosophila eye is formed from neuronal precursors, making it an ideal system to assay the effects of altered gene expression on neuronal tissue formation. The eye-specific expression of RNAi constructs for these genes revealed that both CG15269 and Crol caused neurodegenerative phenotypes, whereas CG15436 produced a phenotype similar to srl-EY. Crol-RNAi expression reduced mean lifespan when expressed in dopaminergic neurons, whereas CG15436-RNAi significantly increased lifespan. CG15436 was PARIS-like in both structure and function, and we characterized the effects of decreased gene expression in both the neuron-rich D. melanogaster eye and in dopaminergic neurons.

Knockdown of the putative Lifeguard homologue CG3814 in neurons of Drosophila melanogaster.

Lifeguard is an integral transmembrane protein that modulates FasL-mediated apoptosis by interfering with the activation of caspase 8. It is evolutionarily conserved, with homologues present in plants, nematodes, zebra fish, frog, chicken, mouse, monkey, and human. The Lifeguard homologue in Drosophila, CG3814, contains the Bax inhibitor-1 family motif of unknown function. Downregulation of Lifeguard disrupts cellular homeostasis and disease by sensitizing neurons to FasL-mediated apoptosis. We used bioinformatic analyses to identify CG3814, a putative homologue of Lifeguard, and knocked down CG3814/LFG expression under the control of the Dopa decarboxylase (Ddc-Gal4) transgene in Drosophila melanogaster neurons to investigate whether it possesses neuroprotective activity. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons resulted in a shortened lifespan and impaired locomotor ability, phenotypes that are strongly associated with the degeneration and loss of dopaminergic neurons. Lifeguard interacts with anti-apoptotic Bcl-2 proteins and possibly pro-apoptotic proteins to exert its neuroprotective function. The co-expression of Buffy, the sole anti-apoptotic Bcl-2 gene family member in Drosophila, and CG3814/LFG by stable inducible RNA interference, suppresses the shortened lifespan and the premature age-dependent loss in climbing ability. Suppression of CG3814/LFG in the Drosophila eye reduces the number of ommatidia and increases disruption of the ommatidial array. Overexpression of Buffy, along with the knockdown of CG3814/LFG, counteracts the eye phenotypes. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons in Drosophila diminishes its neuroprotective ability and results in a shortened lifespan and loss of climbing ability, phenotypes that are improved upon overexpression of the pro-survival Buffy.

Inhibition of Atg6 and Pi3K59F autophagy genes in neurons decreases lifespan and locomotor ability in Drosophila melanogaster.

Autophagy is a cellular mechanism implicated in the pathology of Parkinson's disease. The proteins Atg6 (Beclin 1) and Pi3K59F are involved in autophagosome formation, a key step in the initiation of autophagy. We first used the GMR-Gal4 driver to determine the effect of reducing the expression of the genes encoding these proteins on the developing Drosophila melanogaster eye. Subsequently, we inhibited their expression in D. melanogaster neurons under the direction of a Dopa decarboxylase (Ddc) transgene, and examined the effects on longevity and motor function. Decreased longevity coupled with an age-dependent loss of climbing ability was observed. In addition, we investigated the roles of these genes in the well-studied α-synuclein-induced Drosophila model of Parkinson's disease. In this context, lowered expression of Atg6 or Pi3K59F in Ddc-Gal4-expressing neurons results in decreased longevity and associated age-dependent loss of locomotor ability. Inhibition of Atg6 or Pi3K59F together with overexpression of the sole pro-survival Bcl-2 Drosophila homolog Buffy in Ddc-Gal4-expressing neurons resulted in further decrease in the survival and climbing ability of Atg6-RNAi flies, whereas these measures were ameliorated in Pi3K59F-RNAi flies.

Arm-Gal4 inheritance influences development and lifespan in Drosophila melanogaster.

The UAS-Gal4 ectopic expression system is a widely used and highly valued tool that allows specific gene expression in Drosophila melanogaster. Yeast transcription factor Gal4 can be directed using D. melanogaster transcriptional control elements, and is often assumed to have little effect on the organism. By evaluation of the consequences of maternal and paternal inheritance of a Gal4 transgene under the transcriptional regulation of armadillo control elements (arm-Gal4), we demonstrated that Gal4 expression could be detrimental to development and longevity. Male progeny expressing arm-Gal4 in the presence of UAS-lacZ transgene had reduced numbers and size of ommatidia, compared to flies expressing UAS-lacZ transgene under the control of other Gal4 transgenes. Aged at 25°C, the median life span of male flies with maternally inherited elav-Gal4 was 70 days, without a responding transgene or with UAS-lacZ. The median life span of maternally inherited arm-Gal4 male flies without a responding transgene was 48 days, and 40 days with the UAS-lacZ transgene. A partial rescue of this phenotype was observed with the expression of UAS-lacZ under paternal arm-Gal4 control, having an average median lifespan of 60 days. This data suggests that arm-Gal4 has detrimental effects on Drosophila development and lifespan that are directly dependent upon parental inheritance, and that the benign responder and reporter gene UAS-lacZ may influence D. melanogaster development. These findings should be taken into consideration during the design and execution of UAS-Gal4 expression experiments.

Expression of Pink1 with α-synuclein in the dopaminergic neurons of Drosophila leads to increases in both lifespan and healthspan.

Overexpression of the gene coding for α-synuclein has been shown to be an inherited cause of Parkinson disease. Our laboratory has previously co-expressed the parkin and Pink1 genes to rescue α-synuclein-induced phenotypes within a Drosophila model. To further investigate the effect of Pink1 in this model, we performed longevity and behavioral studies using several drivers to express the α-synuclein and Pink1 genes. Our findings showed that overexpression of Pink1 and overexpression of Pink1 with α-synuclein resulted in an increased lifespan when driven with the TH-Gal4 transgene. This increase in longevity was accompanied by an increased healthspan, as measured by mobility over time, suggesting that this is an example of improved functional aging. Our results indicate that, in the dopaminergic cells targeted by TH-Gal4, increased expression of α-synuclein and Pink1 together have a synergistic effect, allowing for enhanced protection and increased survival of the organism.

Expression of GFP can influence aging and climbing ability in Drosophila.

Green fluorescent protein (GFP) is widely used as a reporter transgene in a variety of organisms. Some of the advantages of using GFP include non-invasive visualization of biological events and/or tissues in live specimens and its benign nature. When GFP is expressed throughout the organism, in neurons and eyes, lifespan and climbing ability of flies are significantly decreased compared to similar crosses with a lacZ reporter. Also, GFP expression can have subtle effects on eye morphology, with neural and ubiquitous expression. Since GAL4/UAS expression of GFP can influence aging and climbing ability in the Drosophila system of directed gene expression, we found that the latter of these advantages, namely its harmless, non-toxic nature, can be conditional, depending upon the mode of expression and the biological endpoint. We suggest that caution should be used when using GFP to visualize cellular events, especially in long-term assays.

Genetic analysis of protein kinase B (AKT) in Drosophila.

The decision between survival and death is an important aspect of cellular regulation during development and malignancy. Central to this regulation is the process of apoptosis, which is conserved in multicellular organisms [1]. A variety of signalling cascades have been implicated in modulation of apoptosis, including the phosphatidylinositol (Pl) 3-kinase pathway. Activation of Pl 3-kinase is protective, and inhibition of this lipid kinase enhances cell death under several conditions including deregulated expression of c-Myc, neurotrophin withdrawal and anoikis [2-7]. Recently, the protective effects of Pl 3-kinase have been linked to its activation of the pleckstrin homology (PH)-domain-containing protein kinase B (PKB or AKT) [8]. PKB/AKT was identified from an oncogene, v-akt, found in a rodent T-cell lymphoma [9]. To initiate a genetic analysis of PKB, we have isolated and characterized a Drosophila PKB/AKT mutant (termed Dakt1) that exhibits ectopic apoptosis during embryogenesis as judged by induction of membrane blebbing, DNA fragmentation and macrophage infiltration. Apoptosis caused by loss of Dakt function is rescued by caspase suppression but is distinct from the previously described reaper/grim/hid functions. These data implicate Dakt1 as a cell survival gene in Drosophila, consistent with cell protection studies in mammals.

Protected P-element termini suggest a role for inverted-repeat-binding protein in transposase-induced gap repair in Drosophila melanogaster.

P-element transposition is thought to occur by a cut-and paste mechanism that generates a double-strand break at the donor site, the repair of which can lead to internally deleted elements. We have generated a series of both phenotypically stronger and weaker allelic derivatives of vg21, a vestigial mutant caused by a P-element insertion in the 5' region of the gene. Virtually all of the new alleles arose by internal deletion of the parental element in vg21, and we have characterized a number of these internally deleted P elements. Depending upon the selection scheme used, we see a very different spectrum of amount and source of P-element sequences in the resultant derivatives. Strikingly, most of the breakpoints occur within the inverted-repeats such that the last 15-17 bp of the termini are retained. This sequence is known to bind the inverted-repeat-binding protein (IRBP). We propose that the IRBP may act to preserve the P-element ends when transposition produces a double-strand gap. This allows the terminus to serve as a template upon which DNA synthesis can act to repair the gap. Filler sequences found at the breakpoints of the internally deleted P elements resemble short stretches, often in tandem arrays, of these terminal sequences. The structure of the filler sequences suggests replication slippage may occur during the process of gap repair.

Genetic organization of the cSOD microregion of Drosophila melanogaster.

Cu/Zn superoxide dismutase (cSOD) is an important enzymatic agent of physiological defense against active oxygen species. Previously, we defined the essential biological role of cSOD in Drosophila through analysis of a cSOD null mutant. In the process of isolating this mutant, we also identified several vital genes in the chromosomal region surrounding the cSOD gene (the cSOD microregion). To further our genetic analysis of cSOD function, we have undertaken a detailed description of the cSOD microregion as defined by the breakpoints of the deficiency Df(3L)lxd9. Examination and correlation of mutations previously recovered with new X-ray induced mutations described in this paper identify a total of 12 vital genes, including cSOD, and one nonvital gene, lxd, in this region. We propose the adoption of a single genetic nomenclature for the genes of this region. Two newly generated hypomorphic alleles of cSOD are described that confer phenotypes similar to cSODn108, confirming the important physiological role of the enzyme in Drosophila.

Phenotypic consequences of copper-zinc superoxide dismutase overexpression in Drosophila melanogaster.

We report here the isolation of a tandem duplication of a small region of the third chromosome of Drosophila melanogaster containing the Cu-Zn superoxide dismutase (cSOD) gene. This duplication is associated with a dosage-dependent increase in cSOD activity. The biological consequences of hypermorphic levels of cSOD in genotypes carrying this duplication have been investigated under diverse conditions of oxygen stress imposed by acute exposure to ionizing radiation, chronic exposure to paraquat, and the normoxia of standard laboratory culture. We find that a 50% increase in cSOD activity above the normal diploid level confers increased resistance to ionizing radiation and, in contrast, confers decreased resistance to the superoxide-generating agent paraquat. The duplication is associated with a minor increase in adult life-span under conditions of normoxia. These results reveal important features of the biological function of cSOD within the context of the overall oxygen defense system of Drosophila.

Targeting of an enhancer trap to vestigial.

The vestigial gene (vg+) is required for normal wing development and is expressed in a spatially distinct pattern in imaginal discs. We have exploited a general property of P element alleles to target an enhancer trap to the 5' region of the gene. By replacing the P element resident at this site in vg21 with a P element carrying a lacZ reporter gene, the vglacZ1 allele was selected on the basis of its increased mutant phenotype. In contrast to vg+ expression, which occurs primarily in the presumptive wing margin and hinge, beta-galactosidase expression in vglacZ1 wing discs is localized to the dorsal wing surface and displays homologous haltere expression. The targeting of P element enhancer traps could be readily extended to other genes with low rates of primary P element insertion.

Genetic evidence that heparin-like glycosaminoglycans are involved in wingless signaling.

We have identified the Drosophila UDP-glucose dehydrogenase gene as being involved in wingless signaling. Mutations in this gene, called kiwi, generate a phenotype identical to that of wingless. UDP-glucose dehydrogenase is required for the biosynthesis of UDP-glucuronate, which in turn is utilized in the biosynthesis of glycosaminoglycans. By rescuing the kiwi phenotype with both UDP-glucuronate and the glycosaminoglycan heparan sulfate, we show that kiwi function in the embryo is crucial for the production of heparan sulfate in the extracellular matrix. Further, injection of heparin degrading enzyme, heparinase (and not chondroitin, dermatan or hyaluronic acid degrading enzyme) into wild-type embryos leads to the degradation of heparin-like glycosaminoglycans and a 'wingless-like' cuticular phenotype. Our study thus provides the first genetic evidence for the involvement of heparin-like glycosaminoglycans in signal transduction.