Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

Uniformed Services University

Parkinson's disease has been linked to altered mitochondrial function. Mutations in parkin (park), the Drosophila ortholog of a human gene that is responsible for many familial cases of Parkinson's disease, shorten life span, abolish fertility and disrupt mitochondrial structure. However, the role played by Park in mitochondrial function remains unclear. Here, we describe a novel Drosophila gene, clueless (clu), which encodes a highly conserved tetratricopeptide repeat protein that is related closely to the CluA protein of Dictyostelium, Clu1 of Saccharomyces cerevisiae and to similar proteins in diverse metazoan eukaryotes from Arabidopsis to humans. Like its orthologs, loss of Drosophila clu causes mitochondria to cluster within cells. We find that strong clu mutations resemble park mutations in their effects on mitochondrial function and that the two genes interact genetically. Conversely, mitochondria in park homozygotes become highly clustered. We propose that Clu functions in a novel pathway that positions mitochondria within the cell based on their physiological state. Disruption of the Clu pathway may enhance oxidative damage, alter gene expression, cause mitochondria to cluster at microtubule plus ends, and lead eventually to mitochondrial failure.

Clueless, a conserved Drosophila gene required for mitochondrial subcellular localization, interacts genetically with parkin.

Mitochondria are critical for neuronal function due to the high demand of ATP in these cell types. During Drosophila development, neuroblasts in the larval brain divide asymmetrically to populate the adult central nervous system. While many of the proteins responsible for maintaining neuroblast cell fate and asymmetric cell divisions are known, little is know about the role of metabolism and mitochondria in neuroblast division and maintenance. The gene clueless (clu) has been previously shown to be important for mitochondrial function. clu mutant adults have severely shortened lifespans and are highly uncoordinated. Part of their lack of coordination is due to defects in muscle, however, in this study we have identified high levels of Clu expression in larval neuroblasts and other regions of the dividing larval brain. We show while mitochondria in clu mutant neuroblasts are mislocalized during the cell cycle, surprisingly, overall brain morphology appears to be normal. This is explained by our observation that clu mutant larvae have normal levels of ATP and do not suffer oxidative damage, in sharp contrast to clu mutant adults. Mutations in two other genes encoding mitochondrial proteins, technical knockout and stress sensitive B, do not cause neuroblast mitochondrial mislocalization, even though technical knockout mutant larvae suffer oxidative damage. These results suggest Clu functions upstream of electron transport and oxidative phosphorylation, has a role in suppressing oxidative damage in the cell, and that lack of Clu's specific function causes mitochondria to mislocalize. These results also support the previous observation that larval development relies on aerobic glycolysis, rather than oxidative phosphorylation. Thus Clu's role in mitochondrial function is not critical during larval development, but is important for pupae and adults.

Drosophila clueless is highly expressed in larval neuroblasts, affects mitochondrial localization and suppresses mitochondrial oxidative damage.

Antigens obtained from the intestinal tract of filarial nematodes have been proposed as potential safe and effective vaccine candidates. Because they may be 'hidden' from the immune response during natural infection, yet accessible by antibodies induced by vaccination, intestinal antigens may have a low potential for eliciting allergic responses when vaccinating previously infected individuals. Despite prior promising data, vaccination with intestinal antigens has yet to be tested in a permissive model of filariasis. In this study we investigated the efficacy of vaccination with filarial intestinal antigens in the permissive Litomosoides sigmodontis BALB/c model of filariasis, and we evaluated the extent to which these antigens are recognized by the immune system during and after infection. Infected BALB/c mice developed lower IgG antibody responses to soluble intestinal antigens (GutAg) than to soluble antigens of whole worms (LsAg). Similarly, GutAg induced less proliferation and less production of IL-4 and IFNγ from splenocytes of infected mice than LsAg. In contrast to these differences, active infection resulted in equivalent levels of circulating GutAg-specific IgE and LsAg-specific IgE levels. Consistent with this, basophil activation, as assessed by flow cytometric staining of intracellular basophil IL-4 expression, was equivalent in response to GutAg and LsAg. Vaccination with GutAg adsorbed to CpG/alum induced GutAg specific IgG1 and IgG2A production, with GutAg specific IgG titers greater than 5-fold higher than those measured in previously infected animals. Despite this response to GutAg vaccination, vaccinated mice harbored similar parasite burdens 8 weeks post infection when compared to non-vaccinated controls. These studies demonstrate that soluble antigens obtained from the intestinal tracts of L. sigmodontis have some qualities of 'hidden' antigens, but they still sensitize mice to allergic reactions and fail to protect against future infection when given as a vaccine adsorbed to alum/CPG.

Vaccination with intestinal tract antigens does not induce protective immunity in a permissive model of filariasis.

There is a close relationship between cardiovascular disease and cardiac energy metabolism, and we have previously demonstrated that palmitate inhibits myocyte contraction by increasing Kv channel activity and decreasing the action potential duration. Glucose and long chain fatty acids are the major fuel sources supporting cardiac function; however, cardiac myocytes can utilize a variety of substrates for energy generation, and previous studies demonstrate the acetate is rapidly taken up and oxidized by the heart. In this study, we tested the effects of acetate on contractile function of isolated mouse ventricular myocytes.

Acetate transiently inhibits myocardial contraction by increasing mitochondrial calcium uptake.

Loss of mitochondrial function often leads to neurodegeneration and is thought to be one of the underlying causes of neurodegenerative diseases such as Parkinson's disease (PD). However, the precise events linking mitochondrial dysfunction to neuronal death remain elusive. PTEN-induced putative kinase 1 (PINK1) and Parkin (Park), either of which, when mutated, are responsible for early-onset PD, mark individual mitochondria for destruction at the mitochondrial outer membrane. The specific molecular pathways that regulate signaling between the nucleus and mitochondria to sense mitochondrial dysfunction under normal physiological conditions are not well understood. Here, we show that Drosophila Clueless (Clu), a highly conserved protein required for normal mitochondrial function, can associate with Translocase of the outer membrane (TOM) 20, Porin and PINK1, and is thus located at the mitochondrial outer membrane. Previously, we found that clu genetically interacts with park in Drosophila female germ cells. Here, we show that clu also genetically interacts with PINK1, and our epistasis analysis places clu downstream of PINK1 and upstream of park. In addition, Clu forms a complex with PINK1 and Park, further supporting that Clu links mitochondrial function with the PINK1-Park pathway. Lack of Clu causes PINK1 and Park to interact with each other, and clu mutants have decreased mitochondrial protein levels, suggesting that Clu can act as a negative regulator of the PINK1-Park pathway. Taken together, these results suggest that Clu directly modulates mitochondrial function, and that Clu's function contributes to the PINK1-Park pathway of mitochondrial quality control.

Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila.

Histone deacetylase inhibitors (HDIs) are under investigation for the treatment of a number of human health problems. HDIs have proven therapeutic value in refractory cases of cutaneous T-cell lymphoma. Electrocardiographic ST segment morphological changes associated with HDIs were observed during development. Because ST segment morphology is typically linked to changes in ATP sensitive potassium (KATP) channel activity, we tested the hypothesis that HDIs affect cardiac KATP channel subunit expression. Two different HDIs, romidepsin and trichostatin A, caused ~20-fold increase in SUR2 (Abcc9) subunit mRNA expression in HL-1 cardiomyocytes. The effect was specific for the SUR2 subunit as neither compound causes a marked change in SUR1 (Abcc8) expression. Moreover, the effect was cell specific as neither HDI markedly altered KATP subunit expression in MIN6 pancreatic β-cells. We observe significant enrichment of the H3K9Ac histone mark specifically at the SUR2 promoter consistent with the conclusion that chromatin remodeling at this locus plays a role in increasing SUR2 gene expression. Unexpectedly, however, we also discovered that HDI-dependent depletion of cellular cholesterol is required for the observed effects on SUR2 expression. Taken together, the data in the present study demonstrate that KATP subunit expression can be epigenetically regulated in cardiomyocytes, defines a role for cholesterol homeostasis in mediating epigenetic regulation and suggests a potential molecular basis for the cardiac effects of the HDIs. 

Histone deacetylase inhibitors modulate KATP subunit transcription in HL-1 cardiomyocytes through effects on cholesterol homeostasis.

Mitochondrial function is tied to the nucleus, in that hundreds of proteins encoded by nuclear genes must be imported into mitochondria. While post-translational import is fairly well understood, emerging evidence supports that mitochondrial site-specific import, or co-translational import, also occurs. However, the mechanism and the extent to which it is used are not fully understood. We have previously shown Clueless (Clu), a conserved multi-domain protein, associates with mitochondrial outer membrane proteins, including Translocase of outer membrane 20, and genetically and physically interacts with the PINK1-Parkin pathway. The human ortholog of Clu, Cluh, was shown to bind nuclear-encoded mitochondrially destined mRNAs. Here we identify the conserved tetratricopeptide domain of Clu as predominantly responsible for binding mRNA. In addition, we show Clu interacts with the ribosome at the mitochondrial outer membrane. Taken together, these data support a model whereby Clu binds to and mitochondrially targets mRNAs to facilitate mRNA localization to the outer mitochondrial membrane, potentially for site-specific or co-translational import. This role may link the presence of efficient mitochondrial protein import to mitochondrial quality control through the PINK1-Parkin pathway. 

Clueless is a conserved ribonucleoprotein that binds the ribosome at the mitochondrial outer membrane.

Proteins encoded by mitochondrial DNA are translated using mitochondrially encoded tRNAs and rRNAs. As with nuclear encoded tRNAs, mitochondrial tRNAs must be processed to become fully functional. The mitochondrial form of ribonuclease P (mt:RNase P) is responsible for 5'-end maturation and is comprised of three proteins; mitochondrial RNase P protein (MRPP) 1 and 2 together with proteinaceous RNase P (PRORP). However, its mechanism and impact on development is not yet known. Using homology searches, we have identified the three proteins composing Drosophila mt:RNase P: Mulder (PRORP), Scully (MRPP2) and Roswell (MRPP1). Here, we show that each protein is essential and localizes with mitochondria. Furthermore, reducing levels of each causes mitochondrial deficits, which appear to be due at least in part to defective mitochondrial tRNA processing. Overexpressing two members of the complex, Mulder and Roswell, is also lethal, and in the case of Mulder, causes abnormal mitochondrial morphology. These data are the first evidence that defective mt:RNase P causes mitochondrial dysfunction, lethality and aberrant mitochondrial tRNA processing in vivo, underscoring its physiological importance. This in vivo mt:RNase P model will advance our understanding of how loss of mitochondrial tRNA processing causes tissue failure, an important aspect of human mitochondrial disease.

Loss of the mitochondrial protein-only ribonuclease P complex causes aberrant tRNA processing and lethality in Drosophila.

Mitochondrial diseases are a prevalent, heterogeneous class of diseases caused by defects in oxidative phosphorylation, whose severity depends upon particular genetic mutations. These diseases can be difficult to diagnose, and current therapeutics have limited efficacy, primarily treating only symptoms. Because mitochondria play a pivotal role in numerous cellular functions, especially ATP production, their diminished activity has dramatic physiological consequences. While this in and of itself makes treating mitochondrial disease complex, these organelles contain their own DNA, mtDNA, whose products are required for ATP production, in addition to the hundreds of nucleus-encoded proteins. Drosophila offers a tractable whole-animal model to understand the mechanisms underlying loss of mitochondrial function, the subsequent cellular and tissue damage that results, and how these organelles are inherited. Human and Drosophila mtDNAs encode the same set of products, and the homologous nucleus-encoded genes required for mitochondrial function are conserved. In addition, Drosophila contain sufficiently complex organ systems to effectively recapitulate many basic symptoms of mitochondrial diseases, yet are relatively easy and fast to genetically manipulate. There are several Drosophila models for specific mitochondrial diseases, which have been recently reviewed (Foriel, Willems, Smeitink, Schenck, & Beyrath, 2015). In this review, we highlight the conservation between human and Drosophila mtDNA, the present and future techniques for creating mtDNA mutations for further study, and how Drosophila has contributed to our current understanding of mitochondrial inheritance.

Fly Models of Human Diseases: Drosophila as a Model for Understanding Human Mitochondrial Mutations and Disease.

Drosophila Clueless (Clu) is a ribonucleoprotein that directly affects mitochondrial function. Loss of clu causes mitochondrial damage, and Clu associates with proteins on the mitochondrial outer membrane. Clu's subcellular pattern is diffuse throughout the cytoplasm, but Clu also forms large mitochondria-associated particles. Clu particles are reminiscent of ribonucleoprotein particles such as stress granules and processing bodies. Ribonucleoprotein particles play critical roles in the cell by regulating mRNAs spatially and temporally. Here, we show that Clu particles are unique, highly dynamic and rapidly disperse in response to stress in contrast to processing bodies and autophagosomes. In addition, Clu particle formation is dependent on diet as ovaries from starved females no longer contain Clu particles, and insulin signaling is necessary and sufficient for Clu particle formation. Oxidative stress also disperses particles. Since Clu particles are only present under optimal conditions, we have termed them "bliss particles". We also demonstrate that many aspects of Clu function are conserved in the yeast homolog Clu1p. These observations identify Clu particles as stress-sensitive cytoplasmic particles whose absence corresponds with altered cell stress and mitochondrial localization.

Rachel T. Cox

Department of Biochemistry and Molecular Biology

Uniformed Services University

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Rachel T. Cox

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Molecular Biology

Uniformed Services University

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Department of Biochemistry and Molecular Biology