The Interactive Fly

Genes involved in tissue and organ development

Mitochondria and mitochondrial function

Mitochondria, apoptosis and autophagy
Mitochondrial fusion
Mitochondria and neural function
Mitochondria, aging, lifespan and diseases
Mitochondria and metabolism
Mitochondria DNA, RNA and protein biology
Mitochondria and evolution
Mitochondria inheritance
Mitochondria and development

Proteins functioning in or associated with mitochondria

The two Drosophila cytochrome C proteins can function in both respiration and caspase activation

Cytochrome C has two apparently separable cellular functions: respiration and caspase activation during apoptosis. While a role of the mitochondria and cytochrome C in the assembly of the apoptosome and caspase activation has been established for mammalian cells, the existence of a comparable function for cytochrome C in invertebrates remains controversial. Drosophila possesses two cytochrome c genes, cyt-c-d and cyt-c-p. Only cyt-c-d is required for caspase activation in an apoptosis-like process during spermatid differentiation, whereas cyt-c-p is required for respiration in the soma. However, both cytochrome C proteins can function interchangeably in respiration and caspase activation, and the difference in their genetic requirements can be attributed to differential expression in the soma and testes. Furthermore, orthologues of the apoptosome components, Ark (Apaf-1) and Dronc (caspase-9), are also required for the proper removal of bulk cytoplasm during spermatogenesis. Finally, several mutants that block caspase activation during spermatogenesis were isolated in a genetic screen, including mutants with defects in spermatid mitochondrial organization. These observations establish a role for the mitochondria in caspase activation during spermatogenesis (Arama, 2006).

Apoptosis is a morphologically distinct form of active cellular suicide that serves to eliminate unwanted and potentially dangerous cells. The key enzymes responsible for the execution of apoptosis are an evolutionarily conserved family of cysteine proteases known as caspases. Caspases are present in an inactive or weakly active state in virtually all cells of higher metazoans, and their activity is carefully regulated by both activators and inhibitors. In vertebrates, the mitochondria play an important role in the control of apoptosis: they release cytochrome C and other pro-apoptotic proteins in response to various death signals. In the cytosol, cytochrome C binds to Apaf-1 (Zou, 1997) which in turn promotes the assembly of a multiprotein complex, termed the 'apoptosome', and caspase-9 activation (Rodriguez, 1999; Adams, 2002; Cain, 2002; Salvesen, 2002). In the ensuing 'caspase cascade', many intracellular substrates are cleaved and apoptosis is executed. However, the exact physiological role of cytochrome C for caspase activation remains to be determined, and a recent report on a mutant cytochrome c that fails to activate Apaf-1 in the mouse (Hao, 2005) suggests that cytochrome C is required for caspase activation in only some mammalian cell types (Arama, 2006).

In invertebrates, any role of cytochrome C for the activation of caspases has remained highly controversial. Whereas RNAi experiments in Drosophila S2 cells have failed to reveal a role for cytochrome C in apoptosis, other reports suggest that cytochrome C may promote caspase activation (Dorstyn, 2002, 2004; Zimmermann, 2002). Drosophila contains two Apaf-1 isoforms: one with a WD40 repeat domain, the target for cytochrome C binding, and another lacking this domain, similar to Caenorhabditis elegans Ced-4. The large isoform can directly bind cytochrome C in vitro and promote cytochrome C-dependent caspase activation in lysates from developing embryos (Kanuka, 1999). Furthermore, an overt alteration in the cytochrome C immuno-staining can be detected in doomed cells in some Drosophila tissues, and the mitochondria from apoptotic cells can activate cytosolic caspases (Varkey, 1999). Finally, disruption of one of the two Drosophila cytochrome c genes, cyt-c-d, is associated with a failure to activate caspases in an apoptosis-like process during sperm terminal differentiation in Drosophila (Arama, 2003). In this process, also known as spermatid individualization, the majority of cytoplasm and cellular organelles are eliminated from the developing spermatids in an apoptosis-like process that requires caspase activity (Arama, 2003). However, it was suggested that the mutants used in s previous study (Huh, 2004) may also affect other genes located in the vicinity of the cyt-c-d locus (Arama, 2006).

In order to rigorously address this issue, a series of genetic and transgenic rescue experiments were conducted that unequivocally establish a role of cytochrome C for caspase activation during Drosophila spermatogenesis. First, a point mutation was isolated in cyt-c-d that is defective in caspase activation. Next, it was demonstrated that transgenic expression of cyt-c-d restores effector caspase activation and rescues all the sterility phenotypes associated with various cyt-c-d mutant alleles. The possibility that cyt-c-p functions specifically in respiration was investigated, whereas cyt-c-d plays a role in caspase regulation. Surprisingly, it was found that expression of either cyt-c-d or cyt-c-p can restore caspase activation in cyt-c-d-deficient spermatids, demonstrating that both proteins are functionally equivalent. Other apoptosome proteins in Drosophila, Ark (Apaf-1) and Dronc (caspase-9) are also required for spermatid individualization, and their mutant phenotypes are similar to spermatids with a block in caspase activity. Surprisingly, however, some active caspase-3 staining can still be detected in these mutant testes, suggesting that cytochrome-C-d may function in yet other unknown pathways to promote caspase-3 activation. Finally, several mutants affecting spermatid mitochondria were identifed that provide a strong link between mitochondrial organization and caspase activation during sperm development (Arama, 2006).

In mammals, mitochondria are important for the regulation of apoptosis, and it has been shown that they can release several proapoptotic proteins into the cytosol in response to apoptotic stimuli. The best-studied case is the release of cytochrome C, an essential component of the respiratory chain. Cytosolic cytochrome C can bind to and activate Apaf-1, which in turn leads to the activation of caspase-9. However, no comparable role of mitochondrial factors for caspase activation has yet been established in invertebrates. The elimination of cytoplasm during terminal differentiation of spermatids in Drosophila involves an apoptosis-like process that requires caspase activity; a P-element insertion (bln1) in one of the two Drosophila cytochrome c genes, cyt-c-d, has been shown to be associated with male-sterility and loss of effector caspase activation during spermatid individualization. This study demonstrates that the defects in caspase activation and spermatid individualization of bln1 mutant males can be rescued by transgenic expression of the ORF of cyt-c-d. Furthermore, from screening more than a thousand male-sterile lines with defects in sperm individualization for defects in active-caspase (CM1) staining, a nonsense point mutation was identified in cyt-c-d, that recapitulates all the phenotypes observed for bln1. Taken together, these results unequivocally demonstrate that cyt-c-d is necessary for effector caspase activation and sperm terminal differentiation in Drosophila (Arama, 2006).

Two decades ago, the mouse cytochrome c gene was used as a probe for screening a Drosophila genomic library and a fragment was isolated that carried two distinct cytochrome c genes. Northern blot analyses indicated high levels of cyt-c-p expression, while cyt-c-d was reported to be expressed at much lower levels in all stages of development. However, neither the exon/intron organization nor the boundaries of the 5' and 3' UTRs of these genes were determined at the time. As a result, the original Northern analyses were performed with a probe corresponding to the untranscribed genomic region between the two cytochrome c genes that was not suitable to properly assess the size and distribution of cytochrome c transcripts. Unfortunately, this has caused considerable confusion in the field from the start, as even the original report noted that the size of the observed cyt-c-d transcript differed more than two-fold from the predicted size. More recently, relying on the incorrect assumption that cyt-c-d is ubiquitously expressed in the fly, it has been suggested that a loss-of-function mutation in cyt-c-d should lead to severe developmental defects and lethality rather than merely male sterility. However, using a specific cyt-c-d 3' UTR probe reveals a transcript of the predicted size that is absent in cyt-c-dbln1 mutants. Furthermore, the RT-PCR and immunofluorescence analyses presented in this study indicate that cyt-c-d is mainly expressed in the male germ line and is completely absent during embryonic and larval development, while cyt-c-p is expressed in the soma during all stages of development. In light of these findings, it is not surprising that loss-of-function mutations in cyt-c-d cause male sterility, whereas cyt-c-p mutations lead to embryonic lethality. RT-PCR results suggest that cyt-c-p is also expressed in the testis, although to a much lower extent than cyt-c-d. This expression is attributed primarily to the somatic cells of the testis, since no cytochrome C protein is detected in cyt-c-dbln1 elongating spermatids, while cyt-c-p RNA is expressed in cyt-c-dbln1 mutant flies. However, the very low cyt-c-d expression detected in the soma of adult females leaves room for the possibility that cyt-c-d might function in caspase activation in some somatic cells as well (Arama, 2006).

In mammalian cells, release of cytochrome C into the cytosol in response to proapoptotic stimuli can be readily demonstrated. However, previous attempts to detect a similar phenomenon in Drosophila have been unsuccessful. In contrast, apoptotic stimuli can lead to increased cytochrome C immuno-reactivity. A possible limitation is that all these studies were conducted using mammalian antibodies with questionable specificity and sensitivity, and only in a small number of cell types and paradigms. Using an antibody that was raised against Drosophila cytochrome C-d, an increase in a 'grainy signal' was detected upon the onset of individualization, with the highest staining observed in the vicinity of the individualization comple (IC). Since it is highly unlikely that additional cytochrome C-d is being transcribed and imported to the mitochondria at this late stage, the explanation is favored that a conformational change or an exposure of a hidden epitope causes the increase in the intensity of the signal. The activation of Dronc, the Drosophila caspase-9 orthologue, also occurs in association with the IC and depends on the presence of the Drosophila Apaf-1 orthologue, Ark. Moreover, the proapoptotic Hid protein is localized in a similar fashion. What are these structures then, which accumulate apoptotic factors in the vicinity of the IC? One plausible suggestion from the literature is that these structures correspond to 'mitochondrial whorls', which result from the extrusion of material from the minor mitochondrial derivative and constitute the leading component of the IC. These 'whorls' can be labeled using a testes-specific mitochondrial-expressed GFP line. Using this GFP marker, it was found that cytochrome C-d is indeed closely associated with mitochondrial whorls. Therefore, it is possible that an active apoptosome forms in the vicinity of the IC in response to dramatic changes in the mitochondrial architecture that occur at this stage of spermatid differentiation. Similarly, studying the response of Drosophila flight muscle cells to oxygen stress, have recently reported that the cristae within individual mitochondria become locally rearranged in a pattern that they termed a 'swirl'. This process was associated with widespread apoptotic cell death in the flight muscle, which was correlated with a conformational change of cytochrome C manifested by the display of an otherwise hidden epitope. Collectively, these observations suggest that apoptosome-like complexes composed of cytochrome C-d, Ark, and Dronc might be associated with unique mitochondrial swirl-like structures. Consistent with this idea, it was found that the long isoform of Ark that contains the WD40 repeats, the target for cytochrome C binding to mammalian Apaf-1, is the major form detectably expressed in testes (Arama, 2006).

The fact that cytochrome C-d immunoreactivity increases in the vicinity of the IC suggests that the extensive mitochondrial organizations preceding individualization may be partially required for caspase activation. Consistent with this idea, several mutants, such as plnZ2-0516, which display defects in Nebenkern differentiation and caspase activation. However, not all mitochondrial differentiation events are required for caspase activation. For example, CM1 staining is seen in fuzzy onions, a mutant defective in the mitochondrial fusion event that generates the Nebenkern. In contrast, analysis of the pln mutant indicates that proper elongation of the Nebenkern is essential for caspase activation. Therefore, characterization of other mitochondrial mutants may shed light on the connection between mitochondrial organization and caspase activation during sperm differentiation (Arama, 2006).

What are the mechanisms by which cytochrome C-d activates caspases during late spermatogenesis? In vertebrate cells, following its release into the cytosol, cytochrome C binds to the WD40 domain of the adaptor molecule Apaf-1, which in turn multimerizes and recruits the initiator caspase, caspase-9 via interaction of their CARD domains. This complex, known as the apoptosome, further cleaves and activates effector caspases like caspase-3. Although this model has become the prevailing dogma in the field, the phenotype of mice mutant for a Cyt c with drastically reduced apoptogenic function ('KA allele') suggests that the mechanisms for caspase activation may be more complex than previously thought. In particular, this study suggests that cytochrome C-independent mechanisms for the activation of Apaf-1 and caspase-9 exist, as well as cytochrome C-dependent but Apaf-1-independent mechanisms for apoptosis. These analyses of ark (Apaf-1) and dronc (caspase-9) loss-of-function mutants demonstrate that both genes are required for spermatid individualization, and that their phenotypes, in particular their failure to properly remove the spermatid cytoplasm into the WB, resemble cyt-c-d mutant spermatids and expression of the caspase inhibitor p35 in the testes. However, some caspase-3-like activity could still be detected in these mutant testes. This may suggest that either the ark and dronc alleles are not null, or that cytochrome C-d also functions in an apoptosome-independent pathway to promote caspase-3 activation. Therefore, the regulation of caspase activation and apoptosis may be more similar between insects and mammals than has been previously appreciated. Further genetic analysis of this pathway in Drosophila may provide general insights into diverse mechanisms of apoptosis activation (Arama, 2006).

Previous observations raised the possibility that the two distinct cytochrome c genes may have evolved to serve distinct functions in respiration and caspase regulation. In order to address this hypothesis, it was asked whether expression of one protein might rescue mutations in the other cytochrome c gene. Surprisingly, it was found that transgenic expression of the cyt-c-p ORF in germ cells rescues caspase activation, spermatid individualization, and sterility of cyt-c-d-/- flies. Therefore, the ability to activate caspases is not restricted to the cytochrome C-d protein, and it is possible that cytochrome C-p functions in apoptosis in at least some somatic cells (Arama, 2006).

Although cyt-c-d is almost exclusively expressed in the male germ cells, ectopic expression of this protein in the soma can rescue the respiration defect and lethality of cyt-c-p-/- mutant flies, demonstrating that cytochrome C-d can function in energy metabolism. This raises the question whether the lack of caspase activation could be due to reduced ATP-levels. Although this is a formal possibility, this explanation is considered very unlikely since mutant spermatids complete many other energy-intensive cellular processes. These include the extensive transformation from round spermatids to 1.8 mm long elongated spermatids, a process that involves extensive remodeling and movement of actin filaments, generation of the axonemal tail, mitochondrial reorganization, plasma/axonemal membranes reorganization, and nuclear condensation and elongation. Since all of these processes can occur in the absence of cytochrome C-d, there is no overt shortage of ATP in cyt-c-d mutants. It is therefore considered very unlikely that ATP has become limiting in these mutant cells. Since earlier stage spermatids express cytochrome C-p, sufficient ATP seems to persist to late developmental stages. In mammalian cells, cellular ATP concentration is sufficiently high (around 2 mM) to keep cultured cell alive for several days upon ATP synthase inhibition. Furthermore, cells in which cytochrome c expression is decreased by RNAi still undergo apoptosis in response to various stimuli. Likewise, it appears that cytochrome C is not essential for the function of mature murine sperm, since mice deficient for the testis specific form of cytochrome C, Cyt cT, are fertile. Taken together, all these observations argue strongly against the possibility that ATP levels in cyt-c-d-/- mutant spermatids would be insufficient for caspase activation (Arama, 2006).

In conclusion, the results presented in this study definitively demonstrate that cytochrome C-d is essential for caspase activation and spermatid individualization. Both cytochrome C proteins of Drosophila are, at least to some extent, functionally interchangeable. The results also indicate that cytochrome C can promote caspase activation in the absence of a functional apoptosome. Given the powerful genetic techniques available, late spermatogenesis of Drosophila promises to be a powerful system to identify novel pathways for mitochondrial regulation of caspase activation (Arama, 2006).

Role of mitochondrial remodeling in programmed cell death in Drosophila melanogaster

The role of mitochondria in Drosophila programmed cell death remains unclear, although certain gene products that regulate cell death seem to be evolutionarily conserved. This study found that developmental programmed cell death stimuli in vivo and multiple apoptotic stimuli ex vivo induce dramatic mitochondrial fragmentation upstream of effector caspase activation, phosphatidylserine exposure, and nuclear condensation in Drosophila cells. Unlike genotoxic stress, a lipid cell death mediator induces an increase in mitochondrial contiguity prior to fragmentation of the mitochondria. Dynamin related protein 1 (Drp1), is important for mitochondrial disruption. Using genetic mutants and RNAi-mediated knockdown of drp-1, it was found that Drp1 not only regulates mitochondrial fission in normal cells, but mediates mitochondrial fragmentation during programmed cell death. Mitochondria in drp-1 mutants fail to fragment, resulting in hyperplasia of tissues in vivo and protection of cells from multiple apoptotic stimuli ex vivo. Thus, mitochondrial remodeling is capable of modifying the propensity of cells to undergo death in Drosophila (Goyal, 2007).

Programmed cell death (PCD) plays an important role in sculpting tissues during animal development. The molecular regulators that are central to this process seem to be evolutionarily conserved from worms to mammals and include autocatalytic initiator caspases, trans-activable effector caspases, cytosolic activating factors (APAF-1), and multidomain Bcl-2 proteins. The proapoptotic Bcl-2-family proteins oligomerize and permeabilize mitochondria, releasing intermembrane space components such as cytochrome-C and Smac/DIABLO into the cytosol, where they activate initiator caspases by an ATP-dependent mechanism. Initiator caspases trans-activate effector caspases that cleave multiple cellular substrates, resulting in DNA degradation, nuclear condensation, and loss of cell integrity (Goyal, 2007 and references therein).

Mitochondrial outer-membrane permeabilization has been proposed to depend on the mitochondrial fission and fusion machinery. Consistent with this, mitochondria undergo dramatic fragmentation very close in time to cytochrome-C release during mammalian cell death. Furthermore, an increase in mitochondrial fragmentation and a block in mitochondrial fusion are essential for cell death progression. In normal cells, the balance in the rates of mitochondrial fission and fusion regulated by Dynamin-related protein-1 (Drp-1), Fis-1 and endophilin (fission), or Mitofusins and Opa-1 (fusion) maintains the dynamic, interconnected mitochondrial tubules. An increase in recruitment of Drp-1 to the mitochondria accentuates staurosporine, lipid, and free oxygen radical stress-induced mitochondrial outer-membrane permeabilization. Moreover, multiple apoptotic stimuli induce mitochondrial recruitment of the proapoptotic Bcl-2-family protein, Bax, to Drp-1 and Mitofusin-2-positive putative mitochondrial fragmentation sites in a Fis-1-dependent manner, consistent with a role for mitochondrial fission and fusion machinery in cell death (Goyal, 2007).

In Drosophila, RHG-family proteins (Reaper, Hid and Grim), genotoxic stresses, and protein synthesis inhibitors antagonize Drosophila inhibitor of apoptosis protein-1 (DIAP-1)-mediated inhibition of the activation of the apical caspase Dronc in an ARK- (Drosophila APAF-1) and ATP-dependent manner, leading to effector caspase activation and cell death. The role of mitochondria in this process is unclear. Cytochrome-C has been shown to be differentially displayed from the mitochondria during cell death. Knockdown of Drosophila cytochrome-C did not affect cell death triggered by genotoxic stress in vitro and ex vivo or developmental stimuli in vivo, although certain nonapoptotic caspase activation pathways utilized during sperm individualization were affected. Furthermore, mitochondrial morphology during Drosophila PCD has not been previously reported (Goyal, 2007 and references therein).

This study shows that multiple apoptotic stimuli result in mitochondrial fragmentation upstream of caspase activation, phosphatidylserine exposure, and nuclear condensation in Drosophila cells. While etoposide induced mitochondrial fragmentation, C6-ceramide resulted in an increase in mitochondrial contiguity prior to its fragmentation. drp-1 mutant or RNAi-treated S2R+ cells are considerably protected from multiple apoptotic stimuli, consistent with reduced mitochondrial fragmentation. Thus, mitochondrial remodeling plays an important role in modifying the propensity of cells to undergo PCD in Drosophila (Goyal, 2007).

Precisely timed ecdysone pulses induce Reaper and Hid expression in the Drosophila larval midgut (0 hr after puparium formation [APF]) or the salivary gland (10 hr APF) and trigger developmental PCD. Mitochondria, visualized by using matrix-targeted GFP (Mito-GFP) in acridine orange-positive, dying prepupal midgut cells (1 hr APF) and salivary glands (minus 4 hr APF), are remarkably fragmented, unlike third-instar larval (-4 hr APF) mitochondria. Quantification revealed a dramatic decrease in the prepupal mitochondrial cross-sectional area (CSA; midgut and salivary gland and a significant increase in the number of mitochondria per cell. Moreover, ecdysone-induced mitochondrial fragmentation is mimicked ex vivo on third-instar larval wing discs by using 1 mM ecdysone for 2 hr. In addition, overexpression of Hid resulted in mitochondrial fragmentation in acridine orange-positive eye disc cells. Thus, mitochondria in Drosophila tissues fragment during PCD, as has been reported in C. elegans and mammalian cells (Goyal, 2007).

To assess the role of mitochondrial remodeling in PCD, mitochondrial morphology was temporally characterized in etoposide-, actinomycin-D-, cycloheximide-, or C6-ceramide (a lipid cell death mediator)-treated larval hemocytes and the S2R+ cell line. A 3- to 4-fold increase in nuclear condensation (6 hr) was preceded by effector caspase activation (5 hr) and phosphatidylserine (PS) exposure in propidium iodide (PI)-negative hemocytes (6 hr). These cells subsequently (10 hr) became characteristically blebbed and PI permeable. The number of etoposide-treated apoptotic hemocytes increased with time. Interestingly, mitochondrial fragmentation, as confirmed by quantifying functionally isolated mitochondria at 3 hr, preceded the onset of PS exposure or nuclear damage. Quantification showed an increase in the number of mitochondria and the contribution of fragmented mitochondria to the mitochondrial cross-sectional area (CSA). Mitochondrial fragmentation was also observed in cycloheximide- or actinomycin-D-treated, Mito-YFP-transfected S2R+ cells (Goyal, 2007).

Surprisingly, mitochondria in C6-ceramide-treated (30-60 min) hemocytes that had normal nuclei were highly contiguous. Quantifying functionally isolated mitochondrial CSA per cell showed a significant increase in the contribution of tubular or extensively tubular mitochondria in these cells when compared with untreated cells. However, by 4 hr, these extensively tubular mitochondria underwent fragmentation in FITC-Annexin V (AnV)-negative hemocytes that had normal nuclei, similar to what was observed with genotoxic stress (Goyal, 2007).

Therefore, genotoxic stresses trigger mitochondrial fragmentation, while the lipid cell death mediator induces increased mitochondrial contiguity and subsequent fragmentation prior to phosphatidylserine exposure, nuclear condensation, and finally plasma membrane permeability during Drosophila cell death (Goyal, 2007).

In hemocytes incubated with an apoptotic stimulus, mitochondrial fragmentation (3-4 hr) preceded any detectable effector caspase activation. Furthermore, inhibiting caspases with zVAD-fmk or by overexpressing DIAP-1 (DIAP-1+) did not affect mitochondrial fragmentation, although hemocyte death was inhibited, as revealed by a lack of apoptotic markers. In addition, overexpression of Dcp-1, a Drosophila effector caspase, did not affect mitochondrial morphology. Thus, mitochondrial fragmentation is upstream of effector caspase activation (Goyal, 2007).

The drp-1 mutants used to study the role of mitochondrial remodeling during Drosophila PCD are functional null alleles, drp-12 (Gly293Ser mutation), picked in a forward screen for genes affecting neurotransmission and drp-1[KG 03815], a P element insertion between the first two exons of drp-1 (13510 in this study) and a hypomorph, nrdD46 (Arg278Trp mutation; 3665 in this study). drp-12, 13510, and the deficiency Df Exel6008 were second-instar larval lethal; however, drp-12 yielded bang-sensitive escapers. The hypomorphic trans-allelic combination of 3665/13510 was third-instar larval lethal, although it yielded a few temperature-sensitive adults. A genomic duplication of drp-1 (Dp [2;1] JS13) completely rescued the lethality associated with drp-12, 13510, and 3665/13510 (Goyal, 2007).

Mitochondria in drp-12 and 3665/13510 hemocytes were extensively tubular when compared with wild-type mitochondria. Quantifying mitochondrial morphology revealed a 2-fold decrease in the number of mitochondria and a significant increase in the contribution of tubular and extensively tubular mitochondria to the total mitochondrial CSA in drp-1 mutant hemocytes when compared with wild-type cells. Interestingly, 13510/+ hemocytes or eye disc cells displayed a dominant mitochondrial fission defect that was completely rescued by a genomic duplication of drp-1. The mitochondrial fission defect in mutant cells could result from a reduced mitochondrial association of Drp-1 (Goyal, 2007).

An increase in mitochondrial contiguity due to a loss of Drp-1 function was also confirmed by measuring fluorescence recovery after photobleaching (FRAP) of Mito-YFP in drp-1 RNAi-treated S2R+ cells that had extensively tubular mitochondria. Relative FRAP of Mito-YFP in a defined mitochondrial region in drp-1 RNAi-treated cells was significantly higher than that observed in mock RNAi-treated cells (Goyal, 2007).

drp-1 mutant hemocytes were protected from etoposide-induced death up to at least 10 hr, as revealed by a lack of caspase activation, PS exposure, or PI permeability in the majority (~80%) of these cells. Furthermore, drp-1 mutant and dsRNA-treated S2R+ cells were significantly protected from cycloheximide-, actinomycin-D-, or UV-B-induced death. Consistent with increased protection, mitochondria in the majority (~98%) of etoposide-treated drp-12 hemocytes failed to fragment. Interestingly, mitochondria in etoposide-treated 3665/13510 hemocytes revealed a tubular, yet beaded and swollen intermediate in mitochondrial fragmentation by 4 hr that yielded some fragmented mitochondria in few (~25%) cells later. Therefore, reduced (drp-12) or delayed (3665/13510) mitochondrial fragmentation decreased effector caspase activation and protected cells from genotoxic stress. Moreover, an increase in expression of Drp-1 in hemocytes resulted in enhancement of etoposide-induced cell death (Goyal, 2007).

The majority (~70%) of the C6-ceramide-treated drp-12 hemocytes did not show effector caspase activation or PS exposure and displayed significant protection, similar to what was observed with etoposide, although hemocytes derived from the weaker allelic combination, 13510/3665, were apoptotic. Unlike 13510/3665 mitochondria, drp-12 mitochondria failed to fragment, consistent with an essential role for Drp-1-mediated mitochondrial fragmentation during apoptosis in Drosophila. Moreover, developmental PCD in drp-12 mutant larvae was considerably reduced, as revealed by the enlarged central nervous system and a prominently elongated ventral ganglion, similar to other PCD-defective mutants reported (Goyal, 2007).

During metamorphosis, the first ecdysone pulse triggers mitochondrial fragmentation in prepupal tissues, although it is after the second ecdysone pulse that salivary gland histolysis occurs. It is likely that DIAP-1 inhibits caspases in these cells that have fragmented mitochondria until it is downregulated at the transcriptional level or degraded after the second ecdysone pulse. Interestingly, this was mimicked ex vivo in etoposide-treated DIAP-1+ hemocytes (Goyal, 2007).

The data presented in this study show involvement of mitochondrial fragmentation for ARK-mediated Dronc activation during cell death. The RHG-family proteins that localize to the mitochondria might activate Drp-1-mediated mitochondrial fragmentation. This could result in exposure of cytochrome-C or release of Peanut, which antagonize DIAP-1-mediated suppression of Dronc. However, since Drosophila PCD is unaffected upon knockdown of cytochrome-C, mitochondrial fragmentation in Drosophila and mammalian cells would increase mitochondrial surface area and perhaps the concentration of bulky head group lipids on the outer mitochondrial membrane, facilitating recruitment of proapoptotic proteins. Drp-1 might organize sites for Drosophila Bcl-2-family protein Debcl function on mitochondria that are similar to mitochondrial sites of Bax recruitment in mammalian cells (Goyal, 2007 and references therein).

These results provide the first evidence that Drp-1-mediated mitochondrial fragmentation upstream of effector caspase activation modifies apoptotic sensitivity. Thus, mitochondrial fragmentation, like caspase activation, plays a conserved and unifying role in diverse cell death pathways from worms to mammals. Although the function of the highly contiguous mitochondria during lipid-induced cell death remains poorly understood, this study brings to the forefront a modulatory role for mitochondrial remodeling in determining the susceptibility of Drosophila cells to death.

Mitochondrial disruption in Drosophila apoptosis

Mitochondrial disruption is a conserved aspect of apoptosis, seen in many species from mammals to nematodes. Despite significant conservation of other elements of the apoptotic pathway in Drosophila, a broad role for mitochondrial changes in apoptosis in flies remains unconfirmed. This study shows that Drosophila mitochondria become permeable in response to the expression of Reaper and Hid, endogenous regulators of developmental apoptosis. Caspase activation in the absence of Reaper and Hid is not sufficient to permeabilize mitochondria, but caspases play a role in Reaper- and Hid-induced mitochondrial changes. Reaper and Hid rapidly localize to mitochondria, resulting in changes in mitochondrial ultrastructure. The dynamin-related protein, Dynamin related protein 1 (Drp1), is important for Reaper- and DNA-damage-induced mitochondrial disruption. Significantly, it was shows that inhibition of Reaper or Hid mitochondrial localization or inhibition of Drp1 significantly inhibits apoptosis, indicating a role for mitochondrial disruption in fly apoptosis (Abdelwahid, 2007).

A role for mitochondria in apoptosis appears to be conserved from mammals to nematodes to yeast. The lack of clear evidence that mitochondria play a role in Drosophila apoptosis has prompted discussion of whether flies represent an evolutionary outlier in this highly conserved process. The data strongly suggests that mitochondrial disruption also plays a role in Drosophila apoptosis (Abdelwahid, 2007).

The data show that mitochondria rapidly become permeable to Cyt c when Rpr or Hid are expressed, both in cultured cells and in vivo. This alteration in mitochondrial permeability was also seen during DNA-damage-induced apoptosis. Importantly, it was demonstrated that the mitochondrial permeabilization during DNA-damage-induced apoptosis is dependent on the genes in the H99 interval. Taken together, these data indicate that Rpr and Hid are both necessary and sufficient for mitochondrial permeabilization (Abdelwahid, 2007).

In contrast, apoptosis induced by Actinomycin D, UV, and DIAP1 RNAi does not result in mitochondrial permeabilization. This indicates that caspase activation alone is not sufficient to induce mitochondrial permeabilization and that the mitochondrial permeabilization seen on Rpr or Hid induction is not simply a general late event in apoptosis. The efficient cell killing by Actinomycin D, UV, and DIAP1 RNAi also implies that mitochondrial permeabilization is not important for all apoptosis in Drosophila cells. Rather, it suggests that the Rpr and Hid proteins have a specific activity on the mitochondria that results in mitochondrial permeabilization to execute apoptosis in a timely manner (Abdelwahid, 2007).

The effects of Rpr and Hid on mitochondria were not limited to permeabilization. It was found that mitochondrial morphology is dramatically altered within 90 min of Rpr or Hid expression, in both S2 cells and embryos. A variety of defects were found in mitochondrial ultrastructure ranging from a rounded appearance, to bulging (and occasional rupture) of the outer mitochondrial membrane, to swelling of the matrix and disruption of the cristae. This was rarely seen with other inducers of apoptosis. Rpr and Hid may directly cause altered mitochondrial morphology or could act indirectly through other proteins localized at the mitochondria (Abdelwahid, 2007).

The absence of mitochondrial permeabilization in cells treated with DIAP1 dsRNA indicates that the mitochondrial function of Rpr and Hid is independent of their ability to inhibit DIAP1. This is confirmed by data showing that expression of DeltaN-Rpr results in mitochondrial permeabilization despite the fact that this protein lacks the necessary motif to inhibit DIAP1 antiapoptotic activity. Taken together, these data demonstrate that Rpr and Hid have dual activities in the cell, both to inhibit DIAP1 and to permeabilize mitochondria. Data from other labs have suggested that Rpr is a multifunctional protein. The data confirm that Rpr has multiple proapoptotic activities in the fly (Abdelwahid, 2007).

The dual functionality of Rpr and Hid parallel the recently described role of C. elegans Egl-1 in mitochondrial damage. Egl-1 induces apoptosis by binding to Ced-9 to promote both the activation of the caspase Ced-3 and mitochondrial fragmentation. Similarly, Rpr and Hid bind to DIAP1, displacing active caspases and act on mitochondria to promote mitochondrial disruption. One difference between C. elegans and flies appears to be the requirement for caspase activity in the mitochondrial disruption. In C. elegans, Ced-3 is not required for fragmentation but is required for apoptosis in response to fragmentation. In Drosophila, caspase activity participates in the mitochondrial changes (Abdelwahid, 2007).

Two lines of evidence support a role for mitochondrial disruption in Drosophila apoptosis. First, Rpr and Hid must localize to mitochondria to elicit a full apoptotic response. Second, if mitochondrial disruption is blocked by inhibiting Dynamin related protein 1 (Drp1) expression, a decrease is seen in apoptosis. These data clearly indicate that mitochondrial localization of Rpr and Hid is required for a full apoptotic response in S2 cells. This agrees with previous data on Rpr and also with studies on a Grim mutant lacking a mitochondrial localization signal. Mitochondrial localization of Hid has been demonstrated in a heterologous system. In the Haining study, Hid killing was not compromised in the absence of mitochondrial localization, in contrast to the current observations in Drosophila cells. A role for mitochondrial localization is also supported by the finding that two mutant forms of Hid that lack mitochondrial localization in mammalian cells behave as weak loss-of-function alleles in the fly (Abdelwahid, 2007).

The mitochondrial fission protein Drp1 is implicated in mitochondrial disruption during apoptosis in yeast, nematodes, and mammals. The current data indicate a role for this protein in Rpr-induced and DNA-damage-induced mitochondrial disruption in S2 cells and in the embryo. Furthermore, the inhibition of mitochondrial disruption after Drp1 knockdown is correlated with a decrease in apoptosis, strongly suggesting that mitochondrial disruption contributes to the apoptotic response. It is interesting to note that Drp1 plays a conserved role in apoptosis in a wide variety of organisms but seems to function downstream of different pathways. In mammals, inhibition of Drp1 blocks apoptosis in response to activation of proapoptotic Bcl-2 family members. In C. elegans, Drp1 inhibition blocks endogenous death downstream of Egl1 and Ced9, also Bcl-2 family proteins. Even in yeast, the role of Drp1 in cell death can be modulated by Bcl-2 family proteins. Surprisingly, in flies, Drp1 appears to be acting downstream of a different family of apoptosis inducers, the RHG proteins. It remains to be seen whether a role for the fly Bcl-2 family proteins can be established in mitochondrial disruption (Abdelwahid, 2007).

Release of apoptogenic factors, most notably Cyt c, from the mitochondria is an essential step in most apoptosis in mammalian systems. However, the current work confirms the findings of others that Cyt c, although released from mitochondria by Rpr and Hid, is not important for Rpr or Hid killing. It should be noted that Cyt c has been shown to be important in some Drosophila developmental apoptosis. In these deaths, Hid is likely to act upstream of Cyt c release. If Cyt c release is required in some cells for Hid-mediated caspase activation, why not in S2 cells? It is possible that there are both Cyt c-dependent and -independent mechanisms for activating caspases, and these may be cell-type dependent. Recent data from mice carrying a nonapoptogenic form of Cyt c supports this model, since this study suggests that there is both Cyt c-dependent and -independent apoptosis during mouse development (Abdelwahid, 2007).

If release of Cyt c is not an essential step in apoptosis in most fly cells, is another apoptosis-inducing factor released during mitochondrial disruption? In mammalian cells, release of other mitochondrial proteins such as SMAC/Diablo, Omi/HTRA2, and AIF are proposed to contribute to apoptosis. There is some evidence that released mitochondrial factors do not contribute to caspase activation in the fly. Unlike in the mammalian system, mitochondrial lysates cannot activate caspases in fly cytoplasmic lysates. An alternative possibility is that mitochondrial disruption per se might contribute to apoptosis in the fly through inhibition of normal mitochondrial functions essential for cell viability. This might serve as a backup system, to maximize apoptosis in cells that express low levels of the RHG proteins. A similar role for mitochondrial disruption has been proposed in C. elegans (Abdelwahid, 2007).

In sum, it is concluded from these studies that Drosophila is not an outlier in evolution with regard to the involvement of mitochondria in the apoptotic process. Rather, the data indicate that mitochondrial changes contribute to Drosophila apoptosis. The findings suggest that the view of the role of mitochondria in cell death has to be broadened beyond the release of proapoptotic factors, to include the general disruption of mitochondria, ensuring that doomed cells have no chance of recovery. Such a model would fit not only the changes seen in mammalian mitochondria, but also those found in yeast, C. elegans, and flies as well (Abdelwahid, 2007).

Mitochondrial fragmentation due to inhibition of fusion increases Cyclin B through mitochondrial superoxide radicals

During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly interesting events are first, mitochondrial hyperfusion during the G1-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented between late G2- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. This study bypassed the 'mitotic mitochondrial fragmentation' checkpoint by inducing fragmented mitochondrial morphology and then measuring the effect on cell cycle progression. Using Drosophila larval hemocytes, Drosophila S2R+ cell and cells in the pouch region of wing imaginal disc of Drosophila larvae it was shown that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. These experiments suggest that the inhibition of mitochondrial fusion increases superoxide radical content and leads to the upregulation of cyclin B that culminates in the observed changes in the cell cycle. Evidence is provided for the importance of mitochondrial superoxide in this process. These results provide an insight into the need for mitofusin-degradation during mitosis and also help in understanding the mechanism by which mitofusins may function as tumor suppressors (Gupte, 2015).

A mitochondrial-associated link between an effector caspase and autophagic flux

It has become evident that caspases function in nonapoptotic cellular processes in addition to the canonical role for caspases in apoptotic cell death. It has been demonstrated that the Drosophila effector caspase Dcp-1 localizes to the mitochondria and positively regulates starvation-induced autophagic flux during mid-oogenesis. Loss of Dcp-1 leads to elongation of the mitochondrial network, increased levels of the adenine nucleotide translocase sesB, increased ATP levels, and a reduction in autophagy. sesB is a negative regulator of autophagic flux, and Dcp-1 interacts with sesB in a nonproteolytic manner to regulate its stability, uncovering a novel mechanism of mitochondrial associated, caspase-mediated regulation of autophagy in vivo (DeVorkin, 2014).

Loss of Drosophila i-AAA protease, dYME1L, causes abnormal mitochondria and apoptotic degeneration

Mitochondrial AAA (ATPases Associated with diverse cellular Activities) proteases i-AAA (intermembrane space-AAA) and m-AAA (matrix-AAA) are closely related and have major roles in inner membrane protein homeostasis. Mutations of m-AAA proteases are associated with neuromuscular disorders in humans. However, the role of i-AAA in metazoans is poorly understood. This study generated a deletion affecting Drosophila i-AAA, dYME1L (dYME1Ldel). Mutant flies exhibited premature aging, progressive locomotor deficiency and neurodegeneration that resemble some key features of m-AAA diseases. dYME1Ldel flies displayed elevated mitochondrial unfolded protein stress and irregular cristae. Aged dYME1Ldel flies had reduced complex I (NADH/ubiquinone oxidoreductase) activity, increased level of reactive oxygen species (ROS), severely disorganized mitochondrial membranes and increased apoptosis. Furthermore, inhibiting apoptosis by targeting dOmi (Drosophila Htra2/Omi) or DIAP1, or reducing ROS accumulation suppressed retinal degeneration. The results suggest that i-AAA is essential for removing unfolded proteins and maintaining mitochondrial membrane architecture. Loss of i-AAA leads to the accumulation of oxidative damage and progressive deterioration of membrane integrity, which might contribute to apoptosis upon the release of proapoptotic molecules such as dOmi. Containing ROS level could be a potential strategy to manage mitochondrial AAA protease deficiency (Qi, 2015).

Loss of dE2F compromises mitochondrial function

E2F/DP transcription factors regulate cell proliferation and apoptosis. This study investigated the mechanism of the resistance of Drosophila dDP mutants to irradiation-induced apoptosis. Contrary to the prevailing view, this is not due to an inability to induce the apoptotic transcriptional program, because this program is induced; rather, this is due to a mitochondrial dysfunction of dDP mutants. This defect is attributed to E2F/DP-dependent control of expression of mitochondria-associated genes. Genetic attenuation of several of these E2F/DP targets mimics the dDP mutant mitochondrial phenotype and protects against irradiation-induced apoptosis. Significantly, the role of E2F/DP in the regulation of mitochondrial function is conserved between flies and humans. Thus, these results uncover a role of E2F/DP in the regulation of mitochondrial function and demonstrate that this aspect of E2F regulation is critical for the normal induction of apoptosis in response to irradiation (Ambrus, 2013).

E2F transcription factors are best understood for their role in controlling the cell cycle, apoptosis, and differentiation. In this report, evidence is presented that E2F is also involved in the regulation of mitochondrial function and identify a specific biological context, DNA damage-induced apoptosis, in which this aspect of E2F control becomes critical. It is suggested that mitochondrial dysfunction, and not the failure to induce the apoptotic geneexpression program, makes E2F-deficient cells refractory to apoptosis (Ambrus, 2013).

In flies and mammals, the conserved mechanism by which E2F triggers apoptosis is transcriptional control of apoptotic targets. Therefore, it is believed that in irradiated cells, dE2f1, like dp53, contributes to the normal transcriptional induction of apoptotic genes. However, the current data do not support such a model since the apoptotic gene expression program was induced properly in irradiated dDP mutants. Thus, in the context of the DNA damage response, the contribution of dE2f1 to the normal transcriptional induction of apoptotic genes is negligible. It is emphasized that the data do not imply that dE2f1 is unimportant. For example, unrestrained dE2f1 activity in rbf mutants has been shown to markedly increase the induction of hid and rpr in response to DNA damage, and this increase determines the elevated sensitivity of rbf mutants to irradiation-induced apoptosis. Since ablation of dp53 completely blocks irradiation-induced apoptosis and induction of apoptotic genes, the irradiation-induced apoptotic program is governed primarily by dp53, and hyperactive dE2f1 can provide additional assistance to dp53 in activating apoptotic genes. This contribution of dE2f1 becomes evident in certain settings, such as in rbf mutants (Ambrus, 2013).

Given that irradiated dDP mutants have a properly induced apoptotic gene expression program that should trigger apoptosis in these cells, their failure to undergo apoptosis is puzzling. It is suggested that the resistance of dDP mutants to apoptosis is the consequence of a mitochondrial dysfunction. In dDP mutants, mitochondria exhibit an abnormal morphology and reduced mitochondrial membrane potential and ATP levels. The lower level of expression of dE2f/dDP mitochondria-associated target genes is a critical event in determining the response of dDP mutants to irradiation, since genetic attenuation of their expression mimics the dDP mutant mitochondrial phenotype and protection from irradiation-induced apoptosis. Significantly, the strongest protection was observed with the genes that exerted the most severe mitochondrial defects upon downregulation. Thus, the response to irradiation-induced apoptosis correlates with the extent of the mitochondrial defects. One possibility is that the mitochondrial dysfunction of dDP-deficient cells lowers their mitochondrial readiness for apoptosis, and therefore the irradiation-induced apoptotic transcriptional program is insufficient to trigger cell death. Intriguingly, 'mitochondrial readiness for apoptosis' is thought to be the molecular basis of a differential response to chemotherapy in cancer patients with acute myelogenous leukemia. Among the mitochondria-associated dE2F/dDP target genes investigated in this work, Mdh2 is particularly interesting. It was previously shown that an Mdh2 mutation prevented apoptosis in another context during ecdysone-induced cell death in salivary glands. Destruction of salivary glands is normally triggered by the induction of rpr and rpr, and both genes were induced in Mdh2 mutants to the level observed in the wild-type. In addition, Mdh2 mutants display a defect in energy production and reduced ATP levels, which is thought to compromise their ability to undergo apoptosis. This setting is highly reminiscent of dDP mutants, which are also remarkably resistant to cell death even in the face of a high level of induction of a DNA damage-dependent apoptotic transcription program (Ambrus, 2013).

The idea that mitochondrial defects could impact execution of apoptosis is consistent with the recently uncovered importance of mitochondria for cell death in Drosophila. Several studies demonstrated that Rpr, Grim, and Hid, the key apoptotic proteins in flies, are localized to mitochondria and that this localization is required for efficient activation of apoptosis. Thus, one possibility is that proapoptotic proteins are not efficiently localized to mitochondria in dDP mutants. It is also possible that the dysfunctional mitochondria of dDP mutants fail to remodel in response to irradiation, which has been shown to be necessary for execution of stress-induced apoptosis. It is noted that the current findings do not imply that dE2F/ dDP normally triggers apoptosis by modulating mitochondrial function, but rather that mitochondrial function is compromised in E2F-deficient cells, which in turn would result in less efficient apoptosis (Ambrus, 2013).

Another important conclusion of this study is that a mechanistic link between the Rb pathway and mitochondria is conserved in mammalian cells. Several Drosophila dE2F/dDP-regulated, mitochondria-associated genes are also E2F targets in mammalian cells, and their expression is similarly reduced in cells when E2F is inactivated. Significantly, this leads to strong mitochondrial defects that are highly reminiscent of the mitochondrial phenotype in Drosophila dDP mutant eye discs. The data are consistent with the recent finding that mammalian E2f1 and pRB regulate expression of oxidative metabolism genes during the adaptive metabolic response in mice. The Rb pathway has been also implicated in the regulation of the mitochondrial biogenesis transcriptional program in erythropoiesis. Intriguingly, a recent study demonstrated that a fraction of endogenous pRB is present at mitochondria, where it directly participates in mitochondrial apoptosis (Hilgendorf, 2013). Given the prominent role of mitochondrial pathways in apoptosis in mammalian cells, it is conceivable that the loss of E2F could impact the efficacy of apoptosis in mammals by a mechanism analogous to that observed in Drosophila. Such an idea is consistent with the finding that inactivation of E2F reduces DNA damage-induced apoptosis in mammalian cells (Ambrus, 2013).

Interestingly, Nicolay (2013) recently demonstrated that an rbf mutation alters cellular metabolism and an abnormal metabolism sensitizes Drosophila to various types of stress. This work, found that inactivation of E2F results in a severe mitochondrial dysfunction, which is the basis for the failure of dDP mutants to undergo DNA damage-induced apoptosis. Thus, a general emerging theme is that perturbation of the Rb pathway may exert profound metabolic changes within the cell that can have a major impact on cell survival (Ambrus, 2013).

Cardiac deficiency of single cytochrome oxidase assembly factor scox induces p53-dependent apoptosis in a Drosophila cardiomyopathy model

The heart is a muscle with high energy demands. Hence, most patients with mitochondrial disease produced by defects in the oxidative phosphorylation (OXPHOS) system are susceptible to cardiac involvement. The presentation of mitochondrial cardiomyopathy includes hypertrophic, dilated and left ventricular noncompaction, but the molecular mechanisms involved in cardiac impairment are unknown. One of the most frequent OXPHOS defects in humans frequently associated with cardiomyopathy is cytochrome c oxidase (COX) deficiency caused by mutations in COX assembly factors such as Sco1 and Sco2. To investigate the molecular mechanisms that underlie the cardiomyopathy associated with Sco deficiency, this study interfered with scox (the single Drosophila Sco orthologue) expression in the heart. Cardiac-specific knockdown of scox reduces fly lifespan, and it severely compromises heart function and structure, producing dilated cardiomyopathy. Cardiomyocytes with low levels of scox have a significant reduction in COX activity and they undergo a metabolic switch from OXPHOS to glycolysis, mimicking the clinical features found in patients harbouring Sco mutations. The major cardiac defects observed are produced by a significant increase in apoptosis, which is dp53-dependent. Genetic and molecular evidence strongly suggest that dp53 is directly involved in the development of the cardiomyopathy induced by scox deficiency. Remarkably, apoptosis is enhanced in the muscle and liver of Sco2 knock-out mice, clearly suggesting that cell death is a key feature of the COX deficiencies produced by mutations in Sco genes in humans (Martínez-Morentin, 2015).

Cardiomyopathies are a collection of myocardial disorders in which the heart muscle is structurally and functionally abnormal. In the past decade, it has become clear that an important proportion of cases of hypertrophic and dilated cardiomyopathies are caused by mutations in genes encoding sarcomeric or desmosomal proteins. In addition, cardiomyopathies (both hypertrophic and dilated) are frequently associated to syndromic and non-syndromic mitochondrial diseases. The importance of oxidative metabolism for cardiac function is supported by the fact that 25–35% of the myocardial volume is taken by mitochondria. The current view of mitochondrial involvement in cardiomyopathy assumes that ETC malfunction results in an increased ROS production, triggering a “ROS-induced ROS release” vicious circle which in turn perpetuates ETC dysfunction via damage in mtDNA and proteins involved in electron transport. Under this view, accumulated mitochondrial damage would eventually trigger apoptosis through mitochondrial permeability transition pore (mPTP) opening other mechanisms. Under normal circumstances, damaged mitochondria would be eliminated through mitophagy. Excessive oxidative damage is supposed to overcome the mitophagic pathway resulting in apoptosis. Nevertheless, although several potential mechanisms have been suggested, including apoptosis deregulation, oxidative stress, disturbed calcium homeostasis or impaired iron metabolism, the molecular basis of the pathogenesis of mitochondrial cardiomyopathy is virtually unknown (Martínez-Morentin, 2015).

Pathogenic mutations in human SCO1 and SCO2 have been reported to cause hypertrophic cardiomyopathy, among other clinical symptoms. However, the molecular mechanisms underlying this cardiac dysfunction have yet to be elucidated. This study reports the first cardiac-specific animal model to study human SCO1/2-mediated cardiomyopathy. Cardiac-specific scox KD in Drosophila provokes a severe dilated cardiomyopathy, as reflected by a significant increase in the conical chamber size, due to mitochondrial dysfunction. It presents a concomitant metabolic switch from glucose oxidation to glycolysis and an increase in ROS levels, leading to p53-dependent cell death. Interestingly, previous studies on patients and rat models have shown that mitochondrial dysfunction is associated with abnormalities in cardiac function and changes in energy metabolism, resulting in glycolysis optimization and lactic acidosis. Furthermore, in the Sco2KI/KO mouse model, where no evidence of cardiomyopathy has been described, partial loss of Sco2 function induces apoptosis in liver and skeletal muscle. In flies scox KD causes a significant reduction in FS and in the DI, as well as cardiac myofibril disorganization. This degenerative process is most likely due to mitochondrial dysfunction rather than to a developmental defect and moreover, the dilated cardiomyopathy developed by flies resembles that caused by mitochondrial fusion defects in flies (Martínez-Morentin, 2015).

The ETC is the major site of ROS production in cells, and aging and many neurodegenerative diseases have been linked to mitochondrial dysfunction that results in excessive oxidative stress. Interestingly, there is an increase in ROS formation associated with oxidative DNA damage in human Sco2−/− cells. Accordingly, it was found that cardiac-specific knockdown of scox increases oxidative stress, although it could not be distinguished whether this increase in free radical accumulation arises from the mitochondria or whether it comes from non-mitochondrial sources due to a loss of cellular homeostasis, as reported in yeast and in a neuro-specific COX-deficient Alzheimer disease mouse model (Martínez-Morentin, 2015).

Sco2 expression is known to be modulated by p53, a transcription factor that participates in many different processes, including cancer development, apoptosis and necrosis. p53 regulates homeostatic cell metabolism by modulating Sco2 expression and contributes to cardiovascular disorders. In addition, p53 activation in response to stress signals, such as increased oxidative stress or high lactic acid production, is well documented. Data from this study, showing that p53 is upregulated in response to scox KD, but not in response to KD of another Complex IV assembly factor, Surf1, suggest a specific genetic interaction between dp53 and scox. This is corroborated by the dramatic effects observed in the heart structure and function when dp53 is overexpressed in scox KD hearts. Furthermore, the functional and structural defects seen in scox KD hearts can be rescued in dp53-DN OE or dp53 null backgrounds, indicating that the scox-induced defects are mediated by increased p53 expression. Interestingly, opposed to scox KD, the heart structure defects induced by dp53 OE can be fully rescued by heart-specific Surf1 KD, further confirming the specificity of the genetic interaction between dp53 and scox (Martínez-Morentin, 2015).

It has recently been shown that SCO2 OE induces p53-mediated apoptosis in tumour xenografts and cancer cells. Furthermore, SCO2 KD sensitizes glioma cells to hypoxia-induced apoptosis in a p53-dependent manner and induces necrosis in tumours expressing WT p53, further linking the SCO2/p53 axis to cell death. In Drosophila, there is a dp53-mediated upregulation of Reaper, Hid and Grim in response to scox KD. This, coupled with the observation that Reaper overexpression in the adult heart enhances the structural defects caused by cardiac-specific scox KD, suggests that scox normally prevents the triggering of dp53-mediated cell death in cardiomyocytes in stress response. Indeed, it was found that there is massive cell death in the skeletal muscle and liver of Sco2KI/KO mice, supporting the hypothesis that Sco proteins might play this role also in mammals (Martínez-Morentin, 2015).

The study provides evidence that scox KD hearts exhibit partial loss of COX activity, with cardiomyocytes undergoing apoptosis. There is evidence from vertebrate and invertebrate models that partial inhibition of mitochondrial respiration promotes longevity and metabolic health due to hormesis. In fact, it has recently been shown that mild interference of the OXPHOS system in Drosophila IFMs preserves mitochondrial function, improves muscle performance and increases lifespan through the activation of the mitochondrial unfolded pathway response and IGF/like signalling pathways. This study speculates that cell death, rather than mitochondrial dysfunction itself, is likely to be the main reason for the profound heart degeneration observed in TinCΔ4-Gal4>scoxi flies. Expression of dominant negative dp53 in scox KD hearts rescues dysfunction and cardiac degeneration, and, most importantly, scox KD in dp53−/− animals causes no apparent heart defects, which could attribute the rescue observed to blockade of the p53 pathway. Indeed, inhibiting apoptosis by p35 or Diap1 OE almost completely rescues the morphological scox KD phenotype. As scox KD in the absence of dp53 causes no symptoms of heart disease, coupled with the inability of p35 and Diap1 to completely rescue the morphological phenotype, suggests that, in addition to inducing apoptosis, dp53 plays a key role in the development of cardiomyopathy (Martínez-Morentin, 2015).

The fact that heart-specific Surf1 KD neither upregulates p53 nor induces apoptosis supports the idea that the partial loss of scox function itself triggers dp53 upregulation and apoptosis, rather than it being a side effect of COX dysfunction and the loss of cellular homeostasis. In this context, it is noteworthy that SCO2 interference in mammalian cells induces p53 re-localization from mitochondria to the nucleus. It is therefore tempting to hypothesize that scox might play another role independent of its function as a COX assembly factor, perhaps in redox regulation as suggested previously and that it may act in conjunction with dp53 to fulfil this role. Another issue deserves further attention, the possibility of this interaction being a tissue-specific response. It may be possible that the threshold of COX deficiency tolerated by the heart might be lower than in other tissues, thus the scox/dp53 genetic interaction may be a tissue-dependent phenomenon or the consequence of a tissue-specific role of scox. In fact, mitochondrial dysfunction in mice is sensed independently from respiratory chain deficiency, leading to tissue-specific activation of cellular stress responses. Thus, more work is necessary to test these hypotheses and try to understand how the partial lack of scox induces cell death through dp53 (Martínez-Morentin, 2015).

Although the role of mitochondria in Drosophila apoptosis remains unclear, there is strong evidence that, as in mammals, mitochondria play an important role in cell death in flies. The localization of Rpr, Hid and Grim in the mitochondria is essential to promote cell death, and fly mitochondria undergo Rpr-, Hid- and Drp1-dependent morphological changes and disruption following apoptotic stimulus. Moreover, the participation of the mitochondrial fission protein Drp1 in cell death is conserved in worms and mammals. It has been proposed that p53 plays a role in the opening of the mPTP that induces necrotic cell death. According to this model, p53 translocates to the mitochondrial matrix upon ROS stimulation, where it binds cyclophilin D (CypD) to induce mPTP opening independent of proapoptotic Bcl-2 family members Bax and Bak, and in contrast to traditional concepts, independent of Ca2+ (Martínez-Morentin, 2015).

Apoptotic and necrotic pathways have a number of common steps and regulatory factors, including mPTP opening that is thought to provoke mitochondrial swelling and posterior delivery of necrotic factors, although Drosophila mPTP activation is not accompanied by mitochondrial swelling. Interestingly, although the p53 protein triggers mitochondrial outer membrane permeabilization (MOMP) in response to cellular stress in mammals, releasing mitochondrial death factors, MOMP in Drosophila is more likely a consequence rather than cause of caspase activation and the release of mitochondrial factors does not appear to play a role in apoptosis. Thus, in cardiac-specific scox KD flies, dp53 might induce mPTP opening to trigger cell death, which in the absence of mitochondrial swelling would result in apoptosis instead of necrosis, as occurs in mammals. Drosophila mPTP has been shown to be cyclosporine A (CsA)-insensitive in vitro, although CsA administration ameliorates the mitochondrial dysfunction with a severely attenuated ATP and enhanced ROS production displayed by collagen XV/XVIII mutants. Interestingly, mice lacking collagen VI display altered mitochondrial structure and spontaneous apoptosis, defects that are caused by mPTP opening and that are normalized in vivo by CsA treatment (Martínez-Morentin, 2015).

Mitochondrial calcium uniporter in Drosophila transfers calcium between the endoplasmic reticulum and mitochondria in oxidative stress-induced cell death

Mitochondrial calcium plays critical roles in diverse cellular processes ranging from energy metabolism to cell death. Previous studies have demonstrated that mitochondrial calcium uptake is mainly mediated by the mitochondrial calcium uniporter (MCU) complex. However, the roles of the MCU complex in calcium transport, signaling, and dysregulation by oxidative stress still remain unclear. This study confirmed that Drosophila MCU contains evolutionarily conserved structures and requires essential MCU regulator (EMRE) for its calcium channel activities. Drosophila MCU loss-of-function mutants, which lacked mitochondrial calcium uptake in response to caffeine stimulation, were generated. Basal metabolic activities were not significantly affected in these MCU mutants as observed in examinations of body weight, food intake, body sugar level, and starvation-induced autophagy. However, oxidative stress-induced increases in mitochondrial calcium, mitochondrial membrane potential depolarization, and cell death were prevented in these mutants. It was also found that inositol 1,4,5-trisphosphate receptor (IP3R) genetically interacts with Drosophila MCU and effectively modulates mitochondrial calcium uptake upon oxidative stress. Taken together, these results support the idea that Drosophila MCU is responsible for ER-to-mitochondrial calcium transfer and for cell death due to mitochondrial dysfunction under oxidative stress (Choi, 2017).

Vps13D encodes a ubiquitin-binding protein that is required for the regulation of mitochondrial size and clearance

The clearance of mitochondria by autophagy, mitophagy, is important for cell and organism health, and known to be regulated by ubiquitin. During Drosophila intestine development, cells undergo a dramatic reduction in cell size and clearance of mitochondria that depends on autophagy, the E1 ubiquitin-activating enzyme Uba1, and ubiquitin. This study screen a collection of putative ubiquitin-binding domain-encoding genes for cell size reduction and autophagy phenotypes. The endosomal sorting complex required for transport (ESCRT) components TSG101 and Vps36, as well as the novel gene Vps13D were identified. Vps13D is an essential gene that is necessary for autophagy, mitochondrial size, and mitochondrial clearance in Drosophila. Interestingly, a similar mitochondrial phenotype is observed in VPS13D mutant human cells. The ubiquitin-associated (UBA) domain of Vps13D binds K63 ubiquitin chains, and mutants lacking the UBA domain have defects in mitochondrial size and clearance and exhibit semi-lethality, highlighting the importance of Vps13D ubiquitin binding in both mitochondrial health and development. VPS13D mutant cells possess phosphorylated DRP1 and mitochondrial fission factor (MFF) as well as DRP1 association with mitochondria, suggesting that VPS13D functions downstream of these known regulators of mitochondrial fission. In addition, the large Vps13D mitochondrial and cell size phenotypes are suppressed by decreased mitochondrial fusion gene function. Thus, these results provide a previously unknown link between ubiquitin, mitochondrial size regulation, and autophagy (Anding, 2018).

Crosstalk between mitochondrial fusion and the Hippo pathway in controlling cell proliferation during Drosophila development

Cell proliferation and tissue growth depend on the coordinated regulation of multiple signaling molecules and pathways during animal development. Previous studies have linked mitochondrial function and the Hippo signaling pathway in growth control. However, the underlying molecular mechanisms are not fully understood. This study identifies a Drosophila mitochondrial inner membrane protein ChChd3 as a novel regulator for tissue growth during larval development. Loss of ChChd3 leads to tissue undergrowth and cell proliferation defects. ChChd3 is required for mitochondrial fusion and removal of ChChd3 increases mitochondrial fragmentation. ChChd3 is another mitochondrial target of the Hippo pathway, although it is only partially required for Hippo pathway mediated overgrowth. Interestingly, lacking of ChChd3 leads to inactivation of Hippo activity under normal development, which is also dependent on the transcriptional co-activator Yorkie (Yki). Furthermore, loss of ChChd3 induces oxidative stress and activates the JNK pathway. In addition, depletion of other mitochondrial fusion components, Opa1 or Marf, inactivates the Hippo pathway as well. Taken together, the study proposes that there is a crosstalk between mitochondrial fusion and the Hippo pathway which is essential in controlling cell proliferation and tissue homeostasis in Drosophila (Deng, 2016).

Mitochondrial fusion but not fission regulates larval growth and synaptic development through steroid hormone production

Mitochondrial fusion and fission affect the distribution and quality control of mitochondria. This study shows that Marf (Mitochondrial associated regulatory factor), is required for mitochondrial fusion and transport in long axons. Moreover, loss of Marf leads to a severe depletion of mitochondria in neuromuscular junctions (NMJs). Marf mutants also fail to maintain proper synaptic transmission at NMJs upon repetitive stimulation, similar to Drp1 fission mutants. However, unlike Drp1, loss of Marf leads to NMJ morphology defects and extended larval lifespan. Marf is required to form contacts between the endoplasmic reticulum and/or lipid droplets (LDs) and for proper storage of cholesterol and ecdysone synthesis in ring glands. Interestingly, human Mitofusin-2 rescues the loss of LD but both Mitofusin-1 and Mitofusin-2 are required for steroid-hormone synthesis. These data show that Marf and Mitofusins share an evolutionarily conserved role in mitochondrial transport, cholesterol ester storage and steroid-hormone synthesis (Sandoval, 2014).

Mitochondrial dynamics plays a critical role in the control of organelle shape, size, number, function and quality control of mitochondria from yeast to mammals. It consists of fusion and fission of mitochondria, which are regulated by several GTPases. Mitochondrial fusion requires the fusion of the outer membrane followed by inner membrane fusion. In mammals, Mitofusin 1 (Mfn1) and Mitofusin 2 (Mfn2) regulate outer mitochondrial fusion whereas inner membrane fusion is controlled by Optic atrophy protein 1 (Opa1). Mitochondrial fission is regulated by Dynamin related protein 1 (Drp1). Decreased fusion results in fragmented round mitochondria, while defective fission leads to fused and enlarged mitochondria (Sandoval, 2014).

Loss of these mitochondrial GTPases results in lethality in worms, flies and mice. Mutations in the human DRP1 gene causes a dominant fatal infantile encephalopathy associated with defective mitochondrial and peroxisomal fission. On the other hand, missense mutations in OPA1 lead to a dominant optic atrophy. Depending on the severity of the mutation, patients may also suffer from ataxia and neuropathy. Also, missense mutations in MFN2 cause Charcot-Marie-Tooth type 2A, a common autosomal dominant peripheral neuropathy associated with axon degeneration. Finally, aberrant levels of mitochondrial GTPases have been associated with Parkinson's, Huntington's and Alzheimers' diseases. These observations in model organisms and human patients suggest that mitochondrial dynamics affects neuronal maintenance in many different contexts (Sandoval and references therein, 2014).

A significant imbalance of mitochondrial fission and fusion may affect the subcellular distribution of mitochondria, especially in neurons since they need to efficiently traffic from the soma to the synapses. Loss of Drosophila Drp1 impairs the delivery of mitochondria to neuromuscular junctions (NMJs), likely because they are large and interconnected. This defect is also associated with a severe depletion of mitochondria in NMJs, which affects local ATP production. This in turn affects the trafficking of synaptic vesicles upon endocytosis during prolonged stimulation. Similarly, in vertebrates, loss of Drp1 leads to an accumulation of mitochondria in the soma and reduced mitochondrial density in dendrites of hippocampal neurons. The Drp1 data in flies and vertebrates indicate that the expanded size of mitochondria affects their mobility (Sandoval, 2014).

Mitochondrial trafficking may also be affected by the physical interaction between the mitochondria and the transport machinery. Recent studies have documented a direct interaction between Mfn2 and a motor adaptor complex for mitochondrial transport, Miro2 (see Drosophila Miro). Moreover, loss of MFN2 in Purkinje cells displayed reduced mitochondrial motility in cerebellar dendrites and reduced mitochondrial transport in axons in cultured dorsal root ganglion neurons. These data suggest that an interaction of Mfn2 with Miro2 may be important for its role in trafficking. Although loss of both Drp1 and MFN2 impair mitochondrial trafficking, a careful comparison of the phenotypes associated with loss of Drosophila Drp1, Mitofusin or Marf, would be useful as the suggested mechanisms by which they impair transport seem very different (Sandoval, 2014).

In addition to their roles in fission and fusion, Drp1, Mfns and Opa1 have been implicated in a variety of other processes. For example, Drp1 has been shown to facilitate the induction of apoptosis whereas Opa1 was shown to affect the stability of cristae junction in inner mitochondrial membrane. Finally, Mfn2 also tethers mitochondria to the endoplasmic reticulum (ER) to mediate Ca2+ uptake (de Brito, 2008). However, the molecular mechanisms underlying these non-canonical functions are less well studied (Sandoval, 2014).

In an unbiased screen designed to identify essential genes that affect neuronal function, the first mutant allelic series was identified of Marf in Drosophila. This study exploits these mutants to determine how loss of Marf affects mitochondrial transport when compared to Drp1 loss. Surprisingly, NMJ defects were identified only in Marf mutants but not in Drp1 mutants. These defects are regulated non-cell autonomously by steroid-hormones produced in ring glands (RG), a major endocrine organ in insects. Through expression of human MFN1 or MFN2 in Marf mutant RG, it was shown that MFN1 and MFN2 have both distinct and complementary roles (Sandoval, 2014).

How does loss of fission or fusion affect mitochondrial function? In the absence of fusion mixing of mitochondrial DNA and proteins may be severely impaired. Given that mitochondrial proteins are in an environment rich in oxygen radicals, lack of fusion may cause more damage than when fission is impaired. Simply stated, loss of fusion proteins like Marf, MFN1 or MFN2 may cause more severe phenotypes than the loss of a fission protein like Drp1. Moreover, proteins like Marf and Drp1 may perform other functions that are not directly related to fusion or fission, and hence affect other processes. Based on a careful phenotypic comparison of loss of Marf and Drp1 in Drosophila many similarities and differences were found (Sandoval, 2014).

Marf mutants display small mitochondria whereas Drp1 mutants exhibit large fused mitochondria. Interestingly, both mutants accumulate mitochondria in the cell body of the neurons and the proximal axonal segments. In Drp1 mutants, the mitochondria seem to be severely elongated in axons where they fail to reach the NMJs, as previously described. The impairment in axonal transport is thought to be due to the fact that the mitochondria are hyperfused and cannot easily be transported. Indeed, loss of Marf in Drp1 mutants can restore mitochondrial trafficking proximally but distal axonal trafficking is still impaired. In Marf mutants, even though mitochondria are small and can enter the axons, the numbers of mitochondria that travel distally toward the NMJs are dramatically reduced. Hence, loss of Marf impairs mitochondrial trafficking and longer axons are more severely affected than shorter axons. Since longer axons are more severely affected in CMT2A patients, defects in mitochondrial trafficking may be at the root of some of the phenotypes associated with the disease (Sandoval, 2014).

Mfn2 has been implicated in axonal transport via binding to Miro2. Indeed, knockdown of MIRO2 in cultured vertebrate neurons affects mitochondrial transport in an identical fashion as loss of MFN2. However, the severity of mitochondrial transport that observed in Marf mutants is much less pronounced than what has been described in dmiro mutants and what was observe when dmiro is lost. Moreover, removal of dmiro in Marf mutants dramatically enhances the Marf phenotype and almost abolishes axonal localization of mitochondria, arguing that Marf cannot be solely responsible for mitochondrial transport in Drosophila (Sandoval, 2014).

A comparison of the presence of mitochondria at NMJ synapses shows that Marf mutants have fewer mitochondria than Drp1 mutants. Moreover, Marf mutants but not Drp1 mutants display a severe increase in small clustered boutons. The small and clustered boutons have also been observed in other mutants like endophilin, synaptojanin, eps15, dap 160, flower and dmiro. However, unlike in Marf mutants, the bouton phenotypes are fully rescued by neuronal expression of the cognate protein within MN in the above mentioned mutants. Moreover, knockdown of Marf in neuron, muscle or glia does not recapitulate the bouton phenotype observe in Marf mutants, suggesting a unique cell non-autonomous requirement of Marf for proper NMJ morphology (Sandoval, 2014).

Marf mutants exhibit two obvious phenotypes at NMJs: a severe depletion of mitochondria and a doubling of the number of boutons combined with a severe reduction in size whereas Drp1 mutants only exhibit a severe reduction in mitochondria. However, electrophysiological studies show that loss of Marf does not affect basal synaptic transmission similar to what is observed in Drp1 mutants. Both respond similarly to wild type NMJs when stimulated at 0.2 Hz and both show a progressive run down at 10 Hz when compared to controls. Moreover, endocytosis using FM1-43 and 60 mM K+ is not impaired in Marf and Drp1 mutants, suggesting a defect in reserve pool mobilization in both mutants. The data also show that the bouton defects observed in Marf mutants do not contribute to the run down in synaptic transmission since Drp1 boutons are normal in number and size yet also have a run down in synaptic transmission (Sandoval, 2014).

Loss of Marf in ring glands (RG) recapitulates the bouton phenotype observed in Marf mutants and expression of Marf in RG fully rescues this phenotype. Interestingly, both Marf and Opa1 are required for steroid hormone production and both lead to extended larval lifespan when knocked down in the RG only (8-10 days), whereas Drp1 mutations do not affect steroid hormone synthesis. Reduction of ecdysone production by knockdown of the prothoracicotropic hormone receptor (torso) in the RG also leads to an extended larval lifespan (9 days) and an increased growth of NMJs. Interestingly, knockdown of Drosophila SUMO (dsmt3) in RG lead to a defect in cholesterol import in the RG, reduced 20E levels and an extended larval lifespan (19 days). Hence, the severe reduction in ecdysone synthesis in Marf mutant RG underlies the prolonged larva stages and NMJ morphological defects (Sandoval, 2014).

The reduction in the number of LDs in RGs when Marf is lost suggests that these RGs are unable to store cholesterol. This storage of cholesterol esters probably permits the RG to produce large amounts of ecdysone when needed, especially at the larval stage and larval to pupal transitions. Cholesterol storage and steroid hormone biosynthesis requires both the ER and mitochondria in vertebrates but loss of MFN1 or MFN2 have not been shown to affect LD synthesis. Defects of anchoring mitochondria to the ER and LDs in Marf RGs argue that these defects lead to the loss of LD and production of ecdysone. In agreement with this hypothesis, expression of human MFN2, which tethers ER to mitochondria, in Marf mutants restores LD synthesis and organelle contacts. Moreover, expression of human MFN2 in RNAi mediated Marf knockdown in neurons and muscles rescues ER morphology and stress. However, MFN2 expression alone in Marf mutant RG did not restore ecydsone synthesis, arguing that there are other mitochondrial defects associated with the loss of Marf (Sandoval, 2014).

The current data show that co-expression of human MFN1 and MFN2 fully rescue the observed phenotypes in Marf mutants. Although RG-specific expression of MFN1 in Marf mutants did not restore LD numbers or organelle contacts, MFN1 is still necessary for ecdysone synthesis together with MFN2, suggesting a role downstream of cholesterol ester storage for both proteins. Moreover, knockdown of Opa1 in RG did not alter LD numbers but causes reduced 20E levels and aberrant NMJs. Opa1 resides within the inner mitochondrial membrane, suggesting its role in ecdysone synthesis is within the mitochondria. Ecdysone synthesis within the mitochondria requires two cytochrome p450 enzymes encoded by disembodied and shadow. Hence, it is likely that impairment in fusion but not fission affects the function of these enzymes (see Model of Marf dual function in steroid synthesis in the ring glands) (Sandoval, 2014).

Opa1 and MFN2 but not Drp1 have been implicated in vertebrate steroidogenesis. Interestingly, in placental trophoblast cells (BeWO) in culture the loss of OPA-1 promotes progesterone production by 70% whereas loss of MFN2 has been reported to lead to a 20% decrease in progesterone production. In contrast, testosterone production in MA-10 Leydig cells was unaffected by loss of OPA1 whereas loss of MFN2 did affect testosterone production by 40% in MA-10 Leydig cells. Hence, in both vertebrate endocrine cells, loss of MFN2 or OPA-1 affected steroids very differently as was observe very similar phenotypes associated with the loss of either protein in the current study. This study also suggests that MFN2 functions upstream of cholesterol entry into the mitochondria at the cholesterol storage stage, since MFN2 restores LD synthesis in Drosophila RG. However, rescuing LD production is not sufficient to restore ecdysone synthesis, suggesting a secondary defect (see Model of Marf dual function in steroid synthesis in the ring glands). In summary, these data indicate that MFN1 and MFN2 have separate functions in vivo that are integrated in a single protein in fly Marf (Sandoval, 2014).

Dissociation of mitochondrial from sarcoplasmic reticular stress in Drosophila cardiomyopathy induced by molecularly distinct mitochondrial fusion defects

Mitochondrial dynamism (fusion and fission) is responsible for remodeling interconnected mitochondrial networks in some cell types. Adult cardiac myocytes lack mitochondrial networks, and their mitochondria are inherently “fragmented”. Mitochondrial fusion/fission is so infrequent in cardiomyocytes as to not be observable under normal conditions, suggesting that mitochondrial dynamism may be dispensable in this cell type. However, it has been previously reported that cardiomyocyte-specific genetic suppression of mitochondrial fusion factors Optic atrophy 1 (Opa1) and Mitofusin (Marf) evokes cardiomyopathy in Drosophila hearts. Fusion-mediated remodeling of mitochondria may be critical for cardiac homeostasis, although never directly observed. Alternately, inner membrane Opa1 and outer membrane mitofusin/MARF might have other as-yet poorly described roles that affect mitochondrial and cardiac function. This study compared heart tube function in three models of mitochondrial fragmentation in Drosophila cardiomyocytes: Drp1 expression, Opa1 RNAi, and mitofusin MARF RNA1. Mitochondrial fragmentation evoked by enhanced Drp1-mediated fission did not adversely impact heart tube function. In contrast, RNAi-mediated suppression of either Opa1 or mitofusin/MARF induced cardiac dysfunction associated with mitochondrial depolarization and ROS production. Inhibiting ROS by overexpressing superoxide dismutase (SOD) or suppressing ROMO1 prevented mitochondrial and heart tube dysfunction provoked by Opa1 RNAi, but not by mitofusin/MARF RNAi. In contrast, enhancing the ability of endoplasmic/sarcoplasmic reticulum to handle stress by expressing Xbp1 rescued the cardiomyopathy of mitofusin/MARF insufficiency without improving that caused by Opa1 deficiency. The study concludes that decreased mitochondrial size is not inherently detrimental to cardiomyocytes. Rather, preservation of mitochondrial function by Opa1 located on the inner mitochondrial membrane, and prevention of ER stress by mitofusin/MARF located on the outer mitochondrial membrane, are central functions of these “mitochondrial fusion proteins” (Bhandari, 2015).

By performing a side-by-side detailed comparison of cardiomyopathies provoked by interrupting fusion of either the outer or inner mitochondrial membranes, this study identified distinct cellular mechanisms for the different molecular lesions. RNAi-mediated suppression of outer mitochondrial membrane mitofusin/MARF and inner mitochondrial membrane Opa1 provokes similar overt phenotypes: in both models mitochondrial size is approximately halved, the proportion of depolarized (sick) mitochondria increases to ~ 40%, mitochondrial ROS production is comparably greater (~ 50%), and the heart tubes exhibit similar reductions in fractional shortening. However, the cardiac defect caused by Opa1 deficiency is readily corrected by attacking the disease at the level of mitochondrial ROS production, through SOD expression or ROMO1 suppression. Indeed, mitochondrial structural and functional abnormalities are also improved by these genetic maneuvers, suggesting that they interrupt a vicious cycle of ROS-induced mitochondrial degeneration provoked by Opa1 insufficiency. Thus, interrupting endogenous mitochondrial ROS production greatly abrogates both the mitotoxicity and the cytotoxicity evoked by Opa1 suppression. This suggests a central role for mitochondrial degeneration in the Opa1-deficient fly heart model and, by extension, other heart diseases caused by defective inner mitochondrial membrane fusion proteins (Bhandari, 2015).

Whereas mitochondrial size and polarization status are similarly impaired in mitofusin/MARF insufficient heart tubes, neither of the mitochondrial-targeted interventions directed at reducing ROS, both of which rescue Opa1 deficient hearts, improve the cardiomyopathy induced by mitofusin/MARF deficiency. Indeed, whereas transgenic expression of SOD1 or SOD2 normalizes both ROS and heart tube function in Parkin-deficient heart tubes and Opa1-deficient heart tubes, it is remarkable that SOD fails to improve ROS levels in the mitofusin/MARF deficient heart tubes. Together with the original observation of transient heart tube functional improvement with SOD1, these observations suggest that there is “ROS escape” in the mitochondrial fusion impaired model that confers resistance to SOD expression or ROMO1 suppression. Instead, heart tube function is normalized without improving either mitochondrial structural or functional abnormalities by genetically enhancing the cardiomyocytes' ability to handle ER stress through XBP1 expression. These results, although surprising, are consistent with an essential role for mitofusin-mediated mitochondrial-ER/SR cross-talk in managing the ER stress response as proposed earlier in heart and in other tissues (Bhandari, 2015).

Previously, the consequences of Opa1 deficiency on Drosophila eye phenotypes have been found to be rescuable with SOD1, which is in accordance with findings of this study. Another study in Drosophila neurons and skeletal muscle also supports an important role for mitofusins, but not Opa1, as modulators of ER stress. However, only human Mfn2, and not human Mfn1, can correct abnormalities induced by mitofusin/MARF suppression in flies. This contrasts with findings in this study that cardiac-specific expression of either human Mfn1 or Mfn2 will fully correct cardiomyopathy induced by cardiomyocyte-specific MARF RNAi. Furthermore, ER dysmorphology has been found in MARF-deficient fly tissues, which was not detected in MARF-deficient heart tubes. It is likely either that the heart has different requirements for mitofusins and ER/SR morphology, or that the powerful tinman gene promoter used for cardiomyocyte-specific gene manipulation confers different expression characteristics to the heart models. Either way, the overall conclusions regarding a role of ER stress in mitofusin deficiency are in agreement (Bhandari, 2015).

Cardiomyocyte mitochondria are the Oompa-Loompas of the heart (with apologies to Roald Dahl): They are diminutive, structurally homogenous, and frequently overlooked despite toiling endlessly behind the scenes to keep the place running. Research has tended to focus on the mitochondrial work product (cardiac metabolism) and the means by which the general mitochondrial population is sustained (through biogenesis), rather than the fate of individual organelles. Indeed, individual cardiomyocyte mitochondria seem hardly worthy of observation, being monotonously similar in appearance and lacking the morphometric remodeling or intra-cellular mobility that has sparked detailed investigations (and visually engaging movie clips) in other cell types. The data presented in this study emphasize that (for mitochondria as well as Oompa-Loompas) size is not the critical determinant of function; it is literally what is inside that counts. Accordingly, one should eschew generalizations and extrapolations of mitochondrial status and dysfunction based strictly on morphometry (Bhandari, 2015).

Regulation of mitochondrial morphology and function by stearoylation of TFR1

Mitochondria are involved in a variety of cellular functions, including ATP production, amino acid and lipid biogenesis and breakdown, signalling and apoptosis. Mitochondrial dysfunction has been linked to neurodegenerative diseases, cancer and ageing. Although transcriptional mechanisms that regulate mitochondrial abundance are known, comparatively little is known about how mitochondrial function is regulated. This study identifies the metabolite stearic acid (C18:0), which was deficient in Elovl6 (Baldspot) mutant flies, and human transferrin receptor 1 (TFR1; also known as TFRC) as mitochondrial regulators. A signalling pathway was elucidated whereby C18:0 stearoylates TFR1, thereby inhibiting its activation of JNK signalling. This leads to reduced ubiquitination of mitofusin via HECT, UBA and WWE domain containing 1, E3 ubiquitin protein ligase (HUWE1), thereby promoting mitochondrial fusion and function. It was found that animal cells are poised to respond to both increases and decreases in C18:0 levels, with increased C18:0 dietary intake boosting mitochondrial fusion in vivo. Intriguingly, dietary C18:0 supplementation can counteract the mitochondrial dysfunction caused by genetic defects such as loss of the Parkinson's disease genes Pink or Parkin in Drosophila. This work identifies the metabolite C18:0 as a signalling molecule regulating mitochondrial function in response to diet (Senyilmaz, 2015).

Vimar is a novel regulator of mitochondrial fission through Miro

As fundamental processes in mitochondrial dynamics, mitochondrial fusion, fission and transport are regulated by several core components, including Miro. As an atypical Rho-like small GTPase with high molecular mass, the exchange of GDP/GTP in Miro may require assistance from a guanine nucleotide exchange factor (GEF). However, the GEF for Miro has not been identified. While studying mitochondrial morphology in Drosophila, it was incidentally observed that the loss of vimar, a gene encoding an atypical GEF, enhanced mitochondrial fission under normal physiological conditions. Because Vimar could co-immunoprecipitate with Miro in vitro, it was speculated that Vimar might be the GEF of Miro. In support of this hypothesis, a loss-of-function (LOF) vimar mutant rescued mitochondrial enlargement induced by a gain-of-function (GOF) Miro transgene; whereas a GOF vimar transgene enhanced Miro function. In addition, vimar lost its effect under the expression of a constitutively GTP-bound or GDP-bound Miro mutant background. These results indicate a genetic dependence of vimar on Miro. Moreover, mitochondrial fission was found to play a functional role in high-calcium induced necrosis, and a LOF vimar mutant rescued the mitochondrial fission defect and cell death. This result can also be explained by vimar's function through Miro, because Miro's effect on mitochondrial morphology is altered upon binding with calcium. In addition, a PINK1 mutant, which induced mitochondrial enlargement and had been considered as a Drosophila model of Parkinson's disease (PD), caused fly muscle defects, and the loss of vimar could rescue these defects. Furthermore, it was found that the mammalian homolog of Vimar, RAP1GDS1, played a similar role in regulating mitochondrial morphology, suggesting a functional conservation of this GEF member. The Miro/Vimar complex may be a promising drug target for diseases in which mitochondrial fission and fusion are dysfunctional (Ding, 2016).

Mitochondrial fission, fusion and transport play important roles for the function of this organelle. The balance between fusion and fission controls mitochondrial morphology, which is mediated by series of large dynamin-related GTPases. Among these GTPases, mitofusin1/mitofusin2 (MFN1/MFN2) and optic atrophy protein1 (OPA1) are the core components that are responsible for mitochondrial fusion, whereas dynamin-related protein 1 (Drp1) is the core component that is responsible for mitochondrial fission. In addition to these GTPases in dynamin-related family, mitochondrial Rho (Miro), an atypical member of the Rho small GTPase family, has a well-known function of transporting the mitochondria along microtubules. Miro also regulates mitochondrial morphology via inhibition of fission under physiological Ca2+ conditions, although the mechanism is not that clear. Large GTPases such as dynamin-like GTPase family members hydrolyze GTP and exchange GTP and GDP without the assistance from other regulators. However, members of the small GTPase family often require other proteins to help release their tightly bound GDP or enhance their low GTPase activities. These proteins are referred to as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively. To date, most small GTPases require unique GEFs or GAPs (Ding, 2016).

An understanding of the regulation of mitochondrial dynamics may help address many human diseases. For instance, mutations in OPA1 or MFN2 result in dominant optic atrophy or Charcot-Marie-Tooth neuropathy type 2A. Abnormal mitochondrial fission also promotes aging and cell death. In necroptosis, the formation of the necrosome promotes mitochondrial fission through dephosphorylation of Drp1. In neuronal excitotoxicity, calcium ions are overloaded, resulting in reduced levels of the MFN2 protein, which enhances mitochondrial fission and leads to neuronal necrosis In addition, other components such as Miro may participate in this process. Miro has two EF hand motifs that bind calcium; thus, Miro can couple calcium increase with reduced mitochondrial motility to meet the locally increased energy demands. Interestingly, Miro also promotes fission in the presence of excess calcium, which is distinct from its inhibitory role in fission under normal calcium concentrations. It is unclear whether Miro plays a functional role in neuronal necrosis (Ding, 2016).

The mitochondrial morphology represents a transient balance between mitochondrial fusion and fission. Using a systematic genetic screen in yeast covering approximately 88% of genes, 117 genes that regulate mitochondrial morphology were identified. Similarly, a screen of 719 genes that are predicted to encode mitochondrial proteins in worms demonstrated that more than 80% of these genes regulate mitochondrial morphology. Although many genes may regulate mitochondrial morphology, their relationships to the core mitochondrial fusion and fission components are unclear (Ding, 2016).

In studying mitochondrial morphology, it was accidently discovered that the loss of vimar (visceral mesodermal armadillo-repeats), which encodes an atypical GEF, promoted mitochondrial fission in Drosophila flight muscle cells. Furthermore, it was found that vimar was capable of interacting with Miro in vitro. Genetically, vimar required normal GDP- or GTP-bound activity of Miro to affect mitochondrial morphology, suggesting vimar is likely the Miro GEF. In addition, it was found that the Miro/Vimar complex suppressed mitochondrial fission during necrosis and mitochondrial fusion in PINK1 mutant model of Parkinson's disease (PD), making vimar a potential drug target (Ding, 2016).

Mitochondrial function can be assessed by the enzymatic activity of citrate synthase (CS), the first enzyme in the Krebs cycle that converts acetyl-CoA and oxaloacetate to citrate. In cultured Drosophila S2 cells, vimar knock down by RNAi resulted in reduced CS activity, indicating that vimar may positively regulate mitochondrial function. Because mitochondrial fission has generally been associated with reduced mitochondrial respiration, the decreased CS activity may be a result of mitochondrial fission. Consistent with this notion, the results demonstrated that the LOF of vimar promoted mitochondrial fission. In addition, a GOF vimar transgene had a minimal effect on mitochondrial morphology, indicating that vimar activity might be saturated under normal physiological conditions (Ding, 2016).

Because Vimar has been predicted to be a GEF, it was hypothesized that Vimar may regulate mitochondrial morphology by affecting a small GTPase, which requires a GEF to help with the GTP/GDP exchange process. Interestingly, Miro is one such small GTPase that is known to play important roles in mitochondrial fission and transport. It is proposed that Vimar and Miro may function as a complex. First, a fraction of the Vimar protein was localized to the mitochondria, possibly indicating a functional role on mitochondria. Interestingly, the mitochondrial localization of Vimar seems not dependent on Miro, because LOF Miro did not affect the mitochondrial fraction of Vimar. This indicates that Vimar may directly bind with mitochondria or through other scaffolding proteins. Second, Vimar and Miro could physically interact with each other, at least in vitro. Their interaction seems not affected by the GTPase activity of Miro, because the constitutively GDP- or GTP-bound Miro mutants did not affect their interactions. Third, vimar genetically interacted with Miro. This included the result demonstrating that the LOF vimar mutant reduced the effect of Miro on mitochondrial fission inhibition and the GOF vimar transgene had the opposite effect. Moreover, in the constitutive GFP-bound or GDP-bound Miro mutants, the effect of the GOF or LOF vimar was abolished. Therefore, Vimar requires the normal GDP/GTP binding activity of Miro to function. It is also known that Miro1 overexpression increase mitochondrial size partially by suppression of the Drp1 function. Consistently, increased mitochondrial fission in the LOF of Miro or vimar was abolished by loss of Drp1, suggesting the Miro/vimar complex depends on Drp1 to regulate mitochondrial morphology (Ding, 2016).

Familial PD caused by mutations in PINK1 or Parkin results in a series of mitochondrial dysfunctions, particularly the failure to eliminate damaged mitochondria through mitophagy. In these PINK1 or Parkin mutants, the key proteins involved in mitochondrial fusion and fission, such as Marf/Mitofusin and Miro, accumulate. In the PINK1 mutant flies, the flight muscle is damaged, resulting in wing posture defects. Similarly, it was observed that Miro overexpression in the flight muscle resulted in a strong wing posture defect. This result may explain the wing posture defect in the PINK1 mutant, in which the levels of the Miro protein are increased. This study demonstrated that the LOF of vimar could rescue the wing defect in the PINK1 mutant, consistent with the hypothesis that vimar functions through Miro (Ding, 2016).

When the intracellular calcium level is high, Miro switches from promoting mitochondrial fission inhibition to enhancing mitochondrial fission. The mechanism for this switch is unclear, although alterations of Drp1 function could be one possibility. Interestingly, Gem1, the yeast homolog of Miro GTPase, has been reported to function as a negative regulator for ER-mitochondria contacts, where Drp1 aggregates and cleaves mitochondria into smaller units. This may serve as the mechanism for Miro to regulate mitochondrial morphology via Drp1. In addition to affect mitochondrial fission, Miro also regulates mitochondrial transport in a calcium dependent manner. For mitochondrial transport, Miro forms protein complexes with Milton, a kinesin adaptor, and with motor proteins, such as kinesin and dynein. In high calcium conditions, Miro alters its binding patterns and results in reduced transport activity. Based on these reports, it is proposed that the Miro/Vimar complex acts together to affect mitochondrial morphology: at normal condition, Miro/Vimar inhibits fission via Drp1; at high calcium state, Ca2+ bound Miro switches its function to promote fission. Indeed, Vimar responds to the calcium change in the same way as Miro. In addition, the data demonstrated that knocking down RAP1GDS1 and Miro1 increased mitochondrial fission and could rescue calcium overload induced necrosis, similar to the loss of Vimar or Miro in Drosophila. These data support the hypothesis that RAP1GDS1 is the mammalian homolog of Vimar, supporting a previous prediction (Ding, 2016).

Mitochondrial fission plays important role in apoptosis by promoting mitochondrial outer-membrane permeabilization (MOMP) to release cytochrome c from the mitochondria. The use of the Drp1 inhibitor mdivi to block fission has been shown to be an effective treatment for stroke, and the function of mitochondrial fission on necrotic cell death has been well documented. The uncertainty lies in the lack of genetic evidence and downstream mechanism of mitochondrial fission in necrosis. The current data demonstrated that mitochondrial fragmentation occurs in necrotic neurons, and the LOF Drp1 and vimar mutants both suppressed neuronal necrosis (Ding, 2016).

Much evidence suggests that the mitochondrial fusion and fission defects are directly linked to many human diseases, and strategies that target the Miro/vimar complex may affect a broad spectrum of diseases. For instance, mutations in the fragile X mental retardation 1 (FMR1) gene, which result from expansion of trinucleotide repeat in the 5' untranslated region, often cause enhanced mitochondrial fission and mental retardation syndrome. Likewise, aberrant mitochondrial fusion was observed in a Drosophila Alzheimer's disease model induced by the ectopic expression of a human tau mutant (tauR406W). In this case, the tau mutant may promote excessive actin stabilization to decrease Drp1 recruitment to the mitochondria, which results in excessive mitochondrial fusion and neurodegeneration. Due to the dual function of the Miro/Vimar complex in high-Ca2+ induced necrosis and PINK1 mutant induced PD, a drug to target this complex may benefit both disease states. As a modulator, it may be safer to target Vimar/ RAP1GDS1 (Ding, 2016).

Genetic analysis in Drosophila reveals a role for the mitochondrial protein p32 in synaptic transmission

Mitochondria located within neuronal presynaptic terminals have been shown to play important roles in the release of chemical neurotransmitters. In the present study, a genetic screen for synaptic transmission mutants of Drosophila has identified the first mutation in a Drosophila homolog of the mitochondrial protein P32. Although P32 is highly conserved and has been studied extensively, its physiological role in mitochondria remains unknown and it has not previously been implicated in neural function. The Drosophila P32 mutant, referred to as dp32EC1, exhibited a temperature-sensitive (TS) paralytic behavioral phenotype. Moreover, electrophysiological analysis at adult neuromuscular synapses revealed a TS reduction in the amplitude of excitatory postsynaptic currents (EPSC) and indicated that dP32 functions in neurotransmitter release. These studies are the first to address P32 function in Drosophila and expand the knowledge of mitochondrial proteins contributing to synaptic transmission (Lutas, 2012).

A genetic screen for synaptic transmission mutants in Drosophila isolated a new mutation in a Drosophila homolog of the mitochondrial protein P32, which represents the first P32 mutation in a multicellular organism. Although P32 is highly conserved and has been studied extensively, its physiological function in mitochondria remains unknown. This new mutant, referred to as dP32EC1, exhibited a temperature-sensitive (TS) paralytic behavioral phenotype. Moreover, electrophysiological analysis at adult neuromuscular synapses revealed a TS reduction in neurotransmitter release, indicating that dP32 serves an important function in synaptic transmission. Immunocytochemical analysis has shown that dP32 is located within presynaptic mitochondria, which are known to be important in ATP production and calcium signaling at synapses. Furthermore, the basic molecular and structural organization of synapses appears to be normal in the dP32 mutant, suggesting a direct role for this protein in synaptic function. At the molecular level, biochemical studies indicated conserved homomultimeric interactions of dP32 subunits. Finally, assessment of presynaptic mitochondrial function was examined in the dP32 mutant through measurement of ATP levels and imaging studies of mitochondrial membrane potential and presynaptic calcium. This work indicated that mitochondrial ATP production and membrane potential in the dP32 mutant resembled wild-type, whereas the mutant exhibited a TS increase in both resting and evoked presynaptic calcium concentration. Taken together, the preceding findings reveal a role for dP32 in synaptic transmission and mitochondrial regulation of presynaptic calcium (Lutas, 2012).

Mitochondrial localization of P32 proteins involves an N-terminal targeting domain that is cleaved from the mature targeted protein. Comparison of Drosophila and vertebrate P32 sequences indicates conservation of the proteolytic cleavage site in dP32. In the present study, an equivalent targeting function for the N-terminal domain of dP32 was demonstrated through its ability to mediate mitochondrial targeting. When the first 71 amino acids of dP32, including the proteolytic cleavage site, was fused to GCaMP3, this fusion protein (mito-GCaMP3) was efficiently targeted to mitochondria. Although only modest sequence conservation was observed between the N-terminal domains of dP32 and vertebrate P32 proteins, previous studies suggest that mitochondrial targeting domains vary in amino acid sequence but often share an amphipathic helical structure (Lutas, 2012).

Structural studies have established that P32 is a homotrimer in which monomers are arranged around a central pore in a donut-like structure. In the present study, homomultimerization of dP32 subunits was demonstrated in co-immunoprecipitation experiments. The trimeric structure of P32 exhibits a highly asymmetric charge distribution that creates a concentration of negatively charged residues along one side of the donut, raising the possibility that P32 may participate in calcium binding within the mitochondrial matrix. Notably, five residues that are spatially clustered to form a pocket on the negatively charged side of human P32, Glu-89, Leu-231, Asp-232, Glu-264, and Tyr-268, are identical in the Drosophila protein. Further genetic analysis may address the importance of these clustered residues in dP32 function at synapses (Lutas, 2012).

Several possible mechanisms of dP32 function in mitochondria and synaptic transmission were considered and investigated in this paper, most notably possible roles in supporting mitochondrial membrane potential, ATP production, and presynaptic calcium signaling. Among these, the observations favor a function for dP32 in mitochondrial mechanisms regulating presynaptic calcium. Although neurotransmitter release was reduced at restrictive temperatures in dP32EC1, the presynaptic calcium concentration was increased both at rest and in response to synaptic stimulation. It is of interest to consider why the increase in presynaptic calcium in dP32EC1 is TS in what appears to be a complete loss-of-function mutant. Previous studies at Drosophila larval neuromuscular synapses at elevated temperatures have observed a TS increase in resting cytosolic calcium and associated inhibition of neurotransmitter release. This calcium increase was enhanced by pharmacological inhibition of presynaptic calcium clearance mechanisms or genetic removal of presynaptic mitochondria, but it remained dependent on temperature. The present findings may reflect a similar TS process involving calcium-dependent inhibition of neurotransmitter release and dP32-dependent mitochondrial mechanisms. Efforts to further address these mechanisms were pursued by employing a calcium indicator targeted to the mitochondrial matrix, mito-GCaMP3. Although this approach was successful for imaging mitochondrial calcium transients elicited by motor axon stimulation in both WT and dP32EC1 at 20°C, robust calcium transients could not be observed in either genotype when the temperature was increased to the restrictive temperatures of 33° or 36° (Lutas, 2012).

The preceding observations suggest that sustained elevation of presynaptic calcium in the dP32 mutant may lead to reduced neurotransmitter release. Such a calcium-dependent mechanism has been reported previously in the squid giant synapse and attributed to calcium-dependent adaptation of the neurotransmitter release apparatus. Understanding the precise mechanism by which loss of dP32 impairs neurotransmitter release will require further investigation. One interesting question is how the absence of dP32 in the mitochondrial matrix leads to increased presynaptic calcium and whether this reflects the putative calcium binding capacity of this protein. Finally, while the present study is focused on the newly discovered role for P32 in neurotransmitter release, the resulting research materials are expected to facilitate in vivo analysis of P32 function in a broad range of biological processes (Lutas, 2012).

PINK1-mediated phosphorylation of Miro inhibits synaptic growth and protects dopaminergic neurons. in Drosophila

Mutations in the mitochondrial Ser/Thr kinase PINK1 cause Parkinson's disease. One of the substrates of PINK1 is the outer mitochondrial membrane protein Miro, which regulates mitochondrial transport. This study uncovered novel physiological functions of PINK1-mediated phosphorylation of Miro, using Drosophila as a model. Endogenous Drosophila Miro (DMiro) was replaced with transgenically expressed wildtype, or mutant DMiro predicted to resist PINK1-mediated phosphorylation. The expression of phospho-resistant DMiro in a DMiro null mutant background was found to phenocopy a subset of phenotypes of PINK1 null. Specifically, phospho-resistant DMiro increased mitochondrial movement and synaptic growth at larval neuromuscular junctions, and decreased the number of dopaminergic neurons in adult brains. Therefore, PINK1 may inhibit synaptic growth and protect dopaminergic neurons by phosphorylating DMiro. Furthermore, muscle degeneration, swollen mitochondria and locomotor defects found in PINK1 null flies were not observed in phospho-resistant DMiro flies. Thus, this study established an in vivo platform to define functional consequences of PINK1-mediated phosphorylation of its substrates (Tsai, 2014).

Mutations in the Ser/Thr kinase PINK1 (PTEN-induced Putative Kinase 1) cause Parkinson's disease (PD), one of the most common neurodegenerative disorders. Emerging evidence suggests that PINK1 functions upstream of another PD-associated protein, the E3 ubiquitin ligase Parkin, to clear damaged mitochondria via mitophagy. How PINK1 primes cytosolic Parkin for mitophagy remains unclear, although PINK1-mediated phosphorylation of Parkin or ubiquitin may be involved. Previous work has shown that PINK1, in cooperation with Parkin, also regulates mitochondrial trafficking by controlling turn-over of Miro (Wang, 2011), an outer mitochondrial membrane (OMM) protein that anchors the kinesin and dynein motors to mitochondria. Work in cultured cells has demonstrated that mitochondrial depolarization or damage stabilizes PINK1 on the OMM. Concomitantly, PINK1 phosphorylates Miro, which then activates proteasomal degradation of Miro in a Parkin-dependent manner and arrests mitochondrial transport. This may serve as a critical step in quarantining damaged mitochondria prior to their degradation via mitophagy. However, the physiological significance of PINK1-mediated phosphorylation of Miro in vivo has not yet been determined (Tsai, 2014).

Recent studies have shown that Mitofusin, another OMM protein, is also a common substrate for both PINK1 and Parkin. Mitofusin facilitates mitochondrial fusion, and mitochondrial damage rapidly degrades Mitofusion causing mitochondria to fragment prior to mitophagy. PINK1 also phosphorylates the anti-apoptotic protein Bcl-xL on the OMM of depolarized mitochondria, not to regulate mitophagy, but to prevent cell death. In addition to the PINK1 OMM substrates Miro, Mitofusin and Bcl-xL, PINK1 mediates phosphorylation of the mitochondrial chaperon TRAP1 and the Serine protease HtrA2, which are both located in the mitochondrial inter-membrane space. This wide range of the potential substrates of PINK1 suggests that it may have multiple cellular functions (Tsai, 2014).

The consensus target sequence for phosphorylation by PINK1 has remained elusive. To date, Miro is the only PINK1 mitochondrial substrate whose phosphorylation residues have been determined (b4 (Wang, 2011). Two Drosophila Miro (DMiro) peptides with a high degree of similarity to the human sequence were identified as potential targets of PINK1-mediated phosphorylation in vitro. This study has determined the critical role of PINK1 phosphorylation sites on DMiro for maintaining neuronal homeostasis and protecting dopaminergic (DA) neurons in vivo.

<>Although several phosphorylation substrates of the mitochondrial Ser/Thr kinase PINK1 have been identified, the precise functional consequences of PINK1-mediated phosphorylation in vivo remain unclear. This study report that in Drosophila PINK1 may inhibit mitochondrial movement and synaptic growth at larval NMJs, and protect DA neurons in adult brains, by phosphorylating the atypical GTPase DMiro (Tsai, 2014).

Drosophila is a robust genetic and cellular tool for modeling human neurodegenerative diseases. Loss of PINK1 in Drosophila mimics many aspects of PD pathology, including a severe loss of DA neurons, which is a hallmark of PD. However, few of the molecular and cellular mechanisms underlying the behavioral and cellular phenotypes of PINK1 null mutant flies have been clearly defined. This study identifies that DMiroS182A,S324A,T325A, which is predicted to resist PINK1-mediated phosphorylation, causes increased mitochondrial movement, synaptic overgrowth, and loss of DA neurons. All three of these defects are also observed in PINK1 null mutant flies. Hence, this work suggests that Miro is a crucial substrate for causing these phenotypes by mutant PINK1. This study opens a new door to fully dissect PINK1 functions by studying its individual substrates. Since PINK1-related hereditary PD shares symptomatic and pathological similarities with the majority of idiopathic PD, such work will advance understanding of the cellular and molecular underpinnings of PD's destructive path (Tsai, 2014).

Extensive studies using cell cultures have established a critical role for PINK1 in damage-induced mitophagy. PINK1/Parkin-dependent regulation of mitochondrial transport by controlling Miro protein levels on mitochondria is likely a key step prior to initiating mitophagy in cultured neurons. This study show that PINK1-mediated phosphorylation of DMiro is required for normal mitochondrial movement in axon terminals, synaptic growth, and the neuroprotection of DA neurons. Importantly, loss of PINK1-mediated phosphorylation of DMiro has no significant effect on the mitochondrial membrane potential, excluding the possibility that the observed phenotypic effects are due to an impairment of mitophagy and an accumulation of damaged mitochondria. Accordingly, under these conditions PINK1-mediated phosphorylation of DMiro may not be required for mitophagy. However, this does not necessarily contradict its mitophagic role; rather, this represents circumstances under which its mitophagic role is dispensable. It is tempting to speculate that an efficient regulation of mitophagy is more critical in aging neurons (Tsai, 2014).

These studies identify a conserved site in human and Drosophila Miro, MiroSer156/DMiroSer182, to be a main residue for PINK1-mediated phosphorylation. Additional conserved sites were found in DMiro that may have a cooperative role. Future studies determining their functions in mammalian systems are warranted to confirm if a similar regulatory mechanism is at play. This study suggests that these PINK1 phosphorylation sites in DMiro are not absolutely required for the subsequent Parkin-dependent degradation of DMiro, because when harsh treatment of mitochondrial uncoupler CCCP is applied, the phospho-resistant DMiroS182A,S324A,T325A is degraded. The failure of DMiroS182A,S324A,T325A to prevent degradation under this condition might be due to PINK1-mediated phosphorylation on other sites that promote DMiro degradation, or due to activation of additional mechanisms. In two recent studies, MiroS156A is significantly degraded by co-expression of PINK1 and Parkin in addition to CCCP treatment in Hela cells, or by overexpression of Parkin together with Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, another mitochondrial uncoupler) treatment in SH-SY5Y cells; whereas in a previous study, MiroS156A is resistant to degradation when only PINK1 or Parkin is individually expressed in HEK293T cells (Wang, 2011). This again suggests that if the PINK1/Parkin pathway is overwhelmingly activated, mutating the few known PINK1-mediated phosphorylation residues in Miro is not sufficient to prevent its degradation (Tsai, 2014).

Why is mitochondrial motility increased in "DMironull, da > DMiroS182A,S324A,T325A"? DMiroS182A,S324A,T325A is resistant to PINK1/Parkin-mediated degradation, which may lead to more DMiroS182A,S324A,T325A accumulation on mitochondria. Unexpectedly, DMiroS182A,S324A,T325A protein level in "DMironull, da > DMiroS182A,S324A,T325A" is not significantly upregulated as compared with DMirowildtype in "DMironull, da > DMirowildtype" using fly whole body lysates. It is likely that PINK1/Parkin-dependent degradation of Miro only occurs in certain cell types, at certain subcellular locations, on certain populations of mitochondria, or under certain circumstances, and thus it is hard to detect a dramatic change using whole body lysates or without overexpression of PINK1/Parkin. Future mechanistic study is needed to test these hypotheses, such as detecting Miro subcellular localization and expression levels in different cell types, in different developmental stages, and with different mitochondrial stresses (Tsai, 2014).

This work highlights the importance of a precise control of mitochondrial movement for neuronal health. Anterograde mitochondrial transport in axons is mediated by a conserved motor/adaptor complex, which includes the motor kinesin heavy chain (KHC), the adaptor protein Milton and the mitochondrial membrane anchor Miro. In the current model, Miro binds to Milton, which in turn binds to KHC recruiting mitochondria to the motors and microtubules. In addition to the transmembrane domain inserted into the OMM, Miro features a pair of EF-hands and two GTPase domains. Miro was also recently found to be a substrate of the Ser/Thr kinase PINK1 and of the E3 ubiquitin ligase Parkin, both mutated in PD. Thus, mitochondrial transport can be regulated by multiple signals upstream of Miro and the motor complex maintaining energy and Ca2+ homeostasis in neuronal processes and terminals. For example, loss of PINK1-mediated phosphorylation of DMiro increases local mitochondrial movement at NMJs. In turn, this may disrupt synaptic homeostasis leading to synaptic overgrowth by mechanisms yet to be identified. Similarly, the loss of DA neurons in adult brains could well be a consequence of impaired synaptic homeostasis together with an accumulation of dysfunctional mitochondria. Local signals that regulate mitochondrial transport through Miro must be crucial to supporting neuronal functions. This study elucidates a fundamental biological mechanism demanded by a healthy neuron (Tsai, 2014).

ERK regulates mitochondrial membrane potential in fission deficient Drosophila follicle cells during differentiation

Mitochondrial morphology regulatory proteins interact with signaling pathways involved in differentiation. In Drosophila oogenesis, EGFR signaling regulates mitochondrial fragmentation in posterior follicle cells (PFCs). EGFR driven oocyte patterning and Notch signaling mediated differentiation are abrogated when PFCs are deficient for the mitochondrial fission protein Drp1. It is not known whether fused mitochondrial morphology in drp1 mutant PFCs exerts its effects on these signaling pathways through a change in mitochondrial electron transport chain (ETC) activity. This study shows that aggregated mitochondria in drp1 mutant PFCs have increased mitochondrial membrane potential. Experiments were performed to assess the signaling pathway regulating mitochondrial membrane potential and how this impacts follicle cell differentiation. drp1 mutant PFCs were found to show an increase in phosphorylated ERK (dpERK) formed downstream of EGFR signaling. ERK regulates high mitochondrial membrane potential in drp1 mutant PFCs. PFCs depleted of ERK and drp1 are able to undergo Notch mediated differentiation. Notably mitochondrial membrane potential decrease via ETC inhibition activates Notch signaling at an earlier stage in wild type and suppresses the Notch signaling defect in drp1 mutant PFCs. Thus, this study shows that the EGFR pathway maintains mitochondrial morphology and mitochondrial membrane potential in follicle cells for its functioning and decrease in mitochondrial membrane potential is needed for Notch mediated differentiation (Tomer, 2018).

Intracellular vesicle trafficking plays an essential role in mitochondrial quality control

The Drosophila gene products Bet1, Slh and CG10144, predicted to function in intracellular vesicle trafficking, were previously found to be essential for mitochondrial nucleoid maintenance. This study shows that Slh and Bet1 co-operate to maintain mitochondrial functions. In their absence, mitochondrial content, membrane potential and respiration became abnormal, accompanied by mitochondrial proteotoxic stress, but without direct effects on mtDNA. Immunocytochemistry showed that both Slh and Bet1 are localized at the Golgi, together with a proportion of Rab5-positive vesicles. Some Bet1, as well as a tiny amount of Slh, co-fractionated with highly purified mitochondria, whilst live-cell imaging showed coincidence of fluorescently tagged Bet1 with most Lysotracker-positive and a small proportion of Mitotracker-positive structures. This 3-way association was disrupted in cells knocked down for Slh, although co-localized lysosomal and mitochondrial signals were still seen. Neither Slh nor Bet1 were required for global mitophagy or endocytosis, but prolonged Slh knockdown resulted in G2 growth arrest, with increased cell diameter. These effects were shared with knockdown of betaCOP but not of CG1044, Snap24 or Syntaxin6. These findings implicate vesicle sorting at the cis-Golgi in mitochondrial quality control (Gerards, 2018).

Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration

Reactive oxygen species (ROS) and mitochondrial defects in neurons are implicated in neurodegenerative disease. This study finds that a key consequence of ROS and neuronal mitochondrial dysfunction is the accumulation of lipid droplets (LD) in glia. In Drosophila, ROS triggers c-Jun-N-terminal Kinase (JNK) and Sterol Regulatory Element Binding Protein (SREBP) activity in neurons leading to LD accumulation in glia prior to or at the onset of neurodegeneration. The accumulated lipids were peroxidated in the presence of ROS. Reducing LD accumulation in glia and lipid peroxidation via targeted lipase overexpression and/or lowering ROS significantly delayed the onset of neurodegeneration. Furthermore, a similar pathway led to glial LD accumulation in Ndufs4 mutant mice with neuronal mitochondrial defects, suggesting that LD accumulation following mitochondrial dysfunction is an evolutionarily conserved phenomenon, and represents an early, transient indicator and promoter of neurodegenerative disease (Liu, 2015).

This study shows that neuronal mitochondrial defects that lead to elevated levels of ROS, induce activation of JNK and SREBP, which in turn elevate lipid synthesis in neurons and formation of LD in glial cells. These LDs contribute to and promote ND through elevated levels of lipid peroxidation. LDs form in glia prior to or at the onset of the appearance of obvious degenerative histological features in Drosophila and mice. Reducing the number and size of LD pharmacologically or genetically delays ND in the fly. This is the first indication that SREBP, lipid droplet biogenesis, and lipid metabolism play a role in the pathogenesis of several neurodegenerative diseases (Liu, 2015).

A growing body of evidence points to the importance of glial health and function in nervous system energy metabolism and homeostasis. Nevertheless, given the number and prevalence of different types of neurodegenerative diseases, very few reports have documented the presence of LDs in either neuron or glia in patients and in animal models. LD accumulation in the brain has been reported in cells that line the ventricles in the globus pallidus and substantia nigra in mutant mice lacking both subunits of the liver X receptor, apolipoprotein E, or a peroxisomal biogenesis factor (Pex5) . In addition, in vitro studies using immortalized cell lines and explants show that LD may form and accumulate in glia under conditions of nutrient deprivation or lipopolysaccharide induced stress. However, LDs have not been shown to play an active role in neurodegenerative processes. Furthermore, LD accumulation has not been reported in patients with or animal models of Leigh syndrome (NDUFS4/Ndufs4, NDUFAF6/sicily), CMT-2A2 or HMSN6 (MFN2/Marf), and ARSAL (MARS2/Aats-met). The lack of neuropathological reports of LDs in animal models or in patients with ND may be attributed to the fact that LD accumulation is transient and mostly occur during presymptomatic stages of the disease (Liu, 2015).

Although these genes/mutants are implicated in very different mitochondrial processes, they exhibit a common phenotype of elevated levels of ROS, leading to LD accumulation. Similar morphological changes of glia have been reported under stress conditions. Interestingly, mid- and late-stage Ndufs4-/- mice exhibit CNS lesions in the same brain regions where the LD accumulate in early stage animals, showing a strong correlative relationship. Similarly, LD accumulation in Drosophila mutants occurs prior to or at the onset of physical signs of ND. Importantly, the delivery of AD4 is able to significantly ameliorate LD accumulation in Drosophila and delay the onset of ND in flies and mice. Hence, the molecular mechanisms underlying these phenotypes are likely to be conserved between these species and potentially also in higher organisms (Liu, 2015).

In the clinical setting, the prescription of antioxidants toward treatment of neurodegenerative diseases has been tested repeatedly on patients with neurodegenerative disorders, without compelling results. The LD accumulation phenotype in these mutants occurs prior to histopathological and physical signs of ND. A brief period of AD4 delivery prior to the onset of symptoms in mutant mice is effective in delaying onset of clinical signs. Thus, therapy with an effective antioxidant that penetrates the blood-brain barrier should be started early and sustained over long periods. In addition, pharmacological manipulation of JNK or lipid levels in the brain may serve as a potential therapy to delay the onset of ND. However, similar to antioxidant treatment, this may need to be administered at an early stage. Hence, early identification of potential ROS related neurological disease based on genetic/genomic diagnosis or by biomarkers may be critical. Since LD accumulation is one of the earliest presymptomatic changes that occurs in the nervous system, detection of LD itself or changes in neurometabolism may be a promising biomarker (Liu, 2015).

In summary, this study provides evidence for the role of altered lipid metabolism and a neuron-glia interplay that promotes ND. In some mitochondrial mutants, an upregulation of SREBP was observed, as well as lipid biogenesis and glial LD formation. The accumulation of LD is not sufficient to promote the ND process itself. However, in the presence of ROS the accumulated lipids are peroxidated and promote ND, possibly by promoting the release of lipids from LD, elevating the cytoplasmic load, and causing a progressive loss of LD. Hence, the synergistic effects of increased lipid synthesis and/or LD accumulation in combination with elevated ROS and lipid peroxidation promote ND. Finally, it was shown that LD accumulation occurs at the onset or precedes ND in flies and mice, suggesting that LD and changes in lipid metabolism in the nervous system may be a promising biomarker to identify brain regions susceptible to but not yet exhibiting symptoms of ND (Liu, 2015).

Vitamin K2 Prevents Lymphoma in Drosophila

Previous studies have established the anticancer effect of vitamin K2 (VK2). However, its effect on lymphoma induced by UBIAD1/heix mutation in Drosophila remains unknown. Therefore, this study aimed to develop an in vivo model of lymphoma for the precise characterization of lymphoma phenotypes. This study also aimed to improve the understanding of the mechanisms that underlie the preventative effects of VK2 on lymphoma. The results demonstrated that VK2 prevents lymphoma by acting as an electron carrier and by correcting the function and structure of mitochondria by inhibiting mitochondrial reactive oxygen species production mtROS. This work identifies mitochondria as a key player in cancer therapy strategies (Dragh, 2017).

Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism

Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. This study sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells (Schell, 2017).

It was first observed almost 100 years ago that, unlike differentiated cells, cancer cells tend to avidly consume glucose, but not fully oxidize the pyruvate that is generated from glycolysis. This was originally proposed to be due to dysfunctional or absent mitochondria, but it has become increasingly clear that mitochondria remain functional and critical. Mitochondria are particularly important in proliferating cells because essential steps in the biosynthesis of amino acids, nucleotide and lipid occur therein. Most proliferating stem cell populations also exhibit a similar glycolytic metabolic program, which transitions to a program of mitochondrial carbohydrate oxidation during differentiation. The first distinct step in carbohydrate oxidation is import of pyruvate into the mitochondrial matrix, where it gains access to the pyruvate dehydrogenase complex (PDH) and enters the tricarboxylic acid (TCA) cycle as acetyl-CoA. The two proteins that assemble to form the mitochondrial pyruvate carrier (MPC) have been recently described. This complex is necessary and sufficient for mitochondrial pyruvate import in yeast, flies and mammals, and thereby serves as the junction between cytoplasmic glycolysis and mitochondrial oxidative phosphorylation. Decreased expression and activity of the MPC underlies the glycolytic program in colon cancer cells in vitro, and forced re-expression of the MPC subunits increased carbohydrate oxidation and impaired the ability of these cells to form colonies in vitro and tumours in vivo. This impairment of tumorigenicity was coincident with a loss of key markers and phenotypes associated with stem cells. This has prompted an examination of whether glycolytic non-transformed stem cells might also exhibit low MPC expression and mitochondrial pyruvate oxidation, which must increase during differentiation (Schell, 2017).

The role of the MPC was studied in the ISCs of the fruit fly Drosophila, which share key aspects of their biology with mammalian ISCs. Both MPC1 and MPC2 are expressed in all four cell types of the intestine, with the lowest level of expression in the ISCs and the highest expression in the differentiated enteroendocrine cells. Confocal imaging of intestines dissected from dMPC1 mutants revealed that the epithelium exhibits multilayering unlike the normal single-cell layer seen in controls. This is a classic overgrowth phenotype that is associated with oncogene mutations in Drosophila. Accordingly, MARCM clonal analysis was used to determine whether a specific loss of the MPC in ISCs leads to an increase in their proliferation. On average, newly divided GFP-marked dMPC1 mutant clones are more than twofold larger than control clones, which were generated in parallel using a wild-type chromosome, indicating that the MPC is required in the ISC lineage to suppress proliferation. Because GFP-marked clones could include cells that differentiate into mature enterocytes or enteroendocrine cells, clonal analysis was conducted in the absence of Notch, thereby blocking ISC differentiation. Under these conditions, an approximately twofold increase was observed in the size of dMPC1 mutant ISC clones. To confirm and extend these results, MPC function was specifically disrupted in the ISCs by using the Dl-GAL4 driver in combination with UAS-GFP, which facilitates stem cell identification. Once again, approximately twofold more GFP-marked stem cells were observed relative to controls when either dMPC1 or dMPC2 expression was disrupted by RNA-mediated interference (RNAi) along with increased ISC proliferation as detected by staining for phosphorylated histone H3 (pHH3). Similar results were obtained when RNAi was targeted to the E1 or E2 subunits of PDH to specifically disrupt the next step in mitochondrial pyruvate oxidation. Importantly, an opposite phenotype was seen when Ldh was reduced by RNAi in the ISCs or progenitor cells. Ldh suppression is known to result in a significant increase in pyruvate levels, which can promote pyruvate oxidation. Taken together with the results with Pdh RNAi, these observations support the model that the MPC limits stem cell proliferation through promoting oxidative pyruvate metabolism in the mitochondria. It also appears to be sufficient as specific overexpression of MPC1 and MPC2 in ISCs or progenitors caused a reduction in stem cell proliferation, the opposite of the loss-of-function phenotype. This can be seen in either Pseudomonas-infected intestines, which undergo rapid stem cell proliferation, or under basal conditions in aged animals. Consistent with this, MPC overexpression under basal conditions had no effects on intestinal morphology, while the intestines from infected flies displayed a fully penetrant size reduction, which is probably due to the inability of ISCs to maintain tissue homeostasis. Taken together, these results demonstrate that mitochondrial pyruvate uptake and metabolism is both necessary and sufficient in a stem cell autonomous manner to regulate ISC proliferation and maintain intestinal homeostasis in Drosophila (Schell, 2017).

Studies in Drosophila, intestinal organoids and mice provide strong evidence that the MPC is necessary and sufficient in a cell autonomous manner to suppress stem cell proliferation. Consistently, this study has demonstrated that ISCs maintain low expression of the subunits that comprise the MPC, which enforces a mode of carbohydrate metabolism wherein glucose is metabolized in the cytosol to pyruvate and other biosynthetic intermediates. This glycolytic metabolic program appears to be sufficient to drive robust and continuous stem cell proliferation. High mitochondrial content was observed in ISCs, which must be geared primarily toward biosynthetic functions and/or oxidation of other substrates such as fatty acids. Increased fatty acids, the metabolism of which is enhanced in MPC-deficient and MPC-inhibited organoids, have been shown to promote ISC expansion and proliferation via enhanced beta-catenin signalling and increasing tumour-initiating capacity. MPC expression increases following differentiation, consistent with the shift in demand from macromolecule biosynthesis to ATP production in support of post-mitotic differentiated cell function. A similar switch in MPC expression can be seen following differentiation of embryonic stem cells, haematopoietic stem cells and trophoblast stem cells. Conversely, MPC expression is reduced after reprogramming fibroblasts to induced pluripotent stem cells. This suggests that the effects of altering pyruvate flux that wad observed in this study might not be restricted to ISCs, but instead be representative of similar effects on multiple stem cell populations. Interestingly, Myc is known to drive a metabolic program that is similar to that observed following MPC loss, characterized by increased glycolysis and reliance on glutamine and fatty acid oxidation with reduced glucose oxidation. This suggests that Myc may play a role in repressing the MPC in stem cells, possibly acting downstream of Wnt/beta-catenin signalling. Consistent with this, Myc and its repressive co-factors localize to the Mpc1 promoter and Myc expression is strongly anti-correlated with Mpc1 expression (Schell, 2017).

Taken together, these studies demonstrate that changes in the MPC and mitochondrial pyruvate metabolism are required to properly orchestrate the proliferation and homeostasis of intestinal stem cells. Importantly, this metabolic program, mediated at least partially by the MPC, appears to be instructive for cell fate, rather than a downstream consequence of cell fate. Future work will define the extent to which the results presented in this study relate to those showing that diet quality and quantity can modulate ISC behaviour. It is tempting to speculate that ISC metabolism is used as a signal for increased or decreased demand for intestinal epithelium. Perhaps of most importance will be to define the mechanisms whereby altered partitioning of pyruvate metabolism affects stem cell proliferation and fate. It is speculated that the robust changes that were observed in fatty acid oxidation and histone acetylation, which are probably downstream of altered metabolite utilization for acetyl-CoA production, play an important role. While the mechanisms are not as yet defined, these studies establish a paradigm wherein mitochondrial metabolism does not merely provide a permissive context for proliferation or differentiation, but rather plays a direct and instructive role in controlling stem cell fate (Schell, 2017).

Iron sulfur and molybdenum cofactor enzymes regulate the Drosophila life cycle by controlling cell metabolism

Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, a hypothesis is formulated that (1) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (2) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (3) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (4) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle (Marelja, 2018).

Mitochondrial retrograde signaling regulates neuronal function

Mitochondria are key regulators of cellular homeostasis, and mitochondrial dysfunction is strongly linked to neurodegenerative diseases, including Alzheimer's and Parkinson's. Mitochondria communicate their bioenergetic status to the cell via mitochondrial retrograde signaling. To investigate the role of mitochondrial retrograde signaling in neurons, mitochondrial dysfunction was induced in the Drosophila nervous system. Neuronal mitochondrial dysfunction causes reduced viability, defects in neuronal function, decreased redox potential, and reduced numbers of presynaptic mitochondria and active zones. Neuronal mitochondrial dysfunction stimulates a retrograde signaling response that controls the expression of several hundred nuclear genes. Drosophila hypoxia inducible factor alpha (HIFalpha) ortholog Similar (Sima) regulates the expression of several of these retrograde genes, suggesting that Sima mediates mitochondrial retrograde signaling. Remarkably, knockdown of Sima restores neuronal function without affecting the primary mitochondrial defect, demonstrating that mitochondrial retrograde signaling is partly responsible for neuronal dysfunction. Sima knockdown also restores function in a Drosophila model of the mitochondrial disease Leigh syndrome and in a Drosophila model of familial Parkinson's disease. Thus, mitochondrial retrograde signaling regulates neuronal activity and can be manipulated to enhance neuronal function, despite mitochondrial impairment (Cagin, 2015).

The human brain constitutes approximately 2% of body weight but consumes 20% of available oxygen because of its high energy demand. Mitochondria are abundant in neurons and generate the majority of cellular ATP through the action of the mitochondrial ATP synthase complex. Mitochondrial disorders are one of the most common inherited disorders of metabolism and have diverse symptoms, but tissues with a high metabolic demand, such as the nervous system, are frequently affected. The primary insult in all mitochondrial diseases is to mitochondrial function, but the etiology of these diseases is highly pleiotropic. This phenomenon is poorly understood, but suggests that the cellular response to mitochondrial dysfunction may be complex and vary between cell types and tissues (Cagin, 2015).

Mitochondrial retrograde signaling is defined as the cellular response to changes in the functional state of mitochondria. Mitochondrial retrograde signaling enables communication of information about changes in processes such as mitochondrial bioenergetic state and redox potential to the rest of the cell and is thus a key mechanism in cellular homeostasis. The best characterized retrograde responses involve mitochondrial dysfunction eliciting changes in nuclear gene transcription. In yeast, mitochondrial dysfunction causes changes in the expression of genes involved in supplying mitochondria with oxaloacetate and acetyl CoA, the precursors of α-ketoglutarate and glutamate, to compensate for failure of the tricarboxylic acid (TCA) cycle (Cagin, 2015).

In proliferating mammalian cell models, mitochondrial retrograde signaling is more diverse and involves increases in cytosolic-free Ca2+, leading to activation of Ca2+-responsive calcineurin, causing the up-regulation of genes controlling Ca2+ storage and transport. In addition to mitochondrial diseases, alterations in mitochondrial function are also associated with late onset neurodegenerative diseases such as Alzheimer's and Parkinson's. Thus, the neuronal response to mitochondrial function may be altered in these diseases and contribute to disease progression. However, neuronal-specific mitochondrial retrograde signaling is poorly understood and its role in neuronal homeostasis is completely unknown (Cagin, 2015).

This study has developed a neuronal-specific model of mitochondrial dysfunction in Drosophila and used this to characterize mitochondrial retrograde signaling in vivo. Retrograde signaling is shown to regulate neuronal function and can be manipulated to alleviate the effects of mitochondrial dysfunction in neurons (Cagin, 2015).

This study shows that the Drosophila HIFα ortholog Sima is potentially a key regulator of the mitochondrial retrograde response in the nervous system and that knockdown of Sima dramatically improves neuronal function in this and other models of mitochondrial dysfunction. Surprisingly, Sima activity in part causes the dysfunction of neurons containing defective mitochondria. Previous studies of Drosophila mutants in the regulatory and catalytic subunits of the mitochondrial DNA polymerase Polγ have demonstrated that loss of mtDNA replication in Drosophila causes mtDNA loss, reduced neuronal stem cell proliferation, and developmental lethality. To avoid the pleiotropic effects of using homozygous mutant animals, this study developed a neuronal-specific model of mitochondrial dysfunction. The phenotypes resulting from TFAM overexpression and expression of a mitochondrially targeted restriction enzyme were characterized, and both of these tools were used to model neuronal-specific mitochondrial dysfunction (Cagin, 2015).

Overexpression of mitochondrial transcription factor A (TFAM) results in mitochondrial dysfunction caused by inhibition of mitochondrial gene expression, rather than an alteration in mtDNA copy number. Overexpression of TFAM has been shown to have different effects depending on the cell type, model system, or ratio of TFAM protein to mtDNA copy number. The current results are consistent with in vitro studies and overexpression of human TFAM in mice and human cells, which have shown that excess TFAM results in the suppression of mitochondrial gene transcription. Ubiquitous expression of mitoXhoI causes early developmental lethality and that, although there was no significant mtDNA loss, the majority of mtDNA was linearized. Given that mtDNA is transcribed as two polycistronic mRNAs, a double-stranded break in coxI would block the transcription of the majority of mitochondrially encoded genes, resulting in severe mitochondrial dysfunction (Cagin, 2015).

Using a Drosophila motor neuron model, mitochondrial dysfunction was found to cause a reduction in the number of active zones, loss of synaptic mitochondria, and locomotor defects. Mitochondrial dysfunction caused by overexpression of PINK1 or Parkin decreases the rate of mitochondrial transport in vitro and in vivo. Furthermore, a recent study using KillerRed demonstrated that local mitochondrial damage results in mitophagy in axons. Therefore, the acute loss of synaptic mitochondria in the current model may result from defects in mitochondrial transport and/or mitophagy (Cagin, 2015).

Previous studies in mice have examined the effects of neuronal mitochondrial dysfunction by using mitoPstI expression, or targeted knockout of TFAM. Knockout of TFAM specifically in mouse dopaminergic neurons (the 'MitoPark' mouse model) causes progressive loss of motor function, intraneuronal inclusions, and eventual neuronal cell death. Interestingly, cell body mitochondria are enlarged and fragmented and striatal mitochondria are reduced in number and size in MitoPark dopaminergic neurons, suggesting that the effects of neuronal mitochondrial dysfunction are conserved in Drosophila and mammals. Larvae mutant for the mitochondrial fission gene drp1 have fused axonal mitochondria and almost completely lack mitochondria at the NMJ, similar to motor neurons overexpressing TFAM or expressing mitoXhoI (Cagin, 2015).

Adult drp1 mutant flies also have severe behavioral defects. Synaptic reserve pool vesicle mobilization is inhibited in drp1 mutant larvae because of the lack of ATP to power the myosin ATPase required for reserve pool tethering and release. Reserve pool vesicle mobilization is likely to be similarly affected in TFAM overexpressing or mitoXhoI-expressing motor neurons, which would result in locomotor defects in these animals (Cagin, 2015).

Interestingly, expression of the Arctic form of β-amyloid1-42 (Aβ) in Drosophila giant fiber neurons also leads to the depletion of synaptic mitochondria and decreased synaptic vesicles. Synaptic loss and alterations in neuronal mitochondrial morphology have also been observed in postmortem tissue from Alzheimer's disease patients. The parallels between these phenotypes and those in the current model suggest a common underlying mechanism (Cagin, 2015).

Using microarray analysis, this study found that mitochondrial dysfunction in neurons regulates the expression of hundreds of nuclear genes. The Drosophila CNS contains different neuronal subtypes, and glial cells, so the results of the microarray are heterogeneous, representing the pooled response to mitochondrial dysfunction throughout the CNS. Mitochondrial dysfunction was phenotypically characterized in motor neurons, but not all of the genes identified from the microarrays are expressed in motor neurons, e.g., Ilp3. The specific genes that are regulated differ depending on whether mitochondrial dysfunction results from TFAM overexpression or knockdown of ATPsynCF6. However, a core group of approximately 140 genes are similarly regulated in both conditions (Cagin, 2015).

Yeast mutants in different components of the TCA cycle result in differing retrograde responses and comparison of somatic cell hybrids (cybrids) carrying the A3243G mtDNA mutation with cybrids completely lacking mtDNA (ρ0 cells) showed overlapping but distinct gene expression profiles. Moreover, another study comparing cybrids with increasing levels of the A3243G mtDNA mutation showed markedly different alterations in nuclear gene expression, depending on the severity of mitochondrial dysfunction (Cagin, 2015).

Taken together, these data suggest that the cellular response to mitochondrial dysfunction is not uniform and adapts to the specific defect and severity of the phenotype. Therapeutic strategies targeting mitochondrial dysfunction in human disease may therefore need to be tailored to the specific mitochondrial insult. Concomitant with the current findings, previous studies have shown that in yeast, Drosophila, and mammalian-proliferating cells, retrograde signaling activates the expression of hypoxic/glycolytic genes and the insulin-like growth factor-1 receptor pathway to compensate for mitochondrial dysfunction. Rtg1 and Rtg3, the transcription factors that coordinate the mitochondrial retrograde response in yeast, are not conserved in metazoans. In mammalian proliferating cellular models, the retrograde response activates the transcription factors nuclear factor of activated T cells (NFAT), CAAT/enhancer binding protein δ (C/EBPδ), cAMP-responsive element binding protein (CREB), and an IκBβ-dependent nuclear factor κB (NFκB) c-Rel/p50. Whether these transcription factors regulate mitochondrial retrograde signaling in the mammalian nervous system is not known (Cagin, 2015).

HIFα/Sima is a direct regulator of LDH expression in flies and mammals, and this study found that Sima also regulates the expression of two other retrograde response genes, Thor and Ilp3, in the Drosophila nervous system. Importantly, Sima is required for the increase in Thor expression in response to mitochondrial dysfunction. Sima has been strongly implicated as a key regulator of mitochondrial retrograde signaling in Drosophila S2 cells knocked down for the gene encoding subunit Va of complex IV. sima, Impl3, and Thor expression were all increased in this model, and there is a significant overlap with the genes regulated in the current model (Cagin, 2015).

These data support the possibility that the Drosophila HIFα ortholog Sima is a key transcriptional regulator of neuronal mitochondrial retrograde signaling. HIFα is stabilized in hypoxia through the action of prolyl hydroxylases and this mechanism was thought to require ROS, but HIFα stabilization may in fact be ROS independent. In mammalian cells carrying the mtDNA A1555G mutation in the 12S rRNA gene, mitochondrial retrograde signaling has been shown to be activated by increased ROS, acting through AMPK and the transcription factor E2F1 to regulate nuclear gene expression. In the Drosophila eye, loss of the complex IV subunit cytochrome c oxidase Va (CoVa) causes decreased ROS. However, retrograde signaling upon loss of CoVa was not mediated by decreased ROS, but by increased AMP activating AMPK. Similarly, the small decrease in redox potential in neurons in response to mitochondrial dysfunction in the current model makes it unlikely that ROS are the mediator of the retrograde signal. Moreover, HIFα physically interacts with several transcriptional regulators including the Drosophila and mammalian estrogen-related receptor and Smad3, as well as its heterodimeric binding partner HIFβ, to regulate gene expression. Mitochondrial retrograde signaling may modulate these or other unidentified HIFα interactors and, thus, control HIF target gene expression without directly regulating HIFα (Cagin, 2015).

In cancer cell models, mitochondrial dysfunction promotes cell proliferation, increased tumourigenicity, invasiveness, and the epithelial-to-mesenchymal transition via retrograde signaling. In these models, inhibition of retrograde signaling prevents these tumourigenic phenotypes. Neuronal mitochondrial dysfunction in the current model causes a cellular response, resulting in a severe deficit in neuronal function. This response may have evolved to protect neurons, through decreased translation and increased glycolysis, from the short-term loss of mitochondrial function. Over longer periods, however, this response may be counterproductive because it results in decreased neuronal activity and locomotor function. Inhibition of neuronal mitochondrial retrograde signaling, through knockdown of Sima, dramatically improves neuronal function. Thus, mitochondrial retrograde signaling contributes to neuronal pathology and can be modified to improve the functional state of the neuron (Cagin, 2015).

Importantly, this intervention works without altering the primary mitochondrial defect. Knockdown of Sima not only abrogates the acute defects in neuronal function, but also suppresses the reduced lifespan caused by neuronal mitochondrial damage. The benefits of reduced Sima expression therefore extend throughout life. In addition to TFAM overexpression, this study also shows that Sima knockdown in neurons rescues a Drosophila model of the mitochondrial disease Leigh syndrome. However, Sima knockdown does not rescue the lethality caused by a temperature-sensitive mutation in coxI (Cagin, 2015).

Mitochondrial diseases are complex, and mutations in different COX assembly factors cause varying levels of COX deficiency in different tissues. The increasing number of Drosophila models of mitochondrial dysfunction will help to unravel the mechanisms underlying the varied pathology of mitochondrial diseases. Ubiquitous knockdown of Sima also partially restores the climbing ability of parkin mutant flies. The ability of reduced Sima expression to rescue both mitochondrial dysfunction and Parkinson's disease models reinforces the link between mitochondrial deficiency and Parkinson's and suggests that retrograde signaling may be a therapeutic target in Parkinson's disease. HIF1α inhibitors are in clinical trials for lymphoma and so, if the current findings can be replicated in mammalian models, HIF1α inhibitors may be candidates for repurposing to treat mitochondrial diseases and neurodegenerative diseases associated with mitochondrial dysfunction, such as Parkinson's disease (Cagin, 2015).

Shawn, the Drosophila homolog of SLC25A39/40, is a mitochondrial carrier that promotes neuronal survival

Mitochondria play an important role in the regulation of neurotransmission, and mitochondrial impairment is a key event in neurodegeneration. Cells rely on mitochondrial carrier proteins of the SLC25 family to shuttle ions, cofactors, and metabolites necessary for enzymatic reactions. Mutations in these carriers often result in rare but severe pathologies in the brain, and some of the genes, including SLC25A39 and SLC25A40, reside in susceptibility loci of severe forms of epilepsy. However, the role of most of these carriers has not been investigated in neurons in vivo. This study identified shawn, the Drosophila homolog of SLC25A39 and SLC25A40, in a genetic screen to identify genes involved in neuronal function. Shawn localizes to mitochondria, and missense mutations result in an accumulation of reactive oxygen species, mitochondrial dysfunction, and neurodegeneration. Shawn regulates metal homeostasis, and shawn mutants exhibit increased levels of manganese, calcium, and mitochondrial free iron. Mitochondrial mutants often cannot maintain synaptic transmission under demanding conditions, but shawn mutants do, and they also do not display endocytic defects. In contrast, shawn mutants harbor a significant increase in neurotransmitter release. These data provide the first functional annotation of these essential mitochondrial carriers in the nervous system, and suggest that metal imbalances and mitochondrial dysfunction may contribute to defects in synaptic transmission and neuronal survival (Slabbaert , 2016).

Cisplatin induces mitochondrial deficits in Drosophila larval segmental nerve
Cisplatin is an effective chemotherapy drug that induces peripheral neuropathy in cancer patients. In rodent dorsal root ganglion neurons, cisplatin binds nuclear and mitochondrial DNA (mtDNA) inducing DNA damage and apoptosis. Platinum-mtDNA adducts inhibit mtDNA replication and transcription leading to mitochondrial degradation. Cisplatin also induces climbing deficiencies associated with neuronal apoptosis in adult Drosophila. This study used Drosophila larvae that express GFP in the mitochondria of motor neurons to observe the effects of cisplatin on mitochondrial dynamics and function. Larvae treated with 10mug/ml cisplatin had normal survival with deficiencies in righting and heat sensing behavior. Behavior was abrogated by, the pan caspase inhibitor, p35. However, active caspase 3 was not detected by immunostaining. There was a 27% decrease in mitochondrial membrane potential and a 42% increase in reactive oxygen species (ROS) in mitochondria along the axon. Examination of mitochondrial axonal trafficking showed no changes in velocity, flux or mitochondrial length. However, cisplatin treatment resulted in a greater number of stationary organelles caused by extended pausing during axonal motility. These results demonstrate that cisplatin induces behavior deficiencies in Drosophila larvae, decreased mitochondrial activity, increased ROS production and mitochondrial pausing without killing the larvae. Thus, this study identified particular aspects of mitochondrial dynamics and function that are affected in cisplatin-induced peripheral neuropathy and may represent key therapeutic targets (Podratz, 2016).

Inhibiting the mitochondrial calcium uniporter during development impairs memory in adult Drosophila

The uptake of cytoplasmic calcium into mitochondria is critical for a variety of physiological processes, including calcium buffering, metabolism, and cell survival. This study demonstrates that inhibiting the mitochondrial calcium uniporter in the Drosophila mushroom body neurons (MBn)-a brain region critical for olfactory memory formation-causes memory impairment without altering the capacity to learn. Inhibiting uniporter activity only during pupation impairs adult memory, whereas the same inhibition during adulthood is without effect. The behavioral impairment is associated with structural defects in MBn, including a decrease in synaptic vesicles and an increased length in the axons of the αβ MBn. These results reveal an in vivo developmental role for the mitochondrial uniporter complex in establishing the necessary structural and functional neuronal substrates for normal memory formation in the adult organism (Drago, 2016).

A mitochondrial ATP synthase subunit interacts with TOR signaling to modulate protein homeostasis and lifespan in Drosophila

Diet composition is a critical determinant of lifespan, and nutrient imbalance is detrimental to health. However, how nutrients interact with genetic factors to modulate lifespan remains elusive. This study investigated how diet composition influences mitochondrial ATP synthase subunit d (ATPsyn-d) in modulating lifespan in Drosophila. ATPsyn-d knockdown extends lifespan in females fed low carbohydrate-to-protein (C:P) diets but not the high C:P ratio diet. This extension is associated with increased resistance to oxidative stress; transcriptional changes in metabolism, proteostasis, and immune genes; reduced protein damage and aggregation, and reduced phosphorylation of S6K and ERK in TOR and mitogen-activated protein kinase (MAPK) signaling, respectively. ATPsyn-d knockdown did not extend lifespan in females with reduced TOR signaling induced genetically by Tsc2 overexpression or pharmacologically by rapamycin. These data reveal a link among diet, mitochondria, and MAPK and TOR signaling in aging and stresses the importance of considering genetic background and diet composition in implementing interventions for promoting healthy aging (Sun, 2014).

Dietary nutrients are among the most critical environmental factors that modulate healthspan and lifespan. Nutrient imbalance is a major risk factor to human health and common among old people. Dietary restriction (DR), by reducing the amount of all or specific nutrients, is a potent nongenetic intervention that promotes longevity in many species. In general, protein restriction is more effective in influencing lifespan than sugar or calorie restriction in Drosophila. However, increasing evidence indicates that the composition of dietary nutrients, such as carbohydrate-to-protein (C:P) ratio, is more critical than individual nutrients in affecting health and lifespan. Optimal lifespan peaks at the C:P ratio 16:1 in Drosophila and 9:1 in Mexican fruit fly. A recent study in mice shows that lifespan is primarily regulated by the C:P ratio in the diet and tends to be longer with higher C:P ratios. Diet composition is also critical for DR to promote longevity in nonhuman primate rhesus monkeys. Two major nutrient-sensing pathways are known to modulate lifespan. One is target-of-rapamycin (TOR) signaling that mostly senses cellular amino acid content and the other is insulin/insulin-like signaling that primarily responds to circulating glucose and energy levels. Excessive carbohydrate and protein intake both contribute to development of insulin resistance and diabetes in animal models and humans. Dietary macronutrients, such as sugar, protein, and fat, may interact with each other to influence nutrient-sensing pathways and consequently health outcome. It is thus critical to take into account diet composition in elucidating molecular mechanisms of aging and in developing effective interventions for promoting healthy aging (Sun, 2014).

Aging is associated with transcriptional and translational changes in many genes and proteins. Some age-related changes are evolutionarily conserved, and many function in nutrient metabolism, such as mitochondrial electron transfer chain (ETC) genes, many of which are downregulated with age in worms, flies, rodents, and humans. Knocking down ETC genes affects lifespan in yeast, worms, and flies. Mitochondrial genes also play a key role in numerous age-related diseases, such as Parkinson's and Alzheimer's disease. However, how mitochondrial genes interact with nutrients to modulate lifespan and health-span remains incompletely elucidated. Understanding gene-environment interactions will be a key to tackle aging and age-related diseases (Sun, 2014).

ATP synthase subunit d (ATPsyn-d) is a component of ATP synthase, ETC complex V, and is known to modulate lifespan in C. elegans. How ATPsyn-d modulates lifespan and whether it functions in modulating lifespan in other species remain to be determined (Sun, 2014).

Given the importance of nutrients as environmental factors in modulating lifespan, this study has investigated whether and how ATPsyn-d interacts with dietary macronutrients to modulate lifespan in Drosophila. ATPsyn-d was found to interact with dietary macronutrients to influence accumulation of oxidative damage and protein aggregates; resistance to oxidative stress; and expression of numerous genes involved in metabolism, proteolysis, and innate immune response and more importantly to modulate lifespan. Moreover, ATPsyn-d affects mitogen-activated protein (MAP) kinase (MAPK) signaling and genetically interacts with TOR signaling to influence lifespan of flies in a diet-composition-dependent manner. This study reveals the critical interaction between mitochondrial genes and nutritional factors and the underlying mechanisms involving TOR signaling in modulating lifespan (Sun, 2014).

Considering the essential role of mitochondrial function in metabolism and aging, this study investigated how diet composition influences the function of ATPsynd, a component of mitochondrial ATP synthase, in aging and the underlying mechanisms. ATPsyn-d knockdown extends lifespan in Drosophila under low sugar-high protein diets, but not under a high sugar-low protein diet. Lifespan extension induced by ATPsyn-d knockdown is associated with increased resistance to oxidative stress and improved protein homeostasis. Furthermore, evidence is provided suggesting ATPsyn-d modulates lifespan through genetically interacting with TOR signaling. Knocking down of atp-5, the worm ATPsynd, extends lifespan in C. elegans, along with the current data suggesting a conserved role of ATPsyn-d in modulating lifespan. Altogether, these findings reveal a connection among diet, mitochondrial ATP synthase, and MAPK and TOR signaling in modulating lifespan and shed light on the molecular mechanisms underlying the impact of diet composition on lifespan (Sun, 2014).

The following model is proposed to explain how ATPsyn-d interacts with dietary macronutrients to modulate lifespan, considering the genetic interaction between ATPsyn-d and TOR signaling and the fact that suppression of TOR signaling by altering expression of its components, such as Tsc1/Tsc2, S6K, and 4E-BP, activates autophagy, improves proteostasis, and promotes longevity in high-protein diets, but not necessarily low-protein diets. It is postulated that TOR signaling is regulated by ATPsyn-d and perhaps other mitochondrial proteins. ATPsyn-d knockdown reduces MAPK signaling and probably affects other signaling pathways, which may consequently decrease TOR signaling to extend lifespan in Drosophila fed high-protein diets, such as SY1:9 and SY1:1, but not low protein diets. It is possible that diet-dependent response is due to knockdown of ATPsyn-d protein to different extent by RNAi under different dietary conditions. This is not likely the case. The amount of ATPsyn-d knockdown is not much different between flies on sugar (S) and yeast (Y) SY1:9 and SY9:1, although lifespan is not increased by ATPsyn-d knockdown for flies under SY9:1. Therefore, variations in ATPsyn-d knockdown under current experimental conditions unlikely contribute significantly to diet-dependent lifespan extension. Consistent with this model, ATPsyn-d knockdown increases resistance to acute oxidative stress, reduces cellular oxidative damage, and improves proteostasis in Drosophila. Reduced oxidative damage by ATPsynd knockdown may lead to decreased MAPK signaling, which in turn modulates TOR signaling and proteostasis (Sun, 2014).

Another likely scenario would be that ATPsyn-d and TOR signaling form a positive but vicious feedback loop through MAPK signaling to induce molecular, metabolic, and physiological changes detrimental to lifespan. This vicious cycle can be disrupted by high-C:P diet, knockdown of mitochondrial genes, or suppression of TOR signaling pharmacologically by rapamycin or genetically by Tsc2 overexpression. Consistent with this possibility is that ATPsyn-d knockdown reduces phosphorylation of S6K, a component of TOR signaling, and increases expression of genes involved in maintaining proteostasis and possibly autophagy, which are regulated by TOR signaling. The level of pS6K reflects the strength of TOR signaling, and reduction- of-function mutants of S6K are known to extend lifespan in several species. ATPsyn-d may genetically interact with TOR signaling to modulate lifespan by influencing protein levels of both S6K and pS6K, although it does not necessarily affect the pS6K/S6K ratio, which may not be a reliable indicator for the strength of TOR signaling under the three SY diets due to the change of S6K level. Furthermore, ATPsyn-d knockdown reduces oxidative damage and polyubiquitinated protein aggregates, which are biomarkers of aging. Rapamycin reduces lifespan extension induced by ATPsyn-d knockdown, which may be due to exacerbation of some detrimental effects of reduced TOR signaling. However, this observation further supports the connection between ATPsyn-d and TOR signaling. Although both rapamycin and ATPsyn-d knockdown reduce pS6K level, ATPsyn-d knockdown, but not rapamycin, decreases S6K level, suggesting ATPsyn-d knockdown and rapamycin affect TOR signaling in different manners. Further studies are warranted to clarify the epistatic relationship between ATPsyn-d and TOR signaling (Sun, 2014).

Increasing evidence has demonstrated the importance of diet composition or carbohydrate to protein ratio in modulating lifespan and health. Nutrient geometry studies conducted in Drosophila have shown that C:P ratio in the diet is far more important in determining lifespan than calorie content or single macronutrient. A recent tour de force nutrient geometry study in mice has confirmed and expanded the view on the critical role of C:P ratio in regulating lifespan and cardiometabolic health to mammals. An important implication from nutrient geometric studies is that diet composition would have a significant impact on the effectiveness of inventions for promoting healthy aging by genetic, pharmaceutical, or nutraceutical approaches (Sun, 2014).

This indeed is the case, although evidence comes from only a handful of studies. Rapamycin feeding extends lifespan in yeast, worms, flies, and mice. Although rapamycin feeding has been shown to extend lifespan of flies under a broad range of diets, some studies have shown that rapamycin feeding does not extend lifespan in flies under high carbohydrate-low protein diets. Supplementation of a nutraceutical derived from cranberry extends lifespan in female flies under a high-C:P-ratio diet, but not a low-C:P-ratio diet. Suppression of TOR signaling by overexpression of Tsc1/Tsc2 extends lifespan in flies under relatively higher-protein diets, but not under low-protein diets, although those studies focused on the variation of protein concentration instead of C:P ratio. Consistent with the link between ATPsyn-d and TOR signaling, ATPsyn-d knockdown extends lifespan in female flies under low sugar-high protein diets, but not high sugar-low protein diet, likely due to the fact that TOR signaling is already low under the high sugar-low protein diet. It was further shown that rapamycin feeding extends lifespan in wild-type female flies, but not in ATPsyn-d knockdown flies (Sun, 2014).

Aging is associated with profound decline in protein homeostasis, and many longevity-related pathways, such as TOR and insulin-like signaling, modulate lifespan through improving proteostasis. Suppression of TOR signaling extends lifespan through decreasing protein translation and increasing autophagy, key processes for maintaining proteostasis. This study found that ATPsyn-d knockdown reduces the level of 4-HNE (an α, β-unsaturated hydroxyalkenal that is produced by lipid peroxidation) protein adducts; a biomarker for lipid protein oxidation; and the level and aggregation of polyubiquitinated protein, a biomarker for proteostasis and aging. ATPsyn-d is a key component of mitochondrial ATP synthase complex. Along with the link between ATPsyn-d and TOR signaling, these data suggest that mitochondrial ATP synthase is critical for maintaining proteostasis and modulating lifespan. This notion is further supported by a recent study showing that α-ketoglutarate, an intermediate in the TCA cycle, suppresses mitochondrial ATP synthase probably by binding to ATP synthase subunit b (ATPsyn-b) and also inhibits TOR signaling to extend lifespan in C. elegans. However, it remains to be determined whether suppression of ATP synthase by α-ketoglutarate results in inhibition of TOR signaling in C. elegans or any other species. It is also likely that ATPsyn-d and ATPsyn-b influences ATP synthase and TOR signaling through different mechanisms, because α-ketoglutarate reduces cellular ATP level in C. elegans, whereas ATPsyn-d knockdown does not significantly change or even increase ATP level in Drosophila. This also suggests that lifespan extension is not necessarily associated with decreased ATP level, which is supported by a study in Drosophila showing that any change of ATP level is not correlated with any change of lifespan induced by knockdown of a number of mitochondrial genes. Nevertheless, these studies suggest that ATP synthase is a key and conserved player linking dietary nutrients from TOR signaling to proteostasis and lifespan (Sun, 2014).

Similar to many longevity-related mutants, lifespan extension induced by ATPsyn-d knockdown is associated with reduced oxidative damage and increased resistance to oxidative stress. ATPsyn-d knockdown increases lifespan and resistance to paraquat, an acute oxidative stress response, under SY1:9 or SY1:1. However, ATPsyn-d knockdown increases resistance to paraquat but does not extend lifespan in female flies under SY9:1. In addition, ATPsyn-d knockdown decreases 4-HNE level, an indicator of accumulated oxidative damage, under SY1:9, but not SY1:1. These indicate that the effect of ATPsyn-d knockdown on oxidative damage and lifespan depends on diet composition, suggesting that oxidative stress resistance is at most partially responsible for lifespan extension. This should not be surprising because it is consistent with numerous studies in the literature showing that stress resistance does not always result in lifespan extension despite the strong link between oxidative stress and aging (Sun, 2014).

The role of mitochondrial genes in modulating lifespan is complex. Knockdown of some electron transfer chain (ETC) genes increases lifespan whereas knockdown of others decreases or does not alter lifespan in C. elegans and Drosophila. This study reveals another layer of complexity regarding the role of ETC genes in lifespan modulation, namely the impact of diet composition. These findings indicate that ATPsyn-d knockdown promotes longevity at least partially through TOR signaling. TOR signaling senses cellular amino acid content and regulates numerous biological processes, including translation, autophagy, and lifespan. 4E-BP, a translational repressor in TOR signaling, mediates lifespan extension induced by dietary restriction (Sun, 2014).

Activated 4E-BP suppresses general translation but selectively increases translation of some mitochondrial ETC genes, the latter of which results in increased mitochondrial biogenesis and potentially lifespan. Lifespan extension induced by dietary restriction is suppressed by knocking down ETC genes regulated by 4E-BP. The findings by Zid suggest that increased protein expression of some ETC genes is associated with lifespan extension induced by dietary restriction. However, unlike those ETC genes, ATPsyn-d knockdown extends instead of decreases lifespan under high-protein diets. Therefore, it is likely that ETC genes can be categorized into two groups: one selectively upregulated by activated 4E-BP and the other insensitive to activated 4E-BP, the latter of which may include ATPsyn-d. The two groups of ETC genes may interact with dietary macronutrients to modulate lifespan perhaps through different modes of action. Future studies are warranted to investigate the dichotomous role of translation of ETC genes in modulating lifespan (Sun, 2014).

Genetic perturbation of key central metabolic genes extends lifespan in Drosophila and affects response to dietary restriction

There is a connection between nutrient inputs, energy-sensing pathways, lifespan variation and aging. Despite the role of metabolic enzymes in energy homeostasis and their metabolites as nutrient signals, little is known about how their gene expression impacts lifespan. This report uses P-element mutagenesis in Drosophila to study the effect on lifespan of reductions in expression of seven central metabolic enzymes and contrasts the effects on normal diet and dietary restriction. The major observation is that for five of seven genes, the reduction of gene expression extends lifespan on one or both diets. Two genes are involved in redox balance, and it was observed that lower activity genotypes significantly extend lifespan. The hexokinases also show extension of lifespan with reduced gene activity. Since both affect the ATP/ADP ratio, this connects with the role of AMP-activated protein kinase as an energy sensor in regulating lifespan and mediating caloric restriction. These genes possess significant expression variation in natural populations, and the experimental genotypes span this level of natural activity variation. These studies link the readout of energy state with the perturbation of the genes of central metabolism and demonstrate their effect on lifespan (Talbert, 2015).

In their lifetime, all organisms experience environments that change both temporally and spatially in their nutrient availability and energy content. The optimal utilization and storage of available energy is a physiological challenge that shapes variation in life-history phenotypes, and in principle sets the trade-off between lifespan and reproduction. Failing to allocate energy optimally to the changing availability of nutrients would be expected to have significant fitness costs. As a consequence, nutrient sensing and response networks are strongly conserved pathways. Since there are optimal physiological responses that reset internal energy balance for different environments, genetic variation associated with these responses would be expected (Talbert, 2015).

As potential modifiers of cell energy state through their action on metabolite levels, the genes of central metabolism are potential sources for this genetic variation. The response to changing nutrient input results in intercellular signals that drive a cascade of downstream gene transcription shifts that facilitate energy utilization and storage. The proximal signal is generally derived from specific metabolite levels as they change under shifting nutrient load. There is considerable precedent for this general mechanism; for example, the well-known action of glucose on insulin secretion in vertebrates, and the signals associated with the secretion and action of adipokinetic hormone (AKH) in insects. The effect of energy balance on longevity in yeast is well known. Since metabolite concentrations act as proximal signals and also appear to correlate with the associated gene expression levels of the component steps in metabolism, this relationship clearly implicates the natural genetic variation in expression (or activity) of the central metabolic genes as potential sources of genetic variation in setting metabolite levels, and thus play a role in sensing and setting responses. Moreover, specific enzymes are expected to emerge as the targets of natural selection where genetic expression or activity level will act as an 'energy-stat'. Different genotypes will bracket and set the nutrient levels involved in triggering downstream responses that affect traits associated with fitness, such as lifespan variation and fecundity (Talbert, 2015).

The well-established observation that nutritional or dietary restriction (DR) extends lifespan in many species is mechanistically related to energy-state signalling. In Drosophila, many studies have shown DR to extend lifespan, and using genetic manipulation, many of the signalling pathways have been identified that extend lifespan and thus effectively mimic DR. Experimental work has shown connections between energy-signalling steps and other energy correlated phenotypes such as starvation resistance, nutrient storage and stress resistance that have connections to DR (Talbert, 2015).

In both plants and animals, it is becoming apparent that many metabolic genes and their enzyme products are associated with roles other than simple processing of metabolites. Based on evidence from RNAi reduction screens of central metabolic genes in Caenorhabditis elegans, it is estimated that as much as 25% of the genes screened implicate metabolism in longevity extension. RNAi knockdown of expression of several genes in the mitochondrial respiratory chain has been shown to extend lifespan in Drosophila. In yeast, the overexpression of several of the genes involved in cofactor shuttles actually extends lifespan. Despite its importance as a model in lifespan studies, and the connection between metabolism and lifespan seen in other models, no studies in Drosophila have examined the mutational perturbation of central metabolic gene activity and their effects on longevity (Talbert, 2015).

Natural populations of Drosophila melanogaster vary in both average lifespan and response of lifespan to dietary challenge. In D. melanogaster, the genes of the central metabolic pathway harbour considerable sequence and expression variation, which often shows change with latitude and season. In this regard, unlike the other experimental models used in aging studies, Drosophila offers the unique opportunity to associate aging and signalling pathways with their population genetics (Talbert, 2015).

The long-term goal of these studies is to integrate geographical and seasonal variation in metabolic genes with life-history phenotypes, such as longevity and its fitness correlates. This report used matched sets of P-element excision alleles in D. melanogaster to create genotypes that possess modest reductions in gene expression and subsequently examined the impact of these perturbations on lifespan under normal and restricted diets. Seven metabolic genes were studied: Idh, Mdh2, Hex-C, Hex-A, Gpdh, Gdh and Men. These enzymes involve possible signalling via glucose, ATP/ADP, NAD/NADH and NADP/NADPH ratios, citrate, pyruvate, malate and glutamate, and they also involve genes with primary expression restricted to the cytoplasm or the mitochondria. A range of effects was observed on lifespan, from none at all to very significant increases in lifespan that can depend on diet (Talbert, 2015).

This study observed that the genetic perturbation of central metabolic genes has significant effects on lifespan. Moreover, the general observation is that low-activity genotypes show extension of lifespan. Perhaps not surprisingly, some genotype effects are also diet dependent. Some genes (Idh, Mdh2, Hex-C) show an increase in lifespan under DR, but no effect of genotype, while others show a strong (Gdh, Men) genotype dependence in their response to DR. Finally, Gpdh shows a strong dependence on genotype activity, yet no effect of DR. These differences are expected because the observations represent seven enzymes that act on different metabolites and cofactors, and are limited to mitochondrial or cytosolic function (Talbert, 2015).

In this work, experimental outcomes across genes are not strictly comparable. First, while single gene effects are being studied within identical genetic backgrounds, the backgrounds differ across the seven gene sets. The P-element progenitor lines differ in the type of element used in the excision series (e.g. KG versus EP), and while the replacement backgrounds possess some chromosomes in common (often the 6326 chromosomes), they generally differ in others. Second, while all of the genotype comparisons involve reductions of activity of 50% or less, the same level of flux control across enzymes cannot be expected. Thus, cytosolic IDH may possess little flux control over NADPH/NADP levels, while MEN may exercise greater control over these metabolites, especially at reduced nutrient levels. It should be pointed out that in the Raleigh population the cytosolic Idh gene bears little molecular polymorphism and the few SNPs seen show little cis-based expression effect or clinal change (Talbert, 2015).

It should also be emphasized that unlike many gene-targeted lifespan studies, this study was not using full knockout genotypes, or genotypes where the relative functional reduction is unknown, and precise estimates of genotype activity are available. Moreover, these activity differences are representative of the range of much of the cis-associated SNP expression variation seen in natural populations. The observation that metabolic genes, when perturbed modestly in activity, have an effect on lifespan is certainly relevant to discussions of the maintenance of genetic variation in these genes in natural populations, especially as nutrient levels vary geographically and seasonally (Talbert, 2015).

Gpdh, Gdh and Hex-A were highlighted in this study study of clinal SNP expression variation in the pathway. These genes, among others, showed significant changes in gene transcript expression with latitude. The observations in this study add a fitness component to the causes of genetic expression variation of metabolic genes in natural populations of Drosophila. Also, the expectation that the gene-specific extension of lifespan can depend on dietary level adds complexity, since nutritional background is expected to shift locally and seasonally in this species. The effect of reduced activity is incrementally small in terms of daily survival, but when integrated over the average lifespan of a fly this can be very significant. This relationship is in contrast to flight metabolism, where similar activity changes have no effects on flight performance (Talbert, 2015).

The enzymes were targeted because they act on different metabolites and cofactors, and are limited to either mitochondrial or cytosolic function. Both IDH and MEN are cytosolic enzymes and NADPH dependent, and, along with the pentose shunt enzymes, provide a significant contribution to the NADPH/NADP pool. Both glutamate (GDH) and malate dehydrogenase (MDH2) are limited to mitochondrial function and, like GPDH, are dependent on NAD/NADH, and will impact that redox balance and its effect on signalling and aging. The two hexokinases, HEX-A and HEX-C, potentially affect the ADP/ATP ratio and have different tissue expressions. They could vary the ADP/ATP content, and in that fashion set energy-state response via regulating AMPK signalling. This AMP-activated kinase is a sensor that has effects in Drosophila, which provides a link between lifespan and caloric restriction (Talbert, 2015).

The observations for the Gpdh and Gdh genes implicate mitochondrial function and the redox balance with lifespan extension. GPDH is part of the essential mitochondrial phosphoglycerol shuttle in insects and is often considered a point of ROS production. The NAD/NADH redox balance is emerging as an important element of lifespan extension in yeast and is often considered a direct readout of metabolic state. Moreover, the lifespan extension associated with these genes might also act through the Sir-like enzymes, which are NAD-dependent histone deacetylases that silence chromatin and thus control transcription in a fashion directly coupled to energy-state imbalance. This relationship is important because starvation in Drosophila has been clearly shown to significantly raise the NAD/NADH ratio, although the role of Sir2 in lifespan extension in Drosophila has been questioned. GDH is also limited to mitochondrial function, and potentially affects the redox balance and NAD/NADH ratio. It connects glutamate, a key energy-state signalling molecule, to metabolic control, and sits at the important crossroads of carbohydrate and amino acid metabolism. As well, glutamate is at the hub of connectivity in the large central metabolic network. Both of these enzymes show significant extension of lifespan with only 40% reductions in whole-body activity. However, all mitochondrial or NAD-dependent genes are not similar in affecting lifespan; comparable activity changes in mitochondrial and NAD-dependent MDH (Mdh2) have little effect on lifespan in either dietary condition (Talbert, 2015).

The dependence of the results on diet is important. Over the past two decades, DR has been shown to impose a trade-off where it extends lifespan and reduces reproduction fecundity. It has gained prominence because of its association with aging research in general, but the phenomenon of DR has obvious relevance to studies of life-history evolution because it will be associated with plastic responses to nutritional challenges in nature, and the potential maintenance of genetic variation. Studies on model organisms have led to the discovery of many genes where mutational perturbation extends lifespan and thus mimics DR restriction. This is most notable in the parallel effects of disruption of genes specifically associated with energy-sensing pathways and signalling of dietary state and DR. Despite its general occurrence in D. melanogaster, an effect of DR on lifespan is not seen in some of the experimental lines. Two (Gpdh, Hex-A) of the seven genes show no DR effect in general. For Gdh, DR is seen just for the low-activity genotype. This different response to DR is suggested by studies where line-by-diet interactions are noted, but is shown more definitively in studies in mice and yeast, where it becomes clear that genetic background affects the response to DR. In another study, 166 single, non-essential genes were made deficient in yeast, and a large proportion of genes showed loss of DR extension capability, as well as an enhanced DR response. Clearly, DR response can be modified genetically and it would not be surprising if genetic variation in natural populations were to reflect this observation (Talbert, 2015).

Does the failure of some expression modified genotypes to respond to DR imply a mechanistic connection to the signalling associated with DR? An interaction between genotype and diet would suggest that the lifespan responses to DR may be coupled to metabolic signals associated with these enzymes or pathways. For example, the full-activity Men genotype shows a significant DR lifespan response, yet the Men low-activity genotype appears resistant to lifespan extension under DR. This may suggest that the NADPH/NADP ratio in the case of MEN is a signal associated with DR. However, the NADPH-dependent IDH shows no genotypic effect on DR, which may contradict this suggestion or simply be because IDH possesses low control over cofactor pool levels. Conversely, the Gdh normal activity genotype shows no DR response, while a genotype reduction in Gdh activity strongly enhances a DR response. Perhaps the reduction in amino acids that is associated with DR in Drosophila is enhanced by reduction of Gdh activity, since glutamate and GDH sit at the crossover of carbohydrate and amino acid metabolism in the mitochondria. However, this interaction must be interpreted with caution, since tests of genotype dependence of DR should be tested by using a range of diet changes (Talbert, 2015).

Where might the mechanism of action reside that extends lifespan or is associated with a genotype-dependent response to DR for these metabolic genes? Discussions of energy-signalling pathways typically start with the statement that nutrient levels are first 'sensed' and then the pathway of interest is addressed (e.g. the insulin receptor insulin/TOR pathway in Drosophila). Presumably, this initial sensing must emanate from direct immediate readouts of the cell's metabolic state (i.e., metabolites). In Drosophila, dietary sugars induce significant metabolite changes. These metabolite levels changes trigger the secretion of neuropeptides from specialized neurosecretory cells. This model of sensing is similar to the regulation of glucagon in mammalian pancreatic cells, and a similar case has been made for the sensing and regulation of AKH by the corpora cardiac cells in Drosophila. It is possible that either these cell-specific or just systemic metabolite levels initiate the signalling process. Genetic variation will regulate these metabolite levels in conjunction with nutrient inputs. In this sense, lifespan extension by some metabolic genes is top-down (Talbert, 2015).

Over the past two decades, a large number of genes in several model species have been observed to extend lifespan when mutated. In Drosophila, most studies have emphasized the signalling cascades emanating from neurosecretory cells. However, the most proximal steps of central metabolism must set the signalling environments because their metabolite levels respond immediately to nutrient inputs. This study places the metabolic pathway in the discussion of energy signalling and looked at the impact of genetic perturbation of some key genes on lifespan. The outcome is that there are numerous examples where reductions in activity extend lifespan. Perhaps not surprisingly, this extension depends on the nutrient environment. It is proposed that this setting of lifespan response to gene expression variation also provides a selective context for naturally segregating metabolic gene variation, and moreover may contribute to unravelling the patterns of genetic variation observed in natural populations (Talbert, 2015).

Increased mitochondrial biogenesis preserves intestinal stem cell homeostasis and contributes to longevity in Indy mutant flies

The Drosophila Indy (I'm not dead yet) gene encodes a plasma membrane transporter of Krebs cycle intermediates, with robust expression in tissues associated with metabolism. Reduced INDY alters metabolism and extends longevity in a manner similar to caloric restriction (CR); however, little is known about the tissue specific physiological effects of INDY reduction. This study focused on the effects of INDY reduction in the Drosophila midgut due to the importance of intestinal tissue homeostasis in healthy aging and longevity. The expression of Indy mRNA in the midgut changes in response to aging and nutrition. Genetic reduction of Indy expression increases midgut expression of the mitochondrial regulator spargel/dPGC-1, which is accompanied by increased mitochondrial biogenesis and reduced reactive oxygen species (ROS). These physiological changes in the Indy mutant midgut preserve intestinal stem cell (ISC) homeostasis and are associated with healthy aging. Genetic studies confirm that dPGC-1 mediates the regulatory effects of INDY, as illustrated by lack of longevity extension and ISC homeostasis in flies with mutations in both Indy and dPGC1. These data suggest INDY may be a physiological regulator that modulates intermediary metabolism in response to changes in nutrient availability and organismal needs by modulating dPGC-1 (Rogers, 2014).

Caloric restriction (CR) extends lifespan in nearly all species and promotes organismal energy balance by affecting intermediary metabolism and mitochondrial biogenesis. Interventions that alter intermediary metabolism are though to extend longevity by preserving the balance between energy production and free radical production Indy (I'm Not Dead Yet) encodes a plasma membrane protein that transports Krebs' cycle intermediates across tissues associated with intermediary metabolism. Reduced Indy-mediated transport extends longevity in worms and flies by decreasing the uptake and utilization of nutrients and altering intermediate nutrient metabolism in a manner similar to CR. Furthermore, it was shown that caloric content of food directly affects Indy expression in fly heads and thoraces, suggesting a direct relationship between INDY and metabolism (Rogers, 2014 and references therein).

dPGC-1/spargel is the Drosophila homolog of mammalian PGC-1, a transcriptional co-activator that promotes mitochondrial biogenesis by increasing the expression of genes encoding mitochondrial proteins. Upregulation of dPGC-1 is a hallmark of CR-mediated longevity and is thought to represent a response mechanism to compensate for energetic deficits caused by limited nutrient availability. Increases in dPGC-1 preserve mitochondrial functional efficiency without consequential changes in ROS. Previous analyses of Indy mutant flies revealed upregulation of mitochondrial biogenesis mediated by increased levels of dPGC-1 in heads and thoraces (Rogers, 2014 and references therein).

Recently, dPGC-1 upregulation in stem and progenitor cells of the digestive tract was shown to preserve intestinal stem cell (ISC) proliferative homeostasis and extend lifespan. The Drosophila midgut is regenerated by multipotent ISCs, which replace damaged epithelial tissue in response to injury, infection or changes in redox environment. Low levels of reactive oxygen species (ROS) maintain stemness, self-renewal and multipotency in ISCs; whereas, age-associated ROS accumulation induces continuous activation marked by ISC hyper-proliferation and loss of intestinal integrity (Rogers, 2014 and references therein).

This study describes a role for Indy as a physiological regulator that modulates expression in response to changes in nutrient availability. This is illustrated by altered Indy expression in flies following changes in caloric content and at later ages suggesting that INDY-mediated transport is adjusted in an effort to meet energetic demands. Further, role was characterized for dPGC-1 in mediating the downstream regulatory effects of INDY reduction, such as the observed changes in Indy mutant mitochondrial physiology, oxidative stress resistance and reduction of ROS levels. Longevity studies support a role for dPGC-1 as a downstream effector of Indy mutations as shown by overlapping longevity pathways and absence of lifespan extension without wild-type levels of dPGC-1. These findings show that Indy mutations affect intermediary metabolism to preserve energy balance in response to altered nutrient availability, which by affecting the redox environment of the midgut promotes healthy aging (Rogers, 2014).

Reduction of Indy gene activity in fruit flies, and homologs in worms, extends lifespan by altering energy metabolism in a manner similar to caloric restriction (CR). Indy mutant flies on regular food share many characteristics with CR flies and do not have further longevity extension when aged on a CR diet. Furthermore, mINDY-/- mice on regular chow share 80% of the transcriptional changes observed in CR mice, supporting a conserved role for INDY in metabolic regulation that mimics CR and promotes healthy aging. This study shifted from systemic to the tissue specific effects of INDY reduction, focusing on the midgut due to the high levels of INDY protein expression in wild type flies and the importance of regulated intestinal homeostasis during aging. The evidence supports a role for INDY as a physiological regulator that senses changes in nutrient availability and alters mitochondrial physiology to sustain tissue-specific energetic requirements (Rogers, 2014).

The age-associated increase in midgut Indy mRNA levels that can be replicated by manipulations that accelerate aging such as increasing the caloric content of food or exposing flies to paraquat. Conversely, it was also shown that CR decreases Indy mRNA in control midgut tissues, which is consistent with previous findings in fly muscle and mouse liver. Diet-induced variation in midgut Indy expression suggests that INDY regulates intermediary metabolism by modifying citrate transport to meet tissue or cell-specific bioenergetic needs. Specifically, as a plasma membrane transporter INDY can regulate cytoplasmic citrate, thereby affecting fat metabolism, respiration, and via conversion to malate, the TCA cycle. Reduced INDY-mediated transport activity in the midgut could prevent age-related ISC-hyperproliferation by decreasing the available energy needed to initiate proliferation, thereby preserving tissue function during aging. This is supported by findings that nutrient availability affects ISC proliferation in adult flies and that CR can affect stem cell quiescence and activation (Rogers, 2014).

One of the hallmarks of CR-mediated longevity extension is increased mitochondrial biogenesis mediated by dPGC-1 (Spargel). Increased dPGC-1 levels and mitochondrial biogenesis have been described in the muscle of Indy mutant flies, the liver of mIndy-/- mice, and this study describes it in the midgut of Indy mutant flies. One possible mechanism for these effects can be attributed to the physiological effects of reduced INDY transport activity. Reduced INDY-mediated transport activity could lead to reduced mitochondrial substrates, an increase in the ADP/ATP ratio, activation of AMPK, and dPGC-1 synthesis. This is consistent with findings in CR flies and the livers of mINDY-/- mice. This study's analysis of mitochondrial physiology in the Indy mutant midgut shows upregulation of respiratory proteins, maintenance of mitochondrial potential and increased mitochondrial biogenesis, all of which are signs of enhanced mitochondrial health. The observed increase in dPGC-1 levels in Indy mutant midgut therefore appears to promote mitochondrial biogenesis and functional efficiency, representing a protective mechanism activated in response to reduced energy availability (Rogers, 2014).

Genetic interventions that conserve mitochondrial energetic capacity have been shown to maintain a favorable redox state and regenerative tissue homeostasis. This is particularly beneficial in the fly midgut, which facilitates nutrient uptake, waste removal and response to bacterial infection. Indy mutant flies have striking increases in the steady-state expression of the GstE1 and GstD5 ROS detoxification genes. As a result, any increase in ROS levels, whether from mitochondrial demise or exposure to external ROS sources can be readily metabolized to prevent accumulation of oxidative damage. Such conditions not only promote oxidative stress resistance, but also preserve ISC homeostasis as demonstrated by consistent proliferation rates throughout Indy mutant lifespan and preserved intestinal architecture in aged Indy mutant midguts. Thus, enhanced ROS detoxification mechanisms induced by Indy reduction and subsequent elevation of dPGC-1 contributes to preservation of ISC functional efficiency, and may be a contributing factor to the long-lived phenotype of Indy mutant flies (Rogers, 2014).

Several lines of evidence indicate that INDY and dPGC-1 are part of the same regulatory network in the midgut, in which dPGC-1 functions as a downstream effector of INDY. The similarity between dPGC-1 mRNA levels and survivorship of flies overexpressing dPGC-1 in esg-positive cells and Indy mutant flies suggests that Indy and dPGC-1 interact to extend lifespan. This is further supported by the lack of additional longevity extension when dPGC-1 is overexpressed in esg-positive cells of Indy mutant flies. Moreover, hypomorphic dPGC-1 flies in an Indy mutant background are similar to controls with respect to life span, declines in mitochondrial activity and ROS-detoxification. Together, these data suggest that dPGC-1 must be present to mediate the downstream physiological benefits and lifespan extension of Indy mutant flies (Rogers, 2014).

There are some physiological differences between the effects of Indy mutation and dPGC-1 overexpression in esg-positive cells. While Indy mutant flies are less resistant to starvation and more resistant to paraquat, a recent report showed that overexpressing dPGC-1 in esg-positive cells has no effect on resistance to starvation or oxidative stress. Additionally, mice lacking skeletal muscle PGC-1α were found to lack mitochondrial changes associated with CR but still showed other CR-mediated metabolic changes. In the fly INDY is predominantly expressed in the midgut, fat body and oenocytes, though there is also low level expression in the malpighian tubules, salivary glands, antenae, heart and female follicle cell membranes. Thus, the effects of INDY on intermediary metabolism and longevity could be partially independent from dPGC-1 or related to changes in tissues other than the midgut (Rogers, 2014).

This study suggests that INDY may function as a physiological regulator of mitochondrial function and related metabolic pathways, by modulating nutrient flux in response to nutrient availability and energetic demands. Given the localization of INDY in metabolic tissues, and importance of regulated tissue homeostasis during aging, these studies highlight INDY as a potential target to improved health and longevity. Reduced Indy expression causes similar physiological changes in flies, worms and mice indicating its regulatory role would be conserved. Further work should examine the interplay between Indy mutation and metabolic pathways, such as insulin signaling, which have been shown to promote stem cell maintenance and healthy aging in flies and mice. In doing so, the molecular mechanisms, which underlie Indy mutant longevity may provide insight for anti-aging therapies (Rogers, 2014).

Rapamycin enhances survival in a Drosophila model of mitochondrial disease

Pediatric mitochondrial disorders are a devastating category of diseases caused by deficiencies in mitochondrial function. Leigh Syndrome (LS) is the most common of these diseases with symptoms typically appearing within the first year of birth and progressing rapidly until death, usually by 6-7 years of age. Genetic inhibition of the mechanistic target of rapamycin (TOR; see Drosophila TOR) has been shown to rescue the short lifespan of yeast mutants with defective mitochondrial function, and pharmacological inhibition of TOR by administration of rapamycin significantly rescues the shortened lifespan, neurological symptoms, and neurodegeneration in a mouse model of LS. However, the mechanism by which TOR inhibition exerts these effects, and the extent to which these effects can extend to other models of mitochondrial deficiency, are unknown. This study probed the effects of TOR inhibition in a Drosophila model of complex I deficiency. Treatment with rapamycin robustly suppresses the lifespan defect in this model of LS, without affecting behavioral phenotypes. Interestingly, this increased lifespan in response to TOR inhibition occurs in an autophagy-independent manner. Further, a fat storage defect was detected in mitochondrial-DNA-encoded NADH dehydrogenase subunit 2 (ND2) mutant flies that is rescued by rapamycin, supporting a model that rapamycin exerts its effects on mitochondrial disease in these animals by altering metabolism (Wang, 2016).

Single nucleotides in the mtDNA sequence modify mitochondrial molecular function and are associated with sex-specific effects on fertility and aging

Mitochondria underpin energy conversion in eukaryotes. Their small genomes have been the subject of increasing attention, and there is evidence that mitochondrial genetic variation can affect evolutionary trajectories and shape the expression of life-history traits considered to be key human health indicators. However, it is not understood how genetic variation across a diminutive genome, which in most species harbors only about a dozen protein-coding genes, can exert broad-scale effects on the organismal phenotype. Such effects are particularly puzzling given that the mitochondrial genes involved are under strong evolutionary constraint and that mitochondrial gene expression is highly conserved across diverse taxa. This study used replicated genetic lines in the fruit fly, Drosophila melanogaster, each characterized by a distinct and naturally occurring mitochondrial haplotype placed alongside an isogenic nuclear background. Sequence variation within the mitochondrial DNA (mtDNA) affects both the copy number of mitochondrial genomes and patterns of gene expression across key mitochondrial protein-coding genes. In several cases, haplotype-mediated patterns of gene expression were gene-specific, even for genes from within the same transcriptional units. This invokes post-transcriptional processing of RNA in the regulation of mitochondrial genetic effects on organismal phenotypes. Notably, the haplotype-mediated effects on gene expression could be traced backward to the level of individual nucleotides and forward to sex-specific effects on fertility and longevity. This study thus elucidates how small-scale sequence changes in the mitochondrial genome can achieve broad-scale regulation of health-related phenotypes and even contribute to sex-related differences in longevity (Camus, 2015).

Mitochondrial genotype modulates mtDNA copy number and organismal phenotype in Drosophila

This study evaluated the role of natural mitochondrial DNA (mtDNA) variation on mtDNA copy number, biochemical features and life history traits in Drosophila cybrid (cytoplasmic hybrid) strains, containing heterologous nuclear-mitochondrial combinations. The effects of both coding region and non-coding A+T region variation on mtDNA copy number is demonstrated; copy number was shown to correlate with mitochondrial biochemistry and metabolically important traits such as development time. For example, high mtDNA copy number correlates with longer development times. These findings support the hypothesis that mtDNA copy number is modulated by mtDNA genome variation and suggest that it affects OXPHOS efficiency through changes in the organization of the respiratory membrane complexes to influence organismal phenotype (Salminen, 2017).

Drosophila mitochondrial topoisomerase III alpha affects the aging process via maintenance of mitochondrial function and genome integrity

Mitochondria play important roles in providing metabolic energy and key metabolites for synthesis of cellular building blocks. Mitochondria have additional functions in other cellular processes, including programmed cell death and aging. A previous study revealed Drosophila mitochondrial topoisomerase III α (Top3α) contributes to the maintenance of the mitochondrial genome and male germ-line stem cells. However, the involvement of mitochondrial Top3α in the mitochondrion-mediated aging process remains unclear. In this study, the M1L flies, in which Top3α protein lacks the mitochondrial import sequence and is thus present in cell nuclei but not in mitochondria, were used as a model system to examine the role of mitochondrial Top3α in the aging of fruit flies. M1L flies were shown to exhibit mitochondrial defects which affect the aging process. First, it was observed that M1L flies have a shorter life span, which was correlated with a significant reduction in the mitochondrial DNA copy number, the mitochondrial membrane potential, and ATP content compared with those of both wild-type and transgene-rescued flies of the same age. Second, a mobility assay and electron microscopic analysis was performed to demonstrate that the locomotion defect and mitophagy of M1L flies were enhanced with age, as compared with the controls. Finally, it was shown that the correlation between the mtDNA deletion level and aging in M1L flies resembles what was reported in mammalian systems. The results demonstrate that mitochondrial Top3α ablation results in mitochondrial genome instability and its dysfunction, thereby accelerating the aging process (Tsai, 2016).

Lamin mutations accelerate aging via defective export of mitochondrial mRNAs through nuclear envelope budding

Defective RNA metabolism and transport are implicated in aging and degeneration, but the underlying mechanisms remain poorly understood. A prevalent feature of aging is mitochondrial deterioration. This study links a novel mechanism for RNA export through nuclear envelope (NE) budding that requires A-type lamin, an inner nuclear membrane-associated protein, to accelerated aging observed in Drosophila LaminC (LamC) mutations. These LamC mutations were modeled after A-lamin (LMNA) mutations causing progeroid syndromes (PSs) in humans. Mitochondrial assembly regulatory factor (Marf), a mitochondrial fusion factor (mitofusin) as well as other transcripts required for mitochondrial integrity and function, were identified in a screen for RNAs that exit the nucleus through NE budding. PS-modeled LamC mutations induced premature aging in adult flight muscles, including decreased levels of specific mitochondrial protein transcripts (RNA) and progressive mitochondrial degradation. PS-modeled LamC mutations also induced the accelerated appearance of other phenotypes associated with aging, including a progressive accumulation of polyubiquitin aggregates and myofibril disorganization. Consistent with these observations, the mutants had progressive jumping and flight defects. Downregulating marf alone induced the above aging defects. Nevertheless, restoring marf was insufficient for rescuing the aging phenotypes in PS-modeled LamC mutations, as other mitochondrial RNAs are affected by inhibition of NE budding. Analysis of NE budding in dominant and recessive PS-modeled LamC mutations suggests a mechanism by which abnormal lamina organization prevents the egress of these RNAs via NE budding. These studies connect defects in RNA export through NE budding to progressive loss of mitochondrial integrity and premature aging (Y. Li, 2016).

Changes of mitochondrial ultrastructure and function during ageing in mice and Drosophila

Ageing is a progressive decline of intrinsic physiological functions. This study examined the impact of ageing on the ultrastructure and function of mitochondria in mouse and fruit flies by electron cryo-tomography and respirometry and discovered distinct age-related changes in both model organisms. Mitochondrial function and ultrastructure are maintained in mouse heart, whereas subpopulations of mitochondria from mouse liver show age-related changes in membrane morphology. Subpopulations of mitochondria from young and old mouse kidney resemble those described for apoptosis. In aged flies, respiratory activity is compromised and the production of peroxide radicals is increased. In about 50% of mitochondria from old flies, the inner membrane organization breaks down. This establishes a clear link between inner membrane architecture and functional decline. Mitochondria were affected by ageing to very different extents, depending on the organism and possibly on the degree to which tissues within the same organism are protected against mitochondrial damage (Brandt, 2017).

Mask loss-of-function rescues mitochondrial impairment and muscle degeneration of Drosophila pink1 and parkin mutants

PTEN-induced kinase 1 (Pink1) and ubiquitin E3 ligase Parkin function in a linear pathway to maintain healthy mitochondria via regulating mitochondrial clearance and trafficking. Mutations in the two enzymes cause the familial form of Parkinson's disease (PD) in humans, as well as accumulation of defective mitochondria and cellular degeneration in flies. This study shows that loss of function of a scaffolding protein Mask, also known as ANKHD1 (Ankyrin repeats and KH domain containing protein 1) in humans, rescues the behavioral, anatomical and cellular defects caused by pink1 or parkin mutations in a cell-autonomous manner. Moreover, similar rescue can also be achieved if Mask knock-down is induced in parkin adult flies when the mitochondrial dystrophy is already manifested. It was found that Mask genetically interacts with Parkin to modulate mitochondrial morphology and negatively regulates the recruitment of Parkin to mitochondria. Also, loss of Mask activity promotes co-localization of the autophagosome marker with mitochondria in developing larval muscle, and that an intact autophagy pathway is required for the rescue of parkin mutant defects by mask loss of function. Together, these data strongly suggest that Mask/ANKHD1 activity can be inhibited in a tissue- and timely-controlled fashion to restore mitochondrial integrity under PD-linked pathological conditions (Zhu, 2015).

Recent studies suggest that PINK1 activates Parkin E3 ubiquitin ligase activity by phosphorylating both Parkin and ubiquitin, and that PINK1 recruits Parkin to the damaged mitochondrial membrane, where Parkin ubiquitinates a pool of outer mitochondrial membrane proteins and promotes mitophagy. These data suggest that mitochondrial dysfunction observed in PD may be the result of compromised mitochondrial quality control mechanisms. Therefore, understanding the pathways of mitochondrial quality control holds the key to unravelling the pathogenesis of PD and other disorders associated with mitochondrial dysfunction (Zhu, 2015).

Flies carrying pink1 or parkin mutations show severe mitochondrial morphological and functional defects in multiple tissues as well as age-dependent  dopaminergic (DA) dysfunction, making it a great genetic model to study mechanisms of mitochondrial homeostasis. Using this model system, previous studies in Drosophila have identified a number of pathways that can be manipulated to rescue the parkin and/or pink1 mutant phenotype. First, increasing mitochondrial fission or decreasing fusion rescues the phenotypes of muscle degeneration and mitochondrial abnormalities in pink1 or parkin mutants. However, manipulation of mitochondrial dynamics causes the opposite effect on loss of parkin or pink1 function in mammalian cells, indicating that Pink1 and Parkin may regulate mitochondrial dynamics in a context-dependent manner. Second, promoting mitochondrial electron transport chain CI activity by overexpressing a yeast NADH dehydrogenase, the CI subunit NDUFA10, the GDNF receptor Ret, Sicily, dNK or Trap1 rescue pink1 mutant mitochondrial defects without affecting parkin mutant phenotypes, suggesting a distinct role of Pink1 in regulating CI activity in addition to its role in Parkin-mediated mitophagy (Zhu, 2015).

This study shows that a highly conserved scaffolding protein Mask, whose normal function is to regulate mitochondrial morphology and selectively inhibit mitophagy, can be targeted in a tissue- and temporal-specific manner to suppress both pink1 and parkin mutant defects in Drosophila. It also shows that such a rescue requires the presence of a functional autophagy pathway. Although tissue- and temporal-specific knock-down of Mask was performed with mainly one mask RNAi line, the mask loss-of-function analysis with mask genetic mutants and another independent RNAi line support the same notion that Mask dynamically regulates mitochondrial morphology. Together, these data suggest that enhancing mitochondrial quality control may serve as a common approach to mitigate mitochondrial dysfunction caused by PD-linked genetic mutations. Consistent with this notion, recent studies show that inhibition of deubiquitinases USP30 and USP15 enhances mitochondrial clearance and quality control, and rescues mitochondrial impairment caused by pink1 or parkin mutations (Zhu, 2015).

It was found that loss of mask function enhances the formation of autophagosome surrounding mitochondria. However, the increase of mCherry-ATG8 did not result in significant increase of free mCherry, suggesting the flux of autophagic degradation is not affected. Further studies are required to elucidate the molecular details by which Mask regulates mitochondrial morphology and function. Recent studies on the connection between Mask and the Hippo pathway demonstrates that Mask physically interacts with the Hippo effector Yorkie, and functions as an essential cofactor of Yorkie in promoting downstream target-gene expression. Interestingly, the Yorkie pathway was also shown to regulate mitochondrial structure and function during fly development. Together, these findings bring up an intriguing possibility that Mask and Yorkie together regulate mitochondrial size during development and disease. It was also shown that reducing Mask activity at the relatively progressed stage of parkin-dependent muscle degeneration mitigates the mitochondrial defects and impairs muscle function, indicating that the human Mask homolog ANKHD1 may serve as a potential therapeutic target for treating PD caused by pink1/parkin mutations (Zhu, 2015).

Mito-nuclear interactions affecting lifespan and neurodegeneration in a Drosophila model of Leigh Syndrome

Proper mitochondrial activity depends upon proteins encoded by genes in the nuclear and mitochondrial genomes that must interact functionally and physically in a precisely coordinated manner. Consequently, mito-nuclear allelic interactions are thought to be of crucial importance on an evolutionary scale, as well as for manifestation of essential biological phenotypes, including those directly relevant to human disease. Nonetheless, detailed molecular understanding of mito-nuclear interactions is still lacking, and definitive examples of such interactions in vivo are sparse. This study describes the characterization of a mutation in Drosophila ND23, a nuclear gene encoding a highly conserved subunit of mitochondrial complex 1. This characterization led to the discovery of a mito-nuclear interaction that affects the ND23 mutant phenotype. ND23 mutants exhibit reduced lifespan, neurodegeneration, abnormal mitochondrial morphology and decreased ATP levels. These phenotypes are similar to those observed in patients with Leigh Syndrome, which is caused by mutations in a number of nuclear genes that encode mitochondrial proteins, including the human ortholog of ND23. A key feature of Leigh Syndrome, and other mitochondrial disorders, is unexpected and unexplained phenotypic variability. It was discovered that the phenotypic severity of ND23 mutations varies depending on the maternally inherited mitochondrial background. Sequence analysis of the relevant mitochondrial genomes identified several variants that are likely candidates for the phenotypic interaction with mutant ND23, including a variant affecting a mitochondrially-encoded component of complex I. Thus, this work provides an in vivo demonstration of the phenotypic importance of mito-nuclear interactions in the context of mitochondrial disease (Loewen, 2018).

Neuron-specific knockdown of Drosophila PDHB induces reduction of lifespan, deficient locomotive ability, abnormal morphology of motor neuron terminals and photoreceptor axon targeting

Pyruvate dehydrogenase complex deficiency (PDCD) is a common primary cause of defects in mitochondrial function and also can lead to peripheral neuropathy. Pyruvate dehydrogenase E1 component subunit β (PDHB) is a subunit of pyruvate dehydrogenase E1, which is a well-known component of PDC. In Drosophila melanogaster, the CG11876 (dPDHB) gene is a homolog of human PDHB. This study established a Drosophila model with neuron-specific knockdown of dPDHB to investigate its role in neuropathy pathogenesis. Knockdown of dPDHB in pan-neurons induced locomotor defects in both larval and adult stages, which was consistent with abnormal morphology of the motor neuron terminals at neuromuscular junctions and mitochondrial fragmentation in brains. Moreover, neuron-specific knockdown of dPDHB also shortened the lifespan of adult flies. In addition, flies with knockdown of dPDHB manifested a rough eye phenotype and aberrant photoreceptor axon targeting. These results with the Drosophila model suggest the involvement of PDHB in peripheral neuropathy (Dung, 2018).

Drosophila melanogaster LRPPRC2 is involved in coordination of mitochondrial translation

Members of the pentatricopeptide repeat domain (PPR) protein family bind RNA and are important for post-transcriptional control of organelle gene expression in unicellular eukaryotes, metazoans and plants. They also have a role in human pathology, as mutations in the leucine-rich PPR-containing (LRPPRC) gene cause severe neurodegeneration. The mammalian LRPPRC protein and its Drosophila melanogaster homolog DmLRPPRC1 (also known as Bicoid stability factor) have been shown to be necessary for mitochondrial translation by controlling stability and polyadenylation of mRNAs. This study reports characterization of DmLRPPRC2 (CG14786), a second fruit fly homolog of LRPPRC, and shows that it has a predominant mitochondrial localization and interacts with a stem-loop interacting RNA binding protein (CG3021/DmSLIRP2). Ubiquitous downregulation of DmLrpprc2 expression causes respiratory chain dysfunction, developmental delay and shortened lifespan. Unexpectedly, decreased DmLRPPRC2 expression does not globally affect steady-state levels or polyadenylation of mitochondrial transcripts. However, some mitochondrial transcripts abnormally associate with the mitochondrial ribosomes and some products are dramatically overproduced and other ones decreased, which, in turn, results in severe deficiency of respiratory chain complexes. The function of DmLRPPRC2 thus seems to be to ensure that mitochondrial transcripts are presented to the mitochondrial ribosomes in an orderly fashion to avoid poorly coordinated translation (Baggio, 2014).

Glucose regulates mitochondrial motility via Milton modification by O-GlcNAc transferase

Cells allocate substantial resources toward monitoring levels of nutrients that can be used for ATP generation by mitochondria. Among the many specialized cell types, neurons are particularly dependent on mitochondria due to their complex morphology and regional energy needs. This study reports a molecular mechanism by which nutrient availability in the form of extracellular glucose and the enzyme O-GlcNAc Transferase (OGT; termed super sex combs by FlyBase), whose activity depends on glucose availability, regulates mitochondrial motility in neurons. Activation of OGT diminishes mitochondrial motility. The mitochondrial motor-adaptor protein Milton was established as a required substrate for OGT to arrest mitochondrial motility by mapping and mutating the key O-GlcNAcylated serine residues. The GlcNAcylation state of Milton was found to be altered by extracellular glucose, and OGT was found to alter mitochondrial motility in vivo. These findings suggest that, by dynamically regulating Milton GlcNAcylation, OGT tailors mitochondrial dynamics in neurons based on nutrient availability (Pekkurnaz, 2014).

The p38 pathway regulates oxidative stress tolerance by phosphorylation of mitochondrial protein IscU

The p38 pathway is an evolutionarily conserved signaling pathway that responds to a variety of stresses. However the underlying mechanisms are largely unknown. This study demonstrates that p38b is a major p38 MAPK involved in the regulation of oxidative stress tolerance in addition to p38a and p38c in Drosophila. The importance of MK2 was shown as a p38-activated downstream kinase in resistance to oxidative stresses. Furthermore, the iron-sulfur cluster scaffold protein IscU was identified as a new substrate of MK2 both in Drosophila and mammalian cells. These results imply a new mechanistic connection between the p38 pathway and mitochondrial iron-sulfur cluster (Tian, 2014).

The atypical cadherin Fat directly regulates mitochondrial function and metabolic state

Fat (Ft) cadherins are enormous cell adhesion molecules that function at the cell surface to regulate the tumor-suppressive Hippo signaling pathway and planar cell polarity (PCP) tissue organization. Mutations in Ft cadherins are found in a variety of tumors, and it is presumed that this is due to defects in either Hippo signaling or PCP. This study shows Drosophila Ft functions in mitochondria to directly regulate mitochondrial electron transport chain integrity and promote oxidative phosphorylation. Proteolytic cleavage releases a soluble 68 kDa fragment (Ftmito) that is imported into mitochondria. Ftmito binds directly to NADH dehydrogenase ubiquinone flavoprotein 2 (Ndufv2), a core component of complex I, stabilizing the holoenzyme. Loss of Ft leads to loss of complex I activity, increases in reactive oxygen species, and a switch to aerobic glycolysis. Defects in mitochondrial activity in ft mutants are independent of Hippo and PCP signaling and are reminiscent of the Warburg effect (Sing, 2014).

Loss of PLA2G6 leads to elevated mitochondrial lipid peroxidation and mitochondrial dysfunction

The PLA2G6 gene encodes a group VIA calcium-independent phospholipase A2 beta enzyme that selectively hydrolyses glycerophospholipids to release free fatty acids. Mutations in PLA2G6 have been associated with disorders such as infantile neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type II and Karak syndrome. More recently, PLA2G6 was identified as the causative gene in a subgroup of patients with autosomal recessive early-onset dystonia-parkinsonism. Neuropathological examination revealed widespread Lewy body pathology and the accumulation of hyperphosphorylated tau, supporting a link between PLA2G6 mutations and parkinsonian disorders. This study shows that knockout of the Drosophila homologue of the PLA2G6 gene, iPLA2-VIA, results in reduced survival, locomotor deficits and organismal hypersensitivity to oxidative stress. Furthermore, it was demonstrated that loss of iPLA2-VIA function leads to a number of mitochondrial abnormalities, including mitochondrial respiratory chain dysfunction, reduced ATP synthesis and abnormal mitochondrial morphology. Moreover, it was shown that loss of iPLA2-VIA is strongly associated with increased lipid peroxidation levels. These findings were confirmed using cultured fibroblasts taken from two patients with mutations in the PLA2G6 gene. Similar abnormalities were seen including elevated mitochondrial lipid peroxidation and mitochondrial membrane defects, as well as raised levels of cytoplasmic and mitochondrial reactive oxygen species. Finally, it was demonstrated that deuterated polyunsaturated fatty acids, which inhibit lipid peroxidation, were able to partially rescue the locomotor abnormalities seen in aged flies lacking iPLA2-VIA gene function, and restore mitochondrial membrane potential in fibroblasts from patients with PLA2G6 mutations. Taken together, these findings demonstrate that loss of normal PLA2G6 gene activity leads to lipid peroxidation, mitochondrial dysfunction and subsequent mitochondrial membrane abnormalities. Furthermore it was shown that the iPLA2-VIA knockout fly model provides a useful platform for the further study of PLA2G6-associated neurodegeneration (Kinghorn, 2015).

Biochemical characterization of a new mitochondrial transporter of dephosphocoenzyme A in Drosophila melanogaster

CoA is an essential cofactor that holds a central role in cell metabolism. Although its biosynthetic pathway is conserved across the three domains of life, the subcellular localization of the eukaryotic biosynthetic enzymes and the mechanism behind the cytosolic and mitochondrial CoA pools compartmentalization are still under debate. In humans, the transport of CoA across the inner mitochondrial membrane has been ascribed to two related genes, SLC25A16 and SLC25A42 whereas in D. melanogaster genome only one gene is present, CG4241, phylogenetically closer to SLC25A42. CG4241 encodes two alternatively spliced isoforms, dPCoAC-A and dPCoAC-B. Both isoforms were expressed in Escherichia coli, but only dPCoAC-A was successfully reconstituted into liposomes, where transported dPCoA and, to a lesser extent, ADP and dADP but not CoA, which was a powerful competitive inhibitor. The expression of both isoforms in a Saccharomyces cerevisiae strain lacking the endogenous putative mitochondrial CoA carrier restored the growth on respiratory carbon sources and the mitochondrial levels of CoA. The results reported in this study and the proposed subcellular localization of some of the enzymes of the fruit fly CoA biosynthetic pathway, suggest that dPCoA may be synthesized and phosphorylated to CoA in the matrix, but it can also be transported by dPCoAC to the cytosol, where it may be phosphorylated to CoA by the monofunctional dPCoA kinase. Thus, dPCoAC may connect the cytosolic and mitochondrial reactions of the CoA biosynthetic pathway without allowing the two CoA pools to get in contact (Vozza, 2016).

Impaired mitochondrial energy production causes light-induced photoreceptor degeneration independent of oxidative stress

In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, mutations were identified in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. ppr was found to be required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. It is proposed that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise (Jaiswal, 2015).

Tumor suppressor WWOX moderates the mitochondrial respiratory complex

Fragile site FRA16D exhibits DNA instability in cancer, resulting in diminished levels of protein from the WWOX gene that spans it. WWOX suppresses tumor growth by an undefined mechanism. WWOX participates in pathways involving aerobic metabolism and reactive oxygen species. WWOX comprises two WW domains as well as a short-chain dehydrogenase/reductase enzyme. This study describes an in vivo genetic analysis in Drosophila melanogaster to identify functional interactions between WWOX and metabolic pathways. Altered WWOX levels modulate variable cellular outgrowths caused by genetic deficiencies of components of the mitochondrial respiratory complexes. This modulation requires the enzyme active site of WWOX, and the defective respiratory complex-induced cellular outgrowths are mediated by reactive oxygen species, dependent upon the Akt pathway and sensitive to levels of autophagy and hypoxia-inducible factor. WWOX is known to contribute to homeostasis by regulating the balance between oxidative phosphorylation and glycolysis. Reduction of WWOX levels results in diminished ability to respond to metabolic perturbation of normal cell growth. Thus, the ability of WWOX to facilitate escape from mitochondrial damage-induced glycolysis (Warburg effect) is, therefore, a plausible mechanism for its tumor suppressor activity (Choo, 2015).

Mitochondrial iron supply is required for the developmental pulse of ecdysone biosynthesis that initiates metamorphosis in Drosophila melanogaster

Synthesis of ecdysone, the key hormone that signals the termination of larval growth and the initiation of metamorphosis in insects, is carried out in the prothoracic gland by an array of iron-containing cytochrome P450s, encoded by the halloween genes. This study shows that mutants in Drosophila mitoferrin (dmfrn), the gene encoding a mitochondrial carrier protein implicated in mitochondrial iron import, fail to grow and initiate metamorphosis under dietary iron depletion or when ferritin function is partially compromised. In mutant dmfrn larvae reared under iron replete conditions, the expression of halloween genes is increased and 20-hydroxyecdysone (20E), the active form of ecdysone, is synthesized. In contrast, addition of an iron chelator to the diet of mutant dmfrn larvae disrupts 20E synthesis. Dietary addition of 20E has little effect on the growth defects, but enables approximately one-third of the iron-deprived dmfrn larvae to successfully turn into pupae and, in a smaller percentage, into adults. This partial rescue is not observed with dietary supply of ecdysone's precursor 7-dehydrocholesterol, a precursor in the ecdysone biosynthetic pathway. The findings reported in this study support the notion that a physiological supply of mitochondrial iron for the synthesis of iron-sulfur clusters and heme is required in the prothoracic glands of insect larvae for steroidogenesis. Furthermore, mitochondrial iron is also essential for normal larval growth (Llorens, 2015).

Evolutionary implications of mitochondrial genetic variation: Mitochondrial genetic effects on OXPHOS respiration and mitochondrial quantity change with age and sex in fruit flies

The ancient acquisition of the mitochondrion into the ancestor of modern-day eukaryotes is thought to have been pivotal in facilitating the evolution of complex life. Mitochondria retain their own diminutive genome, with mitochondrial genes encoding core subunits involved in oxidative phosphorylation. Traditionally, it was assumed there was little scope for genetic variation to accumulate and be maintained within the mitochondrial genome. However, in the past decade, mitochondrial genetic variation has been routinely tied to the expression of life-history traits such as fertility, development, and longevity. To examine whether these broad-scale effects on life-history trait expression might ultimately find their root in mitochondrially-mediated effects on core bioenergetic function, this study measured the effects of genetic variation across twelve different mitochondrial haplotypes on respiratory capacity and mitochondrial quantity in Drosophila. Strains of flies were used that differed only in their mitochondrial haplotype, and each sex was tested separately at two different adult ages. Mitochondrial haplotypes affected both respiratory capacity and mitochondrial quantity. However, these effects were highly context-dependent, with the genetic effects contingent on both the sex and the age of the flies. These sex- and age-specific genetic effects are likely to resonate across the entire organismal life-history, providing insights into how mitochondrial genetic variation may contribute to sex-specific trajectories of life-history evolution (Wolff, 2016a).

Mitochondrial dysfunction plus high-sugar diet provokes a metabolic crisis that inhibits growth

The Drosophila mutant tko25t, a nuclear mis-sense mutation in the gene for mitoribosomal protein Technical knockout, exhibits a deficiency of mitochondrial protein synthesis, leading to a global insufficiency of respiration and oxidative phosphorylation. This entrains an organismal phenotype of developmental delay and sensitivity to seizures induced by mechanical stress. This study found that the mutant phenotype is exacerbated in a dose-dependent fashion by high dietary sugar levels. tko25t larvae were found to exhibit severe metabolic abnormalities that were further accentuated by high-sugar diet. These include elevated pyruvate and lactate, decreased ATP and NADPH. Dietary pyruvate or lactate supplementation phenocopies the effects of high sugar. Based on tissue-specific rescue, the crucial tissue in which this metabolic crisis initiates is the gut. It is accompanied by down-regulation of the apparatus of cytosolic protein synthesis and secretion at both the RNA and post-translational levels, including a novel regulation of S6 kinase at the protein level (Kemppainen, 2016).

Deficiency of succinyl-CoA synthetase α subunit delays development, impairs locomotor activity and reduces survival under starvation in Drosophila

Succinyl-CoA synthetase/ligase (SCS) is a mitochondrial enzyme that catalyzes the reversible process from succinyl-CoA to succinate and free coenzyme A in TCA cycle. SCS deficiencies are implicated in mitochondrial hepatoencephalomyopathy in humans. This study generated a null mutation in Scs α subunit (Scsα). The Drosophila SCS deficiency, designated ScsαKO, contained a high level of succinyl-CoA, a substrate for the enzyme, and altered levels of various metabolites in TCA cycle and glycolysis, indicating that the energy metabolism was impaired. Unlike SCSα deficiencies in humans, there was no reduction in lifespan, indicating that Scsα is not critical for viability in Drosophila. However, they showed developmental delays, locomotor activity defects, and reduced survival under starvation. It was also found that glycogen breakdown occurred during development, suggesting that the mutant flies were unable to produce sufficient energy to promote normal growth. These results suggested that SCSα is essential for proper energy metabolism in Drosophila. The ScsαKO flies should be useful as a model to understand the physiological role of SCSα as well as the pathophysiology of SCSα deficiency (Quan, 2016).

An ancestral role for the mitochondrial pyruvate carrier in glucose-stimulated insulin secretion

Transport of pyruvate into the mitochondrial matrix by the Mitochondrial Pyruvate Carrier (MPC) is an important and rate-limiting step in its metabolism. In pancreatic β-cells, mitochondrial pyruvate metabolism is thought to be important for glucose sensing and glucose-stimulated insulin secretion. To evaluate the role that the MPC plays in maintaining systemic glucose homeostasis, genetically-engineered Drosophila and mice were used with loss of MPC activity in insulin-producing cells. In both species, MPC deficiency results in elevated blood sugar concentrations and glucose intolerance accompanied by impaired glucose-stimulated insulin secretion. In mouse islets, β-cell MPC-deficiency resulted in decreased respiration with glucose, ATP-sensitive potassium (KATP) channel hyperactivity, and impaired insulin release. Moreover, treatment of pancreas-specific MPC knockout mice with glibenclamide, a sulfonylurea KATP channel inhibitor, improved defects in islet insulin secretion and abnormalities in glucose homeostasis in vivo. Finally, using a recently-developed biosensor for MPC activity, it was shown that the MPC is rapidly stimulated by glucose treatment in INS-1 insulinoma cells suggesting that glucose sensing is coupled to mitochondrial pyruvate carrier activity. Altogether, these studies suggest that the MPC plays an important and ancestral role in insulin-secreting cells in mediating glucose sensing, regulating insulin secretion, and controlling systemic glycemia (McCommis, 2016).

Cold acclimation allows Drosophila flies to maintain mitochondrial functioning under cold stress

Environmental stress generally disturbs cellular homeostasis. Researchers have hypothesized that chilling injury is linked to a shortage of ATP. However, previous studies conducted on insects exposed to nonfreezing low temperatures presented conflicting results. This study investigated the mitochondrial bioenergetics of Drosophila melanogaster flies exposed to chronic cold stress (4 degrees C). Mitochondrial oxygen consumption was assessed while monitoring the rate of ATP synthesis at various times (0, 1, 2, and 3 days) during prolonged cold stress and at two assay temperatures (25 and 4 ° C). Organelle responses were compared between cold-susceptible and cold-acclimated phenotypes. Continuous exposure to low temperature provoked temporal declines in the rates of mitochondrial respiration and ATP synthesis. Respiratory control ratios (RCRs) suggested that mitochondria were not critically uncoupled. Nevertheless, after 3 days of continuous cold stress, a sharp decline in the mitochondrial ATP synthesis rate was observed in control flies when they were assayed at low temperature. This change was associated with reduced survival capacity in control flies. In contrast, cold-acclimated flies exhibited high survival and maintained higher rates of mitochondrial ATP synthesis and coupling (i.e., higher RCRs). Adaptive changes due to cold acclimation observed in the whole organism were thus manifested in isolated mitochondria. These observations suggest that cold tolerance is linked to the ability to maintain bioenergetics capacity under cold stress (Colinet, 2016).

Mitonuclear interactions mediate transcriptional responses to hypoxia in Drosophila

Among the major challenges in quantitative genetics and personalized medicine is to understand how gene x gene interactions (G x G: epistasis) and gene x environment interactions (G x E) underlie phenotypic variation. This study used the intimate relationship between mitochondria and oxygen availability to dissect the roles of nuclear DNA (nDNA) variation, mitochondrial DNA (mtDNA) variation, hypoxia, and their interactions on gene expression in Drosophila melanogaster. Mitochondria provide an important evolutionary and medical context for understanding G x G and G x E given their central role in integrating cellular signals. It was hypothesized that hypoxia would alter mitonuclear communication and gene expression patterns. First order nDNA, mtDNA, and hypoxia effects were shown to vary between the sexes, along with mitonuclear epistasis and G x G x E effects. Females were generally more sensitive to genetic and environmental perturbation. While dozens to hundreds of genes are altered by hypoxia in individual genotypes, very little overlap was found among mitonuclear genotypes for genes that were significantly differentially expressed as a consequence of hypoxia; excluding the gene hairy. Oxidative phosphorylation genes were among the most influenced by hypoxia and mtDNA, and exposure to hypoxia increased the signature of mtDNA effects, suggesting retrograde signaling between mtDNA and nDNA. nDNA-encoded genes were identified in the electron transport chain (succinate dehydrogenase) that exhibit female-specific mtDNA effects. These findings have important implications for personalized medicine, the sex-specific nature of mitonuclear communication, and gene x gene coevolution under variable or changing environments (Mossman, 2016).

Leigh syndrome in Drosophila melanogaster: Morphological and biochemical characterization of Surf1 post-transcriptional silencing

Leigh Syndrome (LS) is the most common early-onset, progressive mitochondrial encephalopathy usually leading to early death. The single most prevalent cause of LS is occurrence of mutations in the SURF1 gene, and LSSurf1 patients show a ubiquitous and specific decrease in the activity of mitochondrial respiratory chain complex IV (cytochrome c oxidase, COX). SURF1 encodes an inner membrane mitochondrial protein involved in COX assembly. A D. melanogaster model of LS was establised based on the post-transcriptional silencing of Surfeit 1 (CG9943), the Drosophila homolog of SURF1. Knock down of Surf1 was induced ubiquitously in larvae and adults, which led to lethality; in the mesodermal derivatives, which led to pupal lethality; or in the central nervous system, which allowed survival. A biochemical characterization was carried out in knock down individuals, which revealed that larvae unexpectedly displayed defects in all complexes of the mitochondrial respiratory chain and in the F-ATP synthase, while adults had a COX-selective impairment. Silencing of Surf1 expression in Drosophila S2R+ cells led to selective loss of COX activity associated with decreased oxygen consumption and respiratory reserve. It is concluded that Surf1 is essential for COX activity and mitochondrial function in D. melanogaster, thus providing a new tool that may help clarify the pathogenic mechanisms of LS (Da-Re, 2014).

A Drosophila model for mito-nuclear diseases generated by an incompatible tRNA-tRNA synthetase interaction
Communication between the mitochondrial and nuclear genomes is vital for cellular function. The assembly of mitochondrial enzyme complexes that produce the majority of cellular energy requires the coordinated expression and translation of both mitochondrial and nuclear encoded proteins. The joint genetic architecture of this system complicates the basis of mitochondrial diseases, and mutations in both mtDNA- and nuclear-encoded genes have been implicated in mitochondrial dysfunction. Previously, in a set of mitochondrial-nuclear introgression strains, a dual genome epistasis was characterized in which a naturally occurring mutation in the D. simulans simw501 mtDNA-encoded tRNA for tyrosine interacts with a mutation in the nuclear encoded mitochondrial localized tyrosyl-tRNA synthetase from D. melanogaster. This study shows that the incompatible mitochondrial-nuclear combination results in locomotor defects, reduced mitochondrial respiratory capacity, decreased OXPHOS enzyme activity, and severe alterations in mitochondrial morphology. Transgenic rescue strains containing nuclear variants of the tyrosyl-tRNA synthetase are sufficient to rescue many of the deleterious phenotypes identified when paired with the simw501mtDNA. However, the severity of this defective mito-nuclear interaction varies across traits and genetic backgrounds, suggesting that the impact of mitochondrial dysfunction may be tissue specific. Because mutations in mitochondrial tRNATyr are associated with exercise intolerance in humans, this mitochondrial-nuclear introgression model in Drosophila provides a means to dissect the molecular basis of these, and other mitochondrial diseases that are a consequence of the joint genetic architecture of mitochondrial function (Holmbeck, 2015).

TSPO, a mitochondrial outer membrane protein, controls ethanol-related behaviors in Drosophila

The heavy consumption of ethanol can lead to alcohol use disorders (AUDs) which impact patients, their families, and societies. Yet the genetic and physiological factors that predispose humans to AUDs remain unclear. One hypothesis is that alterations in mitochondrial function modulate neuronal sensitivity to ethanol exposure. Using Drosophila genetics this study reports that inactivation of the mitochondrial outer membrane Translocator protein 18kDa (TSPO), also known as the peripheral benzodiazepine receptor, affects ethanol sedation and tolerance in male flies. Knockdown of dTSPO in adult male neurons results in increased sensitivity to ethanol sedation, and this effect requires the dTSPO depletion-mediated increase in reactive oxygen species (ROS) production and inhibition of caspase activity in fly heads. Systemic loss of dTSPO in male flies blocks the development of tolerance to repeated ethanol exposures, an effect that is not seen when dTSPO is only inactivated in neurons. Female flies are naturally more sensitive to ethanol than males, and female fly heads have strikingly lower levels of dTSPO mRNA than males. Hence, mitochondrial TSPO function plays an important role in ethanol sensitivity and tolerance. Since a large array of benzodiazepine analogues have been developed that interact with the peripheral benzodiazepine receptor, the mitochondrial TSPO might provide an important new target for treating AUDs (Lin, 2015).

SUV3 helicase is required for correct processing of mitochondrial transcripts

Mitochondrial gene expression is largely regulated by post-transcriptional mechanisms that control the amount and translation of each mitochondrial mRNA. Despite its importance for mitochondrial function, the mechanisms and proteins involved in mRNA turnover are still not fully characterized. Studies in yeast and human cell lines have indicated that the mitochondrial helicase SUV3, together with the polynucleotide phosphorylase, PNPase, composes the mitochondrial degradosome. To further investigate the in vivo function of SUV3, the homolog of SUV3 was disrupted in Drosophila melanogaster (Dm). Loss of dmsuv3 led to the accumulation of mitochondrial mRNAs, without increasing rRNA levels, de novo transcription or decay intermediates. Furthermore, a severe decrease was observed in mitochondrial tRNAs accompanied by an accumulation of unprocessed precursor transcripts. These processing defects lead to reduced mitochondrial translation and a severe respiratory chain complex deficiency, resulting in a pupal lethal phenotype. In summary, these results propose that SUV3 is predominantly required for the processing of mitochondrial polycistronic transcripts in metazoan and that this function is independent of PNPase (Clemente, 2015).

tafazzin deficiency in Drosophila disrupts the final stage of spermatogenesis, spermatid individualization, and causes male sterility

Quantitative and qualitative alterations of mitochondrial cardiolipin have been implicated in the pathogenesis of Barth syndrome, an X-linked cardioskeletal myopathy caused by a deficiency in tafazzin, an enzyme in the cardiolipin remodeling pathway. A tafazzin-deficient Drosophila model of Barth syndrome that is characterized by low cardiolipin (CL) concentration, abnormal cardiolipin fatty acyl composition, abnormal mitochondria, and poor motor function has been generated earlier. This study shows that tafazzin deficiency in Drosophila disrupts the final stage of spermatogenesis, spermatid individualization, and causes male sterility. This phenotype can be genetically suppressed by inactivation of the gene encoding a calcium-independent phospholipase A2, iPLA2-VIA, which also prevents cardiolipin depletion/monolysocardiolipin accumulation, although in wild-type flies inactivation of the iPLA2-VIA does not affect the molecular composition of cardiolipin. Furthermore, it was shown that treatment of Barth syndrome patients' lymphoblasts in tissue culture with the iPLA2 inhibitor, bromoenol lactone, partially restores their cardiolipin homeostasis. Taken together, these findings establish a causal role of cardiolipin deficiency in the pathogenesis of Barth syndrome and identify iPLA2-VIA as an important enzyme in cardiolipin deacylation, and as a potential target for therapeutic intervention (Malhotra, 2009).

The cardiolipin metabolism defect associated with Barth syndrome is manifested by the triad of CL depletion, monolyso-CL accumulation, and CL species diversification, i.e., the generation of CL molecules with different fatty acyl compositions. It is not clear whether the abnormal CL homeostasis actually plays a role in the pathogenesis of Barth syndrome, and if so, which aspect is the key factor. This study addresses this issue in a Drosophila model of Barth syndrome (Malhotra, 2009).

It was found that tafazzin deficiency in Drosophila, which alters CL homeostasis and reduces CL levels, also disrupts spermatid individualization during spermatogenesis, resulting in male sterility, and that this male-sterile phenotype can be suppressed by inactivation of the CL-degrading enzyme iPLA2-VIA, which partially restores CL homeostasis in double-mutant flies. These observations suggest that CL content, or at least the MLCL/CL ratio, plays a critical role in Drosophila spermatid individualization. It has been recently shown that the final stage of spermatid differentiation in Drosophila involves an apoptosis-like mechanism, in which the cytochrome c-dependent caspase activation is required for the elimination of excess cytoplasm. Cardiolipin has been shown to play important roles in mitochondria-dependent apoptosis and a recent report demonstrats that CL deficiency increases cells' resistance to apoptosis. Therefore, CL deficiency in Drosophila testes may prevent the syncytial spermatids from initiating the apoptosis-like mechanism required for normal spermatid individualization. (Malhotra, 2009).

The cardinal characteristics of Barth syndrome are cardioskeletal myopathy, exercise intolerance, neutropenia, abnormal mitochondria, and altered CL metabolism. Because in eukaryotes CL is localized exclusively in mitochondria and is required for optimal mitochondrial function, it has been generally assumed that the defective CL metabolism causes the pathophysiology of Barth syndrome. This study tested this hypothesis by genetically manipulating CL metabolism in the Drosophila model. It was found that partial restoration of CL homeostasis through genetic inactivation of iPLA2-VIA suppresses the male-sterile phenotype of tafazzin-deficient flies; this provides the direct evidence that altered CL metabolism is a major contributing factor in Barth syndrome (Malhotra, 2009).

The most abundant CL molecular species from various organisms and tissues contain only 1 or 2 types of fatty acids. In many mammalian tissues, the predominant fatty acyl moiety in CL is linoleic acid (C18:2). For example, 80% of CL molecules in heart and skeletal muscle are tetralinoleoyl CL. However, the role of CL molecular species in vivo remains speculative. The characteristic fatty acyl composition of CL in vivo is achieved through tafazzin-dependent remodeling of nascent CL. However, tafazzin deficiency, such as in Barth syndrome, results not only in abnormal CL acyl composition, but also in CL depletion and monolyso-CL accumulation. Thus, it is unclear which aspect of the CL metabolic disorder contributes to the pathogenesis of Barth syndrome. The finding in this study that the male-sterile phenotype of tafazzin-deficient flies can be suppressed by genetic inactivation of iPLA2-VIA, which prevents CL depletion and monolyso-CL accumulation without correcting the abnormal CL acyl composition, suggests that the abnormal levels of CL and/or monolyso-CL are important pathogenetic factors. Because a cardiolipin synthase mutant of yeast exhibits abnormal mitochondrial function, it is likely that the low CL content is critical in the molecular mechanism of Barth syndrome. Nevertheless, because Barth syndrome is a multisystem disorder, involvement of monolyso-CL accumulation and abnormal CL acyl composition may also play a role in certain tissues and organs (Malhotra, 2009).

The mature acyl chain composition of CL is achieved through a remodeling process, which requires the action of tafazzin. It has been previously shown that tafazzin catalyzes phospholipid-lysophospholipid transacylation that involves both deacylation of a phospholipid such as CL and reacylation of a monolyso-phospholipid, such as monolyso-CL. Unlike the CoA-dependent deacylation-reacylation cycle (Lands cycle), in which a nascent phospholipid is deacylated by a phospholipase A to yield a free fatty acid and a lysophospholipid that is then reacylated by an acyl-CoA-dependent acyltransferase, transacylation does not require acyl-CoA, and proceeds directly by transferring a fatty acyl chain from a phospholipid to a lysophospholipid; no phospholipase is involved and no free fatty acid is generated in the process. It was found that although the calcium-independent phospholipase A2, iPLA2-VIA, is not required for CL remodeling, in the absence of tafazzin, i.e., in the Barth syndrome model, the enzyme plays a major role in the depletion of CL and the accumulation of monolyso-CL (Malhotra, 2009).

The finding that the phenotypic features of tafazzin deficiency can be suppressed by inhibiting iPLA2-VIA activity identifies this enzyme as a potential target for therapeutic intervention in Barth syndrome. Indeed, it was found that treatment of cultured lymphoblasts from Barth patients with the iPLA2 inhibitor BEL partially restores CL homeostasis. The calcium-independent iPLA2-VIA has been implicated in a variety of biological processes, including phospholipid remodeling, arachidonic acid release, apoptosis, and store-operated calcium entry. In addition, iPLA2-VIA knockout mice develop age-dependent neurological impairment and mutations in the iPLA2-VIA gene have been identified in patients with infantile neuroaxonal dystrophy and neurodegeneration with iron accumulation in the brain. Therefore, a therapeutic approach to Barth syndrome based on the inhibition of iPLA2-VIA is likely to require either careful titration of the phospholipase inhibitor, or even its tissue-specific targeting (Malhotra, 2009).

Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase

Characterization of the proteome of organelles and subcellular domains is essential for understanding cellular organization and identifying protein complexes as well as networks of protein interactions. This study established a proteomic mapping platform in live Drosophila tissues using an engineered ascorbate peroxidase (APEX). Upon activation, the APEX enzyme catalyzes the biotinylation of neighboring endogenous proteins that can then be isolated and identified by mass spectrometry. It was demonstrated that APEX labeling functions effectively in multiple fly tissues for different subcellular compartments and maps the mitochondrial matrix proteome of Drosophila muscle to demonstrate the power of APEX for characterizing subcellular proteomes in live cells. Further, the study generated "MitoMax," a database that provides an inventory of Drosophila mitochondrial proteins with subcompartmental annotation. Altogether, APEX labeling in live Drosophila tissues provides an opportunity to characterize the organelle proteome of specific cell types in different physiological conditions (Chen, 2015).

Knockout of Drosophila RNase ZL impairs mitochondrial transcript processing, respiration and cell cycle progression.

RNase ZL is a highly conserved tRNA 3'-end processing endoribonuclease. Similar to its mammalian counterpart, Drosophila RNase ZL (dRNaseZ) has a mitochondria targeting signal (MTS) flanked by two methionines at the N-terminus. Alternative translation initiation yields two protein forms: the long one is mitochondrial, and the short one may localize in the nucleus or cytosol. This study has generated a mitochondria specific knockout of the dRNaseZ gene. In this in vivo model, cells deprived of dRNaseZ activity display impaired mitochondrial polycistronic transcript processing, increased reactive oxygen species (ROS) and a switch to aerobic glycolysis compensating for cellular ATP. Damaged mitochondria impose a cell cycle delay at the G2 phase disrupting cell proliferation without affecting cell viability. Antioxidants attenuate genotoxic stress and rescue cell proliferation, implying a critical role for ROS. It is suggested that under a low-stress condition, ROS activate tumor suppressor p53, which modulates cell cycle progression and promotes cell survival. Transcriptional profiling of p53 targets confirms upregulation of antioxidant and cycB-Cdk1 inhibitor genes without induction of apoptotic genes. This study implicates Drosophila RNase ZL in a novel retrograde signaling pathway initiated by the damage in mitochondria and manifested in a cell cycle delay before the mitotic entry (Xie, 2015)

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

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. Previous studies have shown that 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. This study identified the conserved tetratricopeptide domain of Clu as predominantly responsible for binding mRNA. In addition, Clu was shown to interact 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 (Sen, 2016a).

Clu is a large protein (~160 kDa) that contains several domains. With the exception of the N-terminal ms domain, each domain when deleted fails to rescue the mitochondrial clumping phenotype in S2R+ cells, as well as clu mutant phenotypes in vivo. Given that Clu is able to associate with proteins involved in multiple different processes (ribosomal proteins, mitochondrial outer membrane proteins, and the PINK1-Parkin complex), Clu may act as a type of scaffold that can bring together several mechanisms in order to maintain mitochondrial function and output. Previous work has shown lack of Clu causes an increase in mitochondrial oxidative damage and a decrease in ATP. Over-expressing single-domain deletions significantly reduced ATP levels below clu mutants alone. This could be because too much Clu can have a dominant negative effect by titrating out critical binding partners, thus negatively impacting mitochondrial function. Alternatively, high Clu levels may be toxic in and of themselves, however, overexpressing FL-clu does not appear to be toxic to flies (Sen, 2016a).

While RNAi knockdown of Clu in S2R+ cells causes mitochondrial clumping, the cells themselves do not have reduced levels of ATP compared to control. This may be due to the differences in metabolism between cultured cells and cells in vivo . For example, there was a marked difference between the sedimentation profiles of Clu and Cluh. Cluh is found in the lighter fractions in extract from HeLa cells, whereas Clu from ovary extract was always found in the heaviest fractions in a sucrose gradient. HeLa cells, as well as many other transformed cultured cells, are known to use high amounts of glucose that appears to be used for glycolysis. For tumors, this is known as the Warburg effect. In addition, glucose has been documented to inhibit oxidative phosphorylation, which is known as the Crabtree effect. In fact, the culture media often used, DMEM, contains pre-diabetic levels of glucose, thus providing the cells with high concentrations of glucose. The different metabolism and use of mitochondria could be one explanation for the discrepancy between the sedimentation of Clu and Cluh (Sen, 2016a).

Previously, Gao et al. showed that Cluh co-sediments with light membranes and the ribosomal fraction and is released with high salt, suggesting it is a ribosome associated protein. This analysis has been extended to show that Clu reciprocally co-IPs with three ribosomal proteins (RpLs) and two RpSs (small ribosomal proteins), as well as two eIF3 proteins, and thus does indeed bind the ribosome. Furthermore, addition of EDTA, which dissociates ribosomes, caused Clu to shift from the heaviest to lighter fractions in a sucrose gradient. However, most importantly, Clu is able to bind RpL7a in the mitochondrial fraction but not in the cytoplasm. While there are high levels of homogeneous Clu in the cytoplasm, Clu particles in Drosophila germ cells are clearly juxtaposed to mitochondria but do not co-localize. As was previously shown, Clu associates with mitochondrial outer membrane proteins (Sen, 2015). These data taken together support that Clu predominantly binds the ribosome at the outer mitochondrial membrane in vivo. In addition, Clu associates with TOM20, thus placing Clu in the same location at which mitochondrial protein import occurs (Sen, 2015; Sen, 2016a and references therein).

Drosophila third instar neuroblasts divide frequently (approximately every twenty five minutes) to ultimately create the adult brain. Components of the Myc pathway, which is responsible for ribosome biogenesis, have been shown to have differential expression in neuroblasts. The nucleolar protein Mushroom body miniature (Mbm), which is highly expressed in neuroblasts, is a transcriptional target of Myc and is involved in ribosome biogenesis. Conversely, Brain tumor (Brat) has been shown to be a negative regulator of Myc and becomes asymmetrically localized away from the neuroblast into the daughter cell during cell division. Work involving transcriptome analysis found that genes involved in ribosome biogenesis, as well as other processes, are highly represented in neuroblasts compared to differentiated neurons. Given that an increase in ribosome biogenesis appears intrinsically important for neuroblasts, it follows that ribosomal proteins should also be enriched in neuroblasts. This work shows that RpLs, as well as eIF3-S9, are highly enriched in neuroblasts (Sen, 2016a).

Clu1p and Cluh are ribonucleoproteins, however which Clu domain binds mRNA has not been identified. This study shows the TPR domain of Clu is critical for mRNA binding. TPR domains are well known to facilitate protein-protein interactions. Proteins containing the related HAT domain (half a TPR) and pentatricopeptide repeats (PPRs) have been shown to directly bind RNA. HAT and PPR domains have similar sequence and structure to TPRs. Since UV-cross linking causes covalent bonds between nucleic acid and protein, the current data showing deletion of the TPR greatly decreases the amount of bound mRNA is consistent with it potentially directly binding mRNA (Sen, 2016a).

These data strongly support that Clu normally binds to mRNA in vivo. Combined with data showing Clu binds ribosomal proteins, and can do so in the mitochondrial fraction, suggests that the role of Clu in the cell is to facilitate mRNA binding and association with the ribosome at the mitochondrial outer membrane. In fact, mitochondrial protein levels are globally reduced in Drosophila clu mutants, and specific mitochondrial proteins encoded by Cluh-bound mRNAs are reduced in cluh knockout immortalized mouse embryonic fibroblasts. Thus, Clu may act to provide site-specific translation or co-translational import of mitochondrial proteins. The molecular mechanism of co-translational import is well-established for endoplasmic reticulum bound proteins. It is increasingly clear that co-translational import occurs on mitochondria, however the mechanism is not as clear. Clu forms a complex with the mitophagy proteins PINK1 and Parkin, and PINK1 and TOM20 have been implicated in localized translation of mRNAs encoding respiratory chain proteins. It is possible that Clu may function to link lack of mitochondrial import and activity with mitochondrial damage sensing and destruction (Sen, 2015; Sen, 2016a).

Mitochondrial polyadenylation is a one-step process required for mRNA integrity and tRNA maturation

Polyadenylation has well characterised roles in RNA turnover and translation in a variety of biological systems. While polyadenylation on mitochondrial transcripts has been suggested to be a two-step process required to complete translational stop codons, its involvement in mitochondrial RNA turnover is less well understood. This study analyzed knockdown and knockout models of the mitochondrial poly(A) polymerase (MTPAP) in Drosophila melanogaster. It was demonstrated that polyadenylation of mitochondrial mRNAs is exclusively performed by MTPAP. Further, mitochondrial polyadenylation does not regulate mRNA stability but protects the 3' terminal integrity, and that despite a lack of functioning 3' ends, these trimmed transcripts are translated, suggesting that polyadenylation is not required for mitochondrial translation. Additionally, loss of MTPAP leads to reduced steady-state levels and disturbed maturation of tRNACys, indicating that polyadenylation in mitochondria might be important for the stability and maturation of specific tRNAs (Bratic, 2016). 

Selfish drive can trump function when animal mitochondrial genomes compete

Mitochondrial genomes compete for transmission from mother to progeny. This study explored this competition by introducing a second genome into Drosophila melanogaster to follow transmission. Competitions between closely related genomes favor those functional in electron transport, resulting in a host-beneficial purifying selection. In contrast, matchups between distantly related genomes often favor those with negligible, negative or lethal consequences, indicating selfish selection. Exhibiting powerful selfish selection, a genome carrying a detrimental mutation displaces a complementing genome, leading to population death after several generations. In a different pairing, opposing selfish and purifying selection counterbalances to give stable transmission of two genomes. Sequencing of recombinant mitochondrial genomes shows that the noncoding region, containing origins of replication, governs selfish transmission. Uniparental inheritance prevents encounters between distantly related genomes. Nonetheless, in each maternal lineage, constant competition among sibling genomes selects for super-replicators. The study suggests that this relentless competition drives positive selection, promoting change in the sequences influencing transmission (Ma, 2016).

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

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, this study identified the three proteins composing Drosophila mt:RNase P: Mulder (PRORP), Scully (MRPP2) and Roswell (MRPP1). It was shown 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 understanding of how loss of mitochondrial tRNA processing causes tissue failure, an important aspect of human mitochondrial disease (Sen, 2016b).

Mitonuclear interactions, mtDNA-mediated thermal plasticity, and implications for the Trojan Female Technique for pest control

Pest species pose major challenges to global economies, ecosystems, and health. Unfortunately, most conventional approaches to pest control remain costly, and temporary in effect. As such, a heritable variant of the Sterile Insect Technique (SIT) was proposed, based on the introduction of mitochondrial DNA mutations into pest populations, which impair male fertility but have no effects on females. Evidence for this "Trojan Female Technique" (TFT) was recently provided, in the form of a mutation in the mitochondrial cytochrome b gene (mt:Cyt-b) of Drosophila melanogaster which reduces male fertility across diverse nuclear backgrounds. However, recent studies have shown that the magnitude of mitochondrial genetic effects on the phenotype can vary greatly across environments, with mtDNA polymorphisms commonly entwined in genotype-by-environment (G x E) interactions. This study tested whether the male-sterilizing effects previously associated with the mt:Cyt-b mutation are consistent across three thermal and three nuclear genomic contexts. The effects of this mutation were indeed moderated by the nuclear background and thermal environment, but crucially the fertility of males carrying the mutation was invariably reduced relative to controls. This mutation thus constitutes a promising candidate for the further development of the TFT (Wolff, 2016b).

Mitochondrial-nuclear interactions mediate sex-specific transcriptional profiles in Drosophila

The assembly and function of mitochondria require coordinated expression from two distinct genomes, the mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). This study tested how the transcriptome responds to mtDNA and nDNA variation, along with mitonuclear interactions (mtDNA x nDNA) in Drosophila. Two mtDNA haplotypes that differ in a substantial number of single nucleotide polymorphisms, with >100 amino acid differences, were used. Each haplotype was placed on each of two D. melanogaster nuclear backgrounds and was tested for transcription differences in both sexes. Large numbers of transcripts were found to be differentially expressed between nuclear backgrounds, and mtDNA type altered the expression of nDNA genes, suggesting a retrograde, trans-effect of mitochondrial genotype. Females are generally more sensitive to genetic perturbation than males, and males demonstrate an asymmetrical effect of mtDNA in each nuclear background; mtDNA effects are nuclear background-specific. MtDNA sensitive genes are not enriched in male- or female-limited expression space in either sex. The responses to mitonuclear covariation were shown to be substantially different between the sexes, yet the mtDNA genes are consistently differentially expressed across nuclear backgrounds and sexes. These results provide evidence that mtDNA main effects can be consistent across nuclear backgrounds, but the interactions between mtDNA and nDNA can lead to sex-specific global transcript responses (Mossmann, 2016).

Mutants for Drosophila Isocitrate dehydrogenase 3b are defective in mitochondrial function and larval cell death

The death of larval salivary gland cells during metamorphosis in Drosophila melanogaster has been a key system for studying steroid controlled programmed cell death. This death is induced by a pulse of the steroid hormone ecdysone that takes place at the end of the prepupal period. For many years, it has been thought that the ecdysone direct response gene Eip93F (E93) plays a critical role in initiating salivary gland cell death. This conclusion was based largely on the finding that the three "type" alleles of E93 cause a near-complete block in salivary gland cell death. This study shows that these three mutations are in fact allelic to Idh3b, a nearby gene that encodes the beta subunit of isocitrate dehydrogenase 3, a mitochondrial enzyme of the tricarboxylic acid (TCA) cycle. The strongest of the Idh3b alleles appears to cause a near-complete block in oxidative phosphorylation, as mitochondria are depolarized in mutant larvae, and development arrests early during cleavage in embryos from homozygous-mutant germ line mothers. Idh3b-mutant larval salivary gland cells fail to undergo mitochondrial fragmentation, which normally precedes the death of these cells, and do not initiate autophagy, an early step in the cell death program. These observations suggest a close relationship between the TCA cycle and the initiation of larval cell death. In normal development, tagged Idh3b is released from salivary gland mitochondria during their fragmentation, suggesting that Idh3b may be an apoptogenic factor that functions much like released cytochrome c in mammalian cells (Duncan, 2017).

Sex-specific influences of mtDNA mitotype and diet on mitochondrial functions and physiological traits in Drosophila melanogaster

This study determined the sex-specific influence of mtDNA type (mitotype) and diet on mitochondrial functions and physiology in two Drosophila melanogaster lines. In many species, males and females differ in aspects of their energy production. These sex-specific influences may be caused by differences in evolutionary history and physiological functions. It was predicted the influence of mtDNA mutations should be stronger in males than females as a result of the organelle's maternal mode of inheritance in the majority of metazoans. In contrast, it was predicted the influence of diet would be greater in females due to higher metabolic flexibility. Four diets were included that differed in their protein: carbohydrate (P:C) ratios as they are the two-major energy-yielding macronutrients in the fly diet. Four mitochondrial function traits (Complex I oxidative phosphorylation, reactive oxygen species production, superoxide dismutase activity, and mtDNA copy number) and four physiological traits (fecundity, longevity, lipid content, and starvation resistance) were examined. Traits were assayed at 11 d and 25 d of age. Consistent with predictions it was observe that the mitotype influenced males more than females supporting the hypothesis of a sex-specific selective sieve in the mitochondrial genome caused by the maternal inheritance of mitochondria. Also, consistent with predictions, it was found that the diet influenced females more than males (Aw, 2017).

A unique respiratory adaptation in Drosophila independent of supercomplex formation

Large assemblies of respiratory chain complexes, known as supercomplexes, are present in the mitochondrial membrane. The formation of supercomplexes is thought to contribute to efficient electron transfer, stabilization of each enzyme complex, and inhibition of reactive oxygen species (ROS) generation. In this study, mitochondria from various organisms were solubilized with digitonin, and then the solubilized complexes were separated by blue native PAGE (BN-PAGE). The results revealed a supercomplex consisting of complexes I, III, and IV in mitochondria from bovine and porcine heart, and a supercomplex consisting primarily of complexes I and III in mitochondria from mouse heart and liver. However, supercomplexes were barely detectable in Drosophila flight-muscle mitochondria, and only dimeric complex V was present. Drosophila mitochondria exhibited the highest rates of oxygen consumption and NADH oxidation, and the concentrations of the electron carriers, cytochrome c and quinone were higher than in other species. Respiratory chain complexes were tightly packed in the mitochondrial membrane containing abundant phosphatidylethanol-amine with the fatty acid palmitoleic acid (C16:1), which is relatively high oxidation-resistant as compared to poly-unsaturated fatty acid. These properties presumably allow efficient electron transfer in Drosophila. These findings reveal the existence of a new mechanism of biological adaptation independent of supercomplex formation (Shimada, 2017).

Glial lipid droplets and neurodegeneration in a Drosophila model of complex I deficiency

Mitochondrial defects associated with respiratory chain complex I deficiency lead to heterogeneous fatal syndromes. While the role of NDUFS8, an essential subunit of the core assembly of the complex I, is established in mitochondrial diseases, the mechanisms underlying neuropathology are poorly understood. This study developed a Drosophila model of NDUFS8 deficiency by knocking down the expression of its fly homologue in neurons or in glial cells. Downregulating ND23 in neurons resulted in shortened lifespan, and decreased locomotion. Although total brain ATP levels were decreased, histological analysis did not reveal any signs of neurodegeneration except for photoreceptors of the retina. Interestingly, ND23 deficiency-associated phenotypes were rescued by overexpressing the glucose transporter hGluT3 demonstrating that boosting glucose metabolism in neurons was sufficient to bypass altered mitochondrial functions and to confer neuroprotection. The consequences of ND23 knockdown was then studied in glial cells. In contrast to neuronal knockdown, loss of ND23 in glia did not lead to significant behavioral defects nor to reduced lifespan, but induced brain degeneration, as visualized by numerous vacuoles found all over the nervous tissue. This phenotype was accompanied by the massive accumulation of lipid droplets at the cortex-neuropile boundaries, suggesting an alteration of lipid metabolism in glia. These results demonstrate that complex I deficiency triggers metabolic alterations both in neurons and glial cells which may contribute to the neuropathology (Cabirol-Pol, 2017).

Mitochondrial ribosomal Protein L10 Associates with Cyclin B1/Cdk1 Activity and Mitochondrial Function

Mitochondria ribosomal proteins are important for mitochondrial-encoded protein synthesis and mitochondrial function. In addition to their roles in mitoribosome assembly, several mitochondrial ribosome proteins are also implicated in cellular processes like cell cycle regulation, apoptosis, and mitochondrial homeostasis regulation. This study demonstrate that MRPL10 regulates cyclin B1/Cdk1 (cyclin-dependent kinase 1) activity and mitochondrial protein synthesis in mammalian cells. In Drosophila, inactivation of mRpL10 (the Drosophila ortholog of mammalian MRPL10) in eyes results in abnormal eye development. Furthermore, expression of human cyclin B1 suppresses eye phenotypes and mitochondrial abnormality of mRpL10 knockdown Drosophila. This study identified that the physiological regulatory pathway of MRPL10 and providing new insights into the role of MRPL10 in growth control and mitochondrial function (H. B. Li, 2016).

Duplication of Drosophila melanogaster mitochondrial EF-Tu: pre-adaptation to T-arm truncation and exclusion of bulky aminoacyl residues
Translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA to ribosomes in protein synthesis. EF-Tu generally recognizes aminoacyl moieties and acceptor- and T-stems of aminoacyl-tRNAs. However, nematode mitochondrial (mt) tRNAs frequently lack all or part of the T-arm that is recognized by canonical EF-Tu. It has been reported that two distinct EF-Tu species, EF-Tu1 and EF-Tu2, respectively recognize mt tRNAs lacking T-arms and D-arms in the mitochondria of the chromadorean nematode C. elegans. C. elegans EF-Tu2 specifically recognizes the seryl-moiety of serylated D-armless tRNAs. Mitochondria of the enoplean nematode Trichinella possess three structural types of tRNAs: T-armless tRNAs, D-armless tRNAs, and cloverleaf tRNAs with a short T-arm. Trichinella mt EF-Tu1 binds to all three types and EF-Tu2 binds only to D-armless Ser-tRNAs, showing an evolutionary intermediate state from canonical EF-Tu to chromadorean nematode (e.g. C. elegans) EF-Tu species. This study reports that two EF-Tu species also participate in Drosophila mitochondria. Both Drosophila EF-Tu1 and EF-Tu2 bound to cloverleaf and D-armless tRNAs. Drosophila EF-Tu1 has the ability to recognize T-armless tRNAs that do not evidently exist in Drosophila mitochondria but do exist in related arthropod species. In addition, Drosophila EF-Tu2 preferentially bound to aa-tRNAs carrying small amino acids, but not to aa-tRNAs carrying bulky amino acids. These results suggest that the Drosophila mitochondrial translation system could be another intermediate state between the canonical and nematode mitochondria-type translation systems (Sato, 2017).

The Mitochondrial DNA Polymerase Promotes Elimination of Paternal Mitochondrial Genomes

Mitochondrial DNA (mtDNA) is typically inherited from only one parent. In animals, this is usually the mother. Active programs enforce uniparental inheritance at two levels, eliminating paternal mitochondrial genomes or destroying mitochondria delivered to the zygote by the sperm. Both levels operate in Drosophila. As sperm formation begins, hundreds of doomed mitochondrial genomes are visualized within the two huge mitochondria of each spermatid. These genomes abruptly disappear during spermatogenesis. Genome elimination, which is not in the interests of the restricted genomes, is directed by nuclear genes. Mutation of EndoG, which encodes a mitochondria-targeted endonuclease, retarded elimination. This study shows that knockdown of the nuclear-encoded mtDNA polymerase (Pol gamma-alpha), Tamas, produces a more complete block of mtDNA elimination. Tamas is found in large particles that localize to mtDNA during genome elimination. A simple possible mechanism was discounted by showing that the 3'-exonuclease function of the polymerase is not needed. While DNA elimination is a surprising function for DNA polymerase, it could provide a robust nexus for nuclear control of mitochondrial genome copy number, since use of common interactions for elimination and replication might limit options for the mitochondrial genome to escape restriction. It is suggested that the DNA polymerase may play this role more widely and that inappropriate activation of its elimination ability might underlie association of DNA loss syndromes with mutations of the human mtDNA polymerase (Yu, 2017).

Regulation of mitochondrial complex I biogenesis in Drosophila flight muscles

The flight muscles of Drosophila are highly enriched with mitochondria, but the mechanism by which mitochondrial complex I (CI) is assembled in this tissue has not been described. This study reports the mechanism of CI biogenesis in Drosophila flight muscles and shows that it proceeds via the formation of approximately 315, approximately 550, and approximately 815 kDa CI assembly intermediates. Additionally, specific roles were defined for several CI subunits in the assembly process. In particular, dNDUFS5 was shown to be required for converting an approximately 700 kDa transient CI assembly intermediate into the approximately 815 kDa assembly intermediate. Importantly, incorporation of dNDUFS5 into CI is necessary to stabilize or promote incorporation of dNDUFA10 into the complex. These findings highlight the potential of studies of CI biogenesis in Drosophila to uncover the mechanism of CI assembly in vivo and establish Drosophila as a suitable model organism and resource for addressing questions relevant to CI biogenesis in humans (Garcia, 2017).

PINK1 phosphorylates MIC60/Mitofilin to control structural plasticity of mitochondrial crista junctions

Mitochondrial crista structure partitions vital cellular reactions and is precisely regulated by diverse cellular signals. This study shows that, in Drosophila, mitochondrial cristae undergo dynamic remodeling among distinct subcellular regions and the Parkinson's disease (PD)-linked Ser/Thr kinase PINK1 participates in their regulation. Mitochondria increase crista junctions and numbers in selective subcellular areas, and this remodeling requires PINK1 to phosphorylate the inner mitochondrial membrane protein MIC60/mitofilin, which stabilizes MIC60 oligomerization. Expression of MIC60 restores crista structure and ATP levels of PINK1-null flies and remarkably rescues their behavioral defects and dopaminergic neurodegeneration. In an extension to human relevance, the PINK1-MIC60 pathway was found to be conserved in human neurons, and expression of several MIC60 coding variants in the mitochondrial targeting sequence found in PD patients in Drosophila impairs crista junction formation and causes locomotion deficits. These findings highlight the importance of maintenance and plasticity of crista junctions to cellular homeostasis in vivo (Tsai, 2018).

Drosophila protease ClpXP specifically degrades DmLRPPRC1 controlling mitochondrial mRNA and translation

ClpXP is the major protease in the mitochondrial matrix in eukaryotes, and is well conserved among species. ClpXP is composed of a proteolytic subunit, ClpP, and a chaperone-like subunit, ClpX. Although it has been proposed that ClpXP is required for the mitochondrial unfolded protein response, additional roles for ClpXP in mitochondrial biogenesis are unclear. This study found that Drosophila leucine-rich pentatricopeptide repeat domain-containing protein 1 (DmLRPPRC1) is a specific substrate of ClpXP. Depletion or introduction of catalytically inactive mutation of ClpP increases DmLRPPRC1 and causes non-uniform increases of mitochondrial mRNAs, accumulation of some unprocessed mitochondrial transcripts, and modest repression of mitochondrial translation in Drosophila Schneider S2 cells. Moreover, DmLRPPRC1 over-expression induces the phenotypes similar to those observed when ClpP is depleted. Taken together, ClpXP regulates mitochondrial gene expression by changing the protein level of DmLRPPRC1 in Drosophila Schneider S2 cells (Matsushima, 2017).

ClpXP, Lon and m-AAA are ATP-dependent proteases that contribute to the degradation of all mitochondrial matrix proteins. ClpXP and Lon localize to the matrix space whereas the m-AAA protease is anchored in the membrane with its catalytic site exposed to the matrix space. ClpXP is composed of a proteolytic subunit, ClpP, and a chaperone-like subunit, ClpX, which carries an ATPase associated with diverse cellular activities (AAA+) domain (Matsushima, 2017).

ClpXP is a barrel-shaped, hetero-oligomeric complex in which ClpP forms a two-stack heptameric ring-shaped structure to which two hexameric ClpX rings bind on each side. Lon also carries an AAA+ domain and forms homo-oligomeric, ring-shaped complexes. Lon and ClpP contain serine protease domains and these proteases are well conserved among species. Interestingly, mutations in each protease cause different diseases. Mutations in the gene encoding ClpP cause sensorineural hearing loss and ovarian failure in Perrault syndrome, while mutations in the genes encoding mitochondrial Lon cause cerebral, ocular, dental, auricular, skeletal anomalies (CODAS) syndrome. Moreover, a ClpP knock out in the mouse results in a phenotype similar to the relatively modest phenotype observed in patients with Perrault syndrome whereas deletion of the mitochondrial Lon protease gene causes early lethality (Matsushima, 2017).

ClpXP and Lon function mainly to degrade misfolded or damaged proteins (for example, proteins damaged by oxidation) to prevent the cell from accumulating defective proteins. These protein degradation pathways are collectively called protein quality control. Although there is some overlap in the substrate specificities of mitochondrial proteases, it has been proposed that some mitochondrial matrix proteins are specific substrates for each of them. However, to date, a limited number of proteins has been shown to be specific substrates for each. In addition, ClpXP and Lon also play a key role in mitochondrial biological processes. For instance, we have shown previously that Lon regulates mitochondrial DNA (mtDNA) transcription by regulating the ratio of mitochondrial transcription factor A (TFAM) to mtDNA via degradation of TFAM. In addition, ClpXP modulates mitochondrial unfolded protein responses in C. elegans and mammals (Matsushima, 2017).

Metazoan mtDNA is transcribed as precursor polycistronic RNAs containing rRNAs, tRNAs, and mRNAs. After RNA processing and maturation, each RNA contributes to mitochondrial translation. The pentatricopeptide repeat (PPR) domain is a RNA binding domain and, in metazoans, the PPR protein family participates in mitochondrial RNA biogenesis. Leucine-rich pentatricopeptide repeat domain–containing proteins, called LRPPRCs, have multiple PPR domains. Both mammalian LRPPRC and its Drosophila melanogaster homologue, DmLRPPRC1 are required for polyadenylation of mitochondrial mRNAs (mt-mRNAs). LRPPRCs also control mt-mRNA stability, as loss of LRPPRC or DmLRPPRC1 results in a non-uniform reduction of mitochondrial mRNA abundance. LRPPRC interacts with the non-PPR RNA binding protein, SRA stem-loop interacting RNA binding protein (SLIRP) in ribonucleoprotein complexes. Interestingly, LRPPRC and SLIRP are degraded in mitochondria under some experimental conditions such as inhibition of mitochondrial transcription (Matsushima, 2017).

This study investigated the role of ClpXP protease in regulating the abundance of mitochondrial mRNA, and processing and translation of mitochondrial RNA in Drosophila cells. The results argue strongly that ClpXP modulates mitochondrial RNA biogenesis by the selective degradation of DmLRPPRC1 (Matsushima, 2017).

Mitochondrial transcripts were increased in ClpP knockdown cells, which may result from upregulation of mitochondrial transcription activity because mitochondrial tRNAs, which can be indicators of mitochondrial transcription activity, were increased in addition to upregulation of mtTFB2 protein and mRNA. Previously work that mitochondrial transcripts are also increased in Lon knockdown cells in association with an increase in mtTFB2 protein. These similar events might suggest that upregulation of mtTFB2 is a common response to defects in mitochondrial matrix protein turnover caused by depletion of either Lon or ClpXP. Moreover, mtDNA copy number is also increased in ClpP knockdown cells. These results might suggest that the increase in mtDNA copy number results from the increased levels of mitochondrial transcripts that are required for the synthesis of RNA primers during replication of mtDNA, as was reported previously. Another possible explanation is that the increase in mtDNA is caused by accumulation of other mtDNA replication factors by either direct or indirect effects of ClpP knockdown (Matsushima, 2017).

DmLRPPRC1 overexpression did not change the abundance of mitochondrial tRNAs. Therefore, DmLRPPRC1 may not be involved in mitochondrial transcription, which is in agreement with previous studies showing no link between LRPPRC and transcriptional activity. It has been shown that depletion of DmLRPPRC1 results in the reduction of mitochondrial mRNA levelsand the same phenomenon was observed also in vertebrate cells. In addition, inhibition of mitochondrial transcription reduces LRPPRC and SLIRP levels in vertebrate cells. These results indicate that levels of LRPPRC, SLIRP and mt-mRNAs are interdependent. Likewise, the current study showed that DmLRPPRC1 degradation is facilitated in some conditions, such as inhibition of mitochondrial transcription or depletion of DmSLIRP1 in Drosophila cells. Conversely, DmLRPPRC1 increased when ClpP or ClpX was knocked down, suggesting that DmLRPPRC1 is degraded by ClpXP. In agreement with this hypothesis, ClpP depletion prevented the facilitated degradation of DmLRPPRC1 caused by mitochondrial transcription inhibition or DmSLIRP1 knockdown. DmLRPPRC1 may be specifically degraded by ClpXP because Lon-depletion did not accumulate DmLRPPRC1 or inhibit the facilitated degradation of DmLRPPRC1. Moreover, this study showed that ClpX, which recognize the protein substrates of ClpXP, physically interacts with DmLRPPRC1 in the cells. Collectively, these results strongly suggest that DmLRPPRC1 is a specific substrate of ClpXP in Drosophila cells. By contrast, ClpP knockdown did not prevent the degradation of DmSLIRP1 caused by mitochondrial transcription inhibition or DmLRPPRC1 knockdown, which suggests that the facilitated degradation of DmSLIRP1 is not specifically mediated by ClpXP. DmLRPPRC1 and DmSLIRP1 stabilize each other. It is considered that ClpP knockdown increased DmSLIRP1 protein in control cells due to the DmLRPPRC1 accumulation (Matsushima, 2017).

The preferred amino acid sequences recognized by mitochondrial ClpXP remain unclear. At the same time, proteomic studies of E. coli ClpXP identified some classes of peptide sequences, called preferred degradation tags, and most of the preferred C-terminal degradation tags contain alanine in the C-terminal position. Interestingly, the amino acid in the C-terminal position of DmLRPPRC1 is alanine, suggesting that mitochondrial ClpXP might also recognize the alanine. These results show the possibility that in addition to LONP1, ClpXP may also contribute to the degradation of LRPPRC protein. However, unlike the situation in Schneider S2 cells, ClpXP is not the dominant protease in the degradation of LRPPRC protein in HeLa cells. Moreover, a recent report indicated that knockdown of Lon protease inhibits LRPPRC degradation in mouse cells. Because the C-terminal amino acid in mouse and human LRPPRC is not alanine, these differences in the substrate preference of proteases between species might arise from differential recognition of C-terminal degradation tags (Matsushima, 2017).

It has been shown that overexpressing ClpX in mouse C2C12 cells results in an increase in LRPPRC protein possibly involving the mitochondrial unfolded protein response pathway. This indicates that changes in the protein levels of ClpX may affect the gene expression of LRPPRC in mammalian cells. This study has shown that knockdown of ClpX or ClpP results in an increase of DmLRPPRC1 protein without an increase in the corresponding mRNA. These data indicate that in ClpP or ClpX knockdown Schneider S2 cells, the increase of DmLRPPRC1 likely does not result from signal transduction events (Matsushima, 2017).

This study showed that two unprocessed mRNAs, COXIII-ATP6/8 and ND6-CytB, are accumulated in ClpP knockdown cells or DmLRPPRC1 overexpression cells. Metazoan mtDNA is transcribed as precursor polycistronic transcripts containing mt-mRNAs, mt-rRNAs and mt-tRNAs. Most of the mt-mRNAs and mt-rRNAs are produced when mt-tRNAs are excised by ELAC2 and the RNaseP-complex. However, some processing sites are cleaved without an accompanying mt-tRNA excision, such as COXIII-ATP6/8 and ND6-CytB in Drosophila. The mechanisms involved in these cleavages remain largely unknown. In human cells, the ELAC2 and RNaseP-complexes do not appear to be involved in non-tRNA processing. Recent studies suggest that pentatricopeptide repeat domain 2 protein, which has one PPR domain, contributes to non-tRNA cleavages. DmLRPPRC1 contains multiple PPR domains. Excess DmLRPPRC1 protein may compete with canonical proteins performing the non-tRNA processing of COXIII-ATP6/8 and ND6-CytB (Matsushima, 2017).

An increase of DmLRPPRC1 protein results in a non-uniform increase in mitochondrial mRNA expression. Similar to the previously reported functions of vertebrate LRPPRC, DmLRPPRC1 is responsible for polyadenylation of mitochondrial mRNAs, which affects the stability of mt-mRNAs. However, this study did not detect any difference in migration of mt-mRNAs in northern blot analysis in ClpP RNAi cells. These results suggest that the length of poly(A) tails of mt-mRNAs are not changed dramatically in the presence of excess DmLRPPRC1 protein (Matsushima, 2017).

Excess DmLRPPRC1 resulted in a reduction of mitochondrial translation. These results are consistent a previous finding that shown that depletion of DmLRPPRC1 causes an increase in mitochondrial translation in flies. Similar to the current results, in Schizosaccharomyces pombe, overexpression of a multiple PPR domain-containing protein, PPR5, resulted in inhibition of mitochondrial protein synthesis without an obvious change in steady state levels of mitochondrial transcripts. Mitochondrial protein synthesis is reduced more severely in DmLRPPRC1 overexpressing cells although levels of mitochondrial mRNAs do not differ between ClpP knockdown cells and DmLRPPRC1 overexpression cells. DmLRPPRC1 was increased about two-fold in the former cells while about eight-fold in the latter. A previous study showed that the ratio of TFAM to mtDNA is important for the regulation of mitochondrial transcription and a higher ratio of TFAM to mtDNA causes a reduction in mitochondrial transcript abundance. Analogously, the ratio of DmLRPPRC1 to mtRNAs may be critical for the regulation of mitochondrial translation (Matsushima, 2017).

Ribosome profiling results showed that DmLRPPRC1 overexpression inhibited the formation of fully assembled 55S ribosomes, and enhanced the accumulation of mt-mRNAs in the lower-density fractions that contain mitochondrial small ribosomal subunits. In mitochondrial translation, mt-mRNAs initially interact with the small ribosomal subunit and then are assembled into the full ribosome. Therefore, the results suggest that excess DmLRPPRC1 does not prevent this interaction, but instead interferes with the assembly of mt-mRNA-small ribosomal subunit complexes with large ribosomal subunits (Matsushima, 2017).

In humans, recessive mutations of ClpP have been known to cause Perrault syndrome characterized by sensorineural hearing loss and ovarian failure. Similar to humans, ClpP knockout mice show hearing loss and infertility. Interestingly, knockout of ClpP also causes ineffective mitochondrial translation. A very recent study shows that ClpXP regulates the protein levels of ERAL1, a putative 12S rRNA chaperone. ERAL1 protein was accumulated in ClpP deficient mouse heart and the excess ERAL1 cause ineffective mitochondrial translation by the inhibition of mitochondrial ribosome assembly. To date, many proteins are on the list of candidates of ClpXP substrates. Therefore, some substrate proteins of ClpXP may be involved in mitochondrial translation. Further studies are necessary to clarify the relationship between protein substrates of ClpXP and mitochondrial translation (Matsushima, 2017).

In summary, it is concluded that DmLRPPRC1 is a specific substrate of ClpXP. An increase in DmLRPPRC1 protein, which can result from ClpXP depletion, causes non-uniform increases of mitochondrial mRNAs, accumulation of some unprocessed mitochondrial transcripts and partial inhibition of mitochondrial translation (Matsushima, 2017).

Selections that isolate recombinant mitochondrial genomes in animals

Homologous recombination is widespread and catalyzes evolution. Nonetheless, its existence in animal mitochondrial DNA is questioned. This study designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines. In four experimental settings, recombinant genomes became the sole or dominant genome in the progeny. Thus, selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit. Double-strand breaks enhanced recombination in the germ line and revealed somatic recombination. When the recombination partner was a diverged D. melanogaster genome or a genome from a different species such as D. yakuba, sequencing revealed long continuous stretches of exchange. In addition, the distribution of sequence polymorphisms in recombinants allowed mapping of a selected trait to a particular region in the Drosophila mitochondrial genome. Thus, recombination can be harnessed to dissect function and evolution of mitochondrial genome (Ma, 2015).

Long Oskar controls mitochondrial inheritance in Drosophila melanogaster

Inherited mtDNA mutations cause severe human disease. In most species, mitochondria are inherited maternally through mechanisms that are poorly understood. Genes that specifically control the inheritance of mitochondria in the germline are unknown. This study shows that the long isoform of the protein Oskar regulates the maternal inheritance of mitochondria in Drosophila melanogaster. During oogenesis mitochondria accumulate at the oocyte posterior, concurrent with the bulk streaming and churning of the oocyte cytoplasm. Long Oskar traps and maintains mitochondria at the posterior at the site of primordial germ cell (PGC) formation through an actin-dependent mechanism. Mutating long oskar strongly reduces the number of mtDNA molecules inherited by PGCs. Therefore, Long Oskar ensures germline transmission of mitochondria to the next generation. These results provide molecular insight into how mitochondria are passed from mother to offspring, as well as how they are positioned and asymmetrically partitioned within polarized cells (Hurd, 2016).

Germ cells are the means by which sexually reproducing organisms transmit genetic material to subsequent generations to ensure the continuance of the species. Consequently, the formation and specification of germ cells is one of the most important events in development. PGC formation can occur either through the cytoplasmic inheritance of maternally deposited determinants, called germ plasm, or through inductive cell-signaling events. In D. melanogaster, PGCs are formed by the deposition of germ plasm at the posterior of the embryo. The germ plasm has long been known to be rich in mitochondria. In fact, in mammals one of the names for germline granules is the intermitochondrial cement. The reason for this curious association, however, has been unclear until now (Hurd, 2016).

This study shows that mitochondria accumulate in the germ plasm to ensure the transmission of their genomes to the next generation. In D. melanogaster, most mitochondria are transported to the germ plasm during cytoplasmic streaming in developing oocytes and maintained there by an actin-dependent mechanism. Long Oskar controls mitochondrial anchoring at the posterior and is not only necessary but also sufficient to tether mitochondria wherever it is expressed. Mutating long oskar decreases the number of mitochondrial genomes transmitted to the next generation, demonstrating that Long Oskar is important for mtDNA inheritance. Long Oskar mutants also have reduced numbers of PGCs and frequently impaired oogenesis. The current data suggest that a potential cause of this is a failure to enrich mitochondria at the posterior. However, it remains to be determined whether the reduction in the number of mitochondria at the posterior and in PGCs affects PGC survival, formation, or division. Long Oskar-mediated mitochondrial enrichment could also play a role in the formation, biogenesis, and/or anchoring of germ plasm to the posterior prior to PGC formation. Alternatively, the defects in long oskar mutants could be due to some other function of Long Oskar independent of its role in trapping mitochondria at the posterior (Hurd, 2016).

Previous studies have analyzed mitochondrial distribution during earlier stages of Drosophila oogenesis. They show that mitochondria initially enter the oocyte traveling on microtubules and once there coalesce into a single mass resembling a structure called the Balbiani body. Recent data suggest that selective replication of mtDNA may restrict the transmission of deleterious mtDNA mutations at this time. Further experiments showed that Balbiani body mitochondria associate with the posterior until stage 7, when the oocyte repolarizes its microtubule network. This study analyzed mitochondrial distribution at later stages of oogenesis. The vast majority of mitochondria passed into PGCs accumulate during and after stage 10b, and thus may be predominantly nurse cell derived and Balbiani body independent. In the absence of Long Oskar a small amount of mitochondria do enter the PGCs, however, and it is possible that these could constitute a different pool that entered the oocyte at an earlier stage. Direct visualization of mitochondrial populations are needed to determine whether specific sources of mitochondria reach the posterior pole or whether they are randomly selected from the oocyte pool (Hurd, 2016).

Mitochondrial transport is often an active process in which motor proteins and their adapters move mitochondria along the cytoskeleton. Interestingly, this is not likely the case in D. melanogaster stage 10 oocytes. Instead, it was found that mitochondria move apparently passively, caught in the bulk flow of the oocyte cytoplasm, to localize to the oocyte posterior. This mode of localization is not unique to mitochondria; germ plasm RNAs, such as nanos, also use it to localize to the embryo posterior. Cytoplasmic streaming occurs in a wide variety of other contexts, across a range of organisms and developmental stages. Given the current findings it will be interesting to investigate whether cytoplasmic streaming is used in other contexts as a means of mitochondrial transport or asymmetric localization (Hurd, 2016).

How Long Oskar uses the actin cytoskeleton to anchor mitochondria remains unclear. Oskar is present in two forms, Short and Long. Short Oskar is an integral member of germ plasm and is both necessary and sufficient to form functional PGCs. In stark contrast, Long Oskar is distinctly localized to endocytic membranes and is not required for PGC formation per se (Tanaka, 2011; Vanzo, 2007). Long Oskar may instead function to help anchor the germ plasm by promoting yolk endocytosis and remodeling of the actin cytoskeleton. Unexpectedly, this study did not identify any endocytic proteins in Long Oskar co-immunoprecipitation experiments. Instead, the most abundant Long Oskar interacting proteins identified were actin and actin-binding proteins including surprisingly a number of muscle-specific actinomyosin proteins. This leaves open the possibility that Long Oskar, and more specifically its N-terminal domain, nucleates actin directly or regulates proteins that modify actin. If so, Long Oskar would likely represent a new type of actin-modifying protein, as its N-terminal domain bears no sequence homology to any actin-modifying protein in Drosophila or elsewhere. Overexpression of Long Oskar in S2R+ cells caused gross alteration to the F-actin cytoskeleton, which is also consistent with Long Oskar binding the actin cytoskeleton and possibly competing with other actin cytoskeletal binding proteins. Further experiments will be required to determine exactly how Long Oskar alters the actin cytoskeleton and whether cytoskeletal-mediated mitochondrial localization requires endosomal components (Hurd, 2016).

The actin cytoskeleton is necessary for mitochondrial retention at the posterior pole of the embryo. Defects in mitochondrial interactions with the cytoskeleton are associated with many neurodegenerative diseases. Interestingly, disruption of F-actin with actin-depolymerizing drugs affects mitochondrial retention, but not transport, in Drosophila neurons. Furthermore, in vertebrate axonal neurons mitochondria have been shown to interact with actin microfilaments. As both Oskar and TmII are reported to be expressed in Drosophila neurons, it would be interesting to determine whether these two proteins similarly anchor mitochondria in this cell type. Further high-resolution imaging is also required to determine the regulation and dynamics of this potentially general mechanism of mitochondrial retention (Hurd, 2016).

Long Oskar acts as the main mechanism of mitochondrial inheritance in PGCs. Whether mitochondria that localize to the posterior and represent the majority of those inherited, are chosen at random, or are selected based on fitness, health, or some other attribute remains to be determined. In yeast, such a 'fitness'-based mechanism of inheritance has been observed. There, bundles of F-actin extend from the bud tip to the mother cell and serve as tracks for mitochondrial movement. Far from static, these actin cables are continuously moving away from the bud. Therefore, for mitochondria to be inherited into daughter cells they must 'crawl upstream' against the opposing movement of the actin cables, creating a fitness test such that only the healthiest mitochondria make it and are inherited. It is possible that a similar situation also occurs at the posterior of Drosophila oocytes. Although mitochondria appear to be statically anchored at the posterior in the embryo, the current analysis does not exclude the possibility that they are undergoing short-range movements on actin filaments. Indeed, purifying selection against deleterious mtDNA mutations has been observed in the Drosophila germline. It will be interesting to explore whether the accumulation and inheritance of mitochondria serves as a mechanism to test fitness and/or select against those that carry harmful mutations (Hurd, 2016).

Most organisms inherit mitochondria uniparentally. The reason for this remains unclear. Recent evidence suggests that inheritance of paternal mtDNA can be harmful. Consistent with this, multiple pathways have been described in Drosophila preventing the transmission of paternal mtDNA. Clearly, understanding mechanistically how mitochondria are transmitted and the genes that regulate this process is a key step in ultimately determining why this unusual mode of inheritance is so prevalent in nature (Hurd, 2016).

Incompatibility between mitochondrial and nuclear genomes during oogenesis results in ovarian failure and embryonic lethality

Mitochondrial dysfunction can cause female infertility. An important unresolved issue is the extent to which incompatibility between mitochondrial and nuclear genomes contributes to female infertility. It has previously been shown that a mitochondrial haplotype from D. simulans (simw501) is incompatible with a nuclear genome from the D. melanogaster strain Oregon-R (OreR), resulting in impaired development, which was enhanced at higher temperature. This mito-nuclear incompatibility is between alleles of the nuclear-encoded mitochondrial tyrosyl-tRNA synthetase (Aatm) and the mitochondrial-encoded tyrosyl-tRNA that it aminoacylates. This study shows that this mito-nuclear incompatibility causes a severe temperature-sensitive female infertility. The OreR nuclear genome contributed to death of ovarian germline stem cells and reduced egg production, which was further enhanced by the incompatibility with simw501 mitochondria. Mito-nuclear incompatibility also resulted in aberrant egg morphology and a maternal-effect on embryonic chromosome segregation and survival, which was completely dependent on the temperature and mito-nuclear genotype of the mother. These findings show that maternal mito-nuclear incompatibility during Drosophila oogenesis has severe consequences for egg production and embryonic survival, with important broader relevance to human female infertility and mitochondrial replacement therapy (Zhang, 2017).

Analysis of mitochondrial organization and function in the Drosophila blastoderm embryo

Mitochondria are inherited maternally as globular and immature organelles in metazoan embryos. This study used the Drosophila blastoderm embryo to characterize their morphology, distribution and functions in embryogenesis. Mitochondria are relatively small, dispersed and distinctly distributed along the apico-basal axis in proximity to microtubules by motor protein transport. Live imaging, photobleaching and photoactivation analyses of mitochondrially targeted GFP show that they are mobile in the apico-basal axis along microtubules and are immobile in the lateral plane thereby associating with one syncytial cell. Photoactivated mitochondria distribute equally to daughter cells across the division cycles. ATP depletion by pharmacological and genetic inhibition of the mitochondrial electron transport chain (ETC) activates AMPK and decreases syncytial metaphase furrow extension. In summary, this study shows that small and dispersed mitochondria of the Drosophila blastoderm embryo localize by microtubule transport and provide ATP locally for the fast syncytial division cycles. This study opens the possibility of use of Drosophila embryogenesis as a model system to study the impact of maternal mutations in mitochondrial morphology and metabolism on embryo patterning and differentiation (Chowdhary, 2017).

Activin signaling mediates muscle-to-adipose communication in a mitochondria dysfunction-associated obesity model

Mitochondrial dysfunction has been associated with obesity and metabolic disorders. However, whether mitochondrial perturbation in a single tissue influences mitochondrial function and metabolic status of another distal tissue remains largely unknown. This study analyzed the nonautonomous role of muscular mitochondrial dysfunction in Drosophila. Surprisingly, impaired muscle mitochondrial function via complex I perturbation results in simultaneous mitochondrial dysfunction in the fat body (the fly adipose tissue) and subsequent triglyceride accumulation, the major characteristic of obesity. RNA-sequencing (RNA-seq) analysis, in the context of muscle mitochondrial dysfunction, revealed that target genes of the TGF-beta signaling pathway were induced in the fat body. Strikingly, expression of the TGF-beta family ligand, Activin-&beta& (Act&beta&), was dramatically increased in the muscles by NF-kappaB/Relish (Rel) signaling in response to mitochondrial perturbation, and decreasing Actβ expression in mitochondrial-perturbed muscles rescued both the fat body mitochondrial dysfunction and obesity phenotypes. Thus, perturbation of muscle mitochondrial activity regulates mitochondrial function in the fat body nonautonomously via modulation of Activin signaling (Song, 2017).

Drosophila MIC60/Mitofilin conducts dual roles in mitochondrial motility and crista structure
MIC60/mitofilin constitutes a hetero-oligomeric complex on the inner mitochondrial membranes to maintain crista structure. However, little is known about its physiological functions. By characterizing Drosophila MIC60 mutants, this study defines its roles in vivo. MIC60 performs dual functions to maintain mitochondrial homeostasis. In addition to its canonical role in crista membrane structure, MIC60 regulates mitochondrial motility, likely by influencing protein levels of the outer mitochondrial membrane protein Miro that anchors mitochondria to the microtubule motors. Loss of MIC60 causes loss of Miro and mitochondrial arrest. At a cellular level, loss of MIC60 disrupts synaptic structure and function at the neuromuscular junctions. The double roles of MIC60 in both mitochondrial crista structure and motility position it as a crucial player for cellular integrity and survival (Tsai, 2017).


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Genes involved in organ development

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