Mutations in the mitochondrial GTPase mitofusin 2 (MFN2) cause Charcot-Marie-Tooth disease type 2 (CMT2A), a form of peripheral neuropathy that compromises axonal function. Mitofusins promote mitochondrial fusion and regulate mitochondrial dynamics. They are also reported to be involved in forming contacts between mitochondria and the endoplasmic reticulum. The fruit fly, Drosophila melanogaster, is a powerful tool to model human neurodegenerative diseases, including CMT2A. Here, we have downregulated the expression of the Drosophila mitofusin (dMfn RNAi) in adult flies and showed that this activates mitochondrial retrograde signalling and is associated with an upregulation of genes involved in folic acid (FA) metabolism. Additionally, we demonstrated that pharmacological and genetic interventions designed to increase the FA metabolism pathway suppresses the phenotype of the dMfn RNAi flies. We conclude that strategies to increase FA metabolism may ameliorate diseases, such as peripheral neuropathies, that are associated with loss of mitochondrial function. A video abstract for this article is available at  https://youtu.be/fs1G-QRo6xI .

HtrA2 (high-temperature requirement 2) is a human mitochondrial protease that has a role in apoptosis and Parkinson's disease. The structure of HtrA2 with an intact catalytic triad was determined, revealing a conformational change in the active site loops, involving mainly the regulatory LD loop, which resulted in burial of the catalytic serine relative to the previously reported structure of the proteolytically inactive mutant. Mutations in the loops surrounding the active site that significantly restricted their mobility, reduced proteolytic activity both in vitro and in cells, suggesting that regulation of HtrA2 activity cannot be explained by a simple transition to an activated conformational state with enhanced active site accessibility. Manipulation of solvent viscosity highlighted an unusual bi-phasic behavior of the enzymatic activity, which together with MD calculations supports the importance of motion in the regulation of the activity of HtrA2. HtrA2 is an unusually thermostable enzyme (TM=97.3 °C), a trait often associated with structural rigidity, not dynamic motion. We suggest that this thermostability functions to provide a stable scaffold for the observed loop motions, allowing them a relatively free conformational search within a rather restricted volume.

Colorectal cancer (CRC) is a major contributor to cancer mortality and morbidity. LIM kinase 2 (LIMK2) promotes tumour cell invasion and metastasis. The objectives of this study were to determine how LIMK2 expression is associated with CRC progression and patient outcome, and to use genetically modified Drosophila and mice to determine how LIMK2 deletion affects gastrointestinal stem cell regulation and tumour development.

Mutations in PTEN-induced kinase 1 (PINK1) cause early onset autosomal recessive Parkinson's disease (PD). PINK1 is a 63 kDa protein kinase, which exerts a neuroprotective function and is known to localize to mitochondria. Upon entry into the organelle, PINK1 is cleaved to produce a ∼53 kDa protein (ΔN-PINK1). In this paper, we show that PINK1 is cleaved between amino acids Ala-103 and Phe-104 to generate ΔN-PINK1. We demonstrate that a reduced ability to cleave PINK1, and the consequent accumulation of full-length protein, results in mitochondrial abnormalities reminiscent of those observed in PINK1 knockout cells, including disruption of the mitochondrial network and a reduction in mitochondrial mass. Notably, we assessed three N-terminal PD-associated PINK1 mutations located close to the cleavage site and, while these do not prevent PINK1 cleavage, they alter the ratio of full-length to ΔN-PINK1 protein in cells, resulting in an altered mitochondrial phenotype. Finally, we show that PINK1 interacts with the mitochondrial protease presenilin-associated rhomboid-like protein (PARL) and that loss of PARL results in aberrant PINK1 cleavage in mammalian cells. These combined results suggest that PINK1 cleavage is important for basal mitochondrial health and that PARL cleaves PINK1 to produce the ΔN-PINK1 fragment.

The human brain is a highly complex organ with remarkable energy demands. Although it represents only 2% of the total body weight, it accounts for 20% of all oxygen consumption, reflecting its high rate of metabolic activity. Mitochondria have a crucial role in the supply of energy to the brain. Consequently, their deterioration can have important detrimental consequences on the function and plasticity of neurons, and is thought to have a pivotal role in ageing and in the pathogenesis of several neurological disorders. Owing to their inherent physiological functions, mitochondria are subjected to particularly high levels of stress and have evolved specific molecular quality-control mechanisms to maintain the mitochondrial components. Here, we review some of the most recent advances in the understanding of mitochondrial stress-control pathways, with a particular focus on how defects in such pathways might contribute to neurodegenerative disease.

Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidative stress to maintain cellular homoeostasis and increase cell survival.

The impact of commensal bacteria on the host arises from complex microbial-diet-host interactions. Mapping metabolic interactions in gut microbial communities is therefore key to understand how the microbiome influences the host. Here we use an interdisciplinary approach including isotope-resolved metabolomics to show that in Drosophila melanogaster, Acetobacter pomorum (Ap) and Lactobacillus plantarum (Lp) a syntrophic relationship is established to overcome detrimental host diets and identify Ap as the bacterium altering the host's feeding decisions. Specifically, we show that Ap uses the lactate produced by Lp to supply amino acids that are essential to Lp, allowing it to grow in imbalanced diets. Lactate is also necessary and sufficient for Ap to alter the fly's protein appetite. Our data show that gut bacterial communities use metabolic interactions to become resilient to detrimental host diets. These interactions also ensure the constant flow of metabolites used by the microbiome to alter reproduction and host behaviour.

Eukaryotic Tribbles proteins are pseudoenzymes that regulate multiple aspects of intracellular signalling. Both Drosophila melanogaster and mammalian members of this family of pseudokinases act as negative regulators of insulin signalling. Mammalian tribbles pseudokinase (TRIB) genes have also been linked to insulin resistance and type 2 diabetes mellitus. Type 2 diabetes mellitus is associated with increased body weight, sleep problems and increased long-term mortality. Here, we investigated how manipulating the expression of Tribbles impacts body weight, sleep and mortality. We showed that the overexpression of Drosophila tribbles (trbl) in the fly fat body reduces both body weight and lifespan in adult flies without affecting food intake. Furthermore, it decreases the levels of Drosophila insulin-like peptide 2 (DILP2; ILP2) and increases night-time sleep. The three genes encoding TRIBs of mammals, TRIB1, TRIB2 and TRIB3, show both common and unique features. As the three human TRIB genes share features with Drosophila trbl, we further explored the links between TRIB genetic variants and both body weight and sleep in the human population. We identified associations between the polymorphisms and expression levels of the pseudokinases and markers of body weight and sleep duration. We conclude that Tribbles pseudokinases are involved in the control of body weight, lifespan and sleep.

Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids.

In response to oncogenic stress, the tumour suppressor protein p53 can induce the elimination of cells through induction of cell death or senescence, helping to restrain malignant progression. Conversely, under nutrient stress, p53 can protect cells by supporting metabolic adaptation. Many cancers express mutant p53 proteins that have lost the cell-elimination properties of wild-type p53. However, a previous report showed that a tumour-derived mutant can retain the ability to support cells under glutamine starvation.

Motor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS.

Mitochondrial reactive oxygen species (mtROS) are cellular messengers essential for cellular homeostasis. In response to stress, reverse electron transport (RET) through respiratory complex I generates high levels of mtROS. Suppression of ROS production via RET (ROS-RET) reduces survival under stress, while activation of ROS-RET extends lifespan in basal conditions. Here, we demonstrate that ROS-RET signalling requires increased electron entry and uninterrupted electron flow through the electron transport chain (ETC). We find that in old fruit flies, ROS-RET is abolished when electron flux is decreased and that their mitochondria produce consistently high levels of mtROS. Finally, we demonstrate that in young flies, limiting electron exit, but not entry, from the ETC phenocopies mtROS generation observed in old individuals. Our results elucidate the mechanism by which ROS signalling is lost during ageing.

Parkinson's disease (PD), an age-dependent neurodegenerative disease, is characterised by the selective loss of dopaminergic neurons in the substantia nigra (SN). Mitochondrial dysfunction is a hallmark of PD, and mutations in PINK1, a gene necessary for mitochondrial fitness, cause PD. Drosophila melanogaster flies with pink1 mutations exhibit mitochondrial defects and dopaminergic cell loss and are used as a PD model. To gain an integrated view of the cellular changes caused by defects in the PINK1 pathway of mitochondrial quality control, we combined metabolomics and transcriptomics analysis in pink1-mutant flies with human induced pluripotent stem cell (iPSC)-derived neural precursor cells (NPCs) with a PINK1 mutation. We observed alterations in cysteine metabolism in both the fly and human PD models. Mitochondrial dysfunction in the NPCs resulted in changes in several metabolites that are linked to cysteine synthesis and increased glutathione levels. We conclude that alterations in cysteine metabolism may compensate for increased oxidative stress in PD, revealing a unifying mechanism of early-stage PD pathology that may be targeted for drug development. This article has an associated First Person interview with the first author of the paper.

In Drosophila, endoplasmic reticulum (ER) stress activates the protein kinase R-like endoplasmic reticulum kinase (dPerk). dPerk can also be activated by defective mitochondria in fly models of Parkinson's disease caused by mutations in pink1 or parkin. The Perk branch of the unfolded protein response (UPR) has emerged as a major toxic process in neurodegenerative disorders causing a chronic reduction in vital proteins and neuronal death. In this study, we combined microarray analysis and quantitative proteomics analysis in adult flies overexpressing dPerk to investigate the relationship between the transcriptional and translational response to dPerk activation. We identified tribbles and Heat shock protein 22 as two novel Drosophila activating transcription factor 4 (dAtf4) regulated transcripts. Using a combined bioinformatics tool kit, we demonstrated that the activation of dPerk leads to translational repression of mitochondrial proteins associated with glutathione and nucleotide metabolism, calcium signalling and iron-sulphur cluster biosynthesis. Further efforts to enhance these translationally repressed dPerk targets might offer protection against Perk toxicity.

Metabolic adaptations can allow cancer cells to survive DNA-damaging chemotherapy. This unmet clinical challenge is a potential vulnerability of cancer. Accordingly, there is an intense search for mechanisms that modulate cell metabolism during anti-tumor therapy. We set out to define how colorectal cancer CRC cells alter their metabolism upon DNA replication stress and whether this provides opportunities to eliminate such cells more efficiently.

Serine is a vital amino acid in tumorigenesis. While cells can perform de novo serine synthesis, most transformed cells rely on serine uptake to meet their increased biosynthetic requirements. Solute carriers (SLCs), a family of transmembrane nutrient transport proteins, are the gatekeepers of amino acid acquisition and exchange in mammalian cells and are emerging as anticancer therapeutic targets; however, the SLCs that mediate serine transport in cancer cells remain unknown. Here we perform an arrayed RNAi screen of SLC-encoding genes while monitoring amino acid consumption and cell proliferation in colorectal cancer cells using metabolomics and high-throughput imaging. We identify SLC6A14 and SLC25A15 as major cytoplasmic and mitochondrial serine transporters, respectively. We also observe that SLC12A4 facilitates serine uptake. Dual targeting of SLC6A14 and either SLC25A15 or SLC12A4 diminishes serine uptake and growth of colorectal cancer cells in vitro and in vivo, particularly in cells with compromised de novo serine biosynthesis. Our results provide insight into the mechanisms that contribute to serine uptake and intracellular handling.

Alterations of mitochondrial metabolism and genomic instability have been implicated in tumorigenesis in multiple tissues. High-grade glioma (HGG), one of the most lethal human neoplasms, displays genetic modifications of Krebs cycle components as well as electron transport chain (ETC) alterations. Furthermore, the p53 tumor suppressor, which has emerged as a key regulator of mitochondrial respiration at the expense of glycolysis, is genetically inactivated in a large proportion of HGG cases. Therefore, it is becoming evident that genetic modifications can affect cell metabolism in HGG; however, it is currently unclear whether mitochondrial metabolism alterations could vice versa promote genomic instability as a mechanism for neoplastic transformation. Here, we show that, in neural progenitor/stem cells (NPCs), which can act as HGG cell of origin, inhibition of mitochondrial metabolism leads to p53 genetic inactivation. Impairment of respiration via inhibition of complex I or decreased mitochondrial DNA copy number leads to p53 genetic loss and a glycolytic switch. p53 genetic inactivation in ETC-impaired neural stem cells is caused by increased reactive oxygen species and associated oxidative DNA damage. ETC-impaired cells display a marked growth advantage in the presence or absence of oncogenic RAS, and form undifferentiated tumors when transplanted into the mouse brain. Finally, p53 mutations correlated with alterations in ETC subunit composition and activity in primary glioma-initiating neural stem cells. Together, these findings provide previously unidentified insights into the relationship between mitochondria, genomic stability, and tumor suppressive control, with implications for our understanding of brain cancer pathogenesis.

Drosophila melanogaster is a popular research model organism thanks to its' powerful genetic tools that allow spatial and temporal control of gene expression. The inducible GeneSwitch Gal4 system (GS) system is a modified version of the classic UAS/GAL4 system which allows inducible regulation of gene expression and eliminates background effects. It is widely acknowledged that the GS system is leaky, with low level expression of UAS transgenes in absence of the inducer RU-486 (the progesterone analog that activates the modified GAL4 protein). However, in the course of our experiments, we have observed that the extent of this leak depends on the nature of the transgene being expressed. In the absence of RU-486, when strong drivers are used to express protein coding transgenes, leaky expression is low or negligible, however expression of RNA interference (RNAi) transgenes results in complete depletion of protein levels. The majority of published studies, using the GS system and RNAi transgenes validate knock-down efficiency by comparing target gene mRNA levels between induced and non-induced groups. Here, we demonstrate that this approach is lacking and that both additional control groups and further validation is required at the protein level. Unfortunately, this experimental limitation of the GS system eliminates "the background advantage", but does offer the possibility of performing more complex experiments (e.g. studying depletion and overexpression of different proteins in the same genetic background). The limitations and new possible applications of the GS system are discussed in detail.

Neurons rely on mitochondria as their preferred source of energy. Mutations in PINK1 and PARKIN cause neuronal death in early-onset Parkinson's disease (PD), thought to be due to mitochondrial dysfunction. In Drosophila pink1 and parkin mutants, mitochondrial defects lead to the compensatory upregulation of the mitochondrial one-carbon cycle metabolism genes by an unknown mechanism. Here we uncover that this branch is triggered by the activating transcription factor 4 (ATF4). We show that ATF4 regulates the expression of one-carbon metabolism genes SHMT2 and NMDMC as a protective response to mitochondrial toxicity. Suppressing Shmt2 or Nmdmc caused motor impairment and mitochondrial defects in flies. Epistatic analyses showed that suppressing the upregulation of Shmt2 or Nmdmc deteriorates the phenotype of pink1 or parkin mutants. Conversely, the genetic enhancement of these one-carbon metabolism genes in pink1 or parkin mutants was neuroprotective. We conclude that mitochondrial dysfunction caused by mutations in the Pink1/Parkin pathway engages ATF4-dependent activation of one-carbon metabolism as a protective response. Our findings show a central contribution of ATF4 signalling to PD that may represent a new therapeutic strategy. A video abstract for this article is available at https://youtu.be/cFJJm2YZKKM.

Mucosa-associated Escherichia coli are frequently found in the colonic mucosa of patients with colorectal adenocarcinoma, but rarely in healthy controls. Chronic mucosal E. coli infection has therefore been linked to colonic tumourigenesis. E. coli strains carrying eae (encoding the bacterial adhesion protein intimin) attach intimately to the intestinal mucosa and are classed as attaching and effacing E. coli (AEEC). Enteropathogenic Escherichia coli (EPEC) are the most common form of AEEC identified in man. EPEC utilise a type III secretion system to translocate effector proteins into host cells and infection induces wide-ranging effects on the host cell proteome. We hypothesised that EPEC infection could influence molecular pathways involved in colorectal tumourigenesis.