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Alcohol consumption continues to cause a significant health burden globally. The advent of genome-wide association studies has unraveled many genetic loci associated with alcohol consumption. However, the biological effect of these loci and the pathways involved in alcohol consumption and its health consequences such as alcohol liver disease (ALD) remain to be elucidated. We combined human studies with model organismsDrosophila melanogasterandCaenorhabditis elegansto shed light on the molecular mechanisms underlying alcohol consumption and the health outcomes caused by alcohol intake. Using genetics and metabolite data within the Airwave study, a sample of police forces in the UK, we performed several analyses to identify changes in circulating metabolites that are triggered by alcohol consumption. We selected a set of genes annotated to genetic variants that are (1) known to be implicated in alcohol consumption, (2) are linked to liver function, and (3) are associated with expression (cis-eQTL) of their annotated genes. We used mutations and/or RNA interference (RNAi) to suppress the expression of these genes inC. elegansandDrosophila. We examined the effect of this suppression on ethanol consumption and on the sedative effects of ethanol. We also investigated the alcohol-induced changes in triacylglycerol (TGA) levels inDrosophilaand tested differences in locomotion ofC. elegansafter acute exposure to ethanol. In human population, we found an enrichment of the alcohol-associated metabolites within the linoleic acid (LNA) and alpha linolenic acid (ALA) metabolism pathway. We further showed the effect ofACTR1BandMAPTon locomotionin C. elegansafter exposure to ethanol. We demonstrated that three genes namelyWDPCP, TENM2andGPN1modify TAG levels inDrosophila. Finally, we showed that gene expression ofWDPCPin human population is linked to liver fibrosis and liver cirrhosis. Our results underline the impact of alcohol consumption on metabolism of lipids and pinpointsWDPCPas a gene with potential impact on fat accumulation upon exposure to ethanol suggesting a possible pathway to ALD.

Dysregulated mitochondrial fusion and fission has been implicated in the pathogenesis of numerous diseases. We have identified a novel function of the p53 family protein TAp73 in regulating mitochondrial dynamics. TAp73 regulates the expression of Optic atrophy 1, a protein responsible for controlling mitochondrial fusion, cristae biogenesis and electron transport chain function. Disruption of this axis results in a fragmented mitochondrial network and an impaired capacity for energy productionviaoxidative phosphorylation. Owing to the role of OPA1 in modulating cytochromecrelease, TAp73-/-cells also display an increased sensitivity to apoptotic cell death, e.g.,viaBH3-mimetics. We also show that the TAp73/OPA1 axis has functional relevance in the upper airway, where TAp73 expression is essential for multiciliated cell differentiation and function. Consistently, ciliated epithelial cells ofTrp73-/-(global p73 KO) mice display decreased expression of OPA1 and perturbations of the mitochondrial network, which may drive multiciliated cell loss. In support of this,Trp73andOPA1gene expression is decreased in COPD patients, a disease characterised by alterations in mitochondrial dynamics. We therefore highlight a potential mechanism involving the loss of p73 in COPD pathogenesis. This work also adds to the growing body of evidence for growth-promoting roles of TAp73 isoforms.

The innate immune response mounts a defense against foreign invaders and declines with age. An inappropriate induction of this response can cause diseases. Previous studies showed that mitochondria can be repurposed to promote inflammatory signaling. Damaged mitochondria can also trigger inflammation and promote diseases. Mutations in pink1, a gene required for mitochondrial health, cause Parkinson's disease, and Drosophila melanogaster pink1 mutants accumulate damaged mitochondria. Here, we show that defective mitochondria in pink1 mutants activate Relish targets and demonstrate that inflammatory signaling causes age-dependent intestinal dysfunction in pink1-mutant flies. These effects result in the death of intestinal cells, metabolic reprogramming and neurotoxicity. We found that Relish signaling is activated downstream of a pathway stimulated by cytosolic DNA. Suppression of Relish in the intestinal midgut of pink1-mutant flies restores mitochondrial function and is neuroprotective. We thus conclude that gut-brain communication modulates neurotoxicity in a fly model of Parkinson's disease through a mechanism involving mitochondrial dysfunction.

Mitochondrial Reactive Oxygen Species (mtROS) are cellular messengers essential for cellular homeostasis. In response to stress, reverse electron transport (RET) by respiratory complex I generates high levels of mtROS. Suppression of ROS produced 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 found that ROS-RET is abolished in old fruit flies where electron flux is reduced. Instead, mitochondria in aged flies produce consistently high levels of mtROS. Finally, we demonstrate that in young flies reduction of electron exit from the ETC, but not electron entry, phenocopies mtROS generation observed in old individuals. Our results define the mechanism by which ROS signalling is lost during ageing.HighlightsROS-RET signalling requires an uninterrupted flow of electrons through the ETC.ROS-RET signalling fails during ageing, with mitochondria producing persistently high levels of ROS.Interruption of ROS-RET signalling compromises stress adaptation in old flies.Reducing electron exit suppresses ROS-RET signalling and phenocopies ROS production observed in old mitochondria.

Alzheimer's disease is the most common age-related neurodegenerative disorder. Familial forms of Alzheimer's disease associated with the accumulation of a toxic form of amyloid-β (Aβ) peptides are linked to mitochondrial impairment. The coenzyme nicotinamide adenine dinucleotide (NAD

Alzheimer’s disease is the most common age-related neurodegenerative disorder. Familial forms of Alzheimer’s disease associated with the accumulation of a toxic form of amyloid-β (Aβ) peptides are linked to mitochondrial impairment. The coenzyme nicotinamide adenine dinucleotide (NAD+) is essential for both mitochondrial bioenergetics and nuclear DNA repair through NAD+-consuming poly (ADP-ribose) polymerases (PARPs). Here we analysed the metabolomic changes in flies overexpressing Aβ and showed a decrease of metabolites associated with nicotinate and nicotinamide metabolism, which is critical for mitochondrial function in neurons. We show that increasing the bioavailability of NAD+ protects against Aβ toxicity. Pharmacological supplementation using NAM, a form of vitamin B that acts as a precursor for NAD+ or a genetic mutation of PARP rescues mitochondrial defects, protects neurons against degeneration and reduces behavioural impairments in a fly model of Alzheimer’s disease. Next, we looked at links between PARP polymorphisms and vitamin B intake in patients with Alzheimer’s disease. We show that polymorphisms in the human PARP1 gene or the intake of vitamin B are associated with a decrease in the risk and severity of Alzheimer’s disease. We suggest that enhancing the availability of NAD+ by either vitamin B supplements or the inhibition of NAD+-dependent enzymes such as PARPs are potential therapies for Alzheimer’s disease.

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.

The innate immune response mounts a defence against foreign invaders, but the inappropriate induction of an innate immune response can cause diseases. Previous studies have provided ample evidence showing that mitochondria can be repurposed to promote inflammatory signalling. Damaged mitochondria can also trigger inflammation and promote diseases. Mutations in pink1 cause early-onset Parkinson’s disease (PD), and studies using Drosophila melanogaster have shown that pink1 mutants accumulate damaged mitochondria. Here, we showed that defective mitochondria in pink1 mutants activate Relish targets and demonstrated that inflammatory signalling causes intestinal dysfunction in pink1-mutant flies. These effects result in the death of intestinal cells and metabolic reprogramming, which leads to neurotoxicity. We found that Relish signalling is activated downstream of a pathway stimulated by cytosolic DNA. The suppression of Relish in the intestinal midgut of pink1-mutant flies restores mitochondrial function and protects neurons in the brain. We thus conclude that the gut-brain axis causes neurotoxicity in a fly model of PD through a mechanism involving mitochondrial dysfunction.

Reactive Oxygen Species (ROS) are essential cellular messengers required for cellular homeostasis and regulate the lifespan of several animal species. The main site of ROS production is the mitochondrion, and within it, respiratory complex I (CI) is the main ROS generator. ROS produced by CI trigger several physiological responses that are essential for the survival of neurons, cardiomyocytes and macrophages. Here, we show that CI produces ROS when electrons flow in either the forward (Forward Electron Transport, FET) or reverse direction (Reverse Electron Transport, RET). We demonstrate that ROS production via RET (ROS-RET) is activated under thermal stress conditions and that interruption of ROS-RET production, through ectopic expression of the alternative oxidase AOX, attenuates the activation of pro-survival pathways in response to stress. Accordingly, we find that both suppressing ROS-RET signalling or decreasing levels of mitochondrial H2O2 by overexpressing mitochondrial catalase (mtCAT), reduces survival dramatically in flies under stress. Our results uncover a specific ROS signalling pathway where hydrogen peroxide (H2O2) generated by CI via RET is required to activate adaptive mechanisms, maximising survival under stress conditions., Graphical abstract Image 1, Highlights • Heat stress induces ROS generation through the activation of Reverse electron transport (ROS-RET). • Suppressing ROS-RET attenuates the transcriptional stress response reducing survival under stress. • Mitochondrial catalase reduces survival under stress, identifying H2O2 as the main ROS signalling molecule.

Familial forms of Parkinson's disease (PD) caused by mutations in PINK1 are linked to mitochondrial impairment. Defective mitochondria are also found in Drosophila models of PD with pink1 mutations. The co-enzyme nicotinamide adenine dinucleotide (NAD+) is essential for both generating energy in mitochondria and nuclear DNA repair through NAD+-consuming poly(ADP-ribose) polymerases (PARPs). We found alterations in NAD+ salvage metabolism in Drosophila pink1 mutants and showed that a diet supplemented with the NAD+ precursor nicotinamide rescued mitochondrial defects and protected neurons from degeneration. Additionally, a mutation of Parp improved mitochondrial function and was neuroprotective in the pink1 mutants. We conclude that enhancing the availability of NAD+ by either the use of a diet supplemented with NAD+ precursors or the inhibition of NAD+-dependent enzymes, such as PARPs, which compete with mitochondria for NAD+ is a viable approach to preventing neurotoxicity associated with mitochondrial defects.

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.

Lung cancer is the leading cause of cancer-related death. Two-thirds of cases are diagnosed at an advanced stage that is refractory to curative treatment. Therefore, strategies for the early detection of lung cancer are urgently sought. Total circulating free DNA (cfDNA) and tumour-derived circulating tumour DNA (ctDNA) are emerging as important biomarkers within a ‘liquid biopsy’ for monitoring human disease progression and response to therapy. Owing to the late clinical diagnosis of lung adenocarcinoma, the potential for cfDNA and ctDNA as early detection biomarkers remains unexplored. Here, using a Cre-regulated genetically engineered mouse model of lung adenocarcinoma development, driven by KrasG12D (the KrasLSL-G12D mouse), we serially tracked the release of cfDNA/ctDNA and compared this with tumour burden as determined by micro-computed tomography (CT). To monitor ctDNA, a droplet digital PCR assay was developed to permit discrimination of the KrasLox-G12D allele from the KrasLSL-G12D and KrasWT alleles. We show that micro-CT correlates with endpoint histology and is able to detect pre-malignant tumours with a combined volume larger than 7 mm3. Changes in cfDNA/ctDNA levels correlate with micro-CT measurements in longitudinal sampling and are able to monitor the emergence of lesions before the adenoma-adenocarcinoma transition. Potentially, this work has implications for the early detection of human lung adenocarcinoma using ctDNA/cfDNA profiling. A video abstract for this article is available at https://youtu.be/Ku8xJJyGs3U. This article has an associated First Person interview with the joint first authors of the paper.

Eukaryotic cells are complex systems containing internal compartments with specialised functions. Among these compartments, the endoplasmic reticulum (ER) plays a major role in processing proteins for modification and delivery to other organelles, whereas mitochondria generate energy in the form of ATP. Mitochondria and the ER form physical interactions, defined as mitochondria–ER contact sites (MERCs) to exchange metabolites such as calcium ions (Ca2+) and lipids. Sites of contact between mitochondria and the ER can regulate biological processes such as ATP generation and mitochondrial division. The interactions between mitochondria and the ER are dynamic and respond to the metabolic state of cells. Changes in MERCs have been linked to metabolic pathologies such as diabetes, neurodegenerative diseases and sleep disruption. Here we explored the consequences of increasing contacts between mitochondria and the ER in flies using a synthetic linker. We showed that enhancing MERCs increases locomotion and extends lifespan. We also showed that, in a Drosophila model of Alzheimer's disease linked to toxic amyloid beta (Aβ), linker expression can suppress motor impairment and extend lifespan. We conclude that strategies for increasing contacts between mitochondria and the ER may improve symptoms of diseases associated with mitochondria dysfunction. A video abstract for this article is available at https://youtu.be/_YWA4oKZkes. This article has an associated First Person interview with the first author of the paper., Summary: Enhancing mitochondria–ER contacts ameliorates locomotor phenotypes and extends lifespan in a fly model of Alzheimer's disease.

Lung cancer is the leading cause of cancer-related death. Two-thirds of cases are diagnosed at an advanced stage that is refractory to curative treatment. Therefore, strategies for the early detection of lung cancer are urgently sought. Total circulating free DNA (cfDNA) and tumour-derived circulating tumour DNA (ctDNA) are emerging as important biomarkers within a ‘liquid biopsy’ for monitoring human disease progression and response to therapy. Owing to the late clinical diagnosis of lung adenocarcinoma, the potential for cfDNA and ctDNA as early detection biomarkers remains unexplored. Here, using a Cre-regulated genetically engineered mouse model of lung adenocarcinoma development, driven by KrasG12D (the KrasLSL-G12D mouse), we serially tracked the release of cfDNA/ctDNA and compared this with tumour burden as determined by micro-computed tomography (CT). To monitor ctDNA, a droplet digital PCR assay was developed to permit discrimination of the KrasLox-G12D allele from the KrasLSL-G12D and KrasWT alleles. We show that micro-CT correlates with endpoint histology and is able to detect pre-malignant tumours with a combined volume larger than 7 mm3. Changes in cfDNA/ctDNA levels correlate with micro-CT measurements in longitudinal sampling and are able to monitor the emergence of lesions before the adenoma-adenocarcinoma transition. Potentially, this work has implications for the early detection of human lung adenocarcinoma using ctDNA/cfDNA profiling. A video abstract for this article is available at https://youtu.be/Ku8xJJyGs3U. This article has an associated First Person interview with the joint first authors of the paper., Summary: A liquid biopsy approach is capable of tracking early lung cancer lesions, before the adenoma-adenocarcinoma transition, in an early lung cancer mouse model driven by a KRAS mutation.

This work was funded by the Medical Research Council. Full open access supported by the Velux Foundation, the University of Zurich, and the EPFL School of Life Sciences.

The mitochondrial proteins TRAP1 and HTRA2 have previously been shown to be phosphorylated in the presence of the Parkinson's disease kinase PINK1 but the downstream signalling is unknown. HTRA2 and PINK1 loss of function causes parkinsonism in humans and animals. Here, we identified TRAP1 as an interactor of HTRA2 using an unbiased mass spectrometry approach. In our human cell models, TRAP1 overexpression is protective, rescuing HTRA2 and PINK1-associated mitochondrial dysfunction and suggesting that TRAP1 acts downstream of HTRA2 and PINK1. HTRA2 regulates TRAP1 protein levels, but TRAP1 is not a direct target of HTRA2 protease activity. Following genetic screening of Parkinson's disease patients and healthy controls, we also report the first TRAP1 mutation leading to complete loss of functional protein in a patient with late onset Parkinson's disease. Analysis of fibroblasts derived from the patient reveal that oxygen consumption, ATP output and reactive oxygen speci es are increased compared to healthy individuals. This is coupled with an increased pool of free NADH, increased mitochondrial biogenesis, triggering of the mitochondrial unfolded protein response, loss of mitochondrial membrane potential and sensitivity to mitochondrial removal and apoptosis. These data highlight the role of TRAP1 in the regulation of energy metabolism and mitochondrial quality control. Interestingly, the diabetes drug metformin reverses mutation-associated alterations on energy metabolism, mitochondrial biogenesis and restores mitochondrial membrane potential. In summary, our data show that TRAP1 acts downstream of PINK1 and HTRA2 for mitochondrial fine tuning, whereas TRAP1 loss of function leads to reduced control of energy metabolism, ultimately impacting mitochondrial membrane potential. These findings offer new insight into mitochondrial pathologies in Parkinson's disease and provide new prospects for targeted therapies.

Background Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). Studies in the yeast Saccharomyces cerevisia e have provided valuable insights into the mechanisms of cellular dysfunction associated with the expression of faulty PD genes. Methods We developed a yeast model for full-length LRRK2 studies. We expressed wild-type (wt) LRRK2 and mutations and evaluated their role during oxidative stress conditions. The involvement of mitochondria was assessed by using rho-zero mutants and by evaluating reactive oxygen species (ROS) production and mitochondrial membrane potential by flow cytometry. The involvement of endocytosis was also studied by testing several endocytic mutants and by following the vacuolar delivery of the probe FM4-64. Results Expression of LRRK2 in yeast was associated to increased hydrogen peroxide resistance. This phenotype, which was dependent on mitochondrial function, was not observed for PD-mutants G2019S and R1441C or in the absence of the kinase activity and the WD40 repeat domain. Expression of the pathogenic mutants stimulated ROS production and increased mitochondrial membrane potential. For the PD-mutants, but not for wild-type LRRK2, endocytic defects were also observed. Additionally, several endocytic proteins were required for LRRK2-mediated protection against hydrogen peroxide. Conclusions Our results indicate that LRRK2 confers cellular protection during oxidative stress depending on mitochondrial function and endocytosis. General significance Both the loss of capacity of LRRK2 pathogenic mutants to protect against oxidative stress and their enhancement of dysfunction may be important for the development of PD during the aging process.

The X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti-apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase-mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP-mediated immune response by inducing the BID-dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain-dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization.

Familial forms of Parkinson's disease (PD) caused by mutations in PINK1 are linked to mitochondrial impairment. Defective mitochondria are also found in Drosophila models of PD with pink1 mutations. The co-enzyme nicotinamide adenine dinucleotide (NAD+) is essential for both generating energy in mitochondria and nuclear DNA repair through NAD+-consuming poly(ADP-ribose) polymerases (PARPs). We found alterations in NAD+ salvage metabolism in Drosophila pink1 mutants and showed that a diet supplemented with the NAD+ precursor nicotinamide rescued mitochondrial defects and protected neurons from degeneration. Additionally, a mutation of Parp improved mitochondrial function and was neuroprotective in the pink1 mutants. We conclude that enhancing the availability of NAD+ by either the use of a diet supplemented with NAD+ precursors or the inhibition of NAD+-dependent enzymes, such as PARPs, which compete with mitochondria for NAD+, is a viable approach to preventing neurotoxicity associated with mitochondrial defects., Summary: Dietary supplementation with an NAD+ salvage metabolite or decreasing Parp activity suppress mitochondrial dysfunction and is neuroprotective in a PINK1 model of Parkinson's disease.

Following publication of the article, Dr. Roberta Tufi of the Mitochondrial Biology Unit at the University of Cambridge was concerned to note that her own contribution to the study during her postdoc in Leicester at the MRC Toxicology Unit had not been acknowledged. Specifically, the data in Fig. 1 (panels a, b, and d) were produced though her work.

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.

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.

Recent findings from genetic studies suggest that defective mitochondrial quality control may play an important role in the development of Parkinson's disease (PD). Such defects may result in the impairment of neuronal mitochondria, which leads to both synaptic dysfunction and cell death and results in neurodegeneration. Here, we review state-of-the-art knowledge of how pathways affecting mitochondrial quality control might contribute to PD, with a particular emphasis on the molecular mechanisms employed by PTEN-induced putative kinase 1 (PINK1), HtrA2 and Parkin to regulate mitochondrial quality control.

Intracellular signal transduction networks involving protein kinases are important modulators of cell survival and cell death in multicellular organisms. Functional compromise of these networks has been linked to aberrant apoptosis in diseases such as cancer. To identify novel kinase regulators of cell death, we conducted an RNAi-based screen to identify modulators of the intrinsic apoptosis pathway. Using this approach, we identified MAP4K3 as a novel apoptosis inducer. Here, we present evidence that this pro-apoptotic kinase orchestrates activation of BAX via the concerted posttranscriptional modulation of PUMA, BAD, and BIM. Additionally, we found decreased levels of this kinase in pancreatic cancer samples, suggesting a tumor suppressor role for MAP4K3 in pancreatic tumorigenesis.

HtrA2/Omi is a mitochondrial serine protease that is released into the cytosol and promotes apoptotic processes by binding to several members of the inhibitors of apoptosis protein family. HtrA2/Omi knockout mice show a parkinsonian phenotype, and mutations in the gene encoding HtrA2/Omi have been identified as susceptibility factors for Parkinson disease (PD). These results suggest that HtrA2/Omi may be involved in the pathogenesis of PD. We performed immunohistochemical studies of HtrA2/Omi on brains from patients with alpha-synuclein-related disorders, including PD, dementia with Lewy bodies (DLB), and multiple-system atrophy (MSA); patients with other neurodegenerative diseases; and controls. HtrA2/Omi is expressed in normal brain tissue, and there was some anti-HtrA2/Omi immunostaining of neurons in normal brains as well as those with other neurodegenerative diseases. In PD and DLB brains, both classic (i.e. brainstem-type) and cortical Lewy bodies were intensely immunostained; pale bodies were also strongly immunopositive for HtrA2/Omi. In MSA brains, numerous glial cytoplasmic inclusions, neuronal cytoplasmic inclusions, and dystrophic neurites were also intensely immunoreactive for HtrA2/Omi. These results suggest that widespread accumulation of HtrA2/Omi may occur in pathologic alpha-synuclein-containing inclusions in brains with PD, DLB, or MSA and that HtrA2/Omi may be associated with the pathogenesis of alpha-synucleinopathies.

In mice, targeted deletion of the serine protease HtrA2 (also known as Omi) causes mitochondrial dysfunction leading to a neurodegenerative disorder with parkinsonian features. In humans, point mutations in HtrA2 are a susceptibility factor for Parkinson's disease (PARK13 locus). Mutations in PINK1, a putative mitochondrial protein kinase, are associated with the PARK6 autosomal recessive locus for susceptibility to early-onset Parkinson's disease. Here we determine that HtrA2 interacts with PINK1 and that both are components of the same stress-sensing pathway. HtrA2 is phosphorylated on activation of the p38 pathway, occurring in a PINK1-dependent manner at a residue adjacent to a position found mutated in patients with Parkinson's disease. HtrA2 phosphorylation is decreased in brains of patients with Parkinson's disease carrying mutations in PINK1. We suggest that PINK1-dependent phosphorylation of HtrA2 might modulate its proteolytic activity, thereby contributing to an increased resistance of cells to mitochondrial stress.

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.

The serine protease HtrA2/Omi is released from the mitochondrial intermembrane space following apoptotic stimuli. Once in the cytosol, HtrA2/Omi has been implicated in promoting cell death by binding to inhibitor of apoptosis proteins (IAPs) via its amino-terminal Reaper-related motif, thus inducing caspase activity, and also in mediating caspase-independent death through its own protease activity. We report here the phenotype of mice entirely lacking expression of HtrA2/Omi due to targeted deletion of its gene, Prss25. These animals, or cells derived from them, show no evidence of reduced rates of cell death but on the contrary suffer loss of a population of neurons in the striatum, resulting in a neurodegenerative disorder with a parkinsonian phenotype that leads to death of the mice around 30 days after birth. The phenotype of these mice suggests that it is the protease function of this protein and not its IAP binding motif that is critical. This conclusion is reinforced by the finding that simultaneous deletion of the other major IAP binding protein, Smac/DIABLO, does not obviously alter the phenotype of HtrA2/Omi knockout mice or cells derived from them. Mammalian HtrA2/Omi is therefore likely to function in vivo in a manner similar to that of its bacterial homologues DegS and DegP, which are involved in protection against cell stress, and not like the proapoptotic Reaper family proteins in Drosophila melanogaster.