Knockdown of APOPT1/COA8 Causes Cytochrome c Oxidase Deficiency, Neuromuscular Impairment, and Reduced Resistance to Oxidative Stress in Drosophila melanogaster.

Cytochrome c oxidase (COX) deficiency is the biochemical hallmark of several mitochondrial disorders, including subjects affected by mutations in apoptogenic-1 (APOPT1), recently renamed as COA8 (HGNC:20492). Loss-of-function mutations are responsible for a specific infantile or childhood-onset mitochondrial leukoencephalopathy with a chronic clinical course. Patients deficient in COA8 show specific COX deficiency with distinctive neuroimaging features, i.e., cavitating leukodystrophy. In human cells, COA8 is rapidly degraded by the ubiquitin-proteasome system, but oxidative stress stabilizes the protein, which is then involved in COX assembly, possibly by protecting the complex from oxidative damage. However, its precise function remains unknown. The CG14806 gene (dCOA8) is the Drosophila melanogaster ortholog of human COA8 encoding a highly conserved COA8 protein. We report that dCOA8 knockdown (KD) flies show locomotor defects, and other signs of neurological impairment, reduced COX enzymatic activity, and reduced lifespan under oxidative stress conditions. Our data indicate that KD of dCOA8 in Drosophila phenocopies several features of the human disease, thus being a suitable model to characterize the molecular function/s of this protein in vivo and the pathogenic mechanisms associated with its defects

Inhibition of the deubiquitinase USP8 corrects a Drosophila PINK1 model of mitochondria dysfunction.

Aberrant mitochondrial dynamics disrupts mitochondrial function and contributes to disease conditions. A targeted RNA interference screen for deubiquitinating enzymes (DUBs) affecting protein levels of multifunctional mitochondrial fusion protein Mitofusin (MFN) identified USP8 prominently influencing MFN levels. Genetic and pharmacological inhibition of USP8 normalized the elevated MFN protein levels observed in PINK1 and Parkin-deficient models. This correlated with improved mitochondrial function, locomotor performance and life span, and prevented dopaminergic neurons loss in Drosophila PINK1 KO flies. We identified a novel target antagonizing pathologically elevated MFN levels, mitochondrial dysfunction, and dopaminergic neuron loss of a Drosophila model of mitochondrial dysfunction.his work was supported by grants from the Italian Ministry of Health “Ricerca Finalizzata” (GR-2011-02351151), Rita Levi Montalcini “Brain Gain” program, and Michael J Fox RRIA 2014 (Grant ID 9795) to E Ziviani and by ERC FP7-282280, FP7 CIG PCIG13-GA-2013-618697, and Italian Ministry of Research FIRB RBAP11Z3YA_005 to L Scorrano. AJ Whitworth is funded by MRC Core funding (MC_UU_00015/6)

Modeling human mitochondrial diseases related to MPV17 and APOPT1 in Drosophila melanogaster

Mitochondrial diseases are a clinically heterogeneous group of inherited disorders associated with defects in the oxidative phosphorylation system, with an estimated incidence in between 1:5,000 and 1:10,000 live births. Mitochondrial respiratory chain function depends on the coordinated expression of both mitochondrial and nuclear genomes. Thus, also mutations affecting nuclear-encoded mitochondrial proteins are responsible for mitochondrial disease onset. During the last decades, an increasing number of novel nuclear disease genes have been identified. Among those genes, human MPV17 and APOPT1 have already been linked to mitochondrial diseases but their role in mitochondrial physiology and disease remains still puzzling. Mutations in the human MPV17 nuclear gene, encoding a small hydrophobic mitochondrial inner membrane protein, are a prominent cause of a pediatric hepatocerebral form of mitochondrial DNA depletion syndrome. APOPT1 mutations are responsible for an infantile or childhood mitochondrial encephalopathy hallmarked by profound deficiencies in both COX activity and amount. In order to dissect out the role of these two genes, in this PhD project we focused our attention on the functional and molecular characterization of Drosophila melanogaster orthologs of MPV17 and APOPT1. We found that dMpv17 down-regulation in flies causes a profound mitochondrial DNA depletion in the fat bodies (a Drosophila organ analogous to human liver). Depletion is also detected, albeit moderate, in dMpv17 KD cells. Our results reveal that dMpv17 can form a channel when inserted in an artificial planar lipid bilayer. Moreover, we also show that the Drosophila protein could interact with dMic19, a component of the MICOS complex, as well as dMrp4, that could play a role in dMpv17 gating regulation. The analysis of mitochondrial morphology in dMpv17 down-regulated cells together with the interaction with Mic19 suggest a possible role for dMpv17 in the maintenance of the structural and functional stability of the inner mitochondrial membrane. Further, we confirmed that Drosophila is a reliable model for studying human mitochondrial disease also in the case of APOPT1. Indeed, our preliminary data show that dApopt1 down-regulation in flies causes motor impairment and COX deficiency, characteristic features of the human disease. Not only COX activity but also coxI transcript is decreased in dApopt1 down-regulated flies. Finally, we show that H2O2 treatment and, in turn, oxidative stress induce an increase in dApopt1 transcript. Finally, our data shed new light on the possible role of dMpv17 and dApopt1 in physiological and pathological conditions.Le malattie mitocondriali sono un gruppo ampio e eterogeneo di disordini ereditari causati da difetti del metabolismo energetico mitocondriale attribuibili a un malfunzionamento della catena respiratoria mitocondriale. La loro incidenza è stata stimata tra 1:1500 e 1:10000 nati vivi. Queste sindromi sono il risultato di un gran numero di mutazioni rilevabili sia nel genoma nucleare sia in quello mitocondriale. Negli ultimi decenni, il numero di geni scoperti essere responsabili dell’insorgenza di malattie mitocondriali è enormemente aumentato. Mutazioni nella proteina MPV17, localizzata nella membrana mitocondriale interna, sono state associate ad una particolare forma di sindrome da deplezione di DNA mitocondriale che colpisce primariamente il fegato e il sistema nervoso in età pediatrica. Invece, mutazioni in APOPT1 sono state identificate in pazienti caratterizzati da sintomi neurologici di vari entità associati a perdita della parola e della capacità motoria e accompagnati da un significativo deficit di citocromo C ossidasi a livello muscolare. Dal momento che la funzione di queste due proteine risulta essere ancora sconosciuta, abbiamo cercato di determinare il loro ruolo a livello mitocondriale e nello sviluppo di queste malattie studiando i geni ortologhi in Drosophila, dMpv17 e dApopt1. Abbiamo dimostrato che la down-regolazione dell’espressione di dMpv17 in vivo comporta una diminuzione significativa nel numero di copie di DNA mitocondriale nei fat bodies, un analogo funzionale del fegato dei mammiferi. Inoltre, alcuni risultati ottenuti molto recentemente suggeriscono che dMPV17 sarebbe in grado di formare un canale in un planar lipid bilayer. Infine, abbiamo identificato come suoi possibili interattori un componente del complesso MICOS e dMrp4, una proteina facente parte della famiglia dei trasportatori ABC che potrebbe regolare la sua attività di canale. L’interazione con il complesso MICOS e i risultati della microscopia elettronica sulla morfologia dei mitocondri nelle cellule silenziate per dMpv17, che hanno evidenziato una diminuzione nel numero e nella lunghezza delle creste, fanno ipotizzare un possibile ruolo nel mantenimento della struttura e, quindi, della funzionalità della membrana mitocondriale interna. Studiando l’ortologo di APOPT1, dApopt1, abbiamo osservato che la sua down-regolazione in vivo comporta un marcato difetto locomotorio accompagnato da una significativa riduzione dell’attività della citocromo C ossidasi, sintomi descritti anche nella patologia umana. Infine, poiché la proteina umana sembra avere un ruolo nella risposta allo stress ossidativo, abbiamo dimostrato che l’espressione di dApopt1 è indotta dal trattamento con H2O2. I risultati relativi alla caratterizzazione di dMpv17 e quelli preliminari riguardanti dApopt1 contribuiscono a fare luce sul ruolo di queste proteine sia a livello fisiologico che patologico, e confermano la validità di Drosophila come organismo modello per lo studio delle malattie genetiche uman

Modeling human mitochondrial diseases related to MPV17 and APOPT1 in Drosophila melanogaster

Mitochondrial diseases are a clinically heterogeneous group of inherited disorders associated with defects in the oxidative phosphorylation system, with an estimated incidence in between 1:5,000 and 1:10,000 live births. Mitochondrial respiratory chain function depends on the coordinated expression of both mitochondrial and nuclear genomes. Thus, also mutations affecting nuclear-encoded mitochondrial proteins are responsible for mitochondrial disease onset. During the last decades, an increasing number of novel nuclear disease genes have been identified. Among those genes, human MPV17 and APOPT1 have already been linked to mitochondrial diseases but their role in mitochondrial physiology and disease remains still puzzling. Mutations in the human MPV17 nuclear gene, encoding a small hydrophobic mitochondrial inner membrane protein, are a prominent cause of a pediatric hepatocerebral form of mitochondrial DNA depletion syndrome. APOPT1 mutations are responsible for an infantile or childhood mitochondrial encephalopathy hallmarked by profound deficiencies in both COX activity and amount. In order to dissect out the role of these two genes, in this PhD project we focused our attention on the functional and molecular characterization of Drosophila melanogaster orthologs of MPV17 and APOPT1. We found that dMpv17 down-regulation in flies causes a profound mitochondrial DNA depletion in the fat bodies (a Drosophila organ analogous to human liver). Depletion is also detected, albeit moderate, in dMpv17 KD cells. Our results reveal that dMpv17 can form a channel when inserted in an artificial planar lipid bilayer. Moreover, we also show that the Drosophila protein could interact with dMic19, a component of the MICOS complex, as well as dMrp4, that could play a role in dMpv17 gating regulation. The analysis of mitochondrial morphology in dMpv17 down-regulated cells together with the interaction with Mic19 suggest a possible role for dMpv17 in the maintenance of the structural and functional stability of the inner mitochondrial membrane. Further, we confirmed that Drosophila is a reliable model for studying human mitochondrial disease also in the case of APOPT1. Indeed, our preliminary data show that dApopt1 down-regulation in flies causes motor impairment and COX deficiency, characteristic features of the human disease. Not only COX activity but also coxI transcript is decreased in dApopt1 down-regulated flies. Finally, we show that H2O2 treatment and, in turn, oxidative stress induce an increase in dApopt1 transcript. Finally, our data shed new light on the possible role of dMpv17 and dApopt1 in physiological and pathological conditions

Drosophila Mpv17 forms an ion channel and regulates energy metabolism

Summary: Mutations in MPV17 are a major contributor to mitochondrial DNA (mtDNA) depletion syndromes, a group of inherited genetic conditions due to mtDNA instability. To investigate the role of MPV17 in mtDNA maintenance, we generated and characterized a Drosophila melanogaster Mpv17 (dMpv17) KO model showing that the absence of dMpv17 caused profound mtDNA depletion in the fat body but not in other tissues, increased glycolytic flux and reduced lifespan in starvation. Accordingly, the expression of key genes of glycogenolysis and glycolysis was upregulated in dMpv17 KO flies. In addition, we demonstrated that dMpv17 formed a channel in planar lipid bilayers at physiological ionic conditions, and its electrophysiological hallmarks were affected by pathological mutations. Importantly, the reconstituted channel translocated uridine but not orotate across the membrane. Our results indicate that dMpv17 forms a channel involved in translocation of key metabolites and highlight the importance of dMpv17 in energy homeostasis and mitochondrial function

Inhibition of the deubiquitinase USP8 corrects a Drosophila PINK1 model of mitochondria dysfunction.

Aberrant mitochondrial dynamics disrupts mitochondrial function and contributes to disease conditions. A targeted RNA interference screen for deubiquitinating enzymes (DUBs) affecting protein levels of multifunctional mitochondrial fusion protein Mitofusin (MFN) identified USP8 prominently influencing MFN levels. Genetic and pharmacological inhibition of USP8 normalized the elevated MFN protein levels observed in PINK1 and Parkin-deficient models. This correlated with improved mitochondrial function, locomotor performance and life span, and prevented dopaminergic neurons loss in Drosophila PINK1 KO flies. We identified a novel target antagonizing pathologically elevated MFN levels, mitochondrial dysfunction, and dopaminergic neuron loss of a Drosophila model of mitochondrial dysfunction.his work was supported by grants from the Italian Ministry of Health “Ricerca Finalizzata” (GR-2011-02351151), Rita Levi Montalcini “Brain Gain” program, and Michael J Fox RRIA 2014 (Grant ID 9795) to E Ziviani and by ERC FP7-282280, FP7 CIG PCIG13-GA-2013-618697, and Italian Ministry of Research FIRB RBAP11Z3YA_005 to L Scorrano. AJ Whitworth is funded by MRC Core funding (MC_UU_00015/6)

NAD⁺ repletion with niacin counteracts cancer cachexia

Abstract Cachexia is a debilitating wasting syndrome and highly prevalent comorbidity in cancer patients. It manifests especially with energy and mitochondrial metabolism aberrations that promote tissue wasting. We recently identified nicotinamide adenine dinucleotide (NAD⁺) loss to associate with muscle mitochondrial dysfunction in cancer hosts. In this study we confirm that depletion of NAD⁺ and downregulation of Nrk2, an NAD⁺ biosynthetic enzyme, are common features of severe cachexia in different mouse models. Testing NAD⁺ repletion therapy in cachectic mice reveals that NAD⁺ precursor, vitamin B3 niacin, efficiently corrects tissue NAD⁺ levels, improves mitochondrial metabolism and ameliorates cancer- and chemotherapy-induced cachexia. In a clinical setting, we show that muscle NRK2 is downregulated in cancer patients. The low expression of NRK2 correlates with metabolic abnormalities underscoring the significance of NAD⁺ in the pathophysiology of human cancer cachexia. Overall, our results propose NAD⁺ metabolism as a therapy target for cachectic cancer patients