Rapamycin rescues mitochondrial myopathy via coordinated activation of autophagy and lysosomal biogenesis

Abstract The mTOR inhibitor rapamycin ameliorates the clinical and biochemical phenotype of mouse, worm, and cellular models of mitochondrial disease, via an unclear mechanism. Here, we show that prolonged rapamycin treatment improved motor endurance, corrected morphological abnormalities of muscle, and increased cytochrome c oxidase (COX) activity of a muscle‐specific Cox15 knockout mouse (Cox15sm/sm). Rapamycin treatment restored autophagic flux, which was impaired in naïve Cox15sm/sm muscle, and reduced the number of damaged mitochondria, which accumulated in untreated Cox15sm/sm mice. Conversely, rilmenidine, an mTORC1‐independent autophagy inducer, was ineffective on the myopathic features of Cox15sm/sm animals. This stark difference supports the idea that inhibition of mTORC1 by rapamycin has a key role in the improvement of the mitochondrial function in Cox15sm/sm muscle. In contrast to rilmenidine, rapamycin treatment also activated lysosomal biogenesis in muscle. This effect was associated with increased nuclear localization of TFEB, a master regulator of lysosomal biogenesis, which is inhibited by mTORC1‐dependent phosphorylation. We propose that the coordinated activation of autophagic flux and lysosomal biogenesis contribute to the effective clearance of dysfunctional mitochondria by rapamycin

Rapamycin rescues mitochondrial myopathy via coordinated activation of autophagy and lysosomal biogenesis.

The mTOR inhibitor rapamycin ameliorates the clinical and biochemical phenotype of mouse, worm, and cellular models of mitochondrial disease, via an unclear mechanism. Here, we show that prolonged rapamycin treatment improved motor endurance, corrected morphological abnormalities of muscle, and increased cytochrome c oxidase (COX) activity of a muscle-specific Cox15 knockout mouse (Cox15sm/sm ). Rapamycin treatment restored autophagic flux, which was impaired in naïve Cox15sm/sm muscle, and reduced the number of damaged mitochondria, which accumulated in untreated Cox15sm/sm mice. Conversely, rilmenidine, an mTORC1-independent autophagy inducer, was ineffective on the myopathic features of Cox15sm/sm animals. This stark difference supports the idea that inhibition of mTORC1 by rapamycin has a key role in the improvement of the mitochondrial function in Cox15sm/sm muscle. In contrast to rilmenidine, rapamycin treatment also activated lysosomal biogenesis in muscle. This effect was associated with increased nuclear localization of TFEB, a master regulator of lysosomal biogenesis, which is inhibited by mTORC1-dependent phosphorylation. We propose that the coordinated activation of autophagic flux and lysosomal biogenesis contribute to the effective clearance of dysfunctional mitochondria by rapamycin

SUCLA2 mutations cause global protein succinylation contributing to the pathomechanism of a hereditary mitochondrial disease

Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-beta (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD(+))-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications. The pathomechanism of succinyl-CoA ligase (SCL) deficiency, a hereditary mitochondrial disease, is not fully understood. Here, the authors show that increased succinyl-CoA levels contribute to SCL pathology by causing global protein hyper-succinylation.Peer reviewe

Tissue specific differences in the assembly of mitochondrial complex I is revealed by a novel ENU mutation in ECSIT

Aims: Mitochondrial complex I assembly is a multi-step process which necessitates the involvement of a variety of assembly factors and chaperones to ensure the final active enzyme is correctly assembled. The role of the assembly factor ECSIT was studied across various murine tissues to determine its role in this process and how this varied between tissues of varying energetic demands. We hypothesised that many of the known functions of ECSIT were unhindered by the introduction of an ENU induced mutation, whilst it's role in complex I assembly was affected on a tissue specific basis. Methods and results: Here we describe a mutation in the mitochondrial complex I assembly factor ECSIT which reveals tissue specific requirements for ECSIT in complex I assembly. Mitochondrial complex I assembly is a multi-step process dependent on assembly factors that organise and arrange the individual subunits, allowing for their incorporation into the complete enzyme complex. We have identified an ENU induced mutation in ECSIT (N209I) that exhibits a profound effect on complex I component expression and assembly in heart tissue, resulting in hypertrophic cardiomyopathy in the absence of other phenotypes. The dysfunction of complex I appears to be cardiac specific, leading to a loss of mitochondrial output as measured by Seahorse extracellular flux and various biochemical assays in heart tissue, whilst mitochondria from other tissues were unaffected. Conclusions: These data suggest that the mechanisms underlying complex I assembly and activity may have tissue specific elements tailored to the specific demands of cells and tissues. Our data suggest that tissues with high energy demands, such as the heart, may utilise assembly factors in different ways to low energy tissues in order to improve mitochondrial output. This data have implications for the diagnosis and treatment of various disorders of mitochondrial function as well as cardiac hypertrophy with no identifiable underlying genetic cause. Translational perspective: Mitochondrial diseases often present as multi system disorders with far reaching implications to the health and well being of patients. Diagnoses are often undertaken by characterisation of mitochondrial function from skin or muscle biopsy, with the expectation that any affect on mitochondrial function will be recognisable in all cell types. However, this study demonstrates that mitochondrial function may differ between cell types with the involvement of tissue specific proteins or isoforms, as such, current diagnostic techniques may miss diagnoses of a more specific mitochondrial dysfunction

Tissue specific differences in the assembly of mitochondrial complex I is revealed by a novel ENU mutation in ECSIT

Aims Mitochondrial complex I assembly is a multi-step process which necessitates the involvement of a variety of assembly factors and chaperones to ensure the final active enzyme is correctly assembled. The role of the assembly factor ECSIT was studied across various murine tissues to determine its role in this process and how this varied between tissues of varying energetic demands. We hypothesised that many of the known functions of ECSIT were unhindered by the introduction of an ENU induced mutation, whilst it’s role in complex I assembly was affected on a tissue specific basis. Methods and Results Here we describe a mutation in the mitochondrial complex I assembly factor ECSIT which reveals tissue specific requirements for ECSIT in complex I assembly. Mitochondrial complex I assembly is a multi-step process dependent on assembly factors that organise and arrange the individual subunits, allowing for their incorporation into the complete enzyme complex. We have identified an ENU induced mutation in ECSIT (N209I) that exhibits a profound effect on complex I component expression and assembly in heart tissue, resulting in hypertrophic cardiomyopathy in the absence of other phenotypes. The dysfunction of complex I appears to be cardiac specific, leading to a loss of mitochondrial output as measured by Seahorse extracellular flux and various biochemical assays in heart tissue, whilst mitochondria from other tissues were unaffected. Conclusions These data suggest that the mechanisms underlying complex I assembly and activity may have tissue specific elements tailored to the specific demands of cells and tissues. Our data suggest that tissues with high energy demands, such as the heart, may utilise assembly factors in different ways to low energy tissues in order to improve mitochondrial output. This data have implications for the diagnosis and treatment of various disorders of mitochondrial function as well as cardiac hypertrophy with no identifiable underlying genetic cause. Translational Perspective Mitochondrial diseases often present as multi system disorders with far reaching implications to the health and well being of patients. Diagnoses are often undertaken by characterisation of mitochondrial function from skin or muscle biopsy, with the expectation that any affect on mitochondrial function will be recognisable in all cell types. However, this study demonstrates that mitochondrial function may differ between cell types with the involvement of tissue specific proteins or isoforms, as such, current diagnostic techniques may miss diagnoses of a more specific mitochondrial dysfunction

Dietary supplementation with fish oil and curcumin improves gait speed and mitochondrial function during aging

THEME: Nutrition and AgingBackgroundsSarcopenia is a progressive and generalized skeletal muscle disorder associated with adverse outcomes including falls,fractures, physical disability and mortality. Sarcopenia has multi-factorial causes going from life style changes to metabolic and cellular perturbations.ObjectivesThe objective of this study was to determine the functional benefits of a nutritional intervention with curcumin and fish oil alone or in combination on Sarcopenia and to characterize the underlying mechanisms of action. The concept of this study was to provide combination of ingredients targeting different patho-physiological mechanisms.MethodsTwenty month-old rats received a control diet supplemented with cellulose (CON), or a diet supplemented with either curcumin (CUR), fish oil (OM3) or a combination of both (CUR+OM3). Muscle functionality and metabolism was evaluated after chronic treatment during 3 months and molecular mechanisms were evaluated after short-term treatment over 4 weeks.ResultsWalking speed measured with the catwalk gait analyzer significantly improved in the CUR and CUR+OM3 vs CON groups, and also tended to improve with OM3 alone. These functional benefits involved an activation of the muscle antioxidant capacity by OM3 through SOD and catalase induction. This was associated with synergistic enhancement of mitochondrial bioenergetics by CUR+OM3 through increased activity of citrate synthase and respiratory complexes.ConclusionCurcumin and fish oil supplementation prevent the functional decline of muscle health during aging by directly targeting gait speed independently of muscle mass. The physiological benefits of these two ingredients are associated with the enhancement of muscle antioxidant capacity and the synergistic activation of mitochondrial energy production in aged muscle

AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR

International audienceAMPK is a central regulator of energy homeostasis. AMPK not only elicits acute metabolic responses but also promotes metabolic reprogramming and adaptations in the long-term through regulation of specific transcription factors and coactivators. We performed a whole-genome transcriptome profiling in wild-type (WT) and AMPK-deficient mouse embryonic fibroblasts (MEFs) and primary hepatocytes that had been treated with 2 distinct classes of small-molecule AMPK activators. We identified unique compound-dependent gene expression signatures and several AMPK-regulated genes, including folliculin (Flcn), which encodes the tumor suppressor FLCN. Bioinformatics analysis highlighted the lysosomal pathway and the associated transcription factor EB (TFEB) as a key transcriptional mediator responsible for AMPK responses. AMPK-induced Flcn expression was abolished in MEFs lacking TFEB and transcription factor E3, 2 transcription factors with partially redundant function; additionally, the promoter activity of Flcn was profoundly reduced when its putative TFEB-binding site was mutated. The AMPK-TFEB-FLCN axis is conserved across species; swimming exercise in WT zebrafish induced Flcn expression in muscle, which was significantly reduced in AMPK-deficient zebrafish. Mechanistically, we have found that AMPK promotes dephosphorylation and nuclear localization of TFEB independently of mammalian target of rapamycin activity. Collectively, we identified the novel AMPK-TFEB-FLCN axis, which may function as a key cascade for cellular and metabolic adaptations.-Collodet, C., Foretz, M., Deak, M., Bultot, L., Metairon, S., Viollet, B., Lefebvre, G., Raymond, F., Parisi, A., Civiletto, G., Gut, P., Descombes, P., Sakamoto, K. AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR

PGC1α and exercise adaptations in zebrafish

Fish species display huge differences in physical activity ranging from lethargy to migration of thousands of miles, making them an interesting model to identify determinants of physical fitness. Here, we show a remarkable plasticity of zebrafish in response to exercise and induction of PGC1α (encoded by PPARGC1A), a dominant regulator of mitochondrial biogenesis. Forced expression of human PPARGC1A induces mitochondrial biogenesis, an exercise-like gene expression signature, and physical fitness comparable to wild-type animals trained in counter-current swim tunnels. Quantifying transcriptional and proteomic changes in response to exercise or PGC1α, we identify conserved ‘exercise’ adaptations, including a stoichiometric induction of the electron transport chain (ETC) that re-organizes into respiratory supercomplexes in both conditions. We further show that ndufa4/ndufa4l, previously assigned to complex I, associates to free and supramolecular complex IV in vivo. Thus, zebrafish is a useful and experimentally tractable vertebrate model to study exercise biology, including ETC expression and assembly

In Vivo Correction of COX Deficiency by Activation of the AMPK/PGC-1α Axis

Increased mitochondrial biogenesis by activation of PPAR- or AMPK/PGC-1α-dependent homeostatic pathways has been proposed as a treatment for mitochondrial disease. We tested this hypothesis on three recombinant mouse models characterized by defective cytochrome c-oxidase (COX) activity: a knockout (KO) mouse for Surf1, a knockout/knockin mouse for Sco2, and a muscle-restricted KO mouse for Cox15. First, we demonstrated that double-recombinant animals overexpressing PGC-1α in skeletal muscle on a Surf1 KO background showed robust induction of mitochondrial biogenesis and increase of mitochondrial respiratory chain activities, including COX. No such effect was obtained by treating both Surf1−/− and Cox15−/− mice with the pan-PPAR agonist bezafibrate, which instead showed adverse effects in either model. Contrariwise, treatment with the AMPK agonist AICAR led to partial correction of COX deficiency in all three models, and, importantly, significant motor improvement up to normal in the Sco2KO/KI mouse. These results open new perspectives for therapy of mitochondrial disease