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Mitochondria are dynamic organelles that undergo membrane remodeling events in response to metabolic alterations to generate an adequate mitochondrial network. Here, we investigated the function of mitochondrial fission regulator 1-like protein (MTFR1L), an uncharacterized protein that has been identified in phosphoproteomic screens as a potential AMP-activated protein kinase (AMPK) substrate. We showed that MTFR1L is an outer mitochondrial membrane–localized protein modulating mitochondrial morphology. Loss of MTFR1L led to mitochondrial elongation associated with increased mitochondrial fusion events and levels of the mitochondrial fusion protein, optic atrophy 1. Mechanistically, we show that MTFR1L is phosphorylated by AMPK, which thereby controls the function of MTFR1L in regulating mitochondrial morphology both in mammalian cell lines and in murine cortical neurons in vivo. Furthermore, we demonstrate that MTFR1L is required for stress-induced AMPK-dependent mitochondrial fragmentation. Together, these findings identify MTFR1L as a critical mitochondrial protein transducing AMPK-dependent metabolic changes through regulation of mitochondrial dynamics.

Peer reviewed: True, Funder: Swedish Research Council; FundRef: http://dx.doi.org/10.13039/501100004359, Funder: Ramón Areces Foundation; FundRef: http://dx.doi.org/10.13039/100008054, Mitochondrial dysfunction has been reported in obesity and insulin resistance, but primary genetic mitochondrial dysfunction is generally not associated with these, arguing against a straightforward causal relationship. A rare exception, recently identified in humans, is a syndrome of lower body adipose loss, leptin-deficient severe upper body adipose overgrowth, and insulin resistance caused by the p.Arg707Trp mutation in MFN2, encoding mitofusin 2. How the resulting selective form of mitochondrial dysfunction leads to tissue- and adipose depot-specific growth abnormalities and systemic biochemical perturbation is unknown. To address this, Mfn2R707W/R707W knock-in mice were generated and phenotyped on chow and high fat diets. Electron microscopy revealed adipose-specific mitochondrial morphological abnormalities. Oxidative phosphorylation measured in isolated mitochondria was unperturbed, but the cellular integrated stress response was activated in adipose tissue. Fat mass and distribution, body weight, and systemic glucose and lipid metabolism were unchanged, however serum leptin and adiponectin concentrations, and their secretion from adipose explants were reduced. Pharmacological induction of the integrated stress response in wild-type adipocytes also reduced secretion of leptin and adiponectin, suggesting an explanation for the in vivo findings. These data suggest that the p.Arg707Trp MFN2 mutation selectively perturbs mitochondrial morphology and activates the integrated stress response in adipose tissue. In mice, this does not disrupt most adipocyte functions or systemic metabolism, whereas in humans it is associated with pathological adipose remodelling and metabolic disease. In both species, disproportionate effects on leptin secretion may relate to cell autonomous induction of the integrated stress response.

The inner mitochondria membrane (IMM) is densely packed with proteins necessary for mitochondrial activity, including oxidative phosphorylation (OXPHOS) complexes and transporters. Mitochondrial respiratory chain (MRC) complexes, which generate ATP by OXPHOS, associate in higher-order assemblies, known as supercomplexes (SC), that are structurally interdependent. Numerous patients carrying mutations in a single complex, indeed, present with combined enzyme deficiencies and, in particular, the destabilisation of complex I (CI) has been often described in the absence of complex III (CIII). To clarify the structural and functional relationships between complexes, we have analysed a MTCYB-deficient human cell line, unable to assemble CIII. Our results showed that in this line, CI biogenesis was blocked by preventing the incorporation of the NADH module, the last step of CI assembly, rather than decreasing its stability. Moreover, complex IV (CIV) biogenesis was impaired as well, and CIV subunits appeared sequestered within CIII subassemblies. Therefore, we propose that CIII is central not only for the formation of SC but also for the maturation of the other electron transport chain complexes. These results challenge the previous SC model that described the formation of fully assembled individual complexes before the association in SC. In contrast, they support a cooperative-assembly model in which the main role of CIII in SC is to provide a structural and functional platform for the completion of overall MRC biogenesis. Next, we identified and characterised the interaction between CIV and the mitochondrial calcium uniporter complex (MCUC), responsible for mitochondrial calcium uptake and homeostasis. Our data, indeed, showed a specific physical interaction between this ETC enzyme and various subunits of MCUC in first and second dimension blue native PAGE, SILAC labelling/pulldown, and BioID proteomic-based methods. We then investigated the effects of this association with CIV, measuring enzyme activity and mitochondrial respiration, but also on MCUC and CIV distribution in the IMM, by N-structured illuminated super-resolution microscopy (N-SIM). Our results showed a specific reduction in CIV activity in the absence of MICU1, the main regulator of the uniporter, but surprisingly no effects were observed after MCU downregulation or pharmacological inhibition of mitochondrial Ca2+ entry. Instead, the lack of CIV seems to have an impact on MCU-containing complexes formation and induces a re-localisation of MCU from the cristae membrane to the inner boundary membrane. Further experiments will be performed to shed light on the physiological relevance of this interaction., MRC

Tetracyclines have anticancer properties in addition to their well-known antibacterial properties. It has been proposed that tetracyclines slow metastasis and angiogenesis through inhibition of matrix metalloproteinases. However, we believe that the anticancer effect of tetracyclines is due to their inhibition of mitochondrial protein synthesis, resulting in a decrease of the mitochondrial energy generating capacity. Several groups have developed analogs that are void of antibacterial action. An example is COL-3, which is currently tested for its anticancer effects in clinical trials. We have undertaken a comparative study of the tetracycline analogs COL-3 and doxycycline, which has an antibacterial function, to further investigate the role of the mitochondrial energy generating capacity in the anticancer mechanism and, thereby, evaluate the usefulness of mitochondria as an oncotarget. Our experiments with cultures of the human A549, COLO357 and HT29 cancer cells and fibroblasts indicated that COL-3 is significantly more cytotoxic than doxycycline. Mitochondrial translation assays demonstrated that COL-3 has retained its inhibitory effect on mitochondrial protein synthesis. Both drugs caused a severe decrease in the levels of mitochondrially encoded cytochrome-c oxidase subunits and cytochrome-c oxidase activity. In addition, COL-3 produced a marked drop in the level of nuclear-encoded succinate dehydrogenase subunit A and citrate synthase activity, indicating that COL-3 has multiple inhibitory effects. Contrary to COL-3, the anticancer action of doxycycline appears to be based specifically on inhibition of mitochondrial protein synthesis, which is thought to affect rapidly proliferating cancer cells more than healthy tissue. Doxycycline is likely to cause less side effects that COL-3.

Mitochondria are ubiquitous intracellular organelles found in almost all eukaryotes and involved in various aspects of cellular life, with a primary role in energy production. The interest in this organelle has grown stronger with the discovery of their link to various pathologies, including cancer, aging and neurodegenerative diseases. Indeed, dysfunctional mitochondria cannot provide the required energy to tissues with a high-energy demand, such as heart, brain and muscles, leading to a large spectrum of clinical phenotypes. Mitochondrial defects are at the origin of a group of clinically heterogeneous pathologies, called mitochondrial diseases, with an incidence of 1 in 5000 live births. Primary mitochondrial diseases are associated with genetic mutations both in nuclear and mitochondrial DNA (mtDNA), affecting genes involved in every aspect of the organelle function. As a consequence, it is difficult to find a common cause for mitochondrial diseases and, subsequently, to offer a precise clinical definition of the pathology. Moreover, the complexity of this condition makes it challenging to identify possible therapies or drug targets.