Mitochondrial structure, function and dynamics are temporally controlled by c-Myc

Although the c-Myc (Myc) oncoprotein controls mitochondrial biogenesis and multiple enzymes involved in oxidative phosphorylation (OXPHOS), the coordination of these events and the mechanistic underpinnings of their regulation remain largely unexplored. We show here that re-expression of Myc in myc-/- fibroblasts is accompanied by a gradual accumulation of mitochondrial biomass and by increases in membrane polarization and mitochondrial fusion. A correction of OXPHOS deficiency is also seen, although structural abnormalities in electron transport chain complexes (ETC) are not entirely normalized. Conversely, the down-regulation of Myc leads to a gradual decrease in mitochondrial mass and a more rapid loss of fusion and membrane potential. Increases in the levels of proteins specifically involved in mitochondrial fission and fusion support the idea that Myc affects mitochondrial mass by influencing both of these processes, albeit favoring the latter. The ETC defects that persist following Myc restoration may represent metabolic adaptations, as mitochondrial function is re-directed away from producing ATP to providing a source of metabolic precursors demanded by the transformed cell.     

Initiation of Electron Transport Chain Activity in the Embryonic Heart Coincides with the Activation of Mitochondrial Complex 1 and the Formation of Supercomplexes

Mitochondria provide energy in form of ATP in eukaryotic cells. However, it is not known when, during embryonic cardiac development, mitochondria become able to fulfill this function. To assess this, we measured mitochondrial oxygen consumption and the activity of the complexes (Cx) 1 and 2 of the electron transport chain (ETC) and used immunoprecipitation to follow the generation of mitochondrial supercomplexes. We show that in the heart of mouse embryos at embryonic day (E) 9.5, mitochondrial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the complexes are present. We show that Cx-1 of the ETC is able to accept electrons from the Krebs cycle, but enzyme assays that specifically measure electron flow to ubiquinone or Cx-3 show no activity at this early embryonic stage. At E11.5, mitochondria appear functionally more mature; ETC activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, the assembly of highly efficient respiratory supercomplexes containing Cx-1, -3, and -4, ubiquinone, and cytochrome c begins at E11.5, the exact time when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS of embryonic heart mitochondria are indistinguishable from adult mitochondria. In summary, our data suggest that between E9.5 and E11.5 dramatic changes occur in the mitochondria of the embryonic heart, which result in an increase in OXPHOS due to the activation of complex 1 and the formation of supercomplexes.     

Near-Infrared 808 nm Light Boosts Complex IV-Dependent Respiration and Rescues a Parkinson-Related pink1 Model

Mitochondrial electron transport chain (ETC) defects are observed in Parkinson’s disease (PD) patients and in PD fly- and mouse-models; however it remains to be tested if acute improvement of ETC function alleviates PD-relevant defects. We tested the hypothesis that 808 nm infrared light that effectively penetrates tissues rescues pink1 mutants. We show that irradiating isolated fly or mouse mitochondria with 808 nm light that is absorbed by ETC-Complex IV acutely improves Complex IV-dependent oxygen consumption and ATP production, a feature that is wavelength-specific. Irradiating Drosophila pink1 mutants using a single dose of 808 nm light results in a rescue of major systemic and mitochondrial defects. Time-course experiments indicate mitochondrial membrane potential defects are rescued prior to mitochondrial morphological defects, also in dopaminergic neurons, suggesting mitochondrial functional defects precede mitochondrial swelling. Thus, our data indicate that improvement of mitochondrial function using infrared light stimulation is a viable strategy to alleviate pink1-related defects.     

Mutations in Fis1 disrupt orderly disposal of defective mitochondria

Mitochondrial fission is mediated by the dynamin-related protein Drp1 in metazoans. Drp1 is recruited from the cytosol to mitochondria by the mitochondrial outer membrane protein Mff. A second mitochondrial outer membrane protein, named Fis1, was previously proposed as recruitment factor, but Fis1^−/− cells have mild or no mitochondrial fission defects. Here we show that Fis1 is nevertheless part of the mitochondrial fission complex in metazoan cells. During the fission cycle, Drp1 first binds to Mff on the surface of mitochondria, followed by entry into a complex that includes Fis1 and endoplasmic reticulum (ER) proteins at the ER–mitochondrial interface. Mutations in Fis1 do not normally affect fission, but they can disrupt downstream degradation events when specific mitochondrial toxins are used to induce fission. The disruptions caused by mutations in Fis1 lead to an accumulation of large LC3 aggregates. We conclude that Fis1 can act in sequence with Mff at the ER–mitochondrial interface to couple stress-induced mitochondrial fission with downstream degradation processes.     

Chronic inhibition of the mitochondrial ATP synthase in skeletal muscle triggers sarcoplasmic reticulum distress and tubular aggregates

Tubular aggregates (TA) are honeycomb-like arrays of sarcoplasmic-reticulum (SR) tubules affecting aged glycolytic fibers of male individuals and inducing severe sarcomere disorganization and muscular pain. TA develop in skeletal muscle from Tubular Aggregate Myopathy (TAM) patients as well as in other disorders including endocrine syndromes, diabetes, and ageing, being their primary cause unknown. Nowadays, there is no cure for TA. Intriguingly, both hypoxia and calcium dyshomeostasis prompt TA formation, pointing to a possible role for mitochondria in their setting. However, a functional link between mitochondrial dysfunctions and TA remains unknown. Herein, we investigate the alteration in muscle-proteome of TAM patients, the molecular mechanism of TA onset and a potential therapy in a preclinical mouse model of the disease. We show that in vivo chronic inhibition of the mitochondrial ATP synthase in muscle causes TA. Upon long-term restrained oxidative phosphorylation (OXPHOS), oxidative soleus experiments a metabolic and structural switch towards glycolytic fibers, increases mitochondrial fission, and activates mitophagy to recycle damaged mitochondria. TA result from the overresponse of the fission controller DRP1, that upregulates the Store-Operate-Calcium-Entry and increases the mitochondria-SR interaction in a futile attempt to buffer calcium overloads upon prolonged OXPHOS inhibition. Accordingly, hypoxic muscles cultured ex vivo show an increase in mitochondria/SR contact sites and autophagic/mitophagic zones, where TA clusters grow around defective mitochondria. Moreover, hypoxia triggered a stronger TA formation upon ATP synthase inhibition, and this effect was reduced by the DRP1 inhibitor mDIVI. Remarkably, the muscle proteome of TAM patients displays similar alterations in mitochondrial dynamics and in ATP synthase contents. In vivo edaravone treatment in mice with restrained OXPHOS restored a healthy phenotype by prompting mitogenesis and mitochondrial fusion. Altogether, our data provide a functional link between the ATP synthase/DRP1 axis and the setting of TA, and repurpose edaravone as a possible treatment for TA-associated disorders.Subject terms: Musculoskeletal abnormalities, Energy metabolism     

NIK promotes metabolic adaptation of glioblastoma cells to bioenergetic stress

Cancers, including glioblastoma multiforme (GBM), undergo coordinated reprogramming of metabolic pathways that control glycolysis and oxidative phosphorylation (OXPHOS) to promote tumor growth in diverse tumor microenvironments. Adaptation to limited nutrient availability in the microenvironment is associated with remodeling of mitochondrial morphology and bioenergetic capacity. We recently demonstrated that NF-κB-inducing kinase (NIK) regulates mitochondrial morphology to promote GBM cell invasion. Here, we show that NIK is recruited to the outer membrane of dividing mitochondria with the master fission regulator, Dynamin-related protein1 (DRP1). Moreover, glucose deprivation-mediated metabolic shift to OXPHOS increases fission and mitochondrial localization of both NIK and DRP1. NIK deficiency results in decreased mitochondrial respiration, ATP production, and spare respiratory capacity (SRC), a critical measure of mitochondrial fitness. Although IκB kinase α and β (IKKα/β) and NIK are required for OXPHOS in high glucose media, only NIK is required to increase SRC under glucose deprivation. Consistent with an IKK-independent role for NIK in regulating metabolism, we show that NIK phosphorylates DRP1-S616 in vitro and in vivo. Notably, a constitutively active DRP1-S616E mutant rescues oxidative metabolism, invasiveness, and tumorigenic potential in NIK^−/− cells without inducing IKK. Thus, we establish that NIK is critical for bioenergetic stress responses to promote GBM cell pathogenesis independently of IKK. Our data suggest that targeting NIK may be used to exploit metabolic vulnerabilities and improve therapeutic strategies for GBM.Subject terms: Cancer metabolism, Energy metabolism, Cancer metabolism     

Mitochondrial DNA Mutations Provoke Dominant Inhibition of Mitochondrial Inner Membrane Fusion

Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA). We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS) due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial ATP6 gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP) or to maternally inherited Leigh Syndrome (MILS) in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from the network of functional, fusogenic mitochondria.     

Assaying ATP synthesis in cultured cells: A valuable tool for the diagnosis of patients with mitochondrial disorders

Mitochondrial ATP synthase plays a central role in cell function by synthesising most of the ATP in human tissues. In different cells, active regulation of mitochondrial ATP synthase in response to cellular energy demand has been demonstrated, as well as its alteration under several pathological conditions affecting oxidative phosphorylation (OXPHOS). Traditionally, detection of OXPHOS defects is based on the spectrophotometric measurement of respiratory chain complex activities in muscle biopsies. Considering the broad clinical spectrum of mitochondrial disorders, and the difficulty in arriving at a single diagnostic method, in this study we propose measurement of ATP synthesis in mitochondria from skin fibroblasts as an effective screening tool. In the light of our results this assessment emerges as a useful marker of impaired energy production in primary OXPHOS disorders of childhood and as a tool with the potential to drive further molecular genetic studies.     

Ginkgo Biloba Extract Ameliorates Oxidative Phosphorylation Performance and Rescues Aβ-Induced Failure

Background

Energy deficiency and mitochondrial failure have been recognized as a prominent, early event in Alzheimer''s disease (AD). Recently, we demonstrated that chronic exposure to amyloid-beta (Aβ) in human neuroblastoma cells over-expressing human wild-type amyloid precursor protein (APP) resulted in (i) activity changes of complexes III and IV of the oxidative phosphorylation system (OXPHOS) and in (ii) a drop of ATP levels which may finally instigate loss of synapses and neuronal cell death in AD. Therefore, the aim of the present study was to investigate whether standardized Ginkgo biloba extract LI 1370 (GBE) is able to rescue Aβ-induced defects in energy metabolism.

Methodology/Principal Findings

We used a high-resolution respiratory protocol to evaluate OXPHOS respiratory capacity under physiological condition in control (stably transfected with the empty vector) and APP cells after treatment with GBE. In addition, oxygen consumption of isolated mitochondria, activities of mitochondrial respiratory enzymes, ATP and reactive oxygen species (ROS) levels as well as mitochondrial membrane mass and mitochondrial DNA content were determined. We observed a general antioxidant effect of GBE leading to an increase of the coupling state of mitochondria as well as energy homeostasis and a reduction of ROS levels in control cells and in APP cells. GBE effect on OXPHOS was even preserved in mitochondria after isolation from treated cells. Moreover, these functional data were paralleled by an up-regulation of mitochondrial DNA. Improvement of the OXPHOS efficiency was stronger in APP cells than in control cells. In APP cells, the GBE-induced amelioration of oxygen consumption most likely arose from the modulation and respective normalization of the Aβ-induced disturbance in the activity of mitochondrial complexes III and IV restoring impaired ATP levels possibly through decreasing Aβ and oxidative stress level.

Conclusions/Significance

Although the underlying molecular mechanisms of the mode of action of GBE remain to be determined, our study clearly highlights the beneficial effect of GBE on the cellular OXPHOS performance and restoration of Aβ-induced mitochondrial dysfunction.     

An RMND1 Mutation Causes Encephalopathy Associated with Multiple Oxidative Phosphorylation Complex Deficiencies and a Mitochondrial Translation Defect

Mutations in the genes composing the mitochondrial translation apparatus are an important cause of a heterogeneous group of oxidative phosphorylation (OXPHOS) disorders. We studied the index case in a consanguineous family in which two children presented with severe encephalopathy, lactic acidosis, and intractable seizures leading to an early fatal outcome. Blue native polyacrylamide gel electrophoretic (BN-PAGE) analysis showed assembly defects in all of the OXPHOS complexes with mtDNA-encoded structural subunits, and these defects were associated with a severe deficiency in mitochondrial translation. Immunoblot analysis showed reductions in the steady-state levels of several structural subunits of the mitochondrial ribosome. Whole-exome sequencing identified a homozygous missense mutation (c.1250G>A) in an uncharacterized gene, RMND1 (required for meiotic nuclear division 1). RMND1 localizes to mitochondria and behaves as an integral membrane protein. Retroviral expression of the wild-type RMND1 cDNA rescued the biochemical phenotype in subject cells, and siRNA-mediated knockdown of the protein recapitulated the defect. BN-PAGE, gel filtration, and mass spectrometry analyses showed that RMND1 forms a high-molecular-weight and most likely homopolymeric complex (∼240 kDa) that does not assemble in subject fibroblasts but that is rescued by expression of RMND1 cDNA. The p.Arg417Gln substitution, predicted to be in a coiled-coil domain, which is juxtaposed to a transmembrane domain at the extreme C terminus of the protein, does not alter the steady-state level of RMND1 but might prevent protein-protein interactions in this complex. Our results demonstrate that the RMND1 complex is necessary for mitochondrial translation, possibly by coordinating the assembly or maintenance of the mitochondrial ribosome.     

Glucose Deprivation Converts Poly(ADP-ribose) Polymerase-1 Hyperactivation into a Transient Energy-producing Process

Massive poly(ADP-ribose) formation by poly(ADP-ribose) polymerase-1 (PARP-1) triggers NAD depletion and cell death. These events have been invariantly related to cellular energy failure due to ATP shortage. The latter occurs because of both ATP consumption for NAD resynthesis and impairment of mitochondrial ATP formation caused by an increase of the AMP/ADP ratio. ATP depletion is therefore thought to be an inevitable consequence of NAD loss and a hallmark of PARP-1 activation. Here, we challenge this scenario by showing that PARP-1 hyperactivation in cells cultured in the absence of glucose (Glu⁻ cells) is followed by NAD depletion and an unexpected PARP-1 activity-dependent ATP increase. We found increased ADP content in resting Glu⁻ cells, a condition that counteracts the increase of the AMP/ADP ratio during hyperpoly(ADP-ribosyl)ation and preserves mitochondrial coupling. We also show that the increase of ATP in Glu⁻ cells is due to adenylate kinase activity, transforming AMP into ADP which, in turn, is converted into ATP by coupled mitochondria. Interestingly, PARP-1-dependent mitochondrial release of apoptosis-inducing factor (AIF) and cytochrome complex (Cyt c) is reduced in Glu⁻ cells, even though cell death eventually occurs. Overall, the present study identifies basal ADP content and adenylate kinase as key determinants of bioenergetics during PARP-1 hyperactivation and unequivocally demonstrates that ATP loss is not metabolically related to NAD depletion.     

Mitochondrial morphology and dynamics in <Emphasis Type="Italic">Triticum aestivum</Emphasis> roots in response to rotenone and antimycin A

Mitochondria are dynamic organelles, capable of fusion and fission as a part of cellular responses to various signals, such as the shifts in the redox status of a cell. The mitochondrial electron transport chain (ETC.) is involved in the generation of reactive oxygen species (ROS), with complexes I and III contributing the most to this process. Disruptions of ETC. can lead to increased ROS generation. Here, we demonstrate the appearance of giant mitochondria in wheat roots in response to simultaneous application of the respiratory inhibitors rotenone (complex I of mitochondrial ETC.) and antimycin A (complex III of mitochondrial ETC.). The existence of such megamitochondria was temporary, and following longer treatment with inhibitors mitochondria resumed their conventional size and oval shape. Changes in mitochondrial morphology were accompanied with a decrease in mitochondrial potential and an unexpected increase in oxygen consumption. Changes in mitochondrial morphology and activity may result from the fusion and fission of mitochondria induced by the disruption of mitochondrial ETC. Results from experiments with the inhibitor of mitochondrial fission Mdivi-1 suggest that the retarded fission may facilitate plant mitochondria to appear in a fused shape. The processes of mitochondrial fusion and fission are involved in the regulation of the efficacy of the functions of the respiratory chain complexes and ROS metabolism during stresses. The changes in morphology of mitochondria, along with the changes in their functional activity, can be a part of the strategy of the plant adaptation to stresses.     

IFN‐β rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1

Mitochondrial homeostasis is essential for providing cellular energy, particularly in resource‐demanding neurons, defects in which cause neurodegeneration, but the function of interferons (IFNs) in regulating neuronal mitochondrial homeostasis is unknown. We found that neuronal IFN‐β is indispensable for mitochondrial homeostasis and metabolism, sustaining ATP levels and preventing excessive ROS by controlling mitochondrial fission. IFN‐β induces events that are required for mitochondrial fission, phosphorylating STAT5 and upregulating PGAM5, which phosphorylates serine 622 of Drp1. IFN‐β signaling then recruits Drp1 to mitochondria, oligomerizes it, and engages INF2 to stabilize mitochondria–endoplasmic reticulum (ER) platforms. This process tethers damaged mitochondria to the ER to separate them via fission. Lack of neuronal IFN‐β in the Ifnb ^–/– model of Parkinson disease (PD) disrupts STAT5‐PGAM5‐Drp1 signaling, impairing fission and causing large multibranched, damaged mitochondria with insufficient ATP production and excessive oxidative stress to accumulate. In other PD models, IFN‐β rescues dopaminergic neuronal cell death and pathology, associated with preserved mitochondrial homeostasis. Thus, IFN‐β activates mitochondrial fission in neurons through the pSTAT5/PGAM5/^S622Drp1 pathway to stabilize mitochondria/ER platforms, constituting an essential neuroprotective mechanism.     

The yeast complex I equivalent NADH dehydrogenase rescues pink1 mutants

Pink1 is a mitochondrial kinase involved in Parkinson's disease, and loss of Pink1 function affects mitochondrial morphology via a pathway involving Parkin and components of the mitochondrial remodeling machinery. Pink1 loss also affects the enzymatic activity of isolated Complex I of the electron transport chain (ETC); however, the primary defect in pink1 mutants is unclear. We tested the hypothesis that ETC deficiency is upstream of other pink1-associated phenotypes. We expressed Saccaromyces cerevisiae Ndi1p, an enzyme that bypasses ETC Complex I, or sea squirt Ciona intestinalis AOX, an enzyme that bypasses ETC Complex III and IV, in pink1 mutant Drosophila and find that expression of Ndi1p, but not of AOX, rescues pink1-associated defects. Likewise, loss of function of subunits that encode for Complex I-associated proteins displays many of the pink1-associated phenotypes, and these defects are rescued by Ndi1p expression. Conversely, expression of Ndi1p fails to rescue any of the parkin mutant phenotypes. Additionally, unlike pink1 mutants, fly parkin mutants do not show reduced enzymatic activity of Complex I, indicating that Ndi1p acts downstream or parallel to Pink1, but upstream or independent of Parkin. Furthermore, while increasing mitochondrial fission or decreasing mitochondrial fusion rescues mitochondrial morphological defects in pink1 mutants, these manipulations fail to significantly rescue the reduced enzymatic activity of Complex I, indicating that functional defects observed at the level of Complex I enzymatic activity in pink1 mutant mitochondria do not arise from morphological defects. Our data indicate a central role for Complex I dysfunction in pink1-associated defects, and our genetic analyses with heterologous ETC enzymes suggest that Ndi1p-dependent NADH dehydrogenase activity largely acts downstream of, or in parallel to, Pink1 but upstream of Parkin and mitochondrial remodeling.     

Mitofusin-2 mediates doxorubicin sensitivity and acute resistance in Jurkat leukemia cells

Mitochondria oscillate along a morphological continuum from fragmented individual units to hyperfused tubular networks. Their position at the junction of catabolic and anabolic metabolism couples this morphological plasticity, called mitochondrial dynamics, to larger cellular metabolic programs, which in turn implicate mitochondria in a number of disease states. In many cancers, fragmented mitochondria engage the cell with the biosynthetic capacity of aerobic glycolysis in service of proliferation and progression. Chemo-resistant cancers, however, favor remodeling dynamics that yield fused mitochondrial assemblies utilizing oxidative phosphorylation (OXPHOS) through the electron transport chain (ETC). In this study, expression of Mitofusin-2 (MFN-2), a GTPase protein mediator of mitochondrial fusion, was found to closely correlate to Jurkat leukemia cell survival post doxorubicin (DxR) assault. Moreover, this was accompanied by dramatically increased expression of OXPHOS respiratory complexes and ATP Synthase, as well as a commensurate escalation of state III respiration and respiratory control ratio (RCR). Importantly, CRISPR knockout of MFN-2 resulted in a considerable decrease of doxorubicin (DxR) median lethal dose compared to a treated wildtype control, suggesting an important role of mitochondrial fusion in chemotherapy sensitivity and acute resistance.     

Stoichiometric expression of mtHsp40 and mtHsp70 modulates mitochondrial morphology and cristae structure via Opa1L cleavage

Deregulation of mitochondrial heat-shock protein 40 (mtHsp40) and dysfunction of mtHsp70 are associated with mitochondrial fragmentation, suggesting that mtHsp40 and mtHsp70 may play roles in modulating mitochondrial morphology. However, the mechanism of mitochondrial fragmentation induced by mtHsp40 deregulation and mtHsp70 dysfunction remains unclear. In addition, the functional link between mitochondrial morphology change upon deregulated mtHsp40/mtHsp70 and mitochondrial function has been unexplored. Our coimmunoprecipitation and protein aggregation analysis showed that both overexpression and depletion of mtHsp40 accumulated aggregated proteins in fragmented mitochondria. Moreover, mtHsp70 loss and expression of a mtHsp70 mutant lacking the client-binding domain caused mitochondrial fragmentation. Together the data suggest that the molecular ratio of mtHsp40 to mtHsp70 is important for their chaperone function and mitochondrial morphology. Whereas mitochondrial translocation of Drp1 was not altered, optic atrophy 1 (Opa1) short isoform accumulated in fragmented mitochondria, suggesting that mitochondrial fragmentation in this study results from aberration of mitochondrial inner membrane fusion. Finally, we found that fragmented mitochondria were defective in cristae development, OXPHOS, and ATP production. Taken together, our data suggest that impaired stoichiometry between mtHsp40 and mtHsp70 promotes Opa1_L cleavage, leading to cristae opening, decreased OXPHOS, and triggering of mitochondrial fragmentation after reduction in their chaperone function.     

S6 kinase 1 plays a key role in mitochondrial morphology and cellular energy flow

Mitochondrial morphology, which is associated with changes in metabolism, cell cycle, cell development and cell death, is tightly regulated by the balance between fusion and fission. In this study, we found that S6 kinase 1 (S6K1) contributes to mitochondrial dynamics, homeostasis and function. Mouse embryo fibroblasts lacking S6K1 (S6K1-KO MEFs) exhibited more fragmented mitochondria and a higher level of Dynamin related protein 1 (Drp1) and active Drp1 (pS616) in both whole cell extracts and mitochondrial fraction. In addition, there was no evidence for autophagy and mitophagy induction in S6K1 depleted cells. Glycolysis and mitochondrial respiratory activity was higher in S6K1-KO MEFs, whereas OxPhos ATP production was not altered. However, inhibition of Drp1 by Mdivi1 (Drp1 inhibitor) resulted in higher OxPhos ATP production and lower mitochondrial membrane potential. Taken together the depletion of S6K1 increased Drp1-mediated fission, leading to the enhancement of glycolysis. The fission form of mitochondria resulted in lower yield for OxPhos ATP production as well as in higher mitochondrial membrane potential. Thus, these results have suggested a potential role of S6K1 in energy metabolism by modulating mitochondrial respiratory capacity and mitochondrial morphology.     

Parkin-mediated mitophagy and autophagy flux disruption in cellular models of MERRF syndrome

Mitochondrial diseases are considered rare genetic disorders characterized by defects in oxidative phosphorylation (OXPHOS). They can be provoked by mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). MERRF (Myoclonic Epilepsy with Ragged-Red Fibers) syndrome is one of the most frequent mitochondrial diseases, principally caused by the m.8344A>G mutation in mtDNA, which affects the translation of all mtDNA-encoded proteins and therefore impairs mitochondrial function.In the present work, we evaluated autophagy and mitophagy flux in transmitochondrial cybrids and fibroblasts derived from a MERRF patient, reporting that Parkin-mediated mitophagy is increased in MERRF cell cultures. Our results suggest that supplementation with coenzyme Q₁₀ (CoQ), a component of the electron transport chain (ETC) and lipid antioxidant, prevents Parkin translocation to the mitochondria. In addition, CoQ acts as an enhancer of autophagy and mitophagy flux, which partially improves cell pathophysiology. The significance of Parkin-mediated mitophagy in cell survival was evaluated by silencing the expression of Parkin in MERRF cybrids. Our results show that mitophagy acts as a cell survival mechanism in mutant cells.To confirm these results in one of the main affected cell types in MERRF syndrome, mutant induced neurons (iNs) were generated by direct reprogramming of patients-derived skin fibroblasts. The treatment of MERRF iNs with Guttaquinon CoQ₁₀ (GuttaQ), a water-soluble derivative of CoQ, revealed a significant improvement in cell bioenergetics. These results indicate that iNs, along with fibroblasts and cybrids, can be utilized as reliable cellular models to shed light on disease pathomechanisms as well as for drug screening.