Mitofusins: ubiquitylation promotes fusion

Mitochondrial genes including Mfn2 are at the center of many diseases, underscoring their potential as a therapeutical target. The Chen group now identified 15-oxospiramilactone as a chemical inhibitor of the mammalian deubiquitylase USP30, acting on Mfn1 and Mfn2.Mitofusins, Fzo1 in yeast and Mfn1 and Mfn2 in mammals, are ubiquitylated and this post-translational modification has both positive and negative consequences on mitochondrial fusion¹. The process of ubiquitylation requires enzymes belonging to three classes of proteins called E1, E2 and E3, which catalyze a cascade of successive steps leading to the covalent attachment of the modifier to its target protein². Deubiquitylating enzymes render this modification reversible, thus offering further possibilities for regulation². Ubiquitylation of mitofusins leads to their proteolyic breakdown, inhibiting fusion of mitochondria that consequently undergo fragmentation (Figure 1, left panel)^1,3. For example in response to mitochondrial depolarization or apoptotic stimuli, E3 ligases like Parkin and Huwe1 ubiquitylate and target Mfn1 and Mfn2 to the proteasome (Figure 1, left panel)^3,4. However, ubiquitylation of mitofusins is a dual process and a non-proteolytic role of mitofusin ubiquitylation that promotes mitochondrial fusion is now emerging¹. This opposing mechanism was first described in yeast, where the isopeptidases Ubp12 and Ubp2 that deubiquitylate Fzo1 have been identified⁵. Inhibition and activation of mitochondrial fusion by ubiquitylation enable different morphologies of mitochondria ranging from a multitude of small organelles to a hyperconnected network (Figure 1)⁵. In a recent paper published in Cell Research, Yue et al.⁶ reveal that a similar process is present in mammalian cells. The authors report that the isopeptidase USP30 acts on ubiquitylated forms of Mfn1 and Mfn2 that stimulate mitochondrial fusion (Figure 1, right panel). This discovery identifies for the first time in mammals a positive role of ubiquitylation in the regulation of Mfn1 and Mfn2 fusion activity⁶.Open in a separate windowFigure 1Dual roles of ubiquitylation and deubiquitylation of mitofusins Mfn1 and Mfn2, the key effectors for mitochondrial fusion, in regulating mitochondrial fusion. On one hand, ubiquitylation of Mfn1 and Mfn2 by E3 ligases like Parkin or Huwe1 targets their proteasomal degradation and inhibits mitochondrial fusion, which results in mitochondrial fragmentation due to unopposed fission events. On the other hand, ubiquitylation of Mfn1 and Mfn2 by an unknown E3 ligase enhances their activity and promotes mitochondrial fusion. This positive regulation is counteracted by the deubiquitylase USP30, targeted by the small molecule inhibitor 15-oxospiramilactone.Moreover, Yue et al.⁶ identified the first small molecule inhibitor of mitochondrial fusion, 15-oxospiramilactone, which targets USP30 in both human and mouse cell lines. 15-oxospiramilactone is a semi-synthetic diterpene alkaloid of 330 Da that can be chemically synthetized through an oxidation reaction from spiramines extracted from the roots of a Chinese herbal medicine Spiraea japonica (Rosaceae). Inhibition of USP30 increased ubiquitylation of Mfn1 and Mfn2 and led to an elongation of the mitochondrial network (Figure 1, right panel)^6,7. USP30 is a cysteine ubiquitin isopeptidase N-terminally anchored to the outer membrane of mitochondria, which was previously shown to regulate mitochondrial morphology dependent on Mfn1 and Mfn2⁷. USP30 knockdown leads to mitochondrial elongation, a phenotype rescued by ectopic expression of wild-type USP30, while the catalytically inactive mutant C77S USP30 failed to revert⁷. Yue et al.⁶ show that 15-oxospiramilactone directly interacts with USP30, which also depends on its catalytically active cysteine, and inhibits the DUB activity of USP30 on tetraubiquitin chains. Moreover, they demonstrate that inhibition of USP30 and subsequent mitochondrial elongation are due to stimulated mitochondrial fusion activity, apparently with no influence on mitochondrial fission⁶. Concomitantly, cells showed increased ubiquitylation of Mfn1 and Mfn2 without significant changes in protein turnover of these two proteins⁶. Therefore, in analogy to findings in yeast, ubiquitylation of Mfn1 and Mfn2 can either signal them to activate mitochondrial fusion or in contrast promote their proteasomal degradation, resulting in mitochondrial fission (Figure 1).Importantly, 15-oxospiramilactone reverts the mitochondrial fragmentation phenotype of single Mfn-knockout (Mfn1^−/− or Mfn2^−/−) cells, suggesting that mitochondrial fusion depends on the ubiquitylation of both mitofusin proteins⁶. In yeast, the importance of ubiquitylation was proven by directly attaching a deubiquitylase to Fzo1, which resulted in a non-ubiquitylated and non-functional Fzo1 protein⁵. In addition, the identification and the subsequent mutagenesis study of the ubiquitylation sites in Fzo1 confirmed an interplay between ubiquitylation and oligomerization in mitochondrial fusion in S. cerevisiae⁵. Impairing the yeast E3 ligase SCF^Mdm30 inhibited mitochondrial fusion and, conversely, ablation of UBP12 led to more fusion events^5,8. Given this new identification of USP30 as the functional orthologue of the yeast Ubp12, future studies will certainly aim at the identification of the E3 ligase counterpart of SCF^Mdm30 and ubiquitylation sites in Mfn1 and Mfn2. In addition to USP30 inhibition, other conditions leading to mitochondrial hyperfusion have been previously observed, such as mild stress conditions that increase reactive oxygen species (ROS)⁹. Importantly, oxidative stress and mitochondrial fusion are directly linked as ROS induces disulphide switching of Mfn2 to oligomeric forms that promote mitochondrial fusion⁹. It would be interesting to investigate whether 15-oxospiramilactone also affects the generation of disulphide-mediated mitofusin oligomers, thus activating mitochondrial fusion.Mutations in Mfn2 are causative for the Charcot-Marie-Tooth type 2A neuropathy, an autosomal dominant disorder of the peripheral nervous system that mainly affects axons and lower extremities¹. Deficiencies in Parkin and Mfn2 ubiquitylation were also linked to Parkinson''s disease³. In addition to neuropathies, Mfn2 is associated to other diseases like cardiomyophathies and diabetes¹. Yue et al.⁶ found that 15-oxospiramilactone reverted phenotypes arising from the lack of Mfn1 or Mfn2. It restored the normal distribution of mtDNA, allowed recovery of the ΔΨm and increased the ATP levels and OXPHOS capacity of the rebuilt mitochondrial network. Therefore, this study potentiates 15-oxospiramilactone for therapeutical benefit. The anti-cancer properties of 15-oxospiramilactone, also named S3 or NC043, have been previously reported^10,11. It inhibits Wnt/β-catenin signaling and colon cancer cell tumorigenesis in a xenograft model¹⁰. Moreover, 15-oxospiramilactone increases Bim expression and apoptosis to inhibit tumor growth from Bax^−/−/Bak^−/− cells implanted in mice¹¹. However, Yue et al.⁶ show that the effect of 15-oxospiramilactone in mitochondrial fusion is independent of apoptosis and suggest that the difference is due to drug concentration. Indeed, previous anti-cancer studies used 15-oxospiramilactone at a concentration range of 3.75-15 μM^10,11, whereas 2 μM suffice to inhibit USP30⁶. Further studies are needed to address the clinical relevance of 15-oxospiramilactone and USP30 in Mfn2-associated diseases.     

Muscle-specific Drp1 overexpression impairs skeletal muscle growth via translational attenuation

Mitochondrial fission and fusion are essential processes in the maintenance of the skeletal muscle function. The contribution of these processes to muscle development has not been properly investigated in vivo because of the early lethality of the models generated so far. To define the role of mitochondrial fission in muscle development and repair, we have generated a transgenic mouse line that overexpresses the fission-inducing protein Drp1 specifically in skeletal muscle. These mice displayed a drastic impairment in postnatal muscle growth, with reorganisation of the mitochondrial network and reduction of mtDNA quantity, without the deficiency of mitochondrial bioenergetics. Importantly we found that Drp1 overexpression activates the stress-induced PKR/eIF2α/Fgf21 pathway thus leading to an attenuated protein synthesis and downregulation of the growth hormone pathway. These results reveal for the first time how mitochondrial network dynamics influence muscle growth and shed light on aspects of muscle physiology relevant in human muscle pathologies.Skeletal muscle growth and mitochondrial metabolism are intimately linked. In myogenic precursor cells, mitochondrial mass, mtDNA copy number and mitochondrial respiration increase after the onset of myogenic differentiation;^{1, 2} furthermore, postnatal development of fast-twitch muscle is accompanied by an increase in mtDNA copy number³ and muscle regeneration is impaired when mitochondrial protein synthesis is inhibited with chloramphenicol.^{2, 4} These observations suggest that a change in the mitochondrial metabolism is necessary for proper muscle development. During myogenesis and postnatal development, the shape of mitochondria is also remodelled:^{3, 5, 6} in an elegant mouse model with fluorescent mitochondria it was shown that in young mice mitochondria of the extensor digitorum longus (EDL) muscle are shaped as elongated tubules oriented along the long axis of the muscle fibre, whereas in adult mice mitochondria are punctuated and organised into doublets.¹Mitochondrial network morphology is controlled by the balance between fusion and fission. In mammals, three large GTPases are involved in mitochondrial fusion: mitofusins 1 and 2 (Mfn1 and Mfn2) participate in the early steps of mitochondrial outer-membrane fusion, whereas the optic atrophy 1 protein (Opa1) is essential for inner-membrane fusion.⁷ Mitochondrial fission is mediated by the evolutionarily conserved dynamin-related protein 1 (Drp1).⁸ In humans, mutations in Mfn2 and Opa1 cause two neurodegenerative diseases – Charcot–Marie–Tooth type 2 A and dominant optic atrophy, respectively – and a mutation in Drp1 has been linked to neonatal lethality with multisystem failure.^{9, 10, 11} Moreover, Drp1 expression was reported to increase in a model of cachexia¹² and to contribute to muscle insulin resistance in obese and type 2 diabetic mice.^{13, 14}The importance of mitochondrial dynamics in muscle physiology has become increasingly clear. In skeletal muscle, mitochondria undergo fusion to share matrix content in order to support excitation–contraction coupling.¹⁵ The mitochondrial network is remodelled in atrophic conditions, and denervation and expression of fission machinery in adult myofibres is sufficient to cause muscle wasting.¹⁶ Moreover, mice lacking Mfn1 and 2 in fast-twitch muscles exhibit drastic growth defects and muscle atrophy before dying at 6–8 weeks of age.³ Animal models in which mitochondrial fission proteins are manipulated during skeletal muscle development are not yet available, but in vitro data demonstrate that regulation of Drp1 is critical for myogenesis: myoblasts differentiation requires nitric oxide-dependent inhibition of Drp1⁶ and pharmacological inhibition of Drp1 activity impairs myogenic differentiation.¹⁷To explore in vivo the role of Drp1 and mitochondrial shape in the developing muscle, we generated a transgenic mouse line specifically overexpressing Drp1 in skeletal muscle during myogenesis. These mice display strong impairments in mitochondrial network shape and in muscle growth. We show that the mechanism responsible for the growth defect involves inhibition of protein synthesis and activation of the Atf4 pathway.     

Mitofusins deficiency elicits mitochondrial metabolic reprogramming to pluripotency

Cell reprogramming technology has allowed the in vitro control of cell fate transition, thus allowing for the generation of highly desired cell types to recapitulate in vivo developmental processes and architectures. However, the precise molecular mechanisms underlying the reprogramming process remain to be defined. Here, we show that depleting p53 and p21, which are barriers to reprogramming, yields a high reprogramming efficiency. Deletion of these factors results in a distinct mitochondrial background with low expression of oxidative phosphorylation subunits and mitochondrial fusion proteins, including mitofusin 1 and 2 (Mfn1/2). Importantly, Mfn1/2 depletion reciprocally inhibits the p53-p21 pathway and promotes both the conversion of somatic cells to a pluripotent state and the maintenance of pluripotency. Mfn1/2 depletion facilitates the glycolytic metabolic transition through the activation of the Ras-Raf and hypoxia-inducible factor 1α (HIF1α) signaling at an early stage of reprogramming. HIF1α is required for increased glycolysis and reprogramming by Mfn1/2 depletion. Taken together, these results demonstrate that Mfn1/2 constitutes a new barrier to reprogramming, and that Mfn1/2 ablation facilitates the induction of pluripotency through the restructuring of mitochondrial dynamics and bioenergetics.Cell fate transition occurs under various developmental, physiological, and pathological conditions, including normal embryonic development, aging, and tissue regeneration, as well as tumor initiation and progression. Defining the cellular and molecular mechanisms of cell fate transition and learning to control these mechanisms may be essential for treating abnormal pathological conditions resulting from improper regulation of cell fate. The recent development of induced pluripotent stem cell (iPSC) technology has allowed for the reprogramming of somatic cells to pluripotent stem cells through the use of defined pluripotency factors, and has allowed us to more closely mimic and recapitulate the conditions of cell fate transitions.¹ In studying aspects of somatic cell reprogramming related to pluripotency, dramatic and complex molecular changes at the genetic, epigenetic, and metabolic levels have been observed during the initial stage of reprogramming.² Cell reprogramming faces the challenge of balancing stability and plasticity and must overcome critical barriers, such as cell cycle checkpoints, the mesenchymal–epithelial transition, and metabolic reprogramming, to progress cell fate conversion from a stochastic early phase toward pluripotency.³The p53 pathway limits cell fate transition by inducing classical signaling that leads to cell cycle arrest, senescence, or apoptosis to maintain genome stability in the face of reprogramming-induced stress. Thus, compromising p53 signaling accelerates the reprogramming process.^{4, 5, 6} Recent reports have provided data showing that the fast-cycling population is enriched in p53 knockdown cells, which secures the transition to pluripotency.⁷ It has also been observed that p53 induces the differentiation of damaged embryonic stem cells (ESCs) by suppressing the pluripotency factors, Nanog and Oct4.⁸ Moreover, p53 governs cellular state homeostasis, which constrains the mesenchymal–epithelial transition by inhibiting Klf4-mediated expression of epithelial genes early in the reprogramming process,⁹ and opposes glycolytic metabolic reprogramming, thereby playing an oncosuppressive role.¹⁰ Through the regulation of these canonical and emergent functions, p53 maintains cellular integrity and stability under conditions of cell fate transition.Highly proliferative cells, such as iPSCs and tumor cells, prefer to undergo glycolysis and decrease their dependency on mitochondrial ATP production, which requires the biosynthesis of macromolecules and the alleviation of mitochondrial oxidative stress in rapidly growing cells.¹¹ Furthermore, there are substantial mitochondrial structural changes that interconnected mitochondrial network of somatic cells transforms into an immature phenotype during metabolic reprogramming.¹² These morphological and functional changes in mitochondria are controlled by fusion and fission processes, which are primarily mediated by the dynamin-related GTPases, mitofusins (Mfn) and dynamin-related protein 1 (Drp1), respectively.¹³ Our previous data demonstrated that Drp1 activation via the pluripotency factor Rex1 promotes mitochondrial fragmentation, which contributes to the acquisition and maintenance of stem cell pluripotency.¹⁴ Balancing mitochondrial dynamics is crucial for maintaining cellular homeostasis, and an abnormal mitochondrial dynamic can result in numerous diseases. However, the relevant roles of mitochondrial structural proteins in the cell fate conversion process are not completely understood.Here, we decipher an early stage of cellular reprogramming in a p53 knockout (KO) context related to its function as a cell fate transition checkpoint. p53- and p21-KO cells express low levels of Mfn1/2 at an early stage of reprogramming, and restructuring mitochondrial dynamics and bioenergetics by ablating Mfn promotes the conversion of these cells to a pluripotent cell fate. Our work reveals novel roles of the mitochondrial fusion proteins Mfn1/2 driving entry to and exit from pluripotency by the coordinated integration of p53 signaling.     

Acute focal brain damage alters mitochondrial dynamics and autophagy in axotomized neurons

Mitochondria are key organelles for the maintenance of life and death of the cell, and their morphology is controlled by continual and balanced fission and fusion dynamics. A balance between these events is mandatory for normal mitochondrial and neuronal function, and emerging evidence indicates that mitochondria undergo extensive fission at an early stage during programmed cell death in several neurodegenerative diseases. A pathway for selective degradation of damaged mitochondria by autophagy, known as mitophagy, has been described, and is of particular importance to sustain neuronal viability. In the present work, we analyzed the effect of autophagy stimulation on mitochondrial function and dynamics in a model of remote degeneration after focal cerebellar lesion. We provided evidence that lesion of a cerebellar hemisphere causes mitochondria depolarization in axotomized precerebellar neurons associated with PTEN-induced putative kinase 1 accumulation and Parkin translocation to mitochondria, block of mitochondrial fusion by Mfn1 degradation, increase of calcineurin activity and dynamin-related protein 1 translocation to mitochondria, and consequent mitochondrial fission. Here we suggest that the observed neuroprotective effect of rapamycin is the result of a dual role: (1) stimulation of autophagy leading to damaged mitochondria removal and (2) enhancement of mitochondria fission to allow their elimination by mitophagy. The involvement of mitochondrial dynamics and mitophagy in brain injury, especially in the context of remote degeneration after acute focal brain damage, has not yet been investigated, and these findings may offer new target for therapeutic intervention to improve functional outcomes following acute brain damage.Mitochondria are essential organelles for cell function and viability, and are central to several processes such as energy production, metabolism, calcium buffering, and life/death decisions.¹ Neurons have a high and constant demand for mitochondrial metabolism, to maintain their functions, and contain many mitochondria throughout the cytoplasm, distributed to axons, presynaptic terminals, and dendritic shafts. Mitochondria are highly dynamic organelles that continuously move and change shape. Their morphology is governed by the dynamic equilibrium between fusion and fission processes, both of which are mediated by evolutionarily conserved members of the dynamin family of large GTPases.² Fusion between the outer mitochondrial membranes (OMMs) is mediated by membrane-anchored mitofusins (Mfn1 and Mfn2), whereas that between inner mitochondrial membranes is controlled by optic atrophy 1.³ Mitochondrial fission is regulated by dynamin-related protein 1 (Drp1) and fission protein 1 (Fis1).⁴ Drp1 is predominantly expressed in the cytoplasm and is recruited to mitochondria, where it associates with Fis1 to form a complex that constricts the inner and outer membranes, allowing mitochondria to divide.^{5, 6}Mitochondrial dynamics are crucial to the maintenance of mitochondrial function and neuron survival, as evidenced by findings that pathological imbalances between fusion and fission events develop in many neurodegenerative disorders and brain trauma.^{7, 8} Moreover, mitochondrial fission regulates organelle shape and mediates mitochondria-dependent cell death.⁹ The release of proapoptotic factors, such as cytochrome c (with consequent formation of the apoptosome and caspase activation), from depolarized mitochondria into the cytosol is a significant event in the induction of apoptosis and is associated with Drp1-mediated fragmentation of the mitochondrial network.¹⁰The elimination of dysfunctional mitochondria is therefore a key process with regard to the viability of neurons (and other cell types). Damaged mitochondria that accelerate cell death are removed through autophagy, an evolutionarily conserved lysosome-mediated degradation pathway that maintains the balance between organelle biogenesis, protein synthesis, and degradation of cellular components.¹¹Mitochondria can be selectively degraded by autophagy – a pathway known as mitophagy.¹² Priming of damaged mitochondria can involve several mechanisms, one of which is triggered by Parkin, a cytosolic E3 ubiquitin ligase that is mutated in familial forms of Parkinson''s disease (PD).¹³ Parkin recruitment to impaired mitochondria requires the kinase activity of PINK1 (PTEN-induced putative kinase 1),^{14, 15, 16} a serine/threonine kinase that is also mutated in other autosomal recessive forms of PD.¹⁷ PINK1 levels are very low in polarized mitochondria, to prevent mitophagy of healthy mitochondria; in contrast, when mitochondria are depolarized, full-length PINK1 accumulates rapidly at damaged organelles, crossing the OMM and acting as cellular sensors of damaged mitochondria.¹⁵ PINK1 then recruits Parkin to the mitochondrial surface, where it ubiquitinates several OMM proteins, which in turn recruit other proteins to initiate mitophagy.¹⁸ Mfn1 is a direct substrate of Parkin and its degradation has been suggested to prevent the fusion of damaged and healthy mitochondria.¹⁹ Drp1-dependent fission of mitochondria is also a crucial event that effects their degradation through mitophagy and the inhibition of fission specifically prevents mitochondrial autophagy.²⁰In this study, we examined mitochondrial function and its relationship with autophagy machinery in an in vivo model of acute focal CNS (central nervous system) lesion, focusing on remote changes that are induced by hemicerebellectomy (HCb). Unilateral HCb is a suitable model in which axonal damage-induced neuronal death mechanisms can be studied.^{21, 22} In this model, neuronal degeneration is caused by target deprivation and axonal damage of contralateral precerebellar nuclei of the inferior olive and pontine nuclei. Remote damage is a multifactorial phenomenon in which many components become active in specific time frames²¹ and is significant in determining the overall clinical outcome in many CNS pathologies, including spinal cord injury and traumatic brain injury.^{23, 24, 25}Recently, we demonstrated that autophagy is activated in axotomized neurons after HCb, subsequent to cytochrome c release from mitochondria.²⁶ Further, rapamycin-enhanced autophagy has neuroprotective effects, reducing neuronal death and improving functional recovery. In this study, we examined mitochondrial dynamics and function in axotomized neurons after HCb in mice and analysed the effects of rapamycin on selective elimination of damaged mitochondria.     

NF-κB pathway controls mitochondrial dynamics

The Optic atrophy 1 protein (OPA1) is a key element in the dynamics and morphology of mitochondria. We demonstrated that the absence of IκB kinase-α, which is a key element of the nonclassical NF-κB pathway, has an impact on the mitochondrial network morphology and OPA1 expression. In contrast, the absence of NF-κB essential modulator (NEMO) or IκB kinase-β, both of which are essential for the canonical NF-κB pathway, has no impact on mitochondrial dynamics. Whereas Parkin has been reported to positively regulate the expression of OPA1 through NEMO, herein we found that PARK2 overexpression did not modify the expression of OPA1. PARK2 expression reduced the levels of Bax, and it prevented stress-induced cell death only in Bak-deficient mouse embryonic fibroblast cells. Collectively, our results point out a role of the nonclassical NF-κB pathway in the regulation of mitochondrial dynamics and OPA1 expression.Mitochondria perform multiple functions that are critical to the maintenance of cellular homeostasis. Mitochondrial dysfunctions have been linked to the development of degenerative diseases and aging. Damaged mitochondria are removed by mitophagy, a process partially regulated by the PARK2-encoded E3 ubiquitin ligase (Parkin) in a PTEN-induced putative protein kinase 1 (PINK1)-dependent manner.^{1, 2, 3, 4} During mitophagy, the phosphorylation of mitofusin (Mfn) 2 by PINK1 has been suggested to induce the recruitment of Parkin to the mitochondria in cardiomyocytes.⁵ However, previous groups have shown that that Mfn 1 and 2 are dispensable for Parkin-dependent mitophagy in fibroblasts, whereas the Parkin-dependent degradation of these proteins may impair fusion of damaged mitochondria with the healthy network.^{6, 7, 8} PINK1 and Parkin thus act as a quality control machinery on the outer mitochondrial membrane (OMM) to preserve mitochondrial integrity through the ubiquitination of OMM proteins.^{9, 10} Moreover, through its E3 ubiquitin ligase activity,^{11, 12} Parkin was reported to bind to the linear ubiquitin chain assembly complex (LUBAC) and to increase the ubiquitination of NF-κB essential modulator (NEMO),¹³ a component of the classical NF-κB signaling pathway.¹⁴ Müller–Rischart et al. also proposed that Parkin positively regulates the expression of the mitochondrial guanosine triphosphatase Optic atrophy 1 protein (OPA1) through linear ubiquitination of NEMO.¹³ OPA1 is a regulator of mitochondrial inner membrane fusion and cristae remodeling.^{15, 16, 17} A defect in OPA1 expression is associated with mitochondrial network fragmentation and enhanced sensitivity of the cells to undergo apoptosis by promoting cytochrome c release from the mitochondria.^{18, 19, 20} Because NEMO-deficient mouse embryonic fibroblast (MEF) cells display a normal mitochondrial network morphology, we decided to re-examine the role of Parkin in regulating OPA1 expression through the NF-κB signaling pathway.     

Metabolic regulation of mitochondrial dynamics

IntroductionAs the site for numerous biochemical processes—including oxidative phosphorylation (OXPHOS), the Krebs cycle, β-oxidation of fatty acids, calcium handling, and heme biosynthesis—the mitochondrion plays a central role in cellular metabolism. As a result, the dysfunction of mitochondria, particularly in their metabolic activities, has been associated with many disorders, including metabolic diseases, cancers, and neurodegenerative diseases, as well as the aging process (Carelli and Chan, 2014; Lightowlers et al., 2015).To maintain their health, mitochondria engage in several dynamic behaviors. The main dynamic activities are fusion (the joining of two organelles into one), fission (the division of a single organelle into two), transport (directed movement within a cell), and mitophagy (targeted destruction via the autophagic pathway; Fig. 1). From yeast to mammals, these dynamic behaviors have been shown to be clearly important in both normal physiology and disease states (Labbé et al., 2014; Mishra and Chan, 2014). In an early example, deletion of Fzo1p, a yeast GTPase essential for mitochondrial fusion, resulted in mitochondrial fragmentation, complete loss of mitochondrial DNA (mtDNA), impairment of OXPHOS activity, and inability to grow on nonfermentable carbon sources (Hermann et al., 1998).Open in a separate windowFigure 1.Overview of mitochondrial metabolism and dynamics. The mitochondrion is central to metabolism, being involved in the catabolism of numerous substrates, generation of metabolic signals, and sensing of metabolic cues. The processes diagrammed are not meant to be exhaustive, but to illustrate the diversity of biochemical pathways that impinge on the organelle. Mitochondria participate in macroscopic behaviors (termed dynamics) including fusion, fission, transport, and mitophagy. Although these behaviors are molecularly distinct from the organelle’s bioenergetic reactions, recent studies suggest that metabolism and dynamics are highly linked and regulate one another. ROS, reactive oxygen species.On the surface, these dynamic processes appear mechanistically distinct from the biochemical and metabolic processes occurring within the organelle. However, given the central role of mitochondria in bioenergetics, it is not surprising that in the last several years, multiple lines of evidence have emerged for a strong link between mitochondrial metabolism and dynamics. In this review, we discuss how metabolism regulates the key mitochondrial behaviors of fusion, fission, transport, and mitophagy.

Metabolic control of mitochondrial fusion

Mitochondrial fusion is an evolutionarily conserved process that, in mammals, is mediated by three large GTPases of the dynamin superfamily (Chan, 2012; Labbé et al., 2014): Mitofusin 1 (Mfn1), Mfn2, and Optic Atrophy 1 (Opa1). Because mitochondria have double membranes, mitochondrial fusion is a two-step process requiring outer-membrane fusion followed by inner-membrane fusion. Mfn1 and Mfn2 are integral outer-membrane proteins that mediate outer-membrane fusion, whereas OPA1 has multiple isoforms associated with the inner membrane and mediates inner-membrane fusion. Mitochondrial fusion events occur frequently in numerous cell types cultured in vitro, although fusion rates are cell type dependent and often occur less frequently in tissues (Pham et al., 2012; Eisner et al., 2014). Because the balance between fusion and fission controls mitochondrial morphology, genetic deletion of the fusion genes results in severe fragmentation of the mitochondrial network and abolishes content exchange between mitochondria (Hermann et al., 1998; Chen et al., 2003, 2005). In humans, mutations in Mfn2 cause Charcot–Marie–Tooth disease type 2A, a peripheral neuropathy affecting long motor and sensory neurons (Züchner et al., 2004). Mutations in Opa1 cause dominant optic atrophy, a blindness caused by degeneration of retinal ganglion cells (Alexander et al., 2000; Delettre et al., 2000, 2002).The fusion process is well known to be important for OXPHOS activity, particularly through the regulation of mtDNA levels. The sensitivity of cells to reduced mitochondrial fusion is context dependent. For example, mouse embryonic fibroblasts can tolerate a partial defect in mitochondrial fusion, such as loss of either Mfn1 or Mfn2, without much bioenergetic consequence. However, cerebellar Purkinje neurons cannot survive Mfn2 removal, because of loss of respiratory chain activity (Chen et al., 2007). Moreover, complete loss of mitochondrial fusion caused by removal of both mitofusins or Opa1 results in a dramatic decrease in mtDNA content, heterogeneous loss of mtDNA nucleoids and membrane potential, and reduced respiratory chain function in both cultured cells and mouse tissues (Chen et al., 2005, 2010). Other mechanisms also link these proteins with metabolism: Mfn2 maintains coenzyme Q levels (Mourier et al., 2015), and Opa1 maintains mitochondrial cristae structure and is critical for respiratory chain supercomplex assembly (Cogliati et al., 2013).The energetic states of cells are often associated with specific mitochondrial morphologies. In yeast, nonfermentable culture conditions that force increased OXPHOS activity are accompanied by elongation of the mitochondrial network (Egner et al., 2002; Jakobs et al., 2003). An analogous study with human cells suggested that mitochondria elongate during growth in galactose media, which forces cells to rely more heavily on OXPHOS for ATP production (Rossignol et al., 2004). Elongated mitochondria have also been observed in other conditions associated with increased ATP production (Mitra et al., 2009; Tondera et al., 2009). These observations suggest that high OXPHOS activity correlates with mitochondrial elongation and is consistent with the proposal that elongated mitochondrial networks are more efficient at energy generation and capable of distributing energy through long distances (Amchenkova et al., 1988; Skulachev, 2001).Another possibility, not mutually exclusive, is that increased OXPHOS activity stimulates mitochondrial fusion to cause elongation. The development of in vitro fusion assays using isolated organelles (Meeusen et al., 2004; Hoppins et al., 2011) allowed for more detailed investigations regarding the regulation of mitochondrial fusion. In isolated organelles, addition of respiratory chain substrates that promoted OXPHOS activity led to stimulation of mitochondrial inner-membrane fusion, whereas outer-membrane fusion was unaffected by the metabolic state (Mishra et al., 2014). OXPHOS activity stimulates the metalloprotease Yme1L to proteolytically process Opa1, leading to activation of its fusion activity.Opa1 is expressed as a membrane-integrated long form, which can then be cleaved to a soluble short form by two distinct metalloproteases, the ATP-dependent protease Yme1L and the membrane potential–dependent protease Oma1. It has been well known that the presence of both long and short forms correlates with fusion-competent mitochondria (McQuibban et al., 2003; Song et al., 2007). In mitochondrial fusion intermediates that have undergone outer membrane fusion, proteolytic processing of Opa1 at the Yme1L or Oma1 cleavage site was sufficient to stimulate inner-membrane fusion (Mishra et al., 2014). The processing of Opa1 by two metalloproteases allows differential regulation of inner-membrane fusion. Proteolysis via Yme1L is responsible for OXPHOS-dependent stimulation of inner-membrane fusion (Fig. 2). In contrast, when membrane potential is dissipated, the long isoform of Opa1 is completely cleaved and inactivated (Fig. 2; Ishihara et al., 2006) owing to activation of Oma1 (Ehses et al., 2009; Head et al., 2009). A variety of cellular stresses can activate Oma1 to cleave Opa1 (Baker et al., 2014). This mechanism likely contributes to the mitochondrial fragmentation found in many forms of mitochondrial dysfunction (Duvezin-Caubet et al., 2006). Whereas large-scale depolarization of mitochondria clearly inactivates Opa1, transient depolarizations of mitochondria may partially activate Oma1 and be pro-fusogenic. Transient mitochondrial depolarizations have been reported to occur in cultured cells and are associated with fusion events (Santo-Domingo et al., 2013). It should be noted that cells lacking Yme1L and Oma1 do retain residual mitochondrial fusion activity (Anand et al., 2014) and more work will be needed to dissect how inner-membrane fusion is regulated in this situation.Open in a separate windowFigure 2.Metabolic regulation of mitochondrial fusion. Mitochondrial fusion consists of outer membrane fusion, mediated by mitofusins, followed by inner membrane fusion, mediated by Opa1. Modes of regulation include the following: (1) Oxidative stress and high levels of oxidized glutathione (GSSG) promote trans complexes of mitofusins, facilitated by disulfide bonds (red bars), leading to organelle tethering and enhanced outer-membrane fusion. (2) Inner-membrane fusion is stimulated by OXPHOS activity, which enhances Yme1L-mediated proteolytic processing of Opa1 from the long form to the soluble short form. In isolated organelles, Opa1 proteolysis is necessary and sufficient to activate inner-membrane fusion. (3) Enhanced ATP levels are potentially linked to GTP-loading of Opa1 via the nucleotide diphosphate kinase NM23-H4. GTP loading and hydrolysis by Opa1 are required for inner-membrane fusion. (4) Metabolic stresses, including loss of membrane potential, activate the inner membrane protease Oma1 and result in complete proteolytic processing of Opa1. Short forms of Opa1, by themselves, are inactive for inner-membrane fusion.Together, these studies suggest that the inner membrane proteases Yme1L and Oma1 serve as important sensors to link metabolic conditions to the inner-membrane fusion machinery. In particular, conditions that increase mitochondrial ATP function lead to enhanced fusion, whereas metabolic signals that grossly uncouple the mitochondria result in fusion inhibition. These regulatory modes appear to be in play in normal physiology and disease. In skeletal muscle, the more oxidative fiber types have enhanced mitochondrial fusion, presumably promoting health of the active mitochondrial population (Mishra et al., 2015). In mtDNA disease, defects in OXPHOS result in secondary defects in mitochondrial inner-membrane fusion (Mishra et al., 2014). This regulatory process may help to segregate dysfunctional mitochondria and prevent the spread of mtDNA mutations to wild-type mitochondria. In the case of skeletal muscle, the linkage of fusion to OXPHOS activity may serve to restrict mtDNA defects to a localized region of the muscle fiber, a phenomenon observed in older individuals and some patients with mitochondrial myopathy (Moraes et al., 1992; Elson et al., 2002; Bua et al., 2006). In addition, the inability of defective mitochondria to fuse with the remainder of the organelle population provides a means to segregate mutant organelles into small components ideal for autophagic destruction (Twig et al., 2008), as discussed later.Other metabolic mechanisms to regulate mitochondrial fusion have also been proposed (Fig. 2). Oxidative stress can enhance fusion in both isolated organelles and cells. In this situation, elevated levels of oxidized glutathione promote disulfide-mediated dimerization of mitofusin molecules and organelle tethering, the first step in the fusion process (Shutt et al., 2012). Mfn1 is also regulated by phosphorylation by extracellular signal–regulated kinase, linking the MAPK pathway to mitochondrial fusion (Pyakurel et al., 2015). Other studies have suggested that fusion may be controlled via the local concentration of GTP. NM23-H4 is a mitochondrially localized nucleotide disphosphate kinase that can generate GTP (from GDP) in the presence of ATP. Interestingly, NM23-H4 can promote GTP loading onto Opa1, and knockdown of NM23-H4 leads to fusion defects reminiscent of Opa1 knockdown (Boissan et al., 2014). It remains possible that this mechanism allows cellular ATP levels to be linked to fusion via GTP-loading of Opa1. Thus, multiple steps of the fusion process may be independently targeted via distinct regulatory mechanisms, potentially providing an exquisite level of control over the fusion event (Fig. 2).

Metabolic control of mitochondrial fission

As a complement to fusion, fission of mitochondria is equally critical for cellular and organismal physiology (Chan, 2012; Labbé et al., 2014). Division of mitochondria is mediated by Dynamin-related Protein 1 (Drp1), a large GTPase that is recruited to the mitochondrial outer membrane via a collection of receptor proteins (Mff, Fis1, MiD49, and MiD50). Once on mitochondria, Drp1 assembles around the tubule and constricts it in a GTP-dependent manner to mediate scission. Besides influencing mitochondrial morphology, fission has been implicated in multiple functions, including the facilitation of mitochondrial transport, mitophagy, and apoptosis. In humans, two clinical studies have linked Drp1 mutation to microcephaly, neonatal lethality (Waterham et al., 2007), and refractory epilepsy (Vanstone et al., 2015), and another has linked Mff mutation to two cases of developmental delay with neuromuscular dysfunction (Shamseldin et al., 2012).Perhaps the best-known regulatory mechanism for mitochondrial fission involves phosphorylation of Drp1. Multiple phosphorylation sites and kinases have been identified, and many of these events are linked to signaling pathways activated by metabolic events. In addition, phosphorylation can activate or inhibit Drp1, depending on the site involved. In this review, we focus on Drp1 phosphorylation, although it should be noted that Drp1 regulation is complex, and several other Drp1 posttranslational mechanisms have been identified, including S-nitrosylation, SUMOylation, and acetylation.Studies on Drp1 phosphorylation have centered on two critical sites. Because studies designate these sites differently depending on the species studied, we consolidate the findings and refer to the sites as serine 616 (S616) and S637 based on the sequence of human Drp1, isoform 1. Protein kinase A (PKA) phosphorylation of Drp1 at S637 has been clearly shown to inhibit its activity in vitro, promoting overall elongation of the mitochondrial network in response to pharmacologic activation (e.g., forskolin), β-adrenergic stimulation, or forced exercise (Fig. 3; Chang and Blackstone, 2007; Cribbs and Strack, 2007). Phosphorylation at S637 is counteracted by the phosphatases calcineurin (Cribbs and Strack, 2007; Cereghetti et al., 2008) and PP2A/Bβ2 (Dickey and Strack, 2011). Negative regulation of Drp1 by phosphorylation at S637 also occurs during mTOR inhibition and nitrogen starvation, which increases cAMP levels and activates PKA (Gomes et al., 2011; Rambold et al., 2011). Even though autophagy is activated during starvation, inhibition of Drp1 results in enhanced mitochondrial tubulation that promotes mitochondrial ATP production and spares the organelles from degradation, because of their increased size.Open in a separate windowFigure 3.Metabolic regulation of mitochondrial fission. Fission is mediated by the master regulator Drp1, which must be recruited from a cytosolic pool onto the mitochondrial surface. Receptor proteins on the outer membrane are required for Drp1 recruitment and activation of fission. For simplicity, only two receptor proteins, Mff and MiD51, are shown. Four modes of regulation are color-coded in the diagram: (1) Exercise and nitrogen starvation result in PKA activation, followed by phosphorylation of Drp1 at Ser637, which is inhibitory for fission because of sequestration of Drp1 in the cytosol. (2) Reversal of phosphoS637 can be achieved via calcineurin, which is activated by metabolic uncoupling of the organelle. These events lead to recruitment of Drp1 and rapid activation of fission. (3) Cold exposure and oncogenic Ras^G12V activate fission via Ser616 phosphorylation by PKA or MAPK, respectively. (4) Severe energy depletion can potentially activate fission via elevation of ADP and AMP levels. ADP binding to the MiD51 receptor is necessary for Drp1 recruitment and fission. AMP-sensing by AMPK results in phosphorylated Mff and activated fission.In contrast, drug treatments that inhibit mitochondrial OXPHOS are generally associated with enhancement of fission. The most commonly used are the mitochondrial uncouplers (e.g., CCCP and FCCP), which result in a rapid and dramatic fragmentation of the organellar network in multiple cell types. As noted, uncouplers stimulate Oma1 to cleave Opa1, resulting in inactivation of mitochondrial fusion. On the fission side, dephosphorylation at Drp1 serine 637 via the Ca²⁺-dependent phosphatase calcineurin promotes Drp1 activation and recruitment to the mitochondrial surface (Fig. 3; Cribbs and Strack, 2007; Cereghetti et al., 2008). Calcineurin therefore relays metabolic stimuli associated with calcium changes into alterations in mitochondrial morphology. For example, dysfunction of the calcium-buffering activity of mitochondria would increase cytosolic calcium levels and potentially trigger calcineurin-mediated mitochondrial fission. In addition, mice with calcineurin knocked out in skeletal muscle have recently been shown to exhibit elongated mitochondria, increased respiratory chain activity, resistance to obesity under a high-fat diet, and diminished exercise performance (Pfluger et al., 2015).In brown adipose tissue, thermogenesis has been shown to involve activation of Drp1 and mitochondrial fission. In this cell type, cold exposure results in the oxidation of fatty acids in uncoupled mitochondria to increase heat production, as opposed to ATP synthesis. Intriguingly, the norepinephrine-based signaling events initiated by cold exposure result in PKA activation and Drp1 phosphorylation at S616, which activates mitochondrial fission (Fig. 3; Wikstrom et al., 2014). Through unclear mechanisms, the fission event promotes enhanced uncoupling and sensitivity to fatty acids, thereby aiding heat generation. Thus, mechanisms to enhance fission may decrease OXPHOS efficiency and be useful in times of nutrient excess.Drp1 phosphorylation at S616 has also been implicated in tumorigenesis by oncogenic Ras (Fig. 3; Kashatus et al., 2015; Serasinghe et al., 2015). Up-regulation of the MAPK pathway by Ras induces Erk1-mediated phosphorylation of Drp1 at S616, which results in enhanced mitochondrial fission. Remarkably, inhibition of Drp1 function attenuates the oncogenic activity of Ras in cell and xenograft models of tumorigenesis. In future studies, it will be interesting to determine whether mitochondrial dynamics play an important role in tumor cell metabolism.Finally, regulation of fission may also occur at the level of the Drp1 receptor proteins. In cultured cells, Mff appears to be the primary receptor, because loss of Mff results in dramatic mitochondrial elongation (Gandre-Babbe and van der Bliek, 2008; Otera et al., 2010; Losón et al., 2013). Recent studies show that Mff is a phosphorylation substrate for AMP kinase (AMPK; Ducommun et al., 2015; Toyoma et al., 2016). This phosphorylation event activates Mff and mitochondrial fission, explaining how AMPK links energy deficiency to mitochondrial fragmentation (Fig. 3; Toyoma et al., 2016).Along similar lines, recent crystal structures of the receptor protein MiD51 suggest a potential role for metabolic regulation (Losón et al., 2014; Richter et al., 2014). The cytosolic domain adopts an enzymatically dead nucleotidyltransferase fold, which contains a high-affinity binding site for the dinucleotides ADP and GDP. Indeed, dinucleotide binding appears to be required for receptor function, and the protein potentially serves as a sensor for ADP levels. Thus, MiD51 potentially links metabolic conditions to enhanced organelle fission (Fig. 3). Intriguingly, the paralogous receptor MiD49 does not contain this mode of regulation, as it does not bind nucleotides despite a similar fold and the presence of a binding pocket (Losón et al., 2015). It is unknown whether MiD49 binds an alternative class of ligands that regulates its activity.

Metabolic control of mitochondrial transport

Because of their ability to affect local ATP and calcium concentrations, the subcellular distribution of mitochondria can be extremely important. In a dramatic example, the mitochondria of the sperm cell are concentrated in the proximal region of the flagellum, where they appear ideally positioned to supply ATP for the force-generating motor proteins that drive sperm movement (Woolley, 1970). In a more dynamic example, the mitochondria of neurons can traffic to the axon terminals of neurons to fuel energy-consuming processes such as synaptic vesicle recycling. In many mammalian cells, mitochondrial transport is accomplished via the activity of motor proteins working along the microtubule network, although transport along other cytoskeletal elements can also occur (Saxton and Hollenbeck, 2012). Kinesin motor proteins, particularly the kinesin-1 family, mediate transport in the positive (or anterograde) direction, whereas dyneins mediate transport in the negative (or retrograde) direction (Pilling et al., 2006). The motor proteins are connected to the mitochondrial surface through receptor and adaptor proteins, whose functions were first revealed in Drosophila melanogaster neurons (Stowers et al., 2002; Guo et al., 2005). In mammals, the primary receptor proteins are Miro1 and Miro2, transmembrane GTPases localized in the mitochondrial outer membrane. The Miro proteins interact with kinesin via the Milton proteins, also known as Trak1 and Trak2. Together, the Miro–Milton–kinesin complex mediates anterograde transport of mitochondria along microtubules.Regulation of mitochondrial transport is particularly important in neurons, and experiments in the neuronal system have been fruitful in identifying the relevant mechanisms. Mitochondrial transport behavior in axons is complex and involves a balance between movement and stalling. In axonal segments, a substantial fraction of the mitochondria is immobile at any given time. High calcium levels tend to cause pausing of mitochondria in axons, a phenomenon that may help to retain mitochondria at active sites along the axon. Because Miro contains regulatory EF-hands that bind calcium, it is ideally suited to sense local calcium levels near the mitochondrion. Multiple mechanistic models have been proposed, but they all center on conformational changes triggered by calcium binding to Miro (Macaskill et al., 2009; Wang and Schwarz, 2009; Chen and Sheng, 2013). In one model, high calcium causes the Miro–Milton–kinesin complex to be released from the microtubule (Fig. 4 A; Wang and Schwarz, 2009). In another model, high calcium causes the Miro–Milton complex to release kinesin, thereby freeing mitochondria from the microtubule (Macaskill et al., 2009). In the third, most recent model, mitochondrial pausing in axons is mediated by the protein syntaphilin, which serves to anchor axonal mitochondria to microtubules (Kang et al., 2008; Chen and Sheng, 2013). With high local calcium, kinesin is released by the Miro–Milton complex and binds syntaphilin, which substantially reduces its motor activity (Chen and Sheng, 2013).Open in a separate windowFigure 4.Metabolic regulation of mitochondrial transport and mitophagy. (A) In mammals, mitochondrial transport is primarily mediated by microtubule-dependent motors, such as kinesin for anterograde movement. Kinesin-1 attaches to mitochondria via its adaptor (Milton) and receptor (Miro). The Miro–Milton–kinesin complex allows for organelle movement under basal conditions. (1) At active synapses of neurons, increased Ca²⁺ levels result in pausing of mitochondria to supply local ATP to drive energy-intensive processes such as synaptic vesicle recycling. Depending on the model, Ca²⁺ loading of the EF-hands of Miro is followed by either release of the Miro–Milton–kinesin complex from the microtubule or anchoring of the mitochondrion via syntaphilin. (2) Elevated glucose levels also promote stalling, caused by O-GlcNAc transferase (OGT)-mediated glycosylation of Milton. Although glycosylated Milton is depicted in the syntaphilin model, this is for convenience; the precise method by which glycosylation of Milton mediates stalling is unclear. This regulatory pathway may allow mitochondria to be positioned at locations of nutrient abundance, increasing their efficiency of ATP generation. (B) Multiple mechanisms for regulation of mitophagy have been proposed: (1) Mitochondrial damage leading to loss of membrane potential (ΔΨ_m) causes Pink1 accumulation (not depicted), followed by Parkin recruitment and ubiquitination of multiple outer membrane proteins. These events activate the outer membrane for processing via the proteasome system (UPS), followed by targeting to autophagosome membranes. (2) Severe energy depletion leads to activation of AMPK, followed by phosphorylation and activation of the autophagy regulator ULK1. ULK1 is then able to activate generalized autophagy, including mitophagy. (3) Hypoxia is able to activate mitophagy via the dephosphorylation of FUNDC1 (on the outer membrane) by the PGAM5 phosphatase. Dephosphorylated FUNDC1 serves to recruit LC3 and autophagosomal membranes. (4) Through unknown mechanisms, enhanced OXPHOS activity in the mitochondrion recruits the autophagy regulator Rheb to the outer membrane receptor Nix. Mitochondrially-localized Rheb then promotes autophagy via recruitment of LC3 molecules.Other mechanisms have been suggested to directly link mitochondrial transport to energy status. ATP depletion or hypoxia promotes anterograde mitochondrial movement into axons (Mironov, 2007; Li et al., 2009; Tao et al., 2014) via activation of AMPK or HIF-1α pathways. Alternatively, nutrient status has also been implicated in direct control of organelle transport. Milton interacts with and serves as a substrate for O-GlcNAc transferase, which glycosylates the adaptor at several residues. In the presence of high glucose, glycosylation of Milton results in immobilization of the mitochondria, although the precise mechanism is still unclear (Fig. 4 A; Pekkurnaz et al., 2014). This posttranslational modification may serve to enrich mitochondria at locations with high nutrients, promoting increased efficiency of ATP production.

Metabolic control of mitophagy

The overall mitochondrial mass within a cell is likely regulated by a balance between biogenesis and degradation. When mitochondria are excessive, or become aged or defective, organelle clearance is thought to occur primarily through autophagy, a process termed mitophagy. The removal of mitochondria can be either random or selective. During bulk autophagy, mitochondrial degradation is included as part of a generalized autophagy program activated by the metabolic state of the cell. In other cases, mitophagy is a culling process that selectively degrades only defective mitochondria, thereby maintaining the overall health of the mitochondrial population.Although there is widespread interest in mitophagy as a potential quality control process for mitochondria, it should be noted that in vivo evidence for the importance of mitophagy in mitochondrial homeostasis remains sparse. In particular, studies on the Pink1/Parkin system (described in the next paragraph) have usually relied on overexpressing Parkin and stressing the cells with an uncoupler. Although such approaches are extremely valuable in dissecting the biochemical pathway, further studies are required to determine the in vivo function of mitophagy. The recent development of a mouse reporter for tracking mitophagy in vivo (Sun et al., 2015) will be helpful in this regard.The best-studied mitophagy pathway involves Pink1 and Parkin, genes responsible for some cases of familial Parkinson’s disease. Pink1, a mitochondrially localized kinase, is normally imported and degraded within the organelle. Because protein import is dependent on mitochondrial membrane potential, depolarization results in accumulation of Pink1 on the outer membrane (Matsuda et al., 2010; Narendra et al., 2010). The accumulated Pink1 phosphorylates numerous proteins, including ubiquitin, to recruit and activate Parkin, an E3 ligase (Okatsu et al., 2015). Activated Parkin results in widespread ubiquitination of mitochondrial outer-membrane proteins, whose degradation by the 26S proteasome (Chan et al., 2011; Sarraf et al., 2013) is required for targeting of the mitochondrion to autophagic membranes. Because Parkin is selectively enriched on dysfunctional mitochondria, healthy organelles are spared from autophagic degradation. Pink1 and Parkin have also been implicated in another mitochondrial quality control pathway, distinct from autophagy, in which vesicles bud off from mitochondria and are trafficked to the late endosome and lysosome (McLelland et al., 2014; Sugiura et al., 2014).Mitophagy can be activated under certain cellular stresses. With energy stress, activation of AMPK results in phosphorylation of ULK1 and ULK2, mammalian protein kinases that are orthologues of the autophagy gene ATG1 (Egan et al., 2011). ULK1 and ULK2 promote autophagy, including the degradation of mitochondria. AMPK also inhibits the growth-promoting mTORC pathway, which normally inhibits ULK function. These interlinked mechanisms couple mitophagy to the nutrient status of the cell.Hypoxic conditions are also able to trigger mitophagy via a distinct pathway. Activation of the mitochondrial phosphatase PGAM5 results in dephosphorylation of the mitochondrial autophagy receptor, FUNDC1. The dephosphorylation event promotes the interaction of FUNDC1 with ATG8 (also known as LC3), stimulating formation of the autophagic membrane (Liu et al., 2012; Chen et al., 2014). It is not clear how hypoxia activates PGAM5, and whether this mechanism is selective for individual mitochondria.Finally, metabolic conditions that promote increased mitochondrial function are also associated with increased mitophagy (Melser et al., 2013). Under glucose-free (oxidative) conditions, mitochondrial OXPHOS is up-regulated, and bulk mitophagy is also enhanced. The small GTPase Rheb is proposed to be involved, as it partially localizes to the outer mitochondrial membrane under oxidative conditions and interacts with the mitochondrial autophagy receptor, Nix. The relocalization of Rheb promotes the recruitment of LC3 molecules, thereby enhancing mitophagy. The molecular signals that recruit Rheb are currently unclear. The promotion of mitophagy during oxidative conditions, when mitochondrial function is increased, appears to contrast with previous mitophagy models in which dysfunctional organelles are cleared. However, enhanced respiratory chain activity may promote damage to mitochondria (e.g., through increased production of reactive oxygen species), and it is possible that Rheb is specifically responding to these damaged mitochondria. Alternatively, this mechanism may be in place to increase bulk turnover of the population during conditions of increased functional demand. In either case, the increased mitophagic flux promotes overall energetic efficiency of the organelle population.

Future directions

It is becoming clear that the multiple functions and behaviors of the mitochondrion do not operate independently but instead influence each other and are subject to common regulatory pathways. For instance, depolarization of the organelle triggers fission, inhibits fusion, and promotes mitophagy, whereas hypoxia promotes transport, mitophagy, and fission. Predicting how the cell integrates multiple signals to regulate mitochondrial function is therefore complex and dependent on the specifics of the stimuli, as well as the cell type. It is clear, however, that the organelle is responsive to numerous types of metabolic stimuli, and this likely has resulting effects on the health and function of the organelle population. A future challenge will be to integrate the data from numerous studies, functions, and perturbations to further our understanding of the regulatory biology of mitochondria and its implication in normal physiology and disease states. Understanding how to regulate mitochondrial behavior may provide therapeutic approaches to modulate mitochondrial physiology in diseased states. We have only begun to investigate a few aspects of the dynamic behavior of mitochondria. Additional properties, such as its interaction with other organelles, including lipid droplets and the endoplasmic reticulum, are only beginning to be understood and likely will have clear implications on overall cellular function.     

Mitofusin 1 is degraded at G<Subscript>2</Subscript>/M phase through ubiquitylation by MARCH5

Background

Mitochondria exhibit a dynamic morphology in cells and their biogenesis and function are integrated with the nuclear cell cycle. In mitotic cells, the filamentous network structure of mitochondria takes on a fragmented form. To date, however, whether mitochondrial fusion activity is regulated in mitosis has yet to be elucidated.

Findings

Here, we report that mitochondria were found to be fragmented in G₂ phase prior to mitotic entry. Mitofusin 1 (Mfn1), a mitochondrial fusion protein, interacted with cyclin B1, and their interactions became stronger in G₂/M phase. In addition, MARCH5, a mitochondrial E3 ubiquitin ligase, reduced Mfn1 levels and the MARCH5-mediated Mfn1 ubiquitylation were enhanced in G₂/M phase.

Conclusions

Mfn1 is degraded through the MARCH5-mediated ubiquitylation in G₂/M phase and the cell cycle-dependent degradation of Mfn1 could be facilitated by interaction with cyclin B1/Cdk1 complexes.

     

Hypochlorous Acid Converts the ��-Glutamyl Group of Glutathione Disulfide to 5-Hydroxybutyrolactam, a Potential Marker for Neutrophil Activation

In healthy cells, glutathione disulfide (GSSG) is rapidly reduced back to glutathione (GSH) by glutathione reductase to maintain redox status. The ratio of GSH/GSSG has been used as an indicator of oxidative stress. However, hypochlorous acid (HOCl) generated by the myeloperoxidase-H₂O₂-Cl⁻ system of neutrophils converts GSH to irreversible oxidation products. Although several such products have been identified, yields of these compounds are very low in biological systems, and they cannot account quantitatively for thiol loss. In the current studies, we use liquid chromatography-mass spectrometry (LC-MS) to demonstrate that HOCl and chloramines oxidize GSSG to two irreversible products in high yield. The products, termed M-45 and M-90, are, respectively, 45 or 90 atomic mass units lighter than GSSG. The reaction pathway involves chloramine and aldehyde intermediates, and converts the γ-glutamyl residues of GSSG to 5-hydroxybutyrolactam. Importantly, M-45 and M-90 were resistant to reduction by glutathione reductase. Moreover, the monohydroxylbutyrolactam M-45 accounted for >90% of the endogenous GSH oxidation products generated by activated neutrophils. Because the reaction pathway involves chlorinating intermediates, hydroxylbutyrolactams are likely to be specific products of HOCl, which is generated only by myeloperoxidase. Therefore, our observations implicate M-45 as a potential biomarker for myeloperoxidase activity in vivo.Glutathione (GSH), a tripeptide synthesized in the cytosol from glutamate, cysteine, and glycine, is the predominant antioxidant in mammalian cells. Its concentration ranges from millimolar inside cells to micromolar in plasma (1, 2). In many cells, GSH accounts for >90% of total nonprotein thiol (3, 4). The free thiol group in GSH is responsible for biological activity. As a nucleophilic scavenger, GSH can directly react with electrophilic substances, such as reactive oxygen/nitrogen species, or be oxidized by GSH peroxidase to glutathione disulfide (GSSG). Therefore, it is essential for maintaining intracellular redox status and defending against oxidative injury. Under normal circumstances, GSSG is rapidly reduced back to GSH by glutathione reductase and NADPH. Thus, most of the GSH remains in the reduced form. Under oxidative stress, however, GSH is converted to GSSG, which potentially accumulates (2, 5). Indeed, the GSH/GSSG ratio has been used to evaluate oxidative stress in biological systems. Alterations of this ratio associate with a variety of diseases, including atherosclerosis, cancer, and human immunodeficiency virus infection (6–10).One important source of oxidative stress in humans is myeloperoxidase (MPO),² a heme protein expressed by neutrophils, monocytes, and certain populations of macrophages (11–13). Activation of these inflammatory white blood cells results in the secretion of MPO, which uses hydrogen peroxide (H₂O₂, produced by NADPH oxidase) and chloride anion to generate hypochlorous acid (HOCl) (14). HOCl rapidly reacts with a wide range of functional groups (15–19). At physiological pH, thiol groups and free amino groups are its main targets, and the initial products are oxidized thiols and chloramines.HOCl generates other products in addition to GSSG when it reacts with GSH. Chesney et al. (20) suggested that it oxidizes GSH to a higher oxidation state than the disulfide form because the molar ratio of HOCl consumed to GSH oxidized was 4:1 instead of 1:1 in Escherichia coli. Winterbourn (21) reported that approximately half of the GSH oxidized by HOCl could not be regenerated. These researchers have identified glutathione sulfonamide (GSA), glutathione thiosulfonate, and dehydroglutathione as irreversible higher oxidation products (22, 23). Their observations suggest that the formation of higher order GSSG oxidation products might account in part for the irreversible loss of GSH induced by HOCl. However, activated neutrophils (the source of MPO and therefore of HOCl) generate only low yields of these higher oxidation products, suggesting that the major products of GSH oxidation by MPO remain to be identified.The disulfide and α-amino groups of GSSG are also potential targets of HOCl (17). Disulfides can be oxidized to sulfonic acid via a sulfenyl chloride intermediate (16). α-Amino groups yield chloramines, which undergo decarboxylation, intramolecular H-abstraction, or other reaction pathways to form various products, such as aldehydes and carboxymethyllysine (16, 24). These reactions may be biologically relevant, because carboxymethyllysine production is impaired in mice deficient in the phagocyte NADPH oxidase (25). These observations suggest that GSSG is a potential scavenger of HOCl. Indeed, GSSG reportedly competes for HOCl with its rate constant expected to be 2 × 10⁵ m⁻¹ s⁻¹ (26, 27). Studies from Bast et al. (28) demonstrated that GSSG protects acetylcholinesterase from oxidative inactivation by HOCl. Nagy and Ashby (29) studied the kinetics and mechanism of GSSG oxidation by HOCl. They proposed that HOCl generates the bis-N-chloro-γ-l-glutamyl derivative of GSSG. These studies suggest that GSSG itself may function as an antioxidant.In the current study, we investigated the reaction of GSSG with HOCl and other oxidants. Using liquid chromatography in concert with mass spectrometry (LC-MS), we identified two groups of novel oxidation products, which we termed M-45 and M-90. We characterized their structures and potential reaction pathways. Our results indicate that HOCl and chloramines oxidize the γ-glutamyl moiety of GSSG to 5-hydroxybutyrolactam in high yield.     

Sirtuins and ageing—new findings

Sirtuins are a promising avenue for orally administered drugs that might deliver the anti-aging benefits normally provided by calorie restriction.Calorie (or dietary) restriction was first shown to extend rodent lifespan almost 80 years ago, and remains the most robust longevity-promoting intervention in mammals, genetic or dietary. Sirtuins are NAD-dependent deacylases homologous to yeast Sir2p and were first shown to extend replicative lifespan in budding yeast [1]. Because of their NAD requirement, sirtuins were proposed as mediators of the anti-ageing effects of calorie restriction [1]. Indeed, many studies in yeast, Caenorhabditis elegans, Drosophila melanogaster and mice have supported these ideas [2]. However, a 2011 paper posed a challenge: transgenic strains of C. elegans and Drosophila that overexpress SIR2 were found not to be long-lived [3].Rather than review the extensive sirtuin literature previous to that paper, I focus on a few key studies that have followed it, which underscore a conserved role of sirtuins in slowing ageing. In the first study, two highly divergent budding yeast strains—a lab strain and a clinical isolate—were crossed. A genome-wide quantitative trait locus analysis was then performed to map genes that determine differences in replicative lifespan [4]. The top hit was SIR2, explaining more than one-half of the difference in replicative lifespan between the two strains (due to five codon differences between the SIR2 alleles). In Drosophila, overexpression of dSIR2 in the fat body extended the lifespan of flies on the normal diet, whereas deletion of dSIR2 in the fat body abolished the extension of lifespan by a calorie-restriction-like protocol [5]. This example illustrates the key role of dSIR2 in lifespan determination and its central role in mediating dietary effects on longevity, discussed further below. Another study showed that two transgenic mouse lines that overexpress the mammalian SIRT6—mammals have seven sirtuins—had significantly extended lifespans [6]. Finally, a recent study clearly showed that worm sir2.1 could extend lifespan by regulating two distinct longevity pathways involving insulin-like signalling and the mitochondrial unfolded protein response [7]. All told, this body of work supports the original proposal that sirtuins are conserved mediators of longevity.Many other studies also illustrate that sirtuins can mediate the effects of diet. As an example, calorie restriction completely protected against ageing-induced hearing loss in wild type but not SIRT3^−/− mice [8]. The mitochondrial sirtuin SIRT3 thus helps to protect the neurons of the inner ear against oxidative damage during calorie restriction. Of course, these studies do not imply that sirtuins are the only mediators of calorie restriction effects, but they do indicate that they must be central components.Finally, what about the translational potential of this research, namely using putative SIRT1-activating compounds—resveratrol and newer, synthetic STACs? Two new studies provide strong evidence that the effects of these compounds really do occur through SIRT1. First, acute deletion of SIRT1 in adult mice prevented many of the physiological effects of resveratrol and other STACs [9]. Second, a single mutation adjacent to the SIRT1 catalytic domain abolished the ability of STACs to activate the enzyme in vitro, or to promote the canonical physiological changes in vivo [10].In summary, sirtuins seem to represent a promising avenue by which orally available drugs might deliver anti-ageing benefits normally triggered by calorie restriction. Indeed, the biology of sirtuins is complex and diverse, but this is an indication of their deep reach into key disease processes. Connections between sirtuins and cancer metabolism are but one new example of this. The future path of discovery promises to be exciting and might lead to new drugs that maintain robust health.     

Synaptic dysfunction,memory deficits and hippocampal atrophy due to ablation of mitochondrial fission in adult forebrain neurons

Well-balanced mitochondrial fission and fusion processes are essential for nervous system development. Loss of function of the main mitochondrial fission mediator, dynamin-related protein 1 (Drp1), is lethal early during embryonic development or around birth, but the role of mitochondrial fission in adult neurons remains unclear. Here we show that inducible Drp1 ablation in neurons of the adult mouse forebrain results in progressive, neuronal subtype-specific alterations of mitochondrial morphology in the hippocampus that are marginally responsive to antioxidant treatment. Furthermore, DRP1 loss affects synaptic transmission and memory function. Although these changes culminate in hippocampal atrophy, they are not sufficient to cause neuronal cell death within 10 weeks of genetic Drp1 ablation. Collectively, our in vivo observations clarify the role of mitochondrial fission in neurons, demonstrating that Drp1 ablation in adult forebrain neurons compromises critical neuronal functions without causing overt neurodegeneration.In addition to their crucial importance in energy conversion, mitochondria serve many other housekeeping functions, including calcium buffering, amino-acid and steroid biosynthesis as well as fatty acids beta-oxidation and regulation of cell death. During the past decade, it has become increasingly clear that processes regulating mitochondrial morphology and ultrastructure are influenced by specific cellular requirements upon which mitochondria, in a precisely regulated manner, undergo fusion and division events.¹ Maintaining this balance is especially important for highly energy-consuming, polarized cells such as neurons, where single organellar units sprouting from the mitochondrial network are transported along the cytoskeleton into dendrites and spines to meet local energy requirements.² In addition, elaborate quality-control mechanisms also rely on mitochondrial dynamics: whereas defective organelles are sequestered by fission, enabling their removal from the mitochondrial network,^{3, 4} fusion supports qualitative homogeneity of the syncytium through complementation.⁵Mitochondrial fusion and fission are mediated by large GTPases of the dynamin superfamily.⁶ The outer mitochondrial membrane mitofusins 1 (MFN1) and 2 (MFN2) tether mitochondrial membranes by homodimer or heterodimer formation,⁷ thereby initiating fusion of the organelles, a process that also involves the inner mitochondrial membrane-associated GTPase Optic Atrophy 1.⁸ In addition, MFN2 also mediates contacts between mitochondria and endoplasmic reticulum.⁹ The only known mammalian mitochondrial fission protein, Dynamin-Related Protein 1 (Drp1), translocates upon dephosphorylation by calcineurin¹⁰ to fission sites where it binds to mitochondrial fission factor.¹¹ Drp1 translocation is preceded by ER membranes wrapping around mitochondria to constrict the organelles,¹² thereby facilitating the formation of multimeric Drp1 complexes that, upon GTP hydrolysis, further tighten to complete the process of mitochondrial fission.¹³Genetic evidence in mice and humans indicates that mitochondrial dynamics are crucially important in neurons: in humans, a sporadic dominant-negative DRP1 mutation caused a lethal syndromic defect with abnormal brain development;¹⁴ similarly, constitutive Drp1 knockout in the mouse brain leads to lethal neurodevelopmental defects.^{15, 16} Although the crucial role of Drp1 during brain development is undisputed, studies on Drp1 function in postmitotic (adult) neurons are scarce; likewise, Drp1 ablation studies in primary cultures have so far failed to yield a conclusive picture. In vitro, Drp1 ablation is reported to lead to a super-elongated neuroprotective^{17, 18, 19, 20, 21, 22, 23, 24} or an aggregated mitochondrial phenotype associated with neurodegeneration.^{15, 16, 25, 26, 27} These discrepancies are probably due to different experimental conditions: neuronal health is indeed influenced by the onset and duration of Drp1 inhibition, which varies considerably among the cited reports,²⁸ and different types of neuronal cultures studied display different sensitivity to Drp1 inhibition. In vivo, Drp1 ablation in Purkinje cells results in oxidative stress and neurodegeneration,²⁹ demonstrating that Drp1 is essential for postmitotic neurons'' health. In contrast, transient pharmacological Drp1 inhibition is neuroprotective in several mouse ischemia models, indicating that temporarily blocking mitochondrial fission holds therapeutic potential.^{30, 31, 32}To elucidate the consequences of blocked mitochondrial fission in the central nervous system in vivo, we bypassed the critical role of Drp1 during brain development by generating Drp1^flx/flx mice¹⁵ expressing tamoxifen-inducible Cre recombinase under the control of the CaMKIIα promoter.³³ Upon induced Drp1 deletion in postmitotic adult mouse forebrain neurons, mice develop progressive, neuronal subtype-specific alterations in mitochondrial shape and distribution in the absence of overt neurodegeneration. In addition, respiratory capacity, ATP content, synaptic reserve pool vesicle recruitment as well as spatial working memory are impaired, demonstrating that severely dysregulated mitochondrial dynamics can compromise critical neuronal functions in vivo without causing neuronal cell death.     

Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development

Mitochondrial morphology is determined by a dynamic equilibrium between organelle fusion and fission, but the significance of these processes in vertebrates is unknown. The mitofusins, Mfn1 and Mfn2, have been shown to affect mitochondrial morphology when overexpressed. We find that mice deficient in either Mfn1 or Mfn2 die in midgestation. However, whereas Mfn2 mutant embryos have a specific and severe disruption of the placental trophoblast giant cell layer, Mfn1-deficient giant cells are normal. Embryonic fibroblasts lacking Mfn1 or Mfn2 display distinct types of fragmented mitochondria, a phenotype we determine to be due to a severe reduction in mitochondrial fusion. Moreover, we find that Mfn1 and Mfn2 form homotypic and heterotypic complexes and show, by rescue of mutant cells, that the homotypic complexes are functional for fusion. We conclude that Mfn1 and Mfn2 have both redundant and distinct functions and act in three separate molecular complexes to promote mitochondrial fusion. Strikingly, a subset of mitochondria in mutant cells lose membrane potential. Therefore, mitochondrial fusion is essential for embryonic development, and by enabling cooperation between mitochondria, has protective effects on the mitochondrial population.     

Dynamin-related protein 1 is required for normal mitochondrial bioenergetic and synaptic function in CA1 hippocampal neurons

Disrupting particular mitochondrial fission and fusion proteins leads to the death of specific neuronal populations; however, the normal functions of mitochondrial fission in neurons are poorly understood, especially in vivo, which limits the understanding of mitochondrial changes in disease. Altered activity of the central mitochondrial fission protein dynamin-related protein 1 (Drp1) may contribute to the pathophysiology of several neurologic diseases. To study Drp1 in a neuronal population affected by Alzheimer''s disease (AD), stroke, and seizure disorders, we postnatally deleted Drp1 from CA1 and other forebrain neurons in mice (CamKII-Cre, Drp1^lox/lox (Drp1cKO)). Although most CA1 neurons survived for more than 1 year, their synaptic transmission was impaired, and Drp1cKO mice had impaired memory. In Drp1cKO cell bodies, we observed marked mitochondrial swelling but no change in the number of mitochondria in individual synaptic terminals. Using ATP FRET sensors, we found that cultured neurons lacking Drp1 (Drp1KO) could not maintain normal levels of mitochondrial-derived ATP when energy consumption was increased by neural activity. These deficits occurred specifically at the nerve terminal, but not the cell body, and were sufficient to impair synaptic vesicle cycling. Although Drp1KO increased the distance between axonal mitochondria, mitochondrial-derived ATP still decreased similarly in Drp1KO boutons with and without mitochondria. This indicates that mitochondrial-derived ATP is rapidly dispersed in Drp1KO axons, and that the deficits in axonal bioenergetics and function are not caused by regional energy gradients. Instead, loss of Drp1 compromises the intrinsic bioenergetic function of axonal mitochondria, thus revealing a mechanism by which disrupting mitochondrial dynamics can cause dysfunction of axons.Mitochondrial dynamics – the balance between mitochondrial fission and fusion – regulates mitochondrial quality control by segregating poorly functioning mitochondria for degradation while mixing the contents of healthy mitochondria.^{1, 2} In neurons, fission uniquely facilitates movement of mitochondria down narrow distal axons.^{3, 4} Disruptions of this movement, and of other neuron-specific functions, may explain why systemic mutations in mitochondrial fusion and fission proteins specifically cause death of neurons. However, the roles and requirements of these proteins also differ between neuronal types.¹ For example, mutations in the fusion protein optic atrophy 1 cause degeneration of retinal ganglion neurons,⁵ and mutations in the fusion protein mitofusin-2 or the fission protein ganglioside-induced differentiation-associated protein 1 cause peripheral neuropathy (Charcot-Marie-Tooth types 2A and 4A^{6, 7}).There are several potential reasons why specific neurons have unique requirements for fission–fusion proteins. First, the functions of these proteins may be more critical in vulnerable neuronal populations. Recently, we showed that most midbrain DA neurons are uniquely vulnerable to loss of the central mitochondrial fission protein dynamin-related protein 1 (Drp1),⁴ a GTPase recruited to fission sites on the outer mitochondrial membrane.¹ Loss of Drp1 depletes axonal mitochondria, which is followed by axonal degeneration and neuronal death. However, a subpopulation of midbrain DA neurons survive, despite losing their axonal mitochondria, suggesting that they have lower needs for energy or other mitochondrial functions in their axons.⁴ Do unique requirements for mitochondrial dynamics underlie differential neuronal vulnerability? Do resistant neurons compensate with other fission or fusion mechanisms? Do the functions of fission differ between neurons? Notably, Drp1 may also have mitochondria-independent functions in synaptic vesicle release.⁸ Addressing these issues could help elucidate the physiological functions of mitochondrial dynamics in the nervous system and reveal how shifts in the fission–fusion balance contribute to selective neuronal death in neurodegenerative diseases, including Huntington''s disease, Parkinson''s disease and Alzheimer''s disease (AD),^{1, 4} and in other neurologic disorders, including stroke and epilepsy.^{9, 10, 11}To understand mitochondrial dynamics, it would be useful to know why mitochondrial fission is needed in the nervous system in the first place, and how loss of fission affects mitochondrial functions in specific cell types. Notably, Drp1 knockout did not change respiration or ATP levels in resuspended mouse embryonic fibroblasts (MEFs),^{12, 13} indicating that mitochondrial fission is not required for respiration in these cells. However, neuronal respiration may be more sensitive to Drp1 loss. Indeed, Drp1 loss markedly decreased the number of mitochondria in axons and the cell body in midbrain DA neurons in vivo,⁴ and reduced staining of complex I and IV activity in cerebellar neurons in vivo.¹⁴ However, it is unclear whether these changes translate into decreased ATP levels in neurons and, if so, whether this decrease compromises neuronal function. Furthermore, Drp1 loss caused cell death in cerebellar and most midbrain DA neurons,^{4, 14} which challenges our ability to dissociate the specific effects of Drp1 loss on mitochondrial function from other non-specific changes that accompany cell death.To learn how disrupting mitochondrial fission contributes to selective neurodegeneration, we studied the function of Drp1 in CA1 hippocampal neurons and its role in mitochondrial bioenergetics. Surprisingly, despite losing Drp1, most CA1 neurons survived for more than 1 year in vivo, although their function was compromised, leading to deficits in synaptic transmission and memory. To begin to understand how loss of Drp1 causes neuronal dysfunction, we examined the role of Drp1 in mitochondrial bioenergetics. We found that Drp1 is required to maintain normal mitochondrial-derived ATP levels specifically in axons (but not the cell body), and that the loss of this function is unrelated to the distribution of mitochondria within axons.     

hNOA1 Interacts with Complex I and DAP3 and Regulates Mitochondrial Respiration and Apoptosis

Mitochondria are dynamic organelles that play key roles in metabolism, energy production, and apoptosis. Coordination of these processes is essential to maintain normal cellular functions. Here we characterized hNOA1, the human homologue of AtNOA1 (Arabidopsis thaliana nitric oxide-associated protein 1), a large mitochondrial GTPase. By immunofluorescence, immunoelectron microscopy, and mitochondrial subfractionation, endogenous hNOA1 is localized within mitochondria where it is peripherally associated with the inner mitochondrial membrane facing the mitochondrial matrix. Overexpression and knockdown of hNOA1 led to changes in mitochondrial shape implying effects on mitochondrial dynamics. To identify the interaction partners of hNOA1 and to further understand its cellular functions, we performed immunoprecipitation-mass spectrometry analysis of endogenous hNOA1 from enriched mitochondrial fractions and found that hNOA1 interacts with both Complex I of the electron transport chain and DAP3 (death-associated protein 3), a positive regulator of apoptosis. Knockdown of hNOA1 reduces mitochondrial O₂ consumption ∼20% in a Complex I-dependent manner, supporting a functional link between hNOA1 and Complex I. Moreover, knockdown of hNOA1 renders cells more resistant to apoptotic stimuli such as γ-interferon and staurosporine, supporting a role for hNOA1 in regulating apoptosis. Thus, based on its interactions with both Complex I and DAP3, hNOA1 may play a role in mitochondrial respiration and apoptosis.Emerging evidence indicates that mitochondrial metabolism, apoptosis, and dynamics (fission and fusion) are closely intertwined. Apoptosis and changes in metabolism are associated with morphological changes in mitochondria (1, 2). Conversely, when mitochondrial morphology is altered either by mutations or altered expression of mitochondrial fission or fusion proteins such as the dynamin like large G proteins Drp1 and Opa1, the cell''s susceptibility to apoptotic agents (3) or ability to generate ATP (4, 5) is altered.Apoptosis is controlled by a diverse range of cell signals, which may originate either extracellularly (extrinsic inducers) or intracellularly (intrinsic inducers), and mitochondria play central roles in both pathways (6). The apoptotic pathways involve a growing list of mitochondria-associated proteins, such as Bad, cytochrome c, Smac, AIF, Bcl-2, and others, most of which are located either on the outer mitochondrial membrane (OMM)³ or in the intermembrane space (IMS) (7). Recently, proteins of the mitochondrial matrix such as DAP3, have also been shown to be involved in apoptosis (8). DAP3 has been reported to be involved in both γ-interferon- (9) and tumor necrosis factor-α-induced (10) apoptosis as well as staurosporine-induced mitochondrial fragmentation (11), but the detailed mechanisms involved remain to be elucidated.Besides their role in apoptosis, much more is known about the functions of mitochondria in respiration and generation of ATP. The electron transport chain in the inner mitochondrial membrane (IMM) contains four major enzyme complexes (Complexes I, II, III, and IV) that are involved in transferring electrons from NADH (Complex I-linked) or FADH2 (Complex II-linked) to O₂ and in pumping protons out of the matrix to create an electrochemical proton gradient, which is harnessed by ATP synthase to make ATP (12).Despite the accumulating evidence showing intercommunication between mitochondrial metabolism, apoptosis, and dynamics, how these processes are coordinated remains to be elucidated. In this study we characterize hNOA1, the human homologue of Arabidopsis thaliana nitric oxide-associated protein, 1 (AtNOA1) (13). hNOA1 is a large G protein closely related to dynamin that is associated with the IMM. Perturbation of hNOA1 affects mitochondrial morphology, Complex I-linked O₂ consumption, and the cell''s susceptibility to apoptotic stimuli, possibly through interactions with proteins such as Complex I and DAP3.     

Mitochondrial Toxic Effects of Aβ Through Mitofusins in the Early Pathogenesis of Alzheimer’s Disease

Mitochondrial dysfunction has been implicated in the pathogenesis of Alzheimer’s disease (AD). However, it is obscure how amyloid-beta (Aβ) can impair mitochondria in the early stage of AD pathology. Using PrP-hAPP/hPS1 double-transgenic AD mouse model, we find that abnormal mitochondrial morphology and damaged mitochondrial structure in hippocampal neurons appear in the early stage of AD-like disease development. We also find consistent mitochondrial abnormalities in the SH-SY5Y cells, which express amyloid precursor protein (APP) Swedish mutation (APP_sw) and have been used as a cell model of the early-onset AD. Significant changes of mitofusin GTPases (Mfn1 and Mfn2) were detected both in the PrP-hAPP/hPS1 brains and SH-SY5Y cells. Moreover, our results show that Aβ accumulation in neurons of PrP-hAPP/hPS1 mice can affect the neurogenesis prior to plaque formation. These findings suggest that mitochondrial impairment is a very early event in AD pathogenesis and abnormal expression of Mfn1 and Mfn2 caused by excessive intracellular Aβ is the possible molecular mechanism. Interestingly, l-theanine has significant effects on regulating mitochondrial fusion proteins in SH-SY5Y (APP_sw) cells. Overall, our results not only suggest a new early mechanism of AD pathogenesis but also propose a preventive candidate, l-theanine, for the treatment of AD.