Chaperone-like protein p32 regulates ULK1 stability and autophagy

Mitophagy mediates clearance of dysfunctional mitochondria, and represents one type of mitochondrial quality control, which is essential for optimal mitochondrial bioenergetics. p32, a chaperone-like protein, is crucial for maintaining mitochondrial membrane potential and oxidative phosphorylation. However, the relationship between p32 and mitochondrial homeostasis has not been addressed. Here, we identified p32 as a key regulator of ULK1 stability by forming complex with ULK1. p32 depletion potentiated K48-linked but impaired K63-linked polyubiquitination of ULK1, leading to proteasome-mediated degradation of ULK1. As a result, silencing p32 profoundly impaired starvation-induced autophagic flux and the clearance of damaged mitochondria caused by mitochondrial uncoupler. Importantly, restoring ULK1 expression in p32-depleted cells rescued autophagy and mitophagy defects. Our findings highlight a cytoprotective role of p32 under starvation conditions by regulating ULK1 stability, and uncover a crucial role of the p32–ULK1-autophagy axis in coordinating stress response, cell survival and mitochondrial homeostasis.Mitophagy is a selective form of autophagy by which mitochondria are degraded in autolysosomes. p32 is a critical regulator of mitochondrial bioenergetics.¹ It primarily localizes to the mitochondrial matrix, but has also been reported to be present in other subcellular locations.^{2, 3, 4, 5} Many human tumors exhibit higher p32 expression levels than their nonmalignant counterpart tissues.^{6, 7, 8, 9} Depleting p32 in human cancer cells strongly shifts their metabolism from oxidative phosphorylation to glycolysis.¹ Consistently, p32 knockout causes mid-gestation lethality of knockout embryos and defects in oxidative phosphorylation. Mouse embryonic fibroblasts (MEFs) generated from p32 knockout embryos exhibited impaired ATP production and reduced mitochondrial membrane potential, which is in agreement with the observation that p32 silencing leads to increased mitochondrial fragmentation.^{10, 11} Notably, p32 was found to form protein complex with a variety of molecules^{7, 12, 13} and has been suggested that it may act as a multifunctional chaperone protein.^{12, 13, 14}ULK1 has a crucial role in mitophagy induction.¹⁵ Despite the pivotal role of ULK1 in mitochondrial clearance, little is known as how ULK1 itself is regulated. ULK1 is a relatively stable protein and is subject to proteasome-mediated degradation. Post-translational modifications including K63-linked ubiquitylation^{16, 17} and phosphorylation^{18, 19, 20} have been reported to modulate the rates of ULK1 turnover and kinase activity in different cellular contexts. Hsp90 and Cdc37 have been shown to regulate ULK1 stability and activity by forming complex with ULK1, which subsequently influences Atg13-mediated mitophagy.²¹ Here, we found p32 regulates ULK1 stability by forming protein complex with ULK1. The interaction between ULK1 and p32 is crucial for maintaining the steady-state levels and activity of ULK1. We further show that p32 ablation results in a defect in autophagy in EBSS-starved cells, and impairs clearance of dysfunctional mitochondria in cells exposed to mitochondrial uncoupler. Importantly, these autophagy and mitophagy defects can be restored by re-introducing ULK1 into p32-deficient cells, demonstrating ULK1 functions as a crucial downstream effector of p32.     

Bid chimeras indicate that most BH3-only proteins can directly activate Bak and Bax,and show no preference for Bak versus Bax

The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-x_L according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax.The Bcl-2 family of proteins controls the mitochondrial pathway of apoptosis, a process often dysregulated in cancer and other diseases.^{1, 2, 3} Apoptotic triggers including DNA damage and oncogene activation cause the synthesis or activation of one or more pro-apoptotic Bcl-2 homology region 3 (BH3)-only proteins,^{1, 2, 3, 4} a subfamily that includes Bid, Bim, Puma, Noxa, Bad, Bik, Bmf and Hrk. These proteins then engage via their BH3 domain with other Bcl-2 family members. BH3-only proteins that can directly bind and activate the Bcl-2 effector proteins Bak or Bax are called ‘activators''.⁵ When Bak or Bax become activated and oligomerize in the mitochondrial outer membrane (MOM), the apoptotic ‘switch'' has flipped and the cell is committed to cell death. The prosurvival members (Bcl-2, Bcl-x_L, Mcl-1, Bcl-w, Bfl-1/A1 and Bcl-B) inhibit apoptosis by specifically binding both the BH3-only proteins and activated Bak and Bax.^{6, 7, 8, 9, 10, 11} Thus, the cell''s complement of prosurvival proteins, Bak, and Bax, determines the sensitivity of that cell to each BH3-only protein, and by extension to each type of pro-apoptotic stimulus.A thorough understanding of BH3-only proteins is crucial for the development of cancer therapeutics such as the new class of anti-cancer molecules called BH3 mimetics that are showing significant promise in clinical trials.^{12, 13} The binding of BH3-only proteins to prosurvival proteins has been well-characterized and revealed significant preferences for engaging different members.^{6, 8, 9} How BH3-only proteins bind and activate Bak and Bax remains less understood for several reasons. First, generating stable recombinant BH3-only proteins is difficult because, except for Bid, they are intrinsically disordered^{14, 15, 16} and because most contain hydrophobic C-terminal membrane anchors.¹⁷ Thus, most in vitro studies of BH3-only proteins have used synthetic peptides corresponding to the BH3 domains, C-terminally truncated recombinant proteins or in vitro translated (IVT) proteins. Second, BH3-only reagents bind poorly to recombinant Bak and Bax in the absence of membranes, although detergents and liposomes may substitute for the MOM.^{18, 19, 20} Third, activation of Bak and Bax on mitochondria can be complicated by the presence of other proteins such as prosurvival proteins. Indeed, genetically altering BH3-only protein levels in mice resulted in complex phenotypes due to multiple interactions between family members, precluding firm conclusions as to which BH3-only proteins are direct activators.^{18, 21, 22}Bid and Bim are direct activators according to a variety of approaches,^{5, 8, 9, 23, 24} and were recently proposed to be specific for Bak and Bax, respectively.²⁵ Early studies using Noxa BH3 peptides^{5, 8} and IVT Noxa⁹ concluded that Noxa was not an activator. However, in more recent studies a Noxa BH3 peptide²³ and purified recombinant NoxaΔC²⁰ were found to be activators of both Bak and Bax. Puma has also been described as both an activator^{26, 27} and not an activator.^{8, 28} Du et al.²³ analyzed the full panel of BH3 peptides and classified Bim as a strong activator, Bid, Noxa and Bmf as moderate activators, and Puma, Bik and Hrk as weak activators. The only BH3-only member that has never been described as an activator is Bad.While BH3 peptides and recombinant truncated BH3-only proteins have been useful for in vitro studies, new reagents that target mitochondria may better reflect the behavior of the parent proteins. As Bid is stable as a recombinant protein, we generated chimeras of Bid in which the BH3 domain of Bid was replaced with that of seven other BH3-only proteins. This is a similar approach to the Bim chimeras used for expression in cells¹⁸ and in mice.²⁹ More recently, truncated Bid (tBid) chimeras containing the BH3 domains of Bim, Bak and Bax as well as those of the prosurvival proteins, have been generated as IVT proteins.¹¹To compare the ability of BH3-only proteins to activate Bak and Bax in vitro, we incubated Bid chimeras and BH3 peptides with mitochondria containing either Bak or Bax. We found that the membrane-targeted Bid chimeras were much more potent activators than their related BH3 peptides, and that all BH3 domains except for Bad and Noxa were activators to some extent. We conclude that activation of Bak and Bax may be underestimated by studies using BH3 peptides, and that even BH3-only proteins such as Bik, Bmf and Hrk that are often considered unable to activate Bak or Bax, may act as activators under certain conditions.     

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.     

AMBRA1 is able to induce mitophagy via LC3 binding,regardless of PARKIN and p62/SQSTM1

Damaged mitochondria are eliminated by mitophagy, a selective form of autophagy whose dysfunction associates with neurodegenerative diseases. PINK1, PARKIN and p62/SQTMS1 have been shown to regulate mitophagy, leaving hitherto ill-defined the contribution by key players in ‘general'' autophagy. In basal conditions, a pool of AMBRA1 – an upstream autophagy regulator and a PARKIN interactor – is present at the mitochondria, where its pro-autophagic activity is inhibited by Bcl-2. Here we show that, upon mitophagy induction, AMBRA1 binds the autophagosome adapter LC3 through a LIR (LC3 interacting region) motif, this interaction being crucial for regulating both canonical PARKIN-dependent and -independent mitochondrial clearance. Moreover, forcing AMBRA1 localization to the outer mitochondrial membrane unleashes a massive PARKIN- and p62-independent but LC3-dependent mitophagy. These results highlight a novel role for AMBRA1 as a powerful mitophagy regulator, through both canonical or noncanonical pathways.Autophagy is an important eukaryotic process involved in the lysosomal degradation of cytosolic components in both physiological and pathological conditions. During autophagy, the autophagosomes − specific double-membraned vesicles − engulf a number of different cargoes and then fuse with the lysosomes for subsequent recycling of their content. Several key proteins are involved in autophagosome formation, such as BECLIN 1 and its positive regulator AMBRA1;^{1, 2} a pool of AMBRA1 is localized at the mitochondria, where its pro-autophagic activity is inhibited by mitochondrial resident Bcl-2.³ Interestingly, mitochondria have been described as a source for autophagosome biogenesis;⁴ they play a key role in the cross-talk between autophagy and apoptosis regulation and they are involved in the cell death versus survival decision (reviewed in Strappazzon et al.³).Another mechanistic link exists between autophagy and mitochondria in mammals. Indeed, mitochondria damaged by the uncoupler CCCP (carbonyl cyanide m-chlorophenyl hydrazone) − because of a loss of their mitochondrial membrane potential (ΔΨ_m) − are subjected to a form of selective autophagy, termed mitophagy.^{5, 6, 7} During this process, depolarized mitochondria are ubiquitylated; they then recruit p62 (a protein involved in linking polyubiquitinated protein aggregates to the autophagic machinery) and next they are transported along microtubules to the perinuclear region, where they form rough aggregate structures termed ‘mito-aggresomes'',^{8, 9, 10} a step preceding their lysosomal degradation.Although mitophagy has been described in a number of tissues and in various physiological or pathological conditions (reviewed in Andreux et al.¹¹), very few are the known molecular mechanisms that regulate mitophagy; this is despite the fact that its manipulation may represent a forefront strategy in several human diseases. Thus, rather scarse is yet the availability of chemicals and drug candidates to modulate the process. The autophagy receptor NIX and the kinase Ulk1 mediate developmental removal of mitochondria during retyculocyte differentiation.^{6, 12, 13} Smurf1 has been defined as a new recognized mediator of both viral autophagy and mitophagy.¹⁴ In contrast, the E3 ubiquitin ligase PARKIN and the Ser/Thr kinase PINK1, both found to be mutated in autosomal recessive forms of Parkinson''s disease (PD), regulate mitophagy after mitochondrial damage.⁵ In more detail, PINK1 recruits PARKIN to depolarized mitochondria in order to remove damaged mitochondria. This mitochondrial quality control, driven by PINK1/PARKIN proteins, has recently been better characterized by RNAi screens.¹⁵ In fact, new proteins such as HSPA1L, BAG4 and SIAH3 have been found to modulate translocation of PARKIN to damaged mitochondria, whereas TOMM7 stabilizes PINK1 on the mitochondria. Interestingly, it has been demonstrated that after mitochondrial depolarization, the cytosolic pool of AMBRA1 interacts with PARKIN to enhance mitochondrial clearance.¹⁶In this study, we investigate the molecular mechanism(s) responsible for the AMBRA1-dependent enhancement of PARKIN-mediated mitophagy. We describe for the first time AMBRA1 as a new LIR (LC3 interacting region)-containing protein, and we demonstrate that this motif is essential for the binding between AMBRA1 and LC3, following mitophagy induction. Furthermore, we show that this interaction is crucial in a number of cell systems in order to both amplify PARKIN-mediated mitochondrial clearance and regulate PARKIN-independent mitophagy. In addition, to better understand the role of AMBRA1 at the mitochondria and as AMBRA1 does not possess a clear mitochondrial targeting sequence, we generated and expressed an organelle-targeted mutant of AMBRA1 in two different cell systems. Our data indicate that mitochondrial AMBRA1 induces (1) relocalization of the mitochondrial network around the nucleus, (2) depolarization and ubiquitylation of mitochondria and (3) recruitment of the molecular platform necessary to induce functional mitophagy through a PARKIN/p62-independent pathway.This work thus places AMBRA1 as a central player of mitophagy: we suggest that this molecule facilitates mitochondrial clearance by bringing damaged mitochondria onto autophagosomes via its LIR-mediated LC3 interaction. In addition, we show that high levels of mitochondrial AMBRA1 trigger mitophagy, a finding that could herald new therapies to fight important human disorders, ranging from muscle dystrophy to neurodegeneration.     

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.     

Inhibition of apoptotic Bax translocation to the mitochondria is a central function of parkin

Parkinson''s disease (PD) is the second most prevalent neurodegenerative disorder, affecting 1–3% of the population over 65. Mutations in the ubiquitin E3 ligase parkin are the most common cause of autosomal recessive PD. The parkin protein possesses potent cell-protective properties and has been mechanistically linked to both the regulation of apoptosis and the turnover of damaged mitochondria. Here, we explored these two functions of parkin and the relative scale of these processes in various cell types. While biochemical analyses and subcellular fractionation were sufficient to observe robust parkin-dependent mitophagy in immortalized cells, higher resolution techniques appear to be required for primary culture systems. These approaches, however, did affirm a critical role for parkin in the regulation of apoptosis in primary cultured neurons and all other cells studied. Our prior work demonstrated that parkin-dependent ubiquitination of endogenous Bax inhibits its mitochondrial translocation and can account for the anti-apoptotic effects of parkin. Having found a central role for parkin in the regulation of apoptosis, we further investigated the parkin-Bax interaction. We observed that the BH3 domain of Bax is critical for its recognition by parkin, and identified two lysines that are crucial for parkin-dependent regulation of Bax translocation. Last, a disease-linked mutation in parkin failed to influence Bax translocation to mitochondria after apoptotic stress. Taken together, our data suggest that regulation of apoptosis by the inhibition of Bax translocation is a prevalent physiological function of parkin regardless of the kind of cell stress, preventing overt cell death and supporting cell viability during mitochondrial injury and repair.Loss-of-function mutations in the ubiquitin E3 ligase parkin are the most common cause of autosomal recessive Parkinson''s disease (PD).¹ Multiple functions have been ascribed to parkin, most notably the inhibition of apoptosis^{2, 3, 4, 5, 6, 7} and the induction of autophagic mitochondrial turnover (mitophagy).^{8, 9} However, the relative scale of these effects mediated by endogenous parkin and whether these processes can occur concomitantly or are mutually exclusive, is not known.Bax is a primary effector of cell death that translocates from the cytosol to the mitochondria upon stress, where it facilitates cytochrome c release and the subsequent caspase cascade.¹⁰ We previously identified Bax as a parkin substrate, and found that the anti-apoptotic effects of parkin can be directly linked to the parkin-dependent ubiquitination of Bax and inhibition of its mitochondrial translocation.³ Recent corroborative evidence showed that primary cultured neurons from parkin knock-out (KO) mice accumulate greater levels of activated Bax at the mitochondria than wild-type (WT) neurons after apoptotic stimulation,¹¹ while a separate report showed the parkin-dependent ubiquitination of Bax during mitophagy.¹²In addition to its anti-apoptotic function, parkin facilitates a depolarization-induced and autophagy-dependent turnover of mitochondria. This process is robustly observed in immortalized cell lines expressing human parkin, where exposure to the mitochondrial depolarizing agent carbonyl cyanide 3-chlorophenylhydrazone (CCCP) causes rapid recruitment of parkin from the cytosol to the mitochondrial outer membrane and a coordinated proteasome and autophagosome-mediated turnover of the entire organelle.^{8, 13, 14, 15} Examination of this process in primary neuronal cultures with endogenous parkin expression, however, has been challenging,^{16, 17, 18, 19} and a cooperative role for inhibition of mitochondria-dependent cell death has not been investigated in the context of mitophagy.In this study, we sought further insight into the biological functions of parkin across multiple cell types. Our data showed that whole-cell biochemical techniques were not sufficient to observe the participation of endogenous parkin in mitochondrial turnover but were able to confirm the parkin-dependent regulation of apoptosis. Further examination of the parkin-dependent regulation of apoptosis identified two specific lysines of Bax that are critical for recognition and inhibition of its translocation to the mitochondria by parkin. In addition, the BH3 domain of Bax was critical for its association with parkin. Importantly, we observed parkin-dependent mitophagy and inhibition of apoptotic Bax translocation in the same cell culture systems, suggesting that these two pathways coexist and likely cooperate within neurons. Taken together, our data indicate that the parkin-dependent regulation of Bax is critical for cell survival, irrespective of the nature of cell stress involved.     

Mitochondrial trafficking and anchoring in neurons: New insight and implications

Mitochondria are essential organelles for neuronal growth, survival, and function. Neurons use specialized mechanisms to drive mitochondria transport and to anchor them in axons and at synapses. Stationary mitochondria buffer intracellular Ca²⁺ and serve as a local energy source by supplying ATP. The balance between motile and stationary mitochondria responds quickly to changes in axonal and synaptic physiology. Defects in mitochondrial transport are implicated in the pathogenesis of several major neurological disorders. Recent work has provided new insight in the regulation of microtubule-based mitochondrial trafficking and anchoring, and on how mitochondrial motility influences neuron growth, synaptic function, and mitophagy.

Introduction

Mitochondria are cellular power plants that convert chemicals into ATP through coupled electron transport chain and oxidative phosphorylation. A cortical neuron in human brain utilizes up to ∼4.7 billion ATP molecules per second to power various biological functions (Zhu et al., 2012). Constant ATP supply is essential for nerve cell survival and function (Nicholls and Budd, 2000). Mitochondrial ATP production supports synapse assembly (Lee and Peng, 2008), generation of action potentials (Attwell and Laughlin, 2001), and synaptic transmission (Verstreken et al., 2005). Synaptic mitochondria maintain and regulate neurotransmission by buffering Ca²⁺ (Medler and Gleason, 2002; David and Barrett, 2003). In addition, mitochondria sequester presynaptic Ca²⁺ transients elicited by trains of action potentials and release Ca²⁺ after stimulation, thus inducing certain forms of short-term synaptic plasticity (Werth and Thayer, 1994; Tang and Zucker, 1997; Billups and Forsythe, 2002; Levy et al., 2003; Kang et al., 2008). Removing mitochondria from axon terminals results in aberrant synaptic transmission likely due to insufficient ATP supply or altered Ca²⁺ transients (Stowers et al., 2002; Guo et al., 2005; Ma et al., 2009).Neurons are polarized cells consisting of a relatively small cell body, dendrites with multiple branches and elaborate arbors, and a thin axon that can extend up to a meter long in some peripheral nerves. Due to these extremely varied morphological features, neurons face exceptional challenges to maintain energy homeostasis. Neurons require specialized mechanisms to efficiently distribute mitochondria to far distal areas where energy is in high demand, such as synaptic terminals, active growth cones, and axonal branches (Fig. 1; Morris and Hollenbeck, 1993; Ruthel and Hollenbeck, 2003). Axonal branches and synapses undergo dynamic remodeling during neuronal development and in response to synaptic activity, thereby changing mitochondrial trafficking and distribution. Neurons are postmitotic cells surviving for the lifetime of the organism. A mitochondrion needs to be removed when it becomes aged or dysfunctional. Mitochondria also alter their motility and distribution under certain stress conditions or when their integrity is impaired (Miller and Sheetz, 2004; Chang and Reynolds, 2006; Cai et al., 2012). Therefore, efficient regulation of mitochondrial trafficking and anchoring is essential to: (1) recruit and redistribute mitochondria to meet altered metabolic requirements; and (2) remove aged and damaged mitochondria and replenish healthy ones at distal terminals. Research into neuronal regulation of mitochondrial trafficking and anchoring is thus a very important frontier in neurobiology. This review article focuses on new mechanistic insight into the regulation of microtubule (MT)-based mitochondrial trafficking and anchoring and provides an updated overview of how mitochondrial motility influences neuronal growth, synaptic function, and mitochondrial quality control. Additional insight and overviews from different perspectives can be found in other in-depth reviews (Frederick and Shaw, 2007; Morfini et al., 2009; Hirokawa et al., 2010; MacAskill and Kittler, 2010; Schon and Przedborski, 2011; Court and Coleman, 2012; Saxton and Hollenbeck, 2012; Sheng and Cai, 2012; Birsa et al., 2013; Lovas and Wang, 2013; Schwarz, 2013).Open in a separate windowFigure 1.Mitochondrial trafficking and anchoring in neurons. Due to complex structural features, neurons require specialized mechanisms trafficking mitochondria to their distal destinations and anchoring them in regions where metabolic and calcium homeostatic capacity is in a high demand. The figure highlights transporting mitochondria to a presynaptic bouton (A) and an axonal terminal (B). MT-based long-distance mitochondrial transport relies on MT polarity. In axons, the MT’s plus ends (+) are oriented toward axonal terminals whereas minus ends (−) are directed toward the soma. Thus, KIF5 motors are responsible for anterograde transport to distal synaptic terminals whereas dynein motors return mitochondria to the soma. The motor adaptor Trak proteins can mediate both KIF5- and dynein-driven bi-directional transport of axonal mitochondria (van Spronsen et al., 2013). Myosin motors likely drive short-range mitochondrial movement at presynaptic terminals where enriched actin filaments constitute cytoskeletal architecture. Motile mitochondria can be recruited into stationary pools via dynamic anchoring interactions between syntaphilin and MTs (Kang et al., 2008) or via an unidentified actin-based anchoring receptor (Chada and Hollenbeck, 2004). Such anchoring mechanisms ensure neuronal mitochondria are adequately distributed along axons and at synapses, where constant energy supply is crucial (figure courtesy of Qian Cai, Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ).

Neuronal mitochondria display complex motility patterns

The complex motility patterns of mitochondrial transport in neurons can be visualized by applying time-lapse imaging in live primary neurons. Mitochondria are labeled by expressing DsRed-Mito, a mitochondria-targeted fluorescent protein, or by loading fluorescent dye MitoTracker Green or MitoTracker Red CMXRos, which stains mitochondria in live cells dependent upon membrane potential. Mitochondria move bi-directionally over long distances along processes, pause briefly, and move again, frequently changing direction. Motile mitochondria can become stationary, and stationary ones can be remobilized and redistributed in response to changes in metabolic status and synaptic activity. Given frequent pauses and bi-directional movements, the mean velocities of neuronal mitochondrial transport are highly variable, ranging from 0.32 to 0.91 µm/s (Morris and Hollenbeck, 1995; MacAskill et al., 2009a). In mature neurons, ∼20–30% of axonal mitochondria are motile, some of which pass by presynaptic terminals while the remaining two thirds are stationary (Kang et al., 2008). Using dual-channel imaging of mitochondria and synaptic terminals in hippocampal neurons, a recent study identified five motility patterns of axonal mitochondria including: stationary mitochondria sitting out of synapses (54.07 ± 2.53%) or docking at synapses (16.29 ± 1.66%); motile mitochondria passing through synapses (14.77 ± 1.58%); or pausing at synapses for a short (<200 s, 7.01 ± 1.29%) or a longer period of time (>200 s, 8.30 ± 1.52%; Sun et al., 2013). These findings are consistent with a previous study in primary cortical neurons (Chang et al., 2006).Altered mitochondrial transport is one of the pathogenic changes in major adult-onset neurodegenerative diseases (Sheng and Cai, 2012). Although in vitro live imaging in primary neurons prepared from embryonic or newborn mice provides an important tool to study mechanisms regulating mitochondrial transport, it has obvious limitations for investigating adult-onset disease mechanisms. For example, reduced transport of axonal mitochondria was consistently reported in amyotrophic lateral sclerosis–linked hSOD1^G93A mice (De Vos et al., 2007; Magrané and Manfredi, 2009; Bilsland et al., 2010; Zhu and Sheng, 2011; Marinkovic et al., 2012; Song et al., 2013). Therefore, it is critical to establish spinal motor neuron cultures directly isolated from adult mice, which will provide more reliable models to investigate mitochondrial transport defects underlying adult-onset pathogenesis in motor neurons. Given that the morphology and conditions of neurons grown in culture dishes differ from that of neurons in vivo, an in vivo transport study would better reflect physiological environments. An ex vivo study of mitochondrial transport in acute nerve explants was developed in transgenic mice, in which neuronal mitochondria are labeled with mito-CFP or mito-YFP (Misgeld et al., 2007).

Moving mitochondria along MTs

Mitochondrial transport between the soma and distal processes or synapses depends upon MT-based motors, which drive their cargos via mechanisms requiring ATP hydrolysis (Martin et al., 1999; Hirokawa et al., 2010). MTs are uniformly arranged in axons: their plus ends are oriented distally and minus ends are directed toward the soma. Such a uniform polarity has made axons particularly useful for exploring mechanisms regulating bi-directional transport: dynein motors drive retrograde movement, whereas kinesin motors mediate anterograde transport. Of the 45 kinesin motor genes identified, the kinesin-1 family (KIF5) is the key motor driving mitochondrial transport in neurons (Hurd and Saxton, 1996; Tanaka et al., 1998; Górska-Andrzejak et al., 2003; Cai et al., 2005; Pilling et al., 2006). KIF5 heavy chain (KHC) contains a motor domain with ATPase at the N terminus and a C-terminal tail for binding cargo directly or indirectly via a cargo adaptor. Three isoforms (KIF5A, KIF5B, and KIF5C) of the KIF5 family are found in mammals, of which KIF5B is expressed ubiquitously whereas KIF5A and KIF5C are expressed selectively in neurons (Kanai et al., 2000).KIF5 motors attach to mitochondria through adaptor proteins. The motor–adaptor coupling ensures targeted trafficking and effective regulation of mitochondrial transport. Drosophila protein Milton acts as a KIF5 motor adaptor by binding the KIF5 cargo-binding domain and mitochondrial outer membrane receptor Miro (Stowers et al., 2002; Glater et al., 2006). The Drosophila mutation in Milton reduces mitochondrial trafficking to synapses. Milton appears specific for mitochondrial trafficking because the trafficking of other cargoes such as synaptic vesicles is not affected. Two Milton orthologues, Trak1 and Trak2, are found in mammals and are required for mitochondrial trafficking (Smith et al., 2006; MacAskill et al., 2009b; Koutsopoulos et al., 2010). Elevated Trak2 expression in hippocampal neurons robustly enhances axonal mitochondrial motility (Chen and Sheng, 2013). Conversely, depleting Trak1 or expressing its mutants results in impaired mitochondrial motility in axons (Brickley and Stephenson, 2011). A recent study revealed that mammalian Trak1 and 2 contain one N-terminal KIF5B-binding site and two dynein/dynactin-binding sites, one at the N-terminal domain and the other at the C-terminal domain (van Spronsen et al., 2013). Thus, the Trak proteins can mediate both KIF5- and dynein-driving bi-directional transport of axonal mitochondria (Fig. 1). In contrast to the specific role of Drosophila Milton in mitochondrial transport, mammalian Trak proteins are also recruited to endosomal compartments and associate with GABAA (γ-aminobutyric acid A) receptors and K⁺ channels, highlighting their role in neuronal cargo transport (Grishin et al., 2006; Kirk et al., 2006; Webber et al., 2008).The mitochondria outer membrane receptor Miro is a Rho-GTPase with two Ca²⁺-binding EF-hand motifs and two GTPase domains (Frederick et al., 2004; Fransson et al., 2006). Mutation of the Drosophila miro gene impairs mitochondrial anterograde transport, thus depleting mitochondria at distal synaptic terminals (Guo et al., 2005). The first crystal structures of Drosophila melanogaster Miro comprising the tandem EF-hands and GTPase domains were recently resolved (Klosowiak et al., 2013). From these structures, two additional hidden EF-hands were identified, each one pairing with a canonical EF-hand. These structures will help our understanding of the conformational changes in Miro upon Ca²⁺ binding and nucleotide sensing in regulating mitochondrial motility, dynamics, and function. There are two mammalian Miro isoforms, Miro1 and Miro2. The Miro1–Trak2 adaptor complex regulates mitochondrial transport in hippocampal neurons (MacAskill et al., 2009b). Elevated Miro1 expression increases mitochondrial transport, likely by recruiting more Trak2 and motors to the mitochondria (Chen and Sheng, 2013). Thus, KIF5, Milton (Trak), and Miro assemble into the transport machinery that drives mitochondrial anterograde movement (Fig. 2 A). An alternative KIF5 adaptor for neuronal mitochondria is syntabulin (Fig. 2 B; Cai et al., 2005). Depleting syntabulin or blocking the KIF5–syntabulin coupling reduces mitochondrial anterograde transport in axons. Several other proteins are also suggested as candidate KIF5 motor adaptors for mitochondrial transport, including FEZ1 (fasciculation and elongation protein zeta-1) and RanBP2 (Ran-binding protein 2; Cho et al., 2007; Fujita et al., 2007), although their role in neuronal mitochondrial transport requires further investigation. The existence of multiple motor adaptors may highlight the complex regulation of mitochondrial motility in response to various physiological and pathological signals.Open in a separate windowFigure 2.Activity-dependent regulation of mitochondrial transport. (A) The Miro–Milton (or Miro–Trak) adaptor complex mediates KIF5-driven mitochondrial transport. (B) Syntabulin, FEZ1, and RanBP2 serve as an alternative KIF5 motor adaptor in driving mitochondrial anterograde transport. (C and D) Miro-Ca²⁺ models in regulating mitochondrial motility. Miro contains Ca²⁺-binding EF-hand motifs. The C-terminal cargo-binding domain of KIF5 motors binds to the Miro–Trak adaptor complex. (C) Ca²⁺ binding to Miro’s EF-hands induces the motor domain to disconnect with MTs and thus prevents motor–MT engagement (Wang and Schwarz, 2009). (D) Alternatively, Ca²⁺ binding releases KIF5 motors from mitochondria (MacAskill et al., 2009a). Thus, Ca²⁺ influx after synaptic activity arrests motile mitochondria at activated synapses. (E) Syntaphilin-mediated “engine-switch and brake” model. A Miro-Ca²⁺–sensing pathway triggers the binding switch of KIF5 motors from the Miro–Trak adaptor complex to anchoring protein syntaphilin, which immobilizes axonal mitochondria via inhibiting motor ATPase activity. Thus, syntaphilin turns off the “KIF5 motor engine” by sensing a “stop sign” (elevated Ca²⁺) and putting a brake on mitochondria, thereby anchoring them in place on MTs. When in their stationary status, KIF5 motor–loaded mitochondria remain associated with the MT tracks while KIF5 ATPase is in an inactive state (Chen and Sheng, 2013; Figure courtesy of Qian Cai).Dynein motors are composed of multiple chains including heavy chains (DHC) as the motor domain, intermediate (DIC), light intermediate (DLIC), and light chains (DLC), which function by associating with cargos and regulating motility. Dynein associates with Drosophila mitochondria, and mutations of DHC alter velocity and run length of mitochondrial retrograde transport in axons (Pilling et al., 2006). Miro may serve as an adaptor for both KIF5 and dynein motors in Drosophila (Guo et al., 2005; Russo et al., 2009), as loss of dMiro impairs both kinesin- and dynein-driven transport and overexpressing dMiro also alters mitochondrial transport in both directions. These findings support an attractive model in which the relative activity of the opposite-moving motors is regulated by their cargo–adaptor proteins.

Mitochondrial motility and membrane dynamics

Mitochondrial trafficking, anchoring, and membrane dynamics are coordinated, thus controlling their shape, integrity, and distribution in neurons. It is expected that the size of a mitochondrion may have a direct impact on its mobility. Indeed, interconnected mitochondrial tubules found in fission-deficient neurons are less efficiently transported to distal synapses. Hippocampal neurons expressing defective Drp1, a mitochondrial fission protein, display accumulated mitochondria within the soma and reduced mitochondrial density in dendrites (Li et al., 2004). Drosophila Drp1 is required for delivering mitochondria to neuromuscular junctions (Verstreken et al., 2005). Conversely, mutation of Miro results in mitochondrial fragmentation in addition to impaired motility, whereas overexpressing Miro not only enhances mitochondrial transport but also induces their interconnection (Fransson et al., 2006; Saotome et al., 2008; Liu and Hajnóczky, 2009; MacAskill et al., 2009b). Additionally, yeast Miro, Gem1p, regulates mitochondrial morphology; cells lacking Gem1p contain collapsed, globular, or grape-like mitochondria (Frederick et al., 2004).Mitochondrial fusion events require two organelles to move closer. A “kiss-and-run” interplay is proposed for driving mitochondrial fusion (Liu et al., 2009). Thus, motile mitochondria, particularly in distal axons and at synapses, undergo fusion events at a higher rate than anchored stationary mitochondria. Recent studies highlight the cross talk between the mitochondrial fusion protein Mfn2 and a motor adaptor complex. Mfn2 associates with Miro2 and regulates axonal mitochondrial transport by affecting kinesin motor processivity or by coordinating the motor switch between kinesin and dynein (Misko et al., 2010). Neurons defective in Mfn2 display reduced mitochondrial motility and altered mitochondrial distribution (Chen et al., 2003, 2007; Baloh et al., 2007). Therefore, regulating mitochondrial trafficking directly affects their morphology. Conversely, changing mitochondrial fusion–fission dynamics also impacts their transport and distribution.

Anchoring mitochondria at MTs

Proper axonal and synaptic function requires stationary mitochondria docking in regions where energy is in high demand. The diffusion capacity of intracellular ATP is rather limited (Hubley et al., 1996), particularly within long axonal processes (Sun et al., 2013). Therefore, docked mitochondria ideally serve as stationary power plants for stable and continuous ATP supply necessary to maintain the activity of Na⁺–K⁺ ATPase, fast spike propagation, and synaptic transmission. In addition, mitochondria maintain calcium homeostasis at synapses by sequestering intracellular [Ca²⁺] (Billups and Forsythe, 2002; Kang et al., 2008). Given the fact that two thirds of the mitochondria are stationary for an extended period along axons, neurons require mechanisms dissociating mitochondria from motor proteins or anchoring mitochondria to the cytoskeleton. One intriguing mitochondrial-anchoring protein is syntaphilin, which is a “static anchor” for immobilizing mitochondria specifically in axons (Kang et al., 2008; Chen et al., 2009). Syntaphilin targets axonal mitochondria through its C-terminal mitochondria-targeting domain and axon-sorting sequence. Syntaphilin immobilizes axonal mitochondria by anchoring to MTs. Deleting syntaphilin results in a robust increase of axonal mitochondria in motile pools. Conversely, overexpressing syntaphilin abolishes axonal mitochondrial transport. These findings provide new mechanistic insight that motile mitochondria can be recruited into stationary pools via anchoring interactions between mitochondria-targeted syntaphilin and MTs. Thus, syntaphilin serves as an attractive molecular target for investigations into mechanisms recruiting motile mitochondria into activated synapses. The syntaphilin knockout mouse is an ideal genetic model to examine the impact of enhanced motility of axonal mitochondria on presynaptic function and mitochondrial quality control in distal axons.

Synaptic activity–dependent regulation

Mitochondrial transport and distribution in axons and at synapses are correlated with synaptic activity. Mitochondria are recruited to synapses in response to elevated intracellular Ca²⁺ during sustained synaptic activity. Elevated Ca²⁺ influx, either by activating voltage-dependent calcium channels or NMDA receptors, arrests mitochondrial movement (Rintoul et al., 2003; Yi et al., 2004; Chang et al., 2006; Szabadkai et al., 2006; Ohno et al., 2011). These studies raise a fundamental question: how are mitochondria recruited to synapses in response to Ca²⁺ influx? Recent work from three groups identified Miro as a Ca²⁺ sensor in regulating mitochondrial motility (Saotome et al., 2008; MacAskill et al., 2009a; Wang and Schwarz, 2009). Miro has four Ca²⁺-binding EF-hands (Klosowiak et al., 2013). Elevated cytosolic Ca²⁺ levels, by firing action potentials or activating glutamate receptors, arrest mitochondrial transport through a Miro-Ca²⁺–sensing pathway that inactivates or disassembles the KIF5–Miro–Trak transport machineries. By this mechanism, mitochondria are recruited to activated synapses. A Miro-Ca²⁺–sensing model was proposed in which KIF5 is loaded onto mitochondria through physical coupling via the motor adaptor complex Miro–Milton (or Miro–Trak) at resting Ca²⁺ levels. When motile mitochondria pass by an active synaptic terminal, the local elevated intracellular Ca²⁺ disrupts or inactivates the Miro–Trak–kinesin transport complex by binding to the EF-hand and changing Miro’s conformation; thus, mitochondria are immobilized at activated synapses (Fig. 2, C and D; MacAskill et al., 2009a; Wang and Schwarz, 2009). The two studies disagreed, however, as to whether kinesin remained associated with stationary mitochondria or whether the motor was completely decoupled from the organelle upon immobilization. In addition, Miro-Ca²⁺–dependent regulation affects both anterograde and retrograde transport. When KIF5 transport machinery is disrupted by the Miro-Ca²⁺–sensing mechanism, dynein-driven retrograde transport does not necessarily take over. It is unclear how the Miro-Ca²⁺–sensing pathway inactivates dynein transport machineries. Thus, the role of the Miro-Ca²⁺–sensing pathway in immobilizing mitochondria likely occurs through an anchoring mechanism.A recent study provides evidence to support the above-described hypothesis. Activating the Miro-Ca²⁺ pathway fails to arrest axonal mitochondria in syntaphilin-null hippocampal neurons: axonal mitochondria keep moving during neuronal firing while dendritic mitochondria are effectively immobilized (Chen and Sheng, 2013). This study suggests that syntaphilin-mediated anchoring plays a central role in the activity-dependent immobilization of axonal mitochondria. Syntaphilin competes with Trak2 for binding KIF5 in cells and inhibits the KIF5 motor ATPase activity in vitro, thus suggesting that syntaphilin functions to prevent KIF5 from moving along MTs. Synaptic activity and elevated calcium levels favor the anchoring interaction between syntaphilin and KIF5 by disrupting the Miro–Trak–kinesin transport complex. In addition, sustained neuronal activity induces syntaphilin translocation to axonal mitochondria, although the underlying mechanisms remain elusive. Expressing the syntaphilin mutant lacking this axon-sorting domain results in its distribution into all mitochondria, including those in the soma and dendrites (Kang et al., 2008). These studies collectively suggest a possibility that syntaphilin is delivered to axons before its translocation into mitochondria. One interesting question is how syntaphilin-anchored mitochondria are remobilized. Given the fact that syntaphilin is a cellular target of the E3 ubiquitin ligase Cullin 1 (Yen and Elledge, 2008), syntaphilin may be degraded by ubiquitin-mediated pathways. It is necessary to further investigate the mechanisms underlying activity-induced syntaphilin translocation and its subsequent removal from mitochondria.A new “engine-switch and brake” model (Fig. 2 E) was recently proposed (Chen and Sheng 2013). In response to a “stop” sign (elevated Ca²⁺) at active synapses, syntaphilin switches off the kinesin motor and puts a brake on mitochondria, thereby anchoring them in place on MTs. Thus, KIF5-loaded mitochondria remain associated with the axonal MT track while in their stationary phase. This model may help reconcile the standing disagreements regarding how Miro-Ca²⁺ sensing immobilizes mitochondria in neurons. MacAskill et al. (2009a) propose that Ca²⁺ binding to Miro disconnects KIF5 motors from mitochondria in dendrites by activating glutamate receptors (Fig. 2 D), whereas Wang and Schwarz (2009) suggest that KIF5 remains associated with immobilized mitochondria within axons during elevated Ca²⁺ conditions (Fig. 2 C). These discrepant results are likely due to their selective observations of mitochondrial motility in dendrites versus axons. Upon Miro-Ca²⁺ sensing, KIF5 motors remain associated with axonal mitochondria by binding syntaphilin, which is absent from dendritic mitochondria (Fig. 2 E). This difference may help axonal mitochondria be more responsive to changes in synaptic activity. If the Ca²⁺ signal is removed, the cargo-loaded motor proteins can be quickly reactivated to move mitochondria to new active synapses. This model also suggests that motor loading on mitochondria is insufficient for transport. The release of the anchoring interaction is also required. The latter is supported by a study that both KIF5 and dynein motors remain bound on prion protein vesicles regardless of whether they are motile or stationary (Encalada et al., 2011). A newly reported Alex3 protein, organized in a genomic Armcx cluster encoding mitochondria-targeted proteins, regulates mitochondrial trafficking in neurons by interacting with the KIF5–Miro–Trak complex in a Ca²⁺-dependent manner (López-Doménech et al., 2012). Although the underlying mechanism is unknown, the study highlights the complexity in controlling mitochondrial motility in response to various physiological signals in neurons.

Mitochondrial motility and synaptic variability

Mitochondria are commonly found within synaptic terminals (Shepherd and Harris, 1998), where they power neurotransmission by supplying ATP. A stationary mitochondrion within presynaptic terminals provides a stable and continuous ATP supply and maintains energy homeostasis at synapses. Conversely, a motile mitochondrion passing by presynaptic boutons dynamically alters local ATP levels, thus influencing various ATP-dependent functions at synapses such as: (1) establishing the proton gradient necessary for neurotransmitter loading; (2) removing Ca²⁺ from nerve terminals; and (3) driving synaptic vesicle transport from reserve pools to release sites. For example, Drosophila photoreceptors expressing mutant Milton display impaired synaptic transmission due to reduced mitochondrial trafficking to synapses (Stowers et al., 2002). Mutation of Drosophila Drp1 reduces synaptic localization of mitochondria and results in faster depletion of synaptic vesicles during prolonged stimulation (Verstreken et al., 2005). Addition of ATP to terminals partially rescues these defects, indicating that mitochondrial ATP production is required for maintaining sustained synaptic transmission. Syntabulin loss of function reduces mitochondrial density in axonal terminals, which is accompanied by accelerated synaptic depression and slowed synapse recovery during high-frequency firing (Ma et al., 2009).One of the most notable characteristics of synaptic transmission is the wide variation in synaptic strength in response to repeated stimulations. The effects of synaptic variability on neuronal circuit activity are increasingly recognized. Some degree of synaptic variability may be necessary for signal processing in flexible or adaptive systems (Murthy et al., 1997). A long-standing fundamental question is how the variation in synaptic strength arises. There is general agreement that some structural and molecular stochastic alternations at synapses are the basis for heterogeneity in synaptic transmission from neuron to neuron or from synapse to synapse (Atwood and Karunanithi, 2002; Stein et al., 2005; Marder and Goaillard, 2006; Branco and Staras, 2009; Ribrault et al., 2011). However, it is unknown whether mitochondrial motility at axonal terminals contributes to pulse-to-pulse variability of presynaptic strength at single-bouton levels. This is particularly relevant in hippocampal neurons, where approximately one third of axonal mitochondria are highly motile, some of which dynamically pass through presynaptic boutons.We recently solved this puzzle by combining dual-channel imaging in live neurons and electrophysiological analysis in syntaphilin knockout hippocampal neurons and acute hippocampal slices in which axonal mitochondrial motility is selectively manipulated (Sun et al., 2013). Our study revealed that the motility of axonal mitochondria is one of the primary mechanisms underlying the variability of presynaptic strength. Enhanced motility of axonal mitochondria increases the pulse-to-pulse variability of EPSC amplitudes, whereas immobilizing axonal mitochondria effectively diminishes the variability. The data raise two assumptions: (1) EPSC amplitudes are averaged through summation from multiple synapses; and (2) release at individual boutons changes over time when mitochondrial distribution and motility are dynamically altered. Thus, at any given time these boutons display different patterns of mitochondrial distribution and motility; mitochondria move either in or out of boutons or pass through boutons during stimulation, thus contributing to the pulse-to-pulse variability. By applying dual-channel live imaging of mitochondria and synaptic vesicle release at single-bouton levels, the study further shows that mitochondrial movement, either into or out of presynaptic boutons, significantly influences synaptic vesicle release due to fluctuation of synaptic ATP levels. In the absence of a stationary mitochondrion within an axonal terminal, there is no constant on-site ATP supply. A motile mitochondrion passing through could temporally supply ATP, thus changing synaptic energy levels and influencing ATP-dependent processes including synaptic vesicle cycling, mobilization, and replenishment (Fig. 3). This is further supported by a recent study applying a new quantitative optical presynaptic ATP reporter. Synaptic activity drives large ATP consumption at nerve terminals and synaptic vesicle cycling consumes most presynaptic ATP (Rangaraju et al., 2014). It is possible that all of these ATP-dependent processes collectively contribute to presynaptic variability. Therefore, the fluctuation of synaptic ATP levels resulting from mitochondrial movement is one of the primary sources for the wide variability of EPSC amplitudes. This study revealed, for the first time, that the dynamic movement of axonal mitochondria is one of the primary mechanisms underlying the presynaptic variation in the CNS, thus providing new insight into the fundamental properties of the CNS to ensure the plasticity and reliability of synaptic transmission (Sun et al., 2013).Open in a separate windowFigure 3.Motile mitochondria passing by synapses contribute to presynaptic variation. A stationary mitochondrion within presynaptic terminals powers neurotransmission by stable and continuous ATP supply (left). Conversely, in the absence of a mitochondrion within a presynaptic bouton (right), there is no stable on-site ATP supply; a motile mitochondrion passing through this bouton temporally supplies ATP, thus changing synaptic energy levels and influencing ATP-dependent synaptic functions over time when mitochondrial distribution and motility are altered. Therefore, the fluctuation of synaptic ATP levels resulting from mitochondrial movement is one of the primary sources for the wide variability of synaptic vesicle release and amplitudes of excitatory postsynaptic currents (EPSCs; Sun et al., 2013).

Mitochondrial motility, neuronal morphology, and energy consumption

Proper mitochondrial transport into neurites in developing neurons is tightly regulated to ensure that metabolically active areas are adequately supplied with ATP (Morris and Hollenbeck, 1993). A recent study showed the differential functions of two mitochondrial motor adaptors, Trak1 and Trak2, in driving polarized mitochondrial transport (van Spronsen et al., 2013). Trak1 is prominently localized in axons and is required for axonal mitochondria transport by binding to both kinesin-1 and dynein/dynactin, whereas Trak2 is present in dendrites and mainly transports mitochondria into dendrites by interacting with dynein/dynactin. The folded configuration of Trak2 preferentially binds dynein motors rather than kinesin-1. Consistently, depleting Trak1 inhibits axonal outgrowth, whereas suppressing Trak2 impairs dendrite morphology. Thus, this study established a new role of Trak proteins in polarized mitochondrial targeting necessary to maintain neuronal morphology.Anchored stationary mitochondria ideally serve as local energy power plants. Increased intracellular [ADP]_i slows down mitochondrial movement and recruits mitochondria to subcellular regions correlated with local [ATP]_i depletion (Mironov, 2007). However, the underlying mechanisms coordinating mitochondria transport and energy consumption remain elusive. The balance between motile and stationary mitochondria responds quickly to changes in axonal growth status. Two recent studies demonstrated that generating and maintaining axonal branching depend upon stable and continuous ATP supply by stationary mitochondria captured at branching points. Courchet et al. (2013) revealed that LKB1, the serine/threonine liver kinase B1, regulates terminal axon branching of cortical neurons in both in vitro and in vivo systems through activating downstream kinase NUAK1, an AMPK-like kinase. AMPK is an AMP-activated protein kinase, which is a master regulator of cellular energy homeostasis and is activated in response to stresses that deplete cellular ATP supplies. Deleting LKB1 or NUAK1 results in a threefold decrease in stationary pools of axonal mitochondria, whereas overexpressing LKB1 or NUAK1 conversely increases the proportion of immobilized mitochondria along axons, thus establishing a causal correlation between stationary mitochondria and axonal branching. To test the hypothesis, Courchet et al. (2013) altered the motility of axonal mitochondria by changing expression levels of syntaphilin. Intriguingly, suppressing syntaphilin expression in cortical progenitors at E15.5 enhances axonal mitochondrial motility accompanied by decreased axon branching. Conversely, overexpressing syntaphilin immobilizes axonal mitochondria and increases axon branching, thus highlighting a critical role for syntaphilin-mediated mitochondrial anchoring in AMPK-induced axonal branching. It is possible that LKB1 and NUAK1-mediated signaling pathways regulate axon branch formation and stabilization by recruiting mitochondria to branch points. However, this study raises one mechanistic question: does syntaphilin or the mitochondrial transport motor–adaptor complex serve as a downstream effector of this signaling pathway? Syntaphilin is strictly developmentally regulated in mouse brains, with low expression before postnatal day 7 and peaking two weeks after birth; thus, its expression is hardly detectable in early embryonic stages (Kang et al., 2008). It remains unclear how siRNA application at E15.5 significantly impacts endogenous syntaphilin levels in the brain. Furthermore, it is unknown whether local ATP homeostasis maintained by stationary but not motile mitochondria contributes to the formation and stabilization of axonal branching.As a cellular energy sensor for detecting increased intracellular [AMP]_i or ATP consumption, AMPK activation positively regulates signaling pathways that replenish cellular ATP supplies. Tao et al. (2013) provide further evidence that sustained neuronal depolarization for 20 min depletes cellular ATP and activates AMPK. Activation of AMPK increases anterograde flux of mitochondria into axons and induces axonal branching in regions where mitochondria are docked in an ATP-dependent manner. The mitochondrial uncoupler FCCP blocks axonal branching, even when mitochondria are localized nearby. These studies provide evidence for how the AMPK signaling pathway regulates axon branch formation and stabilization. These studies collectively highlight a critical role for syntaphilin-mediated mitochondrial anchoring in axon branching. One possible mechanism underlying mitochondrial recruiting and anchoring in support of axon branching is through localized intra-axonal protein synthesis (Spillane et al., 2013). Inhibiting mitochondrial respiration and protein synthesis in axons impairs axonal branching. Thus, stationary mitochondria coordinate maturation of axonal filopodia into axon branching likely by supporting active mRNA translation in axonal hot spots. Activity-induced mitochondrial recruitment in axons is further supported by an in vivo study in mouse saphenous nerve axons. Sustained electrical stimulation (50 Hz) enhances anterograde flux of mitochondria into myelinated axons and redistributes them at the peripheral terminals (Sajic et al., 2013).

Mitochondrial motility and mitophagy

Cumulative evidence indicates mitochondrial dysfunction, impaired transport, altered dynamics, and perturbation of mitochondrial turnover are associated with the pathology of major neurodegenerative disorders (Chen and Chan, 2009; Sheng and Cai, 2012). Defective mitochondrial transport accompanied by mitochondrial pathology is one of the most notable pathological changes in several aging-associated neurodegenerative diseases, including Alzheimer’s disease (Rui et al., 2006; Du et al., 2010; Calkins et al., 2011) and amyotrophic lateral sclerosis (De Vos et al., 2007; Magrané and Manfredi, 2009; Bilsland et al., 2010; Shi et al., 2010; Martin, 2011; Zhu and Sheng, 2011; Marinkovic et al., 2012). Dysfunctional mitochondria not only supply less ATP and maintain Ca²⁺ buffering capacity less efficiently, but also release harmful reactive oxygen species (ROS). Defects in mitochondrial transport reduce the delivery of healthy mitochondria to distal processes and also impair removal of damaged mitochondria from synapses, thus causing energy depletion and altered Ca²⁺ transient at synapses. In addition, toxic ROS may further trigger synaptic stress, thereby contributing to neurodegeneration. Mitochondrial quality control involves multiple levels of surveillance to ensure mitochondrial integrity, including repairing dysfunctional mitochondria by fusion with healthy ones (Chen and Chan 2009; Westermann, 2010) and segregating irreversibly damaged mitochondria for mitophagy, a cargo-specific autophagy to remove damaged mitochondria. Effective sequestration of damaged mitochondria into autophagosomes and subsequent clearance within the autophagy–lysosomal system constitute a key cellular pathway in mitochondrial quality control mechanisms. Recent studies indicate that PINK1/Parkin-mediated pathways ensure mitochondrial integrity and function (Clark et al., 2006; Gautier et al., 2008; Narendra et al., 2008). Loss-of-function mutations in PINK1 and Parkin are associated with recessive forms of Parkinson’s disease. When mitochondria are depolarized or their integrity is damaged, PINK1 accumulates on the outer mitochondrial membrane and recruits the E3 ubiquitin ligase Parkin from the cytosol. Parkin ubiquitinates mitochondrial proteins (Geisler et al., 2010; Poole et al., 2010; Ziviani et al., 2010; Yoshii et al., 2011). This process is essential for damaged mitochondria to be engulfed by isolation membranes that then fuse with lysosomes for degradation.Investigating those mechanisms coordinating mitochondrial motility and mitophagy represents an important emerging area for disease-oriented research. Mature acidic lysosomes are mainly located in the soma (Overly and Hollenbeck, 1996; Cai et al., 2010; Lee et al., 2011). Thus, a long-standing question remains: how are aged and damaged mitochondria at distal terminals efficiently eliminated via the autophagy–lysosomal pathway? Cai et al. (2012) recently reported the unique features of spatial and dynamic Parkin-mediated mitophagy in mature cortical neurons, in which mitochondria remain motile after prolonged and chronic dissipation of the mitochondrial membrane potential (Δψ_m) for 24 h. Given the fact that in vitro mitochondrial depolarization will quickly trigger neuron apoptosis, a high-quality culturing system is essential so that the neurons survive long enough to exhibit altered mitochondrial transport and Parkin-mediated mitophagy. Chronically dissipating Δψ_m by prolonged treatment of low concentrations of Δψ_m-uncoupling reagent CCCP (10 µM) results in accumulation of Parkin-targeted mitochondria in the soma and proximal regions. Such compartmental restriction for Parkin-targeted mitochondria is consistently observed in neurons treated with low concentrations of another Δψ_m-dissipating reagent, antimycin A (1 µM), or mitochondrial ATP synthase inhibitor oligomycin (1 µM). This distribution pattern is attributable to altered motility of chronically depolarized mitochondria with reduced anterograde and relatively enhanced retrograde transport, thus reducing anterograde flux of damaged mitochondria into distal processes. Intriguingly, when anchored by syntaphilin before the dissipation of Δψ_m, mitochondria in distal axons also recruit Parkin for mitophagy. Altered motility may be protective under chronic mitochondrial stress conditions; healthy mitochondria remain distally while aged and damaged ones return to the soma for degradation (Fig. 4). This spatial distribution allows neurons to efficiently remove dysfunctional mitochondria distally and then eliminate them via the autophagy–lysosomal pathway in the soma.Open in a separate windowFigure 4.Functional interplay between mitochondrial transport and mitophagy. Chronically dissipating mitochondrial membrane potential (Δψ_m) by prolonged treatment of low concentrations of Δψ_m-uncoupling reagents accumulates Parkin-targeted mitochondria in the soma and proximal regions. Such compartmental restriction is a result of altered motility of depolarized mitochondria with reduced anterograde and relatively enhanced retrograde transport, thus reducing anterograde flux of damaged mitochondria into distal processes (Cai et al., 2012). This spatial process allows neurons to efficiently remove dysfunctional mitochondria from distal axons via the autophagy–lysosomal pathway in the soma, where mature lysosomes are relatively enriched. Damaged mitochondria at axonal terminals can also recruit Parkin for mitophagy once they are anchored by syntaphilin (Cai et al., 2012) or immobilized by turnover of the motor adaptor Miro on the mitochondrial surface (Weihofen et al., 2009; Chan et al., 2011; Wang et al., 2011; Yoshii et al., 2011; Liu et al., 2012; Sarraf et al., 2013). Autophagosomes including those engulfing damaged mitochondria at axonal terminals transport predominantly to the soma for maturation and more efficient degradation of cargoes within acidic lysosomes (Maday et al., 2012).Reduced anterograde transport of depolarized mitochondria is consistent with recent studies showing Parkin-mediated degradation of Miro on the mitochondrial surface upon Δψ_m dissipation–induced mitophagy. In addition to binding to the KIF5–Trak motor complex, the mitochondrial outer membrane protein Miro also interacts with PINK1 and Parkin and is ubiquitinated by Parkin while mitochondria are depolarized (Weihofen et al., 2009; Chan et al., 2011; Wang et al., 2011; Liu et al., 2012; Sarraf et al., 2013). These studies support the current model that altered mitochondrial transport is a critical aspect of the mechanisms by which the PINK1/Parkin pathway governs mitochondrial quality control. Turnover of Miro on the mitochondrial surface may favor their retrograde transport to the soma or immobilize damaged organelles at distal regions for mitophagy (Fig. 4). A recent study reports the predominant retrograde transport of autophagosomes including those engulfing damaged mitochondria from distal axons to the soma (Maday et al., 2012). The retrograde movement is critical for autophagosome maturation and degradation within acidic lysosomes in the proximal regions of neurons. This study highlights the possibility that distal damaged mitochondria, which are either anchored by syntaphilin or immobilized by turning over Miro, may be also degraded in the soma after their retrograde transport in engulfed autophagosomes. Thus, a functional interplay is proposed between mitochondrial motility and mitophagy to ensure proper removal of aged and dysfunctional mitochondria from distal processes. The correlation between mitochondrial Δψ_m and motility is controversial, as demonstrated in two previous studies by acutely treating neurons with high concentrations of Δψ_m-dissipating reagents (Miller and Sheetz, 2004; Verburg and Hollenbeck, 2008). Instead, Cai et al. (2012) examined mitochondrial motility and mitophagy in mature cortical neurons after a 24-h treatment with a much lower concentration of CCCP (10 µM) or antimycin A (1 µM) combined with applying the pan-caspase inhibitor Z-VAD. The majority of neurons survive and mitochondria remain highly motile under such prolonged stress conditions, thus allowing detection of the altered transport event. Therefore, prolonged Δψ_m dissipation in cultured neurons better reflects chronic mitochondria stress under in vivo physiological or pathological conditions.

Moving forward

The recent discoveries of proteins involved in mitochondrial transport and anchoring boosted our understanding of the molecular mechanisms that regulate mitochondrial motility and distribution in response to physiological and pathological signals. However, there are still many mechanistic questions to be addressed in the near future. In particular, how does a Miro-Ca²⁺–sensing pathway arrest both anterograde and retrograde movement of mitochondria at active synapses? How do dynein and KIF5 motors physically or mechanistically interplay to coordinate mitochondrial bi-directional transport? How are stationary mitochondria remobilized, recruited, and redistributed (and vice versa) to sense, integrate, and respond to changes in mitochondrial membrane potential, cellular metabolic status, neuronal growth signals, and pathological stress? Investigating how mitochondrial motility coordinates their function and integrity in neurons represents an important emerging frontier in neurobiology. Mitochondria undergo dynamic membrane fission/fusion and mitophagy to maintain their quality during the neuronal lifetime. Mitochondrial pathology is one of the most notable hallmarks in several major aging-associated neurodegenerative diseases (Chen and Chan, 2009; Schon and Przedborski, 2011; Court and Coleman, 2012; Sheng and Cai, 2012; Itoh et al., 2013). The proper and efficient elimination of damaged mitochondria via mitophagy may serve as an early neuroprotective mechanism. Studying these dynamic processes in live neurons directly isolated from disease-stage adult mice will provide more reliable models for delineating defects in mitochondrial transport and mitophagy underlying adult-onset pathogenesis. Mechanistic insight into these fundamental processes in coordinating mitochondrial trafficking and anchoring, membrane dynamics, and mitophagy will advance our understanding of human neurodegenerative diseases.     

Autophagy and mitochondrial alterations in human retinal pigment epithelial cells induced by ethanol: implications of 4-hydroxy-nonenal

Retinal pigment epithelium has a crucial role in the physiology and pathophysiology of the retina due to its location and metabolism. Oxidative damage has been demonstrated as a pathogenic mechanism in several retinal diseases, and reactive oxygen species are certainly important by-products of ethanol (EtOH) metabolism. Autophagy has been shown to exert a protective effect in different cellular and animal models. Thus, in our model, EtOH treatment increases autophagy flux, in a concentration-dependent manner. Mitochondrial morphology seems to be clearly altered under EtOH exposure, leading to an apparent increase in mitochondrial fission. An increase in 2′,7′-dichlorofluorescein fluorescence and accumulation of lipid peroxidation products, such as 4-hydroxy-nonenal (4-HNE), among others were confirmed. The characterization of these structures confirmed their nature as aggresomes. Hence, autophagy seems to have a cytoprotective role in ARPE-19 cells under EtOH damage, by degrading fragmented mitochondria and 4-HNE aggresomes. Herein, we describe the central implication of autophagy in human retinal pigment epithelial cells upon oxidative stress induced by EtOH, with possible implications for other conditions and diseases.Retinal pigment epithelium (RPE) is a single neuroectodermal layer placed in the outermost part of the eye cup faced to photoreceptors.^{1, 2} Owing to its anatomical location and function, RPE is continuously exposed to potential cell damage caused by oxidative stress, specifically due to oxygen and nitrogen reactive species.³ This is probably one of the reasons why these cells are more resistant to oxidative stress.⁴ Oxidative stress is present as part of the pathophysiology in several retinal degenerations associated with blindness, for example, age-related macular degeneration,³ where RPE is considered a key factor for its development.⁵ Studies with the human-derived cell line ARPE-19 have proven to be very useful in the elucidation of the role of these cells in disease.Autophagy is a catabolic process aimed to degrade damaged organelles, proteins and cellular debris by engulfing them into a double membrane vesicle called the autophagosome and eliminating them by posterior fusion with the lysosome. Activation of macroautophagy, a form of autophagy, has been recently confirmed to be a primary response of ARPE-19 cells to stress.⁶ Furthermore, the two major functions of RPE, phagocytosis of the photoreceptor outer segments and visual cycle performance, have been linked to a noncanonical form of autophagy that is known as LC3 (microtubule-associated protein 1A/1B-light chain 3)-associated phagocytosis and is supposed to contribute to the normal supply of vitamin A and therefore to normal vision.^{7, 8}Despite its negative effects on health, ethanol (EtOH) is consumed daily worldwide, standing as one of the top public health challenges. EtOH induces morphological and physiological changes in the nervous tissue, and most of these changes may be attributed to reactive oxygen species (ROS), as they can be normalized or prevented by antioxidant treatments.^{9, 10, 11, 12, 13} Autophagy has been identified as cytoprotector in nervous and liver cells under EtOH-induced toxicity,^{14, 15} where it seems to degrade damaged organelles, including mitochondria. Recent findings support the idea that there is an increased mitochondrial stress and dysfunction in the RPE cells in AMD patients.^{16, 17} Oxidative-damaged mitochondria, a main source of ROS, seem to be removed by autophagy (known as mitophagy), in order to guarantee cell survival.¹⁸ As a matter of fact, deregulation of mitophagy has been implicated in several neurodegenerative diseases, such as Parkinson''s disease (PD), Alzheimer''s disease (AD) and Huntington''s disease (HD).Peroxidation of polyunsaturated fatty acids is intensified in cells subjected to oxidative stress, and results in the generation of various bioactive compounds, among others 4-hydroxyalkenals (HAE). ROS-induced lipid peroxidation and the resulting HAE markedly contribute to the development and progression of different diseases.¹⁹ Specifically, 4-hydroxy-nonenal (4-HNE), a major α,β-unsaturated aldehyde product of n-6 fatty acid oxidation, has been shown to be involved in a great number of maladies.²⁰ It has been reported that 4-HNE induces apoptosis in ARPE-19 cells²¹ and its ability to form protein adducts, thus it seems to be a key factor in aggresome formation. Aggresome is a term referred to cytoplasmic perinuclear inclusion bodies formed by aggregated proteins.²² Indeed, the presence of aggresomes is a pathological hallmark of most neurodegenerative diseases, and 4-HNE seems to be involved in their formation in AD,²³ PD,²⁴ HD²⁵ and amyotrophic lateral sclerosis.²⁶ These aggresomes depend on the protein type to be cleared,^{27, 28} and their degradation by autophagy, known as aggrephagy, has been proposed to increase cell viability in neurodegeneration models.²⁹ Interestingly, 4-HNE aggregates have been also found in hepatic cells from alcoholic patients.^{30, 31, 32} Recent data provide no clear cut evidence of a link between PD risk and alcohol consumption with both positive³³ and negative³⁴ results.In this study, we report the cellular effects of EtOH on ARPE-19 cells and determine that EtOH exposure induces the formation of 4-HNE-aggresomes, together with other neurodegenerative hallmarks such as mitochondrial damage and autophagy activation. Considering the central role of RPE in retinal physiology and pathophysiology, and its neural origin, these findings render new insights into the mechanism of neurodegeneration caused by alcohol toxicity in retinal cells, and may contribute to the development of therapeutic strategies in several nervous and retinal 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.     

The protease Omi regulates mitochondrial biogenesis through the GSK3β/PGC-1α pathway

Loss of the mitochondrial protease activity of Omi causes mitochondrial dysfunction, neurodegeneration with parkinsonian features and premature death in mnd2 (motor neuron degeneration 2) mice. However, the detailed mechanisms underlying this pathology remain largely unknown. Here, we report that Omi participates in the process of mitochondrial biogenesis, which has been linked to several neurodegenerative diseases. The mitochondrial biogenesis is deficit in mnd2 mice, evidenced by severe decreases of mitochondrial components, mitochondrial DNA and mitochondrial density. Omi cleaves glycogen synthase kinase 3β (GSK3β), a kinase promoting PPARγ coactivator-1α (PGC-1α) degradation, to regulate PGC-1α, a factor important for the mitochondrial biogenesis. In mnd2 mice, GSK3β abundance is increased and PGC-1α abundance is decreased significantly. Inhibition of GSK3β by SB216763 or overexpression of PGC-1α can restore mitochondrial biogenesis in mnd2 mice or Omi-knockdown N2a cells. Furthermore, there is a significant improvement of the movement ability of mnd2 mice after SB216763 treatment. Thus, our study identified Omi as a novel regulator of mitochondrial biogenesis, involving in Omi protease-deficient-induced neurodegeneration.Mitochondria have a vital role in neuronal death and survival.¹ As critical cellular organelles, mitochondria have highly dynamic properties, including mitochondrial fission, fusion, transport, biogenesis and degradation. The changes of those properties affect mitochondrial functions, leading to the occurrence of diseases.^{2, 3} Growing lines of evidence suggest that the mitochondrial dysfunction is involved in aging and neurodegenerative diseases, such as Alzheimer''s disease (AD), Huntington''s disease (HD) and Parkinson''s disease (PD).^{4, 5} Similar to other neurodegenerative diseases, PD is a progressive neurological disorder, which is characterized by the development of cytoplasmic aggregates known as Lewy bodies and degeneration of dopaminergic (DA) neurons in the substantia nigra of midbrain and other brain regions.⁶ In PD, dysfunction of mitochondria has been documented to be associated with disease pathogenesis in PD brains and both genetic- and toxin-induced PD animal models. In PD brains, mutations in mitochondrial DNA (mtDNA) occur more frequently than those in age-matched control; and mutations in the nuclear-encoded mtDNA polymerase-γ gene, which impair mtDNA replication and result in multiple mtDNA deletions, cause PD-like symptoms.⁵ Meanwhile, several PD-associated gene products, including α-synuclein, parkin, DJ-1, PINK1 (PTEN-induced putative kinase 1), leucine-rich repeat kinase 2, ubiquitin carboxy-terminal hydrolase L1 and Omi, have been identified to be associated with PD, and lead to mitochondrial dysfunction with changes in mitochondrial morphology, biogenesis and mitophagy in vivo and in vitro.^{5, 7, 8, 9} Besides, mitochondrial toxins, such as MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and rotenone that inhibit complex I of the mitochondrial respiratory chain, cause clinically parkinsonian phenotype.^{10, 11}The serine protease Omi (also known as HtrA2) belongs to the high-temperature requirement factor A (HtrA) family, and was originally identified as a mammalian homolog of the Escherichia coli heat-shock-induced serine protease HtrA/DegP and DegS.¹² Omi is mainly localized in mitochondria, although a fraction of it is also found in nucleus.¹³ Omi is released from the mitochondria into the cytosol to mediate cell death by caspase-dependent or -independent pathways in response to apoptotic stimuli.^{14, 15} However, the notion that Omi is an apoptosis inducer in the central nervous system was challenged by studies of Omi-overexpressing or -deficient mice. Omi-overexpressing mice show normal development without any sign of apoptotic cell death.¹⁶ On the other hand, mnd2 (motor neuron degeneration 2) mice that harbor protease-deficient Omi S276C mutants, and Omi-knockout mice both suffer from progressive neurodegeneration, especially in striatum, and motor abnormalities similar to PD. Both mice fail to gain weight and die before postnatal day 40 due to neurodegeneration with progressive mitochondrial damage.^{17, 18, 19} Besides, mutations in the Omi gene have also been identified in PD patients.^{20, 21} Previous studies have shown that Omi has a vital role in the mitochondrial integrity, and the loss of protease activity leads to mitochondrial dysfunction, such as abnormal mitochondrial morphology and increased mtDNA mutation and deletions, increased susceptibility of mitochondrial membrane permeabilization, decreased mitochondrial membrane potential, and reduced mitochondrial density in mnd2 mice and Omi-knockout mice.^{17, 18, 22} Omi has been found to act downstream of PINK1, but parallel to parkin, in a mitochondrial stress sensing pathway to sense the different stresses, which may be defective in PD.²³ These findings suggest that the primary function of Omi is involved in neuroprotection, especially in the maintenance of mitochondrial homeostasis.^{23, 24}In this article, we identified that Omi cleaves glycogen synthase kinase 3β (GSK3β) to regulate PPARγ coactivator-1α (PGC-1α) abundance and to ensure mitochondrial biogenesis.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

CHCHD2 inhibits apoptosis by interacting with Bcl-x L to regulate Bax activation

Mitochondrial outer membrane permeabilization (MOMP) is a critical control point during apoptosis that results in the release of pro-apoptotic mitochondrial contents such as cytochrome c. MOMP is largely controlled by Bcl-2 family proteins such as Bax, which under various apoptotic stresses becomes activated and oligomerizes on the outer mitochondrial membrane. Bax oligomerization helps promote the diffusion of the mitochondrial contents into the cytoplasm activating the caspase cascade. In turn, Bax is regulated primarily by anti-apoptotic Bcl-2 proteins including Bcl-xL, which was recently shown to prevent Bax from accumulating at the mitochondria. However, the exact mechanisms by which Bcl-xL regulates Bax and thereby MOMP remain partially understood. In this study, we show that the small CHCH-domain-containing protein CHCHD2 binds to Bcl-xL and inhibits the mitochondrial accumulation and oligomerization of Bax. Our data show that in response to apoptotic stimuli, mitochondrial CHCHD2 decreases prior to MOMP. Furthermore, when CHCHD2 is absent from the mitochondria, the ability of Bcl-xL to inhibit Bax activation and to prevent apoptosis is attenuated, which results in increases in Bax oligomerization, MOMP and apoptosis. Collectively, our findings establish CHCHD2, a previously uncharacterized small mitochondrial protein with no known homology to the Bcl-2 family, as one of the negative regulators of mitochondria-mediated apoptosis.Apoptosis is a tightly regulated form of programmed cell death that is critical for proper embryonic development, tissue homeostasis and immune response. Aberrant regulation of apoptosis contributes to a wide range of ailments including autoimmune disorders, neurodegenerative diseases and cancer. Unlike necrotic cell death, apoptosis is a genetic program that is characterized by distinct morphological features such as membrane blebbing, chromatin condensation, DNA fragmentation and cell shrinkage.¹ In vertebrates, apoptosis can occur through two pathways: extrinsic, or receptor-mediated apoptosis, and intrinsic, or mitochondria-mediated apoptosis. Intrinsic apoptosis is induced by cellular stressors such as DNA damage, which lead to mitochondrial outer membrane permeabilization (MOMP), cytochrome c release from the mitochondrial intermembrane space, activation of cysteine proteases (caspases) and induction of apoptosis. Once MOMP occurs, cell death is thought to be inevitable. Therefore, much research has been devoted to elucidating the mechanisms and signaling pathways that govern this critical regulatory point in apoptosis.MOMP is controlled largely by the B-cell lymphoma 2 (Bcl-2) family of proteins,² all of which contain at least one of four BH (Bcl-2 homology) domains designated BH1–4. During apoptosis, the pro-apoptotic Bcl-2 proteins Bax and/or Bak become activated and oligomerize on the mitochondrial outer membrane³ increasing mitochondrial membrane permeabilization through a mechanism that is not entirely clear. Bax and Bak are activated by BH3-only Bcl-2 family proteins such as Bim, t-Bid and Puma.^{4, 5, 6, 7, 8, 9, 10, 11, 12, 13} Conversely, Bax and Bak are inhibited by pro-survival Bcl-2 family proteins such as Bcl-2, Mcl-1 and Bcl-xL.^{2, 14, 15, 16} Of the pro-survival Bcl-2 family proteins, Bcl-2 is found at the outer mitochondrial membrane, whereas Bcl-xL and Mcl-1 localize to the outer mitochondrial membrane and the mitochondrial matrix.^{17, 18} Matrix-localized Bcl-xL and Mcl-1 have been shown to promote mitochondrial respiration,¹⁹ suggesting that crosstalk exists between apoptotic pathways and other mitochondria-based biological events. Based on this recent discovery, one might reason that other mitochondrial proteins previously characterized as structural proteins or metabolism-associated enzymes could play an additional intermediate role in the regulation of apoptosis by interacting with Bcl-2 family proteins.We identified CHCHD2 in a mass spectrometry-based screen for binding partners of p32, a mitochondrial protein previously shown by our lab to bind and mediate the apoptotic effects of the tumor suppressor p14ARF.²⁰ CHCHD2 was subsequently detected in independent screens for proteins that regulate cellular metabolism and migration;^{21, 22} however, the functions of CHCHD2 remain unknown. CHCHD2 is encoded by the chchd2 gene (coiled-coil helix coiled-coil helix domain-containing 2), which spans 4921 base pairs, contains 4 exons, and is located on human chromosome 7p11.2, a chromosomal region that is often amplified in glioblastomas.²³ The protein encoded by the chchd2 gene is ubiquitously expressed²⁴ and is relatively small, as it codes for only 151 amino acids. CHCHD2 is well-conserved among different species from humans to yeast, and mouse and human CHCHD2 share 87% amino acid sequence identity (Supplementary Figures S1A and S1B). CHCHD2 contains a C-terminal CHCH (coiled-coil helix coiled-coil helix) domain, which is characterized primarily by four cysteine residues spaced 10 amino acids apart from one another (CX(9)C motif).²⁵ The function of the CHCH domain is not well understood, and the few characterized proteins that harbor this domain have diverse functions. Many CHCH domain-containing proteins localize to the mitochondrial inner membrane or the intermembrane space, including Cox12, Cox17, Cox19, Cox23, Mia40 (yeast homolog of human CHCHD4), CHCHD3 and CHCHD6. Cox17 and Cox19 aid in the assembly of the COX complex,^{26, 27} whereas Mia40/Tim40 has been shown to transport proteins into the mitochondrial intermembrane space.^{28, 29} Furthermore, CHCHD3 and CHCHD6 are essential for maintaining the integrity of mitochondrial cristae and thus mitochondrial function.^{30, 31, 32} Interestingly, a recent report has shown that CHCHD6 is regulated by DNA damage stress, and alterations in CHCHD6 expression affect the viability of breast cancer cells in response to genotoxic anticancer drugs.³²Despite advances in our understanding of how MOMP and apoptosis are regulated by the Bcl-2 family of proteins, much remains unknown with respect to the mechanisms that lead to Bax activation and oligomerization particularly concerning the roles that mitochondria-associated proteins play in the process. In this study, we characterize the small, mitochondria-localized protein CHCHD2 as a novel regulator of Bax oligomerization and apoptosis. Furthermore, we show evidence that CHCHD2 binds to Bcl-xL at the mitochondria under unstressed conditions. In response to apoptotic stimuli, CHCHD2 decreases and loses its mitochondria localization, which is accompanied by decreased Bcl-xL–Bax interaction and increased Bax homo-oligomerization and Bax–Bak hetero-oligomerization. Collectively, our results suggest that CHCHD2 negatively regulates the apoptotic cascade upstream of Bax oligomerization.     

Genetic deletion of the mitochondrial phosphate carrier desensitizes the mitochondrial permeability transition pore and causes cardiomyopathy

The mitochondrial phosphate carrier (PiC) is critical for ATP synthesis by serving as the primary means for mitochondrial phosphate import across the inner membrane. In addition to its role in energy production, PiC is hypothesized to have a role in cell death as either a component or a regulator of the mitochondrial permeability transition pore (MPTP) complex. Here, we have generated a mouse model with inducible and cardiac-specific deletion of the Slc25a3 gene (PiC protein). Loss of PiC protein did not prevent MPTP opening, suggesting it is not a direct pore-forming component of this complex. However, Slc25a3 deletion in the heart blunted MPTP opening in response to Ca²⁺ challenge and led to a greater Ca²⁺ uptake capacity. This desensitization of MPTP opening due to loss or reduction in PiC protein attenuated cardiac ischemic-reperfusion injury, as well as partially protected cells in culture from Ca²⁺ overload induced death. Intriguingly, deletion of the Slc25a3 gene from the heart long-term resulted in profound hypertrophy with ventricular dilation and depressed cardiac function, all features that reflect the cardiomyopathy observed in humans with mutations in SLC25A3. Together, these results demonstrate that although the PiC is not a direct component of the MPTP, it can regulate its activity, suggesting a novel therapeutic target for reducing necrotic cell death. In addition, mice lacking Slc25a3 in the heart serve as a novel model of metabolic, mitochondrial-driven cardiomyopathy.The mitochondrial oxidative phosphorylation (OXPHOS) system is the primary source of cellular energy production. Defects in OXPHOS occur with a frequency of 1 in 5000 live births¹ and underlie a wide range of mitochondrial disorders that often affect multiple organ systems and tissues with high oxidative energy demands, such as brain, skeletal muscle, and heart.² Cardiac phenotypes associated with mitochondrial disease are diverse, and can range from cardiomyopathies to cardiac conduction defects.^{3, 4, 5}The mitochondrial phosphate carrier (PiC) is a member of the solute carrier 25A family that has a critical role in OXPHOS, serving as the primary route for inorganic phosphate (Pi) import into the mitochondrial matrix.^{6, 7} PiC, together with the adenine nucleotide translocator (ANT) and the ATP synthase, forms the ATP synthasome whereby all of the metabolites needed to generate ATP are within one immediate microdomain.^{8, 9} The importance of PiC in facilitating energy production is highlighted by the profound disease phenotype observed in patients presenting with mutations in the skeletal muscle-specific isoform of this gene.^{10, 11} Such patients present with a multisystemic disorder characterized by muscle hypotonia, lactic acidosis, severe hypertrophic cardiomyopathy, and shortened lifespan.^{10, 11} Similarly, patients with SLC25A4 (ANT1 protein) deficiency present with cardiomyopathy,¹² as do mice lacking the Slc25a4 gene,¹³ likely due to a similar molecular defect in the efficiency of ATP production within the mitochondria.In addition to its role in mitochondrial energy metabolism, PiC has been implicated in regulating cell death by serving either as a modulator or a direct component of the mitochondrial permeability transition pore (MPTP).^{14, 15, 16} The MPTP is a non-selective channel that forms in response to Ca²⁺ overload and oxidant stress that allows inner-membrane permeability to solutes up to 1500 Da in size, leading to loss of mitochondrial membrane potential, mitochondrial swelling and rupture, and eventually cell death through necrosis.¹⁵ Structurally, the MPTP complex has been proposed to be comprised of the ATP synthase^{17, 18} and to be regulated by cyclophilin D (CypD),^{19, 20} ANT,²¹ and the pro-apoptotic proteins Bax and Bak in the outer mitochondrial membrane.^{22, 23} PiC has also been suggested to be an inner-membrane component of the MPTP because it can form nonspecific channels in lipid membranes and because the MPTP is known to be activated by Pi.^{24, 25, 26, 27} Finally, PiC directly interacts with CypD in the mitochondrial matrix, which is a verified regulator and component of the MPTP.¹⁶ Saccharomyces cerevisiae lacking PiC have altered MPTP characteristics with a smaller pore size, suggesting it might directly participate in the mitochondrial permeability pore.²⁸ However, partial reduction of PiC by siRNAs in cultured cells had no effect on mitochondrial permeability activity, suggesting that PiC is not required for MPTP function.²⁷ Definitive genetic proof of PiC''s involvement in MPTP formation/function is currently lacking.In the present study, we tested the role of PiC in MPTP regulation and cell death in vivo using a mouse model with inducible cardiomyocyte-specific deletion of the Slc25a3 gene (encodes PiC). We found that cardiac mitochondria depleted of PiC were able to undergo permeability transition, suggesting that PiC is not a requisite component of the MPTP. However, the extent of Ca²⁺-induced MPTP opening was blunted, suggesting that PiC serves to regulate this activity. Furthermore, Slc25a3 deletion produced a unique mouse model of mitochondrial-driven hypertrophic cardiomyopathy that recapitulates features observed in human patients with phosphate carrier deficiency and metabolic cardiomyopathy.