Rapamycin attenuates mitochondrial dysfunction via activation of mitophagy in experimental ischemic stroke

Rapamycin has been demonstrated to exhibit neuroprotective functions via the activation of autophagy in a cerebral ischemia model. However, the involvement of mitophagy in this process and its contribution to the protection of mitochondrial function remains unknown. The present study explored the characteristics of mitophagy after cerebral ischemia and the effect of rapamycin on mitochondrial function. Male Sprague–Dawley rats underwent transient middle cerebral artery occlusion (tMCAO). Neurological deficits scores; infarct volumes; mitophagy morphology; and the levels of malondialdehyde (MDA), adenosine triphosphate (ATP) and mitochondrial membrane potentials (Δψm) were examined. The expression of LC3, Beclin-1 and p62 in the mitochondrial fraction combined with transmission electronic microscopy were used to explore mitophagic activity after ischemia. We also blocked autophagosome formation using 3-methyladenine (3-MA) to check the linkage between the mitochondrial protective effect of rapamycin and enhanced mitophagy. We observed that rapamycin significantly enhanced mitophagy, as evidenced by the increase in LC3-II and Beclin-1 expression in the mitochondria and p62 translocation to the mitochondria. Rapamycin reduced infarct volume, improved neurological outcomes and inhibited mitochondrial dysfunction compared with the control animals (p < 0.05). However, these protective effects were reversed by 3-methyladenine treatment after rapamycin. The present study indicates that rapamycin treatment attenuates mitochondrial dysfunction following cerebral ischemia, which is linked to enhanced mitophagy.     

Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs

It has been widely assumed that Atg8 family LC3/GABARAP proteins are essential for the formation of autophagosomes during macroautophagy/autophagy, and the sequestration of cargo during selective autophagy. However, there is little direct evidence on the functional contribution of these proteins to autophagosome biogenesis in mammalian cells. To dissect the functions of LC3/GABARAPs during starvation-induced autophagy and PINK1-PARK2/Parkin-dependent mitophagy, we used CRISPR/Cas9 gene editing to generate knockouts of the LC3 and GABARAP subfamilies, and all 6 Atg8 family proteins in HeLa cells. Unexpectedly, the absence of all LC3/GABARAPs did not prevent the formation of sealed autophagosomes, or selective engulfment of mitochondria during PINK1-PARK2-dependent mitophagy. Despite not being essential for autophagosome formation, the loss of LC3/GABARAPs affected both autophagosome size, and the efficiency at which they are formed. However, the critical autophagy defect in cells lacking LC3/GABARAPs was failure to drive autophagosome-lysosome fusion. Relative to the LC3 subfamily, GABARAPs were found to play a prominent role in autophagosome-lysosome fusion and recruitment of the adaptor protein PLEKHM1. Our work clarifies the essential contribution of Atg8 family proteins to autophagy in promoting autolysosome formation, and reveals the GABARAP subfamily as a key driver of starvation-induced autophagy and PINK1-PARK2-dependent mitophagy. Since LC3/GABARAPs are not essential for mitochondrial cargo sequestration, we propose an additional mechanism of selective autophagy. The model highlights the importance of ubiquitin signals and autophagy receptors for PINK-PARK2-mediated selectivity rather than Atg8 family-LIR-mediated interactions.     

Neferine suppresses autophagy-induced inflammation,oxidative stress and adipocyte differentiation in Graves' orbitopathy

Previous studies in Graves’ orbitopathy (GO) patient-derived fibroblasts showed that inhibition of autophagy suppresses adipogenic differentiation. Autophagy activation is associated with inflammation, production of reactive oxygen species and fibrosis. Neferine is an alkaloid extracted from Nelumbo nucifera, which induces Nrf2 expression and inhibits autophagy. Here, we have elucidated the role of neferine on interleukin (IL)-13-induced autophagy using patient-derived orbital fibroblasts as an in vitro model of GO. GO patient-derived orbital fibroblasts were isolated and cultured to generate an in vitro model of GO. Autophagy was determined by Western blot detection of the markers such as Beclin-1, Atg-5 and LC3 and by immunofluorescence detection of autophagosome formation. Analysis of differentiation towards an adipogenic lineage was performed by Oil red O staining. The expression of inflammatory factors was detected by ELISA and semiquantitative RT-PCR. Neferine inhibited autophagy in GO orbital fibroblasts, as indicated by the suppression of IL-13-induced autophagosome formation, overexpression of autophagy markers, increased LC3-II/LC3-I levels and finally down-regulation of p62. Neferine suppressed IL-13-induced inflammation, ROS generation, fibrosis and adipogenic differentiation in GO patient-derived orbital fibroblasts. The anti-inflammatory, antioxidant and antiadipogenic effects of neferine were accompanied by the up-regulation of Nrf2. These results indicated that orbital tissue remodelling and inflammation in GO may be mediated by autophagy, and neferine suppressed autophagy-related inflammation and adipogenesis through a mechanism involving Nrf2.     

Temporal dynamics of PARK2/parkin and OPTN/optineurin recruitment during the mitophagy of damaged mitochondria

Damaged mitochondria are selectively degraded via autophagy in a regulated pathway known as mitophagy. Parkinson disease-linked proteins PINK1 (PTEN induced putative kinase 1) and PARK2 (parkin RBR E3 ubiquitin protein ligase) are recruited to the outer mitochondrial membrane upon mitochondrial damage, leading to the PARK2-mediated ubiquitination of mitochondrial proteins. Here, we discuss our recent work demonstrating that OPTN (optineurin) is recruited to damaged mitochondria, serving as an autophagy receptor for autophagosome formation around mitochondria. Using high-resolution live-cell imaging, we find that OPTN is recruited to ubiquitinated mitochondria downstream of PARK2, and induces autophagosome assembly around mitochondria via its LC3-interacting region. Mutations in OPTN are linked to both glaucoma and ALS (amyotrophic lateral sclerosis), and an ALS-associated E478G mutation in OPTN''s ubiquitin binding domain leads to defective mitophagy and accumulation of damaged mitochondria. Importantly, our results highlight a role for mitophagy defects in ALS pathogenesis, and demonstrate that defects in the same pathway for mitochondrial homeostasis are causal for both familial Parkinson disease and ALS.     

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.     

Zinc prevents mitochondrial superoxide generation by inducing mitophagy in the setting of hypoxia/reoxygenation in cardiac cells

Zinc plays a role in autophagy and protects cardiac cells from ischemia/reperfusion injury. This study aimed to test if zinc can induce mitophagy leading to attenuation of mitochondrial superoxide generation in the setting of hypoxia/reoxygenation (H/R) in cardiac cells. H9c2 cells were subjected to 4?h hypoxia followed by 2?h reoxygenation. Under normoxic conditions, treatments of cells with ZnCl₂ increased both the LC3-II/LC3-I ratio and GFP-LC3 puncta, implying that zinc induces autophagy. Further experiments showed that endogenous zinc is required for the autophagy induced by starvation and rapamycin. Zinc down-regulated TOM20, TIM23, and COX4 both in normoxic cells and the cells subjected to H/R, indicating that zinc can trigger mitophagy. Zinc increased ERK activity and Beclin1 expression, and zinc-induced mitophagy was inhibited by PD98059 and Beclin1 siRNA during reoxygenation. Zinc-induced Beclin1 expression was reversed by PD98059, implying that zinc promotes Beclin1 expression via ERK. In addition, zinc failed to induce mitophagy in cells transfected with PINK1 siRNA and stabilized PINK1 in mitochondria. Moreover, zinc-induced PINK1 stabilization was inhibited by PD98059. Finally, zinc prevented mitochondrial superoxide generation and dissipation of mitochondrial membrane potential (ΔΨ_m) at reoxygenation, which was blocked by both the Beclin1 and PINK1 siRNAs, suggesting that zinc prevents mitochondrial oxidative stress through mitophagy. In summary, zinc induces mitophagy through PINK1 and Beclin1 via ERK leading to the prevention of mitochondrial superoxide generation in the setting of H/R. Clearance of damaged mitochondria may account for the cardioprotective effect of zinc on H/R injury.     

Mitochondria of highly metastatic breast cancer cell line MDA-MB-231 exhibits increased autophagic properties

Autophagy is a cellular housekeeping process that removes damaged or unwanted cellular components and recycles them to build new constituents. It is essential for tumor growth under adverse environment. Mitochondria play an important role in the formation of autophagosome and its subsequent docking and fusion with lysosome. To understand the contribution of mitochondria to the regulation of homeostatic autophagy in cancer cells, we used the transmitochondrial cytoplasmic hybrid (cybrid) model. Cybrid system allowed us to compare mitochondria from different cell types including highly metastatic breast cancer cell line MDA-MB-231 (c231), less metastatic breast cancer cell lines: MDA-MB-436 (c436) and MDA-MB-468 (c468), as well as non-cancerous mammary epithelial cell MCF-10A (c10A) in a defined nuclear background. The c231 exhibited lower LC3-II levels but higher ratio of LC3-II/LC3-I than c436, c468 and c10A. In addition, c231 displayed more punctate LC3-positive cells and had lower levels of sequestosome 1 (p62/SQSTM1) than other cybrids. These suggested that mitochondria could contribute to the increased autophagy and autophagic flux in metastatic cancer. This increased autophagy was found to be non-selective autophagy instead of selective mitophagy since LC3 puncta in c231 did not co-localize with mitochondria labeled by Mitotracker red or Tomm 20. The promotion of mitochondrial permeability transition (MPT) in c231 also contributed to increased autophagy. Block of MPT by the inhibition of low-conductance stage of MPT pores resulted in a decrease of LC3 puncta in c231. These results suggested that mitochondria from highly metastatic breast cancer cell line MDA-MB-231 can promote homeostatic autophagy of cancer through opening low-conductance MPT pores.     

Involvement of mitophagy in oncogenic K-Ras-induced transformation

Although mitochondrial impairment has often been implicated in carcinogenesis, the mechanisms of its development in cancer remain unknown. We report here that autophagy triggered by oncogenic K-Ras mediates functional loss of mitochondria during cell transformation to overcome an energy deficit resulting from glucose deficiency. When Rat2 cells were infected with a retrovirus harboring constitutively active K-Ras^V12, mitochondrial respiration significantly declined in parallel with the acquisition of transformation characteristics. Decreased respiration was not related to mitochondrial biogenesis but was inversely associated with the increased formation of acidic vesicles enclosing mitochondria, during which autophagy-related proteins such as Beclin 1, Atg5, LC3-II and vacuolar ATPases were induced. Interestingly, blocking autophagy with conventional inhibitors (bafilomycin A, 3-methyladenin) and siRNA-mediated knockdown of autophagy-related genes recovered respiratory protein expression and respiratory activity; JNK was involved in these phenomena as an upstream regulator. The cells transformed by K-Ras^V12 maintained cellular ATP level mainly through glycolytic ATP production without induction of GLUT1, the low K_m glucose transporter. Finally, K-Ras^V12-triggered LC3-II formation was modulated by extracellular glucose levels, and LC3-II formation increased only in hepatocellular carcinoma tissues exhibiting low glucose uptake and increased K-Ras expression. Taken together, our observations suggest that mitochondrial functional loss may be mediated by oncogenic K-Ras-induced mitophagy during early tumorigenesis even in the absence of hypoxia, and that this mitophagic process may be an important strategy to overcome the cellular energy deficit triggered by insufficient glucose.     

Extrusion of mitochondrial contents from lipopolysaccharide-stimulated cells: Involvement of autophagy

Sepsis/endotoxemia is elicited by the circulatory distribution of pathogens/endotoxins into whole bodies, and causes profound effects on human health by causing inflammation in multiple organs. Mitochondrial damage is one of the characteristics of the cellular degeneration observed during sepsis/endotoxemia. Elimination of damaged mitochondria through the autophagy-lysosome system has been reported in the liver, indicating that autophagy should play an important role in liver homeostasis during sepsis/endotoxemia. An increased appearance of mitochondrial DNA and proteins in the plasma is another feature of sepsis/endotoxemia, suggesting that damaged mitochondria are not only eliminated within the cells, but also extruded through currently unknown mechanisms. Here we provide evidence for the secretion of mitochondrial proteins and DNA from lipopolysaccharide (LPS)-stimulated rat hepatocytes as well as mouse embryonic fibroblasts (MEFs). The secretion of mitochondrial contents is accompanied by the secretion of proteins that reside in the lumenal space of autolysosomes (LC3-II and CTSD/cathepsin D), but not by a lysosomal membrane protein (LAMP1). The pharmacological inhibition of autophagy by 3MA blocks the secretion of mitochondrial constituents from LPS-stimulated hepatocytes. LPS also stimulates the secretion of mitochondrial as well as autolysosomal lumenal proteins from wild-type (Atg5^+/+) MEFs, but not from atg5^−/− MEFs. Furthermore, we show that direct exposure of purified mitochondria activates polymorphonuclear leukocytes (PMNs), as evident by the induction of IL1B/interlekin-1β, a pro-inflammatory cytokine. Taken together, the data suggest the active extrusion of mitochondrial contents, which provoke an inflammatory response of immune cells, through the exocytosis of autolysosomes by cells stimulated with LPS.     

Collapsin Response Mediator Protein 5 (CRMP5) Induces Mitophagy,Thereby Regulating Mitochondrion Numbers in Dendrites

Degradation of damaged mitochondria by mitophagy is an essential process to ensure cell homeostasis. Because neurons, which have a high energy demand, are particularly dependent on the mitochondrial dynamics, mitophagy represents a key mechanism to ensure correct neuronal function. Collapsin response mediator proteins 5 (CRMP5) belongs to a family of cytosolic proteins involved in axon guidance and neurite outgrowth signaling during neural development. CRMP5, which is highly expressed during brain development, plays an important role in the regulation of neuronal polarity by inhibiting dendrite outgrowth at early developmental stages. Here, we demonstrated that CRMP5 was present in vivo in brain mitochondria and is targeted to the inner mitochondrial membrane. The mitochondrial localization of CRMP5 induced mitophagy. CRMP5 overexpression triggered a drastic change in mitochondrial morphology, increased the number of lysosomes and double membrane vesicles termed autophagosomes, and enhanced the occurrence of microtubule-associated protein 1 light chain 3 (LC3) at the mitochondrial level. Moreover, the lipidated form of LC3, LC3-II, which triggers autophagy by insertion into autophagosomes, enhanced mitophagy initiation. Lysosomal marker translocates at the mitochondrial level, suggesting autophagosome-lysosome fusion, and induced the reduction of mitochondrial content via lysosomal degradation. We show that during early developmental stages the strong expression of endogenous CRMP5, which inhibits dendrite growth, correlated with a decrease of mitochondrial content. In contrast, the knockdown or a decrease of CRMP5 expression at later stages enhanced mitochondrion numbers in cultured neurons, suggesting that CRMP5 modulated these numbers. Our study elucidates a novel regulatory mechanism that utilizes CRMP5-induced mitophagy to orchestrate proper dendrite outgrowth and neuronal function.     

Autophagy is a protective response to ethanol neurotoxicity

Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II) and upregulated LC3 puncta in SH-SY5Y neuroblastoma cells. It also enhanced the levels of LC3-II and BECN1 in the developing brain; meanwhile, ethanol reduced SQSTM1 (p62) levels. Bafilomycin A₁, an inhibitor of autophagosome and lysosome fusion, increased p62 levels in the presence of ethanol. Bafilomycin A₁ and rapamycin potentiated ethanol-increased LC3 lipidation, whereas wortmannin and a BECN1-specific shRNA inhibited ethanol-promoted LC3 lipidation. Ethanol increased mitophagy, which was also modulated by BECN1 shRNA and rapamycin. The evidence suggested that ethanol promoted autophagic flux. Activation of autophagy by rapamycin reduced ethanol-induced ROS generation and ameliorated ethanol-induced neuronal death in vitro and in the developing brain, whereas inhibition of autophagy by wortmannin and BECN1-specific shRNA potentiated ethanol-induced ROS production and exacerbated ethanol neurotoxicity. Furthermore, ethanol inhibited the MTOR pathway and downregulation of MTOR offered neuroprotection. Taken together, the results suggest that autophagy activation is a neuroprotective response to alleviate ethanol toxicity. Ethanol modulation of autophagic activity may be mediated by the MTOR pathway.     

Pentacyclic triterpene oleanolic acid protects against cardiac aging through regulation of mitophagy and mitochondrial integrity

Advanced aging exhibits altered cardiac geometry and function involving mitochondrial anomaly. Natural compounds display promises in the regulation of cardiac homeostasis via governance of mitochondrial integrity in aging. This study examined the effect of oleanolic acid (OA), a natural pentacyclic triterpenoid with free radical scavenging and P450 cyclooxygenase-regulating properties, on cardiac aging and mechanisms involved with a focus on mitophagy. Young (4–5 month-old) and old (22–24 month-old) mice were treated with OA for 6 weeks prior to assessment of cardiac function, morphology, ultrastructure, mitochondrial integrity, cell death and autophagy. Our data revealed that OA treatment alleviated aging-induced changes in myocardial remodeling (increased heart weight, chamber size, cardiomyocyte area and interstitial fibrosis), contractile function and intracellular Ca²⁺ handling, apoptosis, necroptosis, inflammation, autophagy and mitophagy (LC3B, p62, TOM20 and FUNDC1 but not BNIP3 and Parkin). OA treatment rescued aging-induced anomalies in mitochondrial ultrastructure (loss of myofilament alignment, swollen mitochondria, increased circularity), mitochondrial biogenesis and O₂^? production without any notable effect at young age. Interestingly, OA-offered benefit against cardiomyocyte aging was nullified by deletion of the mitophagy receptor FUNDC1 using FUNDC1 knockout mice, denoting an obligatory role for FUNDC1 in OA-elicited preservation of mitophagy. OA reconciled aging-induced changes in E3 ligase MARCH5 but not FBXL2, and failed to affect aging-induced rises in IP3R3. Taken together, our data indicated a beneficial role for OA in attenuating cardiac remodeling and contractile dysfunction in aging through a FUNDC1-mediated mechanism.     

Protective role of PARK2/Parkin in sepsis-induced cardiac contractile and mitochondrial dysfunction

Mitochondrial quality control plays a vital role in the maintenance of optimal mitochondrial function. However, its roles and regulation remain ill-defined in cardiac pathophysiology. Here, we tested the hypothesis that PARK2/Parkin, an E3-ligase recently described as being involved in the regulation of cardiac mitophagy, is important for (1) the maintenance of normal cardiac mitochondrial function; and (2) adequate recovery from sepsis, a condition known to induce reversible mitochondrial injury through poorly understood mechanisms. Investigations of mitochondrial and cardiac function were thus performed in wild-type and Park2-deficient mice at baseline and at 2 different times following administration of a sublethal dose of E. coli lipopolysaccharide (LPS). LPS injection induced cardiac and mitochondrial dysfunctions that were followed by complete recovery in wild-type mice. Recovery was associated with morphological and biochemical evidence of mitophagy, suggesting that this process is implicated in cardiac recovery from sepsis. Under baseline conditions, multiple cardiac mitochondrial dysfunctions were observed in Park2-deficient mice. These mild dysfunctions did not result in a visibly distinct cardiac phenotype. Importantly, Park2-deficient mice exhibited impaired recovery of cardiac contractility and constant degradation of mitochondrial metabolic functions. Interestingly, autophagic clearance of damaged mitochondria was still possible in the absence of PARK2 likely through compensatory mechanisms implicating PARK2-independent mitophagy and upregulation of macroautophagy. Together, these results thus provide evidence that in vivo, mitochondrial autophagy is activated during sepsis, and that compensation for a lack of PARK2 is only partial and/or that PARK2 exerts additional protective roles in mitochondria.     

Lactate accelerates calcification in VSMCs through suppression of BNIP3-mediated mitophagy

Arterial media calcification is one of the major complications of diabetes mellitus, which is related to oxidative stress and apoptosis. Mitophagy is a special regulation of mitochondrial homeostasis and takes control of intracellular ROS generation and apoptotic pathways. High circulating levels of lactate usually accompanies diabetes. The potential link between lactate, mitophagy and vascular calcification is investigated in this study. Lactate treatment accelerated VSMC calcification, evaluated by measuring the calcium content, ALP activity, RUNX2, BMP-2 protein levels, and Alizarin red S staining. Lactate exposure caused excessive intracellular ROS generation and VSMC apoptosis. Lactate also impaired mitochondrial function, determined by mPTP opening rate, mitochondrial membrane potential and mitochondrial biogenesis markers. Western blot analysis of LC3-II and p62 and mRFP-GFP-LC3 adenovirus detection for autophagy flux revealed that lactate blocked autophagy flux. LC3-II co-staining with LAMP-1 and autophagosome quantification revealed lactate inhibited autophagy. Furthermore, lactate inhibited mitophagy, which was confirmed by TOMM20 and BNIP3 protein levels, LC3-II colocalization with BNIP3 and TEM assays. In addition, BNIP3-mediated mitophagy played a protective role against VSMC calcification in the presence of lactate. This study suggests that lactate accelerates osteoblastic phenotype transition of VSMC and calcium deposition partly through the BNIP3-mediated mitophagy deficiency induced oxidative stress and apoptosis.     

Defects in Mitochondrial Fission Protein Dynamin-Related Protein 1 Are Linked to Apoptotic Resistance and Autophagy in a Lung Cancer Model

Evasion of apoptosis is implicated in almost all aspects of cancer progression, as well as treatment resistance. In this study, resistance to apoptosis was identified in tumorigenic lung epithelial (A549) cells as a consequence of defects in mitochondrial and autophagic function. Mitochondrial function is determined in part by mitochondrial morphology, a process regulated by mitochondrial dynamics whereby the joining of two mitochondria, fusion, inhibits apoptosis while fission, the division of a mitochondrion, initiates apoptosis. Mitochondrial morphology of A549 cells displayed an elongated phenotype–mimicking cells deficient in mitochondrial fission protein, Dynamin-related protein 1 (Drp1). A549 cells had impaired Drp1 mitochondrial recruitment and decreased Drp1-dependent fission. Cytochrome c release and caspase-3 and PARP cleavage were impaired both basally and with apoptotic stimuli in A549 cells. Increased mitochondrial mass was observed in A549 cells, suggesting defects in mitophagy (mitochondrial selective autophagy). A549 cells had decreased LC3-II lipidation and lysosomal inhibition suggesting defects in autophagy occur upstream of lysosomal degradation. Immunostaining indicated mitochondrial localized LC3 punctae in A549 cells increased after mitochondrial uncoupling or with a combination of mitochondrial depolarization and ectopic Drp1 expression. Increased inhibition of apoptosis in A549 cells is correlated with impeded mitochondrial fission and mitophagy. We suggest mitochondrial fission defects contribute to apoptotic resistance in A549 cells.     

Human Atg8-cardiolipin interactions in mitophagy: Specific properties of LC3B,GABARAPL2 and GABARAP

The phospholipid cardiolipin (CL) has been proposed to play a role in selective mitochondrial autophagy, or mitophagy. CL externalization to the outer mitochondrial membrane would act as a signal for the human Atg8 ortholog subfamily, MAP1LC3 (LC3). The latter would mediate both mitochondrial recognition and autophagosome formation, ultimately leading to removal of damaged mitochondria. We have applied quantitative biophysical techniques to the study of CL interaction with various Atg8 human orthologs, namely LC3B, GABARAPL2 and GABARAP. We have found that LC3B interacts preferentially with CL over other di-anionic lipids, that CL-LC3B binding occurs with positive cooperativity, and that the CL-LC3B interaction relies only partially on electrostatic forces. CL-induced increased membrane fluidity appears also as an important factor helping LC3B to bind CL. The LC3B C terminus remains exposed to the hydrophilic environment after protein binding to CL-enriched membranes. In intact U87MG human glioblastoma cells rotenone-induced autophagy leads to LC3B translocation to mitochondria and subsequent delivery of mitochondria to lysosomes. We have also observed that GABARAP, but not GABARAPL2, interacts with CL in vitro. However neither GABARAP nor GABARAPL2 were translocated to mitochondria in rotenone-treated U87MG cells. Thus the various human Atg8 orthologs might play specific roles in different autophagic processes.