miR-218-5p and doxorubicin combination enhances anticancer activity in breast cancer cells through Parkin-dependent mitophagy inhibition.

Breast Cancer (BC) is one of the most common tumours, and is known for its ability to develop resistance to chemotherapeutic treatments. Autophagy has been linked to chemotherapeutic response in several types of cancer, highlighting its contribution to this process. However, the role of mitophagy, a selective form of autophagy responsible for damaged mitochondria degradation, in the response to therapies in BC is still unclear. In order to address this point, we analysed the role of mitophagy in the treatment of the most common anticancer drug, doxorubicin (DXR), in different models of BC, such as a luminal A subtype-BC cell line MCF7 cells, cultured in 2-Dimension (2D) or in 3-Dimension (3D), and the triple negative BC (TNBC) cell line MDA-MB-231. Through a microarray analysis, we identified a relationship between mitophagy gene expressions related to the canonical PINK1/Parkin-mediated pathway and DXR treatment in BC cells. Afterwards, we demonstrated that the PINK1/Parkin-dependent mitophagy is indeed induced following DXR treatment and that exogenous expression of a small non-coding RNA, the miRNA-218-5p, known to target mRNA of Parkin, was sufficient to inhibit the DXR-mediated mitophagy in MCF7 and in MDA-MB-231 cells, thereby increasing their sensitivity to DXR. Considering the current challenges involved in BC refractory to treatment, our work could provide a promising approach to prevent tumour resistance and recurrence, potentially leading to the development of an innovative approach to combine mitophagy inhibition and chemotherapy.

AMBRA1 phosphorylation by CDK1 and PLK1 regulates mitotic spindle orientation.

AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis.

Cobalt chloride has beneficial effects across species through a hormetic mechanism.

Severe oxygen and iron deficiencies have evolutionarily conserved detrimental effects, leading to pathologies in mammals and developmental arrest as well as neuromuscular degeneration in the nematode Caenorhabditis elegans. Yet, similar to the beneficial effects of mild hypoxia, non-toxic levels of iron depletion, achieved with the iron chelator bipyridine or through frataxin silencing, extend C. elegans lifespan through hypoxia-like induction of mitophagy. While the positive health outcomes of hypoxia preconditioning are evident, its practical application is rather challenging. Here, we thus test the potential beneficial effects of non-toxic, preconditioning interventions acting on iron instead of oxygen availability. We find that limiting iron availability through the iron competing agent cobalt chloride has evolutionarily conserved dose-dependent beneficial effects: while high doses of cobalt chloride have toxic effects in mammalian cells, iPS-derived neurospheres, and in C. elegans, sub-lethal doses protect against hypoxia- or cobalt chloride-induced death in mammalian cells and extend lifespan and delay age-associated neuromuscular alterations in C. elegans. The beneficial effects of cobalt chloride are accompanied by the activation of protective mitochondrial stress response pathways.

Ambra1 deficiency impairs mitophagy in skeletal muscle.

BACKGROUND: Maintaining healthy mitochondria is mandatory for muscle viability and function. An essential surveillance mechanism targeting defective and harmful mitochondria to degradation is the selective form of autophagy called mitophagy. Ambra1 is a multifaceted protein with well-known autophagic and mitophagic functions. However, the study of its role in adult tissues has been extremely limited due to the embryonic lethality caused by full-body Ambra1 deficiency. METHODS: To establish the role of Ambra1 as a positive regulator of mitophagy, we exploited in vivo overexpression of a mitochondria-targeted form of Ambra1 in skeletal muscle. To dissect the consequence of Ambra1 inactivation in skeletal muscle, we generated muscle-specific Ambra1 knockout (Ambra1fl/fl :Mlc1f-Cre) mice. Mitochondria-enriched fractions were obtained from muscles of fed and starved animals to investigate the dynamics of the mitophagic flux. RESULTS: Our data show that Ambra1 has a critical role in the mitophagic flux of adult murine skeletal muscle and that its genetic inactivation leads to mitochondria alterations and myofibre remodelling. Ambra1 overexpression in wild-type muscles is sufficient to enhance mitochondria clearance through the autophagy-lysosome system. Consistently with this, Ambra1-deficient muscles display an abnormal accumulation of the mitochondrial marker TOMM20 by +76% (n = 6-7; P < 0.05), a higher presence of myofibres with swollen mitochondria by +173% (n = 4; P < 0.05), and an alteration in the maintenance of the mitochondrial membrane potential and a 34% reduction in the mitochondrial respiratory complex I activity (n = 4; P < 0.05). Lack of Ambra1 in skeletal muscle leads to impaired mitophagic flux, without affecting the bulk autophagic process. This is due to a significantly decreased recruitment of DRP1 (n = 6-7 mice; P < 0.01) and Parkin (n = 6-7 mice; P < 0.05) to the mitochondrial compartment, when compared with controls. Ambra1-deficient muscles also show a marked dysregulation of the endolysosome compartment, as the incidence of myofibres with lysosomal accumulation is 20 times higher than wild-type muscles (n = 4; P < 0.05). Histologically, Ambra1-deficient muscles of both 3- and 6-month-old animals display a significant decrease of myofibre cross-sectional area and a 52% reduction in oxidative fibres (n = 6-7; P < 0.05), thus highlighting a role for Ambra1 in the proper structure and activity of skeletal muscle. CONCLUSIONS: Our study indicates that Ambra1 is critical for skeletal muscle mitophagy and for the proper maintenance of functional mitochondria.

A protective variant of the autophagy receptor CALCOCO2/NDP52 in Multiple Sclerosis (MS).

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, which has been found associated with dysfunctional mitochondria. In order to advance our understanding of the complex molecular mechanisms underlying this disease, we analyzed mitophagy, a process fundamental for the elimination of damaged mitochondria through the autophagic process, in peripheral blood mononuclear cells (PBMCs) of MS patients. Through a genetic analysis carried out on 203 MS patients and 1000 healthy controls, we identified a natural variant of CALCOCO2/NDP52, a well-known autophagic receptor, associated with and protective in MS. Structural modeling of the CALCOCO2 variant and functional studies highlighted an amino acid substitution (G140E) located near the LC3-interacting region (LIR) motif of CALCOCO2, crucial in controlling mitophagy. In addition, we found that among PBMCs, CALCOCO2 is mainly expressed in B cells and, by mediating mitophagy, it reduces pro-inflammatory cytokine production following stimulation of these cells. Here we summarize these recent findings, discuss the putative protective roles of CALCOCO2 in B cells and its novel association with an autoimmune disease such as MS.Abbreviations: LIR: LC3-interacting region; MS: multiple sclerosis; PBMC: peripheral blood mononuclear cells; RR-MS: relapsing-remitting MS; TLR: toll like receptor.

Characterization of a natural variant of human NDP52 and its functional consequences on mitophagy.

The role of mitophagy, a process that allows the removal of damaged mitochondria from cells, remains unknown in multiple sclerosis (MS), a disease that is found associated with dysfunctional mitochondria. Here we have qualitatively and quantitatively studied the main players in PINK1-mediated mitophagy in peripheral blood mononuclear cells (PBMCs) of patients with relapsing-remitting MS. We found the variant c.491G>A (rs550510, p.G140E) of NDP52, one of the major mitophagy receptor genes, associated with a MS cohort. Through the characterization of this variant, we discovered that the residue 140 of human NDP52 is a crucial modulator of NDP52/LC3C binding, promoting the formation of autophagosomes in order to drive efficient mitophagy. In addition, we found that in the PBMC population, NDP52 is mainly expressed in B cells and by ensuring efficient mitophagy, it is able to limit the production of the proinflammatory cytokine TNF-α following cell stimulation. In sum, our results contribute to a better understanding of the role of NDP52 in mitophagy and underline, for the first time, a possible role of NDP52 in MS.

A global view of the miRNA-mitophagy connexion.

Mitochondria are highly dynamics organelles that provide the necessary energy for cellular functions. However, when they are dysfunctional, they can, by contrast, be very harmful for the cell. Mitophagy ensures their recycling and preserves cell performance. This mechanism is particularly important in neurons because they use a lot of energy. Failed mitophagy can thus affect the development of neurons and lead to brain problems. In this regard, a tight regulation of this process is needed. In recent years microRNAs, as regulators of several biological processes, have attracted attention in the field of mitophagy. In this review, we focused on the studies that highlight the miRNAs implicated in the regulation of mitophagic pathways. In particular, we described the first study carried out 7 years ago, in the context of mitophagy during erythroid differentiation. Next, we have cited all the other works to date on microRNAs and mitophagy regulation. Finally, we have underlined the importance of these discoveries in order to define new therapeutic approaches in the context of age-related diseases involving mitochondrial dysfunctions, such as cancers and neurodegenerative diseases.

Mitophagy and iron: two actors sharing the stage in age-associated neuronal pathologies.

Aging is characterized by the deterioration of different cellular and organismal structures and functions. A typical hallmark of the aging process is the accumulation of dysfunctional mitochondria and excess iron, leading to a vicious cycle that promotes cell and tissue damage, which ultimately contribute to organismal aging. Accordingly, altered mitochondrial quality control pathways such as mitochondrial autophagy (mitophagy) as well as altered iron homeostasis, with consequent iron overload, can accelerate the aging process and the development and progression of different age-associated disorders. In this review we first briefly introduce the aging process and summarize molecular mechanisms regulating mitophagy and iron homeostasis. We then provide an overview on how dysfunction of these two processes impact on aging and age-associated neurodegenerative disorders with a focus on Alzheimer's disease, Parkinson's disease and Amyotrophic Lateral Sclerosis. Finally, we summarize some recent evidence showing mechanistic links between iron metabolism and mitophagy and speculate on how regulating the crosstalk between the two processes may provide protective effects against aging and age-associated neuronal pathologies.

miR-218 Inhibits Mitochondrial Clearance by Targeting PRKN E3 Ubiquitin Ligase.

The selective elimination of dysfunctional mitochondria through mitophagy is crucial for preserving mitochondrial quality and cellular homeostasis. The most described mitophagy pathway is regulated by a positive ubiquitylation feedback loop in which the PINK1 (PTEN induced kinase 1) kinase phosphorylates both ubiquitin and the E3 ubiquitin ligase PRKN (Parkin RBR E3 ubiquitin ligase), also known as PARKIN. This event recruits PRKN to the mitochondria, thus amplifying ubiquitylation signal. Here we report that miR-218 targets PRKN and negatively regulates PINK1/PRKN-mediated mitophagy. Overexpression of miR-218 reduces PRKN mRNA levels, thus also reducing protein content and deregulating the E3 ubiquitin ligase action. In fact, following miR-218 overexpression, mitochondria result less ubiquitylated and the autophagy machinery fails to proceed with correct mitochondrial clearance. Since mitophagy defects are associated with various human diseases, these results qualify miR-218 as a promising therapeutic target for human diseases.

Correction to: HUWE1 controls MCL1 stability to unleash AMBRA1-induced mitophagy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

HUWE1 controls MCL1 stability to unleash AMBRA1-induced mitophagy.

Receptor-mediated mitophagy is a crucial process involved in mitochondria quality control. AMBRA1 is a mitophagy receptor for the selective removal of damaged mitochondria in mammalian cells. A critical unresolved issue is how AMBRA1-mediated mitophagy is controlled in response to cellular stress. Here, we investigated the role of BCL2-family proteins on AMBRA1-dependent mitophagy and showed that MCL1 delays AMBRA1-dependent mitophagy. Indeed, MCL1 overexpression is sufficient to inhibit recruitment to mitochondria of the E3 Ubiquitin ligase HUWE1, a crucial dynamic partner of AMBRA1, upon AMBRA1-mediated mitophagy induction. In addition, we found that during mitophagy induced by AMBRA1, MCL1 levels decreased but were sustained by inhibition of the GSK-3ß kinase, which delayed AMBRA1-mediated mitophagy. Also, we showed that MCL1 was phosphorylated by GSK-3ß at a conserved GSK-3 phosphorylation site (S159) during AMBRA1-mediated mitophagy and that this event was accompanied by HUWE1-dependent MCL1 degradation. Altogether, our results demonstrate that MCL1 stability is regulated by the kinase GSK-3ß and the E3 ubiquitin ligase HUWE1 in regulating AMBRA1-mediated mitophagy. Our work thus defines MCL1 as an upstream stress-sensitive protein, functional in AMBRA1-mediated mitophagy.

Reversible induction of mitophagy by an optogenetic bimodular system.

Autophagy-mediated degradation of mitochondria (mitophagy) is a key process in cellular quality control. Although mitophagy impairment is involved in several patho-physiological conditions, valuable methods to induce mitophagy with low toxicity in vivo are still lacking. Herein, we describe a new optogenetic tool to stimulate mitophagy, based on light-dependent recruitment of pro-autophagy protein AMBRA1 to mitochondrial surface. Upon illumination, AMBRA1-RFP-sspB is efficiently relocated from the cytosol to mitochondria, where it reversibly mediates mito-aggresome formation and reduction of mitochondrial mass. Finally, as a proof of concept of the biomedical relevance of this method, we induced mitophagy in an in vitro model of neurotoxicity, fully preventing cell death, as well as in human T lymphocytes and in zebrafish in vivo. Given the unique features of this tool, we think it may turn out to be very useful for a wide range of both therapeutic and research applications.

Mitophagy could fight Parkinson's disease through antioxidant action.

During aging, the process of mitophagy, a system that allows the removal of dysfunctional mitochondria through lysosomal degradation, starts to malfunction. Because of this defect, damaged mitochondria are not removed correctly, and their decomposing components accumulate inside the cells. Dysfunctional mitochondria that are not removed by mitophagy produce high amounts of reactive oxygen species (ROS) and, thus, cause oxidative stress. Oxidative stress, in turn, is very harmful for the cells, neuronal cells, in particular. Consequently, the process of mitophagy plays a crucial role in mitochondria-related disease. Mitochondrial dysfunctions and oxidative stress are well-established factors contributing to Parkinson's disease (PD), one of the most common neurodegenerative disorders. In this review, we report various known antioxidants for PD treatments and describe the stimulation of mitophagy process as a novel and exciting method for reducing oxidative stress in PD patients. We describe the different mechanisms responsible for mitochondria removal through the mitophagy process. In addition, we review the functional connection between mitophagy induction and reduction of oxidative stress in several in vitro models of PD and also agents (drugs and natural compounds) already known to be antioxidants and to be able to activate mitophagy. Finally, we propose that there is an urgent need to test the use of mitophagy-inducing antioxidants in order to fight PD.

AMBRA1 Controls Regulatory T-Cell Differentiation and Homeostasis Upstream of the FOXO3-FOXP3 Axis.

Regulatory T cells (Treg) are necessary to maintain immunological tolerance and are key players in the control of autoimmune disease susceptibility. Expression of the transcription factor FOXP3 is essential for differentiation of Treg cells and indispensable for their suppressive function. However, there is still a lack of knowledge about the mechanisms underlying its regulation. Here, we demonstrate that pro-autophagy protein AMBRA1 is also a key modulator of T cells, regulating the complex network that leads to human Treg differentiation and maintenance. Indeed, through its ability to interact with the phosphatase PP2A, AMBRA1 promotes the stability of the transcriptional activator FOXO3, which, in turn, triggers FOXP3 transcription. Furthermore, we found that AMBRA1 plays a significant role in vivo by regulating Treg cell induction in mouse models of both tumor growth and multiple sclerosis, thus highlighting the role of AMBRA1 in the control of immune homeostasis.

HUWE1 E3 ligase promotes PINK1/PARKIN-independent mitophagy by regulating AMBRA1 activation via IKKα.

The selective removal of undesired or damaged mitochondria by autophagy, known as mitophagy, is crucial for cellular homoeostasis, and prevents tumour diffusion, neurodegeneration and ageing. The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems. Here, we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy, a process that takes place independently of the main mitophagy receptors. Furthermore, we show that mitophagy function of AMBRA1 is post-translationally controlled, upon HUWE1 activity, by a positive phosphorylation on its serine 1014. This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1, thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity. Altogether, these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells.

AMBRA1-Mediated Mitophagy Counteracts Oxidative Stress and Apoptosis Induced by Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells.

Therapeutic strategies are needed to protect dopaminergic neurons in Parkinson's disease (PD) patients. Oxidative stress caused by dopamine may play an important role in PD pathogenesis. Selective autophagy of mitochondria (mitophagy), mainly regulated by PINK1 and PARKIN, plays an important role in the maintenance of cell homeostasis. Mutations in those genes cause accumulation of damaged mitochondria, leading to nigral degeneration and early-onset PD. AMBRA1ActA is a fusion protein specifically expressed at the mitochondria, and whose expression has been shown to induce a powerful mitophagy in mammalian cells. Most importantly, the pro-autophagy factor AMBRA1 is sufficient to restore mitophagy in fibroblasts of PD patients carrying PINK1 and PARKIN mutations. In this study, we investigated the potential neuroprotective effect of AMBRA1-induced mitophagy against 6-hydroxydopamine (6-OHDA)- and rotenone-induced cell death in human neuroblastoma SH-SY5Y cells. We demonstrated that AMBRA1ActA overexpression was sufficient to induce mitochondrial clearance in SH-SY5Y cells. We found that apoptosis induced by 6-OHDA and rotenone was reversed by AMBRA1-induced mitophagy. Finally, transfection of SH-SY5Y cells with a vector encoding AMBRA1ActA significantly reduced 6-OHDA and rotenone-induced generation of reactive oxygen species (ROS). Altogether, our results indicate that AMBRA1ActA is able to induce mitophagy in SH-SY5Y cells in order to suppress oxidative stress and apoptosis induced by both 6-OHDA and rotenone. These results strongly suggest that AMBRA1 may have promising neuroprotective properties with an important role in limiting ROS-induced dopaminergic cell death, and the utmost potential to prevent PD or other neurodegenerative diseases associated with mitochondrial oxidative stress.

ATM kinase sustains breast cancer stem-like cells by promoting ATG4C expression and autophagy.

The efficacy of Ataxia-Telangiectasia Mutated (ATM) kinase signalling inhibition in cancer therapy is tempered by the identification of new emerging functions of ATM, which suggests that the role of this protein in cancer progression is complex. We recently demonstrated that this tumor suppressor gene could act as tumor promoting factor in HER2 (Human Epidermal Growth Factor Receptor 2) positive breast cancer. Herein we put in evidence that ATM expression sustains the proportion of cells with a stem-like phenotype, measured as the capability to form mammospheres, independently of HER2 expression levels. Transcriptomic analyses revealed that, in mammospheres, ATM modulates the expression of cell cycle-, DNA repair- and autophagy-related genes. Among these, the silencing of the autophagic gene, autophagy related 4C cysteine peptidase (ATG4C), impairs mammosphere formation similarly to ATM depletion. Conversely, ATG4C ectopic expression in cells silenced for ATM expression, rescues mammospheres growth. Finally, tumor array analyses, performed using public data, identify a significant correlation between ATM and ATG4C expression levels in all human breast cancer subtypes, except for the basal-like one.Overall, we uncover a new connection between ATM kinase and autophagy regulation in breast cancer. We demonstrate that, in breast cancer cells, ATM and ATG4C are essential drivers of mammosphere formation, suggesting that their targeting may improve current approaches to eradicate breast cancer cells with a stem-like phenotype.

MIR7-3HG, a MYC-dependent modulator of cell proliferation, inhibits autophagy by a regulatory loop involving AMBRA1.

Macroautophagy/autophagy is a tightly regulated intracellular catabolic pathway involving the lysosomal degradation of cytoplasmic organelles and proteins to be recycled into metabolic precursors. AMBRA1 (autophagy and Beclin 1 regulator 1) has a central role in the autophagy signaling network; it acts upstream of MTORC1-dependent autophagy by stabilizing the kinase ULK1 (unc-51 like autophagy activating kinase 1) and by favoring autophagosome core complex formation. AMBRA1 also regulates the cell cycle by modulating the activity of the phosphatase PPP2/PP2A (protein phosphatase 2) and degradation of MYC. Of note, post-transcriptional regulation mediated by noncoding microRNAs (MIRNAs) contributes significantly to control autophagy. Here we describe a new role for the microRNA MIR7-3HG/MIR-7 as a potent autophagy inhibitor. Indeed, MIR7-3HG targets the 3' untranslated region (UTR) of AMBRA1 mRNA, inducing a decrease of both AMBRA1 mRNA and protein levels, and thus causing a block in autophagy. Furthermore, MIR7-3HG, through AMBRA1 downregulation, prevents MYC dephosphorylation, establishing a positive feedback for its own transcription. These data suggest a new and interesting role of MIR7-3HG as an anti-autophagic MIRNA that may affect oncogenesis through the regulation of the tumor suppressor AMBRA1.

Fine-tuning of ULK1 mRNA and protein levels is required for autophagy oscillation.

Autophagy is an intracellular degradation pathway whose levels are tightly controlled to secure cell homeostasis. Unc-51-like kinase 1 (ULK1) is a conserved serine-threonine kinase that plays a central role in the initiation of autophagy. Here, we report that upon autophagy progression, ULK1 protein levels are specifically down-regulated by the E3 ligase NEDD4L, which ubiquitylates ULK1 for degradation by the proteasome. However, whereas ULK1 protein is degraded, ULK1 mRNA is actively transcribed. Upon reactivation of mTOR-dependent protein synthesis, basal levels of ULK1 are promptly restored, but the activity of newly synthesized ULK1 is inhibited by mTOR. This prepares the cell for a new possible round of autophagy stimulation. Our results thus place NEDD4L and ULK1 in a key position to control oscillatory activation of autophagy during prolonged stress to keep the levels of this process under a safe and physiological threshold.

Prosurvival AMBRA1 turns into a proapoptotic BH3-like protein during mitochondrial apoptosis.

Autophagy and apoptosis are 2 stress-response mechanisms that are closely interconnected. However, the molecular interplays between these 2 pathways remain to be clarified. Here we report that the crucial proautophagic factor AMBRA1 can act as a positive mediator of mitochondrial apoptosis. Indeed, we show that, in a proapoptotic positive feedback loop, the C-terminal part of AMBRA1, generated by CASP/CASPASE cleavage upon apoptosis induction, inhibits the antiapoptotic factor BCL2 by a direct binding through its BH3-like domain. The mitochondrial AMBRA1-BCL2 complex is thus at the crossroad between autophagy and cell death and may represent a novel target in development of therapeutic approaches in clinical diseases.