Cancer research is a rapidly expanding field, requiring new methods and techniques to investigate the complex biology of the disease and its characteristics. A pivotal aspect of this research is the evaluation of the effects and efficacy of therapies. Here, we describe a simple protocol to generate mammospheres from MCF-7 cells, a useful model of breast cancer stem cells to evaluate cell death after therapy treatment. This subpopulation of cells, found in all types of tumors, is critically involved in therapeutic response as well as in disease recurrence and relapse, and therefore requires particular attention in oncology. The use of mammospheres represents a valuable approach to obtain preliminary in vitro results that closely resemble in vivo responses, providing an effective means to assess the efficacy of both conventional and next-generation therapeutic agents on breast cancer cell death.

Microtubules acetylation has emerged as a key regulator of cellular homeostasis, but its roles in autophagy remain understudied. Here, we identify α-tubulin acetyltransferase 1 (ATAT1), the enzyme responsible for α-tubulin K40 acetylation, as a critical regulator of NCOA4-mediated ferritinophagy and iron homeostasis in cancer cells. Human cancer cell lines were stably or transiently silenced for ATAT1 expression. Autophagy induction was evaluated by visualizing punctate structures and by analyzing changes in autophagic marker levels. Seahorse and flow cytometry experiments were conducted to study the impact of ATAT1 silencing on cell metabolism. Additionally, analysis of iron homeostasis genes, free iron pool, as well as colocalization of NCO4A and ferritin to autophagosome were analyzed to confirm activation of ferritinophagy. Finally, we treated cells with RSL3 (a ferroptosis inducer) and ferrostatin-1 or chloroquine to understand the connection between ATAT1, autophagy, and ferroptosis-induced cell death. Genetic approaches were used to study the role of NCO4A and K40 acetylation in these pathways. We show that ATAT1 silencing induces an oxidative stress response accompanied by a functional autophagic flux. Notably, ATAT1-silenced cells exhibited reduced ATP production and oxygen consumption rate compared with control cells, as well as altered mitochondrial dynamics under both normal and stress conditions. Importantly, ATAT1 loss leads to intracellular iron overload by inducing NCOA4-mediated ferritinophagy, which targets the degradation of the iron storage protein ferritin, thus maintaining intracellular iron homeostasis. Activation of ferritinophagy, in turn, renders ATAT1-silenced lung cancer cells more susceptible to ferroptotic cell death. Notably, the key phenotypes observed in ATAT1-silenced cells are absent in cells with non-acetylatable α-tubulin, demonstrating a direct role for the loss of ATAT1 protein on the induction of a ferroptosis vulnerability phenotype. These findings challenge the traditional view of ATAT1 as a simple microtubule modifier and position this acetyltransferase as a central node in redox, metabolic, and autophagic regulation.

Selective elimination of early pathological TAU species may be a promising therapeutic strategy to reduce the accumulation of TAU, which contributes to neurodegeneration and is a hallmark of Alzheimer’s disease (AD). Pathological hyper-phosphorylated TAU can be degraded through selective autophagy, and NDP52/CALCOCO2 is one of the autophagy receptors involved in this process. In 2021, we discovered a variant of NDP52, called NDP52GE (rs550510), that is more efficient at promoting autophagy. We here anticipate that this variant could be a powerful factor that could eliminate pathological forms of TAU better than its WT form (NDP52WT). Indeed, we provide evidence that in in vitro systems and in a Drosophila melanogaster model of TAU-induced AD, the NDP52GE variant is much more effective than the NDP52WT in reducing the accumulation of pathological forms of TAU through the autophagic process and rescues typical neurodegenerative phenotypes induced by hTAU toxicity. Mechanistically, we showed that NDP52WT and NDP52GE bind pTAU with comparable efficiency, but that NDP52GE binds the autophagic machinery (LC3C and LC3B) more efficiently than NDP52WT does, which could explain its greater efficiency in removing pTAU. Finally, by performing a genetic analysis of a cohort of 435 AD patients, we defined the NDP52GE variant as a protective factor for AD. Overall, our work highlights the variant NDP52GE as a resilience factor in AD that shows a robust effectiveness in driving pathological TAU degradation.

The devastating neurodegenerative disorder of Alzheimer's disease hallmarks the presence of protein aggregates known as amyloid-β plaques and neurofibrillary tangles, composed of amyloid-β peptides and aberrantly phosphorylated Tau protein, respectively. The accumulation of these inclusions leads to significant alterations in neuronal homeostasis and overall brain function, resulting in a progressive and rapid cognitive decline. Autophagy, the molecular mechanism of cellular waste removal through the lysosomal pathway, accounts for the degradation of both amyloid-β plaques and neurofibrillary tangles in the brain, conferring therefore protection against the pathology. In addition to general autophagy, several lines of evidence have reported the implication of selective autophagy receptors, including sequestosome 1/p62, the neighbor of BRCA1 gene, the nuclear-dot protein 52, and optineurin, in mediating the autophagic clearance of amyloid-β, phosphorylated Tau, or both. Herein, we have highlighted autophagy and selective autophagy as pivotal mechanisms in Alzheimer's disease, underlining selective autophagy receptors as a potential target for treatments in the future.

Autophagy has been associated with responses to chemotherapies in several types of cancer, highlighting its contribution to the development of resistance to treatments. Breast cancer (BC) is one of the most common tumors and is known for its ability to develop resistance to treatments. Doxorubicin (DXR) is a drug commonly used in BC and known to damage mitochondria. Thus, we thought to investigate if DXR treatment induces mitophagy, a selective form of autophagy specifically degrading mitochondria, in BC cells. By performing a global analysis of mitophagy-associated genes, we found a relationship between their expressions and DXR treatment. We revealed that PINK1/PARKIN-mediated mitophagy is induced following DXR treatment in different cellular BC models, such as the luminal subtype A cell line MCF7 and in the triple-negative BC cell line MDA-MB-231. By Interfering the E3 ubiquitin ligase PARKIN using miR-218-5p, we showed the efficacy in specifically targeting mitophagy in the treatment of BC. Indeed, PARKIN depletion improves cancer cells sensitivity to DXR treatment in vitro, in both stem-like and non-stem-like BC cells. Our approach, which combines two tumoricidal methods, mitophagy inhibition and chemotherapy, could therefore represent a new strategy for BC treatment. Abbreviations: BC: breast cancer; DXR: doxorubicin; MFN1: mitofusin 1; MFN2: mitofusin 2; miRNA: micro RNA; NPs: nanoparticles; OMM: outer mitochondrial membrane; PINK1: PTEN induced kinase 1; SPATA18: spermatogenesis Associated 18; TBNC: triple negative breast cancer.

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 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.

AbstractBackgroundMaintaining 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.MethodsTo 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.ResultsOur 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.ConclusionsOur study indicates that Ambra1 is critical for skeletal muscle mitophagy and for the proper maintenance of functional mitochondria.

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.

Severe oxygen and iron deficiency have evolutionarily conserved detrimental effects, leading to pathologies in mammals and developmental arrest as well as neuromuscular degeneration in the nematode C. 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. The positive health outcomes of applying hypoxia preconditioning are obvious but have low feasible application. Here, we thus tested the potential beneficial effects of non-toxic, preconditioning interventions acting on iron instead of oxygen availability. We show that, similar to hypoxia preconditioning, limiting iron availability through frataxin silencing, iron chelating or iron competing agents, has evolutionarily conserved beneficial effects protecting against severe hypoxia-induced cellular and neuromuscular alterations. Moreover, we identify inhibition of ferroptosis - a form of non-apoptotic cell death specifically mediated by iron-induced lipid peroxidation - as a mediator of the beneficial effects elicited by frataxin silencing, most likely through gpx-independent suppression. We find that limitation of iron availability also protects against age-associated neuromuscular deficits, pointing to ferroptosis inhibition as a novel strategy to promote healthy aging.

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.

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.

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 ofmitophagic 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.

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.

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.

Neuroblastoma is the most widespread solid tumor in childhood, presenting a plethora of symptoms and different responses to treatment. One of its features is the production of oxidative stress due to the accumulation of reactive species necessary for cancer metabolism, proliferation, and progression. In recent years, different evidence has shown the role of oxidative stress in neuroblastoma in counteracting cancer cell diffusion. Since neuroblastoma cells are characterized by a high proliferation rate, they are commonly used as an in vitro elective model for mimicking neurons in order to study neurodegenerative pathologies, such as Parkinson's disease or Alzheimer's disease. Accordingly, here, we focused on several molecular mechanisms concerning the role of oxidative stress in the neuroblastoma.

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-3β kinase, which delayed AMBRA1-mediated mitophagy. Also, we showed that MCL1 was phosphorylated by GSK-3β 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-3β 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.

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.

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.

Regulatory T cells (T-reg) 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 T-reg 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 T-reg 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 T-reg cell induction in mouse models of both tumor growth and multiple sclerosis, thus highlighting the role of AMBRA1 in the control of immune homeostasis.