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Jun 10, 2022

Emerging roles of mitotic autophagy

Eugenia Almacellas, Caroline Mauvezin

Lysosomes exert pleiotropic functions to maintain cellular homeostasis and degrade autophagy cargo. Despite the great advances that have boosted our understanding of autophagy and lysosomes in both physiology and pathology, their function in mitosis is still controversial. During mitosis, most organelles are reshaped or repurposed to allow the correct distribution of chromosomes. Mitotic entry is accompanied by a reduction in sites of autophagy initiation, supporting the idea of an inhibition of autophagy to protect the genetic material against harmful degradation. However, there is accumulating evidence revealing the requirement of selective autophagy and functional lysosomes for a faithful chromosome segregation. Degradation is the most-studied lysosomal activity, but recently described alternative functions that operate in mitosis highlight the lysosomes as guardians of mitotic progression. Because the involvement of autophagy in mitosis remains controversial, it is important to consider the specific contribution of signalling cascades, the functions of autophagic proteins and the multiple roles of lysosomes, as three entangled, but independent, factors controlling genomic stability. In this Review, we discuss the latest advances in this area and highlight the therapeutic potential of targeting autophagy for drug development.

Jun 1, 2022

Mechanoautophagy: Synergies Between Autophagy and Cell Mechanotransduction at Adhesive Complexes

Andrea Ravasio, Eugenia Morselli, and Cristina Bertocchi

Cells are exposed and respond to various mechanical forces and physical cues stemming from their environment. This interaction has been seen to differentially regulate various cellular processes for maintenance of homeostasis, of which autophagy represents one of the major players. In addition, autophagy has been suggested to regulate mechanical functions of the cells including their interaction with the environment. In this minireview, we summarize the state of the art of the fascinating interplay between autophagy and the mechanotransduction machinery associated with cell adhesions, that we name ¨Mechanoautophagy¨

May 20, 2022

Ambra1 deficiency impaired mitophagy in skeletal muscle

Lisa Gambarotto, Samuele Metti, Martina Chrisam, Cristina Cerqua, Patrizia Sabatelli, Andrea Armani, Carlo Zanon, Marianna Spizzotin, Silvia Castagnaro, Flavie Strappazzon, Paolo Grumati, Matilde Cescon, Paola Braghetta, Eva Trevisson, Francesco Cecconi, Paolo Bonaldo

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.

Jan 27, 2022

Aberrant upregulation of the glycolytic enzyme
PFKFB3 in CLN7 neuronal ceroid lipofuscinosis

Lopez-Fabuel et al

CLN7 neuronal ceroid lipofuscinosis is an inherited lysosomal storage neurodegenerative disease highly prevalent in children. CLN7/MFSD8 gene encodes a lysosomal membrane glycoprotein, but the biochemical processes affected by CLN7-loss of function are unexplored thus preventing development of potential treatments. Here, we found, in the Cln7∆ex2 mouse model of CLN7 disease, that failure in autophagy causes accumulation of structurally and bioenergetically impaired neuronal mitochondria. In vivo genetic approach reveals elevated mitochondrial reactive oxygen species (mROS) in Cln7∆ex2 neurons that mediates glycolytic enzyme PFKFB3 activation and contributes to CLN7 pathogenesis. Mechanistically, mROS sustains a signaling cascade leading to protein stabilization of PFKFB3, normally unstable in healthy neurons. Administration of the highly selective PFKFB3 inhibitor AZ67 in Cln7∆ex2 mouse brain in vivo and in CLN7 patients-derived cells rectifies key disease hallmarks. Thus, aberrant upregulation of the glycolytic enzyme PFKFB3 in neurons may contribute to CLN7 pathogenesis and targeting PFKFB3 could alleviate this and other lysosomal storage diseases.

Nov 14, 2021

TRAIL Triggers CRAC-Dependent Calcium Influx and Apoptosis through the Recruitment of Autophagy Proteins to Death-Inducing Signaling Complex

Kelly Airiau, Pierre Vacher, Olivier Micheau, Valerie Prouzet-Mauleon, Guido Kroemer, Mohammad Amin Moosavi, and Mojgan Djavaheri-Mergny.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills various cancer cell types, but also leads to the activation of signaling pathways that favor resistance to cell death. Here, we investigated the as yet unknown roles of calcium signaling and autophagy regulatory proteins during TRAIL-induced cell death in leukemia cells. Taking advantage of the Gene Expression Profiling Interactive Analysis (GEPIA) project, we first found that leukemia patients present a unique TRAIL receptor gene expression pattern that may reflect their resistance to TRAIL. The exposure of NB4 acute promyelocytic leukemia cells to TRAIL induces intracellular Ca2+ influx through a calcium release-activated channel (CRAC)-dependent mechanism, leading to an anti-apoptotic response. Mechanistically, we showed that upon TRAIL treatment, two autophagy proteins, ATG7 and p62/SQSTM1, are recruited to the death-inducing signaling complex (DISC) and are essential for TRAIL-induced Ca2+ influx and cell death. Importantly, the treatment of NB4 cells with all-trans retinoic acid (ATRA) led to the upregulation of p62/SQSTM1 and caspase-8 and, when added prior to TRAIL stimulation, significantly enhanced DISC formation and the apoptosis induced by TRAIL. In addition to uncovering new pleiotropic roles for autophagy proteins in controlling the calcium response and apoptosis triggered by TRAIL, our results point to novel therapeutic strategies for sensitizing leukemia cells to TRAIL.

Oct 6, 2021

Targeting CAMKK2 and SOC Channels as a Novel Therapeutic Approach for Sensitizing Acute Promyelocytic Leukemia Cells to All-Trans Retinoic Acid

Faten Merhi, Karla Alvarez-Valadez, Jenifer Trepiana, Claire Lescoat, Alexis Groppi, Jean-William Dupuy, Pierre Soubeyran, Guido Kroemer, Pierre Vacher, and Mojgan Djavaheri-Mergny

Calcium ions (Ca2+) play important and diverse roles in the regulation of autophagy, cell death and differentiation. Here, we investigated the impact of Ca2+ in regulating acute promyelocytic leukemia (APL) cell fate in response to the anti-cancer agent all-trans retinoic acid (ATRA). We observed that ATRA promotes calcium entry through store-operated calcium (SOC) channels into acute promyelocytic leukemia (APL) cells. This response is associated with changes in the expression profiles of ORAI1 and STIM1, two proteins involved in SOC channels activation, as well as with a significant upregulation of several key proteins associated to calcium signaling. Moreover, ATRA treatment of APL cells led to a significant activation of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and its downstream effector AMP-activated protein kinase (AMPK), linking Ca2+ signaling to autophagy. Pharmacological inhibition of SOC channels and CAMKK2 enhanced ATRA-induced cell differentiation and death. Altogether, our results unravel an ATRA-elicited signaling pathway that involves SOC channels/CAMKK2 activation, induction of autophagy, inhibition of cellular differentiation and suppression of cell death. We suggest that SOC channels and CAMKK2 may constitute novel drug targets for potentiating the anti-cancer effect of ATRA in APL patients.

Oct 1, 2021

The Autophagy, Inflammation and Metabolism Center international eSymposium - an early-career investigators' seminar series during the COVID-19 pandemic

Jose L Nieto-Torres, Joanne Durgan, Anais Franco-Romero, Paolo Grumati, Carlos M Guardia, Andrew M Leidal, Michael A Mandell, Christina G Towers, Fei Wang

The Autophagy, Inflammation and Metabolism (AIM) Center organized a globally accessible, virtual eSymposium during the COVID-19 pandemic in 2020. The conference included presentations from scientific leaders, as well as a career discussion panel, and provided a much-needed platform for early-career investigators (ECIs) to showcase their research in autophagy. This Perspective summarizes the science presented by the ECIs during the event and discusses the lessons learned from a virtual meeting of this kind during the pandemic. The meeting was a learning experience for all involved, and the ECI participants herein offer their thoughts on the pros and cons of virtual meetings as a modality, either as standalone or hybrid events, with a view towards the post-pandemic world.

Jul 7, 2021

A Mammalian Target of Rapamycin Perilipin 3 (mTORC1-Plin3) Pathway is essential to Activate Lipophagy and Protects Against Hepatosteatosis

Marina Garcia-Macia et al

NAFLD is the most common hepatic pathology in western countries and no treatment is currently available. NAFLD is characterized by the aberrant hepatocellular accumulation of fatty acids in the form of lipid droplets (LDs). Recently, it was shown that liver LD degradation occurs through a process termed lipophagy, a form of autophagy. However, the molecular mechanisms governing liver lipophagy are elusive. Here, we aimed to ascertain the key molecular players that regulate hepatic lipophagy and their importance in NAFLD.

Jun 17, 2021

Ischemia-induced upregulation of autophagy preludes dysfunctional lysosomal storage and associated synaptic impairments in neurons.

Xia Zhang, Mengping Wei, Jiahui Fan, Weijie Yan, Xu Zha, Huimeng Song, Rongqi Wan, Yanling Yin, Wei Wang.

Macroautophagy/autophagy is vital for neuronal homeostasis and functions. Accumulating evidence suggest that autophagy is impaired during cerebral ischemia, contributing to neuronal dysfunction and neurodegeneration. However, the outcomes after transient modification in autophagy machinery are not fully understood. This study investigated the effects of ischemic stress on autophagy and synaptic structures using a rat model of oxygen-glucose deprivation (OGD) in hippocampal neurons and a mouse model of middle cerebral artery occlusion (MCAO). Upon acute ischemia, an initial autophagy modification occurred in an upregulation manner. Following, the number of lysosomes increased, as well as lysosomal volume, indicating dysfunctional lysosomal storage. These changes were prevented by inhibiting autophagy via 3-methyladenine (3-MA) treatment or ATG7 (autophagy related 7) knockdown, or were mimicked by rapamycin (RAPA), a known activator of autophagy. This suggests that dysfunctional lysosomal storage is associated with the early burst of autophagy. Dysfunctional lysosomal storage contributed to autophagy dysfunction because the basal level of MTOR-dependent lysosomal biogenesis in the reperfusion was not sufficient to clear undegraded cargoes after transient autophagy upregulation. Further investigation revealed that impairment of synaptic ultra-structures, accompanied by dysfunctional lysosomal storage, may result from a failure in dynamic turnover of synaptic proteins. This indicates a vital role of autophagy-lysosomal machinery in the maintenance of synaptic structures. This study supports previous evidence that dysfunctional lysosomal storage may occur following the upregulation of autophagy in neurons. Appropriate autophagosome-lysosomal functioning is vital for maintenance of neuronal synaptic function and impacts more than the few known synaptic proteins.

May 18, 2021

Melatonin Ameliorates Autophagy Impairment in a Metabolic Syndrome Model

Adrián Santos-Ledo et al

Metabolic syndrome is a global health problem in adults and its prevalence among children and adolescents is rising. It is strongly linked to a lifestyle with high-caloric food, which causes obesity and lipid metabolism anomalies. Molecular damage due to excessive oxidative stress plays a major role during the development of metabolic syndrome complications. Among the different hormones, melatonin presents strong antioxidant properties, and it is used to treat metabolic diseases. However, there is not a consensus about its use as a metabolic syndrome treatment. The aim of this study was to identify melatonin effects in a metabolic syndrome model. Golden hamsters were fed with 60% fructose-enriched food to induce metabolic syndrome and were compared to hamsters fed with regular chow diet. Both groups were also treated with melatonin. Fructose-fed hamsters showed altered blood lipid levels (increased cholesterol and LDL) and phenotypes restored with the melatonin treatment. The Harderian gland (HG), which is an ideal model to study autophagy modulation through oxidative stress, was the organ that was most affected by a fructose diet. Redox balance was altered in fructose-fed HG, inducing autophagic activation. However, since LC3-II was not increased, the impairment must be in the last steps of autophagy. Lipophagy HG markers were also disturbed, contributing to the dyslipidemia. Melatonin treatment improved possible oxidative homeostasis through autophagic induction. All these results point to melatonin as a possible treatment of the metabolic syndrome.

May 2, 2021

Autophagy-targeted therapy to modulate age-related diseases: Success, pitfalls, and new directions

Waleska Kerllen Martins et al

Autophagy is a critical metabolic process that supports homeostasis at a basal level and is dynamically regulated in response to various physiological and pathological processes. Autophagy has some etiologic implications that support certain pathological processes due to alterations in the lysosomal-degradative pathway. Some of the conditions related to autophagy play key roles in highly relevant human diseases, e.g., cardiovascular diseases(15.5%), malignant and other neoplasms (9.4%), and neurodegenerative conditions (3.7%). Despite advances in the discovery of new strategies to treat these age-related diseases, autophagy has emerged as a therapeutic option after preclinical and clinical studies. Here, we discuss the pitfalls and success in regulating autophagy initiation and its lysosome-dependent pathway to restore its homeostatic role and mediate therapeutic effects for cancer, neurodegenerative, and cardiac diseases. The main challenge for the development of autophagy regulators for clinical application is the lack of specificity of the repurposed drugs, due to the low pharmacological uniqueness of their target, including those that target the PI3K/AKT/mTOR and AMPK pathway. Then, future efforts must be conducted to deal with this scenery, including the disclosure of key components in the autophagy machinery that may intervene in its therapeutic regulation. Among all efforts, those focusing on the development of novel allosteric inhibitors against autophagy inducers, as well as those targeting autolysosomal function, and their integration into therapeutic regimens should remain a priority for the field.

Feb 26, 2021

Mechanobiology of Autophagy: The Unexplored Side of Cancer

Maria Paz Hernández-Cáceres, Leslie Munoz, Javiera M. Pradenas, Francisco Pena, Pablo Lagos, Pablo Aceiton, Gareth I. Owen, Eugenia Morselli, Alfredo Criollo, Andrea Ravasio, and Cristina Bertocchi

Proper execution of cellular function, maintenance of cellular homeostasis and cell survival depend on functional integration of cellular processes and correct orchestration of cellular responses to stresses. Cancer transformation is a common negative consequence of mismanagement of coordinated response by the cell. In this scenario, by maintaining the balance among synthesis, degradation, and recycling of cytosolic components including proteins, lipids, and organelles the process of autophagy plays a central role. Several environmental stresses activate autophagy, among those hypoxia, DNA damage, inflammation, and metabolic challenges such as starvation. In addition to these chemical challenges, there is a requirement for cells to cope with mechanical stresses stemming from their microenvironment. Cells accomplish this task by activating an intrinsic mechanical response mediated by cytoskeleton active processes and through mechanosensitive protein complexes which interface the cells with their mechano-environment. Despite autophagy and cell mechanics being known to play crucial transforming roles during oncogenesis and malignant progression their interplay is largely overlooked. In this review, we highlight the role of physical forces in autophagy regulation and their potential implications in both physiological as well as pathological conditions. By taking a mechanical perspective, we wish to stimulate novel questions to further the investigation of the mechanical requirements of autophagy and appreciate the extent to which mechanical signals affect this process.