Autophagy, or cellular self-digestion, is a cellular pathway involved in protein and organelle degradation, with an astonishing number of connections to human disease and physiology. For example, autophagic dysfunction is associated with cancer, neurodegeneration, microbial infection and ageing. Paradoxically, although autophagy is primarily a protective process for the cell, it can also play a role in cell death. Understanding autophagy may ultimately allow scientists and clinicians to harness this process for the purpose of improving human health.

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Autophagy fights disease through cellular self-digestion

Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.

/pdf/ampk-and-mtor-regulate-autophagy-through-direct-14wqsz16ke.pdf

AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

Methods in Mammalian Autophagy Research

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA

Autophagy is an evolutionary conserved process that degrades and recycles cellular components through the lysosomal pathway to maintain cellular homeostasis. It has long been considered as a pro-survival mechanism to maintain viability under stressful conditions. However, a growing body of evidence demonstrates that autophagy could also promote or act as a pro-death program. Autophagy plays an ambivalent role in cancer: it can operate either as a pro-tumoral mechanism or paradoxically as an anti-tumoral mechanism depending on the context and the stage of the tumours, making it an interesting field of investigation.

p62/SQSTM1 is an adapter protein playing a key role during the autophagy process. It promotes the selective degradation of ubiquitinated cellular substrates (i.e. proteins and organelles). As a multidomain protein adapter, p62 is involved in the regulation of various signaling pathways including those that control cell death pathways. In this regard, p62 can directly interact with the death receptor DR5 and promotes death receptor induced-apoptosis through caspase 8 stabilization.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by an abnormal proliferation of myeloblasts (precursors of myeloid white blood cells) without differentiation. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the translocation t(15;17)(q22;q12) which results to the expression of the chimeric protein PML-RAR alpha. All-trans retinoic acid (ATRA) induces cancer cells differentiation and results in complete clinical remission in APL patients. It is now used as a first-line therapy. Despite the success of this treatment, some patients are refractory to ATRA or relapse. Thus, new therapeutic strategies are needed.

We previously showed that ATRA-induced APL cell differentiation was associated with autophagy induction and p62/SQSMT1 accumulation, a response that confers a survival advantage to mature APL cells. As recent findings showed several molecular links between p62 and extrinsic apoptosis pathway, we decided to investigate the link between p62/SQSMT1 and cell death in AML cells upon treatment by TRAIL, an inducer of extrinsic apoptosis. We found that p62/SQSTM1 is required for apoptotic responses in APL cells. Our results also reveal the synergic cooperation between TRAIL and ATRA to increase apoptosis. We are currently investigating the molecular mechanisms underlying the role of autophagy as well as the autophagy receptor p62/SQSM1 in TRAIL-induced apoptosis in APL cells.

Citation Format: Kelly Airiau, Olivier Micheau, Anne-Marie Vacher, Faten Mehri, Pierre Vacher, Mojgan Djavaheri-Mergny. Regulation of TRAIL-induced apoptotic signaling by the autophagy receptor p62 in acute promyelocytic leukemia cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3723.

Abstract 3723: Regulation of TRAIL-induced apoptotic signaling by the autophagy receptor p62 in acute promyelocytic leukemia cells

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.

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

Autophagy is a catabolic lysosomal-dependent pathway involved in the degradation of cellular materials, supplying precursor compounds and energy for macromolecule synthesis and metabolic needs [...].

https://www.mdpi.com/2073-4409/12/3/498/pdf?version=1675937112

Exploring the Complex Link between Autophagy, Regulated Cell Death, and Cell Fate Pathways in Cancer Pathogenesis and Therapy

Autophagy (a Greek term meaning self-digestion) and apoptosis (a Greek term meaning “falling off”) constitute two distinct evolutionarily conserved processes that are involved in tissue homeostasis in metazoans. Dysfunction of one of both processes can result in development of human pathologies including cancer, neurodegenerative diseases, or inflammatory-related diseases. Autophagy and apoptosis share several signaling pathways and can mutually regulate each other to ensure cellular homeostasis in response to a given stress signal. Although autophagy has been often considered as a survival mechanism, a body of evidence reveals that autophagy can contribute to both caspase-dependent and caspase-independent programmed cell death. In this chapter, we summarize our current understanding of the molecular interplay between autophagy and death receptor signaling pathways and discuss how autophagy and autophagy-regulatory proteins can control receptor-mediated cell death signaling.

Cross-Talk Between Autophagy and Death Receptor Signaling Pathways

https://www.mdpi.com/2073-4409/10/5/988/pdf

Erratum: Rémy et al. Isolation and Culture of Human Stem Cells from Apical Papilla under Low Oxygen Concentration Highlight Original Properties. Cells 2019, 8, 1485.

Mojgan Djavaheri-Mergny

Papers

Abstract 3723: Regulation of TRAIL-induced apoptotic signaling by the autophagy receptor p62 in acute promyelocytic leukemia cells

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

Exploring the Complex Link between Autophagy, Regulated Cell Death, and Cell Fate Pathways in Cancer Pathogenesis and Therapy

Cross-Talk Between Autophagy and Death Receptor Signaling Pathways

Erratum: Rémy et al. Isolation and Culture of Human Stem Cells from Apical Papilla under Low Oxygen Concentration Highlight Original Properties. Cells 2019, 8, 1485.