Autophagy in the Pathogenesis of Disease

The mechanistic target of rapamycin (mTOR) signaling pathway senses and integrates a variety of environmental cues to regulate organismal growth and homeostasis. The pathway regulates many major cellular processes and is implicated in an increasing number of pathological conditions, including cancer, obesity, type 2 diabetes, and neurodegeneration. Here, we review recent advances in our understanding of the mTOR pathway and its role in health, disease, and aging. We further discuss pharmacological approaches to treat human pathologies linked to mTOR deregulation.

mTOR Signaling in Growth Control and Disease

mTOR signaling in growth control and disease.

https://archive-ouverte.unige.ch/unige:18229/ATTACHMENT01

TOR signaling in growth and metabolism.

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)

Cell growth and proliferation requires an intricate coordination between the stimulatory signals arising from nutrients and growth factors and the inhibitory signals arising from intracellular and extracellular stresses. Alteration of the coordination often causes cancer. In mammals, the mTOR (mammalian target of rapamycin) protein kinase is the central node in nutrient and growth factor signaling, and p53 plays a critical role in sensing genotoxic and other stresses. The results presented here demonstrate that activation of p53 inhibits mTOR activity and regulates its downstream targets, including autophagy, a tumor suppression process. Moreover, the mechanisms by which p53 regulates mTOR involves AMP kinase activation and requires the tuberous sclerosis (TSC) 1/TSC2 complex, both of which respond to energy deprivation in cells. In addition, glucose starvation not only signals to shut down mTOR, but also results in the transient phosphorylation of the p53 protein. Thus, p53 and mTOR signaling machineries can cross-talk and coordinately regulate cell growth, proliferation, and death.

https://www.pnas.org/content/pnas/102/23/8204.full.pdf

The coordinate regulation of the p53 and mTOR pathways in cells.

The origin of the primary membrane of the vaccinia virus, a double-membrane structure that surrounds the immature virions (IV), is not fully understood. Here we investigated whether the primary membrane originates from the autophagic membrane. Morphologic studies by electron microscopy (EM) showed no apparent difference in viral maturation in the autophagy-deficient cell lines, the atg5(-/-) mouse embryonic fibroblasts (MEFs) and the beclin1(-/-) embryonic stem (ES) cells, compared to their isogenic wild-type counterparts. Moreover, viral growth curves demonstrated that vaccinia viruses replicate and mature in the autophagy-deficient cell lines as efficiently as they do in their isogenic wild type counterpart cells. This study indicates that the cellular autophagy machinery is not required for the life-cycle of vaccinia virus, suggesting that the primary vaccinia viral membrane does not originate from the autophagic membrane.

Cellular autophagy machinery is not required for vaccinia virus replication and maturation.

The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12–ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia virus instead actively disrupts the cellular autophagy through a novel molecular mechanism that is associated with aberrant LC3 lipidation and a direct conjugation between ATG12 and ATG3.

https://www.tandfonline.com/doi/pdf/10.4161/auto.7.12.17793

Haiyan Zhang

Papers

The coordinate regulation of the p53 and mTOR pathways in cells.

Cellular autophagy machinery is not required for vaccinia virus replication and maturation.

Vaccinia virus leads to ATG12–ATG3 conjugation and deficiency in autophagosome formation.