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

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms Recent reviews have described the range of assays that have been used for this purpose(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi) Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes This set of guidelines is 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 verify an autophagic response

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-4sx3t3he7d.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

https://www.cell.com/neuron/pdf/S0896-6273(14)01088-5.pdf

The Roles of PINK1, Parkin and Mitochondrial Fidelity in Parkinson's Disease

Autophagy is a catabolic process aimed at recycling cellular components and damaged organelles in response to diverse conditions of stress, such as nutrient deprivation, viral infection and genotoxic stress. A growing amount of evidence in recent years argues for oxidative stress acting as the converging point of these stimuli, with reactive oxygen species (ROS) and reactive nitrogen species (RNS) being among the main intracellular signal transducers sustaining autophagy. This review aims at providing novel insight into the regulatory pathways of autophagy in response to glucose and amino acid deprivation, as well as their tight interconnection with metabolic networks and redox homeostasis. The role of oxidative and nitrosative stress in autophagy is also discussed in the light of its being harmful for both cellular biomolecules and signal mediator through reversible posttranslational modifications of thiol-containing proteins. The redox-independent relationship between autophagy and antioxidant response, occurring through the p62/Keap1/Nrf2 pathway, is also addressed in order to provide a wide perspective upon the interconnection between autophagy and oxidative stress. Herein, we also attempt to afford an overview of the complex crosstalk between autophagy and DNA damage response (DDR), focusing on the main pathways activated upon ROS and RNS overproduction. Along these lines, the direct and indirect role of autophagy in DDR is dissected in depth.

/pdf/oxidative-stress-and-autophagy-the-clash-between-damage-and-49lnde13mj.pdf

Oxidative stress and autophagy: the clash between damage and metabolic needs

Autophagy is a diverse family of processes that transport cytoplasm and organelles into the lysosome/vacuole lumen for degradation. During macroautophagy cargo is packaged in autophagosomes that fuse with the lysosome/vacuole. During microautophagy cargo is directly engulfed by the lysosome/vacuole membrane. Piecemeal microautophagy of the nucleus (PMN) occurs in Saccharomyces cerevisiae at nucleus-vacuole (NV) junctions and results in the pinching-off and release into the vacuole of nonessential portions of the nucleus. Previous studies concluded macroautophagy ATG genes are not absolutely required for PMN. Here we report using two biochemical assays that PMN is efficiently inhibited in atg mutant cells: PMN blebs are produced, but vesicles are rarely released into the vacuole lumen. Electron microscopy of arrested PMN structures in atg7, atg8, and atg9 mutant cells suggests that NV-junction–associated micronuclei may normally be released from the nucleus before their complete enclosure by the vacuole membrane. In this regard PMN is similar to the microautophagy of peroxisomes (micropexophagy), where the side of the peroxisome opposite the engulfing vacuole is capped by a structure called the “micropexophagy-specific membrane apparatus” (MIPA). The MIPA contains Atg proteins and facilitates terminal enclosure and fusion steps. PMN does not require the complete vacuole homotypic fusion genes. We conclude that a spectrum of ATG genes is required for the terminal vacuole enclosure and fusion stages of PMN.

Piecemeal Microautophagy of the Nucleus Requires the Core Macroautophagy Genes

The molecular details of the biogenesis of double-membraned autophagosomes are poorly understood. We identify the Saccharomyces cerevisiae AAA–adenosine triphosphatase Cdc48 and its substrate-recruiting cofactor Shp1/Ubx1 as novel components needed for autophagosome biogenesis. In mammals, the Cdc48 homologue p97/VCP and the Shp1 homologue p47 mediate Golgi reassembly by extracting an unknown monoubiquitinated fusion regulator from a complex. We find no requirement of ubiquitination or the proteasome system for autophagosome biogenesis but detect interaction of Shp1 with the ubiquitin-fold autophagy protein Atg8. Atg8 coupled to phosphatidylethanolamine (PE) is crucial for autophagosome elongation and, in vitro, mediates tethering and hemifusion. Interaction with Shp1 requires an FK motif within the N-terminal non–ubiquitin-like Atg8 domain. Based on our data, we speculate that autophagosome formation, in contrast to Golgi reassembly, requires a complex in which Atg8 functionally substitutes ubiquitin. This, for the first time, would give a rationale for use of the ubiquitin-like Atg8 during macroautophagy and would explain why Atg8-PE delipidation is necessary for efficient macroautophagy.

https://rupress.org/jcb/article-pdf/190/6/965/1347712/jcb_201002075.pdf

Cdc48/p97 and Shp1/p47 regulate autophagosome biogenesis in concert with ubiquitin-like Atg8

Nucleus-vacuole (NV) junctions are formed in Saccharomyces cerevisiae through interactions between Vac8 in the vacuole membrane and Nvj1 in the perinuclear ER. Upon starvation, vesicles containing part of the nucleus emanate from these contact sites and finally pinch off into invaginations of the vacuole. Due to its morphological similarity to microautophagy this process had been termed "piecemeal microautophagy of the nucleus" (PMN). We recently discovered that a number of ATG genes required for macroautophagy and micropexophagy are also required for PMN and accordingly named it micronucleophagy. Therefore, PMN represents a novel model system to investigate the functions of the highly conserved but poorly understood core autophagic apparatus. We here extend the morphological analysis of PMN using immunogold and freeze fracture electron microscopy.

Piecemeal microautophagy of the nucleus: Genetic and morphological traits

Background/aims The aim of our study was to evaluate the diagnostic accuracy of B-Mode ultrasound and Hepatorenal Index (HRI) by high-end devices for the detection and classification of hepatic steatosis in patients with various causes of chronic liver disease (CLD). Methods We retrospectively enrolled patients with CLD who underwent liver biopsy and baseline ultrasound between March 2016 and May 2019. Sonographic graduation of steatosis (0°-III°) using B-Mode criteria and HRI were correlated with the histological graduation (S0 ( 33-66%) and S3 (>66%). Interobserver agreement was calculated. Results 157 patients were evaluated. B-Mode ultrasound had a sensitivity of 75.6% and a specificity of 76.0% to differentiate between steatosis and no steatosis (AUROC 0.758). Using B-Mode criteria for advanced steatosis (≥II°), specificity for presence of histological steatosis was ≥98.7%. For detection of advanced steatosis (≥S2), sensitivity of B-mode criteria was 90.9%. In a subgroup of patients with advanced liver fibrosis, sensitivity of B-mode criteria was 95.0% for detection of advanced steatosis (S≥2). A HRI cut-off-value of 1.46 differentiates between patients with steatosis and patients without steatosis with a sensitivity of 42.7% and a specificity of 90.7% (AUROC 0.680). Interobserver agreement of both B-Mode and HRI was good to excellent. Conclusion B-Mode ultrasound using high-end devices is an excellent method to detect advanced steatosis in patients with various CLD. For diagnosis of mild steatosis, modern ultrasound devices may have higher sensitivity but at the expense of specificity. Stage of fibrosis and etiology of CLD seem not to impact on diagnostic accuracy. The additional calculation of HRI seems to have no additional benefit with regard to detect or grade hepatic steatosis in our study population.

Diagnostic accuracy of B-Mode ultrasound and Hepatorenal Index for graduation of hepatic steatosis in patients with chronic liver disease.

Background Colorectal cancer (CRC) is the leading gastrointestinal malignancy. The development from premalignant intraepithelial lesions leading to invasive cancer is paradigmatic for the stepwise carcinogenesis of epithelial cancers, but the knowledge of the underlying mechanism of carcinogenesis and progression of CRC is still incomplete. The understanding of epigenetic mechanisms of carcinogenesis has led to new therapeutic approaches during the last years. Enhancer of zeste homolog 2 (EZH2) is one central epigenetic silencer of the polycomb repressor complex 2 (PRC2) that is already in clinical use as a novel drug target and is associated with poorer prognosis in several cancer entities. Patients and methods The protein expression of EZH2 and other members of the PRC2 as well as resulting posttranslational modifications were investigated by immunohistochemistry in 187 patients with CRC and in 94 patients with premalignant colorectal lesions and correlated with their clinical outcome. Furthermore, the corresponding mRNA expression levels were analyzed in 217 patients with rectal cancer that were enrolled in a prospective clinical trial. Results We found a weak expression of EZH2 in normal colon mucosa that increased in low grade, peaked in high grade intraepithelial neoplasia, and decreased again in invasive CRC. The posttranslational modification caused by EZH2 as a measure of EZH2 activity showed the same behavior. Strong protein and mRNA expression of EZH2 were significantly correlated with favorable prognosis in both investigated cohorts. Conclusion The expression and activity of EZH2 are associated with colorectal carcinogenesis and most expressed in intraepithelial high-grade lesions. Strong expression of EZH2 is associated with a significantly favorable prognosis in patients suffering from CRC.

Sebastian C B Bremer

Papers

Piecemeal Microautophagy of the Nucleus Requires the Core Macroautophagy Genes

Cdc48/p97 and Shp1/p47 regulate autophagosome biogenesis in concert with ubiquitin-like Atg8

Piecemeal microautophagy of the nucleus: Genetic and morphological traits

Diagnostic accuracy of B-Mode ultrasound and Hepatorenal Index for graduation of hepatic steatosis in patients with chronic liver disease.

Enhancer of Zeste Homolog 2 in Colorectal Cancer Development and Progression.