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. 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 fl ux 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, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost 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 fi eld 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 delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so 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 autophagy assays, we hope to encourage technical innovation in the field

Cognitive Reserve and Anxiety Interactions Play a Fundamental Role in the Response to the Stress

The aims of the present study were to assess the possible interaction between Cognitive Reserve (CR) and State Anxiety (SA) on adrenocortical and physiological responses in coping situations. Forty healthy, middle-aged men completed the Cognitive Reserve Scale and the State-Trait Anxiety Inventory. We used an Observational Fear Conditioning (OFC) paradigm in order to assess emotional learning and to induce stress. Electrodermal activity (EDA) and salivary cortisol concentrations were measured throughout the conditions. Our results indicate that those who indicated having higher state anxiety showed a lower capacity for learning the contingency, along with presenting higher salivary cortisol peak response following the observational fear-conditioning paradigm. The most prominent finding was the interaction between cognitive reserve and state anxiety on cortisol response to the post observational fear-conditioning paradigm. Thus, those who showed a high anxiety-state and, at the same time, a high cognitive reserve did not present an increased salivary cortisol response following the observational fear-conditioning paradigm. Given these results, we postulate that the state anxiety reported by participants, reflects emotional activation that hinders the attention needed to process and associate emotional stimuli. However, cognitive reserve has an indirect relation with conditioning, enabling better emotional learning. In this context, cognitive reserve demonstrated a protective effect on hormonal response in coping situations, when reported anxiety or emotional activation were high. These findings suggest that cognitive reserve could be used as a tool to deal with the effects of stressors in life situations, limiting development of the allostatic load.</jats:p