Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures

Long-Term Clinical Outcome of Cardiogenic Shock Patients Undergoing Impella CP Treatment vs. Standard of Care

The number of patients treated with the mechanical circulatory support device Impella Cardiac Power (CP) for cardiogenic shock is steadily increasing. The aim of this study was to investigate long-term survival and complications related to this modality. Patients undergoing Impella CP treatment for cardiogenic shock were retrospectively enrolled and matched with cardiogenic shock patients not treated with mechanical circulatory support between 2010 and 2020. Data were collected from the cardiogenic shock registry of the university hospital of Munich (DRKS00015860). 70 patients with refractory cardiogenic shock without mechanical circulatory support were matched with 70 patients treated with Impella CP. At presentation, the mean age was 67 ± 15 years with 80% being male in the group without support and 67 ± 14 years (p = 0.97) with 76% being male (p = 0.68) in the group with Impella. There was no significant difference in the rate of cardiac arrest (47% vs. 51%, p = 0.73) and myocardial infarction was the predominant cause of cardiogenic shock in both groups (70% vs. 77%). A total of 41% of patients without cardiocirculatory support and 54% of patients with Impella support died during the first month (p = 0.17). After one year, mortality rates were similar in both groups (55% in conventional vs. 59% in Impella CP group, p = 0.30) as was mortality rate at long-term 5-years follow-up (64% in conventional vs. 73% in Impella CP group, p = 0.33). The rate of clinically significant bleedings during ICU stay was lower in the conventional group than in the Impella support group (15% vs. 43%, p = 0.002). In this small observational and non-randomized analysis no difference in long-term outcome between patients treated with Impella CP vs. guideline directed cardiogenic shock therapy without mechanical circulatory support could be detected. Care must be taken regarding the high rate of bleeding and vascular complications when using Impella CP. Large, adequately powered studies are urgently needed to investigate the efficacy and safety of Impella CP in cardiogenic shock

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field