ATG8ylation of proteins: a way to cope with cell stress?

The ATG8 family of proteins regulates autophagy in a variety of ways. Recently, ATG8s were demonstrated to conjugate directly to cellular proteins in a process termed "ATG8ylation," which is amplified by mitochondrial damage and antagonized by ATG4 proteases. ATG8s may have an emerging role as small protein modifiers.Julian M. Carosi, Thanh N. Nguyen, Michael Lazarou, Sharad Kumar and Timothy J. Sargean

Retromer has a selective function in cargo sorting via endosome transport carriers

Retromer is a peripheral membrane protein complex that coordinates multiple vesicular trafficking events within the endolysosomal system. Here, we demonstrate that retromer is required for the maintenance of normal lysosomal morphology and function. The knockout of retromer subunit Vps35 causes an ultrastructural alteration in lysosomal structure and aberrant lysosome function, leading to impaired autophagy. At the whole-cell level, knockout of retromer Vps35 subunit reduces lysosomal proteolytic capacity as a consequence of the improper processing of lysosomal hydrolases, which is dependent on the trafficking of the cation-independent mannose 6-phosphate receptor (CI-M6PR). Incorporation of CI-M6PR into endosome transport carriers via a retromer-dependent process is restricted to those tethered by GCC88 but not golgin-97 or golgin-245. Finally, we show that this retromer-dependent retrograde cargo trafficking pathway requires SNX3, but not other retromer-associated cargo binding proteins, such as SNX27 or SNX-BAR proteins. Therefore, retromer does contribute to the retrograde trafficking of CI-M6PR required for maturation of lysosomal hydrolases and lysosomal function.Yi Cui, Julian M. Carosi, Zhe Yang, Nicholas Ariotti, Markus C. Kerr, Robert G. Parton, Timothy J. Sargeant, and Rohan D. Teasdal

Track A Basic Science

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138319/1/jia218438.pd

Receptor Recycling by Retromer

The highly conserved retromer complex controls the fate of hundreds of receptors that pass through the endolysosomal system and is a central regulatory node for diverse metabolic programs. More than 20 years ago, retromer was discovered as an essential regulator of endosome-to-Golgi transport in yeast; since then, significant progress has been made to characterize how metazoan retromer components assemble to enable its engagement with endosomal membranes, where it sorts cargo receptors from endosomes to the trans-Golgi network or plasma membrane through recognition of sorting motifs in their cytoplasmic tails. In this review, we examine retromer regulation by exploring its assembled structure with an emphasis on how a range of adaptor proteins shape the process of receptor trafficking. Specifically, we focus on how retromer is recruited to endosomes, selects cargoes, and generates tubulovesicular carriers that deliver cargoes to target membranes. We also examine how cells adapt to distinct metabolic states by coordinating retromer expression and function. We contrast similarities and differences between retromer and its related complexes: retriever and commander/CCC, as well as their interplay in receptor trafficking. We elucidate how loss of retromer regulation is central to the pathology of various neurogenerative and metabolic diseases, as well as microbial infections, and highlight both opportunities and cautions for therapeutics that target retromer. Finally, with a focus on understanding the mechanisms that govern retromer regulation, we outline new directions for the field moving forward.Julian M. Carosi, Donna Denton, Sharad Kumar, Timothy J. Sargean

Retromer dysfunction at the nexus of tauopathies

Tauopathies define a broad range of neurodegenerative diseases that encompass pathological aggregation of the microtubule-associated protein tau. Although tau aggregation is a central feature of these diseases, their underlying pathobiology is remarkably heterogeneous at the molecular level. In this review, we summarize critical differences that account for this heterogeneity and contrast the physiological and pathological functions of tau. We focus on the recent understanding of its prion-like behavior that accounts for its spread in the brain. Moreover, we acknowledge the limited appreciation about how upstream cellular changes influence tauopathy. Dysfunction of the highly conserved endosomal trafficking complex retromer is found in numerous tauopathies such as Alzheimer's disease, Pick's disease, and progressive supranuclear palsy, and we discuss how this has emerged as a major contributor to various aspects of neurodegenerative diseases. In particular, we highlight recent investigations that have elucidated the contribution of retromer dysfunction to distinct measures of tauopathy such as tau hyperphosphorylation, aggregation, and impaired cognition and behavior. Finally, we discuss the potential benefit of targeting retromer for modifying disease burden and identify important considerations with such an approach moving toward clinical translation.Julian M. Carosi, Donna Denton, Sharad Kumar, Timothy J. Sargean

The Break-Fast study protocol: a single arm pre-post study to measure the effect of a protein-rich breakfast on autophagic flux in fasting healthy individuals

Published online: 01 November 2022Background: Autophagy is a cellular process that cleanses cells and is particularly important during ageing. Autophagy has been extensively studied in vitro and in animal models and is known to be sensitive to nutrition. However, human data are limited because autophagic flux (autophagic degradative activity) has been challenging to measure in humans. This protocol paper describes the Break-Fast study, in which autophagic flux will be measured using a recently developed blood test, before and after ingestion of whey protein. This aims to determine whether an acute nutritional intervention can change autophagy in humans. Methods: A minimum of forty healthy participants (both male and female) aged 20–50 years, BMI 18.5–29.9 kg/m2 will be recruited into this single arm pre-post study. Participants will visit the clinic after an overnight fast for a first blood collection after which they will consume a whey protein-rich drink. A second blood collection will be performed 60 minutes after consumption of the drink. The primary outcome is the change in autophagic flux at 60 minutes post drink. Secondary outcomes include changes in blood glucose, autophagy-related proteins and mRNA, plasma hormones (e.g. insulin, C-peptide, adiponectin, GLP-1, GIP, ghrelin), cytokines, amino acids and lipids, protein synthesis, and correlation between molecular cell damage and autophagic flux. Discussion: This study will provide information about whether autophagy responds to nutrients in humans, and if nutritional strategies could be used to treat or prevent autophagy-related diseases such as Alzheimer’s disease or cancer. Trial registration: Australian New Zealand Clinical Trials Registry (ANZCTR), anzctr. org. au ACTRN12621001029886. Registered on 5 August 2021.Julien Bensalem, Leonie K. Heilbronn, Jemima R. Gore, Amy T. Hutchison, Timothy J. Sargeant and Célia Fourrie

Intermittent fasting activates markers of autophagy in mouse liver, but not muscle from mouse or humans

Objectives: Intermittent fasting (IF) activates autophagy in cardiac muscle and pancreatic islets. We examined the effect of IF on markers of autophagy in liver and skeletal muscle in mice and in humans. Methods: Ten-wk-old C57 BL/6 J male mice were ad libitum (AL) fed a high-fat diet (HFD) or chow diet for 8 wk, before randomization to AL or IF (24-h fast, 3 non-consecutive days per week) for 8 wk (8–16 per group). Tissue was collected in the fed or 22-h fasted state. Fifty women (51 ± 2 y, 31.8 ± 4.3 kg/m2) were randomly assigned to one of two IF protocols (24-hfast, 3 non-consecutive days per week) and fed at 70% (IF70) or 100% (IF100) of energy requirements for 8 wk. Vastus lateralis muscle was collected at 0800 after 12- and 24-h fasts. Microtubule-associated protein light chain 1 (Map1 lc3 b), Beclin1 (Becn1), Sequestosome 1 (Sqstm1/p62), and Lysosomal associated membrane protein 2 (Lamp2) were assessed by quantitative polymerase chain reaction and LC3, BECLIN1 and LAMP1 protein content by immunoblotting. Results: Fasting increased hepatic LC3 I protein and Map1 lc3 b mRNA levels in IF mice fed chow or HFD. LAMP1 protein and Beclin1 mRNA levels in liver were also increased by fasting, but only in chow-fed mice. IF did not activate markers of autophagy in mouse muscle. In humans, a 24-h fast increased SQSTM1. BECLIN1, SQSTM1 and LAMP2 mRNA levels were decreased in IF70 after a 12-h overnight fast . Conclusion: Markers of autophagy in liver, but not in muscle, were elevated in response to IF in mice. In humans, autophagy markers in muscle were reduced, likely in response to weight loss.Rajesh Chaudhary, Bo Liu, Julien Bensalem, Timothy J. Sargeant, Amanda J. Page, Gary A. Wittert, Amy T. Hutchison, Leonie K. Heilbron

Basal autophagic flux measured in blood correlates positively with age in adults at increased risk of type 2 diabetes

OnlinePublPreclinical data show that autophagy delays age-related disease. It has been postulated that age-related disease is—at least in part—caused by an age-related decline in autophagy. However, autophagic fux has never been measured in humans across a spectrum of aging in a physiologically relevant context. To address this critical gap in knowledge, the objective of this cross-sectional observational study was to measure basal autophagic fux in whole blood taken from people at elevated risk of developing type 2 diabetes and correlate it with chronological age. During this study, 119 people were recruited and fve people were excluded during sample analysis such that 114 people were included in the fnal analysis. Basal autophagic fux measured in blood and correlations with parameters such as age, body weight, fat mass, AUSDRISK score, blood pressure, glycated hemoglobin HbA1c, blood glucose and insulin, blood lipids, high-sensitivity C-reactive protein, plasma protein carbonylation, and plasma β-hexosaminidase activity were analysed. Despite general consensus in the literature that autophagy decreases with age, we found that basal autophagic flux increased with age in this human cohort. This is the first study to report measurement of basal autophagic flux in a human cohort and its correlation with age. This increase in basal autophagy could represent a stress response to age-related damage. These data are significant not only for their novelty but also because they will inform future clinical studies and show that measurement of basal autophagic flux in a human cohort is feasible.Julien Bensalem, Xiao Tong Teong, Kathryn J. Hattersley, Leanne K. Hein, Célia Fourrier, Kai Liu, Amy T. Hutchison, Leonie K. Heilbronn, Timothy J. Sargean