It is well established that many tumor types possess defective autophagic pathways. Several studies have reported that the transmembrane, autophagic lipid scramblase ATG9B is altered in multiple cancers, suggesting that this dysregulation could contribute to oncogenesis. Therefore, the goal of this study was to define the cellular distribution of ATG9B in two different tumor cell types and to provide insights into its cellular function. Surprisingly, we found that ATG9B shows a modest association with autophagic structures and exhibits a unique and prominent localization to mitochondria, in contrast to its related form ATG9A. Upon expression of tagged ATG9B forms, this mitochondrial distribution was accompanied by aberrant changes in mitochondrial morphology as well as a reduction in the mitochondrial membrane potential and the release of mtDNA. Few indicators for ATG9B-dependent mitophagy were noted. Instead, ATG9B overexpression led to pronounced apoptotic cell death as assessed by a variety of indicators. Further, we find that the N-terminal sequence of ATG9B acts as a mitochondrial targeting domain and that expression of this peptide alone can induce apoptotic cell death. These findings provide new insights into a putative cellular localization and function for ATG9B.
Microautophagy catabolizes lipid droplets in liver hepatocytes via the small GTPase Rab5 and is connected to alcoholic fatty liver
Introduction and Objective Alcohol consumption has increased dramatically in recent years, representing a significant challenge to human health in metabolic disease. The leading cause of alcohol‐related death is alcohol‐associated liver disease (AALD), which is present at early stages (i.e. steatosis) in 90% of heavy drinkers. During early AALD, liver hepatocytes accumulate large stores of triglycerides in the form of lipid droplets (LDs). Under normal conditions, degradative lysosomes form direct contacts with LDs and catabolize lipid via microlipophagy, a specialized autophagic pathway utilizing the lipid‐degrading enzyme lysosomal acid lipase (LAL). However, the trafficking mechanisms that govern LD‐lysosome contacts during microlipophagy are poorly understood. Several promising candidates exist within the Rab family of small GTPases, which are well‐known to direct the trafficking of intracellular vesicles and LDs. Moreover, previous studies suggest that Rab proteins play central roles in LD catabolism. Thus, the OBJECTIVE of this study was to investigate novel Rab proteins that might be impaired by EtOH leading to altered LD dynamics and microautophagy. Materials and Methods To this end, we utilized RNAi gene silencing, subcellular fractionation, Western blot analysis, and high‐resolution microscopy to define Rabs that might target LDs to lysosomes during microlipophagy. To mimic alcoholic fatty liver, cellular and animal models of alcohol consumption were also used. Changes in LD‐lysosome interaction were measured by quantitative confocal microscopy following pharmacological inhibition of LAL by LAListat. Results First, confocal microscopy revealed that GFP‐Rab5, an early endosomal Rab GTPase, localizes to hepatocellular LDs consistent with proteomic and Western blot analyses showing abundant levels of Rab5 in biochemically isolated LDs. Importantly, siRNA knockdown of Rab5 in cultured hepatocytes showed substantial increases in total LD area (2‐fold) and LD number (1.6‐fold), and also blocked the catabolism of LDs in lipid‐loaded cells. Finally, Rab5 knockdown nearly completely abolished microlipophagy as measured by fluorescent LD accumulation in lysosomes following LAListat treatment. The dissociation of LDs from lysosomes in Rab5 knockdown cells was strikingly similar to the defect observed in EtOH‐fed hepatocytes. Together, these findings suggest that Rab5 is a key mediator of targeting LDs to the lysosome compartment for catabolism and may be a target of EtOH insult. Significance/Implication This study sheds light on new molecular mechanisms of microlipophagy that are affected by chronic alcohol consumption. These mechanisms may represent promising avenues for future therapeutic development toward mitigating hepatic steatosis and slowing the progression of AALD.
Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)
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While the life cycles of hepatitis viruses (A, B, C, D, and E) have been modestly characterized, recent intensive studies have provided new insights. Because these viruses “hijack” the membrane trafficking of the host cell machinery during replicative propagation, it is essential to determine and understand these specific cellular pathways. Hepatitis B virus (HBV) and hepatitis C virus are well known as leading causes of liver cirrhosis and hepatocellular carcinoma. While substantial inroads toward treating hepatitis C virus patients have recently been made, patients with HBV continue to require lifelong treatment, which makes a thorough understanding of the HBV life cycle essential. Importantly, these viruses have been observed to “hijack” the secretory and endocytic membrane trafficking machineries of the hepatocyte. These can include the canonical clathrin‐mediated endocytic process that internalizes virus through cell surface receptors. While these receptors are encoded by the host genome for normal hepatocellular functions, they also exhibit virus‐specific recognition. Further, functions provided by the multivesicular body, which include endosomal sorting complexes required for transport, are now known to envelope a variety of different hepatitis viruses. In this review, we summarize the recent findings regarding the cellular membrane trafficking machineries used by HBV in the context of other hepatitis viruses. (Hepatology2018; 00:000‐000).
Mutant Huntingtin Impairs Axonal Trafficking in Mammalian Neurons In Vivo and In Vitro