Hao Zhang

Bio: Hao Zhang is an academic researcher from Indiana University. The author has contributed to research in topics: Autophagy & Lysosome. The author has an hindex of 9, co-authored 13 publications receiving 643 citations. Previous affiliations of Hao Zhang include Indiana University – Purdue University Indianapolis.

Autophagy induced by calcium phosphate precipitates targets damaged endosomes

Abstract: Calcium phosphate precipitates (CPPs) form complexes with DNA, which enter cells via endocytosis. Under this condition CPPs induce autophagy via the canonic autophagy machinery. Here we showed that CPP-induced autophagy was also dependent on endocytosis as the process was significantly inhibited by methyl-β-cyclodextrin and dynasore, which suppress clathrin-dependent endocytosis. Consistently, CPP treatment triggered the formation of filipin-positive intracellular vesicles whose membranes are rich in cholesterol. Unexpectedly, these vesicles were also positive for galectin 3, suggesting that they were damaged and the membrane glycans became accessible to galectins to bind. Endosome damage was caused by endocytosis of CPPs and was reversed by calcium chelators or by endocytosis inhibitors. Notably, CPP-induced LC3-positive autophagosomes were colocalized with galectin 3, ubiquitin, and p62/SQSTM1. Inhibition of galectin 3 reduced p62 puncta and autophagosome formation. Knockdown of p62 additionally inhibited the colocalization of autophagosomes with galectins. Furthermore, most of the galectin 3-positive vesicles were colocalized with Rab7 or LAMP1. Agents that affect endosome/lysosome maturation and function, such as bafilomycin A1, also significantly affected CPP-induced tubulovesicular autophagosome formation. These findings thus indicate that endocytosed CPPs caused endosome damage and recruitment of galectins, particularly at the later endosome stage, which led to the interaction of the autophagosomal membranes with the damaged endosome in the presence of p62. Background: Relationship of autophagosomes with endosomal vesicles varies in different conditions. Results: Calcium phosphate precipitates required endocytosis to induce autophagy, caused endosome damage, and recruited autophagosomes to the damaged vesicles. Conclusion: Damaged endosomes can be targeted by autophagosomes. Significance: Autophagy may play a role in endosomal homeostasis.

Dynamic MTORC1-TFEB feedback signaling regulates hepatic autophagy, steatosis and liver injury in long-term nutrient oversupply.

Abstract: Normal metabolism requires a controlled balance between anabolism and catabolism. It is not completely known how this balance can be retained when the level of nutrient supply changes in the long term. We found that in murine liver anabolism, as represented by the phosphorylation of RPS6KB (ribosomal protein S6 kinase), was soon elevated while catabolism, as represented by TFEB (transcription factor EB)-directed gene transcription and lysosomal activities, was downregulated after the administration of a high-fat diet (HFD). Surprisingly, neither the alteration in RPS6KB phosphorylation nor that in TFEB functions was static over the long course of HFD feeding. Instead, the 2 signals exhibited dynamic alterations in opposite directions, which could be explained by the dependence of MTORC1 (MTOR complex 1) activation on TFEB-supported lysosome function and the feedback suppression of TFEB by MTORC1. Disruption of the dynamics by enforced expression of TFEB in HFD-fed mice at the peaks of MTORC1 activation restored lysosome function. Consistently, interference of MTORC1 activation with rapamycin or with a constitutively activated RRAGA mutant at the peak or nadir of MTORC1 oscillation enhanced or reduced the lysosome function, respectively. These treatments also improved or exacerbated hepatic steatosis and liver injury, respectively. Finally, there was a significant inverse correlation between TFEB activation and steatosis severity in the livers of patients with non-alcohol fatty liver diseases, supporting the clinical relevance of TFEB-regulated events. Thus, maintaining catabolic function through feedback mechanisms during enhanced anabolism, which is caused by nutrient oversupply, is important for reducing liver pathology.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Abstract: 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.

Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion.

Abstract: Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradati...

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

Abstract: 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.

Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1

Abstract: The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase-dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid-sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.

Pharmacological modulation of autophagy: therapeutic potential and persisting obstacles.

Abstract: Autophagy is central to the maintenance of organismal homeostasis in both physiological and pathological situations Accordingly, alterations in autophagy have been linked to clinically relevant conditions as diverse as cancer, neurodegeneration and cardiac disorders Throughout the past decade, autophagy has attracted considerable attention as a target for the development of novel therapeutics However, such efforts have not yet generated clinically viable interventions In this Review, we discuss the therapeutic potential of autophagy modulators, analyse the obstacles that have limited their development and propose strategies that may unlock the full therapeutic potential of autophagy modulation in the clinic