Optineurin mediates delivery of membrane from transferrin receptor-positive endosomes to autophagosomes

Autophagy is a conserved quality control mechanism that removes damaged proteins, organelles and invading bacteria through lysosome-mediated degradation. During autophagy several organelles including endoplasmic reticulum, mitochondria, plasma membrane and endosomes contribute membrane for autophagosome formation. However, the mechanisms and proteins involved in membrane delivery to autophagosomes are not clear. Optineurin (OPTN), a cytoplasmic adaptor protein, is involved in promoting maturation of phagophores into autophagosomes; it is also involved in regulating endocytic trafficking and recycling of transferrin receptor (TFRC). Here, we have examined the role of optineurin in the delivery of membrane from TFRC-positive endosomes to autophagosomes. Only a small fraction of autophagosomes was positive for TFRC, indicating that TFRC-positive endosomes could contribute membrane to a subset of autophagosomes. The percentage of TFRC-positive autophagosomes was reduced in Optineurin knockout mouse embryonic fibroblasts (Optn-/-MEFs) in comparison with normal MEFs. Upon over-expression of optineurin, the percentage of TFRC-positive autophagosomes was increased in Optn-/- MEFs. Unlike wild-type optineurin, a disease-associated mutant, E478G, defective in ubiquitin binding, was not able to enhance formation of TFRC-positive autophagosomes in Optn-/- MEFs. TFRC degradation mediated by autophagy was decreased in optineurin deficient cells. Our results suggest that optineurin mediates delivery of TFRC and perhaps associated membrane from TFRC-positive endosomes to autophagosomes, and this may contribute to autophagosome formation.

Dendropanoxide, a Triterpenoid from Dendropanax morbifera, Ameliorates Hepatic Fibrosis by Inhibiting Activation of Hepatic Stellate Cells through Autophagy Inhibition

Hepatic fibrosis results from chronic liver damage and is characterized by excessive accumulation of extracellular matrix (ECM). In this study, we showed that dendropanoxide (DPX), isolated from Dendropanax morbifera, had anti-fibrotic effects on hepatic fibrosis by inhibiting hepatic stellate cell (HSC) activation. DPX suppressed mRNA and protein expression of α-SMA, fibronectin, and collagen in activated HSCs. Moreover, DPX (40 mg/kg) treatment significantly lowered levels of liver injury markers (aspartate aminotransferase and alanine transaminase), expression of fibrotic markers, and deposition of ECM in a carbon tetrachloride-induced mouse model. Anti-fibrotic effects of DPX were comparable to those of silymarin in a hepatic fibrosis mouse model. As a possible mechanism of anti-fibrotic effects, we showed that DPX inhibited autophagosome formation (LC3B-II) and degradation of p62, which have important roles in HSC activation. These findings suggest that DPX inhibits HSC activation by inhibiting autophagy and can be utilized in hepatic fibrosis therapy.

FOXO1 Is a Critical Switch Molecule for Autophagy and Apoptosis of Sow Endometrial Epithelial Cells Caused by Oxidative Stress

Oxidative stress (OS) is involved in various reproductive diseases and can induce autophagy and apoptosis, which determine the different fates of cells. However, the sequence and the switch mechanism between autophagy and apoptosis are unclear. Here, we reported that chronic restraint stress (CRS) induced OS (decreased T-AOC, T-SOD, CAT and GSH-Px and increased MDA) and then disturbed the endocrine environment of sows during early pregnancy, including the hypothalamic-pituitary-ovarian (HPO) and the hypothalamic-pituitary-adrenal (HPA) axes. Meanwhile, after CRS, the KEAP1/NRF2 pathway was inhibited and attenuated the antioxidative ability to cause OS of the endometrium. The norepinephrine (NE) triggered β2-AR to activate the FOXO1/NF-κB pathway, which induced endometrial inflammation. CRS induced the caspase-dependent apoptosis pathway and caused MAP1LC3-II accumulation, SQSTM1/p62 degradation, and autophagosome formation to initiate autophagy. Furthermore, in vitro, a cellular OS model was established by adding hydrogen peroxide into cells. Low OS maintained the viability of endometrial epithelial cells by triggering autophagy, while high OS induced cell death by initiating caspase-dependent apoptosis. Autophagy preceded the occurrence of apoptosis, which depended on the subcellular localization of FOXO1. In the low OS group, FOXO1 was exported from the nucleus to be modified into Ac-FOXO1 and bound to ATG7 in the cytoplasm, which promoted autophagy to protect cells. In the high OS group, FOXO1 located in the nucleus to promote transcription of proapoptotic proteins and then induce apoptosis. Here, FOXO1, as a redox sensor switch, regulated the transformation of cell autophagy and apoptosis. In summary, the posttranslational modification of FOXO1 may become the target of OS treatment.

Presynaptic Autophagy and the Connection With Neurotransmission

Autophagy is an evolutionary conserved catabolic pathway essential for the maintenance of cellular homeostasis. Defective proteins and organelles are engulfed by autophagosomal membranes which fuse with lysosomes for cargo degradation. In neurons, the orchestrated progression of autophagosome formation and maturation occurs in distinct subcellular compartments. For synapses, the distance from the soma and the oxidative stress generated during intense neuronal activity pose a challenge to maintain protein homeostasis. Autophagy constitutes a crucial mechanism for proper functioning of this unique and vulnerable cellular compartment. We are now beginning to understand how autophagy is regulated at pre-synaptic terminals and how this pathway, when imbalanced, impacts on synaptic function and -ultimately- neuronal survival. We review here the current state of the art of “synaptic autophagy”, with an emphasis on the biogenesis of autophagosomes at the pre-synaptic compartment. We provide an overview of the existing knowledge on the signals inducing autophagy at synapses, highlight the interplay between autophagy and neurotransmission, and provide perspectives for future research.

The caspase-6–p62 axis modulates p62 droplets based autophagy in a dominant-negative manner

SQSTM1/p62, as a major autophagy receptor, forms droplets that are critical for cargo recognition, nucleation, and clearance. p62 droplets also function as liquid assembly platforms to allow the formation of autophagosomes at their surfaces. It is unknown how p62-droplet formation is regulated under physiological or pathological conditions. Here, we report that p62-droplet formation is selectively blocked by inflammatory toxicity, which induces cleavage of p62 by caspase-6 at a novel cleavage site D256, a conserved site across human, mouse, rat, and zebrafish. The N-terminal cleavage product is relatively stable, whereas the C-terminal product appears undetectable. Using a variety of cellular models, we show that the p62 N-terminal caspase-6 cleavage product (p62-N) plays a dominant-negative role to block p62-droplet formation. In vitro p62 phase separation assays confirm this observation. Dominant-negative regulation of p62-droplet formation by caspase-6 cleavage attenuates p62 droplets dependent autophagosome formation. Our study suggests a novel pathway to modulate autophagy through the caspase-6–p62 axis under certain stress stimuli.

Spatial control of avidity regulates initiation and progression of selective autophagy

Autophagosomes form at the endoplasmic reticulum in mammals, and between the vacuole and the endoplasmic reticulum in yeast. However, the roles of these sites and the mechanisms regulating autophagosome formation are incompletely understood. Vac8 is required for autophagy and recruits the Atg1 kinase complex to the vacuole. Here we show that Vac8 acts as a central hub to nucleate the phagophore assembly site at the vacuolar membrane during selective autophagy. Vac8 directly recruits the cargo complex via the Atg11 scaffold. In addition, Vac8 recruits the phosphatidylinositol 3-kinase complex independently of autophagy. Cargo-dependent clustering and Vac8-dependent sequestering of these early autophagy factors, along with local Atg1 activation, promote phagophore assembly site assembly at the vacuole. Importantly, ectopic Vac8 redirects autophagosome formation to the nuclear membrane, indicating that the vacuolar membrane is not specifically required. We propose that multiple avidity-driven interactions drive the initiation and progression of selective autophagy.

Decanoic Acid Stimulates Autophagy in D. discoideum

Ketogenic diets, used in epilepsy treatment, are considered to work through reduced glucose and ketone generation to regulate a range of cellular process including autophagy induction. Recent studies into the medium-chain triglyceride (MCT) ketogenic diet have suggested that medium-chain fatty acids (MCFAs) provided in the diet, decanoic acid and octanoic acid, cause specific therapeutic effects independent of glucose reduction, although a role in autophagy has not been investigated. Both autophagy and MCFAs have been widely studied in Dictyostelium, with findings providing important advances in the study of autophagy-related pathologies such as neurodegenerative diseases. Here, we utilize this model to analyze a role for MCFAs in regulating autophagy. We show that treatment with decanoic acid but not octanoic acid induces autophagosome formation and modulates autophagic flux in high glucose conditions. To investigate this effect, decanoic acid, but not octanoic acid, was found to induce the expression of autophagy-inducing proteins (Atg1 and Atg8), providing a mechanism for this effect. Finally, we demonstrate a range of related fatty acid derivatives with seizure control activity, 4BCCA, 4EOA, and Epilim (valproic acid), also function to induce autophagosome formation in this model. Thus, our data suggest that decanoic acid and related compounds may provide a less-restrictive therapeutic approach to activate autophagy.

Syntaxin 17, an ancient SNARE paralog, plays different and conserved roles in different organisms

Mammalian syntaxin 17 (Stx17) has several functions, other than membrane fusion, including mitochondrial division, autophagosome formation and lipid droplet expansion. Different from conventional syntaxins, Stx17 has a long C-terminal hydrophobic region with a hairpin-like structure flanked by a basic amino acid-enriched C-terminal tail. Although Stx17 is one of the six ancient SNAREs and present in diverse eukaryotic organisms, it has been lost in multiple lineages during evolution. In the present study, we compared the localization and function of fly and nematode Stx17s expressed in HeLa cells with those of human Stx17. We found that fly Stx17 predominantly localizes to the cytosol and mediates autophagy, but not mitochondrial division. Nematode Stx17, on the other hand, is predominantly present in mitochondria and facilitates mitochondrial division, but is irrelevant to autophagy. These differences are likely due to different structures in the C-terminal tail. Non-participation of fly Stx17 and nematode Stx17 in mitochondrial division and autophagy, respectively, was demonstrated in individual organisms. Our results provide an insight into the evolution of Stx17 in metazoa.