Skeletal muscle type-specific mitochondrial adaptation to high-fat diet relies on differential autophagy modulation.

In obesity, skeletal muscle mitochondrial activity changes to cope with increased nutrient availability. Autophagy has been proposed as an essential mechanism involved in the regulation of mitochondrial metabolism. Still, the contribution of autophagy to mitochondrial adaptations in skeletal muscle during obesity is unknown. Here, we show that in response to high-fat diet (HFD) feeding, distinct skeletal muscles in mice exhibit differentially regulated autophagy that may modulate mitochondrial activity. We observed that after 4 and 40 weeks of high-fat diet feeding, OXPHOS subunits and mitochondrial DNA content increased in the oxidative soleus muscle. However, in gastrocnemius muscle, which has a mixed fiber-type composition, the mitochondrial mass increased only after 40 weeks of HFD feeding. Interestingly, fatty acid-supported mitochondrial respiration was enhanced in gastrocnemius, but not in soleus muscle after a 4-week HFD feeding. This increased metabolic profile in gastrocnemius was paralleled by preserving autophagy flux, while autophagy flux in soleus was reduced. To determine the role of autophagy in this differential response, we used an autophagy-deficient mouse model with partial deletion of Atg7 specifically in skeletal muscle (SkM-Atg7+/- mice). We observed that Atg7 reduction resulted in diminished autophagic flux in skeletal muscle, alongside blunting the HFD-induced increase in fatty acid-supported mitochondrial respiration observed in gastrocnemius. Remarkably, SkM-Atg7+/- mice did not present increased mitochondria accumulation. Altogether, our results show that HFD triggers specific mitochondrial adaptations in skeletal muscles with different fiber type compositions, and that Atg7-mediated autophagy modulates mitochondrial respiratory capacity but not its content in response to an obesogenic diet.

Pyrimethamine Modulates Interplay between Apoptosis and Autophagy in Chronic Myelogenous Leukemia Cells.

Pyrimethamine (Pyri) is being used in combination with other medications to treat serious parasitic infections of the body, brain, or eye and to also reduce toxoplasmosis infection in the patients with HIV infection. Additionally, Pyri can display significant anti-cancer potential in different tumor models, but the possible mode of its actions remains unclear. Hence, in this study, the possible anti-tumoral impact of Pyri on human chronic myeloid leukemia (CML) was deciphered. Pyri inhibited cell growth in various types of tumor cells and exhibited a marked inhibitory action on CML cells. In addition to apoptosis, Pyri also triggered sustained autophagy. Targeted inhibition of autophagy sensitized the tumor cells to Pyri-induced apoptotic cell death. Moreover, the activation of signal transducer and activator of transcription 5 (STAT5) and its downstream target gene Bcl-2 was attenuated by Pyri. Accordingly, small interfering RNA (siRNA)-mediated STAT5 knockdown augmented Pyri-induced autophagy and apoptosis and promoted the suppressive action of Pyri on cell viability. Moreover, ectopic overexpression of Bcl-2 protected the cells from Pyri-mediated autophagy and apoptosis. Overall, the data indicated that the attenuation of STAT5-Bcl-2 cascade by Pyri can regulate its growth inhibitory properties by simultaneously targeting both apoptosis and autophagy cell death mechanism(s).

Essential Role of CRIM1 on Endometrial Receptivity in Goat.

In domestic ruminants, endometrial receptivity is related to successful pregnancy and economic efficiency. Despite several molecules having been reported in the past regarding endometrial receptivity regulation, much regarding the mechanism of endometrial receptivity regulation remains unknown due to the complex nature of the trait. In this work, we demonstrated that the cysteine-rich transmembrane bone morphogenetic protein (BMP) regulator 1 (CRIM1) served as a novel regulator in the regulation of goat endometrial receptivity in vitro. Our results showed that hormones and IFN-τ increased the expression of CRIM1 in goat endometrial epithelial cells (EECs). Knockdown of CRIM1 via specific shRNA hindered cell proliferation, cell adhesion and prostaglandins (PGs) secretion and thus derailed normal endometrial receptivity. We further confirmed that receptivity defect phenotypes due to CRIM1 interference were restored by ATG7 overexpression in EECs while a loss of ATG7 further impaired receptivity phenotypes. Moreover, our results showed that changing the expression of ATG7 affected the reactive oxygen species (ROS) production. Moreover, mR-143-5p was shown to be a potential upstream factor of CRIM1-regulated endometrial receptivity in EECs. Overall, these results suggest that CRIM1, as the downstream target of miR-143-5p, has effects on ATG7-dependent autophagy, regulating cell proliferation, cell adhesion and PG secretion, and provides a new target for the diagnosis and treatment of early pregnancy failure and for improving the success rates of artificial reproduction.

Autophagy deficiency modulates microglial lipid homeostasis and aggravates tau pathology and spreading.

The autophagy-lysosomal pathway plays a critical role in intracellular clearance and metabolic homeostasis. While neuronal autophagy is known to participate in the degradation of neurofibrillary tangles composed of hyperphosphorylated and misfolded tau protein in Alzheimer's disease and other tauopathies, how microglial-specific autophagy regulates microglial intrinsic properties and neuronal tau pathology is not well understood. We report here that Atg7, a key mediator of autophagosome biogenesis, plays an essential role in the regulation of microglial lipid metabolism and neuroinflammation. Microglia-specific deletion of Atg7 leads to the transition of microglia to a proinflammatory status in vivo and to inflammasome activation in vitro. Activation of ApoE and lipid efflux attenuates the lipid droplets accumulation and inhibits cytokine production in microglial cells with Atg7 deficiency. Functionally, we show that the absence of microglial Atg7 enhances intraneuronal tau pathology and its spreading. Our results reveal an essential role for microglial autophagy in regulating lipid homeostasis, neuroinflammation, and tau pathology.

Atg7 deficiency in microglia drives an altered transcriptomic profile associated with an impaired neuroinflammatory response.

Microglia, resident immunocompetent cells of the central nervous system, can display a range of reaction states and thereby exhibit distinct biological functions across development, adulthood and under disease conditions. Distinct gene expression profiles are reported to define each of these microglial reaction states. Hence, the identification of modulators of selective microglial transcriptomic signature, which have the potential to regulate unique microglial function has gained interest. Here, we report the identification of ATG7 (Autophagy-related 7) as a selective modulator of an NF-κB-dependent transcriptional program controlling the pro-inflammatory response of microglia. We also uncover that microglial Atg7-deficiency was associated with reduced microglia-mediated neurotoxicity, and thus a loss of biological function associated with the pro-inflammatory microglial reactive state. Further, we show that Atg7-deficiency in microglia did not impact on their ability to respond to alternative stimulus, such as one driving them towards an anti-inflammatory/tumor supportive phenotype. The identification of distinct regulators, such as Atg7, controlling specific microglial transcriptional programs could lead to developing novel therapeutic strategies aiming to manipulate selected microglial phenotypes, instead of the whole microglial population with is associated with several pitfalls.

Influenza A virus infection-induced macroautophagy facilitates MHC class II-restricted endogenous presentation of an immunodominant viral epitope.

CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires de novo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on de novo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed.

Atg7 Knockout Alleviated the Axonal Injury of Neuro-2a Cells Induced by Tri-Ortho-Cresyl Phosphate.

Autophagy is believed to be essential for the maintenance of axonal homeostasis in neurons. However, whether autophagy is causally related to the axon degeneration in organophosphorus-induced delayed neuropathy (OPIDN) still remains unclear. This research was designed to investigate the role of autophagy in axon degeneration following tri-ortho-cresyl phosphate (TOCP) in an in vitro model. Differentiated wild-type and Atg7-/- neuro-2a (N2a) cells were treated with TOCP for 24 h. Axonal degeneration in N2a cells was quantitatively analyzed; the key molecules responsible for axon degeneration and its upstream signaling pathway were determined by Western blotting and real-time PCR. The results found that Atg7-/- cells exhibited a higher resistance to TOCP insult than wild-type cells. Further study revealed that TOCP caused a significant decrease in pro-survival factors NMNATs and SCG10 and a significant increase in pro-degenerative factor SARM1 in both cells. Notably, Atg7-/- cells presented a higher level of pro-survival factors and a lower level of pro-degenerative factors than wild-type cells in the same setting of TOCP administration. Moreover, DLK-MAPK pathway was activated following TOCP. Altogether, our results suggest that autophagy is able to affect TOCP-induced axonal injury via regulating the balance between pro-survival and pro-degenerative factors, providing a promising avenue for the potential therapy for OPIDN patients.

ATG7 is dispensable for LC3-PE conjugation in thioglycolate-elicited mouse peritoneal macrophages.

Thioglycolate-elicited macrophages exhibit abundant conjugation of LC3 with PE (LC3-II). Among other autophagy-related (ATG) proteins, it is proposed that, like in yeast, both ATG5 and ATG7 are essential for LC3 conjugation. Using atg5-deficient (-/-) and atg7-/-macrophages, we provide evidence that loss of ATG5 but not of ATG7 resulted in LC3-II depletion. Accumulation of LC3-II in elicited atg7-/- macrophages in response to bafilomycin A1 validated these data. Furthermore, complete loss of ATG3 in atg7-/- macrophages demonstrated that ATG7 and ATG3 are dispensable for LC3-PE conjugation. In contrast to thioglycolate-elicited macrophages, naïve peritoneal and bone marrow-derived atg7-/- macrophages exhibited no LC3-II, even under inflammatory stimuli in vitro. Hence, the macrophage metabolic status dictates the level of LC3-PE conjugation with a supportive but nonessential role of ATG7, disclosing the eukaryotic exception from the LC3 lipidation model based on yeast data. Abbreviations: ATG: autophagy-related; BM: bone marrow; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PE: phosphatidylethanolamine.

Microglial autophagy-associated phagocytosis is essential for recovery from neuroinflammation.

Multiple sclerosis (MS) is a leading cause of incurable progressive disability in young adults caused by inflammation and neurodegeneration in the central nervous system (CNS). The capacity of microglia to clear tissue debris is essential for maintaining and restoring CNS homeostasis. This capacity diminishes with age, and age strongly associates with MS disease progression, although the underlying mechanisms are still largely elusive. Here, we demonstrate that the recovery from CNS inflammation in a murine model of MS is dependent on the ability of microglia to clear tissue debris. Microglia-specific deletion of the autophagy regulator Atg7, but not the canonical macroautophagy protein Ulk1, led to increased intracellular accumulation of phagocytosed myelin and progressive MS-like disease. This impairment correlated with a microglial phenotype previously associated with neurodegenerative pathologies. Moreover, Atg7-deficient microglia showed notable transcriptional and functional similarities to microglia from aged wild-type mice that were also unable to clear myelin and recover from disease. In contrast, induction of autophagy in aged mice using the disaccharide trehalose found in plants and fungi led to functional myelin clearance and disease remission. Our results demonstrate that a noncanonical form of autophagy in microglia is responsible for myelin degradation and clearance leading to recovery from MS-like disease and that boosting this process has a therapeutic potential for age-related neuroinflammatory conditions.

The LC3-conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles.

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.

Autophagy inhibition blunts PDGFRA adipose progenitors' cell-autonomous fibrogenic response to high-fat diet.

Adipose tissue (AT) fibrosis in obesity compromises adipocyte functions and responses to intervention-induced weight loss. It is driven by AT progenitors with dual fibro/adipogenic potential, but pro-fibrogenic pathways activated in obesity remain to be deciphered. To investigate the role of macroautophagy/autophagy in AT fibrogenesis, we used Pdgfra-CreErt2 transgenic mice to create conditional deletion of Atg7 alleles in AT progenitor cells (atg7 cKO) and examined sex-dependent, depot-specific AT remodeling in high-fat diet (HFD)-fed mice. Mice with atg7 cKO had markedly decreased extracellular matrix (ECM) gene expression in visceral, subcutaneous, and epicardial adipose depots compared to Atg7lox/lox littermates. ECM gene program regulation by autophagy inhibition occurred independently of changes in the mass of fat tissues or adipocyte numbers of specific depots, and cultured preadipocytes treated with pharmacological or siRNA-mediated autophagy disruptors could mimic these effects. We found that autophagy inhibition promotes global cell-autonomous remodeling of the paracrine TGF-BMP family landscape, whereas ECM gene modulation was independent of the autophagic regulation of GTF2IRD1. The progenitor-specific mouse model of ATG7 inhibition confirms the requirement of autophagy for white/beige adipocyte turnover, and combined to in vitro experiments, reveal progenitor autophagy dependence for AT fibrogenic response to HFD, through the paracrine remodeling of TGF-BMP factors balance. Abbreviations: CQ: chloroquine; ECM: extracellular matrix; EpiAT: epididymal adipose tissue; GTF2IRD1: general transcription factor II I repeat domain-containing 1; HFD: high-fat diet; KO: knockout; OvAT: ovarian adipose tissue; PDGFR: platelet derived growth factor receptor; ScAT: subcutaneous adipose tissue; TGF-BMP: transforming growth factor-bone morphogenic protein.

Autophagic death of neural stem cells mediates chronic stress-induced decline of adult hippocampal neurogenesis and cognitive deficits.

Macroautophagy/autophagy is generally regarded as a cytoprotective mechanism, and it remains a matter of controversy whether autophagy can cause cell death in mammals. Here, we show that chronic restraint stress suppresses adult hippocampal neurogenesis in mice by inducing autophagic cell death (ACD) of hippocampal neural stem cells (NSCs). We generated NSC-specific, inducible Atg7 conditional knockout mice and found that they had an intact number of NSCs and neurogenesis level under chronic restraint stress and were resilient to stress- or corticosterone-induced cognitive and mood deficits. Corticosterone treatment of adult hippocampal NSC cultures induced ACD via SGK3 (serum/glucocorticoid regulated kinase 3) without signs of apoptosis. Our results demonstrate that ACD is biologically important in a mammalian system in vivo and would be an attractive target for therapeutic intervention for psychological stress-induced disorders.Abbreviations: AAV: adeno-associated virus; ACD: autophagic cell death; ACTB: actin, beta; Atg: autophagy-related; ASCL1/MASH1: achaete-scute family bHLH transcription factor 1; BafA1: bafilomycin A1; BrdU: Bromodeoxyuridine/5-bromo-2'-deoxyuridine; CASP3: caspase 3; cKO: conditional knockout; CLEM: correlative light and electron microscopy; CORT: corticosterone; CRS: chronic restraint stress; DAB: 3,3'-diaminobenzidine; DCX: doublecortin; DG: dentate gyrus; GC: glucocorticoid; GFAP: glial fibrillary acidic protein; HCN: hippocampal neural stem; i.p.: intraperitoneal; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MKI67/Ki67: antigen identified by monoclonal antibody Ki 67; MWM: Morris water maze; Nec-1: necrostatin-1; NES: nestin; NR3C1/GR: nuclear receptor subfamily 3, group C, member 1; NSC: neural stem cell; PCD: programmed cell death; PFA: paraformaldehyde; PX: Phox homology; PtdIns3P: phosphatidylinositol-3-phosphate; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; SGK: serum/glucocorticoid-regulated kinases; SGZ: subgranular zone; SOX2: SRY (sex determining region Y)-box 2; SQSTM1: sequestosome 1; STS: staurosporine; TAM: tamoxifen; Ulk1: unc-51 like kinase 1; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; VIM: vimentin; WT: wild type; ZFYVE1: zinc finger, FYVE domain containing 1; Z-VAD/Z-VAD-FMK: pan-caspase inhibitor.

Autophagy Deficiency in Renal Proximal Tubular Cells Leads to an Increase in Cellular Injury and Apoptosis under Normal Fed Conditions.

Renal proximal tubular epithelial cells are significantly damaged during acute kidney injury. Renal proximal tubular cell-specific autophagy-deficient mice show increased sensitivity against renal injury, while showing few pathological defects under normal fed conditions. Considering that autophagy protects the proximal tubular cells from acute renal injury, it is reasonable to assume that autophagy contributes to the maintenance of renal tubular cells under normal fed conditions. To clarify this possibility, we generated a knock out mouse model which lacks Atg7, a key autophagosome forming enzyme, in renal proximal tubular cells (Atg7flox/flox;KAP-Cre+). Analysis of renal tissue from two months old Atg7flox/flox;KAP-Cre+ mouse revealed an accumulation of LC3, binding protein p62/sequestosome 1 (a selective substrate for autophagy), and more interestingly, Kim-1, a biomarker for early kidney injury, in the renal proximal tubular cells under normal fed conditions. TUNEL (TdT-mediated dUTP Nick End Labeling)-positive cells were also detected in the autophagy-deficient renal tubular cells. Analysis of renal tissue from Atg7flox/flox;KAP-Cre+ mice at different age points showed that tubular cells positive for p62 and Kim-1 continually increase in number in an age-dependent manner. Ultrastructural analysis of tubular cells from Atg7flox/flox;KAP-Cre+ revealed the presence of intracellular inclusions and abnormal structures. These results indicated that autophagy-deficiency in the renal proximal epithelial tubular cells leads to an increase in injured cells in the kidney even under normal fed conditions.

Blood autophagy defect causes accelerated non-hematopoietic organ aging.

Autophagy has been well studied in regulating aging; however, the impact of autophagy in one organ on the aging of other organs has not been documented. In this study, we used a mouse model with deletion of an autophagy-essential gene Atg7 in hematopoietic system to evaluate the intrinsic role of hematopoietic autophagy on the aging of non-hematopoietic organs. We found that autophagy defect in hematopoietic system causes growth retardation and shortened lifespan, along with aging-like phenotypes including hypertrophic heart, lung and spleen, but atrophic thymus and reduced bone mineral density at organismal level. Hematopoietic autophagy defect also causes increased oxidative stress and mitochondrial mass or aging gene expression at cellular level in multiple non-hematopoietic organs. The organ aging in the Atg7-deleted mice was reversed by anatomic connection to wild-type mice with intact blood autophagy via parabiosis, but not by injection of blood cell-free plasma. Our finding thus highlights an essential role of hematopoietic autophagy for decelerating aging in non-hematopoietic organs.

Autophagy-deficient mice are more susceptible to engrafted leukemogenesis.

Autophagy is primarily considered as an important survival mechanism for both normal cells and cancer cells in response to metabolic stress or chemotherapy; but the role of autophagy in leukemogenesis is not fully understood. The aim of this study is to explore the role of intrinsic autophagy in the leukemogenesis of B-cell acute lymphoblastic leukemia (B-ALL). In this study, conditional knockout mice Atg7f/f;Ubc-Cre, in which an autophagy-essential gene Atg7 is universally deleted, were used as recipients, B-ALL cell line 697 was used as donor cells to generate leukemia mouse model. Compared to wild-type mice, Atg7 knockout mice were more susceptible to engrafted leukemogenesis, shown by increase in white blood cells, lymphocytes, and platelets, decrease in HSPC number and its colony-forming unit (CFU). The liver and spleen displayed hepatosplenomegaly and inflammatory cell infiltration. Furthermore, second competitive transplantation revealed dysfunction of the HSPC in Atg7-knockout leukemia mice represented by destructive self-renew ability (CFU) and reconstitution ability including decreased B220, Ter 119 cells, and increased Gr-1 cell percentage. In summary, Mice with universal deletion of Atg7 are more inclined to the occurrence of engrafted human leukemia, which is largely attributed to the deterioration of the function of HSPC in autophagy deficient mice.

Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype.

Vascular smooth muscle cells (VSMCs) are one of the main cellular determinants in arterial pathology. A large body of evidence indicates that death of VSMCs is associated with features of high-risk/vulnerable atherosclerotic plaques. Mitochondrial turnover is an essential aspect of the mitochondrial quality control in which dysfunctional mitochondria are selectively eliminated through autophagy and replaced through expansion of preexisting mitochondria. Even though successful autophagy promotes VSMC survival, it is unclear whether reduced autophagic flux affects mitochondrial quality control of VSMCs in atherosclerotic plaques. By using apolipoprotein E-deficient (ApoE-/-) mice carrying a VSMC-specific deletion of the essential autophagy gene Atg7, we show in the present study that impaired VSMC autophagy promotes an unstable plaque phenotype, as well as the accumulation of fragmented mitochondria with reduced bioenergetic efficiency and more oxidative stress. Furthermore, we demonstrate that disrupted autophagic flux is linked to defective mitophagy and biogenesis of mitochondria, which exacerbate VSMC apoptosis and in turn plaque vulnerability. Overall, our data indicate that mitochondrial quality control is a promising therapeutic target to stabilize atherosclerotic plaques.

Atg7 mediates renal tubular cell apoptosis in vancomycin nephrotoxicity through activation of PKC-δ.

Recent studies have shown that autophagy exhibits a renoprotective role in various models of acute kidney injury (AKI). However, its role in vancomycin (Van)-induced AKI remains largely unclarified. This study was the first to indicate that autophagy was rapidly activated in both human kidney-2 cells and renal tissues, and mammalian target of rapamycin (mTOR) was inactivated via the suppression of ERK1/2 and mTOR during Van treatment. Interestingly, for both in vitro and in vivo experiments, the suppression of autophagy via chloroquine and PT-Atg7-KO significantly ameliorated Van-induced kidney injury and renal tubular cell apoptosis. Global gene expression analysis indicated that the expression levels of 6159 genes were induced by Van treatment in the kidney cortical tissues of PT-Atg7 wild-type mice, and 18 of them were notably suppressed in PT-Atg7-KO mice. These 18 genes were further classified as programmed cell death, protein binding, signal transduction, E3 ubiquitin ligase, nucleoside diphosphate kinase activity, and E1-like activating enzyme. Unexpectedly, following Van treatment, PKC-δ expression was found to be highest among the 4 genes related to cell death, which was remarkably suppressed in vitro and in PT-Atg7-KO mice. In addition, Atg7 could induce renal cell apoptosis during Van treatment via binding to PKC-δ. Likewise, the inhibition of PKCδ ameliorated Van-induced apoptosis in human kidney-2 cells and kidney tissues. Furthermore, the data showed that PT-Atg7-KO exerted a renoprotective effect against Van-induced nephrotoxicity, but this effect was lost after injection with myc-tagged PKCδ. Taken altogether, these results indicate that Van induces autophagy by suppressing the activation of the ERK1/2 and mTOR signaling pathway. In addition, Atg7 mediates Van-induced AKI through the activation of PKCδ. In sum, autophagy inhibition may serve as a novel therapeutic target for treating nephrotoxic AKI induced by Van.-Xu, X., Pan, J., Li, H., Li, X., Fang, F., Wu, D., Zhou, Y., Zheng, P., Xiong, L., Zhang, D. Atg7 mediates renal tubular cell apoptosis in vancomycin nephrotoxicity through activation of PKC-δ.

Autophagy maintains tumour growth through circulating arginine.

Autophagy captures intracellular components and delivers them to lysosomes, where they are degraded and recycled to sustain metabolism and to enable survival during starvation1-5. Acute, whole-body deletion of the essential autophagy gene Atg7 in adult mice causes a systemic metabolic defect that manifests as starvation intolerance and gradual loss of white adipose tissue, liver glycogen and muscle mass1. Cancer cells also benefit from autophagy. Deletion of essential autophagy genes impairs the metabolism, proliferation, survival and malignancy of spontaneous tumours in models of autochthonous cancer6,7. Acute, systemic deletion of Atg7 or acute, systemic expression of a dominant-negative ATG4b in mice induces greater regression of KRAS-driven cancers than does tumour-specific autophagy deletion, which suggests that host autophagy promotes tumour growth1,8. Here we show that host-specific deletion of Atg7 impairs the growth of multiple allografted tumours, although not all tumour lines were sensitive to host autophagy status. Loss of autophagy in the host was associated with a reduction in circulating arginine, and the sensitive tumour cell lines were arginine auxotrophs owing to the lack of expression of the enzyme argininosuccinate synthase 1. Serum proteomic analysis identified the arginine-degrading enzyme arginase I (ARG1) in the circulation of Atg7-deficient hosts, and in vivo arginine metabolic tracing demonstrated that serum arginine was degraded to ornithine. ARG1 is predominantly expressed in the liver and can be released from hepatocytes into the circulation. Liver-specific deletion of Atg7 produced circulating ARG1, and reduced both serum arginine and tumour growth. Deletion of Atg5 in the host similarly regulated [corrected] circulating arginine and suppressed tumorigenesis, which demonstrates that this phenotype is specific to autophagy function rather than to deletion of Atg7. Dietary supplementation of Atg7-deficient hosts with arginine partially restored levels of circulating arginine and tumour growth. Thus, defective autophagy in the host leads to the release of ARG1 from the liver and the degradation of circulating arginine, which is essential for tumour growth; this identifies a metabolic vulnerability of cancer.

Lipid metabolism in mouse embryonic fibroblast cells in response to autophagy induced by nutrient stress.

Autophagy is of great significance in maintaining cellular homeostasis. Aberrant autophagy has been reported to contribute to the disease aetiology of metabolic syndrome, especially several key lysosomal storage disorders. However, the molecular mechanisms and the correlation between autophagy and lipid metabolism remains unclear. This study was designed and aimed to reveal the alteration of lipid metabolism in response to the autophagy induced by nutrient stress to give new insights into the molecular mechanisms between autophagy and lipid metabolism. An online normal-phase/reversed-phase two-dimensional liquid chromatography-mass spectrometry (NP/RP 2D LC-MS) method was developed to perform the lipidomics analysis of Atg7-/- mouse embryonic fibroblast cells (MEFs) and wild-type MEFs under nutrient stress. 48 and 35 lipid species in wild-type and Atg7-/- MEFs respectively finally meet the screening criteria with p-value less than 0.05 and fold change more than 1.5 in response to nutrient stress. Their alterations indicated that autophagy participated lipid metabolism to generate energy and form autophagosomes with significantly increased free fatty acids and glycerophospholipids, which protected wild-type MEFs from serious damages and delayed cell death. However, in Atg7-/- MEFs, due to the inhibition of autophagy, lipids were continuously consumed and cells suffered from damages even death. These results illustrated the close relationship between autophagy and lipid metabolism comprehensively and revealed diverse lipid targets for the investigation of autophagy.

SNAP23 regulates BAX-dependent adipocyte programmed cell death independently of canonical macroautophagy.

The t-SNARE protein SNAP23 conventionally functions as a component of the cellular machinery required for intracellular transport vesicle fusion with target membranes and has been implicated in the regulation of fasting glucose levels, BMI, and type 2 diabetes. Surprisingly, we observed that adipocyte-specific KO of SNAP23 in mice resulted in a temporal development of severe generalized lipodystrophy associated with adipose tissue inflammation, insulin resistance, hyperglycemia, liver steatosis, and early death. This resulted from adipocyte cell death associated with an inhibition of macroautophagy and lysosomal degradation of the proapoptotic regulator BAX, with increased BAX activation. BAX colocalized with LC3-positive autophagic vacuoles and was increased upon treatment with lysosome inhibitors. Moreover, BAX deficiency suppressed the lipodystrophic phenotype in the adipocyte-specific SNAP23-KO mice and prevented cell death. In addition, ATG9 deficiency phenocopied SNAP23 deficiency, whereas ATG7 deficiency had no effect on BAX protein levels, BAX activation, or apoptotic cell death. These data demonstrate a role for SNAP23 in the control of macroautophagy and programmed cell death through an ATG9-dependent, but ATG7-independent, pathway regulating BAX protein levels and BAX activation.