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Autophagy is a cellular self-degrading process that plays a key role in cellular health and functioning. Since autophagy disorder is related to many diseases, it is highly important to detect autophagy. This study aimed to establish a dual-sensing mechanism-based ratiometric viscosity-sensitive lysosome-targeted two-photon fluorescent probe

Cancer-associated fibroblasts (CAFs) are one of the most prominent and active components in the pancreatic tumor microenvironment. Our data show that CAFs are critical for survival from pancreatic ductal adenocarcinoma (PDAC) on glutamine deprivation. Specifically, we uncovered a role for nucleosides, which are secreted by CAFs through autophagy in a nuclear fragile X mental retardation-interacting protein 1 (NUFIP1)-dependent manner, increased glucose utilization and promoted growth of PDAC. Moreover, we demonstrate that CAF-derived nucleosides induced glucose consumption under glutamine-deprived conditions and displayed a dependence on MYC. Using an orthotopic mouse model of PDAC, we found that inhibiting nucleoside secretion by targeting NUFIP1 in the stroma reduced tumor weight. This finding highlights a previously unappreciated metabolic network within pancreatic tumors in which diverse nutrients are used to promote growth in an austere tumor microenvironment.

Background and Objectives Calcium-sensing receptor (CaSR) is known to regulate hypoxia-induced pulmonary hypertension (HPH) and vascular remodeling via the phenotypic modulation of pulmonary arterial smooth muscle cells (PASMCs) in small pulmonary arteries. Moreover, autophagy is an essential modulator of VSMC phenotype. But it is not clear whether CaSR can regulate autophagy involving the phenotypic modulation under hypoxia. Methods The viability of human PASMCs was detected by cell cycle and BrdU. The expressions of proliferation protein, phenotypic marker protein, and autophagy protein in human PASMCs were determined by western blot. Results Our results showed that hypoxia-induced autophagy was considerable at 24 h. The addition of NPS2390 decreased the expression of autophagy protein and synthetic phenotype marker protein osteopontin and increased the expression of contractile phenotype marker protein SMA-ɑ and calponin via suppressing downstream PI3K/Akt/mTOR signal pathways. Conclusions Our study demonstrates that treatment of NPS2390 was conducive to inhibit the proliferation and reverse phenotypic modulation of PASMCs by regulating autophagy levels.

In recent years, the global incidence and mortality of myocardial infarction (MI) has increased and become one of the important diseases threatening public health. Long non‑coding (lnc)RNAs are a type of ncRNA that serve critical roles in the progression of various types of disease. The present study aimed to investigate the effect and mechanism of lncRNA cardiac autophagy inhibitory factor (CAIF) on cardiac ischemia/reperfusion (I/R) injury. CAIF was downregulated in the myocardium of I/R rats and cardiomyocytes treated with hydrogen peroxide (H2O2). Further experiments demonstrated that CAIF overexpression inhibited I/R‑induced cardiac infarction and apoptosis

Autophagy is a protein degradation process by which intracellular materials are recycled for energy homeostasis. However, the metabolic status and energy source of autophagy-defective tumor cells are poorly understood. Here, our data show that amino acid uptake from the extracellular environment is increased in autophagy-deficient cells upon glutamine deprivation. This elevated amino acid uptake results from activating transcription factor 4 (ATF4)-dependent upregulation of AAT (amino acid transporter) gene expression. Furthermore, we identify SIRT6, a NAD+-dependent histone deacetylase, as a corepressor of ATF4 transcriptional activity. In autophagy-deficient cells, activated NRF2 enhances ATF4 transcriptional activity by disrupting the interaction between SIRT6 and ATF4. In this way, autophagy-deficient cells exhibit increased AAT expression and show increased amino acid uptake. Notably, inhibition of amino acid uptake reduces the viability of glutamine-deprived autophagy-deficient cells, but not significantly in wild-type cells, suggesting reliance of autophagy-deficient tumor cells on extracellular amino acid uptake.

High levels of autophagy exist in Leydig cells of the testis, but its physiological function is unknown. Gao et al. now show that autophagy promotes uptake of cholesterol, an essential precursor for testosterone synthesis, by removing the NHERF2 negative regulator of the high-density lipoprotein receptor SR-BI., Testosterone is indispensable for sexual development and maintaining male characteristics, and deficiency of this hormone results in primary or late-onset hypogonadism (LOH). Testosterone is primarily produced in Leydig cells, where autophagy has been reported to be extremely active. However, the functional role of autophagy in testosterone synthesis remains unknown. In this study, we show that steroidogenic cell–specific disruption of autophagy influenced the sexual behavior of aging male mice because of a reduction in serum testosterone, which is similar to the symptoms of LOH. The decline in testosterone was caused mainly by a defect in cholesterol uptake in autophagy-deficient Leydig cells. Further studies revealed that once autophagic flux was disrupted, Na+/H+ exchanger regulatory factor 2 (NHERF2) accumulated in Leydig cells, resulting in the down-regulation of scavenger receptor class B, type I (SR-BI) and eventually leading to insufficient cholesterol supply. Collectively, these results reveal that autophagy promotes cholesterol uptake into Leydig cells by eliminating NHERF2, suggesting that dysfunction of autophagy might be causal in the loss of testosterone production in some patients.

// Ling Li 1, 2 , Hongli Jin 1, 2 , Hualei Wang 2, 4 , Zengguo Cao 2 , Na Feng 2, 4 , Jianzhong Wang 3 , Yongkun Zhao 2 , Xuexing Zheng 2, 5 , Pengfei Hou 1, 2 , Nan Li 2 , Hang Chi 2 , Pei Huang 2, 3 , Cuicui Jiao 2 , Qian Li 2 , Lina Wang 2, 3 , Tiecheng Wang 2 , Weiyang Sun 2 , Yuwei Gao 2, 4 , Changchun Tu 2 , Guixue Hu 3 , Songtao Yang 2, 4 , Xianzhu Xia 2, 4 1 Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, China 2 Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun, China 3 Department of Preventive Veterinary Medicine, College of Animal Science and Technology, Jilin Agricultural University, Changchun, China 4 Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou, China 5 School of Public Health, Shandong University, Jinan, China Correspondence to: Hualei Wang email: whl831125@163.com Songtao Yang email: yst62041@163.com Guixue Hu email: huguixue901103@163.com Keywords: rabies virus, proteome, proteomics analysis, autophagy, differential virulence Received: May 23, 2016 Accepted: January 16, 2017 Published: February 08, 2017 ABSTRACT Rabies virus (RABV) is a neurotropic virus that causes serious disease in humans and animals worldwide. It has been reported that different RABV strains can result in divergent prognoses in animal model. To identify host factors that affect different infection processes, a kinetic analysis of host proteome alterations in mouse brains infected with different virulent RABV strains was performed using isobaric tags for a relative and absolute quantification (iTRAQ)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach, and this analysis identified 147 differentially expressed proteins (DEPs) between the pathogenic challenge virus standard (CVS)-11 strain and the attenuated SRV9 strain. Bioinformatics analyses of these DEPs revealed that autophagy and several pathways associated with autophagy, such as mammalian target of rapamycin (mTOR) signaling, p70S6K signaling, nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress and superoxide radical degradation, were dysregulated. Validation of the proteomic data showed that attenuated SRV9 induced more autophagosome accumulation than CVS-11 in an in vitro model. Our findings provide new insights into the pathogenesis of RABV and encourage further studies on this topic.

BACKGROUND This study was proposed to compare the efficacy and safety of GTM-1, Rapamycin (Rap), and Carbamazepine (CBZ) in managing Alzheimer disease (AD). The impact of the above mentioned therapeutic drugs on autophagy was also investigated in our study. MATERIAL AND METHODS Firstly, 3×Tg AD mice were randomly allocated into 4 groups (each group with 10 mice), in which AD mice were separately treated with dimethylsulfoxide (DMSO, vehicle group), GTM-1 (6 mg/kg), Rap (1 mg/kg), and CBZ (100 mg/kg). Then spatial memory and learning ability of mice was tested using the Morris water maze. Routine blood tests were performed to evaluate the toxicity of these drugs. Amyloid-β42 (Aβ42) concentration was detected by ELISA and immunohistochemistry. Proteins related to autophagy were detected by Western blot. RESULTS GTM-1, Rap, and CBZ significantly improved the spatial memory of 3×Tg AD mice compared to that in the vehicle group (all P

Manganese (Mn) pollution is an important environmental problem because of the potential toxicity to human and animal health. However, the effects of Mn on energy metabolism and autophagy are not clear. Consequently, we examined the effects of excessive and chronic exposure to Mn on liver function, oxidative stress, respiratory chain complex activity, and autophagy in chicken liver. Our results indicated that the accumulation of Mn in the liver and levels of AST and ALT in the serum of the Mn-exposed group were significantly higher (P < 0.05) than those in the control group at 90 days; the activities of GSH-Px, SOD, CAT, Na+-K+-ATPase, Mg2+-ATPase, Ca2+-ATPase, and respiratory chain complexes (I, II, III) in the Mn-exposed group were significantly decreased (P < 0.05) compared to the control group. However, the MDA content, NO content, iNOS activity, mRNA and protein levels of iNOS, and autophagy-related genes in the Mn-exposed group were significantly increased (P < 0.05) compared to the control group. In contrast, the mRNA level and protein expression of mTOR were significantly decreased (P < 0.05) compared to the control group. Furthermore, the characteristic autophagic vacuolar organelles were observed in the Mn-exposed group. These results suggested that excess Mn exposure can cause a disorder of energy metabolism by mitochondrial injury and induce oxidative stress and autophagy, which eventually lead to liver damage.

The calcium-sensing receptor (CaSR) releases intracellular calcium ([Ca2+]i) by accumulating inositol phosphate. Changes in [Ca2+]i initiate myocardial hypertrophy. Furthermore, autophagy associated with [Ca2+]i. Autophagy has previously been demonstrated to participate in the hypertrophic process. The current study investigated whether suppression of CaSR affects the hypertrophic response via modulating autophagy. Isoproterenol (ISO) was used to induce cardiac hypertrophy in Wistar rats. Hypertrophic status was determined by echocardiographic assessment, hematoxylin and eosin, and Masson's staining. The protein expression levels of CaSR and autophagy level were observed. Changes of hypertrophy and autophagy indicators were observed following intravenous injection of a CaSR inhibitor. An ISO-induced cardiomyocyte hypertrophy model was established and used determine the involvement of GdCl3. [Ca2+]i was determined using Fluo-4/AM dye followed by confocal microscopy. The expression levels of various active proteins were analyzed by western blotting. The size of the heart, expression levels of CaSR and autophagy level were markedly increased in hypertrophic myocardium. In addition, the present study demonstrated that the indicators of hypertrophy and autophagy were effectively suppressed by CaSR inhibitor. Furthermore, similar effects were demonstrated in neonatal rat hypertrophic cardiomyocytes treated with ISO. It was also observed that CaSR regulates the Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ)-AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling pathway induced by ISO in cardiomyocytes. Furthermore, the AMPK inhibition significantly reduced the autophagy level following CaSR stimulation (P

Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the autophagy regulation machinery has been widely studied, the key epigenetic control of autophagy process still remains unknown. Here we report that the methyltransferase EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) epigenetically represses several negative regulators of the MTOR (mechanistic target of rapamycin [serine/threonine kinase]) pathway, such as TSC2, RHOA, DEPTOR, FKBP11, RGS16 and GPI. EZH2 was recruited to these genes promoters via MTA2 (metastasis associated 1 family, member 2), a component of the nucleosome remodeling and histone deacetylase (NuRD) complex. MTA2 was identified as a new chromatin binding protein whose association with chromatin facilitated the subsequent recruitment of EZH2 to silenced targeted genes, especially TSC2. Downregulation of TSC2 (tuberous sclerosis 2) by EZH2 elicited MTOR activation, which in turn modulated subsequent MTOR pathway-related events, including inhibition of autophagy. In human colorectal carcinoma (CRC) tissues, the expression of MTA2 and EZH2 correlated negatively with expression of TSC2, which reveals a novel link among epigenetic regulation, the MTOR pathway, autophagy induction, and tumorigenesis.

Background/Aims: Intracellular calcium concentration ([Ca2+]i) homeostasis, an initial factor of cardiac hypertrophy, is regulated by the calcium-sensing receptor (CaSR) and is associated with the formation of autolysosomes. The aim of this study was to investigate the role of Calhex231, a CaSR inhibitor, on the hypertrophic response via autophagy modulation. Methods: Cardiac hypertrophy was induced by transverse aortic constriction (TAC) in 40 male Wistar rats, while 10 rats underwent a sham operation and served as controls. Cardiac function was monitored by transthoracic echocardiography, and the hypertrophy index was calculated. Cardiac tissue was stained with hematoxylin and eosin (H&E) or Masson's trichrome reagent and examined by transmission electron microscopy. An angiotensin II (Ang II)-induced cardiomyocyte hypertrophy model was established and used to test the involvement of active molecules. Intracellular calcium concentration ([Ca2+]i) was determined by the introduction of Fluo-4/AM dye followed by confocal microscopy. The expression of various active proteins was analyzed by western blot. Results: The rats with TAC-induced hypertrophy had an increased heart size, ratio of heart weight to body weight, myocardial fibrosis, and CaSR and autophagy levels, which were suppressed by Calhex231. Experimental results using Ang II-induced hypertrophic cardiomyocytes confirmed that Calhex231 suppressed CaSR expression and downregulated autophagy by inhibiting the Ca2+/calmodulin-dependent-protein kinase-kinase-β (CaMKKβ)- AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway to ameliorate cardiomyocyte hypertrophy. Conclusions: Calhex231 ameliorates myocardial hypertrophy induced by pressure-overload or Ang II via inhibiting CaSR expression and autophagy. Our results may support the notion that Calhex231 can become a new therapeutic agent for the treatment of cardiac hypertrophy.

Sirt1 is a member of the sirtuin family of proteins and has important roles in numerous biological processes. Sirt1−/- mice display an increased frequency of abnormal spermatozoa, but the mechanism of Sirt1 in spermiogenesis remains largely unknown. Here, we report that Sirt1 might be directly involved in spermiogenesis in germ cells but not in steroidogenic cells. Germ cell-specific Sirt1 knockout mice were almost completely infertile; the early mitotic and meiotic progression of germ cells in spermatogenesis were not obviously affected after Sirt1 depletion, but subsequent spermiogenesis was disrupted due to a defect in acrosome biogenesis, which resulted in a phenotype similar to that observed in human globozoospermia. In addition, LC3 and ATG7 deacetylation was disrupted in the spermatids after knocking out Sirt1, which affected the redistribution of LC3 from the nucleus to the cytoplasm and the activation of autophagy. Furthermore, Sirt1 depletion resulted in the failure of LC3 to be recruited to Golgi apparatus-derived vesicles and in the failure of GOPC and PICK1 to be recruited to nucleus-associated acrosomic vesicles. Taken together, our findings reveal that Sirt1 has a novel physiological function in acrosome biogenesis.

Autophagy is activated to maintain cellular energy homeostasis in response to nutrient starvation. However, autophagy is not persistently activated, which is poorly understood at a mechanistic level. Here, we report that turnover of FoxO1 is involved in the dynamic autophagic process caused by glutamine starvation. X-box-binding protein-1u (XBP-1u) has a critical role in FoxO1 degradation by recruiting FoxO1 to the 20S proteasome. In addition, the phosphorylation of XBP-1u by extracellular regulated protein kinases1/2 (ERK1/2) on Ser61 and Ser176 was found to be critical for the increased interaction between XBP-1u and FoxO1 upon glutamine starvation. Furthermore, knockdown of XBP-1u caused the sustained level of FoxO1 and the persistent activation of autophagy, leading to a significant decrease in cell viability. Finally, the inverse correlation between XBP-1u and FoxO1 expression agrees well with the expression profiles observed in many human cancer tissues. Thus, our findings link the dynamic process of autophagy to XBP-1u-induced FoxO1 degradation.

Cardiovascular disease (CVD) is the leading cause of the death worldwide. An increasing number of studies have found that autophagy is involved in the progression or prevention of CVD. However, the precise mechanism of autophagy in CVD, especially the myocardial ischaemia-reperfusion injury (MI/R injury), is unclear and controversial. Here, we show that the cardiomyocyte-specific disruption of autophagy by conditional knockout of Atg7 leads to severe contractile dysfunction, myofibrillar disarray and vacuolar cardiomyocytes. A negative cytoskeleton organization regulator, CLP36, was found to be accumulated in Atg7 -deficient cardiomyocytes. The cardiomyocyte-specific knockout of Atg7 aggravates the MI/R injury with cardiac hypertrophy, contractile dysfunction, myofibrillar disarray and severe cardiac fibrosis, most probably due to CLP36 accumulation in cardiomyocytes. Altogether, this work reveals autophagy may protect cardiomyocytes from the MI/R injury through the clearance of CLP36, and these findings define a novel relationship between autophagy and the regulation of stress fibre in heart.

The ectoplasmic specialization (ES) is essential for Sertoli-germ cell communication to support all phases of germ cell development and maturity. Its formation and remodeling requires rapid reorganization of the cytoskeleton. However, the molecular mechanism underlying the regulation of ES assembly is still largely unknown. Here, we show that Sertoli cell-specific disruption of autophagy influenced male mouse fertility due to the resulting disorganized seminiferous tubules and spermatozoa with malformed heads. In autophagy-deficient mouse testes, cytoskeleton structures were disordered and ES assembly was disrupted. The disorganization of the cytoskeleton structures might be caused by the accumulation of a negative cytoskeleton organization regulator, PDLIM1, and these defects could be partially rescued by Pdlim1 knockdown in autophagy-deficient Sertoli cells. Altogether, our works reveal that the degradation of PDLIM1 by autophagy in Sertoli cells is important for the proper assembly of the ES, and these findings define a novel role for autophagy in Sertoli cell-germ cell communication.

Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome, and loss of autophagy has been linked to increased genome instability. Here, we report that loss of autophagy is coupled to reduced histone H2A ubiquitination after DNA damage. p62/SQSTM1, which accumulates in autophagy-defective cells, directly binds to and inhibits nuclear RNF168, an E3 ligase essential for histone H2A ubiquitination and DNA damage responses. As a result, DNA repair proteins such as BRCA1, RAP80, and Rad51 cannot be recruited to the sites of DNA double-strand breaks (DSBs), which impairs DSB repair. Moreover, nuclear-localized p62 increased the sensitivity of tumor cells to radiation both in vitro and in vivo, and this required its interaction with RNF168. Our findings indicate that autophagy-deficiency-induced p62 accumulation results in inhibition of histone ubiquitination and highlight the complex relationship between autophagy and the DNA damage response.

Earlier studies have shown that macroautophagy is not a constitutively activated process, however, the mechanism of activation is not fully understood. Here, we report that autophagy is a dynamic process in cancer cells in response to glucose starvation. In addition, we determined that FOXO1 turnover is involved in the regulation of this dynamic process. X-box binding protein 1u (XBP1u) plays a critical role in FOXO1 degradation by recruiting FOXO1 to the 20S proteasome. Moreover, the phosphorylation of XBP1u by mitogen-activated protein kinases 1 and 3 (MAPK1/3, also known as ERK2/1) on serine residues 61 and 176 was found to be essential for the enhancement of the interaction between XBP1u and FOXO1. Thus, our findings support the hypothesis that the turnover of FOXO1 induced by MAPK1/3 and XBP1u is a critical factor regulating the autophagic process.

Pulmonary arterial hypertension (PAH) is a progressive pulmonary vascular disorder with high morbidity and mortality, and is characterized by excessive growth of endothelial cells. Recently, the mammalian target of rapamycin (mTOR) has attracted increasing attention due to its potential as a therapeutic target against certain diseases associated with proliferative and metabolic abnormalities. However, the effect on mTOR on PAH has not yet been elucidated. In the present study, a marked downregulation of mTOR was observed in PAH patients. Following construction of a mouse model of PAH by chronic exposure to hypoxia, adenovirus-mediated upregulation of mTOR significantly attenuated right ventricular systolic pressure, right ventricular hypertrophy and wall thickness of pulmonary arterioles, indicating a protective effect of mTOR on PAH. Further analysis confirmed that mTOR overexpression inhibited autophagy triggered by hypoxia through blocking light chain 3 II expression and increasing p62 levels. In vitro, hypoxia enhanced the proliferation of human pulmonary artery endothelial cells (PAECs), which was markedly abrogated by mTOR overexpression. Of note, upregulation of mTOR inhibited the hypoxia-induced autophagy pathway, which contributed to cell proliferation, while silencing of autophagy by RNA interference with ATG5 significantly inhibited cell proliferation. In conclusion, the results of the present study suggested a potential protective effect of mTOR on the progression of PAH by suppressing PAEC proliferation through blocking the autophagic pathway. Therefore, the present study suggested that mTOR is a promising therapeutic agent against PAH.

Forkhead box O (FOXO) transcriptional protein family members, including FOXO1 and FOXO3, are involved in the modulation of autophagy. However, whether there is redundancy between FOXO1 and FOXO3 in the ability to induce autophagy remains unclear. In this study, we showed that FOXO3 induced a transcription-dependent autophagy, and FOXO1 was required for this process. Overexpression of wild-type FOXO3 (WT) or FOXO3 (3A), which harbors alanine mutations at residues Thr32, Ser253 and Ser315, but not transcription-inactive FOXO3 (∆DB3A), significantly induced autophagy in the human embryonic kidney cell line HEK293T and mouse embryonic fibroblast (MEF) cell lines. Interestingly, depletion of FOXO1 by siRNA attenuated FOXO3-induced autophagy. Our data also showed that FOXO3 overexpression did not increase the expression of FOXO1 at the protein level, although FOXO3 was capable of binding the promoter region of FOXO1 and inducing an increase in the transcription of FOXO1 mRNA. Furthermore, our results showed that FOXO3 promoted the translocation of FOXO1 from the nucleus to the cytoplasm, resulting in an increase in FOXO1-induced autophagy. Moreover, our results supported a mechanism whereby FOXO3 dramatically increased the expression of the class I PtdIns3K catalytic subunit PIK3CA, leading to an increase in AKT1 activity, which resulted in the phosphorylation and nuclear export of FOXO1. To the best of our knowledge, our data are the first to suggest that FOXO1 plays a central role in FOXO3-induced autophagy.

Although Beclin 1 and mTOR are considered to be the main molecules to modulate the autophagic process, searching for other autophagy-regulating molecules is still an ongoing challenge to scientists. Here we demonstrated that FoxO1, a forkhead O family protein, is a mediator of autophagy. Upon oxidative stress or serum starvation, endogenous FoxO1 was required for autophagy in human cancer cell lines, and this process was independent of FoxO1's transcriptional activity as well as mTOR or Beclin 1. In response to stress, FoxO1 dissociated from an NAD+-dependent histone deacetylase SIRT2, and FoxO1 thus became acetylated, and in turn bound to Atg7, an E1-like protein, to influence the autophagic process leading to cell death. In particular, cytosolic FoxO1 suppressed tumor xenograft growth in nude mice in an autophagy-dependent manner. Our studies provide evidence that cytosolic FoxO1-induced autophagy is associated with tumor suppression function.