Autophagy supports survival and phototransduction protein levels in rod photoreceptors

Damage and loss of the postmitotic photoreceptors is a leading cause of blindness in many diseases of the eye. Although the mechanisms of photoreceptor death have been extensively studied, few studies have addressed mechanisms that help sustain these non-replicating neurons for the life of an organism. Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomal pathway for degradation. It is not only a major pathway activated in response to cellular stress, but is also important for cytoplasmic turnover and to supply the structural and energy needs of cells. We examined the importance of autophagy in photoreceptors by deleting the essential autophagy gene Atg5 specifically in rods. Loss of autophagy led to progressive degeneration of rod photoreceptors beginning at 8 weeks of age such that by 44 weeks few rods remained. Cone photoreceptor numbers were only slightly diminished following rod degeneration but their function was significantly decreased. Rod cell death was apoptotic but was not dependent on daily light exposure or accelerated by intense light. Although the light-regulated translocation of the phototransduction proteins arrestin and transducin were unaffected in rods lacking autophagy, Atg5-deficient rods accumulated transducin-α as they degenerated suggesting autophagy might regulate the level of this protein. This was confirmed when the light-induced decrease in transducin was abolished in Atg5-deficient rods and the inhibition of autophagy in retinal explants cultures prevented its degradation. These results demonstrate that basal autophagy is essential to the long-term health of rod photoreceptors and a critical process for maintaining optimal levels of the phototransduction protein transducin-α. As the lack of autophagy is associated with retinal degeneration and altered phototransduction protein degradation in the absence of harmful gene products, this process may be a viable therapeutic target where rod cell loss is the primary pathologic event.Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomes for degradation. Defective autophagy can contribute to the age-dependent accumulation of damaged proteins and organelles leading to altered cellular homeostasis and loss of function.^{1, 2, 3, 4, 5} The metabolic roles of autophagy can be classified into two types, basal and induced. In nutrient-rich conditions, autophagy is suppressed but still occurs at low levels (basal autophagy); however, when cells are subjected to stress (starvation, injury, hypoxia), autophagy is activated immediately (induced autophagy).⁶ Induced autophagy maintains the amino acid pool inside cells to adapt to starvation while constitutive autophagy has been shown to function as a cell-repair mechanism that is important for long-lived postmitotic cells.^{7, 8, 9, 10, 11} Defects in autophagy have been associated with neurodegenerative diseases,^{12, 13, 14, 15} diabetes,^{16, 17} lysosomal storage disease¹⁸ and the loss of vision.¹⁹ In addition to macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) have been described. Although little is known about microautophagy in mammalian cells, macroautophagy (hereafter autophagy) is a major pathway for bulk degradation of cytoplasmic components. CMA is a more selective pathway for degradation of cytosolic proteins that can compensate for the loss of macroautophagy.^{2, 20, 21, 22}Inherited retinal degenerative diseases such as retinitis pigmentosa or Leber''s congenital amaurosis are characterized by premature and progressive death of rod and cone photoreceptor cells.²³ These diseases are characterized by the loss of night vision due to the death of rods followed by the loss of cones leading to diminished visual acuity and a reduction in the quality of life for patients. Disease is typically associated with the production of harmful gene products that promote pathology by inhibiting critical pathways resulting in cell death.^{24, 25, 26} Strategies to prevent photoreceptor death during retinal degenerative disease such as gene replacement therapies or inhibition of cell death pathways have been undertaken with some success;^{27, 28, 29} however, effective treatments for these blinding disorders are lacking.Another strategy that could be used in conjunction with other therapies might be to enhance survival by stimulating autophagy. Augmenting autophagy would increase the supply of nutrients to stressed cells and accelerate removal of damaged proteins thereby prolonging cell survival beyond what would be possible by only preventing cell death. Although canonical^{22, 30, 31, 32, 33} and noncanonical autophagic mechanisms³⁴ have been detected in the eye, our knowledge of basic autophagy functions in this organ is still limited. In order to understand how autophagy maintains retinal homeostasis and function, we undertook studies to examine the consequences of deleting the essential autophagy gene Atg5 in rod photoreceptors.     

The plant metacaspase AtMC1 in pathogen-triggered programmed cell death and aging: functional linkage with autophagy

Autophagy is a major nutrient recycling mechanism in plants. However, its functional connection with programmed cell death (PCD) is a topic of active debate and remains not well understood. Our previous studies established the plant metacaspase AtMC1 as a positive regulator of pathogen-triggered PCD. Here, we explored the linkage between plant autophagy and AtMC1 function in the context of pathogen-triggered PCD and aging. We observed that autophagy acts as a positive regulator of pathogen-triggered PCD in a parallel pathway to AtMC1. In addition, we unveiled an additional, pro-survival homeostatic function of AtMC1 in aging plants that acts in parallel to a similar pro-survival function of autophagy. This novel pro-survival role of AtMC1 may be functionally related to its prodomain-mediated aggregate localization and potential clearance, in agreement with recent findings using the single budding yeast metacaspase YCA1. We propose a unifying model whereby autophagy and AtMC1 are part of parallel pathways, both positively regulating HR cell death in young plants, when these functions are not masked by the cumulative stresses of aging, and negatively regulating senescence in older plants.An emerging theme in cell death research is that cellular processes thought to be regulated by linear signaling pathways are, in fact, complex. Autophagy, initially considered merely a nutrient recycling mechanism necessary for cellular homeostasis, was recently shown to regulate cell death, mechanistically interacting with components that control apoptosis. Deficient autophagy can result in apoptosis^{1, 2, 3} and autophagy hyper-activation can also lead to programmed cell death (PCD).⁴ In addition, the pro-survival function of autophagy is mediated by apoptosis inhibition and apoptosis mediates autophagy, although this cross-regulation is not fully understood.⁵In plants, autophagy can also have both pro-survival and pro-death functions. Autophagy-deficient plants exhibit accelerated senescence,^{6, 7, 8} starvation-induced chlorosis,^{6, 7, 9} hypersensitivity to oxidative stress¹⁰ and endoplasmic reticulum stress.¹¹ Further, autophagy-deficient plants cannot limit the spread of cell death after infection with tissue-destructive microbial infections.^{12, 13} The plant phytohormone salicylic acid (SA) mediates most of these phenotypes.⁸ Autophagy has an essential, pro-survival role in situations where there is an increasing load of damaged proteins and organelles that need to be eliminated, that is, during aging or stress. Autophagy has an opposing, pro-death role during developmentally regulated cell death^{14, 15} or during the pathogen-triggered hypersensitive response PCD (hereafter, HR) that occurs locally at the site of attempted pathogen attack.^{16, 17} The dual pro-death/pro-survival functions of plant autophagy remain a topic of active debate.Also under scrutiny are possible novel functions of caspases and caspase-like proteins as central regulators of pro-survival processes. Caspases were originally defined as executioners of PCD in animals, but increasing evidence indicates that several caspases have non-apoptotic regulatory roles in cellular differentiation, motility and in the mammalian immune system.^{18, 19, 20}Yeast, protozoa and plants do not have canonical caspases, despite the occurrence of morphologically heterogeneous PCDs.²¹ More than a decade ago, distant caspase homologs termed metacaspases were identified in these organisms using structural homology searches.²² Metacaspases were classified into type I or type II metacaspases based on the presence or absence of an N-terminal prodomain, reminiscent of the classification in animals into initiator/inflammatory or executioner caspases, respectively. Despite the architectural analogy between caspases and metacaspases, differences in their structure, function, activation and mode of action exist.^{23, 24, 25}Metacaspases mediate PCD in yeast,^{26, 27, 28, 29, 30, 31} leishmania,^{32, 33} trypanosoma³⁴ and plants.²⁴ We demonstrated that two type I metacaspases, AtMC1 and AtMC2, antagonistically regulate HR in Arabidopsis thaliana.³⁵ Our work showed that AtMC1 is a positive regulator of HR and that this function is mediated by its catalytic activity and negatively regulated by the AtMC1 N-terminal prodomain. AtMC2 antagonizes AtMC1-mediated HR.Besides AtMC2, new examples of metacaspases with a pro-life/non-PCD role are emerging. Protozoan metacaspases are involved in cell cycle dynamics^{34, 36, 37, 38} and cell proliferation.³⁹ The yeast metacaspase Yca1 alters cell cycle dynamics⁴⁰ and interestingly, is required for clearance of insoluble protein aggregates, thus contributing to yeast fitness.⁴¹Here, we explore the linkage between plant autophagy and AtMC1 function in the context of pathogen-triggered HR and aging. Our data support a model wherein autophagy and AtMC1 are part of parallel pathways, both positively regulating HR cell death in young plants and negatively regulating senescence in older plants.     

Epigallocatechin-3-gallate opposes HBV-induced incomplete autophagy by enhancing lysosomal acidification,which is unfavorable for HBV replication

Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, exhibits diverse beneficial properties, including antiviral activity. Autophagy is a cellular process that is involved in the degradation of long-lived proteins and damaged organelles. Recent evidence indicates that modulation of autophagy is a potential therapeutic strategy for various viral diseases. In the present study, we investigated the effect of EGCG on hepatitis B virus (HBV) replication and the possible involvement of autophagy in this process. Our results showed that HBV induced autophagosome formation, which was required for replication of itself. However, although EGCG efficiently inhibited HBV replication, it enhanced, but not inhibited, autophagosome formation in hepatoma cells. Further study showed that HBV induced an incomplete autophagy, while EGCG, similar to starvation, was able to induce a complete autophagic process, which appeared to be unfavorable for HBV replication. Furthermore, it was found that HBV induced an incomplete autophagy by impairing lysosomal acidification, while it lost this ability in the presence of EGCG. Taken together, these data demonstrated that EGCG treatment opposed HBV-induced incomplete autophagy via enhancing lysosomal acidification, which was unfavorable for HBV replication.Macroautophagy (hereafter autophagy) is a conserved cellular process through which cytoplasmic materials are sequestered into double-membrane vacuole called autophagosomes and destined for degradation through fusion with lysosomes.^{1, 2, 3} Accumulating evidence indicates that autophagy is involved in diverse pathophysiological processes, including cancer, neurodegenerative disorders, and cardiovascular diseases.^{4, 5, 6, 7} Recent studies show that autophagy has an important role in regulating the replication of many viruses, including dengue virus, coxsackievirus B3 virus (CVB3), hepatitis C virus (HCV), and influenza virus A.^{8, 9, 10, 11, 12} Several investigations also indicate that autophagy has an important role in hepatitis B virus (HBV) replication: autophagy is induced by HBV and is required for HBV replication; however, the underlying mechanisms remains still unclear.^{13, 14, 15, 16}Green tea is the most commonly consumed beverage worldwide. In traditional Chinese medicine, green tea is considered to have beneficial properties for human health, including antitumorigenic, antioxidant, and anti-inflammatory activities.^{17, 18, 19} Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea and appears to be the primary active ingredient accounting for the latter''s biological effects. In recent years, EGCG is revealed to display inhibitory effect on diverse viruses, such as human immunodeficiency virus type-1, Epstein–Barr virus (EBV), and HCV.^{20, 21, 22, 23, 24, 25} Of interest, EGCG is also found to regulate autophagy formation, although it seems to be cell-type specific.^{26, 27, 28, 29, 30} Given the potential therapeutic effect of EGCG on viral infection and its role in autophagy regulation, we investigated the effect of EGCG on HBV replication and the possible involvement of autophagy in this process.Here we showed that HBV induced an incomplete autophagy that was required for HBV replication; however, a complete autophagic process induced by EGCG appeared to be unfavorable for HBV replication. Further study showed that HBV hampered the autophagic flux by impairing lysosomal acidification, which could be opposed by the treatment of EGCG.     

Pdcd4 deficiency enhances macrophage lipoautophagy and attenuates foam cell formation and atherosclerosis in mice

Macrophage foam cells, a major component of the atherosclerotic lesion, have vital roles in the development of atherosclerosis. Lipoautophagy, a type of autophagy characterized by selective delivery of lipid droplet for lysosomal degradation, may impact atherosclerosis by regulating macrophage foam cell formation. Previously, we reported that programmed cell death 4 (PDCD4), a tumor suppressor, negatively regulated autophagy in tumor cells. However, its roles in macrophage lipoautophagy, foam cell formation and atherosclerosis remain to be established. Here we found that Pdcd4 deficiency clearly improved oxidized low-density lipoproteins-impaired autophagy efflux, promoted autophagy-mediated lipid breakdown in murine macrophages and thus prevented macrophage conversion into foam cells. Importantly, Pdcd4 deficiency in mice significantly upregulated macrophage autophagy in local plaques along with attenuated lipid accumulation and atherosclerotic lesions in high-fat-fed Apolipoprotein E knockout mice. Bone marrow transplantation experiment demonstrated that PDCD4-mediated autophagy in hematopoietic cells contributed to the development of atherosclerosis. These results indicate that endogenous PDCD4 promotes for macrophage foam cell formation and atherosclerosis development via inhibiting autophagy and provides new insights into atherogenesis, suggesting that promoting macrophage autophagy through downregulating PDCD4 expression may be beneficial for treating atherosclerosis.Atherosclerosis is a lipid dysfunction-derived chronic inflammatory process in large and medium arterial wall.¹ Macrophage foam cell, as a major component in the lesion of atherosclerosis, has vital role in the development of atherosclerosis. In the initial step of atherosclerotic development, circulating monocytes migrate into arterial wall via dysfunctional endothelial cells and differentiate into macrophages.^{2, 3, 4} The infiltrated macrophages ingest and digest oxidized low-density lipoprotein (ox-LDL), and then transport lipid out of vascular wall.⁵ However, macrophage with overloaded lipids stored in the form of lipid droplets (LDs) will transform into foam cells. Macrophage foam cell formation could promote the development of atherosclerosis.⁶ Thus, decreasing the formation of macrophage foam cell would be an attractive strategy to reverse plaque lipid buildup.⁷The macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved and well-controlled cellular catabolic process. During the process, cytoplasmic components are sequestered in double-membrane vesicles (which is called autophagosome) and degraded by fusion with lysosomal compartments (autophagolysosome) for recycling application.⁸ The process of autophagy is regulated by several autophagy-related genes (ATGs) encoded proteins, such as ATG5, ATG6 (also known as BECN1), ATG8 (also known as microtubule-associated protein 1 light chain 3, LC3) and ATG12. ATG5 is involved in the early stage of autophagosome formation. ATG5 is conjugated with ATG12 and ATG16L to form ATG12–ATG5–ATG16L complex, which contributes to the elongation and closure of the autophagosomes in the generation of lipidated forms of LC3 family proteins.⁹ Lipoautophagy, a type of autophagy that selectively delivers LDs for lysosomal degradation,¹⁰ regulates lipid metabolism and is involved in the process of atherosclerosis.^{11, 12, 13, 14} In advanced atherosclerosis, macrophage autophagy becomes dysfunctional. However, the basic autophagy deficiency in macrophage by specific Atg5 knockout accelerates atherosclerotic plaques in high-fat-fed ldlr^−/− mice via promoting oxidative stress, plaque necrosis¹² or inflammasome hyperactivation.¹³ More interestingly, autophagy can enhance brokendown of lipid in LD, cholesterol efflux from macrophage foam cells and further inhibit atherogenisis.¹⁴ Stent-based delivery of everolimus (mTOR inhibitor) in atherosclerotic plaques of cholesterol-fed rabbits leads to a marked reduction of macrophages via autophagic cell death.¹⁵ Therefore, regulating the level of macrophage autophagy and macrophage conversion into foam cells would be a potential target for preventing the atherosclerotic plaques formation.¹⁶Programmed cell death 4 (PDCD4), an inhibitor of protein translation, inhibits translation initiation via binding to the translation initiation factor eIF4A or translation elongation by direct or indirectly binding to the coding region of specific RNAs.^{17, 18} Accumulated evidence has demonstrated PDCD4 as a tumor suppressor.¹⁹ PDCD4 can inhibit promotion and progression of tumors, such as lung cancer,²⁰ hepatocellular carcinoma cells,²¹ colon cancer,²² ovarian cancer²³ and glioma.²⁴ In addition, it has been reported that PDCD4 is also involved in the development of inflammatory diseases.^{25, 26, 27, 28, 29, 30} For example, Pdcd4-deficient mice are resistant to experimental allergic encephalitis,²⁵ LPS-induced endotoxin shock²⁶ and type-1 diabetes.²⁷ In addition, Pdcd4-deficient mice are sensitive to LPS/D-galactosamine-induced acute liver injury.²⁸ Recently, we reported that Pdcd4 deficiency attenuated adipocyte foam cells, diet-induced obesity, obesity-associated inflammation and insulin resistance,²⁹ and increased IL-10 expression by macrophages that partly involved in atherosclerosis in hyperlipidemic mice,³⁰ suggesting that PDCD4 may be involved in the metabolism-related diseases. Furthermore, we found that PDCD4 negatively regulated autophagy by inhibiting ATG5 expression in tumor cells.³¹ However, its role in macrophage lipoautophagy and foam formation, and association with atherosclerosis remain to be investigated.In the present study, we found that Pdcd4 deficiency improved ox-LDL-impaired autophagy efflux in murine macrophage and subsequently attenuated macrophage conversion into foam cells in an autophagy-dependent manner and further attenuated the formation of atherosclerotic lesions in hyperlipidemia mice. These results indicate that PDCD4 is critical for macrophage foam cell formation in atherosclerosis development and provides new insights into atherogenesis, and potential therapeutic avenues to treat atherosclerosis-associated diseases.     

Spermidine induces autophagy by inhibiting the acetyltransferase EP300

Several natural compounds found in health-related food items can inhibit acetyltransferases as they induce autophagy. Here we show that this applies to anacardic acid, curcumin, garcinol and spermidine, all of which reduce the acetylation level of cultured human cells as they induce signs of increased autophagic flux (such as the formation of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta and the depletion of sequestosome-1, p62/SQSTM1) coupled to the inhibition of the mammalian target of rapamycin complex 1 (mTORC1). We performed a screen to identify the acetyltransferases whose depletion would activate autophagy and simultaneously inhibit mTORC1. The knockdown of only two acetyltransferases (among 43 candidates) had such effects: EP300 (E1A-binding protein p300), which is a lysine acetyltranferase, and NAA20 (N(α)-acetyltransferase 20, also known as NAT5), which catalyzes the N-terminal acetylation of methionine residues. Subsequent studies validated the capacity of a pharmacological EP300 inhibitor, C646, to induce autophagy in both normal and enucleated cells (cytoplasts), underscoring the capacity of EP300 to repress autophagy by cytoplasmic (non-nuclear) effects. Notably, anacardic acid, curcumin, garcinol and spermidine all inhibited the acetyltransferase activity of recombinant EP300 protein in vitro. Altogether, these results support the idea that EP300 acts as an endogenous repressor of autophagy and that potent autophagy inducers including spermidine de facto act as EP300 inhibitors.Macroautophagy (herein referred to as ‘autophagy'') consist in the sequestration of cytoplasmic material in autophagosomes, followed by their fusion with lysosomes for the bulk degradation of autophagic cargo by lysosomal hydrolases.¹ This phenomenon can be measured by following the redistribution of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) fusion proteins from a diffuse location to autophagosomes (that results in the formation of the so-called GFP-LC3 ‘puncta''), the diminution of the overall abundance of autophagic substrates (such as sequestosome-1, p62/SQSTM1), and the stereotyped activation of proautophagic signals (such as the inhibition of the mammalian target of rapamycin complex 1, mTORC1).²There is growing consensus that the induction of autophagy by nutritional, pharmacological or genetic interventions can reduce age-related pathologies (such as neurodegenerative diseases or type 2 diabetes) and/or extend longevity.^{3, 4, 5, 6} This applies to caloric restriction or intermediate fasting,⁷ continuous or intermittent medication of rapamycin,^{8, 9, 10} administration of the sirtuin 1-activator resveratrol,^{11, 12} external supply of the polyamine spermidine,¹³ or genetic ablation of p53.¹⁴ In all these cases, inhibition of autophagy by deleting or silencing relevant genes abolishes the extension of health span and/or lifespan.^{13, 14, 15, 16, 17} Moreover, direct induction of autophagy by transgenic expression of autophagy-relevant genes such as ATG5 in mice is sufficient to increase lifespan.¹⁸Recently, acetyltransferases have emerged as a potential target for the pharmaceutical induction of autophagy. Thus, depletion of the sole donor of acetyl groups, acetyl-coenzyme A (acetyl-CoA), is sufficient to reduce the acetylation of cytoplasmic and nuclear proteins coupled to the induction of autophagy.^{19, 20, 21, 22} Culture of mammalian cells in nutrient-free (NF) conditions or starvation of mice for 24 h reduced the intracellular nucleocytosolic concentrations of acetyl-CoA at the same time as autophagy was induced, and replenishment of acetyl-CoA by external sources (for instance, by providing a membrane-permeant precursor of α-ketoglutarate for anaplerotic reactions or by microinjection of acetyl-CoA) was sufficient to inhibit starvation-induced autophagy.^{19, 20, 21, 22} Beyond the inhibition of acetyltransferases by acetyl-CoA depletion, direct pharmacological inhibition of acetyltransferases might also contribute to the induction of autophagy. A close correlation between autophagy induction and deacetylation of cytoplasmic proteins was observed in a screen conceived to identify autophagy-stimulating polyphenols²³ as well as in in vivo experiments designed to explore the health-improving effects of coffee.²⁴ Spermidine turned out to be an efficient inhibitor of histone acetyltransferases in vitro¹³ and reduced the global protein acetylation levels in cultured cells.^{25, 26}Driven by these premises, we investigated the hypothesis that several health-related compounds including anacardic acid, curcumin, garcinol and spermidine might induce autophagy by inhibition of acetyltranferases. Here we report results supporting this hypothesis. Moreover, we demonstrate that one particular acetyltransferase, EP300 (E1A-binding protein p300), negatively controls autophagy and that anacardic acid, curcumin, garcinol and spermidine may induce autophagy by directly inhibiting EP300.     

IL10 inhibits starvation-induced autophagy in hypertrophic scar fibroblasts via cross talk between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways

Hypertrophic scar (HS) is a serious skin fibrotic disease characterized by excessive hypercellularity and extracellular matrix (ECM) component deposition. Autophagy is a tightly regulated physiological process essential for cellular maintenance, differentiation, development, and homeostasis. Previous studies show that IL10 has potential therapeutic benefits in terms of preventing and reducing HS formation. However, no studies have examined IL10-mediated autophagy during the pathological process of HS formation. Here, we examined the effect of IL10 on starvation-induced autophagy and investigated the molecular mechanism underlying IL10-mediated inhibition of autophagy in HS-derived fibroblasts (HSFs) under starvation conditions. Immunostaining and PCR analysis revealed that a specific component of the IL10 receptor, IL10 alpha-chain (IL10Rα), is expressed in HSFs. Transmission electron microscopy and western blot analysis revealed that IL10 inhibited starvation-induced autophagy and induced the expression of p-AKT and p-STAT3 in HSFs in a dose-dependent manner. Blocking IL10R, p-AKT, p-mTOR, and p-STAT3 using specific inhibitors (IL10RB, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, rapamycin, and cryptotanshinone, respectively) showed that IL10 inhibited autophagy via IL10Rα-mediated activation of STAT3 (the IL10R-STAT3 pathway) and by directly activating the AKT-mTOR pathway. Notably, these results suggest that IL10-mediated inhibition of autophagy is facilitated by the cross talk between STAT3, AKT, and mTOR; in other words, the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways. Finally, the results also indicate that mTOR-p70S6K is the molecule upon which these two pathways converge to induce IL10-mediated inhibition of autophagy in starved HSFs. In summary, the findings reported herein shed light on the molecular mechanism underlying IL10-mediated inhibition of autophagy and suggest that IL10 is a potential therapeutic agent for the treatment of HS.Autophagy is a degradative process in eukaryotic cells that removes or turns over bulk cytoplasmic constituents through the endosomal and lysosomal fusion system (i.e., autophagosomes).^{1, 2} Autophagy is induced by stressful conditions such as starvation and pathogenic invasion.²Hypertrophic scar (HS) is a major skin fibrotic disorder caused by hypercellularity and extracellular matrix (ECM) component deposition.^{3, 4, 5} HS formation is usually recognized as the consequence of disturbed tissue repair processes and/or disrupted homeostasis in the skin after traumatic injury: HS negatively impacts on patient appearance, skeletal muscle function, and quality of life in general.^{6, 7, 8, 9} About 40–70% of surgeries and over 91% of burn injuries result in HS.¹⁰ A key feature of HS is a metabolic disorder of collagen-based ECM proteins.^{11, 12, 13} Autophagy has an important role in homeostasis of tissue structure and function.^{2, 14, 15} Skin autophagic capability is associated with HS and with the pathogenesis of many human diseases.^{16, 17, 18, 19, 20, 21, 22, 23}Existing studies suggest that cytokines are important regulators of the autophagic process in both immune and non-immune cells.^{24, 25, 26} Interleukin-10 (IL10), expressed by a variety of mammalian cell types, was first described as a cytokine-synthesis-inhibitory factor with immunosuppressive and anti-inflammatory functions.^{27, 28} IL10 has a pivotal role in wound healing^{29, 30} and is a promising therapeutic agent for scar improvement in both animal models and human cutaneous wounds.^{9, 31, 32}Fibroblasts are one of the most important effector cells responsible for HS formation.^{12, 33, 34} Thus, we were prompted to elucidate the mechanisms underlying the interactions among IL10, autophagy, and HS formation, with the aim of providing a molecular foundation for the therapeutic efficacy IL10. We used HS tissue, HS-derived fibroblasts (HSFs), and starvation-induced autophagy in HSFs as our research platform.Here, we report that IL10 inhibited autophagy by interfering with IL10R-mediated activation of IL10R-STAT3, as well as by activating the AKT-mTOR pathway. In addition, cross talk among STAT3, AKT, and mTOR and between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways collaboratively regulated starvation-induced autophagy in HSFs.     

Autophagy and mitochondrial alterations in human retinal pigment epithelial cells induced by ethanol: implications of 4-hydroxy-nonenal

Retinal pigment epithelium has a crucial role in the physiology and pathophysiology of the retina due to its location and metabolism. Oxidative damage has been demonstrated as a pathogenic mechanism in several retinal diseases, and reactive oxygen species are certainly important by-products of ethanol (EtOH) metabolism. Autophagy has been shown to exert a protective effect in different cellular and animal models. Thus, in our model, EtOH treatment increases autophagy flux, in a concentration-dependent manner. Mitochondrial morphology seems to be clearly altered under EtOH exposure, leading to an apparent increase in mitochondrial fission. An increase in 2′,7′-dichlorofluorescein fluorescence and accumulation of lipid peroxidation products, such as 4-hydroxy-nonenal (4-HNE), among others were confirmed. The characterization of these structures confirmed their nature as aggresomes. Hence, autophagy seems to have a cytoprotective role in ARPE-19 cells under EtOH damage, by degrading fragmented mitochondria and 4-HNE aggresomes. Herein, we describe the central implication of autophagy in human retinal pigment epithelial cells upon oxidative stress induced by EtOH, with possible implications for other conditions and diseases.Retinal pigment epithelium (RPE) is a single neuroectodermal layer placed in the outermost part of the eye cup faced to photoreceptors.^{1, 2} Owing to its anatomical location and function, RPE is continuously exposed to potential cell damage caused by oxidative stress, specifically due to oxygen and nitrogen reactive species.³ This is probably one of the reasons why these cells are more resistant to oxidative stress.⁴ Oxidative stress is present as part of the pathophysiology in several retinal degenerations associated with blindness, for example, age-related macular degeneration,³ where RPE is considered a key factor for its development.⁵ Studies with the human-derived cell line ARPE-19 have proven to be very useful in the elucidation of the role of these cells in disease.Autophagy is a catabolic process aimed to degrade damaged organelles, proteins and cellular debris by engulfing them into a double membrane vesicle called the autophagosome and eliminating them by posterior fusion with the lysosome. Activation of macroautophagy, a form of autophagy, has been recently confirmed to be a primary response of ARPE-19 cells to stress.⁶ Furthermore, the two major functions of RPE, phagocytosis of the photoreceptor outer segments and visual cycle performance, have been linked to a noncanonical form of autophagy that is known as LC3 (microtubule-associated protein 1A/1B-light chain 3)-associated phagocytosis and is supposed to contribute to the normal supply of vitamin A and therefore to normal vision.^{7, 8}Despite its negative effects on health, ethanol (EtOH) is consumed daily worldwide, standing as one of the top public health challenges. EtOH induces morphological and physiological changes in the nervous tissue, and most of these changes may be attributed to reactive oxygen species (ROS), as they can be normalized or prevented by antioxidant treatments.^{9, 10, 11, 12, 13} Autophagy has been identified as cytoprotector in nervous and liver cells under EtOH-induced toxicity,^{14, 15} where it seems to degrade damaged organelles, including mitochondria. Recent findings support the idea that there is an increased mitochondrial stress and dysfunction in the RPE cells in AMD patients.^{16, 17} Oxidative-damaged mitochondria, a main source of ROS, seem to be removed by autophagy (known as mitophagy), in order to guarantee cell survival.¹⁸ As a matter of fact, deregulation of mitophagy has been implicated in several neurodegenerative diseases, such as Parkinson''s disease (PD), Alzheimer''s disease (AD) and Huntington''s disease (HD).Peroxidation of polyunsaturated fatty acids is intensified in cells subjected to oxidative stress, and results in the generation of various bioactive compounds, among others 4-hydroxyalkenals (HAE). ROS-induced lipid peroxidation and the resulting HAE markedly contribute to the development and progression of different diseases.¹⁹ Specifically, 4-hydroxy-nonenal (4-HNE), a major α,β-unsaturated aldehyde product of n-6 fatty acid oxidation, has been shown to be involved in a great number of maladies.²⁰ It has been reported that 4-HNE induces apoptosis in ARPE-19 cells²¹ and its ability to form protein adducts, thus it seems to be a key factor in aggresome formation. Aggresome is a term referred to cytoplasmic perinuclear inclusion bodies formed by aggregated proteins.²² Indeed, the presence of aggresomes is a pathological hallmark of most neurodegenerative diseases, and 4-HNE seems to be involved in their formation in AD,²³ PD,²⁴ HD²⁵ and amyotrophic lateral sclerosis.²⁶ These aggresomes depend on the protein type to be cleared,^{27, 28} and their degradation by autophagy, known as aggrephagy, has been proposed to increase cell viability in neurodegeneration models.²⁹ Interestingly, 4-HNE aggregates have been also found in hepatic cells from alcoholic patients.^{30, 31, 32} Recent data provide no clear cut evidence of a link between PD risk and alcohol consumption with both positive³³ and negative³⁴ results.In this study, we report the cellular effects of EtOH on ARPE-19 cells and determine that EtOH exposure induces the formation of 4-HNE-aggresomes, together with other neurodegenerative hallmarks such as mitochondrial damage and autophagy activation. Considering the central role of RPE in retinal physiology and pathophysiology, and its neural origin, these findings render new insights into the mechanism of neurodegeneration caused by alcohol toxicity in retinal cells, and may contribute to the development of therapeutic strategies in several nervous and retinal diseases.     

Shear stress regulates endothelial cell autophagy via redox regulation and Sirt1 expression

Disturbed cell autophagy is found in various cardiovascular disease conditions. Biomechanical stimuli induced by laminar blood flow have important protective actions against the development of various vascular diseases. However, the impacts and underlying mechanisms of shear stress on the autophagic process in vascular endothelial cells (ECs) are not entirely understood. Here we investigated the impacts of shear stress on autophagy in human vascular ECs. We found that shear stress induced by laminar flow, but not that by oscillatory or low-magnitude flow, promoted autophagy. Time-course analysis and flow cessation experiments confirmed that this effect was not a transient adaptive stress response but appeared to be a sustained physiological action. Flow had no effect on the mammalian target of rapamycin-ULK pathway, whereas it significantly upregulated Sirt1 expression. Inhibition of Sirt1 blunted shear stress-induced autophagy. Overexpression of wild-type Sirt1, but not the deacetylase-dead mutant, was sufficient to induce autophagy in ECs. Using both of gain- and loss-of-function experiments, we showed that Sirt1-dependent activation of FoxO1 was critical in mediating shear stress-induced autophagy. Shear stress also induced deacetylation of Atg5 and Atg7. Moreover, shear stress-induced Sirt1 expression and autophagy were redox dependent, whereas Sirt1 might act as a redox-sensitive transducer mediating reactive oxygen species-elicited autophagy. Functionally, we demonstrated that flow-conditioned cells are more resistant to oxidant-induced cell injury, and this cytoprotective effect was abolished after inhibition of autophagy. In summary, these results suggest that Sirt1-mediated autophagy in ECs may be a novel mechanism by which laminar flow produces its vascular-protective actions.Vascular endothelial cells (ECs) are fundamentally important in maintaining structural and functional homeostasis of blood vessels. Normal biological functions of ECs are highly sensitive to the biomechanical stimuli induced by blood flow, of which shear stress acting on the surface of EC has been recognized to be one of the most important vasoactive factors in EC.^{1, 2} A relatively high level of laminar shear stress is cytoprotective, whereas abnormal (low-magnitude or oscillatory) shear stress is a detrimental cellular stress to ECs.¹ Transduction of the mechanical signals involves multiple messenger molecules and signaling proteins, which collectively regulate important endothelial functions, such as gene expression, proliferation, migration, morphogenesis, permeability, thrombogenicity, and inflammation.²Autophagy (also known as macroautophagy) is an evolutionarily conserved cellular stress response.^{3, 4} Autophagy is a cellular self-digestion process, which is responsible for degradation of misfolded proteins and damaged organelles. Autophagic process is mainly mediated by the formation of autophagosome, a double-membrane vacuole structure containing engulfed cellular components. This process requires expression of a group of key genes involved in autophagy, including LC3A, beclin-1, Atg5, Atg7, and Atg12, for example.^{3, 5} Autophagosomes fuse with lysosomes, forming autolysosomes, where the cellular components are degraded by various hydrolases in an acidified environment.^{4, 5} In ECs, an autophagic response can be initiated by different stress stimuli.^{6, 7, 8} It is noted that the cellular outcome following autophagy induction in ECs varies depending on the nature of stimuli and specific experimental settings.^{6, 7, 9, 10} Moreover, there is evidence showing that autophagy may also be involved in modulating other EC functions such as angiogenesis and cellular senescence.^{11, 12} Therefore, understanding the regulatory mechanisms of autophagy in ECs will be important for discovery of strategies to protect normal endothelial functions. Recently, Guo et al. provided some evidence indicating that the autophagic process in EC might be affected by shear stress.¹³ This argument, however, was only based on observations of changed expression levels of LC3 and beclin-1; further experimental evidence is needed to confirm such an effect of shear stress on autophagy. More importantly, the mechanisms underlying this phenomenon are not understood. Different signaling pathways may be involved in modulating autophagy in ECs.^{14, 15, 16} For example, inhibition of the mTOR (mammalian target of rapamycin) pathway by rapamycin-induced endothelial autophagy and prevented energy stress-triggered cell damage.¹⁶ There is also evidence indicating a potential role of Sirt1.¹⁴ Moreover, accumulating evidence has suggested that reactive oxygen species (ROS) are closely implicated in modulating autophagic responses via complex interactions with other autophagy-related factors.¹⁵ Despite of these results, the signaling mechanisms of shear stress-regulated autophagy in EC remain to be defined. Hence, here we aim to delineate the impacts and underlying mechanisms of shear stress on autophagy in human vascular ECs.     

Disrupted autophagy after spinal cord injury is associated with ER stress and neuronal cell death

Autophagy is a catabolic mechanism facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner. Autophagy flux is necessary for normal neuronal homeostasis and its dysfunction contributes to neuronal cell death in several neurodegenerative diseases. Elevated autophagy has been reported after spinal cord injury (SCI); however, its mechanism, cell type specificity and relationship to cell death are unknown. Using a rat model of contusive SCI, we observed accumulation of LC3-II-positive autophagosomes starting at posttrauma day 1. This was accompanied by a pronounced accumulation of autophagy substrate protein p62, indicating that early elevation of autophagy markers reflected disrupted autophagosome degradation. Levels of lysosomal protease cathepsin D and numbers of cathepsin-D-positive lysosomes were also decreased at this time, suggesting that lysosomal damage may contribute to the observed defect in autophagy flux. Normalization of p62 levels started by day 7 after SCI, and was associated with increased cathepsin D levels. At day 1 after SCI, accumulation of autophagosomes was pronounced in ventral horn motor neurons and dorsal column oligodendrocytes and microglia. In motor neurons, disruption of autophagy strongly correlated with evidence of endoplasmic reticulum (ER) stress. As autophagy is thought to protect against ER stress, its disruption after SCI could contribute to ER-stress-induced neuronal apoptosis. Consistently, motor neurons showing disrupted autophagy co-expressed ER-stress-associated initiator caspase 12 and cleaved executioner caspase 3. Together, these findings indicate that SCI causes lysosomal dysfunction that contributes to autophagy disruption and associated ER-stress-induced neuronal apoptosis.In the United States, spinal cord injury (SCI) has an annual incidence of 11 000 and prevalence of nearly 500 000. Neuronal cell death is an important contributor to SCI-induced neurological deficits. Many of the affected neurons do not die because of direct mechanical damage but rather show delayed cell death as a result of injury-induced biochemical changes (secondary injury).^{1, 2, 3, 4} Thus, blocking or attenuating secondary neuronal death may serve to limit posttraumatic disabilities.Macroautophagy (hereafter called autophagy) is a lysosome-dependent catabolic pathway degrading cytoplasmic proteins, protein aggregates and organelles.^{5, 6, 7} Autophagy is initiated by the formation of autophagosomes, double membrane vesicles containing cytoplasmic components that include potentially toxic protein aggregates and damaged organelles. Autophagosomes then fuse with lysosomes to allow degradation of their contents by lysosomal hydrolases.^{8, 9, 10, 11} This progress of cargo, from sequestration in autophagosomes, to their delivery and degradation in lysosomes, is termed autophagy flux. Autophagy flux is important for homeostasis in all cells but appears especially critical in terminally differentiated cells such as neurons.^{12, 13} It is also upregulated, and often plays a protective function, in response to cell injury.^{14, 15} For example, autophagy is activated in response to and can limit effects of homeostasis perturbation in the endoplasmic reticulum (ER stress).^{16, 17} Thus, autophagy plays an important neuroprotective function, while impaired autophagy flux has been implicated in neurodegenerative disorders such as Parkinson''s and Alzheimer''s diseases.^{18, 19, 20, 21}Upregulation of autophagy markers has been observed after SCI,^{22, 23} but its mechanisms and function remain controversial, with both beneficial and detrimental roles proposed. Under certain circumstances, pathologically increased autophagy can contribute to cell death,^{21, 24} particularly when autophagy flux is blocked, for example, because of lysosomal defects. Defects in autophagy flux can also exacerbate ER stress and potentiate ER-stress-induced apoptosis.^{16, 17} ER stress has long been implicated as part of the secondary injury after central nervous system trauma,^{25, 26} but its mechanisms remain unknown.In the current study, we characterized the temporal distribution and cell-type specificity of autophagy following contusive SCI in a rat model. Our data demonstrate that autophagosome accumulation after SCI is not due to increased initiation of autophagy, but rather due to inhibition of autophagy flux. This likely reflects the disruption of lysosomal function after SCI. Pathological accumulation of autophagosomes is prominent in ventral horn (VH) motor neurons, where it is associated with signs of ER stress and related apoptosis. Together, our findings suggest that autophagy is disrupted after SCI and may exacerbate ER stress and neuronal cell death.     

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

Transforming growth factor-β₁ (TGF-β₁) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β₁ may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β₁-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β₁ to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β₁ promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β₁ in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β₁-induced autophagy is required for the fibrogenic response in hATMyofbs.Interstitial fibrosis is common to many cardiovascular disease etiologies including myocardial infarction (MI),¹ diabetic cardiomyopathy² and hypertension.³ Fibrosis may arise due to maladaptive cardiac remodeling following injury and is a complex process resulting from activation of signaling pathways, such as TGF-β₁.⁴ TGF-β₁ signaling has broad-ranging effects that may affect cell growth, differentiation and the production of extracellular matrix (ECM) proteins.^{5, 6} Elevated TGF-β₁ is observed in post-MI rat heart⁷ and is associated with fibroblast-to-myofibroblast phenoconversion and concomitant activation of canonical Smad signaling.⁸ The result is a proliferation of myofibroblasts, which then leads to inappropriate deposition of fibrillar collagens, impaired cardiac function and, ultimately, heart failure.^{9, 10}Autophagy is necessary for cellular homeostasis and is involved in organelle and protein turnover.^{11, 12, 13, 14} Autophagy aids in cell survival by providing primary materials, for example, amino acids and fatty acids for anabolic pathways during starvation conditions.^{15, 16} Alternatively, autophagy may be associated with apoptosis through autodigestive cellular processes, cellular infection with pathogens or extracellular stimuli.^{17, 18, 19, 20} The overall control of cardiac fibrosis is likely due to the complex functioning of an array of regulatory factors, but to date, there is little evidence linking autophagy with fibrogenesis in cardiac tissue.^{11, 12, 13, 14, 15, 16, 17, 18, 21, 22}Recent studies have demonstrated that TGF-β₁ may not only promote autophagy in mouse fibroblasts and human tubular epithelial kidney cells^{15, 23, 24} but can also inhibit this process in fibroblasts extracted from human patients with idiopathic pulmonary fibrosis.²⁵ Moreover, it has recently been reported that autophagy can negatively¹⁵ and positively^{25, 26, 27} regulate the fibrotic process in different model cell systems. In this study, we have explored the putative link between autophagy and TGF-β₁-induced fibrogenesis in human atrial myofibroblasts (hATMyofbs) and in a model of MI rat heart.     

A novel androstenedione derivative induces ROS-mediated autophagy and attenuates drug resistance in osteosarcoma by inhibiting macrophage migration inhibitory factor (MIF)

Osteosarcoma is a common primary bone tumor in children and adolescents. The drug resistance of osteosarcoma leads to high lethality. Macrophage migration inhibitory factor (MIF) is an inflammation-related cytokine implicated in the chemoresistance of breast cancer. In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. The inhibition of MIF by DSTD promoted autophagy by inducing Bcl-2 downregulation and the translocation of HMGB1. N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced ROS-mediated autophagy to rescue cell death. However, in the presence of chemotherapy drugs, DSTD enhanced the chemosensitivity by decreasing the HMGB1 level. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.Osteosarcoma, a common primary bone tumor in children and adolescents, is prone to early metastasis through blood.¹ Treatment with a combination of surgery and aggressive adjuvant chemotherapy has improved the survival rate of osteosarcoma patients. The 5-year-survival rates of non-metastatic patients have reached a plateau of approximately 70%.^{2, 3} However, patients with poor responses to chemotherapeutics will undergo local recurrence and metastasis, which reduce the 5-year-survival rates to only 20% despite additional doses or drugs.^{4, 5} Drug resistance is responsible for the poor prognosis. Attenuating chemoresistance facilitates better treatment of osteosarcoma.^{6, 7} Novel treatment strategies that combine anticancer drugs with adjuvant agents could improve the antitumor effects.^{8, 9}In the 1960s, macrophage migration inhibitory factor (MIF) was identified as a pluripotent protein that modulates inflammation.¹⁰ Increasing evidence suggests that inflammation is closely related to tumorigenesis.¹¹ MIF plays a bridging role between inflammation and tumorigenesis.^{12, 13, 14} MIF triggers the activation of the MAPK and PI3K pathways by binding its membrane receptor CD74, resulting in the inhibition of cell apoptosis.¹⁵ Recently, MIF was demonstrated to be involved in cell proliferation, differentiation, angiogenesis and tumorigenesis.^{16, 17, 18} Some evidence has indicated that MIF is abundantly expressed in various cancers and is significantly associated with tumor invasion and metastasis.^{19, 20, 21} MIF has been well established to be involved in the development of glioblastoma,²² breast cancer,²³ bladder cancer²⁴ and colon cancer.^{20, 25} MIF was also upregulated in osteosarcoma.^{26, 27} The knockdown of MIF blocked osteosarcoma cell proliferation and invasion.²⁶ However, the effect of MIF on drug resistance in osteosarcoma has not yet been investigated. Wu et al. ²³ have revealed that MIF knockdown promoted chemosensitivity by inducing autophagy in breast cancer. In contrast, autophagy reportedly contributed to chemoresistance in osteosarcoma.⁶ These controversial results prompted us to confirm the role of MIF in drug resistance in osteosarcoma.In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. Both N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced reactive oxygen species (ROS)-mediated autophagy to rescue cell death. Furthermore, MIF inhibition by DSTD enhances chemosensitivity by downregulating HMGB1 in osteosarcoma cells. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.     

Oncogenic BRAF induces chronic ER stress condition resulting in increased basal autophagy and apoptotic resistance of cutaneous melanoma

The notorious unresponsiveness of metastatic cutaneous melanoma to current treatment strategies coupled with its increasing incidence constitutes a serious worldwide clinical problem. Moreover, despite recent advances in targeted therapies for patients with BRAF^V600E mutant melanomas, acquired resistance remains a limiting factor and hence emphasises the acute need for comprehensive pre-clinical studies to increase the biological understanding of such tumours in order to develop novel effective and longlasting therapeutic strategies. Autophagy and ER stress both have a role in melanoma development/progression and chemoresistance although their real impact is still unclear. Here, we show that BRAF^V600E induces a chronic ER stress status directly increasing basal cell autophagy. BRAF^V600E-mediated p38 activation stimulates both the IRE1/ASK1/JNK and TRB3 pathways. Bcl-XL/Bcl-2 phosphorylation by active JNK releases Beclin1 whereas TRB3 inhibits the Akt/mTor axes, together resulting in an increase in basal autophagy. Furthermore, we demonstrate chemical chaperones relieve the BRAF^V600E-mediated chronic ER stress status, consequently reducing basal autophagic activity and increasing the sensitivity of melanoma cells to apoptosis. Taken together, these results suggest enhanced basal autophagy, typically observed in BRAF^V600E melanomas, is a consequence of a chronic ER stress status, which ultimately results in the chemoresistance of such tumours. Targeted therapies that attenuate ER stress may therefore represent a novel and more effective therapeutic strategy for BRAF mutant melanoma.Cutaneous melanoma represents one of the most aggressive and difficult to treat forms of human cancer, with a worldwide incidence that has steadily increased over the past 40 years.^{1, 2}Notoriously unresponsive to conventional chemotherapy, metastatic disease is highly invasive and evolves with an extensive repertoire of molecular defences against immunological and cytotoxic attack.³Although linked to exposure to ultraviolet light, it is widely accepted that both genotypic and phenotypic changes in melanocytes predispose to melanocyte transformation and the onset of melanoma.^{4, 5}Surprisingly, p53 mutations are very rare in melanoma, but activity is, however, impaired through direct or indirect inactivation of key elements of this pathway, including through the suppression of APAF-1 expression,⁶ loss of PTEN function,⁷ dysregulation of Bcl-2 expression,⁸ upregulation of the anti-apoptotic protein Mcl-1 together with its altered slice variant expression ^{9, 10} and the ER chaperone GRP78.^{11, 12, 13} Oncogenic mutations, however, in the Ras/Raf pathway are the most well-described genetic mutations associated with melanoma development and progression.¹⁴ Indeed, up to 90% of all melanomas harbour activating NRAS or BRAF mutations, with BRAF^V600E representing more than 90% of BRAF mutations,^{15, 16} the consequence of which is the constitutive activation of RAF-extracellular signal-regulated kinase/ERK signalling promoting melanoma proliferation and resistance to apoptosis.¹⁷ Nevertheless, mutation of NRAS/BRAF alone is not sufficient to initiate melanomagenesis, because these common mutations are also present in benign nevi, thereby highlighting the requirement of other factors to drive melanocyte transformation and melanoma development.^{15, 16} Dysregulation of autophagy has accordingly been postulated as a secondary event contributing to melanoma progression and, importantly, also has a key role in chemoresistance.^{18, 19, 20}Autophagy is the principal catabolic process for the bulk degradation and recycling of aged/damaged cellular components, organelles and proteins through the formation of a double-membraned cytosolic vesicle able to wrap targeted material. The subsequent fusion with lysosomes and degradation of cargo provide nutrients in times of environmental stress, such as nutrient deprivation or hypoxia.²¹ Though essential for the maintenance of cellular homeostasis under conditions of nutrient deprivation, paradoxically, autophagy promotes both tumour suppression and tumour development.²² Although the accumulation of damaged organelles/cytosolic proteins may lead to cellular transformation, autophagy may also sustain tumour growth in a microenvironment which is commonly poor of oxygen and nutrients.²² Thus, not surprisingly, autophagy activation is frequently observed in late-stage malignancy although the molecular mechanisms mediating its activation/regain of function remain unclear.ER stress may also constitute a key secondary event in melanoma development.²³ Primarily a cytoprotective pro-survival process, ER stress is activated as a result of accumulated unfolded proteins, protein overload or depletion of ER calcium stores and mediated through the activity of the master ER chaperone Grp78 and three signalling pathways; PERK/eIF2α/ATF4, IRE-1/Xbp-1 and ATF6 which collectively maintain ER homeostasis through the instigation of an unfolded protein response (UPR)²⁴ or sustained ER stress may lead to the induction of apoptosis.^{25, 26} Increasing evidence indicates that nutrient deprivation and hypoxia lead to activation of the UPR in various solid tumours, frequently correlating with resistance to chemotherapy.²⁷ The accepted hypothesis is thus that activation of the UPR in cancer cells enables their adaption to such ER stress resulting in the resistance to apoptosis through the persistent expression of pro-survival instead of pro-apoptotic proteins.²⁸Although under stress conditions, autophagy and ER stress seem to act in parallel, indeed they are closely related, because one can regulate the other and vice versa. In fact, ER stress is able to promptly stimulate autophagy,²⁶ whereas autophagy selectively removes the membranes of the endoplasmic reticulum at the end of the UPR, although the molecular mechanisms are still largely unclear.²⁹In the present study, we investigated the link between oncogenic BRAF^V600E and increased basal autophagy in melanoma cells, highlighting the pivotal role played by ER stress, possibly responsible for tumour growth and chemoresistance.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.