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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The LRRK2 inhibitor GSK2578215A induces protective autophagy in SH-SY5Y cells: involvement of Drp-1-mediated mitochondrial fission and mitochondrial-derived ROS signaling

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been associated with Parkinson''s disease, and its inhibition opens potential new therapeutic options. Among the drug inhibitors of both wild-type and mutant LRRK2 forms is the 2-arylmethyloxy-5-subtitutent-N-arylbenzamide GSK257815A. Using the well-established dopaminergic cell culture model SH-SY5Y, we have investigated the effects of GSK2578215A on crucial neurodegenerative features such as mitochondrial dynamics and autophagy. GSK2578215A induces mitochondrial fragmentation of an early step preceding autophagy. This increase in autophagosome results from inhibition of fusion rather than increases in synthesis. The observed effects were shared with LRRK2-IN-1, a well-described, structurally distinct kinase inhibitor compound or when knocking down LRRK2 expression using siRNA. Studies using the drug mitochondrial division inhibitor 1 indicated that translocation of the dynamin-related protein-1 has a relevant role in this process. In addition, autophagic inhibitors revealed the participation of autophagy as a cytoprotective response by removing damaged mitochondria. GSK2578215A induced oxidative stress as evidenced by the accumulation of 4-hydroxy-2-nonenal in SH-SY5Y cells. The mitochondrial-targeted reactive oxygen species scavenger MitoQ positioned these species as second messengers between mitochondrial morphologic alterations and autophagy. Altogether, our results demonstrated the relevance of LRRK2 in mitochondrial-activated pathways mediating in autophagy and cell fate, crucial features in neurodegenerative diseases.Nowadays, Parkinson''s disease (PD) constitutes the main motor disorder and the second neurodegenerative disease after Alzheimer''s disease. Etiology of PD remains unknown, but both environmental and genetic factors have been implicated. Among the genes associated with PD is the leucine-rich repeat kinase 2 (LRRK2, PARK8, OMIM 607060) encoding gene encoded by PARK8. Indeed, LRRK2 mutations have been described in a substantial number of idiopathic late-onset PD patients without a known family history of the disease.^{1, 2, 3}The physiologic function remains unknown. It localizes in the cytosol as well as in specific membrane subdomains, including mitochondria, autophagosomes and autolysosomes,⁴ and interacts with a whole array of proteins, including both α- and β-tubulin,^{5, 6} tau,⁷ α-synuclein⁸ and F-actin.⁹ LRRK2 gene mutations, including the most common G2019S,³ are associated with increases in toxic putative kinase activity.^{1, 10} LRRK2 overexpression is toxic to cultured cells,^{11, 12} and LRRK2 loss did not cause neurodegenerative changes (for a review see Tong and Shen¹³). However, LRRK2 transgenic mice lack obvious PD-like behavioral phenotypes.¹⁴ LRRK2-associated PD patients show degeneration of dopaminergic neurons in the substantia nigra.¹⁵ Data from our own group and others have associated mitochondrial apoptotical pathways with PD,^{16, 17, 18} and, in this context, LRRK2 mutant-mediated toxicity could be due to mitochondria-dependent apoptosis.¹⁹ There is considerable evidence for impaired mitochondrial function and morphology in both early-onset, autosomal recessive inherited PD and late-onset sporadic PD.Mitochondrial dynamics include several mechanisms, such as fission, fusion and mitophagy.^{20, 21} Altered fission/fusion dynamics might be a common pathogenic pathway of neurodegenerative diseases. It is well documented that mitochondrial dynamics constitute a relevant issue in some experimental neurodegenerative models.^{20, 22, 23, 24, 25} Mitochondrial dynamics is tightly regulated by cellular pathways including those participated by the dynamin-related protein-1 (Drp1). Drp1 mostly locates in the cytoplasm, but is stimulated after fission stimuli to migrate to the mitochondria. Once there, Drp1 forms ring-like structures, which wrap around the scission site to constrict the mitochondrial membrane resulting in mitochondrial fission.^{26, 27} Interestingly, a functional interaction between PD-associated LRRK2 and members of the dynamin GTPase superfamily has been described.²⁸Macroautophagy (hereafter referred to as autophagy) is an active cellular response, which functions in the intracellular degradation system of cellular debris such as damaged organelles. Whether autophagy promotes cell death or enhances survival is still controversial.^{29, 30} It requires the formation of autophagosomes where cellular content is to be degraded by the action of lysosomal enzymatic content. Autophagosome formation is regulated by an orderly action of >30 autophagy-related (Atg) proteins. Among them is the microtubule-associated protein 1A/1B-light chain 3 (LC3), a homolog of Apg8p, which is essential for autophagy in yeast and is associated with autophagosome membranes.³¹ Interestingly, these vesicles are mostly highly mobile in the cytoplasm.³² Wild-type and mutant LRRK2 expression has been related to autophagy.^{4, 33, 34, 35, 36} Reactive oxygen species (ROS) function as relevant second messengers after several stimuli, including mitochondrial disruption. Exacerbated ROS increases might result in overactivation of antioxidant systems and yield harmful oxidative stress. Among oxidative stress hallmarks is the accumulation of α,β-unsaturated hydroxyalkenal 4-hydroxy-2-nonenal (4-HNE), whose accumulation has been reported in PD post-mortem patient brains,^{37, 38} thus giving a significant relevance to ROS in the pathogenesis of PD.All these results indicate LRRK2 as a promising pharmacologic target in PD treatment.³⁹ Several LRRK2 inhibitor drugs have been synthetized, such as the potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide (GSK2578215A). GSK2578215A exhibits biochemical IC₅₀s of 10.9 nM against wild-type LRRK2, and possesses a high ratio of brain to plasma distribution.⁴⁰ This study provides key insights into the mechanisms downstream of LRRK2 inhibition, and spreads light onto an underexplored, yet potentially tractable therapeutic target for treating LRRK2-associated PD. We demonstrate how inhibition of this kinase results in the activation of cellular death pathways such as the mitochondrial fission machinery, and how cells reply by activating a protective autophagic response. Our results show the presence of oxidative stress hallmarks, thus pointing to a key function for ROS, placed downstream of mitochondrial fission.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.     

Nuclear ULK1 promotes cell death in response to oxidative stress through PARP1

Reactive oxygen species (ROS) may cause cellular damage and oxidative stress-induced cell death. Autophagy, an evolutionarily conserved intracellular catabolic process, is executed by autophagy (ATG) proteins, including the autophagy initiation kinase Unc-51-like kinase (ULK1)/ATG1. Although autophagy has been implicated to have both cytoprotective and cytotoxic roles in the response to ROS, the role of individual ATG proteins, including ULK1, remains poorly characterized. In this study, we demonstrate that ULK1 sensitizes cells to necrotic cell death induced by hydrogen peroxide (H₂O₂). Moreover, we demonstrate that ULK1 localizes to the nucleus and regulates the activity of the DNA damage repair protein poly (ADP-ribose) polymerase 1 (PARP1) in a kinase-dependent manner. By enhancing PARP1 activity, ULK1 contributes to ATP depletion and death of H₂O₂-treated cells. Our study provides the first evidence of an autophagy-independent prodeath role for nuclear ULK1 in response to ROS-induced damage. On the basis of our data, we propose that the subcellular distribution of ULK1 has an important role in deciding whether a cell lives or dies on exposure to adverse environmental or intracellular conditions.Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide (H₂O₂), are formed by the incomplete reduction of oxygen during oxidative phosphorylation and other enzymatic processes. ROS are signaling molecules that regulate cell proliferation, differentiation, and survival.^{1, 2, 3} Accumulation of ROS (i.e., oxidative stress) on exposure to xenobiotic agents or environmental toxins can cause cellular damage and death via apoptotic or nonapoptotic pathways.^{4, 5, 6} Oxidative stress-induced cellular damage and death have been implicated in aging, ischemia-reperfusion injury, inflammation, and the pathogenesis of diseases (e.g., neurodegeneration and cancer).⁷ Oxidative stress also contributes to the antitumor effects of many chemotherapeutic drugs, including camptothecin^{8, 9} and selenite.^{10, 11}Autophagy, an evolutionarily conserved intracellular catabolic process, involves lysosome-dependent degradation of superfluous and damaged cytosolic organelles and proteins.¹² It is typically upregulated under conditions of perceived stress and in response to cellular damage. The consequence of autophagy activation – whether cytoprotective or cytotoxic – appears to depend on the cell type and the nature and extent of stress. Although most studies indicate a cytoprotective role for autophagy, some evidence suggests that it contributes to cell death in response to oxidative stress.^{13, 14, 15, 16, 17} Studies have also indicated that autophagy may be suppressed in response to oxidative stress, thereby sensitizing certain cells to apoptosis.^{18, 19} Unc-51-like kinase/autophagy 1 (ULK1/ATG1) is a mammalian serine–threonine kinase that regulates flux through the autophagy pathway by activating the VPS34 PI(3) kinase complex and facilitating ATG9-dependent membrane recycling.²⁰ Results from two studies suggest that ULK1 expression is altered in response to oxidative stress, and that the corresponding effects on autophagy contribute to cell death.^{18, 21}For example, p53-mediated upregulation of ULK1 and increase in autophagy promote cell death in osteosarcoma cells exposed to sublethal doses of camptothecin,²¹ yet mutant p53-mediated suppression of ULK1 impairs autophagic flux and promotes apoptosis in selenite-treated NB4 cells.¹⁸ Here we investigated the role of ULK1 in cells exposed to H₂O₂.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.