Stimulation Of Autophagy Research Articles

The roles of PKCs in regulating autophagy.

Autophagy, as a highly conserved cellular degradation and recycling process, plays an important part in maintaining cellular homeostasis. PKC signaling is involved in multiple pathways including cell cycle progression, tumorigenesis, migration and autophagy. Literatures about PKC and autophagy from PubMed databases were reviewed in this study. Studies regarding the association of PKC and autophagy remain debatable. Different duration of the stimulation of autophagy and distinct cell contexts result in different function of PKC in regulating autophagy. The subcellular localization of PKCs and their downstream regulators may influence the autophagy regulation as well. As important intracellular components, the mitochondria play an important role in regulating autophagy, by metabolic modulation and structural derangement. Phase II studies regarding PKC-β inhibitor, enzastaurin, showed promising results in MCL, DLBCL and recurrent high-grade gliomas. However, the detailed mechanism is still in need. The mechanism of PKC-β in mediating autophagy in lymphoma and high-grade gliomas remains elusive as well. Moreover, several studies were in agreement that rottlerin enhanced autophagy in breast cancer cells, which warrants further clinical studies to verify PKC-δ as a therapeutic target. Thus, identifying the function of PKC in modulating autophagy and conducting related clinical studies help find novel target for chemotherapy.

Regulation of human mitogen-activated protein kinase 15 (extracellular signal-regulated kinase 7/8) and its functions: A recent update.

Mitogen-activated protein kinase 15 (MAPK15), originally also known as extracellular signal-regulated kinase 7/8, is the most recently identified atypical MAPK and the least studied so far. Examinations of the role of MAPK15 in various cell lines and model systems indicate that MAPK15 participates in a variety of cellular activities such as promoting cell proliferation, cell transformation, and apoptosis; stimulating autophagy; regulating cell division, ciliogenesis, and protein secretion; and maintaining genome stability. As multiple roles of MAPK15 were observed among these studies, therefore, it remains unclear whether MAPK15 acts as a proto-oncogene or tumor suppressor. Here, the recent literature on human MAPK15 and the resulting functions will be discussed.

GD2 ganglioside-binding antibody 14G2a and specific aurora A kinase inhibitor MK-5108 induce autophagy in IMR-32 neuroblastoma cells

The process of autophagy and its role in survival of human neuroblastoma cell cultures was studied upon addition of an anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (14G2a mAb) and an aurora A kinase specific inhibitor, MK-5108. It was recently shown that combination of these agents significantly potentiates cytotoxicity against IMR-32 and CHP-134 neuroblastoma cells in vitro, as compared to the inhibitor used alone. In this study we gained mechanistic insights on autophagy in the observed cytotoxic effects exerted by both agents using cytotoxicity assays, RT-qPCR, immunoblotting, and autophagy detection methods. Enhancement of the autophagy process in the 14G2a mAb- and MK-5108-treated IMR-32 cells was documented by assessing autophagic flux. Application of a lysosomotropic agent—chloroquine (CQ) affected the 14G2a mAb- and MK-5108-stimulated autophagic flux. It is our conclusion that the 14G2a mAb (40 μg/ml) and MK-5108 inhibitor (0.1 μM) induce autophagy in IMR-32 cells. Moreover, the combinatorial treatment of IMR-32 cells with the 14G2a mAb and CQ significantly potentiates cytotoxic effect, as compared to CQ used alone. Most importantly, we showed that interfering with autophagy at its early and late step augments the 14G2a mAb-induced apoptosis, therefore we can conclude that inhibition of autophagy is the primary mechanism of the CQ-mediated sensitization to the 14G2a mAb-induced apoptosis. Although, there was no virtual stimulation of autophagy in the 14G2a mAb-treated CHP-134 neuroblastoma cells, we were able to show that PHLDA1 protein positively regulates autophagy and this process exists in a mutually exclusive manner with apoptosis in PHLDA1-silenced CHP-134 cells.

Autophagy as a promoter of longevity: insights from model organisms.

Autophagy is a conserved process that catabolizes intracellular components to maintain energy homeostasis and to protect cells against stress. Autophagy has crucial roles during development and disease, and evidence accumulated over the past decade indicates that autophagy also has a direct role in modulating ageing. In particular, elegant studies using yeasts, worms, flies and mice have demonstrated a broad requirement for autophagy-related genes in the lifespan extension observed in a number of conserved longevity paradigms. Moreover, several new and interesting concepts relevant to autophagy and its role in modulating longevity have emerged. First, select tissues may require or benefit from autophagy activation in longevity paradigms, as tissue-specific overexpression of single autophagy genes is sufficient to extend lifespan. Second, selective types of autophagy may be crucial for longevity by specifically targeting dysfunctional cellular components and preventing their accumulation. And third, autophagy can influence organismal health and ageing even non-cell autonomously, and thus, autophagy stimulation in select tissues can have beneficial, systemic effects on lifespan. Understanding these mechanisms will be important for the development of approaches to improve human healthspan that are based on the modulation of autophagy.

Autophagy plays a protective role against apoptosis induced by toxicarioside N via the Akt/mTOR pathway in human gastric cancer SGC-7901 cells.

Toxicarioside N (Tox N), a natural product extract from Antiaris toxicaria, has been reported to induce apoptosis in human gastric cancer cells. However, the mechanism and actual role of autophagy in Tox N-induced apoptosis of human gastric cancer cells remains poorly understood. In the current study, we demonstrated that Tox N could induce autophagy by inhibiting the Akt/mTOR signaling pathway in SGC-7901 cells. Moreover, we found that the inhibition of autophagy by 3-methyladenine, an autophagy inhibitor, enhanced Tox N-induced apoptotic cell death. However, the stimulation of autophagy by rapamycin, an autophagy activator, remarkably suppressed Tox N-induced apoptosis, suggesting that autophagy plays a protective role in Tox N-induced apoptosis. Thus, the results from this study suggested that Tox N combination with an autophagy inhibitor might be a promising strategy to enhance the anticancer activity of Tox N for the treatment of human gastric cancer.

Inhibiting autophagy reduces retinal degeneration caused by protein misfolding

ABSTRACTMutations in the genes necessary for the structure and function of vertebrate photoreceptor cells are associated with multiple forms of inherited retinal degeneration. Mutations in the gene encoding RHO (rhodopsin) are a common cause of autosomal dominant retinitis pigmentosa (adRP), with the Pro23His variant of RHO resulting in a misfolded protein that activates endoplasmic reticulum stress and the unfolded protein response. Stimulating macroautophagy/autophagy has been proposed as a strategy for clearing misfolded RHO and reducing photoreceptor death. We found that retinas from mice heterozygous for the gene encoding the RHOP23H variant (hereafter called P23H) exhibited elevated levels of autophagy flux, and that pharmacological stimulation of autophagy accelerated retinal degeneration. In contrast, reducing autophagy flux pharmacologically or by rod-specific deletion of the autophagy-activating gene Atg5, improved photoreceptor structure and function. Furthermore, proteasome levels and activity were reduced in the P23H retina, and increased when Atg5 was deleted. Our findings suggest that autophagy contributes to photoreceptor cell death in P23H mice, and that decreasing autophagy shifts the degradation of misfolded RHO protein to the proteasome and is protective. These observations suggest that modulating the flux of misfolded proteins from autophagy to the proteasome may represent an important therapeutic strategy for reducing proteotoxicity in adRP and other diseases caused by protein folding defects.

High-Throughput Screens To Identify Autophagy Inducers That Function by Disrupting Beclin 1/Bcl-2 Binding.

Autophagy, a lysosomal degradation pathway, plays a crucial role in cellular homeostasis, development, immunity, tumor suppression, metabolism, prevention of neurodegeneration, and lifespan extension. Thus, pharmacological stimulation of autophagy may be an effective approach for preventing or treating certain human diseases and/or aging. We sought to establish a method for developing new chemical compounds that specifically induce autophagy. To do this, we developed two assays to identify compounds that target a key regulatory node of autophagy induction-specifically, the binding of Bcl-2 (a negative regulator of autophagy) to Beclin 1 (an allosteric modulator of the Beclin 1/VPS34 lipid kinase complex that functions in autophagy initiation). These assays use either a split-luciferase assay to measure Beclin 1/Bcl-2 binding in cells or an AlphaLISA assay to directly measure direct Beclin 1/Bcl-2 binding in vitro. We screened two different chemical compound libraries, comprising ∼300 K compounds, to identify small molecules that disrupt Beclin 1/Bcl-2 binding and induce autophagy. Three novel compounds were identified that directly inhibit Beclin 1/Bcl-2 interaction with an IC50 in the micromolar range and increase autophagic flux. These compounds do not demonstrate significant cytotoxicity, and they exert selectivity for disruption of Bcl-2 binding to the BH3 domain of Beclin 1 compared with the BH3 domain of the pro-apoptotic Bcl-2 family members, Bax and Bim. Thus, we have identified candidate molecules that serve as lead templates for developing potent and selective Beclin 1/Bcl-2 inhibitors that may be clinically useful as autophagy-inducing agents.

Alisol A 24-acetate ameliorates nonalcoholic steatohepatitis by inhibiting oxidative stress and stimulating autophagy through the AMPK/mTOR pathway

Alisol A 24-acetate (AA), a natural triterpenoid isolated from the traditional Chinese medicine Rhizoma Alismatis, has various therapeutic effects. We investigated the anti-nonalcoholic steatohepatitis (NASH) effect of AA and its underlying mechanisms in vitro and in vivo. C57BL/6 mice were fed a methionine and choline-deficient (MCD) diet for 4 weeks to induce NASH. The mice were simultaneously treated with a daily dose of AA (15, 30, and 60 mg kg−1, ig) for 4 weeks. On the last day, the animals were sacrificed and plasma and liver tissue were collected. Serum and liver tissue biochemical analyses and histological observation were performed. The human hepatic stellate cell line LX-2 was used to build NASH models by culturing with conditioned medium from WRL-68 liver cells after exposure to MCD medium in vitro. Liver oxidative stress and inflammatory indices and autophagy markers were examined. The results showed that AA suppressed reactive oxygen species (ROS) and inflammation in a NASH mouse model and inhibited the expression of inflammatory cytokines and ROS in LX-2 cells in MCD medium. Furthermore, we found AA stimulated autophagy in mice liver and LX-2, which could be the underlying mechanism of AA in NASH. To further investigate the role of autophagy in LX-2 cells, we found that AA regulated autophagy via the AMPK/mTOR/ULK1 pathway and dorsomorphin, a selective AMPK inhibitor, led to the suppression of AA-induced autophagy. Taken together, our results indicate that AA could be a possible therapy for NASH by inhibiting oxidative stress and stimulating autophagy.

Autophagy regulates exosomal release of prions in neuronal cells

Prions are protein-based infectious agents that autocatalytically convert the cellular prion protein PrPC to its pathological isoform PrPSc. Subsequent aggregation and accumulation of PrPSc in nervous tissues causes several invariably fatal neurodegenerative diseases in humans and animals. Prions can infect recipient cells when packaged into endosome-derived nanoparticles called exosomes, which are present in biological fluids such as blood, urine, and saliva. Autophagy is a basic cellular degradation and recycling machinery that also affects exosomal processing, but whether autophagy controls release of prions in exosomes is unclear. Our work investigated the effect of autophagy modulation on exosomal release of prions and how this interplay affects cellular prion infection. Exosomes isolated from cultured murine central neuronal cells (CAD5) and peripheral neuronal cells (N2a) contained prions as shown by immunoblotting for PrPSc, prion-conversion activity, and cell culture infection. We observed that autophagy stimulation with the mTOR inhibitor rapamycin strongly inhibited exosomal prion release. In contrast, inhibition of autophagy by wortmannin or CRISPR/Cas9-mediated knockout of the autophagy protein Atg5 (autophagy-related 5) greatly increased the release of exosomes and exosome-associated prions. We also show that a difference in exosomal prion release between CAD5 and N2a cells is related to differences at the level of basal autophagy. Taken together, our results indicate that autophagy modulation can control lateral transfer of prions by interfering with their exosomal release. We describe a novel role of autophagy in the prion life cycle, an understanding that may provide useful targets for containing prion diseases.

The role of autophagy in survival response induced by 27-hydroxycholesterol in human promonocytic cells

Autophagy has been shown to be stimulated in advanced atherosclerotic plaques by metabolic stress, inflammation and oxidized lipids. The lack of published studies addressing the potential stimulation of pro-survival autophagy by oxysterols, a family of cholesterol oxidation products, has prompted our study. Thus, the goal of the current study is to elucidate the molecular mechanism of the autophagy induced by 27-hydroxycholesterol (27-OH), that is one of the most abundant oxysterols in advanced atherosclerotic lesions, and to assess whether the pro-oxidant effect of the oxysterol is involved in the given response. Here we showed that 27-OH, in a low micromolar range, activates a pro-survival autophagic response in terms of increased LC3 II/LC3 I ratio and Beclin 1, that depends on the up-regulation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt pathways as a potential result of an intracellular reactive oxygen species increase provoked by the oxysterol in human promonocytic U937 cells. Moreover, 27-OH induced autophagy is dependent on the relation between nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-dependent antioxidant response and p62. The data obtained highlight the involvement of cholesterol oxidation products in the pathogenesis of oxidative stress related chronic diseases like atherosclerosis. Therefore, deeply understanding the complex mechanism and generating synthetic or natural molecules targeting this survival mechanism might be very promising tools in the prevention of such diseases.

Resveratrol interrupts the pro-invasive communication between cancer associated fibroblasts and cholangiocarcinoma cells

Cholangiocarcinoma (CCA), the cancer arising from the epithelial cells of bile ducts, is a prototype of inflammatory-driven cancer. Cytokines released by cancer associated fibroblasts (CAFs) play a pivotal role in CCA progression, driving the epigenetic Epithelial-to-Mesenchymal transition and the growth and metastasization of CCA cells. Consistently, the conditioned medium from CCA-derived CAFs further stimulated the secretion of IL-6, and to a lesser extent of IL-8, by CCA cells. CCA has a poor prognosis, because of late diagnosis and of high resistance to radio- and chemo-therapy of CCA cells. Targeting the CAFs and their secretion could be an alternative option. We found that while IL-6 indeed promoted the cell migration of invasive CCA cells, the nutraceutical Resveratrol strongly counteracted this effect both in CCA cells and in immortalized cholangiocytes. More importantly, here we show that Resveratrol has the potential to abrogate the secretion of IL-6 by CAFs. While the conditioned medium from CAFs strongly induced IL-6 mediated motility of CCA cells, the conditioned medium from CAFs pre-treated with Resveratrol completely halted cancer cell motility and reverted the N-to E-cadherin switch in migrating cells. This effect was associated with stimulation of autophagy in the cancer cells. This is the first demonstration that CAFs secretory products directly affect the regulation of autophagy and consequently the behavior of CCA cells, and that a nutraceutical may revert the malignant phenotype of cancer cells by acting on CAFs metabolism and secretion.

Autophagy-A key pathway for cardiac health and longevity.

As average life expectancy continues to rise in the developed world, age-associated pathologies are increasing in prevalence. The hallmarks of cardiac ageing include cardiomyocyte loss, fibrosis and hypertrophy, all of which contribute to an increased incidence of cardiac disease. At the molecular level, cellular ageing is characterized by increased ROS production, mitochondrial dysfunction and the accumulation of damaged proteins and organelles. Cardiomyocytes and other senescent cell types rely upon autophagy, a lysosome-mediated degradation pathway, to remove potentially toxic protein aggregates and damaged organelles from the cellular milieu. However, increasing lines of evidence point to an age-associated decrease in cardiomyocyte autophagy, with predictably negative consequences for cardiac function and health. Conversely, stimulation of autophagy has been shown to improve cellular health and cardiac function and to increase lifespan in numerous model organisms. Clearly, autophagy represents a critical pathway for cellular vitality, as well as a promising therapeutic target for the treatment of age-related cardiac pathologies. In this review, we will discuss the mechanism of autophagy and its regulation in the cell, the role of autophagy in the ageing heart, and how the autophagy pathway might be targeted to improve cardiac health.

Pancreatic \u03b2 cells overexpressing hIAPP impaired mitophagy and unbalanced mitochondrial dynamics

Human islet amyloid polypeptide (hIAPP), or amylin, has the tendency to aggregate into insoluble amyloid fibrils, a typical feature of islets from type 2 diabetes individuals. Thus, we investigated comparatively the impact of hIAPP on key pathways involved in pancreatic beta survival. INS1E-hIAPP cells present a hyperactivation of MTORC1 and an inhibition of autophagy signaling, those cells showing an increase in cell size. Resveratrol, a MTORC1 inhibitor, can reverse TSC2 degradation that occurs in INS1E-hIAPP cells and diminished MTORC1 hyperactivation with concomitant autophagy stimulation. At the same time, a blockade in mitophagy was found in INS1E-hIAPP cells, as compared with control or INS1E-rIAPP cells. Consistently, human amylin overexpression generates a basal induction of nitrotyrosine levels and polyubiquitinated aggregates. Failure of the protein degradation machinery finally results in an accumulation of damaged and fissioned mitochondria, ROS production, and increased susceptibility to endoplasmic reticulum (ER)-stress-induced apoptosis. Overall, hIAPP overexpression in INS1E cells induced MTORC1 activation and mitophagy inhibition, favoring a pro-fission scenario of damaged mitochondria, these cells turn out to be more susceptible to the ER-stress-induced apoptosis and malfunction.

Autophagy in glioma cells: An identity crisis with a clinical perspective

Over the last decade, autophagy has emerged as one of the critical cellular systems that control homeostasis. Besides management of normal homeostatic processes, autophagy can also be induced by tissue damage stress or by rapidly progressing tumors. During tumor progression, autophagy mediates a cellular reaction to the changes inside and outside of cells, which leads to tumor adaptation. Even though the regulation of autophagy seems universal and is a well-described process, its dysregulation and role in glioma progression remain an important topic of investigation. In this review, we summarize recent evidence of autophagy regulation in brain tumor tissues and possible interconnection between signaling pathways that govern cellular responses. This perspective may help to assess the qualitative differences and various outcomes in response to autophagy stimulation.

Lysosomal damage after spinal cord injury causes accumulation of RIPK1 and RIPK3 proteins and potentiation of necroptosis

Necroptosis, a regulated necrosis pathway mediated by the receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3), is induced following spinal cord injury (SCI) and thought to contribute to neuronal and glial cell death. However, mechanisms leading to activation of necroptosis after SCI remain unclear. We have previously shown that autophagy, a catabolic pathway facilitating degradation of cytoplasmic proteins and organelles in a lysosome-dependent manner, is inhibited following SCI in rats. Our current data confirm that inhibition of autophagy also occurs after thoracic contusive SCI in the mouse model, as indicated by accumulation of both the autophagosome marker, LC3-II and autophagy cargo protein, p62/SQSTM1. This was most pronounced in the ventral horn neurons and was caused by rapid inhibition of lysosomal function after SCI. Interestingly, RIPK1, RIPK3, and the necroptosis effector protein MLKL also rapidly accumulated after SCI and localized to neurons with disrupted autophagy, suggesting that these events may be related. To determine if lysosomal dysfunction could contribute to induction of necroptosis, we treated PC12 cells and primary rat cortical neurons with lysosomal inhibitors. This led to rapid accumulation of RIPK1 and RIPK3, confirming that they are normally degraded by the lysosomal pathway. In PC12 cells lysosomal inhibition also sensitized cells to necroptosis induced by tumor necrosis factor α (TNFα) and caspase inhibitor. Imaging studies confirmed that RIPK1 partially localized to lysosomes in both untreated and lysosomal inhibitor treated cells. Similarly, we detected presence of RIPK1, RIPK3 and MLKL in both cytosol and at lysosomes after SCI in vivo. Furthermore, stimulation of autophagy and lysosomal function with rapamycin treatment led to decreased accumulation of RIPK1 and attenuated cell death after SCI. These data suggest that lysosomal dysfunction after SCI may contribute to both inhibition of autophagy and sensitize cells to necroptosis by promoting RIPK1 and RIPK3 accumulation.

Inflammation, Autophagy, and Apoptosis After Myocardial Infarction.

BackgroundThere is evidence for inflammation, autophagy, and apoptosis in the ischemic heart. Autophagy is a physiologic process for tissue survival. Apoptosis, on the other hand, is a mechanism that serves to clear the debris in the setting of tissue injury. The balance between autophagy and apoptosis may be important in cell survival and cardiac function.Methods and ResultsWe examined the interplay of inflammation and myocyte autophagy and apoptosis during the ischemic process. We subjected mice to total left coronary artery ligation and studied these animals for up to 4 weeks. The inflammatory (tumor necrosis factor [TNF]‐α, monocyte chemoattractant protein‐1, interleukin‐6, and interleukin‐1β) and autophagic signals (light chain‐3 and beclin‐1) were strongest during the first week and then began to decline. However, the apoptotic signals peaked at week 2 after left coronary artery ligation, and the elevated levels persisted until the end of the fourth week. To elucidate the role of inflammation in the regulation of myocyte autophagy and apoptosis, we administered TNF‐α inhibitor (CAS1049741‐03‐8, Millipore, Burlington, MA) to the mice daily during the first week of myocardial infarction. Anti‐TNF‐α therapy reduced the levels of inflammatory cytokines and the inflammatory cell infiltration in and around the infarct area. However, cardiac function measured by echocardiography (fractional shortening and ejection fraction) worsened with anti‐TNF‐α therapy. More importantly, application of TNF‐α inhibitor markedly inhibited autophagy and promoted myocyte apoptosis in the border zone.ConclusionsThese observations suggest that inflammatory response may be protective in the early stage of the myocardial infarction through stimulation of myocyte autophagy. Anti‐inflammatory treatment early after coronary occlusion may have an adverse effect.

Inhibition of ghrelin<em> o</em>-acyltransferase attenuated lipotoxicity by inducing autophagy via AMPK–mTOR pathway

BackgroundNonalcoholic fatty liver disease (NAFLD) has been considered the most commonly occurring chronic hepatopathy in the world. Ghrelin o-acyltransferase (GOAT) is an acylation enzyme which has an acylated position 3 serine on ghrelin. Recent investigation revealed that activated autophagy could attenuate liver steatosis. The aim of this study was to explore therapeutic roles that inhibit GOAT exerted in NAFLD, and its potential association with autophagy.Materials and methodsHuman LO2 cells were pretreated with siRNA-GOAT to induce liver steatosis using free fatty acids (FFAs). A chronic NAFLD model was established by feeding male mice C57bl/6 with high-fat diet (HFD) for 56 days with GO-CoA-Tat administrated subcutaneously. Lipid droplets were identified by Oil Red O stains. Body weight (BW) of mice was measured every week. Autophagy, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), serum biochemical indicators (glucose [Glu], total cholesterol [TC], triglyceride [TG], aspartate aminotransferase [AST], alanine aminotransferase [ALT]) and signaling pathway proteins of phosphorylated AMPK–mTOR were measured.ResultsThe TG contents of the FFA and HFD groups were decreased by the inhibition of GOAT. Among mice treated with GO-CoA-Tat and siRNA-GOAT, IL-6 and TNF-α concentrations were remarkably decreased. Indicators of liver injury such as ALT and AST were also remarkably decreased among mice treated with GO-CoA-Tat. Likewise, GO-CoA-Tat significantly reduced the BW of mice and serum TG, TC and Glu. Autophagy was induced along with reduced lipids in the cells of the FFA and HFD groups. The inhibition of GOAT upregulated autophagy via AMPK–mTOR restoration.ConclusionThese results indicate that the inhibition of GOAT attenuates lipotoxicity by autophagy stimulation via AMPK–mTOR restoration and offers innovative evidence for using GO-CoA-Tat or siRNA-GOAT in NAFLD clinically.

The autophagy inducers AR‐12 and AR‐14 control prion infection

Prion diseases are fatal infectious neurodegenerative disorders that affect both man and animals. The autocatalytic conversion of the cellular prion protein (PrPC) into the pathologic isoform PrPSc is a key feature in prion pathogenesis. Loss of neurons, astrogliosis and mild microglia activation are the main pathological features of prion diseases. This results in a progressive spongiform degeneration of the central nervous system, leading to ataxia, behavioral changes and, in humans, highly progressive loss of intellectual abilities. In the last two decades, great efforts have been made to establish treatment options for prion diseases. These included testing existing drugs for anti‐prion activity in experimental models with only a few agents progressing to human studies of patients with prion diseases. Investigations to date have not resulted in a recognized/proven treatment for prion diseases. AR‐12 is an IND‐approved derivative of celecoxib that demonstrated preclinical activity against several microbial diseases. Recently, AR‐12 has been shown to enhance clearance of misfolded proteins through autophagy stimulation. The latter proposes AR‐12 to be a potential therapeutic agent for neurodegenerative disorders. In the present study, we evaluated the role of AR‐12 and its derivatives in controlling prion infection. We tested AR‐12 in prion infected neuronal and non‐neuronal cell lines. Immunoblotting and confocal microscopy results showed that AR‐12 and its analogue AR‐14 reduced PrPSc levels after only 72 hours of treatment. Furthermore, infected cells were cured of PrPSc after exposure of AR‐12 or AR‐14 for only two weeks. We partially attribute the influence of the AR compounds on prion propagation to autophagy stimulation. Currently, we are investigating the effect of AR‐compounds in vivo. We are also evaluating the effect of combining AR‐ compounds with other anti‐prion agents to investigate the effect on PrPSc and prion seeding activity. Taken together, this study demonstrates that AR‐12 and the AR‐14 analogue are potential new therapeutic agents for prion diseases and possibly protein misfolding disorders involving prion‐like mechanisms.Support or Funding InformationThis work was supported by grants from the Alberta Prion Research Institute (201200002), the National Institute of Health (R01 NS076853‐01A1), the Canada Foundation for Innovation (Leaders Opportunity Fund project #32212) and was performed within the framework of the Calgary Prion Research Unit (CPRU; APRI grant 201600010). B.A.A. is an Alberta Innovates Health Solutions postgraduate fellow, D. A. is an Eyes High postgraduate fellow. S. T. is an Eyes High and Killam PhD student. S.G. is supported by the Canada Research Chair program. There are no conflicts of interest. There was no financial support from ARNO Therapeutics for performing this study. U.S. Patent Application entitled “COMPOSITIONS AND METHODS FOR REDUCING PRION LEVELS” filed February 28, 2017 as 15/445,964; VLP Ref: 2242‐00‐018U01.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Spermidine promotes nucleus pulposus autophagy as a protective mechanism against apoptosis and ameliorates disc degeneration.

Spermidine has therapeutic effects in many diseases including as heart diastolic function, myopathic defects and neurodegenerative disorders via autophagy activation. Autophagy has been found to mitigate cell apoptosis in intervertebral disc degeneration (IDD). Accordingly, we theorize that spermidine may have beneficial effects on IDD via autophagy stimulation. In this study, spermidine's effect on IDD was evaluated in tert‐butyl hydroperoxide (TBHP)‐treated nucleus pulposus cells of SD rats in vitro as well as in a puncture‐induced rat IDD model. We found that autophagy was actuated by spermidine in nucleus pulposus cells. In addition, spermidine treatment weakened the apoptotic effects of TBHP in nucleus pulposus cells. Spermidine increased the expression of anabolic proteins including Collagen‐II and aggrecan and decreased the expression of catabolic proteins including MMP13 and Adamts‐5. Additionally, autophagy blockade using 3‐MA reversed the beneficial impact of spermidine against nucleus pulposus cell apoptosis. Autophagy was thus important for spermidine's therapeutic effect on IDD. Spermidine‐treated rats had an accentuated T2‐weighted signal and a diminished histological degenerative grade than vehicle‐treated rats, showing that spermidine inhibited intervertebral disc degeneration in vivo. Thus, spermidine protects nucleus pulposus cells against apoptosis through autophagy activation and improves disc, which may be beneficial for the treatment of IDD.

Modulation of autophagy as a strategy for development of new vaccine candidates against tuberculosis

Effective prevention of tuberculosis (Tb) would undoubtedly be of paramount relevance in the control of its global burden, which resulted in more than 6 million new cases in 2016. Research aimed to improve the current vaccine, Bacillus Calmette- Guérin (BCG), or directed to develop new candidates, has taken into account the interaction between the host and Mycobacterium tuberculosis (Mtb). Recently, autophagy, an intracellular process of the host, has been shown to act as a mechanism that contributes to bacilli clearance in vitro and in vivo. Stimulation of autophagy, if correctly balanced, is an approach that has the potential to enhance the immune response of the host, and offers new avenues for developing immunogens that may give an improved protection upon immunization, given that in fact, some recent rBCG vaccine candidates have been shown to modulate autophagy. In this Discussion, we analyze the role of autophagy in the context of mycobacterial infection, its modulation via mycobacterial elements, and the management of host response as an alternative to develop new, hopefully improved, Tb-vaccine candidates.