Upregulation Of Autophagy Research Articles

The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods.

Photoperiod is an important factor of mammalian seasonal rhythm. Here, we studied morphological differences in the Harderian gland (HG), a vital photosensitive organ, in male striped dwarf hamsters (Cricetulus barabensis) under different photoperiods (short photoperiod, SP; moderate photoperiod, MP; long photoperiod, LP), and investigated the underlying molecular mechanisms related to these morphological differences. Results showed that carcass weight and HG weight were lower under SP and LP conditions. There was an inverse correlation between blood melatonin levels and photoperiod in the order SP > MP > LP. Protein expression of hydroxyindole-O-methyltransferase (HIOMT), a MT synthesis-related enzyme, was highest in the SP group. Protein expression of bax/bcl2 showed no significant differences, indicating that the level of apoptosis remained stable. Protein expression of LC3II/LC3I was higher in the SP group than that in the MP group. Furthermore, comparison of changes in the HG ultrastructure demonstrated autolysosome formation in the LP, suggesting the lowest autophagy level in under MP. Furthermore, the protein expression levels of ATP synthase and mitochondrial fission factor were highest in the MP group, whereas citrate synthase, dynamin-related protein1, and fission1 remained unchanged in the three groups. The change trends of ATP synthase and citrate synthase activity were similar to that of protein expression among the three groups. In summary, the up-regulation of autophagy under SP and LP may be a primary factor leading to loss of HG weight and reduced mitochondrial energy supply capacity.

Autophagy Plays a Role in the Prolongation of the Life Span of Caenorhabditis elegans by Astaxanthin

Astaxanthin (AST), a xanthophyll belonging to the family of carotenoids, is a potent antioxidant. The effect of AST on longevity and its physiological and molecular mechanism are still unclear. In this study, we proved that AST could prolong the life span of Caenorhabditis elegans. To uncover whether AST could delay aging by upregulating autophagy, we measured the expression of autophagy gene and the life span of autophagy gene bec-1 mutant nematodes, and the results showed that the expression of autophagy gene was upregulated after AST intervention and the disruption of bec-1 weakened the extension of the life span. To explore the molecular mechanism of AST-induced autophagy upregulation, we knocked out the daf-16 or hlh-30 (key genes of insulin/insulin growth factor-1 [IGF-1] signal pathway or target of rapamycin [TOR] signal pathway) by RNA interference, and the expression of autophagy gene lgg-1 decreased. Collectively, our results strongly suggest that autophagy, which is both the insulin/IGF-1 signal pathway dependent and TOR signal pathway dependent, plays a role in the prolongation of the life span of Caenorhabditis elegans by AST.

SS-3 Tumor Treating Fields: from the Petri dish to the patient

Tumor Treating Fields (TTFields) are a non-invasive, loco-regional, antineoplastic treatment modality targeting rapidly dividing cancer cells using low intensity, alternating electric fields at cell-type-specific intermediate frequencies (100–500 kHz). TTFields therapy is approved for the treatment of newly-diagnosed and recurrent glioblastoma as well as malignant pleural mesothelioma, following clinical trials demonstrating efficacy and a favorable safety profile. Using novel in vitro and in vivo systems for TTFields application, research activities are being conducted to extend the understanding of the underlying mechanisms of action (MoA) and to assess additional means to improve treatment outcomes. The demonstrated antimitotic effects of TTFields discerned the positive combinatorial potential of TTFields with other agents targeting the division process. Subsequent to elucidation of anti-mitotic effects, other downstream effects of TTFields include induction of endoplasmic reticulum stress, up-regulation of autophagy and cell death, thus driving immunogenic cell death. Indeed, in several preclinical models, combining TTFields with immunotherapeutics demonstrated enhanced efficacy. Recently, additional novel effects of TTFields were characterized, including inhibition of DNA damage repair responses and induction of transient and reversible permeabilization of the blood brain barrier (BBB). These new findings offer potentially innovative means to optimize treatment outcomes by combining TTFields with radiation therapy and DNA damaging agents, as well as improved delivery of impermeant agents across the BBB. These scientific findings were instrumental in advancing the clinical pipeline of TTFields, which includes conduct of ongoing trials combining TTFields with a variety of modalities, in approved indications and in other solid malignant tumor types. The aim of this talk is to describe TTFields’ preclinical research activities and tools, and to specify how these study outcomes have defined and advanced the clinical pipeline.

Autophagy compensates for Lkb1 loss to maintain adult mice homeostasis and survival.

Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11) is the major energy sensor for cells to respond to metabolic stress. Autophagy degrades and recycles proteins, macromolecules, and organelles for cells to survive starvation. To assess the role and cross-talk between autophagy and Lkb1 in normal tissue homeostasis, we generated genetically engineered mouse models where we can conditionally delete Stk11 and autophagy essential gene, Atg7, respectively or simultaneously, throughout the adult mice. We found that Lkb1 was essential for the survival of adult mice, and autophagy activation could temporarily compensate for the acute loss of Lkb1 and extend mouse life span. We further found that acute deletion of Lkb1 in adult mice led to impaired intestinal barrier function, hypoglycemia, and abnormal serum metabolism, which was partly rescued by the Lkb1 loss-induced autophagy upregulation via inhibiting p53 induction. Taken together, we demonstrated that autophagy and Lkb1 work synergistically to maintain adult mouse homeostasis and survival.

Abstract 14893: Light Activation in Plaque Macrophages Enhances Efferocytosis of Apoptotic Cells via Upregulation of Autophagy and Mertk Expression

Introduction: Autophagy plays a protective role in atherosclerosis and can promote efferocytosis of apoptotic cells. Photoactivation is a promising tool for treating cardiovascular disease, and induces autophagy in macrophages-derived foam cells, however, regulation of autophagy and efferocytosis by photoactivation in atherosclerotic plaque remains unknown. Objective: The present study aims to investigate whether macrophage targeted light activation induces autophagy and enhances efferocytosis in inflamed plaques Methods and Results: Targeted photoactivatable agent showed specific binding affinity and phototoxicity to scavenger receptor-expressing macrophages. Confocal microscopy analysis over time after irradiation demonstrated the induction of apoptosis and autophagy flux in macrophage-derived foam cells. The theranostic agent localized to macrophage scavenger receptors within atherosclerotic plaques, and intravital fluorescence microscopy imaged plaque inflammation and was used to monitor photobleaching after irradiation. Serial in vivo imaging analysis demonstrated that macrophage-specific photoactivation reduced plaque burden and inflammation after 1 week (Figure A). Mechanistically, targeted light activation induced autophagy within atheroma as early as 1 day (Figure B), and increased phagocytic receptor Mer tyrosine kinase (MerTK) expression. Targeted photoactivation had 2-fold-increase in macrophage-associated apoptotic bodies at one week after laser irradiation, indicating enhanced efferocytosis (Figure C). Conclusions: As a sensitive multifunctional therapeutic and imaging strategy, macrophage targeted photoactivation with autophagy induction could confer a promising theranostic strategy for the high-risk plaques.

Ischemia-induced upregulation of autophagy preludes dysfunctional lysosomal storage and associated synaptic impairments in neurons

ABSTRACT Macroautophagy/autophagy is vital for neuronal homeostasis and functions. Accumulating evidence suggest that autophagy is impaired during cerebral ischemia, contributing to neuronal dysfunction and neurodegeneration. However, the outcomes after transient modification in autophagy machinery are not fully understood. This study investigated the effects of ischemic stress on autophagy and synaptic structures using a rat model of oxygen-glucose deprivation (OGD) in hippocampal neurons and a mouse model of middle cerebral artery occlusion (MCAO). Upon acute ischemia, an initial autophagy modification occurred in an upregulation manner. Following, the number of lysosomes increased, as well as lysosomal volume, indicating dysfunctional lysosomal storage. These changes were prevented by inhibiting autophagy via 3-methyladenine (3-MA) treatment or ATG7 (autophagy related 7) knockdown, or were mimicked by rapamycin (RAPA), a known activator of autophagy. This suggests that dysfunctional lysosomal storage is associated with the early burst of autophagy. Dysfunctional lysosomal storage contributed to autophagy dysfunction because the basal level of MTOR-dependent lysosomal biogenesis in the reperfusion was not sufficient to clear undegraded cargoes after transient autophagy upregulation. Further investigation revealed that impairment of synaptic ultra-structures, accompanied by dysfunctional lysosomal storage, may result from a failure in dynamic turnover of synaptic proteins. This indicates a vital role of autophagy-lysosomal machinery in the maintenance of synaptic structures. This study supports previous evidence that dysfunctional lysosomal storage may occur following the upregulation of autophagy in neurons. Appropriate autophagosome-lysosomal functioning is vital for maintenance of neuronal synaptic function and impacts more than the few known synaptic proteins. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AD: Alzheimer disease; ALR: autophagic lysosome reformation; ATG7: autophagy related 7; CTSB: cathepsin B; CTSD: cathepsin D; DAPI: 4ʹ,6-diamidino-2-phenylindole; DEGs: differentially expressed genes; DMEM: Dulbecco’s modified Eagle’s medium; DMSO: dimethyl sulfoxide; GO: Gene Ontology; HBSS: Hanks’ balanced salt solution; HPCA: hippocalcin; i.c.v: intracerebroventricular; KEGG: kyoto encyclopedia of genes and genomes; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; LSDs: lysosomal storage disorders; MAP2: microtubule-associated protein 2; MCAO: middle cerebral artery occlusion; mCTSB: mature CTSB; mCTSD: mature CTSD; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; OGD/R: oxygen-glucose deprivation/reoxygenation; PBS: phosphate-buffered saline; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; proCTSD: pro-cathepsin D; RAPA: rapamycin; RNA-seq: RNA sequencing; RPS6KB/p70S6K: ribosomal protein S6 kinase; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SIM: Structured Illumination Microscopy; SNAP25: synaptosomal-associated protein 25; SQSTM1/p62: sequestosome 1; SYN1: synapsin I; SYT1: synaptotagmin I; TBST: tris-buffered saline Tween-20; TEM: transmission electron microscopy; TFEB: transcription factor EB; tMCAO: transient middle cerebral artery occlusion; TTC: 2,3,5-triphenyltetrazolium chloride; TUBB3: tubulin, beta 3 class III.

Autophagy Induction Contributes to the Neuroprotective Impact of Intermittent Fasting on the Acutely Injured Spinal Cord.

Spinal cord injury (SCI) is one of the leading causes of neurological disability and death. So far, there is no satisfactory treatment for SCI, because of its complex and ill-defined pathophysiology. Recently, autophagy has been implicated as protective in acute SCI rat models. Here, we investigated the therapeutic value of a dietary intervention, namely, intermittent fasting (IF), on neuronal survival after acute SCI in rats, and its underlying mechanism related to autophagy regulation. We found remarkable improvement in both behavioral performance and neuronal survival at the injured segment of the spinal cord of animals previously subjected to IF. Western blotting revealed a marked decrease in apoptosis-related markers such as cleaved caspase 3 levels and the bax/bcl-2 ratio in the IF group, which suggested an inhibition of the intrinsic apoptosis pathway. In addition, the expression of the autophagy markers LC3-II and beclin 1 was also increased in the IF group compared with ad libitum fed animals. In parallel, IF decreased the levels of the substrate protein of autophagy, p62, indicative of an upregulation of the autophagic processes. Treatment with 3-methyladenine (3-MA), a selective inhibitor of autophagy, reversed the downregulated apoptosis-related markers by IF. Finally, IF could activate the adenosine monophosphate (AMP)-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway and enhance lysosome function by upregulating transcription factor (TF)EB expression. Altogether, the present findings suggest that IF exerts a neuroprotective effect after acute SCI via the upregulation of autophagy, and further points to dietary interventions as a promising combinatorial treatment for SCI.

Paeonol inhibits apoptosis of vascular smooth muscle cells via up-regulation of autophagy by activating class III PI3K/Beclin-1 signaling pathway

AimsThe cross talk between autophagy and apoptosis of vascular smooth muscle cells (VSMCs) plays a vital role in the development of atherosclerosis (AS). Paeonol is isolated from the radix of Cortex Moutan with anti-atherosclerotic and anti-apoptosis effects. However, the mechanisms of paeonol on VSMCs apoptosis are still not fully understood. In this study, we aimed to explore whether paeonol could inhibit VSMCs apoptosis though modulating VSMCs autophagy. Materials and methodsThe proteins expressions were detected by western blotting. Autophagosomes and apoptoticbody formation in VSMCs was observed by transmission electron microscopy (TEM). VSMCs autophagy was detected by monodansylcadaverine (MDC) staining using fluorescence microscopy, while VSMCs apoptosis was determined by 4′,6-diamidino-2-phenylindole (DAPI) and flow cytometry. Key findingsWe found that paeonol could significantly increase LC3II protein level, decrease p62 and cleaved caspase-3 proteins levels in aorta of AS mice and ox-LDL-injured VSMCs. Paeonol could augment the number of autophagosomes and reduce the amount of apoptotic bodies in ox-LDL-injured VSMCs. Moreover, paeonol obviously induced VSMCs autophagy compared to ox-LDL group and remarkably suppressed VSMCs apoptosis. However, the effects of paeonol on VSMCs apoptosis could be reversed obviously by 3-MA, the autophagy inhibitor. Furthermore, paeonol could activate class III PI3K-Beclin-1 pathway significantly. Gene silencing of either class III PI3K or Beclin-1 could reverse the effects of paeonol on VSMCs autophagy and apoptosis. SignificanceBased on our results, paeonol could induce VSMCs autophagy by activating class III PI3K/Beclin-1 signaling pathway, thus ultimately inhibiting VSMCs apoptosis.

Amyotrophic Lateral Sclerosis and Autophagy: Dysfunction and Therapeutic Targeting.

Over the past 20 years, there has been a drastically increased understanding of the genetic basis of Amyotrophic Lateral Sclerosis. Despite the identification of more than 40 different ALS-causing mutations, the accumulation of neurotoxic misfolded proteins, inclusions, and aggregates within motor neurons is the main pathological hallmark in all cases of ALS. These protein aggregates are proposed to disrupt cellular processes and ultimately result in neurodegeneration. One of the main reasons implicated in the accumulation of protein aggregates may be defective autophagy, a highly conserved intracellular “clearance” system delivering misfolded proteins, aggregates, and damaged organelles to lysosomes for degradation. Autophagy is one of the primary stress response mechanisms activated in highly sensitive and specialised neurons following insult to ensure their survival. The upregulation of autophagy through pharmacological autophagy-inducing agents has largely been shown to reduce intracellular protein aggregate levels and disease phenotypes in different in vitro and in vivo models of neurodegenerative diseases. In this review, we explore the intriguing interface between ALS and autophagy, provide a most comprehensive summary of autophagy-targeted drugs that have been examined or are being developed as potential treatments for ALS to date, and discuss potential therapeutic strategies for targeting autophagy in ALS.

Effect of silibinin on ethanol- or acetaldehyde-induced damge of mouse primary hepatocytes in vitro

Silibinin, one of the flavonoids isolated from milk thistle seeds of Silybum marianum, has hepatoprotective properties against toxins in clinical. However, the detailed mechanisms have remained unclear. This study investigates the underlying mechanism of silibinin in the protection against ethanol- or acetaldehyde-induced damage of neonatal mouse primary hepatocytes in vitro. The results show that ethanol inhibited proliferation of hepatocytes in a time (12, 24, 36 h) and dose-dependent (0–800 mM) manner. However, silibinin did not show protective effect on ethanol (500 mM)-induced suppression of hepatocyte proliferation. Acetaldehyde, the toxic metabolite of ethanol, appearing immediately in individuals after drink also inhibited the proliferation of hepatocytes in a dose-dependent (0–12 mM) manner. Surprisingly, silibinin significantly increased the cell viability and reduced the leakage of alanine amino transferase (ALT) and aspartate amino transferase (AST) in acetaldehyde-treated hepatocytes, suggesting that silibinin protected cell injury caused by acetaldehyde treatment. The apoptosis-inducing effect of acetaldehyde was demonstrated by the increased number of cells in sub-G1 phase as well as caspase-3 activation. Further study shows that acetaldehyde induced autophagy in the hepatocytes. The autophagy inhibitors, 3-Methyladenine (3-MA) and chloroquine (CQ), further decreased the viability of cells treated with acetaldehyde, suggesting that autophagy plays a protective role against apoptosis. Consistently, silibinin (20 μM) significantly reduced the activation of caspase 3 or apoptosis and increased the conversion of LC3-I to LC3-II or autophagy. Taken together, it is concluded that silibinin does not repress the ethanol- induced hepatocyte injury, whereas silibinin reduces acetaldehyde-caused hepatocyte injury through down-regulation of apoptosis and up-regulation of autophagy.

Epigallocatechin-3-Gallate Reduces Visceral Adiposity Partly through the Regulation of Beclin1-Dependent Autophagy in White Adipose Tissues.

Epigallocatechin-3-gallate (EGCG) is a primary bioactive phytochemical in green tea. Its therapeutic potential in metabolic diseases has been reported; however, the molecular mechanisms of the anti-obesity effect of EGCG have not been fully elucidated. In this study, we examined the effects of EGCG on lipid metabolism and autophagy in adipose tissue. After 8 weeks of high-fat diet feeding, mice were treated with EGCG (20 mg/kg/day) for 2 weeks to test in vivo anti-obesity effects of EGCG. EGCG treatment improved glucose tolerance and caused body weight loss. Interestingly, reduced adipose tissue mass was more prominent in visceral compared to subcutaneous white adipose tissue. Mechanistically, EGCG treatment increased autophagic flux in white adipose tissue through the AMP-activated protein kinase-mediated signaling pathway. Adipocyte-specific knockout of Beclin1 mitigated the effects of EGCG on visceral adipose tissue mass and glucose tolerance, indicating that the anti-obesity effect of EGCG requires Beclin1-dependent autophagy. Collectively, our data demonstrated that EGCG has anti-obesity effects through the upregulation of Beclin1-dependent autophagy and lipid catabolism in white adipose tissue (WAT).

Abstract A06: Differential response to autophagy in primary versus melanoma cells

Abstract Autophagy is a cellular recycling mechanism triggered by nutrient scarcity. Upregulation of autophagy is associated with lifespan extension, DNA repair, and improved cancer immune recognition. Cancer cells can also utilize this mechanism to cope with nutrient-limited conditions. Chloroquine, an inhibitor of autophagy, has shown promising results alone and in combination with other anticancer agents in various types of cancer. However, several promoters of autophagy have also been shown to display potent anticancer properties. To better understand the relationship between autophagy and cancer, we tested promoters and inhibitors of autophagy in a metastatic melanoma cell line (B16F10) and compared the results with primary human dermal fibroblasts (HDF). After 48h, all tested promoters and inhibitors of autophagy decreased the number of live cells in the B16F10 melanoma cell line compared to untreated controls. In primary cells, promotion of autophagy with methionine restriction (25 μM versus 100 μM in control), complete methionine replacement with homocysteine, and 50 μM of the polyamine spermidine did not affect the number of live cells. Sulforaphane (10 μM) and serum starvation significantly decreased the number of live primary cells. Inhibition of autophagy with 6.25 μM chloroquine did not affect primary cell numbers, while there was a significant decrease in cell numbers associated with a dose of 10 μM of chloroquine and 0.1 μM bafilomycin A1. Cell numbers remained unaffected by longer incubations (96h with replenishment after 48h) of HDF cells with methionine restriction, replacement of methionine with homocysteine, and 6.25 μM chloroquine. These data suggest that cancer cells are more sensitive to both promotion and inhibition of autophagy than primary cells. Interesting compound-specific differences were noted in primary cells. Ongoing preclinical studies evaluate the potential of modulating autophagy as an adjuvant cancer therapy for immune checkpoint inhibitors in metastatic melanoma. Citation Format: Lauren C. Morehead, Alan J. Tackett, Isabelle R. Miousse. Differential response to autophagy in primary versus melanoma cells [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A06.

The Tumorigenic Effect of Sphingosine Kinase 1 and Its Potential Therapeutic Target.

Sphingosine kinase 1 (SPHK1) regulates cell proliferation and survival by converting sphingosine to the signaling mediator sphingosine 1-phosphate (S1P). SPHK1 is widely overexpressed in most cancers, promoting tumor progression and is associated with clinical prognosis. Numerous studies have explored SPHK1 as a promising target for cancer therapy. However, due to insufficient knowledge of SPHK1 oncogenic mechanisms, its inhibitors’ therapeutic potential in preventing and treating cancer still needs further investigation. In this review, we summarized the metabolic balance regulated by the SPHK1/S1P signaling pathway and highlighted the oncogenic mechanisms of SPHK1 via the upregulation of autophagy, proliferation, and survival, migration, angiogenesis and inflammation, and inhibition of apoptosis. Drug candidates targeting SPHK1 were also discussed at the end. This review provides new insights into the oncogenic effect of SPHK1 and sheds light on the future direction for targeting SPHK1 as cancer therapy.

Peptide-Based TNF-α-Binding Decoy Therapy Mitigates Lipopolysaccharide-Induced Liver Injury in Mice.

A peptide named SEM18, possessing structural similarity to the binding site of tumor necrosis factor (TNF)-α to TNF receptor 1 (TNFR1), was designed. We investigated whether the SEM18 peptide can mitigate lipopolysaccharide (LPS)-induced liver injury in mice. Adult male Balb/cJ mice received LPS (15 mg/kg; LPS group) or LPS plus SEM18 (LSEM group). Control groups were run simultaneously. At 2 h after LPS, the first dose of SEM18 (0.3 mg/kg) was administered, followed by three supplemental doses of SEM18 (0.15 mg/kg, every 2 h). At 24 h after LPS, surviving mice were euthanized for analyses. Compared with the LPS group, binding of TNF-α to TNFR1 in liver tissues was significantly lower in the LSEM group (p < 0.001). Plasma concentrations of aspartate transaminase and alanine transaminase, as well as Suzuki’s scores (liver damage assessment), wet/dry weight ratios, levels of polymorphonuclear neutrophil infiltration, and levels of mitochondrial injury in liver tissues, of the LSEM group were significantly lower than in the LPS group (all p < 0.05). Levels of necroptosis, pyroptosis, apoptosis, and autophagy upregulation in liver tissues in the LSEM group were also significantly lower than in the LPS group (all p < 0.05). Notably, exogenous TNF-α counteracted these effects of SEM18. SEM18 peptide mitigates LPS-induced liver injury in mice.

Irisin promotes osteogenic differentiation of bone marrow mesenchymal stem cells by activating autophagy via the Wnt//β-catenin signal pathway

Osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) plays a crucial role in osteoporosis. Irisin, an exercise-induced muscle-dependent myokine, has been reported to stimulate the development of brown adipose tissue and regulate energy expenditure. The present study aimed to investigate the effects of irisin on autophagy in BMSCs. Furthermore, the osteogenic differentiation ability was evaluated, as well as the activation of autophagy. It was found that 40 μM irisin for 48 h was an appropriate concentration and time period, with regards to cell viability, which was measured with a Cell Counting Kit-8. Moreover, the increasing expression levels of microtubule-associated protein light chain 3 (Lc3)-I/II and autophagy related 5 (Atg5) by irisin demonstrated the upregulation of autophagy. Mechanistically, bafilomycin A1 and Atg5 small interfering RNA were used to evaluate the possible mechanism of autophagy activated by irisin, and it was identified that irisin may upregulate autophagy by increasing the Atg12-Atg5-Atg16L complex. In addition, with the increasing level of autophagy, osteogenesis and the Wnt/β-catenin signal pathway were also enhanced. However, inhibition of autophagy by bafilomycin A1 negatively regulated osteogenic differentiation. Collectively, the present results suggested that irisin may stimulate autophagy in BMSCs and that osteogenic differentiation may be enhanced by stimulating autophagy.

The GTPase Arl8B Plays a Principle Role in the Positioning of Interstitial Axon Branches by Spatially Controlling Autophagosome and Lysosome Location.

Interstitial axon branching is an essential step during the establishment of neuronal connectivity. However, the exact mechanisms on how the number and position of branches are determined are still not fully understood. Here, we investigated the role of Arl8B, an adaptor molecule between lysosomes and kinesins. In chick retinal ganglion cells (RGCs), downregulation of Arl8B reduces axon branch density and shifts their location more proximally, while Arl8B overexpression leads to increased density and more distal positions of branches. These alterations correlate with changes in the location and density of lysosomes and autophagosomes along the axon shaft. Diminishing autophagy directly by knock-down of atg7, a key autophagy gene, reduces branch density, while induction of autophagy by rapamycin increases axon branching, indicating that autophagy plays a prominent role in axon branch formation. In vivo, local inactivation of autophagy in the retina using a mouse conditional knock-out approach disturbs retino-collicular map formation which is dependent on the formation of interstitial axon branches. These data suggest that Arl8B plays a principal role in the positioning of axon branches by spatially controlling autophagy, thus directly controlling formation of neural connectivity in the brain.SIGNIFICANCE STATEMENT The formation of interstitial axonal branches plays a prominent role in numerous places of the developing brain during neural circuit establishment. We show here that the GTPase Arl8B controls density and location of interstitial axon branches, and at the same time controls also density and location of the autophagy machinery. Upregulation or downregulation of autophagy in vitro promotes or inhibits axon branching. Local disruption of autophagy in vivo disturbs retino-collicular mapping. Our data suggest that Arl8B controls axon branching by controlling locally autophagy. This work is one of the first reports showing a role of autophagy during early neural circuit development and suggests that autophagy in general plays a much more prominent role during brain development than previously anticipated.

Autophagy in Health and Food Science

In recent years, the aging population in society has been increasing on a global scale, leading to concerns regarding increasing health problems. Preventive medicine plays a major role in extending healthy life expectancy in aging societies, and there is an increasing demand for functional foods that can be ingested for a long period of time before health is impaired. The aim of the present review is to briefly elucidate the role of autophagy in disease prevention and anti-aging. The application of autophagy in health promotion using functional foods has also been discussed. Autophagy is an intracellular degradation system that contributes to the maintenance of health and prevention of disease onset by recycling intracellular components. Recent evidence has shown that autophagy plays a potential role in extending health span. Autophagic activity decline has been associated with aging, leading to the onset of various age-associated diseases such as neurodegeneration. Conversely, animal models with artificially enhanced autophagic activity have demonstrated an extended lifespan while maintaining the ability to perform exercise. Therefore, the enhancement of autophagy is anticipated to contribute to health promotion. Although prior studies have described the upregulation of autophagy by food, several of them have not been evaluated using an appropriate method. This paper focuses on autophagy, which is closely related to health maintenance and disease prevention, and summarizes recent studies on the function of food in autophagy and on extending health span from the viewpoint of health and food science.

Up-regulation of autophagy by etanercept treatment results in TNF-induced apoptosis reduction in EA.hy926 endothelial cell line

Rheumatoid arthritis (RA) is an autoimmune systemic inflammatory disease associated with a high prevalence of atherosclerosis. Endothelial dysfunction has emerged as a potentially valuable prognostic tool in predicting the development of atherosclerosis. Tumour necrosis factor (TNF) is the main cytokine involved in RA pathogenesis, exerting a pro-atherogenic role. TNF-inhibitors are effective treatments in RA, also improving endothelial function. Regarding this, no experimental data are known about the involvement of etanercept. We investigated the contribution of TNF to endothelial dysfunction and the effect of in vitro treatment with etanercept, with a special focus on autophagy and apoptosis pathways. Autophagy and apoptosis were evaluated by Western blot and flow cytometry in EA.hy926 endothelial cells treated with TNF alone or in combination with etanercept for 24h. Blocking autophagy, TNF was able to induce endothelial cell apoptosis. Co-treatment with etanercept reverted this effect, up-regulating the autophagy pathway. Our results confirm the protective role of etanercept, by restoring autophagy on TNF-induced endothelial damage.

TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants.

Background & AimsTNFSF15 genetic variants leading to increased TNF superfamily member 15 (TNFSF15) expression confer risk for inflammatory bowel disease (IBD), and TNFSF15 is being explored as a therapeutic target in IBD patients. Although the focus for TNFSF15-mediated inflammatory outcomes has been predominantly on its action on T cells, TNFSF15 also promotes inflammatory outcomes in human macrophages. Given the critical role for macrophages in bacterial clearance, we hypothesized that TNFSF15 promotes antimicrobial pathways in human macrophages and that macrophages from TNFSF15 IBD risk carriers with higher TNFSF15 expression have an advantage in these antimicrobial outcomes.MethodsWe analyzed protein expression, signaling, bacterial uptake, and intracellular bacterial clearance in human monocyte-derived macrophages through flow cytometry, enzyme-linked immunosorbent assay, and gentamicin protection.ResultsAutocrine/paracrine TNFSF15 interactions with death receptor 3 (DR3) were required for optimal levels of pattern-recognition-receptor (PRR)-induced bacterial clearance in human macrophages. TNFSF15 induced pyruvate dehydrogenase kinase 1-dependent bacterial uptake and promoted intracellular bacterial clearance through reactive oxygen species, nitric oxide synthase 2, and autophagy up-regulation. The TNFSF15-initiated TNF receptor-associated factor 2/receptor-interacting protein kinase 1/RIP3 pathway was required for mitogen-activated protein kinase and nuclear factor-κB activation, and, in turn, induction of each of the antimicrobial pathways; the TNFSF15-initiated Fas-associated protein with death domain/mucosa-associated lymphoid tissue lymphoma translocation protein 1/caspase-8 pathway played a less prominent role in antimicrobial functions, despite its key role in TNFSF15-induced cytokine secretion. Complementation of signaling pathways or antimicrobial pathways restored bacterial uptake and clearance in PRR-stimulated macrophages where TNFSF15:DR3 interactions were inhibited. Monocyte-derived macrophages from high TNFSF15-expressing rs6478108 TT IBD risk carriers in the TNFSF15 region showed increased levels of the identified antimicrobial pathways.ConclusionsWe identify that autocrine/paracrine TNFSF15 is required for optimal PRR-enhanced antimicrobial pathways in macrophages, define mechanisms regulating TNFSF15-dependent bacterial clearance, and determine how the TNFSF15 IBD risk genotype modulates these outcomes.

NLRC5 Inhibits Inflammation of Secretory Phase Ectopic Endometrial Stromal Cells by Up-Regulating Autophagy in Ovarian Endometriosis.

Nod-like receptor (NLR) family caspase activation and recruitment domain containing 5 (NLRC5) is a newly identified sub-class of the NLR family. It regulates inflammation and has a key function in innate and adaptive immunologic reactions. Autophagy has been reported to be crucially linked to the pathogenesis of endometriosis. Our recent study identify there is a negative correlation between NLRC5 and autophagy in endometriosis, indicating that NLRC5 and autophagy together act as promising predictors in endometriosis patients. However, the mechanism associating NLRC5 and autophagy in endometriosis is still not completely understood. We hypothesize that autophagy could be involved in NLRC5-mediated inflammation in endometriosis. In order to validate the assumption, we evaluate the effects of NLRC5 and autophagy in the inflammation of ectopic endometrial stromal cells (EESCs) of ovarian endometriosis patients, to specifically determine whether autophagy is involved in NLRC5-mediated inflammation in EESCs. Our results show that over-expression of NLRC5 results in the up-regulation of autophagy in EESCs and inhibition of NLRC5 restricts the level of autophagy in EESCs. Furthermore, over-expression of NLRC5 and promotion of autophagy inhibit interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expressions, whereas inhibition of NLRC5 and autophagy up-regulate IL-6 and TNF-α expressions in EESCs. Additionally, promotion of autophagy contributes to the NLRC5-mediated inhibition of IL-6 and TNF-α expressions in EESCs; inhibition of autophagy restricts NLRC5-mediated inhibition of IL-6 and TNF-α expressions in EESCs. Our results suggest that over-expression of NLRC5 promotes autophagy, thereby inhibiting inflammation in ovarian endometriosis.