Increased Autophagy Flux Research Articles

Knockdown of astrocytic Grin2a exacerbated sleep deprivation-induced cognitive impairments and elevation of amyloid-beta

Sleep disorders are associated with cognitive impairments, greater amyloid-β (Aβ) burden and increased risk of developing Alzheimer’s disease, while the underlying mechanism is unclear. N-methyl-d-aspartate receptors (NMDARs), as vital modulators of cognition, are sensitive to sleep disturbance. Sleep deprivation (SD) could induce the alterations of neuronal NMDAR subunits expression, however the alterations of astrocytic NMDARs in SD have not been reported. Our previous study has demonstrated knockdown of astrocytic Grin2a (gene encoding NMDAR subunit GluN2A) could aggravate Aβ-induced cognitive impairments, but what role astrocytic GluN2A may play in SD is unknown. Here we focused on the changes and roles of hippocampal astrocytic GluN2A in SD. Our results showed SD increased the expression of astrocytic GluN2A. Specific knockdown of hippocampal astrocytic Grin2a aggravated SD-induced cognitive decline, elevated Aβ, and attenuated the SD-induced increase in autophagy flux. Our finding, for the first time, revealed a novel neuroprotective role for astrocytic GluN2A in SD, which may be helpful for developing new preventive and therapeutic targets to sleep disorders.

Semaglutide Protects against 6-OHDA Toxicity by Enhancing Autophagy and Inhibiting Oxidative Stress.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder for which no effective treatment is available. Studies have demonstrated that improving insulin resistance in type 2 diabetes mellitus (T2DM) can benefit patients with PD. In addition, a neuroprotective effect of glucagon-like peptide-1 (GLP-1) receptor agonists was demonstrated in experimental models of PD. In addition, there are some clinical trials to study the neuroprotective effect of GLP-1 analog on PD patients. Semaglutide is a long-acting, once-a-week injection treatment and the only available oral form of GLP-1 analog. In the present study, we treated the human neuroblastoma SH-SY5Y cell line with 6-hydroxydopamine (6-OHDA) as a PD in vitro model to explore the neuroprotective effects and potential mechanisms of semaglutide to protect against PD. Moreover, we compared the effect of semaglutide with liraglutide given at the same dose. We demonstrated that both semaglutide and liraglutide protect against 6-OHDA cytotoxicity by increasing autophagy flux and decreasing oxidative stress as well as mitochondrial dysfunction in SH-SY5Y cells. Moreover, by comparing the neuroprotective effects of semaglutide and liraglutide on PD cell models at the same dose, we found that semaglutide was superior to liraglutide for most parameters measured. Our results indicate that semaglutide, the new long-acting and only oral GLP-1 analog, may be represent a promising treatment for PD.

Progranulin Preserves Autophagy Flux and Mitochondrial Function in Rat Cortical Neurons Under High Glucose Stress.

Chronic hyperglycemia in type II diabetes results in impaired autophagy function, accumulation of protein aggregates, and neurodegeneration. However, little is known about how to preserve autophagy function under hyperglycemic conditions. In this study, we tested whether progranulin (PGRN), a neurotrophic factor required for proper lysosome function, can restore autophagy function in neurons under high-glucose stress. We cultured primary cortical neurons derived from E18 Sprague-Dawley rat pups to maturity at 10 days in vitro (DIV) before incubation in high glucose medium and PGRN for 24-72 h before testing for autophagy flux, protein turnover, and mitochondrial function. We found that although PGRN by itself did not upregulate autophagy, it attenuated impairments in autophagy seen under high-glucose conditions. Additionally, buildup of the autophagosome marker light chain 3B (LC3B) and lysosome marker lysosome-associated membrane protein 2A (LAMP2A) changed in both neurons and astrocytes, indicating a possible role for glia in autophagy flux. Protein turnover, assessed by remaining advanced glycation end-product levels after a 6-h incubation, was preserved with PGRN treatment. Mitochondrial activity differed by complex, although PGRN appeared to increase overall activity in high glucose. We also found that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase 3β (GSK3β), kinases implicated in autophagy function, increased with PGRN treatment under stress. Together, our data suggest that PGRN prevents hyperglycemia-induced decreases in autophagy by increasing autophagy flux via increased ERK1/2 kinase activity in primary rat cortical neurons.

Autophagy triggered by the ROS/ERK signaling pathway protects mouse embryonic palatal cells from apoptosis induced by nicotine.

Maternal cigarette smoking during pregnancy is a known high-risk factor for having a child with a cleft lip and/or palate (CLP), a common congenital malformation. Nicotine is the major teratogen component of cigarettes and e-cigarettes, and nicotine plays an important role in the development of CLP. However, the mechanism underlying nicotine's effect on CLP remains unclear. Here, we aimed to determine the role and molecular mechanisms of nicotine-induced autophagy, an important process involved in regulating the cellular stress response in mouse embryonic palatal cells (MEPCs). First, we found that nicotine promoted MEPCs proliferation and inhibited their apoptosis from 0 to 12h. After 12h, the proliferation was inhibited, and apoptosis was promoted. The migration of MEPCs was also inhibited by nicotine. Simultaneously, long-term nicotine stimulation inhibited the osteogenic differentiation of MEPCs. We then found that nicotine significantly increased autophagy flux in MEPCs at 12h by increasing the expression of microtubule-associated protein light chain 3 (LC3) and reducing P62 expression levels. After nicotine exposure, intracellular reactive oxygen species (ROS) and extracellular signal-regulated kinase-1/2 (ERK1/2) expression significantly increased, and the expression of ERK1/2 was reversed by the ROS scavenging agent N-acetylcysteine (NAC). Moreover, the autophagy induced by nicotine was reversed by SCH772984, a specific inhibitor of ERK1/2, and the autophagy inhibitor chloroquine (CQ). These results suggest that in the early stage of nicotine exposure, MEPCs may trigger autophagy through the ROS/ERK1/2 signaling pathway to avoid cell damage caused by nicotine.

GJA1 reverses arsenic-induced EMT via modulating MAPK/ERK signaling pathway

Arsenic is known as a well-established human carcinogen. Gap Junction Protein Alpha 1 (GJA1) is a multifunction protein that forms gap junction channels and is important for intercellular communication. Recently, its aberrant expression has been shown to associate with cancer recurrence and metastatic spread. However, whether GJA1 plays a role in arsenic carcinogenesis remains unknown. Here, we demonstrated that chronic exposure of human bronchial epithelial BEAS-2B cells to sodium arsenite promoted epithelial-mesenchymal transition (EMT) via increasing the expression of EMT inducer S100A4 and activation of MAPK/ERK signaling. In vitro and in vivo experiments showed that chronic exposure to sodium arsenite reduced GJA1 expression. Forced expression of GJA1 inhibited sodium arsenite-induced EMT via suppressing MAPK/ERK signaling whereas GJA1 knockdown produced an opposite effect. Intriguingly, chronic exposure to sodium arsenite increased autophagy flux. Inhibition of autophagy by pharmacological intervention or genetic deletion of autophagy core gene Beclin-1 upregulated GJA1 expression. These results suggested that GJA1 restrained the carcinogenic effect of sodium arsenite by limiting MAPK/ERK signaling, and GJA1 expression was decreased by arsenic-activated autophagy. In addition, interventions directed at enhancing the level or functional activity of GJA1 could be of preventive and therapeutic interest.

Abstract 5826: Autophagy regulates cancer stem cell properties in triple negative breast cancer via miR-181a-mediated regulation of ATG5/ATG2B

Abstract Autophagy has a dual role in the maintenance of cancer stem cells (CSCs), but the precise relationship between autophagy and cancer stemness requires further investigation. In this study, it was found that luminal and triple-negative breast cancer require distinct therapeutic approaches because of their different expressions of autophagy flux. We identified that autophagy flux was inhibited in triple-negative breast cancer (TNBC) CSCs. Moreover, miRNA-181a (miR-181a) is upregulated both in TNBC CSCs and patients. ATG5 and ATG2B participate in the early formation of autophagosomes and were revealed as targets of miR-181a. Inhibition of miR-181a expression led to attenuation of TNBC cancer stemness and an increase in autophagy flux. Furthermore, treatment with curcumin led to attenuation of cancer stemness in TNBC CSCs; the expression of ATG5 and ATG2B was enhanced and there was an increase of autophagy flux. These results indicated that ATG5 and ATG2B are involved in the suppression of cancer stemness in TNBC. In summary, autophagy inhibits cancer stemness through the miR-181a-regulated mechanism in TNBC. Promoting tumor-suppressive autophagy using curcumin may be a potential method for the treatment of TNBC. Citation Format: Jong Hoon Park, Jee Won Park, Yesol Kim, Jaehee Jun, Yejin Ahn. Autophagy regulates cancer stem cell properties in triple negative breast cancer via miR-181a-mediated regulation of ATG5/ATG2B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5826.

Itch and autophagy-mediated NF-κB activation contributes to inhibition of cathepsin D-induced sensitizing effect on anticancer drugs

Inhibition of cathepsin D (Cat D) sensitizes cancer cells to anticancer drugs via RNF183-mediated downregulation of Bcl-xL expression. Although NF-κB activation is involved in the upregulation of RNF183 expression, the molecular mechanism of NF-κB activation by Cat D inhibition is unknown. We conducted this study to investigate the molecular mechanism underlying Cat D-mediated NF-κB activation. Interestingly, Cat D inhibition-induced IκB degradation in an autophagy-dependent manner. Knockdown of autophagy-related genes (ATG7 and Beclin1) and lysosome inhibitors (chloroquine and bafilomycin A1) blocked IκB degradation via Cat D inhibition. Itch induced K63-linked ubiquitination of IκB and then modulated the protein stability of IκB by Cat D inhibition. Inhibition of Cat D-mediated Itch activation was modulated by the JNK signaling pathway, and phosphorylated Itch could bind to IκB, resulting in polyubiquitination of IκB. Additionally, inhibition of Cat D increased autophagy flux via activation of the LKB1-AMPK-ULK1 pathway. Therefore, our results suggested that Cat D inhibition activated NF-κB signaling via degradation of autophagy-dependent IκB, which is associated with the upregulation of RNF183, an E3 ligase of Bcl-xL. Cat D inhibition enhances TRAIL-induced apoptosis through Bcl-xL degradation via upregulation of RNF183.

STING controls energy stress-induced autophagy and energy metabolism via STX17.

The stimulator of interferon genes (STING) plays a critical role in innate immunity. Emerging evidence suggests that STING is important for DNA or cGAMP-induced non-canonical autophagy, which is independent of a large part of canonical autophagy machineries. Here, we report that, in the absence of STING, energy stress-induced autophagy is upregulated rather than downregulated. Depletion of STING in Drosophila fat cells enhances basal- and starvation-induced autophagic flux. During acute exercise, STING knockout mice show increased autophagy flux, exercise endurance, and altered glucose metabolism. Mechanistically, these observations could be explained by the STING-STX17 interaction. STING physically interacts with STX17, a SNARE that is essential for autophagosome biogenesis and autophagosome-lysosome fusion. Energy crisis and TBK1-mediated phosphorylation both disrupt the STING-STX17 interaction, allow different pools of STX17 to translocate to phagophores and mature autophagosomes, and promote autophagic flux. Taken together, we demonstrate a heretofore unexpected function of STING in energy stress-induced autophagy through spatial regulation of autophagic SNARE STX17.

The TSPO ligand 3,17,19‐androsten‐5‐triol attenuates NASH by increasing FXR expression and autophagy

Translocator protein (TSPO; 18 kDa) is a ubiquitous high‐affinity cholesterol‐ and drug‐binding protein located in the outer mitochondrial membrane. 3,17,19‐androsten‐5‐triol (19‐Atriol) is a ligand binding at the cholesterol recognition amino acid consensus domain of TSPO. Our previous study suggested that TSPO deficiency ameliorated methionine‐choline‐deficient induced non‐alcoholic steatohepatitis (NASH) through downregulation of bile acid synthesis (Li et al., iScience 24: 102457, 2021). We examined the role of cholesterol binding to TSPO in NASH using the 19‐Atriol on high‐fat‐fructose‐cholesterol diet (HFFC)‐induced NASH in rats. After HFFC‐feeding for 13weeks, the rats were subcutaneously administered daily 19‐Atriol (20mg/kg) for 2 weeks (weeks 13‐15). The ratio of liver/body weight and serum triacylglycerol content were significantly reduced in 19‐Atriol‐ compared to vehicle‐treated rats. Immunoblot analyses indicated that liver inflammation (TNFα) and fibrosis markers (COL1A1, ACTA2) were reduced following 19‐Atriol treatment. Farnesoid X receptor (FXR), a bile acid receptor, was upregulated in response to 19‐Atriol treatment in HFFC‐fed rats. In search of the mechanism mediating the effects of 19‐Atriol we observed an increase in autophagy flux and the levels of phosphorylated transcription factor EB (TFEB), a major regulator of autophagy and lysosomal biogenesis. Taken together these results suggest that blocking cholesterol binding to TSPO by 19‐Atriol leads to the attenuation of NASH by increasing FXR expression levels and activating of autophagic degradation as was the case in TSPO‐deficient rats.

Autophagy in muscle regeneration: potential therapies for myopathies.

Autophagy classically functions as a physiological process to degrade cytoplasmic components, protein aggregates, and/or organelles, as a mechanism for nutrient breakdown, and as a regulator of cellular architecture. Its biological functions include metabolic stress adaptation, stem cell differentiation, immunomodulation and diseases regulation, and so on. Current researches have proved that autophagy dysfunction may contribute to the pathogenesis of some myopathies through impairment of myofibres regeneration. Studies of autophagy inhibition also indicate the importance of autophagy in muscle regeneration, while activation of autophagy can restore muscle function in some myopathies. In this review, we aim to report the mechanisms of action of autophagy on muscle regeneration to provide relevant references for the treatment of regenerating defective myopathies by regulating autophagy. Results have shown that one key mechanism of autophagy regulating the muscle regeneration is to affect the differentiation fate of muscle stem cells (MuSCs), including quiescence maintenance, activation and differentiation. The roles of autophagy (organelle/protein degradation, energy facilitation, and/or other) vary at different myogenic stages of the repair process. When the muscle is in homeostasis, basal autophagy can maintain the quiescence state and stemness of MuSCs by renewing organelle and protein. After injury, the increased autophagy flux contributes to meet biological energy demand of MuSCs during activation and proliferation. By mitochondrial remodelling, autophagy during differentiation can promote the metabolic transformation and balance mitochondrial‐mediated apoptosis signals in myoblasts. Autophagy in mature myofibres is also essential for the degradation of necrotic myofibres, and may affect the dynamics of MuSCs by affecting the secretion spectrum of myofibres or the recruitment of supporting cells. Except for myogenic cells, autophagy also plays an important role in regulating the function of non‐myogenic cells in the muscle microenvironment, which is also essential for successful muscle recovery. Autophagy can regulate the immune microenvironment during muscle regeneration through the recruitment and polarization of macrophages, while autophagy in endothelial cells can regulate muscle regeneration in an angiogenic or angiogenesis‐independent manner. Drug or nutrition targeted autophagy has been preliminarily proved to restore muscle function in myopathies by promoting muscle regeneration, and further understanding the role and mechanism of autophagy in various cell types during muscle regeneration will enable more effective combinatorial therapeutic strategies.

Super-resolution analyzing spatial organization of lysosomes with an organic fluorescent probe.

Lysosomes are multifunctional organelles involved in macromolecule degradation, nutrient sensing and autophagy. Live imaging has revealed lysosome subpopulations with dynamics and characteristic cellular localization. An as-yet unanswered question is whether lysosomes are spatially organized to coordinate and integrate their functions. Combined with super-resolution microscopy, we designed a small organic fluorescent probe, TPAE, that targeted lysosomes with a large Stokes shift. When we analyzed the spatial organization of lysosomes against mitochondria in different cell lines with this probe, we discovered different distance distribution patterns between lysosomes and mitochondria during increased autophagy flux. By using SLC25A46 mutation fibroblasts derived from patients containing highly fused mitochondria with low oxidative phosphorylation, we concluded that unhealthy mitochondria redistributed the subcellular localization of lysosomes, which implies a strong connection between mitochondria and lysosomes.

Exercise and Training Regulation of Autophagy Markers in Human and Rat Skeletal Muscle.

Autophagy is a key intracellular mechanism by which cells degrade old or dysfunctional proteins and organelles. In skeletal muscle, evidence suggests that exercise increases autophagosome content and autophagy flux. However, the exercise-induced response seems to differ between rodents and humans, and little is known about how different exercise prescription parameters may affect these results. The present study utilised skeletal muscle samples obtained from four different experimental studies using rats and humans. Here, we show that, following exercise, in the soleus muscle of Wistar rats, there is an increase in LC3B-I protein levels immediately after exercise (+109%), and a subsequent increase in LC3B-II protein levels 3 h into the recovery (+97%), despite no change in Map1lc3b mRNA levels. Conversely, in human skeletal muscle, there is an immediate exercise-induced decrease in LC3B-II protein levels (−24%), independent of whether exercise is performed below or above the maximal lactate steady state, which returns to baseline 3.5 h following recovery, while no change in LC3B-I protein levels or MAP1LC3B mRNA levels is observed. SQSTM1/p62 protein and mRNA levels did not change in either rats or humans following exercise. By employing an ex vivo autophagy flux assay previously used in rodents we demonstrate that the exercise-induced decrease in LC3B-II protein levels in humans does not reflect a decreased autophagy flux. Instead, effect size analyses suggest a modest-to-large increase in autophagy flux following exercise that lasts up to 24 h. Our findings suggest that exercise-induced changes in autophagosome content markers differ between rodents and humans, and that exercise-induced decreases in LC3B-II protein levels do not reflect autophagy flux level.

DRAM-4 and DRAM-5 are compensatory regulators of autophagy and cell survival in nutrient-deprived conditions.

Macroautophagy is a membrane‐trafficking process that delivers cytoplasmic material to lysosomes for degradation. The process preserves cellular integrity by removing damaged cellular constituents and can promote cell survival by providing substrates for energy production during hiatuses of nutrient availability. The process is also highly responsive to other forms of cellular stress. For example, DNA damage can induce autophagy and this involves up‐regulation of the Damage‐Regulated Autophagy Modulator‐1 (DRAM‐1) by the tumor suppressor p53. DRAM‐1 belongs to an evolutionarily conserved protein family, which has five members in humans and we describe here the initial characterization of two members of this family, which we term DRAM‐4 and DRAM‐5 for DRAM‐Related/Associated Member 4/5. We show that the genes encoding these proteins are not regulated by p53, but instead are induced by nutrient deprivation. Similar to other DRAM family proteins, however, DRAM‐4 principally localizes to endosomes and DRAM‐5 to the plasma membrane and both modulate autophagy flux when over‐expressed. Deletion of DRAM‐4 using CRISPR/Cas‐9 also increased autophagy flux, but we found that DRAM‐4 and DRAM‐5 undergo compensatory regulation, such that deletion of DRAM‐4 does not affect autophagy flux in the absence of DRAM‐5. Similarly, deletion of DRAM‐4 also promotes cell survival following growth of cells in the absence of amino acids, serum, or glucose, but this effect is also impacted by the absence of DRAM‐5. In summary, DRAM‐4 and DRAM‐5 are nutrient‐responsive members of the DRAM family that exhibit interconnected roles in the regulation of autophagy and cell survival under nutrient‐deprived conditions.

Lipophagy-Related Protein Perilipin-3 and Resistance of Prostate Cancer to Radiation Therapy

Radiation therapy is a principal treatment modality for localized and locally advanced prostate cancer (PCa). Metabolic alterations, including lipid metabolism, may reduce treatment efficacy, resulting in tumor relapse and poor therapeutic outcome. In the current study, we investigated the role of the lipophagy-related protein perilipin-3 (PLIN3) and the lysosomal acid lipase (LAL) in PCa response to radiation therapy. We explored the in vitro and xenograft (in NOD SCID and R2G2 mice) response to radiation of either PLIN3-depleted or LAL-depleted hormone-refractory (DU145, PC3) and hormone-responsive (22Rv1) PCa cell lines. Moreover, we evaluated the clinical role of PLIN3 and LAL protein expression in a series of PCa tissue specimens from patients treated with radical radiation therapy. In vitro and in vivo experiments showed reduced proliferation and strong radiosensitization of all studied PCa cell lines upon PLIN3 depletion. In vivo experiments demonstrated the significantly augmented radiation therapy efficacy upon PLIN3 depletion, resulting in extensive tissue necrosis. Overexpression of PLIN3 in tissue specimens was correlated with an increased MIB1 proliferation index, increased autophagy flux, reduced response to radiation therapy, and poor prognosis. The effect of LAL depletion on radiation therapy was of lesser importance. Assessment of PLIN3 expression may identify subgroups of patients with PCa who are less responsive to radiation therapy and at high risk of relapse after irradiation. Whether radiation therapy efficacy may be enhanced by concurrent autophagy or PLIN3 inhibition in this subgroup of patients demands clinical evaluation.

Autophagy inhibits cancer stemness in triple-negative breast cancer via miR-181a-mediated regulation of ATG5 and/or ATG2B.

Autophagy has a dual role in the maintenance of cancer stem cells (CSCs), but the precise relationship between autophagy and cancer stemness requires further investigation. In this study, it was found that luminal and triple‐negative breast cancers require distinct therapeutic approaches because of their different amounts of autophagy flux. We identified that autophagy flux was inhibited in triple‐negative breast cancer (TNBC) CSCs. Moreover, miRNA‐181a (miR‐181a) expression is upregulated in both TNBC CSCs and patient tissues. Autophagy‐related 5 (ATG5) and autophagy‐related 2B (ATG2B) participate in the early formation of autophagosomes and were revealed as targets of miR‐181a. Inhibition of miR‐181a expression led to attenuation of TNBC stemness and an increase in autophagy flux. Furthermore, treatment with curcumin led to attenuation of cancer stemness in TNBC CSCs; the expression of ATG5 and ATG2B was enhanced and there was an increase of autophagy flux. These results indicated that ATG5 and ATG2B are involved in the suppression of cancer stemness in TNBC. In summary, autophagy inhibits cancer stemness through the miR‐181a‐regulated mechanism in TNBC. Promoting tumor‐suppressive autophagy using curcumin may be a potential method for the treatment of TNBC.

NUMB facilitates autophagy initiation through targeting SCFβ-TrCP2 complex.

Given the critical role of SCF E3 ligases in autophagy by modulating the protein stability of various autophagic components, the activity of SCF should be tightly controlled to maintain the autophagic flux. We here showed that Numb, a multifunctional adaptor protein, increased the protein abundance of DEPTOR, which is an inhibitor of mTORC1, leading to increased autophagy flux. In vitro ubiquitination assay demonstrated that Numb inhibited SCFβ-TrCP2 mediated ubiquitination of DEPTOR. Mechanistically, Numb interrupted the interaction between β-TrCP2 and SKP1 by directly binding with SKP1. In the presence of wild type β-TrCP2, Numb overexpression inhibited DEPTOR degradation. Whereas, in the presence of the mutant β-TrCP2 which lacks the F-box domain, Numb overexpression did not affect the protein abundance of DEPTOR. In mouse model of renal fibrosis induced by unilateral ureteral obstruction, the expression of Numb was significantly increased. Consistently, the upregulation of Numb was observed in renal fibrotic lesions of chronic kidney disease patients. Specifically depleting Numb in proximal renal tubules decreased the protein abundance of DEPTOR, attenuated autophagy in fibrotic lesions and protected the kidney from development of renal fibrosis in vivo. Taken together, both in vitro and in vivo data indicated that Numb functions as a novel regulator to fine tuning the activity of SCFβ-TrCP2 in modulating autophagy.

The Novel Inducer of Innate Immunity HO53 Stimulates Autophagy in Human Airway Epithelial Cells

Aroylated phenylenediamines (APDs) are novel modulators of innate immunity with respect to enhancing the expression of antimicrobial peptides and maintaining epithelial barrier integrity. Here, we present a new study on induction of autophagy in human lung epithelial cells by the APD HO53. Interestingly, HO53 affected autophagy in a dose-dependent manner, demonstrated by increased microtubule-associated proteins 1A/1B light-chain 3B (LC3B) processing in mature polarized bronchial epithelial cells. The quantification of LC3B puncta showed increased autophagy flux and formation of autophagosomes visualized by transmission electron microscopy. The phenotypic changes indicated that autophagy induction was associated with activation of 5′ adenosine monophosphate-activated protein kinase (AMPK), nuclear translocation of transcription factor EB (TFEB), and changes in expression of autophagy-related genes. The kinetics of the explored signaling pathways indicated on activation of AMPK followed by the nuclear translocation of TFEB. Moreover, our data suggest that HO53 modulates epigenetic changes related to induction of autophagy manifested by transcriptional regulation of histone-modifying enzymes. These changes were reflected by decreased ubiquitination of histone 2B at the lysine 120 residue that is associated with autophagy induction. Taken together, HO53 modulates autophagy, a part of the host defense system, through a complex mechanism involving several pathways and epigenetic events.

Abstract 14447: Increased Autophagy Flux May Underline Cardiomyocyte Remodeling in Atrial Fibrillation

Atrial fibrillation (AF), the most common progressive tachyarrhythmia, results in structural remodeling that impairs electrical activation of the atria. Studies have shown that mitochondrial dysfunction underlies cardiomyocyte remodeling. Here, we report a 50% reduction in total mitochondrial DNA in the atria tissue of RAP dogs as compared to control dogs, as assessed by qPCR the levels of mitochondrial DNA including mt-NT-1, mt-NT-4, mt-NT 5, mt-NT 6. This reduction in mitochondrial DNA suggests reduced mitochondria number in RAP dogs. Autophagic flux (LC3 II protein levels) was markedly increased in the atria tissue of the AF dog model (vs control dog).Furthermore, we examined autophagy flux in isolated cardiomyocytes. Results showed that LC3II was remarkably activated when subject to electrical stimulation for 6 hours under 3 Hertz condition, suggesting the direct effect of electrical stimulation on increasing autophagy. There was no change in the mRNA levels of autophagosome genes including Belin 1, ATG8, LC3, Lamp1 in the RAP dog tissue as compared to control, however, ATG7 increased by 2 fold and GNG2 increased by 5 fold. In conclusion, our study suggests that electrical stimulation-induced autophagy, and reduced mitochondria number and function, may underlie cardiomyocyte remodeling in AF. Targeting the autophagy pathway may present new therapeutic targets for AF. Ongoing AAV-mediated knockdown experiments in isolated cardiomyocytes will further elucidate the mechanism of electrical stimulation-induced autophagy.

Knockdown of PEX16 Induces Autophagic Degradation of Peroxisomes.

Peroxisome abundance is regulated by homeostasis between the peroxisomal biogenesis and degradation processes. Peroxin 16 (PEX16) is a peroxisomal protein involved in trafficking membrane proteins for de novo peroxisome biogenesis. The present study demonstrates that PEX16 also modulates peroxisome abundance through pexophagic degradation. PEX16 knockdown in human retinal pigment epithelial-1 cells decreased peroxisome abundance and function, represented by reductions in the expression of peroxisome membrane protein ABCD3 and the levels of cholesterol and plasmalogens, respectively. The activation of pexophagy under PEX16 knockdown was shown by (i) abrogated peroxisome loss under PEX16 knockdown in autophagy-deficient ATG5 knockout cell lines, and (ii) increased autophagy flux and co-localization of p62—an autophagy adaptor protein—with ABCD3 in the presence of the autophagy inhibitor chloroquine. However, the levels of cholesterol and plasmalogens did not recover despite the restoration of peroxisome abundance following chloroquine treatment. Thus, PEX16 is indispensable for maintaining peroxisome homeostasis by regulating not only the commonly known biogenesis pathway but also the autophagic degradation of peroxisomes.

Glucose starvation induces autophagy via ULK1-mediated activation of PIKfyve in an AMPK-dependent manner

Autophagy is an essential catabolic process induced to provide cellular energy sources in response to nutrient limitation through the activation of kinases, like AMP-activated protein kinase (AMPK) and ULK1. Although glucose starvation induces autophagy, the exact mechanism underlying this signaling has yet to be elucidated. Here, we reveal a role for ULK1 in non-canonical autophagy signaling using diverse cell lines. ULK1 activated by AMPK during glucose starvation phosphorylates the lipid kinase PIKfyve on S1548, thereby increasing its activity and the synthesis of the phospholipid PI(5)P without changing the levels of PI(3,5)P2. ULK1-mediated activation of PIKfyve enhances the formation of PI(5)P-containing autophagosomes upon glucose starvation, resulting in an increase in autophagy flux. Phospho-mimic PIKfyve S1548D drives autophagy upregulation and lowers autophagy substrate levels. Our study has identified how ULK1 upregulates autophagy upon glucose starvation and induces the formation of PI(5)P-containing autophagosomes by activating PIKfyve.