Autophagosome-like Structures Research Articles

Regulatory mechanism of perinatal nonylphenol exposure on cardiac mitochondrial autophagy and the PINK1/Parkin signaling pathway in male offspring rats

ObjectiveThis study investigated whether perinatal exposure to nonylphenol (NP) induces mitochondrial autophagy (i.e., mitophagy) damage in neonatal rat cardiomyocytes (NRCMs) and whether the PINK1/Parkin signaling pathway is involved in NP-induced primary cardiomyocyte injury. Methods and resultsIn vivo: Perinatal NP exposure increased apoptosis and mitochondrial damage in NRCMs. Mitochondrial swelling and autophagosome-like structures with multiple concentric membranes were observed in the 100 mg/kg NP group, with an increase in the number of autophagosomes. Disorganized fiber arrangement and elevated serum myocardial enzyme levels were observed with increasing NP dosage. Additionally, NP exposure led to increased MDA levels and decreased SOD activity and ATP levels in myocardial tissue. The mRNA expression levels of autophagy-related genes, including Beclin-1, p62, and LC3B, as well as the expression of mitochondrial autophagy–related proteins (PINK1, p-Parkin, Parkin, Beclin-1, p62, LC3-I, LC3-II, and LC3-II/I) and apoptosis-related proteins (Bax and caspase-3), increased, whereas the expression levels of the mitochondrial membrane protein TOMM20 and the anti-apoptotic protein Bcl-2 decreased. In vitro: NP increased ROS levels, LDH release, and decreased ATP levels in NRCMs. CsA treatment significantly inhibited the expression of autophagy-related proteins (Beclin-1, LC3-II/I, and p62) and apoptosis-related proteins (caspase-3 and Bax), increased the expression levels of TOMM20 and Bcl-2 proteins, increased cellular ATP levels, and inhibited LDH release. The inhibition of the PINK1/Parkin signaling pathway suppressed the expression of mitochondrial autophagy–related proteins (PINK1, p-Parkin, Parkin, Beclin-1, LC3-II/I, and p62) and apoptosis-related proteins (caspase-3 and Bax), increased TOMM20 and Bcl-2 protein expression, increased ATP levels, and decreased LDH levels in NRCMs. ConclusionsThis study is novel in reporting that perinatal NP exposure induced myocardial injury in male neonatal rats, thereby inducing mitophagy. The PINK1/Parkin signaling pathway was involved in this injury by regulating mitophagy.

Circadian clock protein BMAL1 broadly influences autophagy and endolysosomal function in astrocytes

An emerging role for the circadian clock in autophagy and lysosome function has opened new avenues for exploration in the field of neurodegeneration. The daily rhythms of circadian clock proteins may coordinate gene expression programs involved not only in daily rhythms but in many cellular processes. In the brain, astrocytes are critical for sensing and responding to extracellular cues to support neurons. The core clock protein BMAL1 serves as the primary positive circadian transcriptional regulator and its depletion in astrocytes not only disrupts circadian function but also leads to a unique cell-autonomous activation phenotype. We report here that astrocyte-specific deletion of Bmal1 influences endolysosome function, autophagy, and protein degradation dynamics. In vitro, Bmal1-deficient astrocytes exhibit increased endocytosis, lysosome-dependent protein cleavage, and accumulation of LAMP1- and RAB7-positive organelles. In vivo, astrocyte-specific Bmal1 knockout (aKO) brains show accumulation of autophagosome-like structures within astrocytes by electron microscopy. Transcriptional analysis of isolated astrocytes from young and aged Bmal1 aKO mice indicates broad dysregulation of pathways involved in lysosome function which occur independently of TFEB activation. Since a clear link has been established between neurodegeneration and endolysosome dysfunction over the course of aging, this work implicates BMAL1 as a key regulator of these crucial astrocyte functions in health and disease.

Autophagic and phytochemical aspects of color changes in white petals of snapdragon flower during development and senescence.

Color change in petals is a clever strategy to attract more pollinators and one of the attractive features of edible flowers for consumers. Several physiological, phytochemical, and ultrastructural factors are involved in this process. However, this phenomenon is well underexplored in white petals. In this study, we investigated the color changes of the white petals of the snapdragon (Antirrhinum majus 'Legend White') flower from different aspects during development and senescence. In the ultrastructural analysis, both epidermal and mesophyll cells were examined. During flower development, plastid transition and autophagy processes led to the fading of the green color of young petals and the reduction of starch content, chlorophyll, and carotenoids. The piecemeal chlorophagy was observed in the degradation of starch granules. Leucoplasts were converted into autophagosome-like structures and then disappeared. The presence of these structures was evidence of the transformation of the plastid to the vacuole. As the green color faded, phytochemical compounds were synthesized. With partial flower opening and progression of senescence, pH and phenolic compounds were responsible for color changes. The highest amount of phenolic compound was observed after the flower opening stages. However, Phenolic colored compounds or total anthocyanins became colorless under the influence of low pH. The decrease in starch content caused an increase in the lightness parameter, and the petal color changed to pale yellow.

Host autophagy limits Toxoplasma gondii proliferation in the absence of IFN-γ by affecting the hijack of Rab11A-positive vesicles.

Autophagy has been recognized as a bona fide immunological process. Evidence has shown that this process in IFN-γ stimulated cells controls Toxoplasma gondii proliferation or eliminates its infection. However, little is known about the effect of T. gondii infection on the host cell autophagy in the absence of IFN-γ. Multiple autophagy detection methods and CRISPR/CAS9 technology were used to study T. gondii-induced autophagy in HeLa and several other mammalian cell lines. Here, we report increased LC3 II, autophagosome-like membrane structures, enhanced autophagic flux, and decreased lysosomes in a range of mammalian cell lines without IFN-γ treatment after T. gondii infection. Specifically, disruption of host atg5 (a necessary gene for autophagy) in HeLa cells promoted the intracellular replication of T. gondii, with the transcript level of rab11a increased, compared with that in wild-type cells. Further, after T. gondii infection, the abundance of Rab11A remained stable in wild-type HeLa cells but decreased in atg5 -/- mutant. Disruption of rab11a in the HeLa cells compromised the proliferation of T. gondii, and increased the transcription of gra2 in the parasite. Compared to the T. gondii wild-type RH∆ku80 strain, the ∆gra2 mutant induces enhanced host autophagy in HeLa cells, and results in slower replication of the parasite. Collectively, these results indicate that host cell autophagy can limit T. gondii proliferation in an IFN-γ independent manner, possibly by affecting the hijack of host Rab11A-positive vesicles by the parasite which involved TgGRA2. The findings provide novel insights into T. gondii infection in host cells and toxoplasmosis research.

Unprocessed wheat γ-gliadin reduces gluten accumulation associated with the endoplasmic reticulum stress and elevated cell death.

Summary Along with increasing demands for high yield, elite processing quality and improved nutrient value in wheat, concerns have emerged around the effects of gluten in wheat‐based foods on human health. However, knowledge of the mechanisms regulating gluten accumulation remains largely unexplored.Here we report the identification and characterization of a wheat low gluten protein 1 (lgp1) mutant that shows extremely low levels of gliadins and glutenins.The lgp1 mutation in a single γ‐gliadin gene causes defective signal peptide cleavage, resulting in the accumulation of an excessive amount of unprocessed γ‐gliadin and a reduced level of gluten, which alters the endoplasmic reticulum (ER) structure, forms the autophagosome‐like structures, leads to the delivery of seed storage proteins to the extracellular space and causes a reduction in starch biosynthesis. Physiologically, these effects trigger ER stress and cell death.This study unravels a unique mechanism that unprocessed γ‐gliadin reduces gluten accumulation associated with ER stress and elevated cell death in wheat. Moreover, the reduced gluten level in the lgp1 mutant makes it a good candidate for specific diets for patients with diabetes or kidney diease.

Investigation of the cell structure and organelles during autolytic PCD of Antirrhinum majus "Legend White" petals.

One of the classes of the plant developmental programmed cell death (PCD) is vacuolar cell death or autolysis. The results of the transmission electron microscope (TEM) studies indicated that this type of PCD occurs during the petal senescence of Antirrhinum majus "Legend White" flowers. The major hallmarks of the process related to the ultrastructure of the cells involved chloroplast degradation, vacuolation, chromatin condensation, cell wall swelling, degradation of Golgi apparatus, protoplasmic shrinkage, degradation of the endoplasmic reticulum, nuclear fragmentation, rupture of tonoplast, and plasma membrane. Macroautophagy and microautophagy processes were also clearly observed during vacuole formation. As in yeasts, in the present study, Golgi apparatus became autophagosome-like structures during degradation that had autophagy activity and then disappeared. Our results revealed a type of selective microautophagy, piecemeal microautophagy of the nucleus (PMN), in nuclear degradation during PCD of petals that has not previously been reported in plants. Moreover, vesicular structures, such as paramural and multilamellar bodies, were observed in some stages.

Combined exposure to fine particulate matter and high glucose aggravates endothelial damage by increasing inflammation and mitophagy: the involvement of vitamin D

BackgroundCardiovascular diseases (CVDs) are related to particulate matter (PM2.5) exposure. Researchers have not clearly determined whether hyperglycemia, a hallmark of diabetes, exacerbates PM2.5-induced endothelial damage. Thus, this study aimed to investigate the combined effects of PM2.5 and high glucose on endothelial damage.ResultsHere, we treated human umbilical vein endothelial cells (HUVECs) with 30 mM high glucose and 50 μg/mL PM (HG + PM) to simulate endothelial cells exposed to hyperglycemia and air pollution. First, we showed that HUVECs exposed to PM under high glucose conditions exhibited significant increases in cell damage and apoptosis compared with HUVECs exposed to PM or HG alone. In addition, PM significantly increased the production of reactive oxygen species (ROS) in HUVECs and mitochondria treated with HG and decreased the expression of superoxide dismutase 1 (SOD1), a free radical scavenging enzyme. The coexposure group exhibited significantly increased ROS production in cells and mitochondria, a lower mitochondrial membrane potential, and increased levels of the autophagy-related proteins p62, microtubule-associated protein 1 light chain 3β (LC3B), and mitophagy-related protein BCL2 interacting protein 3 (Bnip3). Moreover, autophagosome-like structures were observed in the HG + PM group using transmission electron microscopy. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were also increased through the JNK/p38 signaling pathway in the HG + PM group. As a ROS scavenger, vitamin D treatment effectively protected cells under HG and PM conditions by increasing cell viability, reducing mitochondrial ROS production, and suppressing the formation of mitophagy and inflammation. Furthermore, diabetes was induced in mice by administering streptozotocin (STZ). Mice were treated with PM by intratracheal injection. Vitamin D effectively alleviated oxidative stress, mitophagy, and inflammation in the aortas of mice treated with STZ and PM.ConclusionTaken together, simultaneous exposure to PM and high glucose exerts significant harmful effects on endothelial cells by inducing ROS production, mitophagy, and inflammation, while vitamin D reverses these effects.

Pulmonary inflammation and cellular responses following exposure to benzalkonium chloride: Potential impact of disrupted pulmonary surfactant homeostasis

Benzalkonium chloride (BKC) is a prototypical quaternary ammonium disinfectant. Previously, we suggested a no lethal dose level (0.005%) and an LD50 range (0.5–0.05%) of BKC following a single pharyngeal aspiration. Herein, we exposed BKC repeatedly by pharyngeal aspiration for 14 days (0.005 and 0.01%, female mice, total five times with interval of two days, 5 mice/group) and 28 days (0, 0.001, 0.005, and 0.01%, male and female mice, weekly, 16 mice/sex/group). Death following 14 days-repeated exposure did not occur. Meanwhile, chronic pathological lesions were observed in the lung tissues of mice exposed to BKC for 28 days. The total number of bronchial alveolar lavage cells increased, and pulmonary homeostasis of immunologic messenger molecules was disturbed. Following, we investigated BKC-induced cellular responses using human bronchial epithelial cells. The cytotoxicity increased rapidly with concentration. Lysosomal volume, NO production, and lipid peroxidation increased in BKC-treated cells, whereas intracellular ROS level decreased accompanying structural and functional damage of mitochondria. We also found that BKC affected the expression level of immune response, DNA damage, and amino acid biosynthesis-related molecules. More interestingly, lamellar body- and autophagosome-like structures were notably observed in cells exposed to BKC, and necrotic and apoptotic cell death were identified accompanying cell accumulation in the G2/M phase. Therefore, we suggest that repeated respiratory exposure of BKC causes pulmonary inflammation and lung tissue damage and that dead and damaged cells may contribute to the inflammatory response. In addition, the formation process of lamellar body-like structures may function as a key toxicity mechanism.

Clostridium difficile toxin B induces morphological changes consistent with autophagy in the human adenocarcinoma cell line (HT-29)

Purpose: Toxin B (TcdB) is a product of the bacteria Clostridium difficile, and can trigger apoptosis or necrosis. In the present manuscript, we proposed that TcdBVPI and TcdB8864 would cause morphological changes in cultured cells that are characteristic of autophagy, a constitutive cellular event closely linked with apoptosis or necrosis.
 Methods: We examined an in vitro model of cultured HT-29 cells to determine the occurrence of autophagy. The cells were incubated separately with TcdBVPI and TcdB8864 for 2 to 24 hours, and then examined using transmission electron microscopy.
 Results: In HT-29 cells, ultrastructural changes were observed following 8 hours of treatment with TcdBVPI and TcdB8864. Upon exposure to both toxins, complex changes occurred that affected the cellular framework, including the dilated regions of the granular endoplasmic reticulum, the Golgi apparatus, as well as electron-lucent and electron-dense autophagosome-like structures. Additionally, lipid droplets were present in the cytoplasm of HT-29 cells.
 Conclusion: Our findings indicate that TcdBVPI and TcdB8864 indeed induce ultrastructural changes in HT-29 cells. The presence of these autophagic structures in the cytoplasm of the HT-29 cells suggests that autophagy may be induced during treatment by both toxins. Therefore, this effect could represent an important protective mechanism for cell survival.

GFS9 Affects Piecemeal Autophagy of Plastids in Young Seedlings of Arabidopsis thaliana.

Chloroplasts, and plastids in general, contain abundant protein pools that can be major sources of carbon and nitrogen for recycling. We have previously shown that chloroplasts are partially and sequentially degraded by piecemeal autophagy via the Rubisco-containing body. This degradation occurs during plant development and in response to the environment; however, little is known about the fundamental underlying mechanisms. To discover the mechanisms of piecemeal autophagy of chloroplasts/plastids, we conducted a forward-genetics screen following ethyl-methanesulfonate mutagenesis of an Arabidopsis (Arabidopsis thaliana) transgenic line expressing chloroplast-targeted green fluorescent protein (CT-GFP). This screen allowed us to isolate a mutant, gfs9-5, which hyperaccumulated cytoplasmic bodies labeled with CT-GFP of up to 1.0 μm in diameter in the young seedlings. We termed these structures plastid bodies (PBs). The mutant was defective in a membrane-trafficking factor, green fluorescent seed 9 (GFS9), and PB accumulation in gfs9-5 was promoted by darkness and nutrient deficiency. Transmission electron microscopy indicated that gfs9-5 hyperaccumulated structures corresponding to autophagosomes and PBs. gfs9-5 hyperaccumulated membrane-bound endogenous ATG8 proteins, transgenic yellow fluorescent protein (YFP)-ATG8e proteins and autophagosome-like structures labeled with YFP-ATG8e. The YFP-ATG8e signal was associated with the surface of plastids and their protrusions in gfs9-5. Double mutants of gfs9 and autophagy-defective 5 did not accumulate PBs. In gfs9-5, the YFP-ATG8e proteins and PBs could be delivered to the vacuole and autophagic flux was increased. We discuss a possible connection between GFS9 and autophagy and propose a potential use of gfs9-5 as a new tool to study piecemeal plastid autophagy.

High-throughput quantitative analysis of axonal transport in cultured neurons from SOD1H46R ALS mice by using a microfluidic device

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective loss of motor neurons. We have previously shown that autophagosome-like vesicular structures are progressively accumulated in the spinal axons of an ALS mouse model, overexpressing human Cu/Zn superoxide dismutase (SOD1) mutant, prior to the onset of motor symptoms. This suggests that axonal transport perturbation can be an early sign of neuronal dysfunction. However, the exact causal relationship between axonal transport deficits and neurodegeneration is not fully understood. To clarify whether axonal transport of organelles even in neurons at early developmental stages was affected by overexpression of mutant SOD1, we conducted a microfluidic device-based high-throughput quantitative analysis of the axonal transport of acidic vesicles and mitochondria in primary cultured cortical neurons established from SOD1H46R transgenic mice. Compared to wild-type (WT), a significantly increased number of motile acidic vesicles, i.e., autophagosomes and/or late-endosomes, was observed in the axons of SOD1H46R neurons. By contrast, mitochondria moving along the axons were significantly decreased in SOD1H46R compared to WT. Since such phenotypes, where the axonal transport of these organelles is differently affected by mutant SOD1 expression, emerge before axonal degeneration, axonal transport deficits could dysregulate axon homeostasis, thereby ultimately accelerating neurodegeneration.

Protein homeostasis in LGMDR9 (LGMD2I) - The role of ubiquitin-proteasome and autophagy-lysosomal system.

Limb-girdle muscular dystrophy R9 (LGMDR9) is an autosomal recessive disorder caused by mutations in the fukutin-related protein gene (FKRP), encoding a glycosyltransferase involved in α-dystroglycan modification. Muscle atrophy, a significant feature of LGMDR9, occurs by a change in the normal balance between protein synthesis and protein degradation. The ubiquitin-proteasome system (UPS) and autophagy-lysosomal system play a key role in protein degradation in skeletal muscle cells, but their involvement in the pathology of LGMDR9 is still largely unknown. We have aimed at clarifying whether proteolysis through the UPS and the autophagy-lysosomal pathway is dysregulated in LGMDR9 patients. Vastus lateralis biopsies from 8 normal controls and 12 LGMDR9 patients harbouring the c.826C>A/c.826C>A FKRP genotype were assessed for protein markers related to UPS, the autophagy-lysosomal system and endoplasmic reticulum (ER) stress/unfolded protein response (UPR), followed by ultrastructural analysis by transmission electron microscopy (TEM). Protein levels of E3 ubiquitin ligases Atrogin-1 and MuRF1 showed a pattern similar to normal controls. Elevation of the autophagy markers Atg7, LC3B-II, decreased level of p62 as well as downregulation of the negative autophagy regulator mTORC1, indicated an activation of autophagy in LGMDR9. Mitophagy markers Bnip3 and Parkin were decreased. TEM analysis demonstrated accumulation of autophagosome-like structures in LGMDR9 muscle. There was also an increase in the expression of ER stress/UPR markers PDI, peIF2α and CHOP and a decrease in IRE1α. However, GRP94, Bip and Calnexin remained unchanged. Our findings indicate that autophagy and ER stress are induced in LGMDR9 muscle.

Phagophore Closure.

Phagophore closure is a critical step during macroautophagy. However, the proteins and mechanisms to regulate this step have been elusive for a long time. In 2017, Rab5 was affirmed to play a role in phagophore closure in yeast. Furthermore, in mammalian cells, ESCRT III was reported to have roles in phagophore closure and mitophagosome closure in vivo in 2018 and 2019, respectively. The role of ESCRT in phagophore closure was confirmed in yeast, both in vivo and in vitro, in 2019. Most importantly, the latter paper found that Atg17 recruited the ESCRT III subunit Snf7 to the phagophore to close it under the control of Rab5. To determine the closure characteristics of autophagosome-like membrane structures in ESCRT mutants, a traditional protease protection assay with immunoblotting was used, accompanied by new techniques that were developed, including immunofluorescence assays, autophagosome completion assays, and the optogenetic closure assay. This study delivered our current understanding of phagophore closure and provided more reference methods to detect membrane closure.

Aberrant autophagosome formation occurs upon small molecule inhibition of ULK1 kinase activity.

Autophagy is a crucial homeostatic mechanism that mediates the degradation of damaged or excess intracellular components. Such components are engulfed and sequestered into double membrane autophagosomes, which deliver their contents to lysosomes for degradation. Autophagy plays a role in numerous human disorders and its pharmacological targeting by small molecules offers therapeutic potential. The serine/threonine kinase ULK1 (and its homologue ULK2) is the most upstream component of the autophagic machinery and is required for autophagy initiation. Here, we use the most selective and potent published ULK1 inhibitors to gain insights into ULK1 kinase function during autophagy. Treatment with all inhibitors blocked autophagy but also resulted in the limited formation of initial autophagosome-like structures, which appeared abnormal in size and did not traffic to lysosomes. We found that upon ULK1 inhibition, phosphatidylinositol-3-phosphate-binding proteins are still recruited to forming autophagosomes, implying that ULK1 activity is not essential for VPS34 activation. We conclude that the kinase activity of ULK1 is important in regulating autophagosome maturation, by the phosphorylation of currently unidentified key substrates.

Involvement of autophagy in realgar quantum dots (RQDs) inhibition of human endometrial cancer JEC cells.

Realgar (As4S4) has been used in traditional Chinese medicines for treatment of malignancies. The poor solubility of As4S4 hampered its clinical applications. Realgar quantum dots (RQDs) were developed to overcome these problems. Previous studies revealed that the RQDs were effective against endometrial cancer JEC cells and hepatocarcinoma HepG2 cells via inducing apoptosis.Apoptosis and autophagy are important programmed cell death pathways leading to anticancer effects. This study further examined effects of RQDs on autophagy, focusing on the formation of the autophagosome in JEC cells. CCK8 assay was used to examine cell proliferation. Flow cytometry was used to analyze cell cycle. Transmission electron microscopy (TEM) was used to examine the autophagy, cells were transfected with pEGFP-C3-MAP1LC3B plasmid to examine effects of RQDs on autophagosome via confocal microscope. Autophagy-related proteins were examined by Western blot. RQDs exhibited cytotoxicity in JEC cells in a concentration- and time- dependent manner. RQDs induced G2 and S phase arrest in JEC cells. RQDs significantly induced autophagy, with the double-membrane and autophagosome-like structures by TEM. The diffused distribution of pEGFP-C3-MAP1LC3B green fluorescence were become the punctuate pattern fluorescence after treatment with RQDs in cells transfected with pEGFP-C3-MAP1LC3B plasmid RQDs increased the expression of autophagyregulatory proteins LC3 I/II, Beclin-1, p62 and Atg12 in a concentration-dependent manner, similar to autophagy induced by serum starvation, except for p62, as induction of p62 is a characteristic of arsenic compounds. Taken together, the present study clearly demonstrated that RQDs can induce autophagy in JEC cells as one of mechanisms of anticancer effects, and indicated that RQDs may be developed as an autophagy inducer.

Exocyst Subcomplex Functions in Autophagosome Biogenesis by Regulating Atg9 Trafficking

During autophagy, double-membrane vesicles called autophagosomes capture and degrade the intracellular cargo. The de novo formation of autophagosomes requires several vesicle transport and membrane fusion events which are not completely understood. We studied the involvement of exocyst, an octameric tethering complex, which has a primary function in tethering post-Golgi secretory vesicles to plasma membrane, in autophagy. Our findings indicate that not all subunits of exocyst are involved in selective and general autophagy. We show that in the absence of autophagy specific subunits, autophagy arrest is accompanied by accumulation of incomplete autophagosome-like structures. In these mutants, impaired Atg9 trafficking leads to decreased delivery of membrane to the site of autophagosome biogenesis thereby impeding the elongation and completion of the autophagosomes. The subunits of exocyst, which are dispensable for autophagic function, do not associate with the autophagy specific subcomplex of exocyst.

Lipopolysaccharide-Induced Autophagy Mediates Retinal Pigment Epithelium Cells Survival. Modulation by the Phospholipase D Pathway.

Inflammation and oxidative stress are common factors involved in the pathogenesis of retinal diseases, such as aged-related macular degeneration (AMD) and diabetic retinopathy (DR). Autophagy is a catabolic process essential to cell survival in response to stress. This process is highly active in retinal pigment epithelium (RPE) cells. Our previous findings demonstrated that lipopolysaccharide (LPS) induces an inflammatory response of RPE cells that implies classical phospholipases D (PLD1 and 2) activation, cyclooxygenase-2 (COX-2) expression, prostaglandin E2 (PGE2) production and reduced cell viability. In this work, we studied the autophagic process and its modulation by the PLD pathway in D407 and ARPE-19 RPE cells exposed to LPS. LPS (10 μg/ml or 25 μg/ml) exposure for 24 h increased light chain 3B-II (LC3B-II) content (an autophagy marker) and LC3B-positive punctate structures in both RPE cell lines studied. Next, the drug bafilomycin A1 (BAF, 50 nM) was used to block the autophagic flux. In cells pre-incubated with BAF, LC3B-II and sequestosome 1 (SQSTM1/p62) levels and autophagosome-like structures were increased by LPS, demonstrating that the inflammatory injury increases the autophagic process in RPE cells. To study the role of the PLD pathway, cells were pre-incubated for 1 h with selective PLD1 (VU0359595) or PLD2 (VU0285655-1) inhibitors prior to LPS addition. Under control condition, LC3B-positive punctate structures were increased in cells pre-incubated with PLD2 inhibitor while with PLD1 inhibitor were increased in cells exposed to LPS. MTT reduction assays showed that early autophagy inhibitors, 3-methyladenin (3-MA) or LY294002, enhanced the loss in cell viability induced by LPS exposure for 48 h. On the contrary, the inhibition of PLD1 and PLD2 prevented the loss in cell viability induced by LPS. In conclusion, our results show that even though LPS treatment promotes an inflammatory response in RPE cells, it also triggers the activation of the autophagic process which in turn may serve as a protective mechanism for the cells. In addition, we demonstrate that the PLD pathway modulates the autophagic process in RPE cells. Our findings contribute to the knowledge of the molecular basis of retinal inflammatory and degenerative diseases and open new avenues for potential therapeutic exploration.

Niclosamide Triggers Non-Canonical LC3 Lipidation.

Autophagy is a highly- evolutionarily-conserved catabolic pathway activated by various cellular stresses. Recently, non-canonical autophagy (NCA), which does not require all of the ATG proteins to form autophagosome or autophagosome-like structures, has been found in various conditions. Moreover, mounting evidence has indicated that non-canonical LC3 lipidation (NCLL) may reflect NCA. We and others have reported that niclosamide (Nic), an anti-helminthic drug approved by the Food and Drug Administration, could induce canonical autophagy via a feedback downregulation of mTOR complex 1. In this study, we found that Nic could also induce NCLL, which is independent of the ULK1 complex and Beclin 1 complex, but dependent on ubiquitin-like conjugation systems. Although bafilomycin A1 and concanamycin A, two known V-ATPase inhibitors, significantly inhibited Nic-induced NCLL, Nic-induced NCLL was demonstrated to be independent of V-ATPase. In addition, the Golgi complex and vimentin were involved in Nic-induced NCLL, which might be a platform or membrane source for Nic-induced LC3-positive structures. These results would be helpful to broaden our understanding of the working mechanisms of Nic and evaluate its pharmacological activities in diseases.

AMPK-dependent autophagy upregulation serves as a survival mechanism in response to Tumor Treating Fields (TTFields)

Tumor Treating Fields (TTFields), an approved treatment modality for glioblastoma, are delivered via non-invasive application of low-intensity, intermediate-frequency, alternating electric fields. TTFields application leads to abnormal mitosis, aneuploidy, and increased cell granularity, which are often associated with enhancement of autophagy. In this work, we evaluated whether TTFields effected the regulation of autophagy in glioma cells. We found that autophagy is upregulated in glioma cells treated with TTFields as demonstrated by immunoblot analysis of the lipidated microtubule-associated protein light chain 3 (LC3-II). Fluorescence and transmission electron microscopy demonstrated the presence of LC3 puncta and typical autophagosome-like structures in TTFields-treated cells. Utilizing time-lapse microscopy, we found that the significant increase in the formation of LC3 puncta was specific to cells that divided during TTFields application. Evaluation of selected cell stress parameters revealed an increase in the expression of the endoplasmic reticulum (ER) stress marker GRP78 and decreased intracellular ATP levels, both of which are indicative of increased proteotoxic stress. Pathway analysis demonstrated that TTFields-induced upregulation of autophagy is dependent on AMP-activated protein kinase (AMPK) activation. Depletion of AMPK or autophagy-related protein 7 (ATG7) inhibited the upregulation of autophagy in response to TTFields, as well as sensitized cells to the treatment, suggesting that cancer cells utilize autophagy as a resistance mechanism to TTFields. Combining TTFields with the autophagy inhibitor chloroquine (CQ) resulted in a significant dose-dependent reduction in cell growth compared with either TTFields or CQ alone. These results suggest that dividing cells upregulate autophagy in response to aneuploidy and ER stress induced by TTFields, and that AMPK serves as a key regulator of this process.

NBR1 is involved in selective pexophagy in filamentous ascomycetes and can be functionally replaced by a tagged version of its human homolog

ABSTRACTMacroautophagy/autophagy is a conserved degradation process in eukaryotic cells involving the sequestration of proteins and organelles within double-membrane vesicles termed autophagosomes. In filamentous fungi, its main purposes are the regulation of starvation adaptation and developmental processes. In contrast to nonselective bulk autophagy, selective autophagy is characterized by cargo receptors, which bind specific cargos such as superfluous organelles, damaged or harmful proteins, or microbes, and target them for autophagic degradation. Herein, using the core autophagy protein ATG8 as bait, GFP-Trap analysis followed by liquid chromatography mass spectrometry (LC/MS) identified a putative homolog of the human autophagy cargo receptor NBR1 (NBR1, autophagy cargo receptor) in the filamentous ascomycete Sordaria macrospora (Sm). Fluorescence microscopy revealed that SmNBR1 colocalizes with SmATG8 at autophagosome-like structures and in the lumen of vacuoles. Delivery of SmNBR1 to the vacuoles requires SmATG8. Both proteins interact in an LC3 interacting region (LIR)-dependent manner. Deletion of Smnbr1 leads to impaired vegetative growth under starvation conditions and reduced sexual spore production under non-starvation conditions. The human NBR1 homolog partially rescues the phenotypic defects of the fungal Smnbr1 deletion mutant. The Smnbr1 mutant can neither use fatty acids as a sole carbon source nor form fruiting bodies under oxidative stress conditions. Fluorescence microscopy revealed that degradation of a peroxisomal reporter protein is impaired in the Smnbr1 deletion mutant. Thus, SmNBR1 is a cargo receptor for pexophagy in filamentous ascomycetes.