Autophagic Substrate P62 Research Articles

Dysregulation of mitochondria, apoptosis and mitophagy in Leber’s hereditary optic neuropathy with MT-ND1 3635G>A mutation

Leber’s hereditary optic neuropathy (LHON) is a maternal inherited disorder, primarily due to mitochondrial DNA (mtDNA) mutations. This investigation aimed to assess the pathogenicity of m.3635G>A alteration known to confer susceptibility to LHON. The disruption of electrostatic interactions among S110 of the MT-ND1 and the side chain of E4, along with the carbonyl backbone of M1 in the NDUFA1, was observed in complex I of cybrids with m.3635G>A. This disturbance affected the complex I assembly activity by changing the mitochondrial respiratory chain composition and function. In addition, the affected cybrids exhibited notable deficiencies in complex I activities, including impaired mitochondrial respiration and depolarization of its membrane potential. Apoptosis was also stimulated in the mutant group, as witnessed by the secretion of cytochrome c and activation of PARP, caspase 3, 7, and 9 compared to the control. Furthermore, the mutant group exhibited decreased levels of autophagy protein light chain 3, accumulation of autophagic substrate P62, and impaired PINK1/Parkin-dependent mitophagy. Overall, the current study has confirmed the crucial involvement of the alteration of the m.3635G>A gene in the development of LHON. These findings contribute to a deeper comprehension of the pathophysiological mechanisms underlying LHON, providing a fundamental basis for further research.

CREBH promotes autophagy to ameliorate NASH by regulating Coro1a

Dysfunctional autophagy aggravates oxidative stress and inflammation in hepatocytes and accelerates the progression of nonalcoholic steatohepatitis (NASH). Here, we demonstrated that cAMP-responsive element-binding protein H (CREBH) is a transcriptional regulator of hepatic autophagy in response to diet-induced NASH. The results showed that the upregulation of CREBH in lipid-overloaded hepatocytes improves cell damage, dysfunction of autophagic flux and associated abnormal accumulation of the autophagosome marker LC3-II and autophagic substrate p62. CREBH deficiency aggravated the dysfunctional autophagy and liver injury and even caused NASH-associated liver fibrosis. In addition, the changing trend of autolysosomes and lysosome membrane-associated protein (LAMP1) was consistent with the expression level of CREBH. This result indicated that CREBH might promote autophagic degradation by restoring the formation of autolysosomes, thereby improving the blocked autophagic flux. Moreover, we observed that CREBH inhibited the expression of Coronin 1a (Coro1a), an autophagosome-lysosome fusion-related gene, through transcriptional regulation. The overexpression of Coro1a in LO2 liver cells inhibited autophagic flux and elevated inflammatory cytokine levels upon palmitic acid (PA) stimulation. Overall, our findings provide new insights into the regulatory role of CREBH in the progression of NASH and reveal that Coro1a is a novel target gene of CREBH based on the autophagy pathway.

Exogenous galanin alleviates hepatic steatosis by promoting autophagy via the AMPK-mTOR pathway

Defective autophagy-induced intracellular lipid degradation is causally associated with non-alcoholic fatty liver disease (NAFLD) development. Therefore, agents that can restore autophagy may have potential clinical application prospects on this public health issue. Galanin (GAL) is a pleiotropic peptide that regulates autophagy and is a potential drug for the treatment of NAFLD. In this study, we used an MCD-induced NAFLD mouse model in vivo and an FFA-induced HepG2 hepatocyte model in vitro to evaluate the anti-NAFLD effect of GAL. Exogenous GAL supplementation significantly attenuated lipid droplet accumulation and suppressed hepatocyte TG levels in mice and cell models. Mechanistically, Galanin-mediated reduction of lipid accumulation was positively correlated with upregulated p-AMPK, as evidenced by upregulated protein expressions of fatty acid oxidation-related gene markers (PPAR-α and CPT1A), upregulated expressions of the autophagy-related marker (LC3B), and downregulated autophagic substrate p62 levels. In FFA-treated HepG2 cells, activation of fatty acid oxidation and autophagy-related proteins by galanin was reversed by autophagy inhibitors, chloroquine, and the AMPK inhibitor. Galanin ameliorates hepatic fat accumulation by inducing autophagy and fatty acid oxidation via the AMPK/mTOR pathway.

De novo lipogenesis fuels adipocyte autophagosome and lysosome membrane dynamics

Adipocytes robustly synthesize fatty acids (FA) from carbohydrate through the de novo lipogenesis (DNL) pathway, yet surprisingly DNL contributes little to their abundant triglyceride stored in lipid droplets. This conundrum raises the hypothesis that adipocyte DNL instead enables membrane expansions to occur in processes like autophagy, which requires an abundant supply of phospholipids. We report here that adipocyte Fasn deficiency in vitro and in vivo markedly impairs autophagy, evident by autophagosome accumulation and severely compromised degradation of the autophagic substrate p62. Our data indicate the impairment occurs at the level of autophagosome-lysosome fusion, and indeed, loss of Fasn decreases certain membrane phosphoinositides necessary for autophagosome and lysosome maturation and fusion. Autophagy dependence on FA produced by Fasn is not fully alleviated by exogenous FA in cultured adipocytes, and interestingly, imaging studies reveal that Fasn colocalizes with nascent autophagosomes. Together, our studies identify DNL as a critical source of FAs to fuel autophagosome and lysosome maturation and fusion in adipocytes.

4.8% sevoflurane induces activation of autophagy in human neuroblastoma SH-SY5Y cells by the AMPK/mTOR signaling pathway

Prolonged sevoflurane exposure leads to neurotoxicity. Autophagy plays an important role in promoting cell survival in different conditions. However, the role and mechanism of autophagy in sevoflurane-induced neurotoxicity were not fully elucidated. We attempted to indicate whether sevoflurane could activate the AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR)-mediated autophagy to attenuate anesthetics-induced neuronal injury in this study. Sevoflurane treatment significantly decreased the cell viability and induced apoptosis of SH-SY5Y cells. The expression level of Bcl-2 decreased, while that of Bax remarkably increased. Meanwhile, autophagy was activated by sevoflurane exposure as evidenced by increased expression levels of autophagy-related proteins (LC3-II and Atg5), decreased expression level of autophagic substrate P62, and increased autophagosomes and autolysosomes. Further autophagosomes and fewer autolysosomes were observed in the presence of Bafilomycin A1, an autolysosomes degradation inhibitor, suggesting that sevoflurane induced autophagic flux rather than inhibiting degradation of autophagy. Activation of autophagy by rapamycin partly reversed the sevoflurane-decreased cell viability. In contrast, inhibition of autophagy by 3-Methyladenine (3-MA) or Atg5-targeted small interfering RNA (siRNA) aggravated the sevoflurane-induced neurotoxicity. Further examination revealed that sevoflurane-induced autophagy was mediated by the AMPK/mTOR signaling pathway, with increased p-AMPK expression and decreased p-mTOR expression. Collectively, these results indicated that sevoflurane activates autophagy by regulating the AMPK/mTOR signaling pathway, which is protective against sevoflurane-induced damage in SH-SY5Y cells. Our results may assist clinicians to develop further promising therapeutic strategies for the neurotoxicity induced by inhaled anesthetics.

A mutation in desmin makes skeletal muscle less vulnerable to acute muscle damage after eccentric loading in rats.

Desminopathy is the most common intermediate filament disease in humans. The most frequent mutation causing desminopathy in patients is a R350P DES missense mutation. We have developed a rat model with an analogous mutation in R349P Des. To investigate the role of R349P Des in mechanical loading, we stimulated the sciatic nerve of wild‐type littermates (WT) (n = 6) and animals carrying the mutation (MUT) (n = 6) causing a lengthening contraction of the dorsi flexor muscles. MUT animals showed signs of ongoing regeneration at baseline as indicated by a higher number of central nuclei (genotype: P < .0001). While stimulation did not impact central nuclei, we found an increased number of IgG positive fibers (membrane damage indicator) after eccentric contractions with both genotypes (stimulation: P < .01). Interestingly, WT animals displayed a more pronounced increase in IgG positive fibers with stimulation compared to MUT (interaction: P < .05). In addition to altered histology, molecular signaling on the protein level differed between WT and MUT. The membrane repair protein dysferlin decreased with eccentric loading in WT but increased in MUT (interaction: P < .05). The autophagic substrate p62 was increased in both genotypes with loading (stimulation: P < .05) but tended to be more elevated in WT (interaction: P = .05). Caspase 3 levels, a central regulator of apoptotic cell death, was increased with stimulation in both genotypes (stimulation: P < .01) but more so in WT animals (interaction: P < .0001). Overall, our data indicate that R349P Des rats have a lower susceptibility to structural muscle damage of the cytoskeleton and sarcolemma with acute eccentric loading.

Assocation Between Leber's Hereditary Optic Neuropathy and MT-ND1 3460G>A Mutation-Induced Alterations in Mitochondrial Function, Apoptosis, and Mitophagy.

PurposeTo investigate the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-linked MT-ND1 3460G>A mutation.MethodsCybrid cell models were generated by fusing mitochondrial DNA-less ρ0 cells with enucleated cells from a patient carrying the m.3460G>A mutation and a control subject. The impact of m.3460G>A mutations on oxidative phosphorylation was evaluated using Blue Native gel electrophoresis, and measurements of oxygen consumption were made with an extracellular flux analyzer. Assessment of reactive oxygen species (ROS) production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Assays for apoptosis and mitophagy were undertaken via immunofluorescence analysis.ResultsNineteen Chinese Han pedigrees bearing the m.3460G>A mutation exhibited variable penetrance and expression of LHON. The m.3460G>A mutation altered the structure and function of MT-ND1, as evidenced by reduced MT-ND1 levels in mutant cybrids bearing the mutation. The instability of mutated MT-ND1 manifested as defects in the assembly and activity of complex I, respiratory deficiency, diminished mitochondrial adenosine triphosphate production, and decreased membrane potential, in addition to increased production of mitochondrial ROS in the mutant cybrids carrying the m.3460G>A mutation. The m.3460G>A mutation mediated apoptosis, as evidenced by the elevated release of cytochrome c into the cytosol and increasing levels of the apoptotic-associated proteins BAK, BAX, and PARP, as well as cleaved caspases 3, 7, and 9, in the mutant cybrids. The cybrids bearing the m.3460G>A mutation exhibited reduced levels of autophagy protein light chain 3, accumulation of autophagic substrate P62, and impaired PTEN-induced kinase 1/parkin-dependent mitophagy.ConclusionsOur findings highlight the critical role of m.3460G>A mutation in the pathogenesis of LHON, manifested by mitochondrial dysfunction and alterations in apoptosis and mitophagy.

RIP3 impedes transcription factor EB to suppress autophagic degradation in septic acute kidney injury

Autophagy is an important renal-protective mechanism in septic acute kidney injury (AKI). Receptor interacting protein kinase 3 (RIP3) has been implicated in the renal tubular injury and renal dysfunction during septic AKI. Here we investigated the role and mechanism of RIP3 on autophagy in septic AKI. We showed an activation of RIP3, accompanied by an accumulation of the autophagosome marker LC3II and the autophagic substrate p62, in the kidneys of lipopolysaccharide (LPS)-induced septic AKI mice and LPS-treated cultured renal proximal tubular epithelial cells (PTECs). The lysosome inhibitor did not further increase the levels of LCII or p62 in LPS-treated PTECs. Moreover, inhibition of RIP3 attenuated the aberrant accumulation of LC3II and p62 under LPS treatment in vivo and in vitro. By utilizing mCherry-GFP-LC3 autophagy reporter mice in vivo and PTECs overexpression mRFP-GFP-LC3 in vitro, we observed that inhibition of RIP3 restored the formation of autolysosomes and eliminated the accumulated autophagosomes under LPS treatment. These results indicated that RIP3 impaired autophagic degradation, contributing to the accumulation of autophagosomes. Mechanistically, the nuclear translocation of transcription factor EB (TFEB), a master regulator of the lysosome and autophagy pathway, was inhibited in LPS-induced mice and LPS-treated PTECs. Inhibition of RIP3 restored the nuclear translocation of TFEB in vivo and in vitro. Co-immunoprecipitation further showed an interaction of RIP3 and TFEB in LPS-treated PTECs. Also, the expression of LAMP1 and cathepsin B, two potential target genes of TFEB involved in lysosome function, were decreased under LPS treatment in vivo and in vitro, and this decrease was rescued by inhibiting RIP3. Finally, overexpression of TFEB restored the autophagic degradation in LPS-treated PTECs. Together, the present study has identified a pivotal role of RIP3 in suppressing autophagic degradation through impeding the TFEB-lysosome pathway in septic AKI, providing potential therapeutic targets for the prevention and treatment of septic AKI.

Inhibition of autophagy aggravates DNA damage response and gastric tumorigenesis via Rad51 ubiquitination in response to H. pylori infection

ABSTRACT Helicobacter pylori (H. pylori) infection is the strongest known risk factor for the development of gastric cancer. DNA damage response (DDR) and autophagy play key roles in tumorigenic transformation. However, it remains unclear how H. pylori modulate DDR and autophagy in gastric carcinogenesis. Here we report that H. pylori infection promotes DNA damage via suppression of Rad51 expression through inhibition of autophagy and accumulation of p62 in gastric carcinogenesis. We find that H. pylori activated DNA damage pathway in concert with downregulation of repair protein Rad51 in gastric cells, C57BL/6 mice and Mongolian gerbils. In addition, autophagy was increased early and then decreased gradually during the duration of H. pylori infection in vitro in a CagA-dependent manner. Moreover, loss of autophagy led to promotion of DNA damage in H. pylori-infected cells. Furthermore, knockdown of autophagic substrate p62 upregulated Rad51 expression, and p62 promoted Rad51 ubiquitination via the direct interaction of its UBA domain. Finally, H. pylori infection was associated with elevated levels of p62 in gastric intestinal metaplasia and decreased levels of Rad51 in dysplasia compared to their H. pylori- counterparts. Our findings provide a novel mechanism into the linkage of H. pylori infection, autophagy, DNA damage and gastric tumorigenesis.

The interrupted effect of autophagic flux and lysosomal function induced by graphene oxide in p62-dependent apoptosis of F98 cells

BackgroundGraphene oxide (GO) nanoparticles (NPs) have been widely applied in various fields, especially in biomedical applications. Extensive studies have suggested that GO can pass through the blood–brain barrier (BBB) and induce abnormal autophagy and cytotoxicity in the central nervous system (CNS). However, the effect and specific mechanism of GO on astrocytes, the most abundant cells in the brain still has not been extensively investigated.ResultsIn this study, we systematically explored the toxicity and mechanism of GO exposure in the rat astroglioma-derived F98 cell line using molecular biological techniques (immunofluorescence staining, flow cytometry and Western blot) at the subcellular level and the signaling pathway level. Cells exposed to GO exhibited decreased cell viability and increased lactate dehydrogenase (LDH) release in a concentration- and time-dependent manner. GO-induced autophagy was evidenced by transmission electron microscopy (TEM) and immunofluorescence staining. Western blots showed that LC3II/I and p62 were upregulated and PI3K/Akt/mTOR was downregulated. Detection of lysosomal acidity and cathepsin B activity assay indicated the impairment of lysosomal function. Annexin V-FITC-PI detection showed the occurrence of apoptosis after GO exposure. The decrease in mitochondrial membrane potential (MMP) with an accompanying upregulation of cleaved caspase-3 and Bax/Bcl-2 further suggested that endogenous signaling pathways were involved in GO-induced apoptosis.ConclusionThe exposure of F98 cells to GO can elicit concentration- and time-dependent toxicological effects. Additionally, increased autophagic response can be triggered after GO treatment and that the blocking of autophagy flux plays a vital role in GO cytotoxicity, which was determined to be related to dysfunction of lysosomal degradation. Importantly, the abnormal accumulation of autophagic substrate p62 protein can induce capase-3-mediated apoptosis. Inhibition of abnormal accumulation of autophagic cargo could alleviate the occurrence of GO-induced apoptosis in F98 cells.

Excessive apoptosis and disordered autophagy flux contribute to the neurotoxicity induced by high iodine in Sprague-Dawley rat

In recent years, the detrimental effects of high iodine on intelligence are gaining tons of attention, but the relationship between high iodine and neurotoxicity is controversial. This study aimed to explore whether high iodine intake may impair intelligence and the roles of apoptosis and autophagy in high iodine-induced neurotoxicity. The results showed that high iodine exposure reduced brain coefficient and intelligence of rats, and caused histopathological abnormalities in hippocampus. Moreover, high iodine increased hippocampal apoptosis, as confirmed by elevation of apoptotic proteins and TUNEL-positive incidence. Further study showed that high iodine impaired mitochondrial ultrastructure and caused elevation of Bax, cytochrome c and decline of Bcl2, indicating the participation of mitochondrial apoptotic pathway. Simultaneously, high iodine also increased the number of autophagosomes. Intriguingly, the expression of autophagosomes formation protein Atg7, Beclin1 and autophagic substrate p62 were elevated, suggesting that the accumulated autophagosomes is not only due to the enhancement of formation but also the decline of clearance. These, together with the numerous damaged organelles observed in hippocampal ultrastructure, reveal the crucial role of disordered autophagy flux in high iodine-elicited neurotoxicity. Collectively, these findings suggest that excessive apoptosis and disordered autophagy flux contribute to high iodine-elicited neurotoxicity.

Autophagy Suppresses Invasiveness of Endometrial Cells through Reduction of Fascin-1.

Objective Autophagy has been reported to be involved in the development of various disorders such as neurodegenerative and metabolic diseases and tumors. Autophagy activators and inhibitors are also potential therapeutics for these diseases. However, the mechanism of autophagic involvement in different diseases is not the same, and the role of autophagy in endometriosis (EM) has not yet been elucidated. This research investigated the mechanism by which autophagy acts in EM, with the aim of establishing a theoretical basis for its prevention and treatment through the targeted interference with autophagy. Methods We used an RNA interference fragment targeting ATG5, the autophagy activator rapamycin, and the autophagy inhibitor 3-MA or overexpression of filopodia-related protein fascin-1, in conjunction with clonogenic assays, growth curves, and scratch assay to investigate the influence of autophagy on cellular growth, proliferation, and invasiveness. We collected specimens from 20 clinical cases of EM and investigated the protein expression of the autophagic marker LC3-II, the autophagic substrate p62, and fascin-1. Results Rapamycin was able to inhibit the proliferation and colony formation of the endometriotic cell line CRL-7566, whereas the autophagy inhibitor 3-MA as well as the interference with the autophagy-related gene ATG5 had the opposite effect. More importantly, the autophagy activator rapamycin was able to inhibit the growth of filopodia in the endometriotic cells, and the overexpression of the fascin-1 restored the rapamycin-induced decrease of invasiveness. We found that the expression of the autophagy marker LC3-II was significantly reduced among the clinical EM specimens compared to the control group, while the expressions of fascin-1 and autophagic substrate p62 were increased. Conclusion Our results indicate that the inhibition of autophagy and exogenous expression of fascin-1 may promote the invasiveness of endometrial cells. As a corollary, autophagy represents a potential target for the treatment of EM.

Leber's hereditary optic neuropathy (LHON)-associated ND5 12338T > C mutation altered the assembly and function of complex I, apoptosis and mitophagy.

Mutations in mitochondrial DNA (mtDNA) have been associated with Leber's hereditary optic neuropathy (LHON) and their pathophysiology remains poorly understood. In this study, we demonstrated that a missense mutation (m.12338T>C, p.1M>T) in the ND5 gene contributed to the pathogenesis of LHON. The m.12338T>C mutation affected the first methionine (Met1) with a threonine and shortened two amino acids of ND5. We therefore hypothesized that the mutated ND5 perturbed the structure and function of complex I. Using the cybrid cell models, generated by fusing mtDNA-less (ρ°) cells with enucleated cells from LHON patients carrying the m.12338T>C mutation and a control subject belonging to the same mtDNA haplogroup, we demonstrated that the m.12338T>C mutation caused the reduction of ND5 polypeptide, perturbed assemble and activity of complex I. Furthermore, the m.12338T>C mutation caused respiratory deficiency, diminished mitochondrial adenosine triphosphate levels and membrane potential and increased the production of reactive oxygen species. The m.12338T>C mutation promoted apoptosis, evidenced by elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 9, 3, 7 and Poly ADP ribose polymerase in the cybrids carrying the m.12338T>C mutation, as compared with control cybrids. Moreover, we also document the involvement of m.12338T>C mutation in decreased mitophagy, as showed by reduced levels of autophagy protein light chain 3 and accumulation of autophagic substrate p62 in the in mutant cybrids as compared with control cybrids. These data demonstrated the direct link between mitochondrial dysfunction caused by complex I mutation and apoptosis or mitophagy. Our findings may provide new insights into the pathophysiology of LHON.

Natural autophagy blockers, dauricine (DAC) and daurisoline (DAS), sensitize cancer cells to camptothecin-induced toxicity.

Autophagy is a cellular bulk degradation pathway implicated in various diseases. Inhibition of autophagy has been regarded as a new therapeutic strategy for cancer treatment, especially in combination with chemotherapy. In our study, we identified two natural compounds, dauricine (DAC) and daurisoline (DAS), as two potent autophagy blockers through a high-content screening. DAC and DAS are alkaloids isolated from traditional Chinese medicine Rhizoma Menispermi. We systematically examined the effects of DAC and DAS on autophagy function in HeLa cells and found that DAC and DAS induced massive formation of autophagic vacuoles and lipidation of LC3. The accumulation of autophagic vacuoles and LC3 lipidation are due to blockage of autophagosome maturation as evidenced by interrupted colocalization of autophagsosome and lysosome, increased GFP-LC3/RFP-LC3 ratio and accumulation of autophagic substrate p62. Moreover, DAC and DAS impaired lysosomal function, as indicated by reduced lysosomal protease activity and increased lysosomal pH values. Importantly, we showed that DAC and DAS strongly inhibited the lysosome V-type ATPase activity. For the therapeutic potential, we found that DAC and DAS blocked the campothecin (CPT)-induced protective autophagy in HeLa cells, and dramatically sensitized the multiple cancer cells to CPT-induced cell death. In conclusion, our result shows that DAC and DAS are autophagy inhibitors which inhibit the lysosomal degradation of auophagic vacuoles, and sensitize the CPT-induced cancer cell death. The study implies the therapeutic potential of DAC and DAS in the treatment of cancers in combination of chemotherapy by inhibiting autophagy.

Evidence that the essential oil of bergamot modulates autophagy flux in human tumor cell lines

The long standing folk use of the essential oil (EO) of bergamot (Citrus bergamia Risso et Poiteau) as antiseptic is consistent with the observed antifungal and antimicrobial activities in conjunction with the most recently described increase of oxidative metabolism in human polimorphonuclear leukocytes [1]. We have previously reported that in SH-SY5Y human neuroblastoma cultures the EO of bergamot causes concentration-dependent cell death [2]. Here we now report compelling evidence demonstrating that in these cell cultures bergamot modulates autophagy in a concentration-dependent fashion, not linked to effects on cell survival and via mechanisms mTOR-independent. Enhanced LC3 conversion and degradation of the autophagic substrate p62 are still observed following suppression of Beclin-1 expression by siRNA. The observed effects are not cell line specific because enhanced LC3 lipidation and reduced p62 levels were also observed in bergamot exposed MCF7 human breast cancer cells. In depth investigation on the principle responsible for autophagy activation shows that limonene, the main terpene of the EO, is responsible for such an important effect of the phytocomplex. Autophagy is a complex and finely tuned cellular process that plays fundamental roles under physiological as well as pathological conditions; in fact, it has been implicated in severe human diseases, including cancer. Accordingly, our original observation is of great importance for putative clinical translation in chemotherapy.

Low Shear Stress Inhibited Endothelial Cell Autophagy Through TET2 Downregulation.

Low shear stress plays a crucial role in the initiation and progression of atherosclerotic lesions. However, the detailed mechanisms of these processes remain unclear. In this study, the effect of low shear stress on endothelial cell autophagy and its potential mechanism were investigated. Results showed autophagy dysfunction and ten-eleven translocation 2 (TET2) protein downregulation during atherosclerotic lesion progression. Autophagic markers BECLIN 1 and LC3II/LC3I under low shear stress (5dyne/cm(2)) obviously decreased compared with those under physiological shear stress (15dyne/cm(2)), whereas autophagic substrate p62 increased. TET2 expression was also downregulated under low shear stress. Endothelial cell autophagy was improved with TET2 overexpression but was impaired by TET2 siRNA treatment. Moreover, TET2 overexpression upregulated the expression of endothelial cell nitric oxide synthase (eNOS) and downregulated the expression of endothelin-1 (ET-1). TET2siRNA further attenuated eNOS expression and stimulated ET-1 expression. Overall, the results showed that low shear stress downregulated endothelial cell autophagy by impaired TET2 expression, which might contribute to theatherogenic process.

Regulation of autophagic flux by CHIP.

Autophagy is a major degradation system which processes substrates through the steps of autophagosome formation, autophagosome-lysosome fusion, and substrate degradation. Aberrant autophagic flux is present in many pathological conditions including neurodegeneration and tumors. CHIP/STUB1, an E3 ligase, plays an important role in neurodegeneration. In this study, we identified the regulation of autophagic flux by CHIP (carboxy-terminus of Hsc70-interacting protein). Knockdown of CHIP induced autophagosome formation through increasing the PTEN protein level and decreasing the AKT/mTOR activity as well as decreasing phosphorylation of ULK1 on Ser757. However, degradation of the autophagic substrate p62 was disturbed by knockdown of CHIP, suggesting an abnormality of autophagic flux. Furthermore, knockdown of CHIP increased the susceptibility of cells to autophagic cell death induced by bafilomycin A1. Thus, our data suggest that CHIP plays roles in the regulation of autophagic flux.

Fructose supplementation impairs rat liver autophagy through mTORC activation without inducing endoplasmic reticulum stress

Supplementation with 10% liquid fructose to female rats for 2weeks caused hepatic steatosis through increased lipogenesis and reduced peroxisome proliferator activated receptor (PPAR) α activity and fatty acid catabolism, together with increased expression of the spliced form of X-binding protein-1 (Rebollo et al., 2014). In the present study, we show that some of these effects are preserved after sub-chronic (8weeks) fructose supplementation, specifically increased hepatic expression of lipid synthesis-related genes (stearoyl-CoA desaturase, ×6.7-fold; acetyl-CoA carboxylase, ×1.6-fold; glycerol-3-phosphate acyltransferase, ×1.65-fold), and reduced fatty acid β-oxidation (×0.77-fold), resulting in increased liver triglyceride content (×1.69-fold) and hepatic steatosis. However, hepatic expression of PPARα and its target genes was not modified and, further, livers of 8-week fructose-supplemented rats showed no sign of unfolded protein response activation, except for an increase in p-IRE1 levels. Hepatic mTOR phosphorylation was enhanced (×1.74-fold), causing an increase in the phosphorylation of UNC-51-like kinase 1 (ULK-1) (×2.8-fold), leading to a decrease in the ratio of LC3B-II/LC3B-I protein expression (×0.39-fold) and an increase in the amount of the autophagic substrate p62, indicative of decreased autophagy activity. A harmful cycle may be established in the liver of 8-week fructose-supplemented rats where lipid accumulation may cause defective autophagy, and reduced autophagy may result in decreased free fatty acid formation from triglyceride depots, thus reducing the substrates for β-oxidation and further increasing hepatic steatosis. In summary, the length of supplementation is a key factor in the metabolic disturbances induced by fructose: in short-term studies, PPARα inhibition and ER stress induction are critical events, whereas after sub-chronic supplementation, mTOR activation and autophagy inhibition are crucial.

Beclin 1 knockdown retards re-endothelialization and exacerbates neointimal formation via a crosstalk between autophagy and apoptosis

ObjectiveEndothelial regeneration is an essential process for the prevention of excessive neointimal formation following endothelial denudation. Beclin 1, a mammalian autophagy gene, is a link between autophagy and apoptosis. We hypothesized that the interference of Beclin 1 can influence re-endothelialization and ultimately affect neointimal formation by regulating autophagy and apoptosis. MethodsA rat carotid injury model of endothelial denudation was used, and small interfering RNA of Beclin 1 was perivascularly administered. Neointima was evaluated by morphological analysis. von Willebrand factor, Beclin 1, LC3, autophagic substrate p62 and caspase-3 levels were detected by immunofluorescence or Western blotting. Terminal deoxynucleotidyl transferase-mediated digoxigenin–dUTP–biotin nick-end labeling assay was performed to evaluate apoptosis. ResultsCarotid injury induced an upregulation of Beclin 1 protein which was down regulated by more than 50% with small RNA interference. Beclin 1 knockdown significantly retarded re-endothelialization 7 days after injury and subsequently augmented neointima by more than 2 folds at 14 and 21 days. Autophagy and apoptosis were detected to reveal the regulatory effect of Beclin 1. The injury-activated autophagy, shown by the increased levels of punctate LC3 and LC3II as well as decreased p62 expression, was significantly inhibited by Beclin 1 knockdown. Meanwhile, the apoptotic endothelial cell number was increased and caspase-3 was up-regulated, though the expression of truncated BID was not significantly influenced. ConclusionBeclin 1 knockdown exacerbated neointimal formation after rat carotid injury, associated with retarded re-endothelialization due to enhanced apoptosis, while simultaneously prohibiting autophagic activation. The data suggested an essential role of Beclin 1 as a regulator between autophagy and apoptosis in the setting of neointimal formation.

Autophagic Impairment Contributes to Systemic Inflammation-Induced Dopaminergic Neuron Loss in the Midbrain

BackgroundNeuroinflammation plays an important role in the pathogenesis of Parkinson’s disease (PD), inducing and accelerating dopaminergic (DA) neuron loss. Autophagy, a critical mechanism for clearing misfolded or aggregated proteins such as α-synuclein (α-SYN), may affect DA neuron survival in the midbrain. However, whether autophagy contributes to neuroinflammation-induced toxicity in DA neurons remains unknown.ResultsIntraperitoneal injection of lipopolysaccharide (LPS, 5 mg/kg) into young (3-month-old) and aged (16-month-old) male C57BL/6J mice was observed to cause persistent neuroinflammation that was associated with a delayed and progressive loss of DA neurons and accumulation of α-SYN in the midbrain. The autophagic substrate-p62 (SQSTM1) persistently increased, whereas LC3-II and HDAC6 exhibited early increases followed by a decline. In vitro studies further demonstrated that TNF-α induced cell death in PC12 cells. Moreover, a sublethal dose of TNF-α (50 ng/ml) increased the expression of LC3-II, p62, and α-SYN, implying that TNF-α triggered autophagic impairment in cells.ConclusionNeuroinflammation may cause autophagic impairment, which could in turn result in DA neuron degeneration in midbrain.