Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNK signaling

Fluoride is an effective caries prophylactic, but at high doses can also be an environmental health hazard. Acute or chronic exposure to high fluoride doses can result in dental enamel and skeletal and soft tissue fluorosis. Dental fluorosis is manifested as mottled, discolored, porous enamel that is susceptible to dental caries. Fluoride induces cell stress, including endoplasmic reticulum stress and oxidative stress, which leads to impairment of ameloblasts responsible for dental enamel formation. Recently we reported that fluoride activates SIRT1 and autophagy as an adaptive response to protect cells from stress. However, it still remains unclear how SIRT1/autophagy is regulated in dental fluorosis. In this study, we demonstrate that fluoride exposure generates reactive oxygen species (ROS) and the resulting oxidative damage is counteracted by SIRT1/autophagy induction through c-Jun N-terminal kinase (JNK) signaling in ameloblasts. In the mouse-ameloblast-derived cell line LS8, fluoride induced ROS, mitochondrial damage including cytochrome-c release, up-regulation of UCP2, attenuation of ATP synthesis, and H2AX phosphorylation (γH2AX), which is a marker of DNA damage. We evaluated the effects of the ROS inhibitor N-acetylcysteine (NAC) and the JNK inhibitor SP600125 on fluoride-induced SIRT1/autophagy activation. NAC decreased fluoride-induced ROS generation and attenuated JNK and c-Jun phosphorylation. NAC decreased SIRT1 phosphorylation and formation of the autophagy marker LC3II, which resulted in an increase in the apoptosis mediators γH2AX and cleaved/activated caspase-3. SP600125 attenuated fluoride-induced SIRT1 phosphorylation, indicating that fluoride activates SIRT1/autophagy via the ROS-mediated JNK pathway. In enamel organs from rats or mice treated with 50, 100, or 125 ppm fluoride for 6 weeks, cytochrome-c release and the DNA damage markers 8-oxoguanine, p-ATM, and γH2AX were increased compared to those in controls (0 ppm fluoride). These results suggest that fluoride-induced ROS generation causes mitochondrial damage and DNA damage, which may lead to impairment of ameloblast function. To counteract this impairment, SIRT1/autophagy is induced via JNK signaling to protect cells/ameloblasts from fluoride-induced oxidative damage that may cause dental fluorosis.     

Inhibition of autophagy reduces the rate of fluoride-induced LS8 apoptosis via regulating ATG5 and ATG7

Excessive fluoride affects ameloblast differentiation and tooth development. The fate of fluorinated ameloblasts is determined by multiple signaling pathways in response to a range of stimuli. Both autophagy and apoptosis are involved in the regulation of dental fluorosis as well as in protein synthesis and enamel mineralization. Emerging evidence suggests that autophagy and apoptosis are interconnected and that their interaction greatly influences cell death. However, the effect of autophagy on apoptosis in fluoride-treated ameloblasts is unclear. Here, we employed an in vitro cellular model of fluorosis in mouse ameloblast-like LS8 cells and induced autophagy using sodium fluoride (NaF). Our findings suggest that NaF treatment induces autophagy in LS8 cells, and ATG5 and ATG7 are important molecules involved in this process. We also showed that NaF treatment reduced cell viability in Atg5/7 siRNA and autophagy inhibitor-treated LS8 cells. More importantly, NaF-induced apoptosis can be reversed by inhibiting early stage of autophagy. In conclusion, our study shows that autophagy is closely related to dental fluorosis, and inhibition of autophagy, especially ATG5/7, reduces fluoride-induced cell death and apoptosis.     

Regulation of mitochondrial FoF1ATPase activity by Sirt3-catalyzed deacetylation and its deficiency in human cells harboring 4977 bp deletion of mitochondrial DNA

Sirt3, a mitochondrial NAD⁺-dependent deacetylase, is regarded as a potential regulator in cellular metabolism. However, the role of Sirt3 in the regulation of mitochondrial F_oF₁ATPase and the linkage to mitochondrial diseases is unclear. In this study, we demonstrated a role of Sirt3 in the regulation of F_oF₁ATPase activity in human cells. Knockdown of Sirt3 in 143B cells by shRNA transfection caused increased acetylation levels of the α and OSCP subunits of F_oF₁ATPase. We showed that Sirt3 physically interacted with the OSCP and led to its subsequent deacetylation. By incubation of mitochondria with the purified Sirt3 protein, Sirt3 could regulate F_oF₁ATPase activity through its deacetylase activity. Moreover, suppression of Sirt3 reduced the F_oF₁ATPase activity, consequently decreased the intracellular ATP level, diminished the capacity of mitochondrial respiration, and compromised metabolic adaptability of 143B cells to the use of galactose as the energy source. In human cells harboring ? 85% of mtDNA with 4977 bp deletion, we showed that oxidative stress induced a reduction of Sirt3 expression, and an increased acetylation of the OSCP subunit of F_oF₁ATPase. Importantly, the expression of Sirt3 was also decreased in the skin fibroblasts from patients with CPEO syndrome. We further demonstrated that oxidative stress induced by 5–10 μM of menadione impaired the Sirt3-mediated deacetylation and activation on F_oF₁ATPase activity through decreasing the protein level of Sirt3. Our findings suggest that increased intracellular ROS levels might modulate the expression of Sirt3 which deacetylates and activates F_oF₁ATPase in human cells with mitochondrial dysfunction caused by a pathogenic mtDNA mutation.     

Protein Kinase CK2 Is Upregulated by Calorie Restriction and Induces Autophagy

Calorie restriction (CR) and the activation of autophagy extend healthspan by delaying the onset of age-associated diseases in most living organisms. Because protein kinase CK2 (CK2) downregulation induces cellular senescence and nematode aging, we investigated CK2’s role in CR and autophagy. This study indicated that CR upregulated CK2’s expression, thereby causing SIRT1 and AMP-activated protein kinase (AMPK) activation. CK2α overexpression, including antisense inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760, stimulated autophagy initiation and nucleation markers (increase in ATG5, ATG7, LC3BII, beclin-1, and Ulk1, and decrease in SQSTM1/p62). The SIRT1 deacetylase, AKT, mammalian target of rapamycin (mTOR), AMPK, and forkhead homeobox type O (FoxO) 3a were involved in CK2-mediated autophagy. The treatment with the AKT inhibitor triciribine, the AMPK activator AICAR, or the SIRT1 activator resveratrol rescued a reduction in the expression of lgg-1 (the Caenorhabditis elegans ortholog of LC3B), bec-1 (the C. elegans ortholog of beclin-1), and unc-51 (the C. elegans ortholog of Ulk1), mediated by kin-10 (the C. elegans ortholog of CK2β) knockdown in nematodes. Thus, this study indicated that CK2 acted as a positive regulator in CR and autophagy, thereby suggesting that these four miRs’ antisense inhibitors can be used as CR mimetics or autophagy inducers.     

Atg5 deficiency-mediated mitophagy aggravates cardiac inflammation and injury in response to angiotensin II

ObjectiveHypertension induces end-organ damage through inflammation, and autophagy plays a crucial role in the regulation of cellular homeostasis. In the present study, we aimed to define the role of autophagy in the development of inflammation and cardiac injury induced by angiotensin II (Ang II).Methods and ResultsAutophagy protein 5 (Atg5) haplodeficiency (Atg5^+/−) and age-matched wild-type (WT) C57BL/6 J mice were infused with Ang II (1500 ng/kg/min) or saline for 7 days. Heart sections were stained with hematoxylin and eosin (H&E), Masson's trichrome, and immunohistochemical stains. Cytokine and LC3 levels were measured using real-time PCR or western blot analysis. After Ang II infusion, the WT mice exhibited marked macrophage accumulation, cytokine expression, and reactive oxygen species (ROS) production compared with saline-infused controls. However, these effects induced by Ang II infusion were aggravated in Atg5^+/− mice. These effects were associated with Atg5-mediated impaired autophagy, accompanied by increased production of ROS and activation of nuclear factor-κB (NF-κB) in macrophages. Finally, increased cardiac inflammation in Atg5 haplodeficient mice was associated with increased cardiac fibrosis.ConclusionAtg5 deficiency-mediated autophagy increases ROS production and NF-κB activity in macrophages, thereby contributing to cardiac inflammation and injury. Thus, improving autophagy may be a novel therapeutic strategy to ameliorate hypertension-induced inflammation and organ damage.     

Regulation of Autophagy by the p300 Acetyltransferase

Autophagy is a regulated process of intracellular catabolism required for normal cellular maintenance, as well as serving as an adaptive response under various stress conditions, including starvation. The molecular regulation of autophagy in mammalian cells remains incompletely understood. Here we demonstrate a role for protein acetylation in the execution and regulation of autophagy. In particular, we demonstrate that the p300 acetyltransferase can regulate the acetylation of various known components of the autophagy machinery. Knockdown of p300 reduces acetylation of Atg5, Atg7, Atg8, and Atg12, although overexpressed p300 increases the acetylation of these same proteins. Furthermore, p300 and Atg7 colocalize within cells, and the two proteins physically interact. The interaction between p300 and Atg7 is dependent on nutrient availability. Finally, we demonstrate that knockdown of p300 can stimulate autophagy, whereas overexpression of p300 inhibits starvation-induced autophagy. These results demonstrate a role for protein acetylation and particularly p300 in the regulation of autophagy under conditions of limited nutrient availability.Macro-autophagy, herein referred to as autophagy, is an evolutionary conserved process first characterized in lower organisms (1). In yeast, over 20 separate genes (designated ATG1, ATG2, etc.) have been demonstrated to be essential to carry out the autophagy program. This process is thought to provide a mechanism for the efficient removal of both long lived proteins and damaged cellular organelles. This regulated degradation provides several essential functions for the cell. First, it allows for the removal of damaged and potentially harmful cellular contents. In addition, in breaking down various intracellular components, the autophagy process provides essential building blocks for the cell to use in the re-synthesis of necessary macromolecules. To accomplish this recycling effort, the coordinated actions of various Atg gene products are required. In particular, the Atg gene products together orchestrate the formation of a double membrane structure known as the autophagosome that engulfs the intended cellular cargo targeted for degradation. The autophagosome eventually fuses with the vacuole in yeast or the lysosome in mammals.In both yeast and mammalian cells, autophagy can be stimulated by the withdrawal of nutrients. Under these conditions, autophagic degradation of nonessential components may be essential to meet ongoing energetic needs in the presence of limited extracellular nutrients. This point was underscored by the analysis of mice containing a targeted deletion of Atg5 (2). In the absence of Atg5, there is a lack of both basal and starvation-induced autophagy. Mice lacking Atg5 are born normally but succumb within the 1st day of life. This post-natal lethality is thought to be due in large part for the requirement of autophagy to supply the energetic needs of neonates. These needs are particularly critical during the small window of time where the animal no longer has a placental circulation and before the pup can begin to nurse and thus obtain external nutrients.Relatively little is known regarding how signals such as nutrient availability are able to be transduced to ultimately regulate the level of cellular autophagy. One important pathway that impinges on the process is signaling thorough the target of rapamycin (TOR)2 network (3). Evidence suggests that TOR signaling inhibits autophagy, and indeed agents such as rapamycin that can inhibit TOR are known to result in increased autophagy. We recently have observed that in addition to this mode of regulation, the NAD-dependent deacetylase Sirt1 is also a regulator of autophagy in mammalian cells and tissues (4). In particular, we demonstrated that in the absence of Sirt1 levels of acetylation for various components of the autophagy machinery are increased and that starvation-induced autophagy is impaired. Interestingly, like the Atg5 knock-out animals, Sirt1^-/- mice are also born normally but die within the few hours to days after birth. Consistent with a defect in autophagy, electron micrographs of hearts from Sirt1^-/- mice demonstrated an accumulation of abnormal appearing organelles, including mitochondria, a phenotype previously observed in Atg-deficient animals (5). Here we have further characterized the role of acetylation in the regulation of autophagy, and in particular, we demonstrate a role for the p300 acetyltransferase in this process.     

Intermittent-hypoxia induced autophagy attenuates contractile dysfunction and myocardial injury in rat heart

Sleep apnea syndrome (SAS) is considered to be associated with heart failure (HF). It is known that autophagy is induced in various heart diseases thereby promotes survival, but its excess may be maladaptive. Intermittent hypoxia (IH) plays pivotal role in the pathogenesis of SAS. We aimed to clarify the relationships among IH, autophagy, and HF. Rats underwent IH at a rate of 20 cycles/h (nadir of 4% O2 to peak of 21% O2 with 0% CO2) or normal air breathing (control) for 8 h/d for 3 weeks. IH increased the cardiac LC3II/LC3I ratio. The IH induced upregulation of LC3II was attenuated by the administration of an inhibitor of autophagosome formation 3-methyladenine (3-MA), but enhanced by an inhibitor of autophagosome–lysosome fusion chloroquine (CQ), showing enhanced autophagic flux in IH hearts. Electron microscopy confirmed an increase in autophagosomes and lysosomes in IH. With 3-MA or CQ, IH induced progressive deterioration of fractional shortening (FS) on echocardiography over 3 weeks, although IH, 3-MA, or CQ alone had no effects. With CQ, IH for 4 weeks increased serum troponin T levels, reflecting necrosis. Western blotting analyses showed dephosphorylation of Akt and mammalian target of rapamycin (mTOR) at Akt (Ser2448, 2481) sites, suggesting the activation of autophagy via Akt inactivation. Conclusions. IH-mediated autophagy maintains contractile function, whereas when autophagy is inhibited, IH induces systolic dysfunction due to myocyte necrosis. General significance. This study highlighted the potential implications of autophagy in cardio-protection in early SAS patients without comorbidity, reproduced in normal rats by 3 ~ 4 weeks of IH.     

SIRT1 Regulates the Chemoresistance and Invasiveness of Ovarian Carcinoma Cells

BACKGROUND: SIRT1 is a longevity gene that forestalls aging and age-related diseases including cancer, and has recently attracted widespread attention due to its overexpression in some cancers. We previously identified the overexpression of SIRT1 in ovarian carcinoma (OvCa) as a poor prognostic factor. However, mechanistic insights into the function of SIRT1 in OvCa have yet to be elucidated. METHODS: Quantitative real-time reverse PCR (qRT-PCR) and Western blotting were employed to examine the expression of SIRT1 in a panel of human OvCa cell lines. si-RNA or sh-RNA and cDNA technologies were utilized to knockdown or overexpress SIRT1, respectively. The effects of SIRT1 on proliferation and chemoresistance were examined using a WST-1 assay, and the underlying mechanisms were confirmed using an apoptotic assay, and the quantification of glutathione (GSH), and reactive oxygen species (ROS). The aggressiveness of SIRT1 was analyzed using in vitro invasion and migration assays. RESULTS: SIRT1 was more strongly expressed in OvCa cell lines than in the immortalized ovarian epithelium at the gene and protein levels. Stress up-regulated the expression of SIRT1 in dose- and time-dependent manners. SIRT1 significantly enhanced the proliferation (P < .05), chemoresistance (P < .05), and aggressiveness of OvCa cells by up-regulating multiple antioxidant pathways to inhibit oxidative stress. Further study into the overexpression of SIRT1 demonstrated the up-regulation of several stemness-associated genes and enrichment of CD44v9 via an as-yet-unidentified pathway. CONCLUSIONS: Our results suggest that SIRT1 plays a role in the acquisition of aggressiveness and chemoresistance by OvCa, and has potential as a therapeutic target for OvCa.     

Concentration-dependent effects of resveratrol and metabolites on the redox status of human erythrocytes in single-dose studies

Dietary trans-resveratrol (RES) is rapidly metabolized into sulfated and glucuronated conjugates in humans. This study focused on the in vitro determination of the antioxidant capacity of RES and its main physiological metabolites and on its relevance in vivo. In vitro, RES, RES-3-O-sulfate (R3S) and 3-O-glucuronide (R3G) showed antioxidant activities at a concentration of 1 mM when compared to Trolox using an assay in which the antioxidant inhibits iron-induced linoleic acid oxidation: 0.87±0.08 mM Trolox equivalents (TE) for RES, 0.52±0.01 mM TE for R3S and 0.36±0.02 mM TE for R3G. At a concentration of 1 μM, compounds promoted linoleic acid peroxidation (RES −0.30±0.09 mM TE, R3S −0.48±0.05 mM TE and R3G −0.57±0.07 mM TE). To elucidate whether these effects were reflected in vivo, total antioxidant capacity, reactive oxygen species (ROS), conjugated fatty acid dienes (CD), superoxide dismutase (SOD) and catalase (CAT) activities were determined in human plasma and erythrocytes over 24 h, after oral intake of either 0.05 g RES as piceid or 5 g RES. Oral administration of RES did not show an impact on total antioxidant capacity, ROS or CD. However, enzymatic activities of ROS scavenging SOD and CAT were significantly lower after high-dose compared to low-dose administration of RES (P<.03 and P<.01). In conclusion, in healthy subjects, neither 0.05 g nor 5 g RES changed blood oxidative state, although our in vitro data point to a prooxidative activity of low concentrations of RES and its metabolites, which could be important in vivo for individuals with compromised antioxidant defense capacity.     

Adipose tissue conditioned media support macrophage lipid-droplet biogenesis by interfering with autophagic flux

Obesity promotes the biogenesis of adipose tissue (AT) foam cells (FC), which contribute to AT insulin resistance. Autophagy, an evolutionarily-conserved house-keeping process, was implicated in cellular lipid handling by either feeding and/or degrading lipid-droplets (LDs). We hypothesized that beyond phagocytosis of dead adipocytes, AT-FC biogenesis is supported by the AT microenvironment by regulating autophagy. Non-polarized (“M0”) RAW264.7 macrophages exposed to AT conditioned media (AT-CM) exhibited a markedly enhanced LDs biogenesis rate compared to control cells (8.3 Vs 0.3 LDs/cells/h, p < 0.005). Autophagic flux was decreased by AT-CM, and fluorescently following autophagosomes over time revealed ~ 20% decline in new autophagic vesicles' formation rate, and 60–70% decrease in autophagosomal growth rate, without marked alternations in the acidic lysosomal compartment. Suppressing autophagy by either targeting autophagosome formation (pharmacologically, with 3-methyladenine or genetically, with Atg12 ± Atg7-siRNA), decreased the rate of LD formation induced by oleic acid. Conversely, interfering with late autophago-lysosomal function, either pharmacologically with bafilomycin-A1, chloroquine or leupeptin, enhanced LD formation in macrophages without affecting LD degradation rate. Similarly enhanced LD biogenesis rate was induced by siRNA targeting Lamp-1 or the V-ATPase. Collectively, we propose that secreted products from AT interrupt late autophagosome maturation in macrophages, supporting enhanced LDs biogenesis and AT-FC formation, thereby contributing to AT dysfunction in obesity.     

Chronic Akt activation attenuated lipopolysaccharide-induced cardiac dysfunction via Akt/GSK3β-dependent inhibition of apoptosis and ER stress

Sepsis is characterized by systematic inflammation and contributes to cardiac dysfunction. This study was designed to examine the effect of protein kinase B (Akt) activation on lipopolysaccharide-induced cardiac anomalies and underlying mechanism(s) involved. Mechanical and intracellular Ca^2 + properties were examined in myocardium from wild-type and transgenic mice with cardiac-specific chronic Akt overexpression following LPS (4 mg/kg, i.p.) challenge. Akt signaling cascade (Akt, phosphatase and tensin homologue deleted on chromosome ten, glycogen synthase kinase 3 beta), stress signal (extracellular-signal-regulated kinases, c-Jun N-terminal kinases, p38), apoptotic markers (Bcl-2 associated X protein, caspase-3/-9), endoplasmic reticulum (ER) stress markers (glucose-regulated protein 78, growth arrest and DNA damage induced gene-153, eukaryotic initiation factor 2α), inflammatory markers (tumor necrosis factor α, interleukin-1β, interleukin-6) and autophagic markers (Beclin-1, light chain 3B, autophagy-related gene 7 and sequestosome 1) were evaluated. Our results revealed that LPS induced marked decrease in ejection fraction, fractional shortening, cardiomyocyte contractile capacity with dampened intracellular Ca^2 + release and clearance, elevated reactive oxygen species (ROS) generation and decreased glutathione and glutathione disulfide (GSH/GSSG) ratio, increased ERK, JNK, p38, GRP78, Gadd153, eIF2α, BAX, caspase-3 and -9, downregulated B cell lymphoma 2 (Bcl-2), the effects of which were significantly attenuated or obliterated by Akt activation. Akt activation itself did not affect cardiac contractile and intracellular Ca^2 + properties, ROS production, oxidative stress, apoptosis and ER stress. In addition, LPS upregulated levels of Beclin-1, LC3B and Atg7, while suppressing p62 accumulation. Akt activation did not affect Beclin-1, LC3B, Atg7 and p62 in the presence or absence of LPS. Akt overexpression promoted phosphorylation of Akt and GSK3β. In vitro study using the GSK3β inhibitor SB216763 mimicked the response elicited by chronic Akt activation. Taken together, these data showed that Akt activation ameliorated LPS-induced cardiac contractile and intracellular Ca^2 + anomalies through inhibition of apoptosis and ER stress, possibly involving an Akt/GSK3β-dependent mechanism.     

Mitochondrial aldehyde dehydrogenase obliterates endoplasmic reticulum stress-induced cardiac contractile dysfunction via correction of autophagy

ER stress triggers myocardial contractile dysfunction while effective therapeutic regimen is still lacking. Mitochondrial aldehyde dehydrogenase (ALDH2), an essential mitochondrial enzyme governing mitochondrial and cardiac function, displays distinct beneficial effect on the heart. This study was designed to evaluate the effect of ALDH2 on ER stress-induced cardiac anomalies and the underlying mechanism involved with a special focus on autophagy. WT and ALDH2 transgenic mice were subjected to the ER stress inducer thapsigargin (1 mg/kg, i.p., 48 h). Echocardiographic, cardiomyocyte contractile and intracellular Ca^2 + properties as well as myocardial histology, autophagy and autophagy regulatory proteins were evaluated. ER stress led to compromised echocardiographic indices (elevated LVESD, reduced fractional shortening and cardiac output), cardiomyocyte contractile and intracellular Ca^2 + properties and cell survival, associated with upregulated autophagy, dampened phosphorylation of Akt and its downstream signal molecules TSC2 and mTOR, the effects of which were alleviated or mitigated by ALDH2. Thapsigargin promoted ER stress proteins Gadd153 and GRP78 without altering cardiomyocyte size and interstitial fibrosis, the effects of which were unaffected by ALDH2. Treatment with thapsigargin in vitro mimicked in vivo ER stress-induced cardiomyocyte contractile anomalies including depressed peak shortening and maximal velocity of shortening/relengthening as well as prolonged relengthening duration, the effect of which was abrogated by the autophagy inhibitor 3-methyladenine and the ALDH2 activator Alda-1. Interestingly, Alda-1-induced beneficial effect against ER stress was obliterated by autophagy inducer rapamycin, Akt inhibitor AktI and mTOR inhibitor RAD001. These data suggest a beneficial role of ALDH2 against ER stress-induced cardiac anomalies possibly through autophagy reduction.     

Cdc14 Phosphatase Promotes TORC1-Regulated Autophagy in Yeast

Cdc14 protein phosphatase is critical for late mitosis progression in budding yeast, although its orthologs in other organisms, including mammalian cells, function as stress-responsive phosphatases. We found herein unexpected roles of Cdc14 in autophagy induction after nutrient starvation and target of rapamycin complex 1 (TORC1) kinase inactivation. TORC1 kinase phosphorylates Atg13 to repress autophagy under nutrient-rich conditions, but if TORC1 becomes inactive upon nutrient starvation or rapamycin treatment, Atg13 is rapidly dephosphorylated and autophagy is induced. Cdc14 phosphatase was required for optimal Atg13 dephosphorylation, pre-autophagosomal structure formation, and autophagy induction after TORC1 inactivation. In addition, Cdc14 was required for sufficient induction of ATG8 and ATG13 expression. Moreover, Cdc14 activation provoked autophagy even under normal conditions. This study identified a novel role of Cdc14 as the stress-responsive phosphatase for autophagy induction in budding yeast.     

CNS SIRT3 Expression Is Altered by Reactive Oxygen Species and in Alzheimer’s Disease

Progressive mitochondrial dysfunction contributes to neuronal degeneration in age-mediated disease. An essential regulator of mitochondrial function is the deacetylase, sirtuin 3 (SIRT3). Here we investigate a role for CNS Sirt3 in mitochondrial responses to reactive oxygen species (ROS)- and Alzheimer’s disease (AD)-mediated stress. Pharmacological augmentation of mitochondrial ROS increases Sirt3 expression in primary hippocampal culture with SIRT3 over-expression being neuroprotective. Furthermore, Sirt3 expression mirrors spatiotemporal deposition of β-amyloid in an AD mouse model and is also upregulated in AD patient temporal neocortex. Thus, our data suggest a role for SIRT3 in mechanisms sensing and tackling ROS- and AD-mediated mitochondrial stress.     

Altered cellular redox status,sirtuin abundance and clock gene expression in a mouse model of developmentally primed NASH

BackgroundWe have previously shown that high fat (HF) feeding during pregnancy primes the development of non-alcoholic steatohepatits (NASH) in the adult offspring. However, the underlying mechanisms are unclear.AimsSince the endogenous molecular clock can regulate hepatic lipid metabolism, we investigated whether exposure to a HF diet during development could alter hepatic clock gene expression and contribute to NASH onset in later life.MethodsFemale mice were fed either a control (C, 7% kcal fat) or HF (45% kcal fat) diet. Offspring were fed either a C or HF diet resulting in four offspring groups: C/C, C/HF, HF/C and HF/HF. NAFLD progression, cellular redox status, sirtuin expression (Sirt1, Sirt3), and the expression of core clock genes (Clock, Bmal1, Per2, Cry2) and clock-controlled genes involved in lipid metabolism (Rev-Erbα, Rev-Erbβ, RORα, and Srebp1c) were measured in offspring livers.ResultsOffspring fed a HF diet developed NAFLD. However HF fed offspring of mothers fed a HF diet developed NASH, coupled with significantly reduced NAD⁺/NADH (p < 0.05, HF/HF vs C/C), Sirt1 (p < 0.001, HF/HF vs C/C), Sirt3 (p < 0.01, HF/HF vs C/C), perturbed clock gene expression, and elevated expression of genes involved lipid metabolism, such as Srebp1c (p < 0.05, C/HF and HF/HF vs C/C).ConclusionOur results suggest that exposure to excess dietary fat during early and post-natal life increases the susceptibility to develop NASH in adulthood, involving altered cellular redox status, reduced sirtuin abundance, and desynchronized clock gene expression.     

One step closer to understanding mammalian macroautophagy initiation: Interplay of 2 HORMA architectures in the ULK1 complex

ULK1 and ATG13 assemble with RB1CC1/FIP200 and ATG101 to form a macroautophagy (hereafter autophagy) induction (ULK1) complex in higher eukaryotes. The yeast counterpart, the Atg1 complex, is comprised of Atg1 and Atg13 (ULK1 and ATG13 homologs), Atg17 (a proposed functional homolog of RB1CC1), and either the Atg101 subunit (in Schizosaccharomyces pombe) or the Atg29-Atg31 heterodimer (in Saccharomyces cerevisiae). With mutual exclusivity of, and no detectable homology between, the Atg29-Atg31 dimer and Atg101, knowledge about the roles of these proteins in autophagy induction is an important piece in the puzzle of understanding the molecular mechanism of autophagy initiation. A recent study reporting the structure of the S. pombe homolog Atg101 bound to the Atg13^HORMA domain is a notable contribution to this knowledge (see the punctum in this issue of the journal).     

Sirtuin 6 protects human retinal pigment epithelium cells from LPS-induced inflammation and apoptosis partly by regulating autophagy

ABSTRACT

Lipopolysaccharides (LPS)-induced retinal inflammation is an important factor in retinal diseases. This study was aimed to investigate the effect of Sirt6 on LPS-induced retinal injury. ARPE-19 cells were incubated with LPS to induce inflammation. The cell viability was determined using CCK-8 assay. The mRNA level and protein expression of corresponding genes was detected using qRT-PCR and western blot, respectively. The production of inflammatory cytokines was measured using ELISA kit. The levels of oxidative stress-related factors were measured using their detection kits. Cell apoptosis was observed using TUNEL assay. The results showed that Sirt6 was downregulated after LPS treatment. Sirt6 strengthened LPS-induced autophagy by promoting the expression of LC3II/I, beclin1 and ATG5. Sirt6 treatment significantly inhibited LPS-induced inflammation, oxidative stress and cell apoptosis, which was then partly abolished by 3 MA. These results suggest Sirt6 to be an important regulator for LPS-induced inflammation, oxidative stress, and apoptosis partly by regulating cell autophagy.     

Autophagy inhibition uncovers the neurotoxic action of the antipsychotic drug olanzapine

We investigated the role of autophagy, a controlled cellular self-digestion process, in regulating survival of neurons exposed to atypical antipsychotic olanzapine. Olanzapine induced autophagy in human SH-SY5Y neuronal cell line, as confirmed by the increase in autophagic flux and presence of autophagic vesicles, fusion of autophagosomes with lysosomes, and increase in the expression of autophagy-related (ATG) genes ATG4B, ATG5, and ATG7. The production of reactive oxygen species, but not modulation of the main autophagy repressor MTOR or its upstream regulators AMP-activated protein kinase and AKT1, was responsible for olanzapine-triggered autophagy. Olanzapine-mediated oxidative stress also induced mitochondrial depolarization and damage, and the autophagic clearance of dysfunctional mitochondria was confirmed by electron microscopy, colocalization of autophagosome-associated MAP1LC3B (LC3B henceforth) and mitochondria, and mitochondrial association with the autophagic cargo receptor SQSTM1/p62. While olanzapine-triggered mitochondrial damage was not overtly toxic to SH-SY5Y cells, their death was readily initiated upon the inhibition of autophagy with pharmacological inhibitors, RNA interference knockdown of BECN1 and LC3B, or biological free radical nitric oxide. The treatment of mice with olanzapine for 14 d increased the brain levels of autophagosome-associated LC3B-II and mRNA encoding Atg4b, Atg5, Atg7, Atg12, Gabarap, and Becn1. The administration of the autophagy inhibitor chloroquine significantly increased the expression of proapoptotic genes (Trp53, Bax, Bak1, Pmaip1, Bcl2l11, Cdkn1a, and Cdkn1b) and DNA fragmentation in the frontal brain region of olanzapine-exposed animals. These data indicate that olanzapine-triggered autophagy protects neurons from otherwise fatal mitochondrial damage, and that inhibition of autophagy might unmask the neurotoxic action of the drug.