LC3 Overexpression Research Articles

Autophagy induction promotes renal cyst growth in polycystic kidney disease.

BackgroundPolycystic kidney disease (PKD) involves renal cysts arising from proliferating tubular cells. Autophagy has been recently suggested as a potential therapeutic target in PKD, and mammalian target of rapamycin (mTOR) is a key negative regulator of autophagy. However, the effect of autophagy regulation on cystogenesis has not been elucidated in PKD mice.MethodsClinical validation was performed using GEO datasets and autosomal dominant polycystic kidney disease (ADPKD) patient samples. Newly established PKD and LC3 transgenic mice were used for in vivo verifications, and additional tests were performed in vitro and in vivo using multiple autophagy drugs.FindingsNeither autophagy stimulation nor LC3 overexpression alleviated PKD. Furthermore, we observed the inhibitory effect of an autophagy inhibitor on cysts, indicating its possible therapeutic use in a specific group of patients with ADPKD.InterpretationOur findings provide a novel insight into the pathogenesis related to autophagy in PKD, suggesting that drugs related to autophagy regulation should be considered with caution for treating PKD.Funding SourcesThis work was supported by grants from the Bio & Medical Technology Development Program; the Collaborative Genome Program for Fostering New Post-Genome Industry of the NRF; the Basic Science Program.

LC3 and ATG5 overexpression and neuronal cell death in the prefrontal cortex of postmortem chronic methamphetamine users

Methamphetamine (METH) abuse is accompanied by oxidative stress, METH-induced neurotoxicity, and apoptosis. Oxidative stress has devastating effects on the structure of proteins and cells. Autophagy is an evolutionarily conserved intracellular regulated mechanism for orderly degradation of dysfunctional proteins or removing damaged organelles. The precise role of autophagy in oxidative stress-induced apoptosis of dopaminergic neuronal cells caused by METH has not clarified completely. In this study, we sought to evaluate the effects of METH abuse on autophagy in the prefrontal cortex of postmortem users, mainly focusing on the ATG5 and LC3 during neuroinflammation. Postmortem molecular and histological examination was done for two groups containing 12 non-addicted and 14 METH addicted cases. ATG5 and LC3 expression were analyzed by real-time PCR and immunohistochemistry (IHC) methods. Histopathological analysis was performed by stereological cell counting of neuronal cells using Hematoxylin and Eosin (H & E) staining technique. In order to detect DNA damage in the prefrontal lobe, Tunnel staining was performed. Real-time PCR and IHC assay showed overexpression of ATG5 and LC3 protein in the prefrontal cortex of Meth users. The cell death and neuronal degeneration were increased significantly based on Tunel assay and the stereological analysis in the Prefrontal cortex. Chronic METH exposure probably induces ATG5 and LC3 overexpression and neuronal cell death in the Prefrontal cortex of the postmortem cases.

Evaluation of ATG7 and Light Chain 3 (LC3) Autophagy Genes Expression in AML Patients.

Background and aim: Autophagy, known as cell death type II, is a housekeeping pathway that currently has been worked on in matters of tumorigenesis and leukomogenesis. Therefore, expression levels of ATG7 and LC3 as two key genes in AML patients are targeted in this study.Material and method: This study was performed on 55 de novo AML patients against 17 healthy volunteers, acquired samples from bone marrow (BM) and peripheral blood (PB) sources in different ages and gender. The evaluation was executed by mRNA extraction, cDNA synthesis, real-time PCR and data was analyzed by SPSS. Results: Analyzed data indicate a significant decrease between expression of ATG7 and LC3 in AML patients against control (Pv < 0.05). Decrease in both genes expression was detected in most of the patients, 81.81% and 75.55%, respectively. Also LC3 overexpression was detected in 11.33% of AML patients. Moreover, a positive significant correlation between ATG7 and LC3 genes was detected (r = 0.481; Pv = 0.001). Conclusion: This study showed that significant reduction of autophagy genes in de novo AML patients is important to overcome this system and initiate leukomogenesis. It seems a new insight is required for new achievements in diagnosis, prognosis, treatment and monitoring AML patients.

Prognostic relevance of autophagy-related markers LC3, p62/sequestosome 1, Beclin-1 and ULK1 in colorectal cancer patients with respect to KRAS mutational status.

BackgroundAutophagy is a cellular pathway that regulates transportation of cytoplasmic macromolecules and organelles to lysosomes for degradation. Autophagy is involved in both tumorigenesis and tumour suppression. Here we investigated the potential prognostic value of the autophagy-related proteins Beclin-1, p62, LC3 and uncoordinated (UNC) 51-like kinase 1 (ULK1) in a cohort of colorectal cancer (CRC) specimens.MethodsIn this study, we analysed the immunoexpression of the autophagy-related proteins p62, LC3, Beclin-1 and ULK1 in 127 CRC patients with known KRAS mutational status and detailed clinical follow-up.ResultsSurvival analysis of p62 staining showed a significant correlation of cytoplasmic (not nuclear) p62 expression with a favourable tumour-specific overall survival (OS). The prognostic power of cytoplasmic p62 was found in the KRAS-mutated subgroup but was lost in the KRAS wildtype subgroup. Survival analysis of Beclin-1 staining did not show an association with OS in the complete cohort. LC3 overexpression demonstrated a slight, though not significant, association with decreased OS. Upon stratifying cases by KRAS mutational status, nuclear (not cytoplasmic) Beclin-1 staining was associated with a significantly decreased OS in the KRAS-mutated subgroup but not in the KRAS wildtype CRCs. In addition, LC3 overexpression was significantly associated with decreased OS in the KRAS-mutated CRC subgroup. ULK1 expression was not correlated to survival.ConclusionsImmunohistochemical analyses of LC3, p62 and Beclin-1 may constitute promising novel prognostic markers in CRC, especially in KRAS-mutated CRCs. This strategy might help in identifying high-risk patients who would benefit from autophagy-related anticancer drugs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12957-016-0946-x) contains supplementary material, which is available to authorized users.

Defects in MAP1S-mediated autophagy cause reduction in mouse lifespans especially when fibronectin is overexpressed.

Autophagy is a cellular process that executes the turnover of dysfunctional organelles and misfolded or abnormally aggregated proteins. Microtubule-associated protein MAP1S interacts with autophagy marker LC3 and positively regulates autophagy flux. LC3 binds with fibronectinmRNA and facilitates its translation. The synthesized fibronectin protein is exported to cell surface to initiate the assembly of fibronectin extracellular matrix. Fibronectin is degraded in lysosomes after it is engulfed into cytosol via endocytosis. Here, we show that defects in MAP1S-mediated autophagy trigger oxidative stress, sinusoidal dilation, and lifespan reduction. Overexpression of LC3 in wild-type mice increases the levels of fibronectin and γ-H2 AX, a marker of DNA double-strand breakage. LC3-induced fibronectin is efficiently degraded in lysosomes to maintain a balance of fibronectin levels in wild-type mice so that the mice live a normal term of lifespan. In the LC3 transgenic mice with MAP1S deleted, LC3 enhances the synthesis of fibronectin but the MAP1S depletion causes an impairment of the lysosomal degradation of fibronectin. The accumulation of fibronectin protein promotes liver fibrosis, induces an accumulation of cell population at the G0/G1 stage, and further intensifies oxidative stress and sinusoidal dilatation. The LC3-induced overexpression of fibronectin imposes stresses on MAP1S-deficient mice and dramatically reduces their lifespans. Therefore, MAP1S-mediated autophagy plays an important role in maintaining mouse lifespan especially in the presence of extra amount of fibronectin.

Autophagy is deficient and inversely correlated with COX-2 expression in nasal polyps: a novel insight into the inflammation mechanism.

Nasal polyposis is characterised by persistent inflammation of the upper airways. Autophagy has been implicated in many chronic inflammatory diseases. Whether autophagy plays a role in nasal polyp (NP) inflammation is completely unknown and deserves investigation. LC3 and COX-2 expression, the common autophagy and inflammation indicators, respectively, was analysed by immunoblotting in fresh tissues of NP and control nasal mucosa (NM). Primary cultures of NP-derived fibroblasts (NPDFs) and NMDFs were established for in vitro studies. Autophagy was induced by amino acid starvation and LC3 ectopic overexpression or inhibited by 3-methyladenine in the fibroblasts. Inflammation was induced by IL1-β and TNF-α. LC3 and COX-2 expression was confirmed in NP specimens by immunohistochemistry. LC3 expression was decreased while COX-2 expression was significantly increased in fresh NP tissues compared with the NM control. In NMDFs and NPDFs, autophagy induction by starvation and LC3 overexpression downregulated COX-2 expression. Conversely, autophagy inhibition by 3-methyladenine enhanced COX-2 expression. However, IL1-β and TNF-α had no effect on autophagy. Immunohistochemical studies on the NP specimens showed that most displayed low LC3 expression, whereas COX-2 was highly expressed in >50% of the specimens. Examination of two consecutive NP sections from the same tissue blocks revealed a negative correlation between LC3 and COX-2 expression. Autophagy is deficient in NP tissues and COX-2 is negatively regulated by autophagy in NP-derived fibroblasts. Since COX-2 is essential for the production of pro-inflammatory mediators, this study might help interpret persistent mucosal inflammation in NP. Attenuation of inflammation by restoring autophagy might be a therapeutic strategy for treating NP.

LC3 overexpression reduces Aβ neurotoxicity through increasing α7nAchR expression and autophagic activity in neurons and mice

Autophagy is an intracellular degradation pathway with dynamic interactions for eliminating damaged organelles and protein aggregates by lysosomal digestion. The EGFP-conjugated microtubule-associated protein 1 light chain 3 (EGFP-LC3) serves to monitor autophagic process. Extracellular β-amyloid peptide accumulation is reported as a major cause in Alzheimer's disease (AD) pathogenesis; large numbers of autophagic vacuoles accumulate in patients' brains. We previously demonstrated that extracellular Aβ (eAβ) induces strong autophagic response and α7nAChR acts as a carrier to bind with eAβ; which further inhibits Aβ-induced neurotoxicity via autophagic degradation. In the present study, we overexpressed LC3 in both neuroblastoma cells (SH-SY5Y/pEGFP-LC3) and mice (TgEGFP-LC3) to assess the effect of LC3 overexpression on Aβ neurotoxicity. SH-SY5Y/pEGFP-LC3 cells and primary cortical neuron cultures derived from E17 (embryonic day 17) TgEGFP-LC3 mice showed not only better resistance against Aβ neurotoxicity but also higher α7nAChR expression and autophagic activity than control. Administration of α-bungarotoxin (α-BTX) to block α7nAChR antagonized the neuroprotective action of SH-SY5Y/pECGF-LC3 cells, suggesting that eAβ binding with α7nAChR is an important step in Aβ detoxification. LC3 overexpression thus exerts neuroprotection through increasing α7nAChR expression for eAβ binding and further enhancing autophagic activity for Aβ clearance in vitro and in vivo.

Autophagy is deficient and inversely correlated with COX-2 expression in nasal polyps: a novel insight into the inflammation mechanism

Nasal polyposis is characterised by persistent inflammation of the upper airways. Autophagy has been implicated in many chronic inflammatory diseases. Whether autophagy plays a role in nasal polyp (NP) inflammation is completely unknown and deserves investigation. LC3 and COX-2 expression, the common autophagy and inflammation indicators, respectively, was analysed by immunoblotting in fresh tissues of NP and control nasal mucosa (NM). Primary cultures of NP-derived fibroblasts (NPDFs) and NMDFs were established for in vitro studies. Autophagy was induced by amino acid starvation and LC3 ectopic overexpression or inhibited by 3-methyladenine in the fibroblasts. Inflammation was induced by IL1-β and TNF-α. LC3 and COX-2 expression was confirmed in NP specimens by immunohistochemistry. LC3 expression was decreased while COX-2 expression was significantly increased in fresh NP tissues compared with the NM control. In NMDFs and NPDFs, autophagy induction by starvation and LC3 overexpression downregulated COX-2 expression. Conversely, autophagy inhibition by 3-methyladenine enhanced COX-2 expression. However, IL1-β and TNF-α had no effect on autophagy. Immunohistochemical studies on the NP specimens showed that most displayed low LC3 expression, whereas COX-2 was highly expressed in >50% of the specimens. Examination of two consecutive NP sections from the same tissue blocks revealed a negative correlation between LC3 and COX-2 expression. Autophagy is deficient in NP tissues and COX-2 is negatively regulated by autophagy in NP-derived fibroblasts. Since COX-2 is essential for the production of pro-inflammatory mediators, this study might help interpret persistent mucosal inflammation in NP. Attenuation of inflammation by restoring autophagy might be a therapeutic strategy for treating NP.

Loss of PINK1 impairs stress-induced autophagy and cell survival.

The mitochondrial kinase PINK1 and the ubiquitin ligase Parkin are participating in quality control after CCCP- or ROS-induced mitochondrial damage, and their dysfunction is associated with the development and progression of Parkinson's disease. Furthermore, PINK1 expression is also induced by starvation indicating an additional role for PINK1 in stress response. Therefore, the effects of PINK1 deficiency on the autophago-lysosomal pathway during stress were investigated. Under trophic deprivation SH-SY5Y cells with stable PINK1 knockdown showed downregulation of key autophagic genes, including Beclin, LC3 and LAMP-2. In good agreement, protein levels of LC3-II and LAMP-2 but not of LAMP-1 were reduced in different cell model systems with PINK1 knockdown or knockout after addition of different stressors. This downregulation of autophagic factors caused increased apoptosis, which could be rescued by overexpression of LC3 or PINK1. Taken together, the PINK1-mediated reduction of autophagic key factors during stress resulted in increased cell death, thus defining an additional pathway that could contribute to the progression of Parkinson's disease in patients with PINK1 mutations.

Angiogenesis impairment in diabetes: role of methylglyoxal-induced receptor for advanced glycation endproducts, autophagy and vascular endothelial growth factor receptor 2.

Diabetes impairs physiological angiogenesis by molecular mechanisms that are not fully understood. Methylglyoxal (MGO), a metabolite of glycolysis, is increased in patients with diabetes. This study defined the role of MGO in angiogenesis impairment and tested the mechanism in diabetic animals. Endothelial cells and mouse aortas were subjected to Western blot analysis of vascular endothelial growth factor receptor 2 (VEGFR2) protein levels and angiogenesis evaluation by endothelial cell tube formation/migration and aortic ring assays. Incubation with MGO reduced VEGFR2 protein, but not mRNA, levels in a time and dose dependent manner. Genetic knockdown of the receptor for advanced glycation endproducts (RAGE) attenuated the reduction of VEGFR2. Overexpression of Glyoxalase 1, the enzyme that detoxifies MGO, reduced the MGO-protein adducts and prevented VEGFR2 reduction. The VEGFR2 reduction was associated with impaired angiogenesis. Suppression of autophagy either by inhibitors or siRNA, but not of the proteasome and caspase, normalized both the VEGFR2 protein levels and angiogenesis. Conversely, induction of autophagy either by rapamycin or overexpression of LC3 and Beclin-1 reduced VEGFR2 and angiogenesis. MGO increased endothelial LC3B and Beclin-1, markers of autophagy, which were accompanied by an increase of both autophagic flux (LC3 punctae) and co-immunoprecipitation of VEGFR2 with LC3. Pharmacological or genetic suppression of peroxynitrite (ONOO−) generation not only blocked the autophagy but also reversed the reduction of VEGFR2 and angiogenesis. Like MGO-treated aortas from normglycemic C57BL/6J mice, aortas from diabetic db/db and Akita mice presented reductions of angiogenesis or VEGFR2. Administration of either autophagy inhibitor ex vivo or superoxide scavenger in vivo abolished the reductions. Taken together, MGO reduces endothelial angiogenesis through RAGE-mediated, ONOO–dependent and autophagy-induced VEGFR2 degradation, which may represent a new mechanism for diabetic angiogenesis impairment.

Copper compound induces autophagy and apoptosis of glioma cells by reactive oxygen species and jnk activation

BackgroundGlioblastoma multiforme (GBM) is the most aggressive of the primary brain tumors, with a grim prognosis despite intensive treatment. In the past decades, progress in research has not significantly increased overall survival rate.MethodsThe in vitro antineoplastic effect and mechanism of action of Casiopeina III-ia (Cas III-ia), a copper compound, on rat malignant glioma C6 cells was investigated.ResultsCas III-ia significantly inhibited cell proliferation, inducing autophagy and apoptosis, which correlated with the formation of autophagic vacuoles, overexpression of LC3, Beclin 1, Atg 7, Bax and Bid proteins. A decrease was detected in the mitochondrial membrane potential and in the activity of caspase 3 and 8, together with the generation of intracellular reactive oxygen species (ROS) and increased activity of c-jun NH2-terminal kinase (JNK). The presence of 3-methyladenine (as selective autophagy inhibitor) increased the antineoplastic effect of Cas III-ia, while Z-VAD-FMK only showed partial protection from the antineoplastic effect induced by Cas III-ia, and ROS antioxidants (N-acetylcysteine) decreased apoptosis, autophagy and JNK activity. Moreover, the JNK –specific inhibitor SP600125 prevented Cas III-ia-induced cell death.ConclusionsOur data suggest that Cas III-ia induces cell death by autophagy and apoptosis, in part due to the activation of ROS –dependent JNK signaling. These findings support further studies of Cas III-ia as candidate for treatment of human malignant glioma.

Role of Rapamycin-Induced Autophagy in Pancreatic Islets

We recently published our work in this journal on the ef-fectsofrapamycinonpancreaticb-cellsdysfunctioninisletgrafts, including autophagy (1). The comment followed byPallet(2)providesfurtheropportunitytoemphasizeseveralpoints discussed in our paper.Autophagy is a tightly regulated degradation process thatdelivers cytoplasmic material to the lysosomes via theautophagosomes. We reported previously that rapamycinupregulates autophagy in islet b-cells and that this ef-fect results in cell death and impaired insulin potency.Autophagosomes contain microtubule-associated protein-light-chain-3(LC3),whichisusedasamarkerofautophagy.The rapamycin-induced autophagy in pancreatic islets wasconfirmed by two widely used methods. First, the uncon-jugated (LC3-I) and conjugated forms (LC3-II) of LC3 wereseparated by SDS-PAGE; then immunoblotting showed ac-cumulation of LC3-II, which correlates with the numberof autophagosomes relative to the amount of endoge-nous LC3-II protein in islets. Second, LC3 distribution wasmappedinGFP-LC3transgenicmice.ThismethodisbasedondetectionofLC3-positivestructuresbyfluorescencemi-croscopy, although it has certain limitations. Under certainconditions, e.g. LC3 overexpression, LC3 easily incorpo-rates into the protein aggregates, hindering accurate lo-calization. Although this limitation is independent of au-tophagy, any LC3 localization by this method should beinterpreted with caution. In our study (1), we did not per-form transient transfection with GFP-LC3 gene but usedstable GFP-LC3 transgenic mice. The data showed in-creased autophagosome production in islets, though thiswas not confirmed by electron microscopy.A major problem inautophagyresearch isthelack of highlyspecific autophagy inhibitors. PI3-kinase inhibitors such aswortmannin and 3-MA (3) are the most commonly usedautophagy inhibitors, but they inhibit the activities of bothclass I and III PI3-kinase. Indeed, 3-MA, which was used inour study (1), targets other kinases and affects other cel-lular processes, especially at high concentrations. Accord-ingly, we used this regent at 10 mM, which is the recom-mended concentration for inhibition of autophagy. While3-MA and wortmannin block the formation of autophago-somes at early stages of autophagy, other PI3-kinase in-hibitors (e.g. bafilomycin A1), which was recommended byPallet (2), block later stages of autophagy. Bafilomycin A1was reported to inhibit autophagosome-lysosome fusion,and also altered intralysosomal degradation by inhibitingacidification (4).Recent loss-of-function studies of Atg genes have ana-lyzedtheparadoxicalfunctionsofautophagyincellsurvivaland death. We also examined this issue in our rapamycin-treated islets (1). Another study demonstrated that in-duction of autophagy over 48 h resulted in cell death (5)whereas induction over 12 h had no effect on cell viabil-ity. These findings support the more rational belief that au-tophagyfacilitatestheremovalofdamagedproteinsandor-ganelles, thus providing temporary protection during tran-sient stress; but can also lead to “autophagic cell death”under prolonged stimulation. Perhaps this death-inducingfunctionofautophagyistriggeredbythedigestionofacriti-calamountofcytoplasmiccontentsnecessaryforsurvival,with subsequent activation of death-promoting molecules.Further research is required in this field including crosstalkbetween autophagic and apoptotic cell death.

Methamphetamine-induced neurotoxicity linked to ubiquitin-proteasome system dysfunction and autophagy-related changes that can be modulated by protein kinase C delta in dopaminergic neuronal cells

A compromised protein degradation machinery has been implicated in methamphetamine (MA)-induced neurodegeneration. However, the signaling mechanisms that induce autophagy and ubiquitin-proteasome system (UPS) dysfunction are not well understood. The present study investigates the contributions of protein kinase C delta (PKCδ)-mediated signaling events in MA-induced autophagy, UPS dysfunction, and cell death. Using an in vitro mesencephalic dopaminergic cell culture model, we demonstrate that MA-induced early induction of autophagy is associated with reduction in proteasomal function and concomitant dissipation of mitochondrial membrane potential (MMP), followed by significantly increased PKCδ activation, caspase-3 activation, accumulation of ubiquitin-positive aggregates and microtubule-associated light chain-3 (LC3-II) levels. Interestingly, siRNA-mediated knockdown of PKCδ or overexpression of cleavage-resistant mutant of PKCδ dramatically reduced MA-induced autophagy, proteasomal function, and associated accumulation of ubiquitinated protein aggregates, which closely paralleled cell survival. Importantly, when autophagy was inhibited either pharmacologically (3-MA) or genetically (siRNA-mediated silencing of LC3), the dopaminergic cells became sensitized to MA-induced apoptosis through caspase-3 activation. Conversely, overexpression of LC3 partially protected against MA-induced apoptotic cell death, suggesting a neuroprotective role for autophagy in MA-induced neurotoxicity. Notably, rat striatal tissue isolated from MA-treated rats also exhibited elevated LC3-II, ubiquitinated protein levels, and PKCδ cleavage. Taken together, our data demonstrate that MA-induced autophagy serves as an adaptive strategy for inhibiting mitochondria-mediated apoptotic cell death and degradation of aggregated proteins. Our results also suggest that the sustained activation of PKCδ leads to UPS dysfunction, resulting in the activation of caspase-3-mediated apoptotic cell death in the nigrostriatal dopaminergic system.

The Ubiquitin-like Protein LC3 Regulates the Rho-GEF Activity of AKAP-Lbc

AKAP-Lbc is a member of the A-kinase anchoring protein (AKAP) family that has been recently associated with the development of pathologies, such as cardiac hypertrophy and cancer. We have previously demonstrated that, at the molecular level, AKAP-Lbc functions as a guanine nucleotide exchange factor (GEF) that promotes the specific activation of RhoA. In the present study, we identified the ubiquitin-like protein LC3 as a novel regulatory protein interacting with AKAP-Lbc. Mutagenesis studies revealed that LC3, through its NH(2)-terminal alpha-helical domain, interacts with two binding sites located within the NH(2)-terminal regulatory region of AKAP-Lbc. Interestingly, LC3 overexpression strongly reduced the ability of AKAP-Lbc to interact with RhoA, profoundly impairing the Rho-GEF activity of the anchoring protein and, as a consequence, its ability to promote cytoskeletal rearrangements associated with the formation of actin stress fibers. Moreover, AKAP-Lbc mutants that fail to interact with LC3 show a higher basal Rho-GEF activity as compared with the wild type protein and become refractory to the inhibitory effect of LC3. This suggests that LC3 binding maintains AKAP-Lbc in an inactive state that displays a reduced ability to promote downstream signaling. Collectively, these findings provide evidence for a previously uncharacterized role of LC3 in the regulation of Rho signaling and in the reorganization of the actin cytoskeleton.

Autophagy in MHC Class II Presentation: Sampling from Within

MHC class II molecules usually present exogenous antigens, but peptidome analyses have also identified many antigens from cytosolic or nuclear sources. In this issue of Immunity, Schmid et al. show that MHC class II molecules can present these through autophagosomes.