Aβ1-42 monomers or oligomers have different effects on autophagy and apoptosis

The role of autophagy and its relationship with apoptosis in Alzheimer disease (AD) pathogenesis is poorly understood. Disruption of autophagy leads to buildup of incompletely digested substrates, amyloid-β (Aβ) peptide accumulation in vacuoles and cell death. Aβ, in turn, has been found to affect autophagy. Thus, Aβ might be part of a loop in which it is both the substrate of altered autophagy and its cause. Given the relevance of different soluble forms of Aβ1-42 in AD, we have investigated whether monomers and oligomers of the peptide have a differential role in causing altered autophagy and cell death. Using differentiated SK-N-BE neuroblastoma cells, we found that monomers hamper the formation of the autophagic BCL2-BECN1/Beclin 1 complex and activate the MAPK8/JNK1-MAPK9/JNK2 pathway phosphorylating BCL2. Monomers also inhibit apoptosis and allow autophagy with intracellular accumulation of autophagosomes and elevation of levels of BECN1 and LC3-II, resulting in an inhibition of substrate degradation due to an inhibitory action on lysosomal activity. Oligomers, in turn, favor the formation of the BCL2-BECN1 complex favoring apoptosis. In addition, they cause a less profound increase in BECN1 and LC3-II levels than monomers without affecting the autophagic flux. Thus, data presented in this work show a link for autophagy and apoptosis with monomers and oligomers, respectively. These studies are likely to help the design of novel disease modifying therapies.     

S6K1 Negatively Regulates TAK1 Activity in the Toll-Like Receptor Signaling Pathway

Transforming growth factor β (TGF-β)-activated kinase 1 (TAK1) is a key regulator in the signals transduced by proinflammatory cytokines and Toll-like receptors (TLRs). The regulatory mechanism of TAK1 in response to various tissue types and stimuli remains incompletely understood. Here, we show that ribosomal S6 kinase 1 (S6K1) negatively regulates TLR-mediated signals by inhibiting TAK1 activity. S6K1 overexpression causes a marked reduction in NF-κB and AP-1 activity induced by stimulation of TLR2 or TLR4. In contrast, S6K1^−/− and S6K1 knockdown cells display enhanced production of inflammatory cytokines. Moreover, S6K1^−/− mice exhibit decreased survival in response to challenge with lipopolysaccharide (LPS). We found that S6K1 inhibits TAK1 kinase activity by interfering with the interaction between TAK1 and TAB1, which is a key regulator protein for TAK1 catalytic function. Upon stimulation with TLR ligands, S6K1 deficiency causes a marked increase in TAK1 kinase activity that in turn induces a substantial enhancement of NF-κB-dependent gene expression, indicating that S6K1 is negatively involved in the TLR signaling pathway by the inhibition of TAK1 activity. Our findings contribute to understanding the molecular pathogenesis of the impaired immune responses seen in type 2 diabetes, where S6K1 plays a key role both in driving insulin resistance and modulating TLR signaling.     

The Metastasis Suppressor,N-myc Downstream-regulated Gene 1 (NDRG1), Inhibits Stress-induced Autophagy in Cancer Cells

N-myc downstream regulated gene 1 (NDRG1) is a potent metastasis suppressor with an undefined role in the stress response. Autophagy is a pro-survival pathway and can be regulated via the protein kinase-like endoplasmic reticulum kinase (PERK)/eIF2α-mediated endoplasmic reticulum (ER) stress pathway. Hence, we investigated the role of NDRG1 in stress-induced autophagy as a mechanism of inhibiting metastasis via the induction of apoptosis. As thiosemicarbazone chelators induce stress and up-regulate NDRG1 to inhibit metastasis, we studied their effects on the ER stress response and autophagy. This was important to assess, as little is understood regarding the role of the stress induced by iron depletion and its role in autophagy. We observed that the chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), which forms redox-active iron and copper complexes, effectively induced ER stress as shown by activation of the PERK/eIF2α pathway. Dp44mT also increased the expression of the autophagic marker, LC3-II, and this was dependent on activation of the PERK/eIF2α axis, as silencing PERK prevented LC3-II accumulation. The effect of Dp44mT on LC3-II expression was at least partially due to iron-depletion, as this effect was also demonstrated with the classical iron chelator, desferrioxamine (DFO), and was not observed for the DFO-iron complex. NDRG1 overexpression also inhibited basal autophagic initiation and the ER stress-mediated autophagic pathway via suppression of the PERK/eIF2α axis. Moreover, NDRG1-mediated suppression of the pro-survival autophagic pathway probably plays a role in its anti-metastatic effects by inducing apoptosis. In fact, multiple pro-apoptotic markers were increased, whereas anti-apoptotic Bcl-2 was decreased upon NDRG1 overexpression. This study demonstrates the role of NDRG1 as an autophagic inhibitor that is important for understanding its mechanism of action.     

Activation of CREB‐mediated autophagy by thioperamide ameliorates β‐amyloid pathology and cognition in Alzheimer’s disease

Alzheimer''s disease (AD) is an age‐related neurodegenerative disease, and the imbalance between production and clearance of β‐amyloid (Aβ) is involved in its pathogenesis. Autophagy is an intracellular degradation pathway whereby leads to removal of aggregated proteins, up‐regulation of which may be a plausible therapeutic strategy for the treatment of AD. Histamine H3 receptor (H3R) is a presynaptic autoreceptor regulating histamine release via negative feedback way. Our previous study showed that thioperamide, as an antagonist of H3R, enhances autophagy and protects against ischemic injury. However, the effect of thioperamide on autophagic function and Aβ pathology in AD remains unknown. In this study, we found that thioperamide promoted cognitive function, ameliorated neuronal loss, and Aβ pathology in APP/PS1 transgenic (Tg) mice. Interestingly, thioperamide up‐regulated autophagic level and lysosomal function both in APP/PS1 Tg mice and in primary neurons under Aβ‐induced injury. The neuroprotection by thioperamide against AD was reversed by 3‐MA, inhibitor of autophagy, and siRNA of Atg7, key autophagic‐related gene. Furthermore, inhibition of activity of CREB, H3R downstream signaling, by H89 reversed the effect of thioperamide on promoted cell viability, activated autophagic flux, and increased autophagic‐lysosomal proteins expression, including Atg7, TFEB, and LAMP1, suggesting a CREB‐dependent autophagic activation by thioperamide in AD. Taken together, these results suggested that H3R antagonist thioperamide improved cognitive impairment in APP/PS1 Tg mice via modulation of the CREB‐mediated autophagy and lysosomal pathway, which contributed to Aβ clearance. This study uncovered a novel mechanism involving autophagic regulating behind the therapeutic effect of thioperamide in AD.     

TGF-β1 Protects against Mesangial Cell Apoptosis via Induction of Autophagy

Autophagy can lead to cell death in response to stress, but it can also act as a protective mechanism for cell survival. We show that TGF-β1 induces autophagy and protects glomerular mesangial cells from undergoing apoptosis during serum deprivation. Serum withdrawal rapidly induced autophagy within 1 h in mouse mesangial cells (MMC) as determined by increased microtubule-associated protein 1 light chain 3 (LC3) levels and punctate distribution of the autophagic vesicle-associated-form LC3-II. We demonstrate that after 1 h there was a time-dependent decrease in LC3 levels that was accompanied by induction of apoptosis, evidenced by increases in cleaved caspase 3. However, treatment with TGF-β1 resulted in induction of the autophagy protein LC3 while suppressing caspase 3 activation. TGF-β1 failed to rescue MMC from serum deprivation-induced apoptosis upon knockdown of LC3 by siRNA and in MMC from LC3 null (LC3^−/−) mice. We show that TGF-β1 induced autophagy through TAK1 and Akt activation, and inhibition of PI3K-Akt pathway by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or dominant-negative Akt suppressed LC3 levels and enhanced caspase 3 activation. TGF-β1 also up-regulated cyclin D1 and E protein levels while down-regulating p27, thus stimulating cell cycle progression. Bafilomycin A1, but not MG132, blocked TGF-β1 down-regulation of p27, suggesting that p27 levels were regulated through autophagy. Taken together, our data indicate that TGF-β1 rescues MMC from serum deprivation-induced apoptosis via induction of autophagy through activation of the Akt pathway. The autophagic process may constitute an adaptive mechanism to glomerular injury by inhibiting apoptosis and promoting mesangial cell survival.     

Autophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome

Accumulation of β-amyloid (Aβ) and resultant inflammation are critical pathological features of Alzheimer disease (AD). Microglia, a primary immune cell in brain, ingests and degrades extracellular Aβ fibrils via the lysosomal system. Autophagy is a catabolic process that degrades native cellular components, however, the role of autophagy in Aβ degradation by microglia and its effects on AD are unknown. Here we demonstrate a novel role for autophagy in the clearance of extracellular Aβ fibrils by microglia and in the regulation of the Aβ-induced NLRP3 (NLR family, pyrin domain containing 3) inflammasome using microglia specific atg7 knockout mice and cell cultures. We found in microglial cultures that Aβ interacts with MAP1LC3B-II via OPTN/optineurin and is degraded by an autophagic process mediated by the PRKAA1 pathway. We anticipate that enhancing microglial autophagy may be a promising new therapeutic strategy for AD.     

Adiponectin up‐regulates the decrease of myocardial autophagic flux induced by β1‐adrenergic receptor autoantibody partly dependent on AMPK

Cardiomyocytes autophagy is essential for maintaining cardiac function. Our previous studies have found that β₁‐adrenergic receptor autoantibody (β₁‐AA) induced the decreased myocardial autophagic flux, which resulted in cardiomyocyte death and cardiac dysfunction. And other studies demonstrated that β₁‐AA induced the decrease of AMPK phosphorylation, the key hub of autophagy pathway, while adiponectin up‐regulated autophagic flux mediated by AMPK. However, it is not clear whether adiponectin improves the inhibition of myocardial autophagic flux induced by β₁‐AA by up‐regulating the level of AMPK phosphorylation. In this study, it has been confirmed that β₁‐AA induced the decrease of AMPK phosphorylation level in both vivo and vitro. Moreover, pretreatment of cardiomyocytes with AMPK inhibitor Compound C could further reduce the autophagic flux induced by β₁‐AA. Adiponectin deficiency could aggravate the decrease of myocardial AMPK phosphorylation level, autophagic flux and cardiac function induced by β₁‐AA. Further, exogenous adiponectin could reverse the decline of AMPK phosphorylation level and autophagic flux induced by β₁‐AA and even reduce cardiomyocyte death. While pretreated with the Compound C, the adiponectin treatment did not improve the decreased autophagosome formation, but still improved the decreased autophagosome clearance induced by β₁‐AA in cardiomyocytes. This study is the first time to confirm that β₁‐AA could inhibit myocardial autophagic flux by down‐regulating AMPK phosphorylation level. Adiponectin could improve the inhibition of myocardial autophagic flux induced by β₁‐AA partly dependent on AMPK, so as to provide an experimental basis for the treatment of patients with β₁‐AA‐positive cardiac dysfunction.     

TAK1 Is Required for Survival of Mouse Fibroblasts Treated with TRAIL,and Does So by NF-κB Dependent Induction of cFLIPL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a “death ligand”—a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-κB and JNK signalling pathways. To determine the role of TGF-β-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1−/− MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-κB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-κB, protected TAK1−/− MEFs against TRAIL killing, suggesting that TAK1 activation of NF-κB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-κB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK1−/− MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1–NF-κB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance.     

Inhibition of the sonic hedgehog pathway activates TGF-β-activated kinase (TAK1) to induce autophagy and suppress apoptosis in thyroid tumor cells

The sonic hedgehog (Shh) pathway is highly activated in a variety of malignancies and plays important roles in tumorigenesis, tumor growth, drug resistance, and metastasis. Our recent study showed that the inhibitors of the Shh pathway such as cyclopamine (CP), a Smothened (SMO) inhibitor, and GANT61, a Gli1 inhibitor, have modest inhibitory effects on thyroid tumor cell proliferation and tumor growth. The objective of this study was to determine whether autophagy was induced by inhibition of the Shh pathway and could negatively regulate GANT61-induced apoptosis. Here we report that inhibition of the Shh pathway by Gli1 siRNA or by cyclopamine and GANT61 induced autophagy in SW1736 and KAT-18 cells, two anaplastic thyroid cancer cell lines; whereas Gli1 overexpression suppressed autophagy. Mechanistic investigation revealed that inhibition of the Shh pathway activated TAK1 and its two downstream kinases, the c-Jun-terminal kinase (JNK) and AMP-activated protein kinase (AMPK). GANT61-induced autophagy was blocked by TAK1 siRNA and the inhibitors of TAK1 (5Z-7-oxozeaenol, 5Z), JNK (SP600125), and AMPK (Compound C, CC). Inhibition of autophagy by chloroquine and 5Z and by TAK1 and Beclin-1 siRNA enhanced GANT61-induced apoptosis and its antiproliferative activity. Our study has shown that inhibition of the Shh pathway induces autophagy by activating TAK1, whereas autophagy in turn suppresses GANT61-induced apoptosis. We have uncovered a previously unrecognized role of TAK1 in Shh pathway inhibition-induced autophagy and apoptosis.Subject terms: Thyroid cancer, Macroautophagy, Apoptosis, Drug development     

Macropinocytosis and TAK1 mediate anti-inflammatory to pro-inflammatory macrophage differentiation by HIV-1 Nef

Macrophages (MΦ) are functionally classified into two types, anti-inflammatory M2 and pro-inflammatory M1. Importantly, we recently revealed that soluble HIV-1 proteins, particularly the pathogenetic protein Nef, preferentially activate M2-MΦ and drive them towards an M1-like MΦ, which might explain the sustained immune activation seen in HIV-1-infected patients. Here, we show that the preferential effect of Nef on M2-MΦ is mediated by TAK1 (TGF-β-activated kinase 1) and macropinocytosis. As with MAP kinases and NF-κB pathway, Nef markedly activated TAK1 in M-CSF-derived M2-MΦ but not in GM-CSF-derived M1-MΦ. Two Nef mutants, which were unable to activate MAP kinases and NF-κB pathway, failed to activate TAK1. Indeed, the TAK1 inhibitor 5Z-7-oxozeaenol as well as the ectopic expression of a dominant-negative mutant of TAK1 or TRAF2, an upstream molecule of TAK1, inhibited Nef-induced signaling activation and M1-like phenotypic differentiation of M2-MΦ. Meanwhile, the preferential effect of Nef on M2-MΦ correlated with the fact the Nef entered M2-MΦ more efficiently than M1-MΦ. Importantly, the macropinosome formation inhibitor EIPA completely blocked the internalization of Nef into M2-MΦ. Because the macropinocytosis activity of M2-MΦ was higher than that of M1-MΦ, our findings indicate that Nef enters M2-MΦ efficiently by exploiting their higher macropinocytosis activity and drives them towards M1-like MΦ by activating TAK1.     

Positive regulation of TNFR1 signaling via SH3 recognition motif

TNF is a pleiotropic cytokine and shows its biological function by binding to its receptors called TNFR1 and TNFR2. While TNFR1 induces apoptosis by activation of caspase-8 via the “death domain”, it also activates IKKα/β, MKK3/6, MKK4/7 by activation of TAK1. Although the TNFR1 signaling pathway is known by in large, it is not known how AKT and MAPKs p38, ERK1/2, and JNK1/2 are activated. The presence of a proline-rich PPAP region, (P448PAP451, a binding site for the SH3 domain-containing proteins) very close to the C-terminus promoted us to determine whether this region has any role in the TNFR1 signal transduction. To test this, the codons of P448 and P451 were changed to that of Alanin, GCG, via site-directed mutagenesis, and this plasmid was named as TNFR1-SH3-P/A. Subsequently, ectopically expressed the wild type TNFR1 and TNFR1-SH3-P/A in 293T cells and determined the levels of TNF-α-mediated phosphorylations of ERK, p38, JNK and AKT, NF-kB, and caspase-8 activation. While ectopic expression of our mutant diminished TNFα-mediated phosphorylations of p38, JNK, ERK and AKT, it increased NF-kB, and caspase-8 activations. In conclusion, TNFα-mediated ERK, AKT, JNK, p38 activations are affected by TNFR1 SH3 domain modifications.     

Restarting stalled autophagy a potential therapeutic approach for the lipid storage disorder,Niemann-Pick type C1 disease

Autophagy is essential for cellular homeostasis and its dysfunction in human diseases has been implicated in the accumulation of misfolded protein and in cellular toxicity. We have recently shown impairment in autophagic flux in the lipid storage disorder, Niemann-Pick type C1 (NPC1) disease associated with abnormal cholesterol sequestration, where maturation of autophagosomes is impaired due to defective amphisome formation caused by failure in SNARE machinery. Abrogation of autophagy also causes cholesterol accumulation, suggesting that defective autophagic flux in NPC1 disease may act as a primary causative factor not only by imparting its deleterious effects, but also by increasing cholesterol load. However, cholesterol depletion treatment with HP-β-cyclodextrin impedes autophagy, whereas pharmacologically stimulating autophagy restores its function independent of amphisome formation. Of potential therapeutic relevance is that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may rescue both the cholesterol and autophagy defects in NPC1 disease.     

HTLV-1 Tax Stimulates Ubiquitin E3 Ligase,Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation

Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechanisms remain obscure. IKK and its upstream kinase, TGFβ-activated kinase 1 (TAK1), contain ubiquitin-binding subunits, NEMO and TAB2/3 respectively, which interact with K63-linked polyubiquitin (K63-pUb) chains. Recruitment to K63-pUb allows cross auto-phosphorylation and activation of TAK1 to occur, followed by TAK1-catalyzed IKK phosphorylation and activation. Using cytosolic extracts of HeLa and Jurkat T cells supplemented with purified proteins we have identified ubiquitin E3 ligase, ring finger protein 8 (RNF8), and E2 conjugating enzymes, Ubc13:Uev1A and Ubc13:Uev2, to be the cellular factors utilized by Tax for TAK1 and IKK activation. In vitro, the combination of Tax and RNF8 greatly stimulated TAK1, IKK, IκBα and JNK phosphorylation. In vivo, RNF8 over-expression augmented while RNF8 ablation drastically reduced canonical NF-κB activation by Tax. Activation of the non-canonical NF-κB pathway by Tax, however, is unaffected by the loss of RNF8. Using purified components, we further demonstrated biochemically that Tax greatly stimulated RNF8 and Ubc13:Uev1A/Uev2 to assemble long K63-pUb chains. Finally, co-transfection of Tax with increasing amounts of RNF8 greatly induced K63-pUb assembly in a dose-dependent manner. Thus, Tax targets RNF8 and Ubc13:Uev1A/Uev2 to promote the assembly of K63-pUb chains, which signal the activation of TAK1 and multiple downstream kinases including IKK and JNK. Because of the roles RNF8 and K63-pUb chains play in DNA damage repair and cytokinesis, this mechanism may also explain the genomic instability of HTLV-1-transformed T cells and ATL cells.     

In Vivo Knockdown of TAK1 Accelerates Bone Marrow Proliferation/Differentiation and Induces Systemic Inflammation

TAK1 (TGF-β Activated Kinase 1) is a MAPK kinase kinase, which activates the p38- and JNK-MAPK and NF-κB pathways downstream of receptors such as Toll-Like-, cytokine- and T-cell and B-cell receptors. Representing such an important node in the pro-inflammatory signal-transduction network, the function of TAK1 has been studied extensively. TAK1 knock-out mice are embryonic lethal, while conditional knock-out mice demonstrated either a pro- or anti-inflammatory function. To study the function of TAK1 protein in the adult immune system, we generated and characterized a transgenic mouse expressing TAK1 shRNA under the control of a doxycycline-inducible promoter. Following treatment of TAK-1 shRNA transgenic mice with doxycycline an effective knockdown of TAK1 protein levels was observed in lymphoid organs and cells in the peritoneal cavity (>50% down regulation). TAK1 knockdown resulted in significant changes in leukocyte populations in blood, bone marrow, spleen and peritoneal cavity. Upon TAK1 knockdown mice demonstrated splenomegaly, signs of systemic inflammation (increased levels of circulating cytokines and increase in cellularity of the B-cell areas and in germinal center development in the follicles) and degenerative changes in heart, kidneys and liver. Not surprisingly, TAK1-Tg mice treated with LPS or anti-CD3 antibodies showed enhanced cytokine/chemokine secretion. Finally, analysis of progenitor cells in the bone marrow upon doxycycline treatment showed increased proliferation and differentiation of myeloid progenitor cells. Given the similarity of the phenotype with TGF-β genetic models, our data suggest that in our model the function of TAK1 in TGF-β signal-transduction is overruling its function in pro-inflammatory signaling.     

Inhibition of Autophagic Turnover in β-Cells by Fatty Acids and Glucose Leads to Apoptotic Cell Death

Autophagy, a cellular recycling process responsible for turnover of cytoplasmic contents, is critical for maintenance of health. Defects in this process have been linked to diabetes. Diabetes-associated glucotoxicity/lipotoxicity contribute to impaired β-cell function and have been implicated as contributing factors to this disease. We tested the hypothesis that these two conditions affect β-cell function by modulating autophagy. We report that exposure of β-cell lines and human pancreatic islets to high levels of glucose and lipids blocks autophagic flux and leads to apoptotic cell death. EM analysis showed accumulation of autophagy intermediates (autophagosomes), with abundant engulfed cargo in palmitic acid (PA)- or glucose-treated cells, indicating suppressed autophagic turnover. EM studies also showed accumulation of damaged mitochondria, endoplasmic reticulum distention, and vacuolar changes in PA-treated cells. Pulse-chase experiments indicated decreased protein turnover in β-cells treated with PA/glucose. Expression of mTORC1, an inhibitor of autophagy, was elevated in β-cells treated with PA/glucose. mTORC1 inhibition, by treatment with rapamycin, reversed changes in autophagic flux, and cell death induced by glucose/PA. Our results indicate that nutrient toxicity-induced cell death occurs via impaired autophagy and is mediated by activation of mTORC1 in β-cells, contributing to β-cell failure in the presence of metabolic stress.