An insight into the mechanistic role of p53-mediated autophagy induction in response to proteasomal inhibition-induced neurotoxicity

The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the two most important components of cellular mechanisms for protein degradation. In the present study we investigated the functional relationship of the two systems and the interactional role of p53 in vitro. Our study showed that the proteasome inhibitor lactacystin induced an increase in p53 level and autophagy activity, whereas inhibition of p53 by pifithrin-α or small interference RNA (siRNA) of p53 attenuated the autophagy induction and increased protein aggregation. Furthermore, we found that the pretreatment with the autophagy inhibitor 3-methyladenine or Beclin 1 siRNA further activated p53 and its downstream apoptotic pathways, while the autophagy inducer rapamycin showed the opposite effects. Moreover, we demonstrated that rapamycin pretreatment increased tyrosine hydroxylase (TH) protein level in dopamine (DA) neurons, which was associated with its induction of autophagy to degrade aggregated proteins. Our results suggest that p53 can mediate proteasomal inhibition-induced autophagy enhancement which in turn can partially block p53 or its downstream mitochondria-dependent apoptotic pathways. Further autophagy induction with rapamycin protects DA neurons from lactacystin-mediated cell death by downregulating p53 and its related apoptotic pathways and by inducing autophagy to degrade aggregated proteins. Therefore, rapamycin may be a promising drug for protection against neuronal injury relevant to Parkinson’s disease (PD). Our studies thus provide a mechanistic insight into the functional link between the two protein degradation systems.     

The role of autophagy in the pathogenesis of exposure keratitis

Incomplete tear film spreading and eyelid closure can cause defective renewal of the ocular surface and air exposure‐induced epithelial keratopathy (EK). In this study, we characterized the role of autophagy in mediating the ocular surface changes leading to EK. Human corneal epithelial cells (HCECs) and C57BL/6 mice were employed as EK models, respectively. Transmission electron microscopy (TEM) evaluated changes in HCECs after air exposure. Each of these models was treated with either an autophagy inhibitor [chloroquine (CQ) or 3‐methyladenine (3‐MA)] or activator [Rapamycin (Rapa)]. Immunohistochemistry assessed autophagy‐related proteins, LC3 and p62 expression levels. Western blotting confirmed the expression levels of the autophagy‐related proteins [Beclin1 and mammalian target of rapamycin (mTOR)], the endoplasmic reticulum (ER) stress‐related proteins (PERK, eIF2α and CHOP) and the PI3K/Akt/mTOR signalling pathway‐related proteins. Real‐time quantitative PCR (qRT‐PCR) determined IL‐1β, IL‐6 and MMP9 gene expression levels. The TUNEL assay detected apoptotic cells. TEM identified autophagic vacuoles in both EK models. Increased LC3 puncta formation and decreased p62 immunofluorescent staining and Western blotting confirmed autophagy induction. CQ treatment increased TUNEL positive staining in HCECs, while Rapa had an opposite effect. Similarly, CQ injection enhanced air exposure‐induced apoptosis and inflammation in the mouse corneal epithelium, which was inhibited by Rapa treatment. Furthermore, the phosphorylation status of PERK and eIF2α and CHOP expression increased in both EK models indicating that ER stress‐induced autophagy promoted cell survival. Taken together, air exposure‐induced autophagy is indispensable for the maintenance of corneal epithelial physiology and cell survival.     

Upregulated autophagy protects cardiomyocytes from oxidative stress-induced toxicity

Autophagy is a cellular self-digestion process that mediates protein quality control and serves to protect against neurodegenerative disorders, infections, inflammatory diseases and cancer. Current evidence suggests that autophagy can selectively remove damaged organelles such as the mitochondria. Mitochondria-induced oxidative stress has been shown to play a major role in a wide range of pathologies in several organs, including the heart. Few studies have investigated whether enhanced autophagy can offer protection against mitochondrially-generated oxidative stress. We induced mitochondrial stress in cardiomyocytes using antimycin A (AMA), which increased mitochondrial superoxide generation, decreased mitochondrial membrane potential and depressed cellular respiration. In addition, AMA augmented nuclear DNA oxidation and cell death in cardiomyocytes. Interestingly, although oxidative stress has been proposed to induce autophagy, treatment with AMA did not result in stimulation of autophagy or mitophagy in cardiomyocytes. Our results showed that the MTOR inhibitor rapamycin induced autophagy, promoted mitochondrial clearance and protected cardiomyocytes from the cytotoxic effects of AMA, as assessed by apoptotic marker activation and viability assays in both mouse atrial HL-1 cardiomyocytes and human ventricular AC16 cells. Importantly, rapamycin improved mitochondrial function, as determined by cellular respiration, mitochondrial membrane potential and morphology analysis. Furthermore, autophagy induction by rapamycin suppressed the accumulation of ubiquitinylated proteins induced by AMA. Inhibition of rapamycin-induced autophagy by pharmacological or genetic interventions attenuated the cytoprotective effects of rapamycin against AMA. We propose that rapamycin offers cytoprotection against oxidative stress by a combined approach of removing dysfunctional mitochondria as well as by degrading damaged, ubiquitinated proteins. We conclude that autophagy induction by rapamycin could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes.     

Inhibition of the mammalian target of rapamycin promotes cyclic AMP-induced differentiation of NG108-15 cells

To clarify the involvement of autophagy in neuronal differentiation, the effect of rapamycin, an mTOR complex inhibitor, on the dibutyryl cAMP (dbcAMP)-induced differentiation of NG108-15 cells was examined. Treatment of NG108-15 cells with 1 mM dbcAMP resulted in induction of differentiation, including neurite outgrowth and varicosity formation, enhanced voltage-sensitive Ca2+ channel activity and expression of microtubule-associated protein 2, and these effects involved phosphorylation of cAMP-response element binding protein (CREB) and extracellular signal regulated kinase (ERK). Simultaneous application of dbcAMP and rapamycin synergistically increased and accelerated differentiation. mTOR or raptor silencing with siRNA had a similar effect to rapamycin. Rapamycin and silencing of mTOR or raptor evoked autophagy, while blockade of autophagy by addition of 3-methyladenine or beclin 1 or Atg5 silencing prevented the potentiation of differentiation. Silencing of rictor also evokes autophagy, at a level 55% of that induced by raptor silencing and enhancement of differentiation is proportional. Rapamycin also caused increased ATP generation and cell cycle arrest in G0/G1 phase, but had no effect on CREB and ERK phosphorylation. dbcAMP also induced ATP generation, but not autophagy or cell cycle arrest. These results suggest that the increased autophagy, ATP generation and cell cycle arrest caused by mTOR inhibition promotes the dbcAMP-induced differentiation of NG108-15 cells.     

Insulin receptor substrate-1 prevents autophagy-dependent cell death caused by oxidative stress in mouse NIH/3T3 cells

Background

Insulin receptor substrate (IRS)-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS) are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels.

Methods and results

In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expression levels; we found that the IRS-1 overexpressing cells grow more rapidly than control cells do. Treatment of cells with glucose oxidase (GO) provided a continuous source of ROS; low dosages of GO promoted cell growth, while high doses induced cell death. Evidence for GO induced autophagy includes increased levels of isoform B-II microtubule-associated protein 1 light chain 3 (LC3), aggregation of green fluorescence protein-tagged LC3, and increased numbers of autophagic vacuoles in cells. Overexpression of IRS-1 resulted in inhibition of basal autophagy, and reduced oxidative stress-induced autophagy and cell death. ROS decreased the mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase signaling, while overexpression of IRS-1 attenuated this inhibition. Knockdown of autophagy-related gene 5 inhibited basal autophagy and diminished oxidative stress-induced autophagy and cell death.

Conclusion

Our results suggest that overexpression of IRS-1 promotes cells growth, inhibits basal autophagy, reduces oxidative stress-induced autophagy, and diminishes oxidative stress-mediated autophagy-dependent cell death. ROS-mediated autophagy may occur via inhibition of IRS-1/phosphatidylinositol 3-kinase/mTOR signaling. Our data afford a plausible explanation for IRS-1 involvement in tumor initiation and progression.     

Novel targets for Huntington's disease in an mTOR-independent autophagy pathway

Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases such as Huntington's disease. Autophagy induction with the mTOR inhibitor rapamycin accelerates clearance of these toxic substrates. As rapamycin has nontrivial side effects, we screened FDA-approved drugs to identify new autophagy-inducing pathways. We found that L-type Ca2+ channel antagonists, the K+ATP channel opener minoxidil, and the G(i) signaling activator clonidine induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, in which cAMP regulates IP3 levels, influencing calpain activity, which completes the cycle by cleaving and activating G(s)alpha, which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced, and we provide proof of principle for therapeutic relevance in Huntington's disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca2+ (like excitotoxicity) inhibit autophagy, thus retarding clearance of aggregate-prone proteins.     

Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells

The role of the main intracellular energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK) in the induction of autophagic response and cell death was investigated in SH-SY5Y human neuroblastoma cells exposed to the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The induction of autophagy in SH-SY5Y cells was demonstrated by acridine orange staining of intracellular acidic vesicles, the presence of autophagosome- and autophagolysosome-like vesicles confirmed by transmission electron microscopy, as well as by microtubule-associated protein 1 light-chain 3 (LC3) conversion and p62 degradation detected by immunoblotting. 6-OHDA induced phosphorylation of AMPK and its target Raptor, followed by the dephosphorylation of the major autophagy inhibitor mammalian target of rapamycin (mTOR) and its substrate p70S6 kinase (S6K). 6-OHDA treatment failed to suppress mTOR/S6K phosphorylation and to increase LC3 conversion, p62 degradation and cytoplasmatic acidification in neuroblastoma cells in which AMPK expression was downregulated by RNA interference. Transfection of SH-SY5Y cells with AMPK or LC3β shRNA, as well as treatment with pharmacological autophagy inhibitors suppressed, while mTOR inhibitor rapamycin potentiated 6-OHDA-induced oxidative stress and apoptotic cell death. 6-OHDA induced phosphorylation of p38 mitogen-activated protein (MAP) kinase in an AMPK-dependent manner, and pharmacological inhibition of p38 MAP kinase reduced neurotoxicity, but not AMPK activation and autophagy triggered by 6-OHDA. Finally, the antioxidant N-acetyl cysteine antagonized 6-OHDA-induced activation of AMPK, p38 and autophagy. These data suggest that oxidative stress-mediated AMPK/mTOR-dependent autophagy and AMPK/p38-dependent apoptosis could be valid therapeutic targets for neuroprotection.     

Inhibition of the mammalian target of rapamycin promotes cyclic AMP-induced differentiation of NG108-15 cells

To clarify the involvement of autophagy in neuronal differentiation, the effect of rapamycin, an mTOR complex inhibitor, on the dibutyryl cAMP (dbcAMP)-induced differentiation of NG108-15 cells was examined. Treatment of NG108-15 cells with 1 mM dbcAMP resulted in induction of differentiation, including neurite outgrowth and varicosity formation, enhanced voltage-sensitive Ca²⁺ channel activity and expression of microtubule-associated protein 2, and these effects involved phosphorylation of cAMP-response element binding protein (CREB) and extracellular signal regulated kinase (ERK). Simultaneous application of dbcAMP and rapamycin synergistically increased and accelerated differentiation. mTOR or raptor silencing with siRNA had a similar effect to rapamycin. Rapamycin and silencing of mTOR or raptor evoked autophagy, while blockade of autophagy by addition of 3-methyladenine or beclin 1 or Atg5 silencing prevented the potentiation of differentiation. Silencing of rictor also evokes autophagy, at a level 55% of that induced by raptor silencing and enhancement of differentiation is proportional. Rapamycin also caused increased ATP generation and cell cycle arrest in G₀/G₁ phase, but had no effect on CREB and ERK phosphorylation. dbcAMP also induced ATP generation, but not autophagy or cell cycle arrest. These results suggest that the increased autophagy, ATP generation and cell cycle arrest caused by mTOR inhibition promotes the dbcAMP-induced differentiation of NG108-15 cells.     

Rapamycin treatment inhibits CHO cell death in a serum‐free suspension culture by autophagy induction

Rapamycin, a specific mTOR inhibitor, has been used as a chemical activator in autophagy research both in vitro and in vivo. Recently, autophagy has received attention as an anti‐cell death engineering target in addition to apoptosis in the Chinese hamster ovary (CHO) cell engineering field. Here, the effect of rapamycin and the subsequent autophagy induction is investigated on two CHO cell lines, DG44 host and an antibody‐producing recombinant CHO (rCHO), in a serum‐free suspension culture. In both cell lines, the rapamycin treatment delayed the viability drop and apoptosis induction. In particular, the improved cell viability of the antibody‐producing rCHO cell line resulting from the rapamycin treatment led to a 21% increase in the maximum antibody concentration. From observations that a rapamycin derivative, everolimus, demonstrated similar positive effects in both cell lines, but not FK‐506, which forms the same complex as rapamycin, but does not inhibit mTOR, it was demonstrated that the positive effects of rapamycin appear to be mTOR‐dependent. In addition, the cultivation with rapamycin and/or an autophagy inhibitor, bafilomycin A1, indicated that the autophagy induction is related to the positive effects of rapamycin. The genetic perturbation of the autophagy pathway through the regulation of the expression level of Beclin‐1, an important autophagy regulator, resulted in a delayed autophagy induction and apoptosis inhibition in response to the rapamycin treatment in the DG44 host cell line. Taken together, the results obtained in this study imply a positive role for autophagy and predict the usefulness of pro‐autophagy engineering in CHO cell cultures. Biotechnol. Bioeng. 2012; 109: 3093–3102. © 2012 Wiley Periodicals, Inc.     

Autophagy Reduces Neuronal Damage and Promotes Locomotor Recovery via Inhibition of Apoptosis After Spinal Cord Injury in Rats

Autophagy is an intracellular catabolic mechanism that maintains the balance of proteins, lipids and aging organelles. 3-Methyladenine (3-MA) is a selective inhibitor of autophagy, whereas rapamycin, an antifungal agent, is a specific inducer of autophagy, inhibiting the protein mammalian target of rapamycin. In the present study, we examined the role of autophagy, inhibited by 3-MA and enhanced by rapamycin, in a model of acute spinal cord injury in rats. We found that rapamycin could significantly increase the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin1 at the injury site. At the same time, the number of neurons and astrocytes with LC3 positive in the spinal cord was upregulated with time. In addition, administration of rapamycin produced an increase in the Basso, Beattie and Bresnahan scores of injured rats, indicating high recovery of locomotor function. Furthermore, expression of the proteins Bcl-2 and Bax was upregulated and downregulated, respectively. By contrast, the results for rats treated with 3-MA, which inhibits autophagy, were the opposite of those seen with the rapamycin-treated rats. These results show that induction of autophagy can produce neuroprotective effects in acute spinal cord injury in rats via inhibition of apoptosis.     

Effects of autophagy inducers on recombinant antibody production in insect cells

Insect cells have recently proven to be an excellent platform for the high-level production of functional recombinant proteins. Autophagy is an important mechanism that promotes cell survival by eliminating damaged organelles and protein aggregates, and it also may influence recombinant protein production. In the present study, we compared the effects that autophagy inducers rapamycin, everolimus, and lithium chloride exert on recombinant lepidopteran insect cells that secrete an engineered antibody molecule. Compared with nontreatment, treatment with either rapamycin or everolimus prolonged cell growth to allow high cell density, improved viability in the declining phase, and then increased the yield of secreted antibodies. These positive effects appeared to be induced via autophagy since autophagosomes were clearly detected, particularly in cells treated with rapamycin or everolimus. Unlike rapamycin, another autophagy inducer, FK506, was ineffective in insect cells. The addition of an appropriate autophagy inducer may be effective in increasing the productivity of recombinant proteins in insect cells.

     

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.     

Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies

The formation of intra-neuronal mutant protein aggregates is a characteristic of several human neurodegenerative disorders, like Alzheimer's disease, Parkinson's disease (PD) and polyglutamine disorders, including Huntington's disease (HD). Autophagy is a major clearance pathway for the removal of mutant huntingtin associated with HD, and many other disease-causing, cytoplasmic, aggregate-prone proteins. Autophagy is negatively regulated by the mammalian target of rapamycin (mTOR) and can be induced in all mammalian cell types by the mTOR inhibitor rapamycin. It can also be induced by a recently described cyclical mTOR-independent pathway, which has multiple drug targets, involving links between Ca(2+)-calpain-G(salpha) and cAMP-Epac-PLC-epsilon-IP(3) signalling. Both pathways enhance the clearance of mutant huntingtin fragments and attenuate polyglutamine toxicity in cell and animal models. The protective effects of rapamycin in vivo are autophagy-dependent. In Drosophila models of various diseases, the benefits of rapamycin are lost when the expression of different autophagy genes is reduced, implicating that its effects are not mediated by autophagy-independent processes (like mild translation suppression). Also, the mTOR-independent autophagy enhancers have no effects on mutant protein clearance in autophagy-deficient cells. In this review, we describe various drugs and pathways inducing autophagy, which may be potential therapeutic approaches for HD and related conditions.     

Calorie restriction combined with resveratrol induces autophagy and protects 26-month-old rat hearts from doxorubicin-induced toxicity

The multiple beneficial effects of calorie restriction (CR) on several organs, including the heart, are widely known. Recently, the plant polyphenol resveratrol has been shown to possess several beneficial effects similar to those of CR. Among the host of effects on cardiac muscle, a cellular self-eating process called autophagy has been shown to be induced by both CR and resveratrol. Autophagy is vital in removing dysfunctional organelles and damaged proteins from the cell, thereby maintaining cellular quality control. In this study, we explored whether short-term moderate CR (20%), either alone or in combination with resveratrol, can induce autophagy in the hearts of 26-month-old Fischer 344 × Brown Norway rats. Autophagy stimulation was investigated by measuring the protein expression levels of the autophagy proteins beclin-1, Atg5, and p62 and the LC3-II/LC3-I ratio. We found that 20% CR or resveratrol alone for 6 weeks could not induce autophagy, but 20% CR in combination with 50 mg/kg/day resveratrol resulted in an induction of autophagy in the hearts of 26-month-old rats. Although oxidative stress has been proposed to be an inducer of autophagy, treatment with the chemotherapeutic drug doxorubicin was unable to stimulate autophagy. The enhanced autophagy due to CR + resveratrol was associated with protection from doxorubicin-induced damage, as measured by cardiac apoptotic levels and serum creatine kinase and lactate dehydrogenase activity. We propose that a combinatorial approach of low-dose CR and resveratrol has the potential to be used therapeutically to induce autophagy and provides protection against doxorubicin-mediated toxicity.     

Antimony trichloride induces a loss of cell viability via reactive oxygen species-dependent autophagy in A549 cells

Antimony (Sb) is one of the most prevalent heavy metals and frequently leads to biological toxicity. Although autophagy is believed to be involved in metal-associated cytotoxicity, there is no evidence of its involvement following exposure. Moreover, the underlying mechanism of autophagy remains unclear. In this study, treatment with antimony trichloride caused autophagy in a dose- and time-dependent manner in A549 cells but did not affect the level of Atg5 or Atg7 mRNA expression. Furthermore, Sb enhanced autophagic flux while upregulating p62 gene and protein levels. The classic mechanistic target of rapamycin (mTOR) pathway is not involved in Sb-induced autophagy. However, Sb-induced autophagy and the upregulation of p62 were inhibited by treatment with the antioxidant N-acetylcysteine (NAC). Subsequent analyses demonstrated that the inhibition of autophagy protected A549 cells from a loss of cell viability, while the activation of autophagy by rapamycin had the opposite effect. These data suggest that reactive oxygen species-dependent autophagy mediates Sb-stimulated cell viability loss in A549 cells.     

人参根提取物增强巨噬细胞RAW264.7的自噬水平并提高其增殖和吞噬能力

研究人参根提取物对巨噬细胞RAW264.7的增殖能力、吞噬能力和自噬水平的影响以及其相关性.用细胞计数试剂(CCK-8)检测不同浓度的人参根以及加入对巨噬细胞RAW264.7增殖的影响;采用中性红吞噬实验检测人参根提取物对巨噬细胞吞噬活性的影响;采用吖啶橙染色法(AO染色法)检测自噬体的形成;采用免疫印迹法(Weste...     

Stimulation of autophagy suppresses the intracellular survival of Burkholderia pseudomallei in mammalian cell lines

Burkholderia pseudomallei is the causative agent of melioidosis, a tropical infection of humans and other animals. The bacterium is an intracellular pathogen that can escape from endosomes into the host cytoplasm, where it replicates and infects adjacent cells. We investigated the role played by autophagy in the intracellular survival of B. pseudomallei in phagocytic and non-phagocytic cell lines. Autophagy was induced in response to B. pseudomallei invasion of murine macrophage (RAW 264.7) cells and a proportion of the bacteria co-localized with the autophagy effector protein LC3, a marker for autophagosome formation. Pharmacological stimulation of autophagy in RAW 264.7 and murine embryonic fibroblast (MEF) cell lines resulted in increased co-localization of B. pseudomallei with LC3 while basal levels of co-localization could be abrogated using inhibitors of the autophagic pathway. Furthermore, induction of autophagy decreased the intracellular survival of B. pseudomallei in these cell lines, but bacterial survival was not affected in MEF cell lines deficient in autophagy. Treatment of infected macrophages with chloramphenicol increased the proportion of bacteria within autophagosomes indicating that autophagic evasion is an active process relying on bacterial protein synthesis. Consistent with this hypothesis, we identified a B. pseudomallei type III secreted protein, BopA, which plays a role in mediating bacterial evasion of autophagy. We conclude that the autophagic pathway is a component of the innate defense system against invading B. pseudomallei, but which the bacteria can actively evade. However, when autophagy is pharmacologically induced using rapamycin, bacteria are actively sequestered in autophagosomes, ultimately decreasing their survival.     

GADD34 mediates cytoprotective autophagy in mutant huntingtin expressing cells via the mTOR pathway

Increased protein aggregation and altered cell signaling accompany many neurodegenerative diseases including Huntington's disease (HD). Cell stress is counterbalanced by signals mediating cell repair but the identity of these are not fully understood. We show here that the mammalian target of rapamycin (mTOR) pathway is inhibited and cytoprotective autophagy is activated in neuronal PC6.3 cells at 24 h after expression of mutant huntingtin proteins. Tuberous sclerosis complex (TSC) 1/2 interacted with growth arrest and DNA damage protein 34 (GADD34), which caused TSC2 dephosphorylation and induction of autophagy in mutant huntingtin expressing cells. However, GADD34 and autophagy decreased at later time points, after 48 h of transfection with the concomitant increase in mTOR activity. Overexpression of GADD34 counteracted these effects and increased cytoprotective autophagy and cell survival. These results show that GADD34 plays an important role in cell protection in mutant huntingtin expressing cells. Modulation of GADD34 and the TSC pathway may prove useful in counteracting cell degeneration accompanying HD and other neurodegenerative diseases.     

The protective role of metformin in autophagic status in peripheral blood mononuclear cells of type 2 diabetic patients

Autophagy plays an important role in the pathophysiology of type 2 diabetes (T2D). Metformin is the most common antidiabetic drug. The main objective of this study was to explore the molecular mechanism of metformin in starvation‐induced autophagy in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic patients. PBMCs were isolated from 10 diabetic patients and 7 non‐diabetic healthy volunteers. The autophagic puncta and markers were measured with the help of monodansylcadaverine staining and western blot. Additionally, transmission electron microscopy was also performed. No significant changes were observed in the initial autophagy marker protein levels in PBMCs of T2D after metformin treatment though diabetic PBMCs showed a high level of phospho‐mammalian target of rapamycin, p62 and reduced expression of phospho‐AMP‐activated protein kinase and lysosomal membrane‐associated protein 2, indicating a defect in autophagy. Also, induction of autophagy by tunicamycin resulted in apoptosis in diabetic PBMCs as observed by caspase‐3 cleavage and reduced expression of Bcl2. Inhibition of autophagy by bafilomycin rendered consistent expression of p62 indicating a defect in the final process of autophagy. Further, electron microscopic studies also confirmed massive vacuole overload and a sign of apoptotic cell death in PBMCs of diabetic patients, whereas metformin treatment reduced the number of autophagic vacuoles perhaps by lysosomal fusion. Thus, our results indicate that defective autophagy in T2D is associated with the fusion process of lysosomes which could be overcome by metformin.