Andrographolide sulfonate improves Alzheimer-associated phenotypes and mitochondrial dysfunction in APP/PS1 transgenic mice.
Geng Ji,Liu Wen,Xiong Yuyun,Ding Hongqun,Jiang Chunhong,Yang Xiaoling,Li Xiang,Elgehama Ahmed,Sun Yang,Xu Qiang,Guo Wenjie,Gao Jing
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Alzheimer's disease is a neurodegenerative disorder with Amyloid-β plaques onset, synaptic damage, and cognitive decline. Aβ deposits cause pathological events including oxidative stress, mitochondrial dysfunction, and neuron death. In this study, APPswe/PSENΔ9 double transgenic mice model was used to imitate Alzheimer's disease and the effect and possible mechanism of Andrographolide sulfonate were examined. Andrographolide sulfonate was given to the mice for 7 months before the onset of Aβ plaque. Spatial memory test showed that Andrographolide sulfonate treatment prevented cognitive decline. Aβ deposits were not affected while hippocampus and synapse damage was significantly alleviated. Mechanism studies showed that oxidative stress and mitochondrial swelling was reduced after Andrographolide sulfonate administration. These findings suggest that Andrographolide sulfonate, which has been applied in clinical medicine, might be a promising therapeutic agent for AD therapy via mitochondria protection.
The diverse role of TIGAR in cellular homeostasis and cancer.
Geng Ji,Yuan Xiao,Wei Mingzhen,Wu Junchao,Qin Zheng-Hong
Free radical research
TP53-induced glycolysis and apoptosis regulator (TIGAR) is a p53 target protein that plays critical roles in glycolysis and redox balance. Accumulating evidence shows that TIGAR is highly expressed in cancer. TIGAR redirects glycolysis and promotes carcinoma growth by providing metabolic intermediates and reductive power derived from pentose phosphate pathway (PPP). The expression of TIGAR in cancer is positively associated with chemotherapy resistance, suggesting that TIGAR could be a novel therapeutic target. In this review, we briefly presented the function of TIGAR in metabolic homeostasis in normal and cancer cells. Finally, we discussed the future directions of TIGAR research in cancer.
TIGAR regulates mitochondrial functions through SIRT1-PGC1α pathway and translocation of TIGAR into mitochondria in skeletal muscle.
Geng Ji,Wei Mingzhen,Yuan Xiao,Liu Ziqi,Wang Xinxin,Zhang Dingmei,Luo Li,Wu Junchao,Guo Wenjie,Qin Zheng-Hong
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
TP53-induced glycolysis and apoptosis regulator (TIGAR), a glycolytic inhibitor, plays vital roles in regulating cellular metabolism and oxidative stress. However, the role of highly expressed TIGAR in skeletal muscle remains unexplored. In the present study, TIGAR levels varied in different skeletal muscles and fibers. An exhaustive swimming test with a load corresponding to 5% of body weight was utilized in mice to assess the effects of TIGAR on exercise-induced fatigue and muscle damage. The running time and metabolic indicators were significantly greater in wild-type (WT) mice compared with TIGAR knockout (KO) mice. Poor exercise capacity was accompanied by decreased type IIA fibers in TIGAR KO mice. Decreased mitochondrial number and mitochondrial oxidative phosphorylation were observed more in TIGAR KO mice than in WT mice, which were involved in sirtuin 1 (SIRT1)-mediated deacetylation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), and resveratrol treatment in TIGAR KO mice can increase mitochondrial content and exercise time. Much more TIGAR was also detected in mitochondria during exhaustive exercise. In addition, TIGAR, rather than mitochondria-targeted TIGAR achieved by plasmid transfection, promoted SIRT1-PGC1α pathway. Glutathione -transferase-TIGAR pull-down assay followed by liquid chromatography mass spectrometry found that TIGAR interacted with ATP synthase F1 subunit α (ATP5A1), and its binding to ATP5A1 increased during exhaustive exercise. Overexpression of mitochondrial-TIGAR enhanced ATP generation, maintained mitochondrial membrane potential and reduced mitochondrial oxidative stress under hypoxia condition. Taken together, our results uncovered a novel role for TIGAR in mitochondrial regulation in fast-twitch oxidative skeletal muscle through SIRT1-PGC1α and translocation into mitochondria, which contribute to the increase in exercise endurance of mice.-Geng, J., Wei, M., Yuan, X., Liu, Z., Wang, X., Zhang, D., Luo, L., Wu, J., Guo, W., Qin, Z.-H. TIGAR regulates mitochondrial functions through SIRT1-PGC1α pathway and translocation of TIGAR into mitochondria in skeletal muscle.
Andrographolide triggers autophagy-mediated inflammation inhibition and attenuates chronic unpredictable mild stress (CUMS)-induced depressive-like behavior in mice.
Geng Ji,Liu Jia,Yuan Xiao,Liu Wen,Guo Wenjie
Toxicology and applied pharmacology
Depression is one of the most common psychiatric disorders in the world. Andrographolide is a natural product that displays evident anti-inflammatory activities. The purpose of the present study was to explore the antidepressant potential of andrographolide in chronic unpredictable mild stress (CUMS)-induced depressive-like behavior in mice. Performance in behavioral tests such as the forced swim test, sucrose preference test, tail suspension test and Y-maze was improved following andrographolide administration. The pro-inflammatory mediator NO and cytokines IL-1β, IL-6 as well as TNF-α were measured in the prefrontal cortex using a reagent kit, ELISA and real-time PCR. NF-κB signaling, NLRP3 inflammasome assembly and autophagy process were examined in the prefrontal cortex using western blotting. It was observed that 5 mg/kg andrographolide treatment obviously improved depressive-like behavior. In addition, 5 mg/kg andrographolide treatment also decreased the expression of pro-inflammatory mediators and cytokines (NO, COX-2, iNOS, IL-1β, IL-6 and TNF-α), NF-κB signaling (p-p65, p-IκBα) and NLRP3 inflammasome assembly (NLRP3, ASC and caspase-1) in the prefrontal cortex. Moreover, autophagy levels increased after andrographolide treatment. Finally, the antidepressant and anti-inflammatory effects of andrographolide were compromised by the application of chloroquine (CQ), which suggested that andrographolide-induced autophagy was mainly affected by the initiation rather than the blocking of autophagic flux. In conclusion, these results suggest that andrographolide produces antidepressant-like and anti-inflammatory effects in CUMS-induced mice which maybe mediated by the upregulation of autophagy.
DRAM1 plays a tumor suppressor role in NSCLC cells by promoting lysosomal degradation of EGFR.
Geng Ji,Zhang Rong,Yuan Xiao,Xu Haidong,Zhu Zhou,Wang Xinxin,Wang Yan,Xu Guoqiang,Guo Wenjie,Wu Junchao,Qin Zheng-Hong
Cell death & disease
Lung cancer is the leading cause of cancer-associated mortality worldwide. DNA damage-regulated autophagy modulator 1 (DRAM1) plays an important roles in autophagy and tumor progression. However, the mechanisms by which DRAM1 inhibits tumor growth are not fully understood. Here, we report that DRAM1 was decreased in nonsmall-cell lung carcinoma (NSCLC) and was associated with poor prognosis. We confirmed that DRAM1 inhibited the growth, migration, and invasion of NSCLC cells in vitro. Furthermore, overexpression of DRAM1 suppressed xenografted NSCLC tumors in vivo. DRAM1 increased EGFR endocytosis and lysosomal degradation, downregulating EGFR signaling pathway. On one side, DRAM1 interacted with EPS15 to promote EGFR endocytosis, as evidence by the results of proximity labeling followed by proteomics; on the other, DRAM1 recruited V-ATP6V1 subunit to lysosomes, thereby increasing the assemble of the V-ATPase complex, resulting in decreased lysosomal pH and increased activation of lysosomal proteases. These two actions of DRAM1 results in acceleration of EGFR degradation. In summary, these in vitro and in vivo studies uncover a novel mechanism through which DRAM1 suppresses oncogenic properties of NSCLC by regulating EGFR trafficking and degradation and highlights the potential value of DRAM1 as a prognostic biomarker in lung cancers.
DRAM1 deficiency affects the organization and function of the Golgi apparatus.
Wei Mingzhen,Zhu Zhou,Wu Junchao,Wang Yan,Geng Ji,Qin Zheng-Hong
DRAM1 (DNA damage-regulated autophagy modulator 1) is a transmembrane protein that predominantly localizes to the lysosome but is also found in other membranous organelles; however, its function in these organelles remains largely unknown. We found that DRAM1 was partially located in the Golgi apparatus, and knockdown of DRAM1 caused fragmentation of the Golgi apparatus in cells. The phenomenon of fragmented Golgi was not related to microtubule organization, and there was no direct interaction between DRAM1 and Golgi structural proteins (ARF1, GM130, syntaxin 6 and GRASP55). Moreover, Golgi-targeting DRAM1 failed to rescue the fragmentation of Golgi in DRAM1-deficient cells. The transport of ts045-VSVG-GFP, an indicator of movement from the Golgi apparatus to the plasma membrane, was delayed in DRAM1-knockdown cells. Moreover, the trafficking of CI-MPR from the plasma membrane to the Golgi was also impeded in DRAM1-knockdown cells. These results indicated that DRAM1 regulated the structure of the Golgi apparatus and affected Golgi apparatus-associated vesicular transport.
Andrographolide alleviates Parkinsonism in MPTP-PD mice via targeting mitochondrial fission mediated by dynamin-related protein 1.
Geng Ji,Liu Wen,Gao Jian,Jiang Chunhong,Fan Ting,Sun Yang,Qin Zheng-Hong,Xu Qiang,Guo Wenjie,Gao Jing
British journal of pharmacology
BACKGROUND AND PURPOSE:Accumulating evidence indicates that mitochondrial dynamics play an important role in the progressive deterioration of dopaminergic neurons. Andrographolide has been found to exert neuroprotective effects in several models of neurological diseases. However, the mechanism of how andrographolide protects neurons in Parkinson's disease (PD) remains not fully understood. EXPERIMENTAL APPROACH:Behavioural experiments were performed to examine the effect of andrographolide in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-PD mice. Mitochondrial mass and morphology were visualized using transmission electron microscopy (TEM). SH-SY5Y cells and primary mouse neurons were exposed to rotenone to mimic PD in vitro. Western blotting, co-immunoprecipitation and immunofluorescence were performed. The target protein of andrographolide was identified by biotin-andrographolide pulldown assay as well as drug affinity responsive target stability (DARTS), cellular thermal shift (CETSA), and surface plasmon resonance (SPR) assays. KEY RESULTS:Andrographolide administration improved behavioural deficits and attenuated loss of dopaminergic neurons in MPTP-exposed mice and reduced cell death induced by rotenone in vitro. An increased mitochondrial mass, and decreased surface area were found in the striatum from MPTP-PD mice, as well as in rotenone-treated primary neurons and SH-SY5Y cells, while andrographolide treatment preserved mitochondrial mass and morphology. Dynamin-related protein 1 (DRP1) was identified as a target protein of andrographolide. Andrographolide bound to DRP1 and inhibited its GTPase activity, thereby preventing excessive mitochondria fission and neuronal damage in PD. CONCLUSIONS AND IMPLICATIONS:Our findings suggest that andrographolide may protect neurons against rotenone- or MPTP-induced damage in vitro and in vivo through inhibiting mitochondrial fission.
Synergistic effects of prostaglandin E1 and lithium in a rat model of cerebral ischemia.
Han Rong,Gao Bo,Sheng Rui,Zhang Li-sha,Zhang Hui-lin,Gu Zhen-lun,Qin Zheng-hong
Acta pharmacologica Sinica
AIM:Heat shock proteins (HSPs) are important regulators of cellular survival and exert neuroprotective effects against cerebral ischemia. Both prostaglandin E1 (PGE1) and lithium have been reported to protect neurons against ischemic injury. The present study was undertaken to examine if lithium could potentiate the neuroprotection of PGE1 against cerebral ischemia, and if the synergetic effects take place at the level of HSPs. METHODS:Brain ischemia was induced by a permanent middle cerebral artery occlusion (pMCAO) in rats. Rats were pretreated with subcutaneous injection of lithium for 2 d and a single intravenous administration of PGE1 immediately after ischemic insult. Cerebrocortical blood flow of each group was closely monitored prior to onset of ischemia, 5 min, 15 min, 30 min and 60 min after surgical operation. Body temperature was measured before, 5 min, 2 h and 24 h after the onset of pMCAO. The infarct volume, brain edema and motor behavior deficits were analyzed 24 h after ischemic insult. Cytoprotective HSP70 and heme oxygenase-1 (HO-1) in the striatum of the ipsilateral hemisphere were detected by immunoblotting. Brain sections from the striatum of the ipsilateral hemisphere were double-labeled with the anti-HSP70 antibody and 4,6-diamidino-2-phenylindole (DAPI). RESULTS:Treatment with PGE1 (8 and 16 microg/kg, iv) or lithium (0.5 mEq/kg, sc) alone reduced infarct volume, neurological deficits and brain edema induced by focal cerebral ischemia in rats. Moreover, a greater neuroprotection was observed when PGE1 and lithium were given together. Co-administration of PGE1 and lithium significantly upregulated cytoprotective HSP70 and HO-1 protein levels. CONCLUSION:Lithium and PGE1 may exert synergistic effects in treatment of cerebral ischemia and thus may have potential clinical value for the treatment of stroke.
Reduced Nicotinamide Adenine Dinucleotide Phosphate, a Pentose Phosphate Pathway Product, Might Be a Novel Drug Candidate for Ischemic Stroke.
Li Mei,Zhou Zhi-Peng,Sun Meiling,Cao Lijuan,Chen Jieyu,Qin Yuan-Yuan,Gu Jin-Hua,Han Feng,Sheng Rui,Wu Jun-Chao,Ding Yuqiang,Qin Zheng-Hong
BACKGROUND AND PURPOSE:Our previous study has defined a role of TP53-induced glycolysis and apoptosis regulator in neuroprotection against ischemic injury through increasing the flow of pentose phosphate pathway. We hypothesized that the pentose phosphate pathway product nicotinamide adenine dinucleotide phosphate (NADPH) could be a novel drug for treatment of ischemic stroke. METHODS:The NADPH was given before, at the onset, or after stroke onset with single or repeated intravenous (mice and rats) or intraperitoneal injections (monkey). The short- and long-term therapeutic effects of NADPH were evaluated in male adult ICR mice (total=614) with transient middle cerebral artery occlusion, in male adult Sprague-Dawley rats (total=114) with permanent middle cerebral artery occlusion, and in male adult rhesus monkey (total=12) with thrombotic middle cerebral artery occlusion. RESULTS:Administration of NADPH led to a dramatic increase in the levels of ATP and reduced form of glutathione, whereas it decreased the levels of reactive oxygen species. NADPH significantly reduced infarct volume, improved poststroke survival, and recovery of neurological functions in mouse and rat models of stroke. Robust neuroprotection of a single dose of NADPH was seen when it was administered within 5 hours after reperfusion; however, repeat administration of NADPH twice a day for 7 days starting 24 hours after the onset of stroke also offered therapeutic effects. Pretreatment with NADPH also significantly improved the outcome of stroke insult. CONCLUSIONS:Administration of exogenous NADPH significantly protected neurons against ischemia/reperfusion-induced injury in 2 rodent stroke models. Thus, NADPH might be a promising drug candidate for treatment of ischemic stroke.
Combined NADPH and the NOX inhibitor apocynin provides greater anti-inflammatory and neuroprotective effects in a mouse model of stroke.
Qin Yuan-Yuan,Li Mei,Feng Xing,Wang Jian,Cao Lijuan,Shen Xi-Kui,Chen Jieyu,Sun Meiling,Sheng Rui,Han Feng,Qin Zheng-Hong
Free radical biology & medicine
Our previous study has reported that the pentose phosphate pathway product nicotinamide adenine dinucleotide phosphate (NADPH) protected neurons against ischemia/reperfusion-induced brain injury. NADPH can either act as a co-enzyme to produce GSH or a substrate of NADPH oxidase (NOX) to generate ROS. This study was designed to elucidate the effects of co-treatment with NADPH and NOX inhibitor apocynin on ischemia/reperfusion-induced brain inflammation and neuronal injury. The results showed that both NADPH and apocynin markedly attenuated ischemia/reperfusion-induced increases in the levels of NOX2, NOX4 and ROS. NADPH and apocynin significantly inhibited the phosphorylation and degradation of IκBα, NF-κBp65 nuclear localization, and the expression of NF-κB target gene cyclooxygenase (COX2) and inducible nitric oxide synthase (iNOS). Furthermore, both NADPH and apocynin suppressed the expression of inflammasome proteins including NLRP3 ASC, caspase-1, interleukin (IL)-1β and IL-18 in the ischemic cortex as revealed by Western blot analysis and immunofluorescence. Moreover, all these effects were greatly amplified by combination of NADPH and apocynin. Both NADPH and apocynin significantly reduced infarct volume, improved post-stroke survival, and recovery of neurological functions in mouse model of stroke. Consistently, the combination of NADPH and apocynin produced greater beneficial effects in against ischemic brain damage. These studies suggest that, beyond anti-oxidative effects, NADPH may also have anti-inflammatory effects and combination of NADPH and NOX inhibitors could produce a greater neuroprotective effect in ischemic stroke.
TIGAR inhibits ischemia/reperfusion-induced inflammatory response of astrocytes.
Chen Jieyu,Zhang Ding-Mei,Feng Xing,Wang Jian,Qin Yuan-Yuan,Zhang Tian,Huang Qiao,Sheng Rui,Chen Zhong,Li Mei,Qin Zheng-Hong
The inflammatory response of glial cells contributes to neuronal damage or repair after brain ischemia/reperfusion insult. We previously demonstrated a protective role of TP53-induced glycolysis and apoptosis regulator (TIGAR) in ischemic neuronal injury through increasing the flow of pentose phosphate pathway (PPP). The present study investigated the possible role of TIGAR in ischemia/reperfusion-induced inflammatory response of astrocytes. Male ICR mice were subjected to middle cerebral artery occlusion for 2 h followed by 24 h reperfusion and cultured primary astrocytes were subjected to oxygen glucose deprivation for 9 h followed by 24 h reoxygenation (OGD/R). Adenoviral vectors were used to alter the levels of TIGAR protein in brain and in culture primary astrocytes. We showed that during the OGD/R insult the protein levels of TIGAR were rapidly increased in astrocytes. Overexpression of TIGAR mediated increased the viability, levels of NADPH and rGSH, and reduced intracellular reactive oxygen species (ROS) in cultured primary astrocytes. Overexpression of TIGAR not only significantly reduced infarct volume after stroke insult but also markedly reduced long-term mortality and improved recovery of neurological functions. Overexpression of TIGAR tempered OGD/R- or ischemia/reperfusion-induced the upregulation of inducible nitric oxide synthase (iNOS), cyclooxygenases COX2 and the release of pro-inflammatory cytokines interleukin 1 beta (IL-1β) and tumor necrosis factor-α (TNF-α), while TIGAR knockdown produced opposite effects on these parameters. Moreover, Overexpression of TIGAR suppressed OGD/R-induced degradation of IκBα and NF-κB nuclear translocation in cultured primary astrocytes. The present study elucidates a novel mechanism by which TIGAR protects neurons against ischemia/reperfusion injury.
Neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways.
Wen Ya-Dan,Sheng Rui,Zhang Li-Sha,Han Rong,Zhang Xuan,Zhang Xing-Ding,Han Feng,Fukunaga Kohji,Qin Zheng-Hong
It has been reported that ischemic insult increases the formation of autophagosomes and activates autophagy. However, the role of autophagy in ischemic neuronal damage remains elusive. This study was taken to assess the role of autophagy in ischemic brain damage. Focal cerebral ischemia was introduced by permanent middle cerebral artery occlusion (pMCAO). Activation of autophagy was assessed by morphological and biochemical examinations. To determine the contribution of autophagy/lysosome to ischemic neuronal death, rats were pretreated with a single intracerebral ventricle injection of the autophagy inhibitors 3-methyl-adenine (3-MA) and bafliomycin A1 (BFA) or the cathepsin B inhibitor Z-FA-fmk after pMCAO. The effects of 3-MA and Z-FA-fmk on brain damage, expression of proteins involved in regulation of autophagy and apoptosis were assessed with 2,3,5-triphenyltetrazolium chloride (TTC) staining and immunoblotting. The results showed that pMACO increased the formation of autophagosomes and autolysosomes, the mRNA and protein levels of LC3-II and the protein levels of cathepsin B. 3-MA, BFA and Z-FA-fmk significantly reduced infarct volume, brain edema and motor deficits. The neuroprotective effects of 3-MA and Z-FA-fmk were associated with an inhibition on ischemia-induced upregulation of LC3-II and cathepsin B and a partial reversion of ischemia-induced downregulation of cytoprotective Bcl-2. These results demonstrate that ischemic insult activates autophagy and an autophagic mechanism may contribute to ischemic neuronal injury. Thus, autophagy may be a potential target for developing a novel therapy for stroke.
Reduced nicotinamide adenine dinucleotide phosphate inhibits rat platelet aggregation and p38 phosphorylation.
Gu Yi,Sheng Rui,Wu Junchao,Zhou Ying,Qin Zheng-Hong
Previous studies found that reduced nicotinamide adenine dinucleotide phosphate (NADPH) protected neurons against ischemia/reperfusion-induced injury. In addition to ROS reduction and ATP increment, preliminary data suggested that NADPH inhibited ADP and thrombin-induced platelet aggregation. As the effect of NADPH on platelet function was not reported by other investigators, the actions of NADPH on platelet function and mechanisms of actions were investigated in the present study. In vitro studies, the effects of different concentrations of NADPH on platelet aggregation induced by ADP (10 μM), thrombin (0.05 U/mL) or AA (50 μM) were determined. The results showed that NADPH could inhibit platelet aggregation induced by ADP, thrombin or AA in a concentration dependent manner. When the inhibitory effects of NAD, NADH, NADP and NADPH on platelet aggregation were compared, NADPH demonstrated the relatively best effect on platelet aggregation. In vivo studies, the effects of NADPH on platelet aggregation, tail bleeding time, coagulation response and ferric chloride-induced thrombosis were determined in mice or rats. The maximum aggregation rate of platelets of rats injected with NADPH (5 mg/kg) was lower than platelets from control rats. NADPH transiently prolonged tail bleeding time in mice at 30 min after the injection of NADPH (7.5 mg/kg), while aspirin (15 mg/kg) significantly prolonged the tail bleeding time in mice at all time points examined. NADPH (5 mg/kg), as well as aspirin (10 mg/kg), had no effect on coagulation response in rats. Using a FeCl-induced abdominal aorta injury thrombosis model, administration of NADPH (5 mg/kg) significantly delayed the onset of vessel occlusion, while aspirin (10 mg/kg) almost completely prevented the vessel occlusion. With microscopic examination the thrombi in injured vessel sections of rats received NADPH were much smaller and less dense than that of rats received vehicle treatment. ADP induced an increase in phosphorylation of p38 and the effect was markedly inhibited by the p38 inhibitor SB203580. Similarly, NADPH also inhibited ADP-induced phosphorylation of p38. Similar to NADPH, SB203580 robustly inhibited ADP- and thrombin-induced platelet aggregation. In addition, NADPH also reduced ADP-induced increases in ROS in platelets. The current results demonstrated that NADPH inhibited platelet aggregation, oxidative stress and p38 phosphorylation, suggesting that NADPH might be a novel compound for management of high risk of cardiovascular disease.
Autophagy activation is associated with neuroprotection in a rat model of focal cerebral ischemic preconditioning.
Sheng Rui,Zhang Li-Sha,Han Rong,Liu Xiao-Qian,Gao Bo,Qin Zheng-Hong
Several recent studies have showed that autophagy is involved in ischemic brain damage, but it may also play a pro-survival role in ischemic preconditioning. This study was taken to determine the role of autophagy in an animal model of cerebral ischemic preconditioning (IPC). Focal cerebral IPC was produced in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The rats were pretreated with intracerebral ventricle infusion of the autophagy inhibitors 3-methyladenine (3-MA) and bafliomycin A1 (Baf A1) or the autophagy inducer rapamycin to evaluate the contribution of autophagy to IPC-induced neuroprotection. The results from electron microscopic examinations and immunofluorescence showed that both IPC and PFI induced autophagy activation, but the extent and persistence of autophagy activation were varied. IPC treatment significantly reduced infarct volume, brain edema and motor deficits after subsequent PFI, whereas 3-MA and Baf A1 suppressed the neuroprotection induced by IPC. 3-MA pretreatment also significantly attenuated upregulation of LC3-II, beclin 1 and HSP70 and downregulation of p62. To further determine if autophagy induction is responsible for IPC-induced neuroprotection, rats were treated with rapamycin 24 h before the onset of PFI. The results showed that rapamycin reduced infarct volume, brain edema and motor deficits induced by PFI. Rapamycin pretreatment also increased the protein levels of LC3-II and beclin 1. These results demonstrate that autophagy activation during IPC offers a remarkable tolerance to a subsequent fatal ischemic insult, and IPC's neuroprotective effects can be mimicked by autophagy inducers.
Sestrin2 Protects Dopaminergic Cells against Rotenone Toxicity through AMPK-Dependent Autophagy Activation.
Hou Yi-Sheng,Guan Jun-Jie,Xu Hai-Dong,Wu Feng,Sheng Rui,Qin Zheng-Hong
Molecular and cellular biology
Dysfunction of the autophagy-lysosomal pathway (ALP) and the ubiquitin-proteasome system (UPS) was thought to be an important pathogenic mechanism in synuclein pathology and Parkinson's disease (PD). In the present study, we investigated the role of sestrin2 in autophagic degradation of α-synuclein and preservation of cell viability in a rotenone-induced cellular model of PD. We speculated that AMP-activated protein kinase (AMPK) was involved in regulation of autophagy and protection of dopaminergic cells against rotenone toxicity by sestrin2. The results showed that both the mRNA and protein levels of sestrin2 were increased in a TP53-dependent manner in Mes 23.5 cells after treatment with rotenone. Genetic knockdown of sestrin2 compromised the autophagy induction in response to rotenone, while overexpression of sestrin2 increased the basal autophagy activity. Sestrin2 presumably enhanced autophagy in an AMPK-dependent fashion, as sestrin2 overexpression activated AMPK, and genetic knockdown of AMPK abrogated autophagy induction by rotenone. Restoration of AMPK activity by metformin after sestrin2 knockdown recovered the autophagy activity. Sestrin2 overexpression ameliorated α-synuclein accumulation, inhibited caspase 3 activation, and reduced the cytotoxicity of rotenone. These results suggest that sestrin2 upregulation attempts to maintain autophagy activity and suppress rotenone cytotoxicity through activation of AMPK, and that sestrin2 exerts a protective effect on dopaminergic cells.
Syntaxin 17 inhibits ischemic neuronal injury by resuming autophagy flux and ameliorating endoplasmic reticulum stress.
Chen Lei,Xia Yun-Fei,Shen Shu-Fang,Tang Jie,Chen Jia-Li,Qian Ke,Chen Zhong,Qin Zheng-Hong,Sheng Rui
Free radical biology & medicine
Previous studies have shown that syntaxin 17 (STX17) is involved in mediating the fusion of autophagosomes and lysosomes. This study aimed to investigate the role and mechanism of STX17 in neuronal injury following cerebral ischemia/reperfusion. The ischemia/reperfusion (I/R) models were established by transient middle cerebral artery occlusion (tMCAO) in mice and oxygen glucose deprivation/reperfusion (O/R) in primary cultured cortical neurons and HT22 cells. Cerebral ischemia/reperfusion significantly up-regulated the expression of STX17 in neurons. Lentivirus mediated knockdown of STX17 in neurons reduced neuronal viability and increased LDH leakage. Injection of AAV9-shSTX17 into the brain of mice then subjected to tMCAO also significantly augmented the infarct area and exacerbated neurobehavioral deficits and mortality. Depletion of STX17 caused accumulation of autophagic marker/substrate LC3 II and p62, blockade of the autophagic flux, and the accumulation of dysfunctional lysosomes. Knockdown of STX17 also aggravated endoplasmic reticulum (ER) stress-dependent neuronal apoptosis induced by ischemia/reperfusion. Importantly, induction of autophagy-lysosomal pathway and alleviation of ER stress partially rescued STX17 knockdown-induced neuronal damage. These results suggest that STX17 may ameliorate ischemia/reperfusion-induced neuronal damage by enhancing autophagy flux and reducing ER stress-dependent neuronal apoptosis.
Autophagy regulates endoplasmic reticulum stress in ischemic preconditioning.
Sheng Rui,Liu Xiao-Qian,Zhang Li-Sha,Gao Bo,Han Rong,Wu Ying-Qiu,Zhang Xiang-Yang,Qin Zheng-Hong
Recent studies have suggested that autophagy plays a prosurvival role in ischemic preconditioning (IPC). This study was taken to assess the linkage between autophagy and endoplasmic reticulum (ER) stress during the process of IPC. The effects of IPC on ER stress and neuronal injury were determined by exposure of primary cultured murine cortical neurons to 30 min of OGD 24 h prior to a subsequent lethal OGD. The effects of IPC on ER stress and ischemic brain damage were evaluated in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The results showed that both IPC and lethal OGD increased the LC3-II expression and decreased p62 protein levels, but the extent of autophagy activation was varied. IPC treatment ameliorated OGD-induced cell damage in cultured cortical neurons, whereas 3-MA (5-20 mM) and bafilomycin A 1 (75-150 nM) suppressed the neuroprotection induced by IPC. 3-MA, at the dose blocking autophagy, significantly inhibited IPC-induced HSP70, HSP60 and GRP78 upregulation; meanwhile, it also aggregated the ER stress and increased activated caspase-12, caspase-3 and CHOP protein levels both in vitro and in vivo models. The ER stress inhibitor Sal (75 pmol) recovered IPC-induced neuroprotection in the presence of 3-MA. Rapamycin 50-200 nM in vitro and 35 pmol in vivo 24 h before the onset of lethal ischemia reduced ER stress and ischemia-induced neuronal damage. These results demonstrated that pre-activation of autophagy by ischemic preconditioning can boost endogenous defense mechanisms to upregulate molecular chaperones, and hence reduce excessive ER stress during fatal ischemia.
NADPH ameliorates MPTP-induced dopaminergic neurodegeneration through inhibiting p38MAPK activation.
Zhou Jing-Si,Zhu Zhou,Wu Feng,Zhou Ying,Sheng Rui,Wu Jun-Chao,Qin Zheng-Hong
Acta pharmacologica Sinica
Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). Although the pathogenic mechanism underlying PD remains largely unknown, decreased nigral glutathione (GSH) in postmortem brains of PD patients supports the presence of oxidative stress in PD. We found that Nicotinamide adenine dinucleotide phosphate (NADPH), which is important for maintaining the level of GSH, protected dopaminergic (DA) neurons from neurotoxicity of MPTP/MPP. In the present study, NADPH prevented DA neurons from MPTP toxicity with increased GSH and decreased reactive oxygen species (ROS) levels in the ventral midbrain of mice, and improved motor activity. Our present results demonstrated that NADPH inhibited the phosphorylation of p38MAPK, decreased the level of TP53 protein, and inhibited TP53 nuclear translocation in DA neurons of SNpc and in MES23.5 cells. Furthermore, NADPH decreased the protein level of TP53 target gene, Bax, cleavage of PARP, and nuclei condensation. Taken together, NADPH abrogated MPTP-induced p38MAPK phosphorylation, TP53 nuclear translocation, and Bax induction, and finally, MPTP/MPP-induced apoptosis of DA neurons. This study suggests that NADPH may be a novel therapeutic candidate for PD.
Endoplasmic reticulum chaperone GRP78 is involved in autophagy activation induced by ischemic preconditioning in neural cells.
Zhang Xiang-Yang,Zhang Tong-Tong,Song Dan-Dan,Zhou Jun- Hao,Han Rong,Qin Zheng-Hong,Sheng Rui
BACKGROUND:Our previous finding showed that brain ischemic preconditioning mediates neuroprotection through endoplasmic reticulum (ER) stress-induced autophagy. This study was aimed at exploring the role of ER chaperone GRP78 in IPC induced autophagy activation in neural cells. RESULTS:Ischemic preconditioning (IPC) and oxygen glucose deprivation (OGD) models were established in rat pheochromocytoma (PC12) cells and primary cultured murine cortical neurons. IPC exerted neuroprotection against subsequent OGD injury in both PC12 cells and primary cortical neurons. IPC increased GRP78 expression and activated autophagy, as evidenced by upregulated LC3 and Beclin1, increased autophagic flux and formation of autophagosomes. BAPTA(dibromo-1,2-bis(aminophenoxy)ethane N,N,N9,N9 - tetra acetic acid, 0.125-2 μM) and small interfering RNA targeted GRP78 abrogated IPC induced neuroprotection and decreased the expression of GRP78, LC3II/LC3I and Beclin1. In contrast, lentiviral vector mediated GRP78 overexpression (LV-GRP78) strengthened resistance of PC12 cells to OGD injury and increased LC3 and Beclin1 expression. Moreover, knockdown of GRP78 in stable GRP78 overexpressing PC12 cells abolished the upregulation of LC3II/LC3I. GRP78 might activate autophagy through AMPK - mTOR pathway. CONCLUSION:These results suggest that IPC- induced GRP78 upregulation is involved in autophagy activation, and hence exerts protection against ischemic injury in neural cells.
TIGAR contributes to ischemic tolerance induced by cerebral preconditioning through scavenging of reactive oxygen species and inhibition of apoptosis.
Zhou Jun-Hao,Zhang Tong-Tong,Song Dan-Dan,Xia Yun-Fei,Qin Zheng-Hong,Sheng Rui
Previous study showed that TIGAR (TP53-induced glycolysis and apoptosis regulator) protected ischemic brain injury via enhancing pentose phosphate pathway (PPP) flux and preserving mitochondria function. This study was aimed to study the role of TIGAR in cerebral preconditioning. The ischemic preconditioning (IPC) and isoflurane preconditioning (ISO) models were established in primary cultured cortical neurons and in mice. Both IPC and ISO increased TIGAR expression in cortical neurons. Preconditioning might upregulate TIGAR through SP1 transcription factor. Lentivirus mediated knockdown of TIGAR significantly abolished the ischemic tolerance induced by IPC and ISO. ISO also increased TIGAR in mouse cortex and hippocampus and alleviated subsequent brain ischemia-reperfusion injury, while the ischemic tolerance induced by ISO was eliminated with TIGAR knockdown in mouse brain. ISO increased the production of NADPH and glutathione (GSH), and scavenged reactive oxygen species (ROS), while TIGAR knockdown decreased GSH and NADPH production and increased the level of ROS. Supplementation of ROS scavenger NAC and PPP product NADPH effectively rescue the neuronal injury caused by TIGAR deficiency. Notably, TIGAR knockdown inhibited ISO-induced anti-apoptotic effects in cortical neurons. These results suggest that TIGAR participates in the cerebral preconditioning through reduction of ROS and subsequent cell apoptosis.
G6PD plays a neuroprotective role in brain ischemia through promoting pentose phosphate pathway.
Cao Lijuan,Zhang Dingmei,Chen Jieyu,Qin Yuan-Yuan,Sheng Rui,Feng Xing,Chen Zhong,Ding Yuqiang,Li Mei,Qin Zheng-Hong
Free radical biology & medicine
TIGAR-regulated pentose phosphate pathway (PPP) plays a critical role in the neuronal survival during cerebral ischemia/reperfusion. Glucose-6-phosphate dehydrogenase (G6PD) is a rate-limiting enzyme in PPP and thus, we hypothesized that it plays an essential role in anti-oxidative defense through producing NADPH. The present study investigated the regulation and the role of G6PD in ischemia/reperfusion-induced neuronal injury with in vivo and in vitro models of ischemic stroke. The results showed that the levels of G6PD mRNA and protein were increased after ischemia/reperfusion. In vivo, lentivirus-mediated G6PD overexpression in mice markedly reduced neuronal damage after ischemia/reperfusion insult, while lentivirus-mediated G6PD knockdown exacerbated it. In vitro, overexpression of G6PD in cultured primary neurons decreased neuronal injury under oxygen and glucose deprivation/reoxygenation (OGD/R) condition, whereas knockdown of G6PD aggravated it. Overexpression of G6PD increased levels of NADPH and reduced form of glutathione (rGSH), and ameliorated ROS-induced macromolecular damage. On the contrary, knockdown of G6PD executed the opposite effects in mice and in primary neurons. Supplementation of exogenous NADPH alleviated the detrimental effects of G6PD knockdown, whereas further enhanced the beneficial effects of G6PD overexpression in ischemic injury. Therefore, our results suggest that G6PD protects ischemic brain injury through increasing PPP. Thus G6PD may be considered as potential therapeutic target for treatment of ischemic brain injury.
A sphingosine kinase 2-mimicking TAT-peptide protects neurons against ischemia-reperfusion injury by activating BNIP3-mediated mitophagy.
Chen Jia-Li,Wang Xin-Xin,Chen Lei,Tang Jie,Xia Yun-Fei,Qian Ke,Qin Zheng-Hong,Waeber Christian,Sheng Rui
We have previously shown that sphingosine kinase 2 (SPK2) interacts with Bcl-2 via its BH3 domain, activating autophagy by inducing the dissociation of Beclin-1/Bcl-2 complexes, and that a TAT-SPK2 peptide containing the BH3 domain of SPK2 protects neurons against ischemic injury. The goals of the present study were to establish the functional significance of these findings, by testing whether TAT-SPK2 was effective in a mouse model of ischemic stroke, and to explore potential underlying mechanisms. Mice were administered with TAT-SPK2 by intraperitoneal injection before or after transient middle cerebral artery occlusion (tMCAO). Infarct volume, neurological deficit and brain water content were assessed 24 h after reperfusion. Mitophagy inhibitor Mdivi-1 and BNIP3 siRNAs were used to examine the involvement of BNIP3-dependent mitophagy in the neuroprotection of TAT-SPK2. Mitophagy was quantified by immunoblotting, immunofluorescence and electron microscopy. The interaction between TAT-SPK2 and Bcl-2, Bcl-2 and BNIP3 was detected by co-immunoprecipitation. In the tMCAO model, pre-treatment with TAT-SPK2 significantly reduced infarct volume, improved neurological function and decreased brain edema. Neuroprotection by TAT-SPK2 was still seen when the peptide was administered 3 h after reperfusion. TAT-SPK2 also significantly improved functional recovery and reduced long-term brain atrophy of the ischemic hemisphere 30 days after administration. Our studies further showed that TAT-SPK2 directly binds to Bcl-2 and disrupts Bcl-2/Beclin-1 or Bcl-2/BNIP3 complexes to induce mitophagy. These results suggest that TAT-SPK2 protects neurons against ischemia reperfusion injury by activating BNIP3-mediated mitophagy. Agents exploiting this molecular mechanism are potential candidates for the treatment of ischemic stroke.
Endogenous level of TIGAR in brain is associated with vulnerability of neurons to ischemic injury.
Cao Lijuan,Chen Jieyu,Li Mei,Qin Yuan-Yuan,Sun Meiling,Sheng Rui,Han Feng,Wang Guanghui,Qin Zheng-Hong
In previous studies, we showed that TP53-induced glycolysis and apoptosis regulator (TIGAR) protects neurons against ischemic brain injury. In the present study, we investigated the developmental changes of TIGAR level in mouse brain and the correlation of TIGAR expression with the vulnerability of neurons to ischemic injury. We found that the TIGAR level was high in the embryonic stage, dropped at birth, partially recovered in the early postnatal period, and then continued to decline to a lower level in early adult and aged mice. The TIGAR expression was higher after ischemia/reperfusion in mouse brain 8 and 12 weeks after birth. Four-week-old mice had smaller infarct volumes, lower neurological scores, and lower mortality rates after ischemia than 8- and 12-week-old mice. TIGAR expression also increased in response to oxygen glucose deprivation (OGD)/reoxygenation insult or H2O2 treatment in cultured primary neurons from different embryonic stages (E16 and E20). The neurons cultured from the early embryonic period had a greater resistance to OGD and oxidative insult. Higher TIGAR levels correlated with higher pentose phosphate pathway activity and less oxidative stress. Older mice and more mature neurons had more severe DNA and mitochondrial damage than younger mice and less mature neurons in response to ischemia/reperfusion or OGD/reoxygenation insult. Supplementation of cultured neurons with nicotinamide adenine dinuclectide phosphate (NADPH) significantly reduced ischemic injury. These results suggest that TIGAR expression changes during development and its expression level may be correlated with the vulnerability of neurons to ischemic injury.
Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).
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Weiqiang,Chen Xin-Ming,Chen Xiong-Wen,Chen Xu,Chen Yan,Chen Ye-Guang,Chen Yingyu,Chen Yongqiang,Chen Yu-Jen,Chen Yue-Qin,Chen Zhefan Stephen,Chen Zhi,Chen Zhi-Hua,Chen Zhijian J,Chen Zhixiang,Cheng Hanhua,Cheng Jun,Cheng Shi-Yuan,Cheng Wei,Cheng Xiaodong,Cheng Xiu-Tang,Cheng Yiyun,Cheng Zhiyong,Chen Zhong,Cheong Heesun,Cheong Jit Kong,Chernyak Boris V,Cherry Sara,Cheung Chi Fai Randy,Cheung Chun Hei Antonio,Cheung King-Ho,Chevet Eric,Chi Richard J,Chiang Alan Kwok Shing,Chiaradonna Ferdinando,Chiarelli Roberto,Chiariello Mario,Chica Nathalia,Chiocca Susanna,Chiong Mario,Chiou Shih-Hwa,Chiramel Abhilash I,Chiurchiù Valerio,Cho Dong-Hyung,Choe Seong-Kyu,Choi Augustine M K,Choi Mary E,Choudhury Kamalika Roy,Chow Norman S,Chu Charleen T,Chua Jason P,Chua John Jia En,Chung Hyewon,Chung Kin Pan,Chung Seockhoon,Chung So-Hyang,Chung Yuen-Li,Cianfanelli Valentina,Ciechomska Iwona A,Cifuentes Mariana,Cinque Laura,Cirak Sebahattin,Cirone Mara,Clague Michael J,Clarke Robert,Clementi Emilio,Coccia Eliana M,Codogno Patrice,Cohen Ehud,Cohen Mickael M,Colasanti Tania,Colasuonno Fiorella,Colbert Robert A,Colell Anna,Čolić Miodrag,Coll Nuria S,Collins Mark O,Colombo María I,Colón-Ramos Daniel A,Combaret Lydie,Comincini Sergio,Cominetti Márcia R,Consiglio Antonella,Conte Andrea,Conti Fabrizio,Contu Viorica Raluca,Cookson Mark R,Coombs Kevin M,Coppens Isabelle,Corasaniti Maria Tiziana,Corkery Dale P,Cordes Nils,Cortese Katia,Costa Maria do Carmo,Costantino Sarah,Costelli Paola,Coto-Montes Ana,Crack Peter J,Crespo Jose L,Criollo Alfredo,Crippa Valeria,Cristofani Riccardo,Csizmadia Tamas,Cuadrado Antonio,Cui Bing,Cui Jun,Cui Yixian,Cui Yong,Culetto Emmanuel,Cumino Andrea C,Cybulsky Andrey V,Czaja Mark J,Czuczwar Stanislaw J,D'Adamo Stefania,D'Amelio Marcello,D'Arcangelo Daniela,D'Lugos Andrew C,D'Orazi Gabriella,da Silva James A,Dafsari Hormos Salimi,Dagda Ruben K,Dagdas Yasin,Daglia Maria,Dai Xiaoxia,Dai Yun,Dai Yuyuan,Dal Col Jessica,Dalhaimer Paul,Dalla Valle Luisa,Dallenga 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R,Gomez Rodrigo,Gómez-Sánchez Rubén,Gomez-Puerto Maria Catalina,Gomez-Sintes Raquel,Gong Qingqiu,Goni Felix M,González-Gallego Javier,Gonzalez-Hernandez Tomas,Gonzalez-Polo Rosa A,Gonzalez-Reyes Jose A,González-Rodríguez Patricia,Goping Ing Swie,Gorbatyuk Marina S,Gorbunov Nikolai V,Görgülü Kıvanç,Gorojod Roxana M,Gorski Sharon M,Goruppi Sandro,Gotor Cecilia,Gottlieb Roberta A,Gozes Illana,Gozuacik Devrim,Graef Martin,Gräler Markus H,Granatiero Veronica,Grasso Daniel,Gray Joshua P,Green Douglas R,Greenhough Alexander,Gregory Stephen L,Griffin Edward F,Grinstaff Mark W,Gros Frederic,Grose Charles,Gross Angelina S,Gruber Florian,Grumati Paolo,Grune Tilman,Gu Xueyan,Guan Jun-Lin,Guardia Carlos M,Guda Kishore,Guerra Flora,Guerri Consuelo,Guha Prasun,Guillén Carlos,Gujar Shashi,Gukovskaya Anna,Gukovsky Ilya,Gunst Jan,Günther Andreas,Guntur Anyonya R,Guo Chuanyong,Guo Chun,Guo Hongqing,Guo Lian-Wang,Guo Ming,Gupta Pawan,Gupta Shashi Kumar,Gupta Swapnil,Gupta Veer Bala,Gupta Vivek,Gustafsson Asa B,Gutterman David D,H B Ranjitha,Haapasalo Annakaisa,Haber James E,Hać Aleksandra,Hadano Shinji,Hafrén Anders J,Haidar Mansour,Hall Belinda S,Halldén Gunnel,Hamacher-Brady Anne,Hamann Andrea,Hamasaki Maho,Han Weidong,Hansen Malene,Hanson Phyllis I,Hao Zijian,Harada Masaru,Harhaji-Trajkovic Ljubica,Hariharan Nirmala,Haroon Nigil,Harris James,Hasegawa Takafumi,Hasima Nagoor Noor,Haspel Jeffrey A,Haucke Volker,Hawkins Wayne D,Hay Bruce A,Haynes Cole M,Hayrabedyan Soren B,Hays Thomas S,He Congcong,He Qin,He Rong-Rong,He You-Wen,He Yu-Ying,Heakal Yasser,Heberle Alexander M,Hejtmancik J Fielding,Helgason Gudmundur Vignir,Henkel Vanessa,Herb Marc,Hergovich Alexander,Herman-Antosiewicz Anna,Hernández Agustín,Hernandez Carlos,Hernandez-Diaz Sergio,Hernandez-Gea Virginia,Herpin Amaury,Herreros Judit,Hervás Javier H,Hesselson Daniel,Hetz Claudio,Heussler Volker T,Higuchi Yujiro,Hilfiker Sabine,Hill Joseph A,Hlavacek William S,Ho Emmanuel A,Ho Idy H T,Ho Philip Wing-Lok,Ho Shu-Leong,Ho Wan Yun,Hobbs G Aaron,Hochstrasser Mark,Hoet Peter H M,Hofius Daniel,Hofman Paul,Höhn Annika,Holmberg Carina I,Hombrebueno Jose R,Yi-Ren Hong Chang-Won Hong,Hooper Lora V,Hoppe Thorsten,Horos Rastislav,Hoshida Yujin,Hsin I-Lun,Hsu Hsin-Yun,Hu Bing,Hu Dong,Hu Li-Fang,Hu Ming Chang,Hu Ronggui,Hu Wei,Hu Yu-Chen,Hu Zhuo-Wei,Hua Fang,Hua Jinlian,Hua Yingqi,Huan Chongmin,Huang Canhua,Huang Chuanshu,Huang Chuanxin,Huang Chunling,Huang Haishan,Huang Kun,Huang Michael L H,Huang Rui,Huang Shan,Huang Tianzhi,Huang Xing,Huang Yuxiang Jack,Huber Tobias B,Hubert Virginie,Hubner Christian A,Hughes Stephanie M,Hughes William E,Humbert Magali,Hummer Gerhard,Hurley James H,Hussain Sabah,Hussain Salik,Hussey Patrick J,Hutabarat Martina,Hwang Hui-Yun,Hwang Seungmin,Ieni Antonio,Ikeda Fumiyo,Imagawa Yusuke,Imai Yuzuru,Imbriano Carol,Imoto Masaya,Inman Denise M,Inoki Ken,Iovanna Juan,Iozzo Renato V,Ippolito Giuseppe,Irazoqui Javier E,Iribarren Pablo,Ishaq Mohd,Ishikawa Makoto,Ishimwe Nestor,Isidoro Ciro,Ismail Nahed,Issazadeh-Navikas Shohreh,Itakura Eisuke,Ito Daisuke,Ivankovic Davor,Ivanova Saška,Iyer Anand Krishnan V,Izquierdo José M,Izumi Masanori,Jäättelä Marja,Jabir Majid Sakhi,Jackson William T,Jacobo-Herrera Nadia,Jacomin Anne-Claire,Jacquin Elise,Jadiya Pooja,Jaeschke Hartmut,Jagannath Chinnaswamy,Jakobi Arjen J,Jakobsson Johan,Janji Bassam,Jansen-Dürr Pidder,Jansson Patric J,Jantsch Jonathan,Januszewski Sławomir,Jassey Alagie,Jean Steve,Jeltsch-David Hélène,Jendelova Pavla,Jenny Andreas,Jensen Thomas E,Jessen Niels,Jewell Jenna L,Ji Jing,Jia Lijun,Jia Rui,Jiang Liwen,Jiang Qing,Jiang Richeng,Jiang Teng,Jiang Xuejun,Jiang Yu,Jimenez-Sanchez Maria,Jin Eun-Jung,Jin Fengyan,Jin Hongchuan,Jin Li,Jin Luqi,Jin Meiyan,Jin Si,Jo Eun-Kyeong,Joffre Carine,Johansen Terje,Johnson Gail V W,Johnston Simon A,Jokitalo Eija,Jolly Mohit Kumar,Joosten Leo A B,Jordan Joaquin,Joseph Bertrand,Ju Dianwen,Ju Jeong-Sun,Ju Jingfang,Juárez Esmeralda,Judith Delphine,Juhász Gábor,Jun Youngsoo,Jung Chang Hwa,Jung Sung-Chul,Jung Yong Keun,Jungbluth Heinz,Jungverdorben Johannes,Just Steffen,Kaarniranta Kai,Kaasik Allen,Kabuta Tomohiro,Kaganovich Daniel,Kahana Alon,Kain Renate,Kajimura Shinjo,Kalamvoki Maria,Kalia Manjula,Kalinowski Danuta S,Kaludercic Nina,Kalvari Ioanna,Kaminska Joanna,Kaminskyy Vitaliy O,Kanamori Hiromitsu,Kanasaki Keizo,Kang Chanhee,Kang Rui,Kang Sang Sun,Kaniyappan Senthilvelrajan,Kanki Tomotake,Kanneganti Thirumala-Devi,Kanthasamy Anumantha G,Kanthasamy Arthi,Kantorow Marc,Kapuy Orsolya,Karamouzis Michalis V,Karim Md Razaul,Karmakar Parimal,Katare Rajesh G,Kato Masaru,Kaufmann Stefan H E,Kauppinen Anu,Kaushal Gur P,Kaushik Susmita,Kawasaki Kiyoshi,Kazan Kemal,Ke Po-Yuan,Keating Damien J,Keber Ursula,Kehrl John H,Keller Kate E,Keller Christian W,Kemper Jongsook Kim,Kenific Candia M,Kepp Oliver,Kermorgant Stephanie,Kern Andreas,Ketteler Robin,Keulers Tom G,Khalfin Boris,Khalil Hany,Khambu Bilon,Khan Shahid Y,Khandelwal Vinoth Kumar Megraj,Khandia Rekha,Kho Widuri,Khobrekar Noopur V,Khuansuwan Sataree,Khundadze Mukhran,Killackey Samuel A,Kim Dasol,Kim Deok Ryong,Kim Do-Hyung,Kim Dong-Eun,Kim Eun Young,Kim Eun-Kyoung,Kim Hak-Rim,Kim Hee-Sik,Hyung-Ryong Kim ,Kim Jeong Hun,Kim Jin Kyung,Kim Jin-Hoi,Kim Joungmok,Kim Ju Hwan,Kim Keun Il,Kim Peter K,Kim Seong-Jun,Kimball Scot R,Kimchi Adi,Kimmelman Alec C,Kimura Tomonori,King Matthew A,Kinghorn Kerri J,Kinsey Conan G,Kirkin Vladimir,Kirshenbaum Lorrie A,Kiselev Sergey L,Kishi Shuji,Kitamoto Katsuhiko,Kitaoka Yasushi,Kitazato Kaio,Kitsis Richard N,Kittler Josef T,Kjaerulff Ole,Klein Peter S,Klopstock Thomas,Klucken Jochen,Knævelsrud Helene,Knorr Roland L,Ko Ben C B,Ko Fred,Ko Jiunn-Liang,Kobayashi Hotaka,Kobayashi Satoru,Koch Ina,Koch Jan C,Koenig Ulrich,Kögel Donat,Koh Young Ho,Koike Masato,Kohlwein Sepp D,Kocaturk Nur M,Komatsu Masaaki,König Jeannette,Kono Toru,Kopp Benjamin T,Korcsmaros Tamas,Korkmaz Gözde,Korolchuk Viktor I,Korsnes Mónica Suárez,Koskela Ali,Kota Janaiah,Kotake Yaichiro,Kotler Monica L,Kou Yanjun,Koukourakis Michael I,Koustas Evangelos,Kovacs Attila L,Kovács Tibor,Koya Daisuke,Kozako Tomohiro,Kraft Claudine,Krainc Dimitri,Krämer Helmut,Krasnodembskaya Anna D,Kretz-Remy Carole,Kroemer Guido,Ktistakis Nicholas T,Kuchitsu Kazuyuki,Kuenen Sabine,Kuerschner Lars,Kukar Thomas,Kumar Ajay,Kumar Ashok,Kumar Deepak,Kumar Dhiraj,Kumar Sharad,Kume Shinji,Kumsta Caroline,Kundu Chanakya N,Kundu Mondira,Kunnumakkara Ajaikumar B,Kurgan Lukasz,Kutateladze Tatiana G,Kutlu Ozlem,Kwak SeongAe,Kwon Ho Jeong,Kwon Taeg Kyu,Kwon Yong Tae,Kyrmizi Irene,La Spada Albert,Labonté Patrick,Ladoire Sylvain,Laface Ilaria,Lafont Frank,Lagace Diane C,Lahiri Vikramjit,Lai Zhibing,Laird Angela S,Lakkaraju Aparna,Lamark Trond,Lan Sheng-Hui,Landajuela Ane,Lane Darius J R,Lane Jon D,Lang Charles H,Lange Carsten,Langel Ülo,Langer Rupert,Lapaquette Pierre,Laporte Jocelyn,LaRusso Nicholas F,Lastres-Becker Isabel,Lau Wilson Chun Yu,Laurie Gordon W,Lavandero Sergio,Law Betty Yuen Kwan,Law Helen Ka-Wai,Layfield Rob,Le Weidong,Le Stunff Herve,Leary Alexandre Y,Lebrun Jean-Jacques,Leck Lionel Y W,Leduc-Gaudet Jean-Philippe,Lee Changwook,Lee Chung-Pei,Lee Da-Hye,Lee Edward B,Lee Erinna F,Lee Gyun Min,Lee He-Jin,Lee Heung Kyu,Lee Jae Man,Lee Jason S,Lee Jin-A,Lee Joo-Yong,Lee Jun Hee,Lee Michael,Lee Min Goo,Lee Min Jae,Lee Myung-Shik,Lee Sang Yoon,Lee Seung-Jae,Lee Stella Y,Lee Sung Bae,Lee Won Hee,Lee Ying-Ray,Lee Yong-Ho,Lee Youngil,Lefebvre Christophe,Legouis Renaud,Lei Yu L,Lei Yuchen,Leikin Sergey,Leitinger Gerd,Lemus Leticia,Leng Shuilong,Lenoir Olivia,Lenz Guido,Lenz Heinz Josef,Lenzi Paola,León Yolanda,Leopoldino Andréia M,Leschczyk Christoph,Leskelä Stina,Letellier Elisabeth,Leung Chi-Ting,Leung Po Sing,Leventhal Jeremy S,Levine Beth,Lewis Patrick A,Ley Klaus,Li Bin,Li Da-Qiang,Li Jianming,Li Jing,Li Jiong,Li Ke,Li Liwu,Li Mei,Li Min,Li Min,Li Ming,Li Mingchuan,Li Pin-Lan,Li Ming-Qing,Li Qing,Li Sheng,Li Tiangang,Li Wei,Li Wenming,Li Xue,Li Yi-Ping,Li Yuan,Li Zhiqiang,Li Zhiyong,Li Zhiyuan,Lian Jiqin,Liang Chengyu,Liang Qiangrong,Liang Weicheng,Liang Yongheng,Liang YongTian,Liao Guanghong,Liao Lujian,Liao Mingzhi,Liao Yung-Feng,Librizzi Mariangela,Lie Pearl P Y,Lilly Mary A,Lim Hyunjung J,Lima Thania R R,Limana Federica,Lin Chao,Lin Chih-Wen,Lin Dar-Shong,Lin Fu-Cheng,Lin Jiandie D,Lin Kurt M,Lin Kwang-Huei,Lin Liang-Tzung,Lin Pei-Hui,Lin Qiong,Lin Shaofeng,Lin Su-Ju,Lin Wenyu,Lin Xueying,Lin Yao-Xin,Lin Yee-Shin,Linden Rafael,Lindner Paula,Ling Shuo-Chien,Lingor Paul,Linnemann Amelia K,Liou Yih-Cherng,Lipinski Marta M,Lipovšek Saška,Lira Vitor A,Lisiak Natalia,Liton Paloma B,Liu Chao,Liu Ching-Hsuan,Liu Chun-Feng,Liu Cui Hua,Liu Fang,Liu Hao,Liu Hsiao-Sheng,Liu Hua-Feng,Liu Huifang,Liu Jia,Liu Jing,Liu Julia,Liu Leyuan,Liu Longhua,Liu Meilian,Liu Qin,Liu Wei,Liu Wende,Liu Xiao-Hong,Liu Xiaodong,Liu Xingguo,Liu Xu,Liu Xuedong,Liu Yanfen,Liu Yang,Liu Yang,Liu Yueyang,Liu Yule,Livingston J Andrew,Lizard Gerard,Lizcano Jose M,Ljubojevic-Holzer Senka,LLeonart Matilde E,Llobet-Navàs David,Llorente Alicia,Lo Chih Hung,Lobato-Márquez Damián,Long Qi,Long Yun Chau,Loos Ben,Loos Julia A,López Manuela G,López-Doménech Guillermo,López-Guerrero José Antonio,López-Jiménez Ana T,López-Pérez Óscar,López-Valero Israel,Lorenowicz Magdalena J,Lorente Mar,Lorincz Peter,Lossi Laura,Lotersztajn Sophie,Lovat Penny E,Lovell Jonathan F,Lovy Alenka,Lőw Péter,Lu Guang,Lu Haocheng,Lu Jia-Hong,Lu Jin-Jian,Lu Mengji,Lu Shuyan,Luciani Alessandro,Lucocq John M,Ludovico Paula,Luftig Micah A,Luhr Morten,Luis-Ravelo Diego,Lum Julian J,Luna-Dulcey Liany,Lund Anders H,Lund Viktor K,Lünemann Jan D,Lüningschrör Patrick,Luo Honglin,Luo Rongcan,Luo Shouqing,Luo Zhi,Luparello Claudio,Lüscher Bernhard,Luu Luan,Lyakhovich Alex,Lyamzaev Konstantin G,Lystad Alf Håkon,Lytvynchuk Lyubomyr,Ma Alvin C,Ma Changle,Ma Mengxiao,Ma Ning-Fang,Ma Quan-Hong,Ma Xinliang,Ma Yueyun,Ma Zhenyi,MacDougald Ormond A,Macian Fernando,MacIntosh Gustavo C,MacKeigan Jeffrey P,Macleod Kay F,Maday Sandra,Madeo Frank,Madesh Muniswamy,Madl Tobias,Madrigal-Matute Julio,Maeda Akiko,Maejima Yasuhiro,Magarinos Marta,Mahavadi Poornima,Maiani Emiliano,Maiese Kenneth,Maiti Panchanan,Maiuri Maria Chiara,Majello Barbara,Major Michael B,Makareeva Elena,Malik Fayaz,Mallilankaraman Karthik,Malorni Walter,Maloyan Alina,Mammadova Najiba,Man Gene Chi Wai,Manai Federico,Mancias Joseph D,Mandelkow Eva-Maria,Mandell Michael A,Manfredi Angelo A,Manjili Masoud H,Manjithaya Ravi,Manque Patricio,Manshian Bella B,Manzano Raquel,Manzoni Claudia,Mao Kai,Marchese Cinzia,Marchetti Sandrine,Marconi Anna Maria,Marcucci Fabrizio,Mardente Stefania,Mareninova Olga A,Margeta Marta,Mari Muriel,Marinelli Sara,Marinelli Oliviero,Mariño Guillermo,Mariotto Sofia,Marshall Richard S,Marten Mark R,Martens Sascha,Martin Alexandre P J,Martin Katie R,Martin Sara,Martin Shaun,Martín-Segura Adrián,Martín-Acebes Miguel A,Martin-Burriel Inmaculada,Martin-Rincon Marcos,Martin-Sanz Paloma,Martina José A,Martinet Wim,Martinez Aitor,Martinez Ana,Martinez Jennifer,Martinez Velazquez Moises,Martinez-Lopez Nuria,Martinez-Vicente Marta,Martins Daniel O,Martins Joilson O,Martins Waleska K,Martins-Marques Tania,Marzetti Emanuele,Masaldan Shashank,Masclaux-Daubresse Celine,Mashek Douglas G,Massa Valentina,Massieu Lourdes,Masson Glenn R,Masuelli Laura,Masyuk Anatoliy I,Masyuk Tetyana V,Matarrese Paola,Matheu Ander,Matoba Satoaki,Matsuzaki Sachiko,Mattar Pamela,Matte Alessandro,Mattoscio Domenico,Mauriz José L,Mauthe Mario,Mauvezin Caroline,Maverakis Emanual,Maycotte Paola,Mayer Johanna,Mazzoccoli Gianluigi,Mazzoni Cristina,Mazzulli Joseph R,McCarty Nami,McDonald Christine,McGill Mitchell R,McKenna Sharon L,McLaughlin BethAnn,McLoughlin Fionn,McNiven Mark A,McWilliams Thomas G,Mechta-Grigoriou Fatima,Medeiros Tania Catarina,Medina Diego L,Megeney Lynn A,Megyeri Klara,Mehrpour Maryam,Mehta Jawahar L,Meijer Alfred J,Meijer Annemarie H,Mejlvang Jakob,Meléndez Alicia,Melk Annette,Memisoglu Gonen,Mendes Alexandrina F,Meng Delong,Meng Fei,Meng Tian,Menna-Barreto Rubem,Menon Manoj B,Mercer Carol,Mercier Anne E,Mergny Jean-Louis,Merighi Adalberto,Merkley Seth D,Merla Giuseppe,Meske Volker,Mestre Ana Cecilia,Metur Shree Padma,Meyer Christian,Meyer Hemmo,Mi Wenyi,Mialet-Perez Jeanne,Miao Junying,Micale Lucia,Miki Yasuo,Milan Enrico,Milczarek Małgorzata,Miller Dana L,Miller Samuel I,Miller Silke,Millward Steven W,Milosevic Ira,Minina Elena A,Mirzaei Hamed,Mirzaei Hamid Reza,Mirzaei Mehdi,Mishra Amit,Mishra Nandita,Mishra Paras Kumar,Misirkic Marjanovic Maja,Misasi Roberta,Misra Amit,Misso Gabriella,Mitchell Claire,Mitou Geraldine,Miura Tetsuji,Miyamoto Shigeki,Miyazaki Makoto,Miyazaki Mitsunori,Miyazaki Taiga,Miyazawa Keisuke,Mizushima Noboru,Mogensen Trine H,Mograbi Baharia,Mohammadinejad Reza,Mohamud Yasir,Mohanty Abhishek,Mohapatra Sipra,Möhlmann Torsten,Mohmmed Asif,Moles Anna,Moley Kelle H,Molinari Maurizio,Mollace Vincenzo,Møller Andreas Buch,Mollereau Bertrand,Mollinedo Faustino,Montagna Costanza,Monteiro Mervyn J,Montella Andrea,Montes L Ruth,Montico Barbara,Mony Vinod K,Monzio Compagnoni Giacomo,Moore Michael N,Moosavi Mohammad A,Mora Ana L,Mora Marina,Morales-Alamo David,Moratalla Rosario,Moreira Paula I,Morelli Elena,Moreno Sandra,Moreno-Blas Daniel,Moresi Viviana,Morga Benjamin,Morgan Alwena H,Morin Fabrice,Morishita Hideaki,Moritz Orson L,Moriyama Mariko,Moriyasu Yuji,Morleo Manuela,Morselli Eugenia,Moruno-Manchon Jose F,Moscat Jorge,Mostowy Serge,Motori Elisa,Moura Andrea Felinto,Moustaid-Moussa Naima,Mrakovcic Maria,Muciño-Hernández Gabriel,Mukherjee Anupam,Mukhopadhyay Subhadip,Mulcahy Levy Jean M,Mulero Victoriano,Muller Sylviane,Münch Christian,Munjal Ashok,Munoz-Canoves Pura,Muñoz-Galdeano Teresa,Münz Christian,Murakawa Tomokazu,Muratori Claudia,Murphy Brona M,Murphy J Patrick,Murthy Aditya,Myöhänen Timo T,Mysorekar Indira U,Mytych Jennifer,Nabavi Seyed Mohammad,Nabissi Massimo,Nagy Péter,Nah Jihoon,Nahimana Aimable,Nakagawa Ichiro,Nakamura Ken,Nakatogawa Hitoshi,Nandi Shyam S,Nanjundan Meera,Nanni Monica,Napolitano Gennaro,Nardacci Roberta,Narita Masashi,Nassif Melissa,Nathan Ilana,Natsumeda Manabu,Naude Ryno J,Naumann Christin,Naveiras Olaia,Navid Fatemeh,Nawrocki Steffan T,Nazarko Taras Y,Nazio Francesca,Negoita Florentina,Neill Thomas,Neisch Amanda L,Neri Luca M,Netea Mihai G,Neubert Patrick,Neufeld Thomas P,Neumann Dietbert,Neutzner Albert,Newton Phillip T,Ney Paul A,Nezis Ioannis P,Ng Charlene C W,Ng Tzi Bun,Nguyen Hang T T,Nguyen Long T,Ni Hong-Min,Ní Cheallaigh Clíona,Ni Zhenhong,Nicolao M Celeste,Nicoli Francesco,Nieto-Diaz Manuel,Nilsson Per,Ning Shunbin,Niranjan Rituraj,Nishimune Hiroshi,Niso-Santano Mireia,Nixon Ralph A,Nobili Annalisa,Nobrega Clevio,Noda Takeshi,Nogueira-Recalde Uxía,Nolan Trevor M,Nombela Ivan,Novak Ivana,Novoa Beatriz,Nozawa Takashi,Nukina Nobuyuki,Nussbaum-Krammer Carmen,Nylandsted Jesper,O'Donovan Tracey R,O'Leary Seónadh M,O'Rourke Eyleen J,O'Sullivan Mary P,O'Sullivan Timothy E,Oddo Salvatore,Oehme Ina,Ogawa Michinaga,Ogier-Denis Eric,Ogmundsdottir Margret H,Ogretmen Besim,Oh Goo Taeg,Oh Seon-Hee,Oh Young J,Ohama Takashi,Ohashi Yohei,Ohmuraya Masaki,Oikonomou Vasileios,Ojha Rani,Okamoto Koji,Okazawa Hitoshi,Oku Masahide,Oliván Sara,Oliveira Jorge M A,Ollmann Michael,Olzmann James A,Omari Shakib,Omary M Bishr,Önal Gizem,Ondrej Martin,Ong Sang-Bing,Ong Sang-Ging,Onnis Anna,Orellana Juan A,Orellana-Muñoz Sara,Ortega-Villaizan Maria Del Mar,Ortiz-Gonzalez Xilma R,Ortona Elena,Osiewacz Heinz D,Osman Abdel-Hamid K,Osta Rosario,Otegui Marisa S,Otsu Kinya,Ott Christiane,Ottobrini Luisa,Ou Jing-Hsiung James,Outeiro Tiago F,Oynebraten Inger,Ozturk Melek,Pagès Gilles,Pahari Susanta,Pajares Marta,Pajvani Utpal B,Pal Rituraj,Paladino Simona,Pallet Nicolas,Palmieri Michela,Palmisano Giuseppe,Palumbo Camilla,Pampaloni Francesco,Pan Lifeng,Pan Qingjun,Pan Wenliang,Pan Xin,Panasyuk Ganna,Pandey Rahul,Pandey Udai B,Pandya Vrajesh,Paneni Francesco,Pang Shirley Y,Panzarini Elisa,Papademetrio Daniela L,Papaleo Elena,Papinski Daniel,Papp Diana,Park Eun Chan,Park Hwan Tae,Park Ji-Man,Park Jong-In,Park Joon Tae,Park Junsoo,Park Sang Chul,Park Sang-Youel,Parola Abraham H,Parys Jan B,Pasquier Adrien,Pasquier Benoit,Passos João F,Pastore Nunzia,Patel Hemal H,Patschan Daniel,Pattingre Sophie,Pedraza-Alva Gustavo,Pedraza-Chaverri Jose,Pedrozo Zully,Pei Gang,Pei Jianming,Peled-Zehavi Hadas,Pellegrini Joaquín M,Pelletier Joffrey,Peñalva Miguel A,Peng Di,Peng Ying,Penna Fabio,Pennuto Maria,Pentimalli Francesca,Pereira Cláudia Mf,Pereira Gustavo J S,Pereira Lilian C,Pereira de Almeida Luis,Perera Nirma D,Pérez-Lara Ángel,Perez-Oliva Ana B,Pérez-Pérez María Esther,Periyasamy Palsamy,Perl Andras,Perrotta Cristiana,Perrotta Ida,Pestell Richard G,Petersen Morten,Petrache Irina,Petrovski Goran,Pfirrmann Thorsten,Pfister Astrid S,Philips Jennifer A,Pi Huifeng,Picca Anna,Pickrell Alicia M,Picot Sandy,Pierantoni Giovanna M,Pierdominici Marina,Pierre Philippe,Pierrefite-Carle Valérie,Pierzynowska Karolina,Pietrocola Federico,Pietruczuk Miroslawa,Pignata Claudio,Pimentel-Muiños Felipe X,Pinar Mario,Pinheiro Roberta O,Pinkas-Kramarski Ronit,Pinton Paolo,Pircs Karolina,Piya Sujan,Pizzo Paola,Plantinga Theo S,Platta Harald W,Plaza-Zabala Ainhoa,Plomann Markus,Plotnikov Egor Y,Plun-Favreau Helene,Pluta Ryszard,Pocock Roger,Pöggeler Stefanie,Pohl Christian,Poirot Marc,Poletti Angelo,Ponpuak Marisa,Popelka Hana,Popova Blagovesta,Porta Helena,Porte Alcon Soledad,Portilla-Fernandez Eliana,Post Martin,Potts Malia B,Poulton Joanna,Powers Ted,Prahlad Veena,Prajsnar Tomasz K,Praticò Domenico,Prencipe Rosaria,Priault Muriel,Proikas-Cezanne Tassula,Promponas Vasilis J,Proud Christopher G,Puertollano Rosa,Puglielli Luigi,Pulinilkunnil Thomas,Puri Deepika,Puri Rajat,Puyal Julien,Qi Xiaopeng,Qi Yongmei,Qian Wenbin,Qiang Lei,Qiu Yu,Quadrilatero Joe,Quarleri Jorge,Raben Nina,Rabinowich Hannah,Ragona Debora,Ragusa Michael J,Rahimi Nader,Rahmati Marveh,Raia Valeria,Raimundo Nuno,Rajasekaran Namakkal-Soorappan,Ramachandra Rao Sriganesh,Rami Abdelhaq,Ramírez-Pardo Ignacio,Ramsden David B,Randow Felix,Rangarajan Pundi N,Ranieri Danilo,Rao Hai,Rao Lang,Rao Rekha,Rathore Sumit,Ratnayaka J Arjuna,Ratovitski Edward A,Ravanan Palaniyandi,Ravegnini Gloria,Ray Swapan K,Razani Babak,Rebecca Vito,Reggiori Fulvio,Régnier-Vigouroux Anne,Reichert Andreas S,Reigada David,Reiling Jan H,Rein Theo,Reipert Siegfried,Rekha Rokeya Sultana,Ren Hongmei,Ren Jun,Ren Weichao,Renault Tristan,Renga Giorgia,Reue Karen,Rewitz Kim,Ribeiro de Andrade Ramos Bruna,Riazuddin S Amer,Ribeiro-Rodrigues Teresa M,Ricci Jean-Ehrland,Ricci Romeo,Riccio Victoria,Richardson Des R,Rikihisa Yasuko,Risbud Makarand V,Risueño Ruth M,Ritis Konstantinos,Rizza Salvatore,Rizzuto Rosario,Roberts Helen C,Roberts Luke D,Robinson Katherine J,Roccheri Maria Carmela,Rocchi Stephane,Rodney George G,Rodrigues Tiago,Rodrigues Silva Vagner Ramon,Rodriguez Amaia,Rodriguez-Barrueco Ruth,Rodriguez-Henche Nieves,Rodriguez-Rocha Humberto,Roelofs Jeroen,Rogers Robert S,Rogov Vladimir V,Rojo Ana I,Rolka Krzysztof,Romanello Vanina,Romani Luigina,Romano Alessandra,Romano Patricia S,Romeo-Guitart David,Romero Luis C,Romero Montserrat,Roney Joseph C,Rongo Christopher,Roperto Sante,Rosenfeldt Mathias T,Rosenstiel Philip,Rosenwald Anne G,Roth Kevin A,Roth Lynn,Roth Steven,Rouschop Kasper M A,Roussel Benoit D,Roux Sophie,Rovere-Querini Patrizia,Roy Ajit,Rozieres Aurore,Ruano Diego,Rubinsztein David C,Rubtsova Maria P,Ruckdeschel Klaus,Ruckenstuhl Christoph,Rudolf Emil,Rudolf Rüdiger,Ruggieri Alessandra,Ruparelia Avnika Ashok,Rusmini Paola,Russell Ryan R,Russo Gian Luigi,Russo Maria,Russo Rossella,Ryabaya Oxana O,Ryan Kevin M,Ryu Kwon-Yul,Sabater-Arcis Maria,Sachdev Ulka,Sacher Michael,Sachse Carsten,Sadhu Abhishek,Sadoshima Junichi,Safren Nathaniel,Saftig Paul,Sagona Antonia P,Sahay Gaurav,Sahebkar Amirhossein,Sahin Mustafa,Sahin Ozgur,Sahni Sumit,Saito Nayuta,Saito Shigeru,Saito Tsunenori,Sakai Ryohei,Sakai Yasuyoshi,Sakamaki Jun-Ichi,Saksela Kalle,Salazar Gloria,Salazar-Degracia Anna,Salekdeh Ghasem H,Saluja Ashok K,Sampaio-Marques Belém,Sanchez Maria Cecilia,Sanchez-Alcazar Jose A,Sanchez-Vera Victoria,Sancho-Shimizu Vanessa,Sanderson J Thomas,Sandri Marco,Santaguida Stefano,Santambrogio Laura,Santana Magda M,Santoni Giorgio,Sanz Alberto,Sanz Pascual,Saran Shweta,Sardiello Marco,Sargeant Timothy J,Sarin Apurva,Sarkar Chinmoy,Sarkar Sovan,Sarrias Maria-Rosa,Sarkar Surajit,Sarmah Dipanka Tanu,Sarparanta Jaakko,Sathyanarayan Aishwarya,Sathyanarayanan Ranganayaki,Scaglione K Matthew,Scatozza Francesca,Schaefer Liliana,Schafer Zachary T,Schaible Ulrich E,Schapira Anthony H V,Scharl Michael,Schatzl Hermann M,Schein Catherine H,Scheper Wiep,Scheuring David,Schiaffino Maria Vittoria,Schiappacassi Monica,Schindl Rainer,Schlattner Uwe,Schmidt Oliver,Schmitt Roland,Schmidt Stephen D,Schmitz Ingo,Schmukler Eran,Schneider Anja,Schneider Bianca E,Schober Romana,Schoijet Alejandra C,Schott Micah 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Satoru,Torres-López Liliana,Torriglia Alicia,Towers Christina G,Towns Roberto,Toyokuni Shinya,Trajkovic Vladimir,Tramontano Donatella,Tran Quynh-Giao,Travassos Leonardo H,Trelford Charles B,Tremel Shirley,Trougakos Ioannis P,Tsao Betty P,Tschan Mario P,Tse Hung-Fat,Tse Tak Fu,Tsugawa Hitoshi,Tsvetkov Andrey S,Tumbarello David A,Tumtas Yasin,Tuñón María J,Turcotte Sandra,Turk Boris,Turk Vito,Turner Bradley J,Tuxworth Richard I,Tyler Jessica K,Tyutereva Elena V,Uchiyama Yasuo,Ugun-Klusek Aslihan,Uhlig Holm H,Ułamek-Kozioł Marzena,Ulasov Ilya V,Umekawa Midori,Ungermann Christian,Unno Rei,Urbe Sylvie,Uribe-Carretero Elisabet,Üstün Suayib,Uversky Vladimir N,Vaccari Thomas,Vaccaro Maria I,Vahsen Björn F,Vakifahmetoglu-Norberg Helin,Valdor Rut,Valente Maria J,Valko Ayelén,Vallee Richard B,Valverde Angela M,Van den Berghe Greet,van der Veen Stijn,Van Kaer Luc,van Loosdregt Jorg,van Wijk Sjoerd J L,Vandenberghe Wim,Vanhorebeek Ilse,Vannier-Santos Marcos A,Vannini Nicola,Vanrell M 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Jochen,Wandosell Francisco G,Wang Bo,Wang Chao-Yung,Wang Chen,Wang Chenran,Wang Chenwei,Wang Cun-Yu,Wang Dong,Wang Fangyang,Wang Feng,Wang Fengming,Wang Guansong,Wang Han,Wang Hao,Wang Hexiang,Wang Hong-Gang,Wang Jianrong,Wang Jigang,Wang Jiou,Wang Jundong,Wang Kui,Wang Lianrong,Wang Liming,Wang Maggie Haitian,Wang Meiqing,Wang Nanbu,Wang Pengwei,Wang Peipei,Wang Ping,Wang Ping,Wang Qing Jun,Wang Qing,Wang Qing Kenneth,Wang Qiong A,Wang Wen-Tao,Wang Wuyang,Wang Xinnan,Wang Xuejun,Wang Yan,Wang Yanchang,Wang Yanzhuang,Wang Yen-Yun,Wang Yihua,Wang Yipeng,Wang Yu,Wang Yuqi,Wang Zhe,Wang Zhenyu,Wang Zhouguang,Warnes Gary,Warnsmann Verena,Watada Hirotaka,Watanabe Eizo,Watchon Maxinne,Wawrzyńska Anna,Weaver Timothy E,Wegrzyn Grzegorz,Wehman Ann M,Wei Huafeng,Wei Lei,Wei Taotao,Wei Yongjie,Weiergräber Oliver H,Weihl Conrad C,Weindl Günther,Weiskirchen Ralf,Wells Alan,Wen Runxia H,Wen Xin,Werner Antonia,Weykopf Beatrice,Wheatley Sally P,Whitton J Lindsay,Whitworth Alexander J,Wiktorska 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Scott O,Zeng Jialiu,Zeng Ju-Deng,Žerovnik Eva,Zhan Lixuan,Zhang Bin,Zhang Donna D,Zhang Hanlin,Zhang Hong,Zhang Hong,Zhang Honghe,Zhang Huafeng,Zhang Huaye,Zhang Hui,Zhang Hui-Ling,Zhang Jianbin,Zhang Jianhua,Zhang Jing-Pu,Zhang Kalin Y B,Zhang Leshuai W,Zhang Lin,Zhang Lisheng,Zhang Lu,Zhang Luoying,Zhang Menghuan,Zhang Peng,Zhang Sheng,Zhang Wei,Zhang Xiangnan,Zhang Xiao-Wei,Zhang Xiaolei,Zhang Xiaoyan,Zhang Xin,Zhang Xinxin,Zhang Xu Dong,Zhang Yang,Zhang Yanjin,Zhang Yi,Zhang Ying-Dong,Zhang Yingmei,Zhang Yuan-Yuan,Zhang Yuchen,Zhang Zhe,Zhang Zhengguang,Zhang Zhibing,Zhang Zhihai,Zhang Zhiyong,Zhang Zili,Zhao Haobin,Zhao Lei,Zhao Shuang,Zhao Tongbiao,Zhao Xiao-Fan,Zhao Ying,Zhao Yongchao,Zhao Yongliang,Zhao Yuting,Zheng Guoping,Zheng Kai,Zheng Ling,Zheng Shizhong,Zheng Xi-Long,Zheng Yi,Zheng Zu-Guo,Zhivotovsky Boris,Zhong Qing,Zhou Ao,Zhou Ben,Zhou Cefan,Zhou Gang,Zhou Hao,Zhou Hong,Zhou Hongbo,Zhou Jie,Zhou Jing,Zhou Jing,Zhou Jiyong,Zhou Kailiang,Zhou Rongjia,Zhou Xu-Jie,Zhou 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In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
Lysosomal Proteolysis Is Associated With Exercise-Induced Improvement of Mitochondrial Quality Control in Aged Hippocampus.
Luo Li,Dai Jia-Ru,Guo Shan-Shan,Lu A-Ming,Gao Xiao-Fang,Gu Yan-Rong,Zhang Xiao-Fei,Xu Hai-Dong,Wang Yan,Zhu Zhou,Wood Lisa J,Qin Zheng-Hong
The journals of gerontology. Series A, Biological sciences and medical sciences
Exercise improves cognitive function in older adults, but the underlying mechanism is largely unknown. Both lysosomal degradation and mitochondrial quality control decline with age. We hypothesized that exercise ameliorates age-related cognitive decline through the improvement of mitochondrial quality control in aged hippocampus, and this effect is associated with lysosomal proteolysis. Sixteen to eighteen-month old male Sprague Dawley rats underwent swim exercise training for 10 weeks. The exercise regimen prevented cognitive decline in aged rats, reduced oxidative stress, and rejuvenated mitochondria in the aged hippocampus. Exercise training promoted mitochondrial biogenesis, increased mitochondrial fusion and fission, and activated autophagy/mitophagy in aged hippocampal neurons. Lysosomal inhibitor chloroquine partly blocked beneficial effects of exercise on cognitive function, oxidative stress, autophagy/mitophagy, and mitochondrial quality control in aged rats. These results suggest that preservation of cognitive function by long-term exercise is associated with improvement of mitochondrial quality control in aged hippocampus and that lysosomal degradation is required for this process. Our findings suggest that exercise training or pharmacological regulation of mitochondrial quality control and lysosomal degradation may be effective strategies for slowing down age-related cognitive decline.
p53 mediates mitochondria dysfunction-triggered autophagy activation and cell death in rat striatum.
Zhang Xing-Ding,Wang Ye,Wang Yan,Zhang Xuan,Han Rong,Wu Jun-Chao,Liang Zhong-Qin,Gu Zhen-Lun,Han Feng,Fukunaga Kohji,Qin Zheng-Hong
In vivo administration of the mitochondrial inhibitor 3-nitropropionic acid (3-NP) produces striatal pathology mimicking Huntington disease (HD). However, the mechanisms of cell death induced by metabolic impairment are not fully understood. The present study investigated contributions of p53 signaling pathway to autophagy activation and cell death induced by 3-NP. Rat striatum was intoxicated with 3-NP by stereotaxic injection. Morphological and biochemical analyses demonstrated activation of autophagy in striatal cells as evidenced by increased formation of autophagosomes, the expression of active lysosomal cathepsin B and D, microtubule associate protein light chain 3 (LC3) and conversion of LC3-I to LC3-II. 3-NP upregulated the expression of tumor suppressor protein 53 (p53) and its target genes including Bax, p53-upregulated modulator of apoptosis (PUMA) and damage-regulated autophagy modulator (DRAM). 3-NP-induced elevations in pro-apoptotic proteins Bax and PUMA, autophagic proteins LC3-II and DRAM were significantly reduced by the p53 specific inhibitor pifithrin-alpha (PFT). PFT also significantly inhibited 3-NP-induced striatal damage. Similarly, 3-NP-induced DNA fragmentation and striatal cell death were robustly attenuated by the autophagy inhibitor 3-methyladenine (3-MA) and bafilomycin A1 (BFA). These results suggest that p53 plays roles in signaling both autophagy and apoptosis. Autophagy, at least partially, contributes to neurodegeneration induced by mitochondria dysfunction.
Molecular and cellular mechanisms of excitotoxic neuronal death.
Wang Yan,Qin Zheng-Hong
Apoptosis : an international journal on programmed cell death
Glutamate receptor-mediated excitatory neurotransmission plays a key role in neural development, differentiation and synaptic plasticity. However, excessive stimulation of glutamate receptors induces neurotoxicity, a process that has been defined as excitotoxicity. Excitotoxicity is considered to be a major mechanism of cell death in a number of central nervous system diseases including stroke, brain trauma, epilepsy and chronic neurodegenerative disorders. Unfortunately clinical trials with glutamate receptor antagonists, that would logically prevent the effects of excessive receptor activation, have been associated with untoward side effects or little clinical benefit. Therefore, uncovering molecular pathways involved in excitotoxic neuronal death is of critical importance to future development of clinical treatment of many neurodegenerative disorders where excitotoxicity has been implicated. This review discusses the current understanding of the molecular and cellular mechanisms of excitotoxicity and their roles in the pathogenesis of diseases of the central nervous system.
p53 induction contributes to excitotoxic neuronal death in rat striatum through apoptotic and autophagic mechanisms.
Wang Yan,Dong Xiao-Xia,Cao Yi,Liang Zhong-Qin,Han Rong,Wu Jun-Chao,Gu Zhen-Lun,Qin Zhen-Hong
The European journal of neuroscience
The present study sought to investigate mechanisms by which p53 induction contributes to excitotoxic neuronal injury. Rats were intrastriatally administered the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid (QA), the changes in the expression of p53 and its target genes involved in apoptosis and autophagy, including p53-upregulated modulator of apoptosis (PUMA), Bax, Bcl-2, damage-regulated autophagy modulator (DRAM) and other autophagic proteins including microtubule-associated protein 1 light chain 3 (LC3) and beclin 1 were assessed. The contribution of p53-mediated autophagy activation to apoptotic death of striatal neurons was assessed with co-administration of the nuclear factor-kappaB (NF-kappaB) inhibitor SN50, the p53 inhibitor Pifithrin-alpha (PFT-alpha) or the autophagy inhibitor 3-methyladenine (3-MA). The increased formation of autophagosomes and secondary lysosomes were observed with transmission electron microscope after excitotoxin exposure. QA induced increases in the expression of p53, PUMA, Bax and a decrease in Bcl-2. These changes were significantly attenuated by pre-treatment with SN50, PFT-alpha or 3-MA. SN50, PFT-alpha or 3-MA also reversed QA-induced upregulation of DRAM, the ratio of LC3-II/LC3-I and beclin 1 protein levels in the striatum. QA-induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by SN50, PFT-alpha or 3-MA. These results suggest that overstimulation of NMDA receptors can induce NF-kappaB-dependent expression of p53. p53 participates in excitotoxic neuronal death probably through both apoptotic and autophagic mechanisms.
Reduced Nicotinamide Adenine Dinucleotide Phosphate Inhibits MPTP-Induced Neuroinflammation and Neurotoxicity.
Zhou Ying,Wu Junchao,Sheng Rui,Li Mei,Wang Yan,Han Rong,Han Feng,Chen Zhong,Qin Zheng-Hong
It is generally believed that oxidative stress and neuroinflammation are implicated in the pathogenesis of Parkinson's disease (PD). Reduced nicotinamide adenine dinucleotide phosphate (NADPH) has been demonstrated to have potent neuroprotective effects against oxidative stress. In the present research, we investigated if NADPH could offer neuroprotection by inhibiting glia-mediated neuroinflammation induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mechanism contributing to PD pathogenesis. The current data demonstrated that MPTP/MPP increased levels of reactive oxygen species (ROS), activated glial cells, and inflammasome proteins in the substantia nigra (SNpc), in addition to inducing the nuclear translocation of nuclear factor-κB (NF-κB) and phosphorylation of p38 MAPK. These responses were inhibited by supplementation of exogenous NADPH. Moreover, NADPH effectively decreased MPP-induced excessive production of ROS, p38 phosphorylation and inflammatory protein of Cyclooxygenase2 (COX2) in cultured microglial BV-2 cells in vitro studies. Similarly, the p38 MAPK inhibitor SB203580 suppressed the upregulation of MPP-induced p38 phosphorylation and COX2 protein levels. Co-culture of neuronal cells with MPP-primed BV-2 cells increased the levels of tumor necrosis factor-alpha (TNF-α) and induced cell death of neuronal cells. These effects were diminished by TNF-α neutralizing antibody and NADPH. NADPH reduced motor dysfunction and the loss of dopaminergic (DA) cells induced by MPTP. Therefore, the present study demonstrates that NADPH protects DA neurons by inhibiting oxidative stress and glia-mediated neuroinflammation both in vitro and in vivo, thus suggesting a potential of clinical application for PD and other neurodegenerative diseases.
The regulation of N-terminal Huntingtin (Htt552) accumulation by Beclin1.
Wu Jun-chao,Qi Lin,Wang Yan,Kegel Kimberly B,Yoder Jennifer,Difiglia Marian,Qin Zheng-hong,Lin Fang
Acta pharmacologica Sinica
AIM:Huntingtin protein (Htt) was a neuropathological hallmark in human Huntington's Disease. The study aimed to investigate whether the macroautophagy regulator, Beclin1, was involved in the degradation of Htt. METHODS:PC12 cells and primary cultured brain neurons of rats were examined. pDC316 adenovirus shuttle plasmid was used to mediate the expression of wild-type Htt-18Q-552 or mutant Htt-100Q-552 in PC12 cells. The expression of the autophagy-related proteins LC3 II and Beclin1, as well as the lysosome-associated enzymes Cathepsin B and L was evaluated using Western blotting. The locations of Beclin1 and Htt were observed with immunofluorescence and confocal microscope. RESULTS:Htt552 expression increased the expression of LC3 II, Beclin1, cathepsin B and L in autophagy/lysosomal degradation pathway. Treatment with the autophagy inhibitor 3-MA or the proteasome inhibitors lactacystin and MG-132 increased Htt552 levels in PC12 cells infected with Ad-Htt-18Q-552 or Ad-Htt-100Q-552. The proteasome inhibitor caused a higher accumulation of Htt552-18Q than Htt552-100Q, and the autophagy inhibitor resulted in a higher accumulation of Htt552-100Q than Htt552-18Q. Similar results were observed in primary cultured neurons infected with adenovirus. In Htt552-expressing cells, Beclin1 was redistributed from the nucleus to the cytoplasm. Htt siRNA prevented Beclin1 redistribution in starvation conditions. Blockade of Beclin1 nuclear export by leptomycin B or Beclin1 deficiency caused by RNA interference induced the formation of mHtt552 aggregates. CONCLUSION:Beclin1 regulates the accumulation of Htt via macroautophagy.
NADPH and Mito-Apocynin Treatment Protects Against KA-Induced Excitotoxic Injury Through Autophagy Pathway.
Liu Na,Lin Miao-Miao,Huang Si-Si,Liu Zi-Qi,Wu Jun-Chao,Liang Zhong-Qin,Qin Zheng-Hong,Wang Yan
Frontiers in cell and developmental biology
Aim:Previous research recognizes that NADPH can produce reduced glutathione (GSH) as a coenzyme and produce ROS as a substrate of NADPH oxidase (NOX). Besides, excessive activation of glutamate receptors results in mitochondrial impairment. The study aims at spelling out the effects of NADPH and Mito-apocynin, a NOX inhibitor which specifically targets the mitochondria, on the excitotoxicity induced by Kainic acid (KA) and its mechanism. Methods:The neuronal excitotoxicity model was constructed by stereotypically injecting KA into the unilateral striatum of mice. Administrated NADPH (, intravenous) 30 min prior and Mito-apocynin (, intragastric) 1 day prior, respectively, then kept administrating daily until mice were sacrificed 14 days later. Nissl staining measured the lesion of striatum and survival status of neurons. Cylinder test of forelimb asymmetry and the adhesive removal test reflected the behavioral deficit caused by neural dysfunction. Determined Total superoxide dismutase (T-SOD), malondialdehyde (MDA), and GSH indicated oxidative stress. Western blot presented the expression levels of LC3-II/LC3-I, SQSTM1/p62, TIGAR, and NOX4. Assessed oxygen consumption rate using High-Resolution Respirometry. , the MitoSOX Indicator reflected superoxide released by neuron mitochondria. JC-1 and ATP assay Kit were used to detect mitochondrial membrane potential (MMP) and energy metabolism, respectively. Results:In this study, we have successfully established excitotoxic model by KA and . KA induced decreased SOD activity and increased MDA concentration. KA cause the change of LC3-II/LC3-I, SQSTM1/p62, and TIGAR expression, indicating the autophagy activation. NADPH plays a protective role and . It reversed the KA-mediated changes in LC3, SQSTM1/p62, TIGAR, and NOX4 protein expression. Mito-apocynin inhibited KA-induced increases in mitochondrial NOX4 expression and activity. Compared with NADPH, the combination showed more significant neuroprotective effects, presenting more neurons survive and better motor function recovery. The combination also better inhibited the over-activated autophagy. , combination of NADPH and Mito-apocynin performed better in restoring mitochondria membrane potential. Conclusion:In summary, combined administration of NADPH and NOX inhibitors offers better neuroprotection by reducing NADPH as a NOX substrate to generate ROS. The combined use of NADPH and Mito-apocynin can better restore neurons and mitochondrial function through autophagy pathway.
TIGAR alleviates ischemia/reperfusion-induced autophagy and ischemic brain injury.
Zhang Ding-Mei,Zhang Tian,Wang Ming-Ming,Wang Xin-Xin,Qin Yuan-Yuan,Wu Junchao,Han Rong,Sheng Rui,Wang Yan,Chen Zhong,Han Feng,Ding Yuqiang,Li Mei,Qin Zheng-Hong
Free radical biology & medicine
Autophagy has been reported to play protective and pathogenetic roles in cerebral ischemia/reperfusion (I/R)-induced neuronal injury. Our previous studies have shown that TP53-induced glycolysis and apoptosis regulator (TIGAR) ameliorates I/R-induced brain injury and reduces anti-cancer drug-induced autophagy activation. However, if TIGAR plays a regulatory role on autophagy in cerebral I/R injury is still unclear. The purpose of the present study is to investigate the role of TIGAR on I/R-induced autophagy activation and ischemic neuronal injury in vivo and in vitro stroke models using TIGAR-transgenic (tg-TIGAR) mice and TIGAR-knockout (ko-TIGAR) mice. The present study confirmed that autophagy was activated after I/R. Overexpression of TIGAR in tg-TIGAR mice significantly reduced I/R-induced autophagy activation and alleviated brain damage, while knockout of TIGAR in ko-TIGAR mice enhanced I/R-induced autophagy activation and exacerbated brain injury in vivo and in vitro. The different activity of autophagy in tg-TIGAR and ko-TIGAR primary neurons after OGD/R were largely reversed by knockdown or re-expression of TIGAR in these neurons. The autophagy inhibitor 3-methyladenine (3-MA) partly prevented exacerbation of brain damage induced by ko-TIGAR, whereas the autophagy inducer rapamycin partially abolished the neuroprotective effect of tg-TIGAR. Knockout of TIGAR reduced the levels of phosphorylated mTOR and S6KP70, which were blocked by 3-MA and NADPH after I/R and OGD/R in vivo and in vitro, respectively. Overexpression of TIGAR increased the levels of phosphorylated mTOR and S6KP70 under OGD/R condition, this enhancement effect was suppressed by rapamycin. In conclusion, our current data suggest that TIGAR protected against neuronal injury partly through inhibiting autophagy by regulating the mTOR-S6KP70 signaling pathway.
An autophagic mechanism is involved in apoptotic death of rat striatal neurons induced by the non-N-methyl-D-aspartate receptor agonist kainic acid.
Wang Yan,Han Rong,Liang Zhong-Qin,Wu Jun-Chao,Zhang Xing-Ding,Gu Zhen-Lun,Qin Zheng-Hong
Previous studies found that kainic acid (KA)-induced apoptosis involved the lysosomal enzyme cathepsin B, suggesting a possible mechanism of autophagy in excitotoxicity. The present study was sought to investigate activation and contribution of autophagy to excitotoxic neuronal injury mediated by KA receptors. The formation of autophagosomes was observed with transmission electron microscope after excitotoxin exposure. The contribution of autophagic mechanisms to KA-induced upregulation of microtubule-associated protein 1A/1B light chain 3 (LC3), lysosome- associated membrane protein 2 (LAMP2) and cathepsin B, release of cytochrome c, activation of caspase-3, down-regulation of Bcl-2, upregulation of Bax, p53, puma and apoptotic death of striatal neurons were assessed with co-administration of the autophagy inhibitor 3-methyladenine (3-MA). These studies showed that KA brought about an increase in the formation of autophagosomes and autolysosomes in the cytoplasm of striatal cells. KA-induced increases in the ratio of LC3-II/LC3-I, LAMP2, cathepsin B, release of cytochrome c and activation of caspase-3 were blocked by pre-treatment with 3-MA. 3-MA also reversed KA-induced down-regulation of Bcl-2 and upregulation of Bax protein levels, LC3, p53 and puma mRNA levels in the striatum. KA-induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by 3-MA. These results suggest that over-stimulation of KA receptors can activate autophagy. The autophagic mechanism participates in programmed cell death through regulating the mitochondria-mediated apoptotic pathway.
Microglia activation contributes to quinolinic acid-induced neuronal excitotoxicity through TNF-α.
Feng Wei,Wang Yan,Liu Zi-Qi,Zhang Xuan,Han Rong,Miao You-Zhu,Qin Zheng-Hong
Apoptosis : an international journal on programmed cell death
It has been reported that activation of NF-κB is involved in excitotoxicity; however, it is not fully understood how NF-κB contributes to excitotoxicity. The aim of this study is to investigate if NF-κB contributes to quinolinic acid (QA)-mediated excitotoxicity through activation of microglia. In the cultured primary cortical neurons and microglia BV-2 cells, the effects of QA on cell survival, NF-κB expression and cytokines production were investigated. The effects of BV-2-conditioned medium (BCM) on primary cortical neurons were examined. The effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, and minocycline (MC), an inhibitor of microglia activation, on QA-induced excitotoxicity were assessed. QA-induced NF-κB activation and TNF-α secretion, and the roles of TNF-α in excitotoxicity were studied. QA at the concentration below 1 mM had no apparent toxic effects on cultured primary neurons or BV-2 cells. However, addition of QA-primed BCM to primary neurons did aggravate QA-induced excitotoxicity. The exacerbation of QA-induced excitotoxicity by BCM was partially ameliorated by inhibiting NF-κB and microglia activation. QA induced activation of NF-κB and upregulation of TNF-α in BV-2 cells. Addition of recombinant TNF-α mimicked QA-induced excitotoxic effects on neurons, and neutralizing TNF-α with specific antibodies partially abolished exacerbation of QA-induced excitotoxicity by BCM. These studies suggested that QA activated microglia and upregulated TNF-α through NF-κB pathway in microglia. The microglia-mediated inflammatory pathway contributed, at least in part, to QA-induced excitotoxicity.
Cathepsin L plays a role in quinolinic acid-induced NF-Κb activation and excitotoxicity in rat striatal neurons.
Wang Yan-Ru,Qin Shu,Han Rong,Wu Jun-Chao,Liang Zhong-Qin,Qin Zheng-Hong,Wang Yan
The present study seeks to investigate the role of cathepsin L in glutamate receptor-induced transcription factor nuclear factor-kappa B (NF-κB) activation and excitotoxicity in rats striatal neurons. Stereotaxic administration of the N-methyl-d-aspartate (NMDA) receptor agonist Quinolinic acid (QA) into the unilateral striatum was used to produce the in vivo excitotoxic model. Co-administration of QA and the cathepsin L inhibitor Z-FF-FMK or 1-Naphthalenesulfonyl-IW-CHO (NaphthaCHO) was used to assess the contribution of cathepsin L to QA-induced striatal neuron death. Western blot analysis and cathepsin L activity assay were used to assess the changes in the levels of cathepsin L after QA treatment. Western blot analysis was used to assess the changes in the protein levels of inhibitor of NF-κB alpha isoform (IκB-α) and phospho-IκB alpha (p-IκBα) after QA treatment. Immunohistochemical analysis was used to detect the effects of Z-FF-FMK or NaphthaCHO on QA-induced NF-κB. Western blot analysis was used to detect the effects of Z-FF-FMK or NaphthaCHO on QA-induced IκB-α phosphorylation and degradation, changes in the levels of IKKα, p-IKKα, TP53, caspase-3, beclin1, p62, and LC3II/LC3I. The results show that QA-induced loss of striatal neurons were strongly inhibited by Z-FF-FMK or NaphthaCHO. QA-induced degradation of IκB-α, NF-κB nuclear translocation, up-regulation of NF-κB responsive gene TP53, and activation of caspase-3 was strongly inhibited by Z-FF-FMK or NaphthaCHO. QA-induced increases in beclin 1, LC3II/LC3I, and down-regulation of p62 were reduced by Z-FF-FMK or NaphthaCHO. These results suggest that cathepsin L is involved in glutamate receptor-induced NF-κB activation. Cathepsin L inhibitors have neuroprotective effects by inhibiting glutamate receptor-induced IκB-α degradation and NF-κB activation.
NADPH protects against kainic acid-induced excitotoxicity via autophagy-lysosome pathway in rat striatum and primary cortical neurons.
Liu Zi-Qi,Liu Na,Huang Si-Si,Lin Miao-Miao,Qin Shu,Wu Jun-Chao,Liang Zhong-Qin,Qin Zheng-Hong,Wang Yan
PURPOSE:To investigate the effects and mechanisms of NADPH on Kainic acid (KA)-induced excitotoxicity. METHODS:KA, a non-N-methyl-d-aspartate glutamate receptor agonist, was exposed to adult SD rats via intrastriatal injection and rat primary cortical neurons to establish excitotoxic models in vivo and in vitro, respectively. To determine the effects of NADPH on KA-induced excitotoxicity, neuronal survival, neurologically behavioral score and oxidative stress were evaluated. To explore the mechanisms of neuroprotective effects of NADPH, the autophagy-lysosome pathway related proteins were detected. RESULTS:In vivo, NADPH (1 mg/kg or 2 mg/kg) diminished KA (2.5 nmol)-induced enlargement of lesion size in striatum, improved KA-induced dyskinesia and reversed KA-induced activation of glial cells. Nevertheless, the neuroprotective effect of NADPH was not significant under the condition of autophagy activation. NADPH (2 mg/kg) inhibited KA (2.5 nmol)-induced down-regulation of TP-53 induced glycolysis and apoptosis regulator (TIGAR) and p62, and up-regulation of the protein levels of LC3-II/LC3-I, Beclin-1 and Atg5. In vitro, the excitotoxic neuronal injury was induced after KA (50 μM, 100 μM or 200 μM) treatment as demonstrated by decreased cell viability. Moreover, KA (100 μM) increased the intracellular levels of calcium and reactive oxygen species (ROS) and declined the levels of the reduced form of glutathione (GSH). Pretreatment of NADPH (10 μM) effectively reversed these changes. Meanwhile NADPH (10 μM) inhibited KA (100 μM)-induced down-regulation of TIGAR and p62, and up-regulation of the ratio of LC3-II/LC3-I, Beclin-1, Atg5, active-cathepsin B and active-cathepsin D. CONCLUSIONS:Our data provide a possible mechanism that NADPH ameliorates KA-induced excitotoxicity by blocking the autophagy-lysosome pathway and up-regulating TIGAR along with its antioxidant properties.
Huntingtin cleavage induced by thrombin in vitro.
Lin Fang,Wu Junchao,Wang Yan,Qin Zhenghong
Acta biochimica et biophysica Sinica
Huntingtin (Htt) mutation causes Huntington's disease. Sequence analysis of Htt revealed a possible thrombin cleavage site in the N-terminal region of Htt. In order to investigate if thrombin can cleave Htt, we expressed the N-terminal fragment (1-969) of wild-type (wt) Htt (Htt 1-969) in MCF-7 cells and studied its cleavage pattern by thrombin in vitro. An expression plasmid pcDNA3-Htt-18Q-969 was used to transfect MCF-cells and Htt 1-969 expression was confirmed with immunofluorescence. Cell lysates were incubated with thrombin (1 U/ml, 10 U/ml, and 30 U/ml) for 1 h in the presence or absence of hirudin, a thrombin inhibitor. Htt fragments were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected with anti-Htt antibodies. An Htt fragment with molecular mass of approximately 80 kDa was produced after incubation with thrombin. The size of this Htt fragment was anticipated by molecular mass generated from thrombin-mediated cleavage at the amino acid 183 in the Htt. Production of an 80 kDa fragment was inhibited by hirudin. This study provides the first evidence that Htt is cleaved by thrombin in vitro at amino acid 183. If endogenous thrombin cleaves Htt in vivo, the physiological significance of thrombin-mediated cleavage of Htt should be further investigated.
Sequestration of glyceraldehyde-3-phosphate dehydrogenase to aggregates formed by mutant huntingtin.
Wu Junchao,Lin Fang,Qin Zhenghong
Acta biochimica et biophysica Sinica
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been reported to interact with proteins containing the polyglutamine (polyQ) domain. The present study was undertaken to evaluate the potential contributions of the polyQ and polyproline (polyP) domains to the co-localization of mutant huntingtin (htt) and GAPDH. Overexpression of N-terminal htt (1-969 amino acids) with 100Q and 46Q (htt1-969-100Q and httl-969-46Q, mutant htt) in human mammary gland carcinoma MCF-7 cells formed more htt aggregates than that of htt1-969-18Q (wild-type htt). The co-localization of GAPDH with htt aggregates was found in the cells expressing mutant but not wild-type htt. Deletion of the polyP region in the N-terminal htt had no effect on the co-localization of GAPDH and mutant htt aggregates. These results suggest that the polyQ domain, but not the polyP domain, plays a role in the sequestration of GAPDH to aggregates by mutant htt. This effect might contribute to the dysfunction of neurons caused by mutant htt in Huntington's disease.
High efficiency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes.
Wang Linhui,Lin Fang,Wu Junchao,Qin Zhenghong
Acta biochimica et biophysica Sinica
Huntington's disease (HD) is caused by an expansion of polyglutamine tract in N-terminus of huntingtin (htt). The mutation of htt leads to dysfunction and premature death of striatal and cortical neurons. However, the effects of htt mutation on glia remain largely unknown. This study aimed to establish a glia HD model using an adenoviral vector to express wild-type and mutant N-terminal huntingtin fragment 1-552 amino acids (htt552) in rat primary cortical astrocytes. We have evaluated optimal conditions for the infection of astrocytes with adenoviral vectors, and the kinetics of the expression of htt552 in astrocytes. The majority of astrocytes expressed the transgene after infection. At 24 h postinfection, the highest rate of infection was 89+/-3% for the wild-type (htt552-18Q) with a multiplicity of infection (m.o.i.) of 80, and the highest rate of infection was 91+/-4% for the mutant type (htt552-100Q) with the same viral dose. The duration of expression of htt552 lasted for about 7 days with a relatively high level from 1 to 4 days post-infection. Mutant huntingtin (htt552-100Q) produced the characteristic HD pathology after 3 days by the appearance of cytoplasmic aggregates and intranuclear inclusions. The result of MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay showed that the inhibition of viability by virus on astrocytes was also dose-dependent. To obtain high infection rate and low toxicity, the viral dose with an m.o.i. of 40 was optimal to our cell model. The present study demonstrates that adenoviral-mediated expression of mutant htt provides an advantageous system for histological and biochemical analysis of HD pathogenesis in primary cortical astrocyte cultures.
The lysosome and neurodegenerative diseases.
Zhang Lisha,Sheng Rui,Qin Zhenghong
Acta biochimica et biophysica Sinica
It has long been believed that the lysosome is an important digestive organelle. There is increasing evidence that the lysosome is also involved in pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Abnormal protein degradation and deposition induced by lysosomal dysfunction may be the primary contributor to age-related neurodegeneration. In this review, the possible relationship between lysosome and various neurodegenerative diseases is described.
Autophagy impairment inhibits differentiation of glioma stem/progenitor cells.
Zhao Yaodong,Huang Qiang,Yang Jicheng,Lou Meiqing,Wang Aidong,Dong Jun,Qin Zhenghong,Zhang Tianyi
Despite of similarities between glioma stem/progenitor cells (GSPCs) and neural stem/progenitor cells (NSPCs), inhibition of differentiation is a distinct characteristic of GSPCs. In this study, we investigated the effects of autophagy impairment on inhibition of differentiation of GSPC, and its molecular mechanism. GSPCs were kept by our laboratory; NSPCs were isolated from human fetal brain tissue. We found that the autophagic activity in GSPCs was significantly lower than that in NSPCs. However, the autophagic activity markedly increased after GSPCs were induced to differentiate by fetal calf serum (FCS). The autophagy inhibitors 3-methyladenine and Bafilomycin A1 (BFA) inhibited the FSC-induced differentiation of GSPCs. And autophagy activator Rapamycin could promote differentiation of GSPCs. In order to disclose whether the loss of PTEN in GSPC is related to the deficiency of autophagic activity in GSPCs (for PTEN being lost in the GSPCs studied by us), we introduced the wild type gene of PTEN into GSPCs, and found that the autophagic activity was restored significantly after the gene transduction. The low autophagic activity in GSPCs leads to the inhibition of differentiation of GSPCs, and the loss of PTEN in GSPCs probably is an underlying mechanism for the low autophagic activity in GSPCs. These results suggest that bust autophagic activity target at PTEN might be a potential therapy target for glioma therapy.
Glioma stem cells involved in tumor tissue remodeling in a xenograft model.
Dong Jun,Zhang Quanbin,Huang Qiang,Chen Hua,Shen Yuntian,Fei Xifeng,Zhang Tianyi,Diao Yi,Wu Zicheng,Qin Zhenghong,Lan Qing,Gu Xiaosong
Journal of neurosurgery
OBJECT:Although tissue remodeling plays a crucial role in the tumorigenesis and progression of human gliomas, its mechanisms remain largely uncertain. In the current study, the authors investigated the potential role of human glioma stem cells (hGSCs) in the tissue remodeling of gliomas. METHODS:Transgenic nude mice with ubiquitous green fluorescent protein (GFP) expression were obtained by crossing nontransgenic NC athymic nude mice with the GFP transgenic C57BL/6J mice. As a result, GFP was expressed in essentially all tissues in the offspring. Human glioma stem cells were then orthotopically implanted into the GFP nude mice in an effort to assess the hGSC-host brain interactions and thereby elucidate the roles of tissue remodeling during tumorigenesis and progression of human gliomas. RESULTS:All of the essential tissues in the GFP transgenic nude mice, including the brain, fluoresced green under an excitation light; therefore, tumor remodeling by hGSCs can be unambiguously distinguished from a bright green background composed of adjacent host GFP-expressing components. This technique enabled the authors to address the following concerns: 1) hGSCs were involved in the invasiveness of gliomas and adjacent stroma degradation of the host. 2) An in vivo study demonstrated that cell fusion occurred between hGSCs and host cells. 3) Vasculogenic mimicry--the formation of patterned, tubular networks of vascular channels by transdifferentiated hGSCs--could be observed. 4) Differentiation mimicry--namely, the differentiation direction of hGSCs bearing multidifferentiation potentials--seemed to be decided by the local host cellular microenviroment. CONCLUSIONS:The results of this study indicated that the GFP transgenic nude mice model with GFP expression in essentially all tissues could be obtained by crossing nontransgenic athymic nude mice with transgenic GFP mice. This model should greatly expand our knowledge of glioma-host interactions. The data indicated that hGSCs might play a decisive role in tissue remodeling of gliomas as well.
Activation of M3 muscarinic receptors inhibits T-type Ca(2+) channel currents via pertussis toxin-sensitive novel protein kinase C pathway in small dorsal root ganglion neurons.
Zhang Yiming,Zhang Ling,Wang Fen,Zhang Yi,Wang Jiangong,Qin Zhenghong,Jiang Xinghong,Tao Jin
Cobrotoxin (CbT), a short-chain postsynaptic α-neurotoxin, has been reported to play a role in analgesia. However, to date, the detailed mechanisms still remain unknown. In the present study, we identify a novel functional role of CbT in modulating T-type Ca(2+) channel currents (T-currents) in small dorsal root ganglia (DRG) neurons as well as pain behaviors in mice. We found that CbT inhibited T-currents in a dose-dependent manner. CbT at 1μM reversibly inhibited T-currents by ~26.3%. This inhibitory effect was abolished by the non-selective muscarinic acetylcholine receptor (mAChR) antagonist atropine, or the selective M3 mAChR antagonist 4-DAMP, while naloxone, an opioid receptor antagonist had no effect. Intracellular infusion of GDP-β-S or pretreatment of the cells with pertussis toxin (PTX) completely blocked the inhibitory effects of CbT. Using depolarizing prepulse, we found the absence of direct binding between G-protein βγ subunits and T-type Ca(2+) channels in CbT-induced T-current inhibition. CbT responses were abolished by the phospholipase C inhibitor U73122 (but not the inactive analog U73343). The classical and novel protein kinase C (nPKC) antagonist chelerythrine chlorid or GF109203X abolished CbT responses, whereas the classical PKC antagonist Ro31-8820 or inhibition of PKA elicited no such effects. Intrathecal administration of CbT (5μg/kg) produced antinociceptive effects in mechanical, thermal, and inflammatory pain models. Moreover, CbT-induced antinociception could be abrogated by 4-DAMP. Taken together, these results suggest that CbT acting through M3 mAChR inhibits T-currents via a PTX-sensitive nPKC pathway in small DRG neurons, which could contribute to its analgesic effects in mice.
Alpha-cobratoxin inhibits T-type calcium currents through muscarinic M4 receptor and Gο-protein βγ subunits-dependent protein kinase A pathway in dorsal root ganglion neurons.
Zhang Ling,Zhang Yiming,Jiang Dongsheng,Reid Paul F,Jiang Xinghong,Qin Zhenghong,Tao Jin
The long-chain neurotoxic protein, alpha-cobratoxin (α-CTx), has been shown to have analgesic effects. However, the underlying mechanisms still remain unclear. In this study, we examined the effects of α-CTx on T-type calcium channel currents (T-currents) and elucidated the relevant mechanisms in mouse dorsal root ganglion (DRG) neurons. Our results showed that α-CTx reversibly inhibited T-currents in a dose-dependent manner. This inhibitory effect was blocked by the selective muscarinic M4 receptor antagonist tropicamide, while methyllycaconitine, a specific antagonist for the α7 subtype of nicotinic receptor had no effect. siRNA targeting the M4 receptor in small DRG neurons abolished α-CTx-induced T-current inhibition. Intracellular application of GDP-β-S or a selective antibody against the G(o)α-protein, as well as pretreatment of the cells with pertussis toxin, abolished the inhibitory effects of α-CTx. The M4 receptor-mediated response was blocked by dialyzing cells with QEHA peptide or anti-G(β) antibody. Pretreatment of the cells with protein kinase A (PKA) inhibitor H89 or intracellular application of PKI 6-22 abolished α-CTx-induced T-current inhibition in small DRG neurons, whereas inhibition of phosphatidylinositol 3-kinase or PKC elicited no such effects. In addition, α-CTx significantly increased PKA activity in DRG neurons, whereas pretreatment of the cells with tropicamide abolished this effect. In summary, our results suggest that activation of muscarinic M4 receptor by α-CTx inhibits T-currents via the G(βγ) of G(o)-protein and PKA-dependent pathway. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.
Truncated N-terminal huntingtin fragment with expanded-polyglutamine (htt552-100Q) suppresses brain-derived neurotrophic factor transcription in astrocytes.
Wang Linhui,Lin Fang,Wang Jin,Wu Junchao,Han Rong,Zhu Lujia,Zhang Guoxing,DiFiglia Marian,Qin Zhenghong
Acta biochimica et biophysica Sinica
Although huntingtin (htt) can be cleaved at many sites by caspases, calpains, and aspartyl proteases, amino acid (aa) 552 was defined as a preferred site for cleavage in human Huntington disease (HD) brains in vivo. To date, the normal function of wild-type N-terminal htt fragment 1-552 aa (htt552) and its pathological roles of mutant htt552 are still unknown. Although mutant htt (mhtt) is also expressed in astrocytes, whether and how mhtt contributes to the neurodegeneration through astrocytes in HD remains largely unknown. In this study, a glia HD model, using an adenoviral vector to express wild-type htt552 (htt552-18Q) and its mutation (htt552-100Q) in rat primary cortical astrocytes, was generated to investigate the influence of htt552 on the transcription of brain-derived neurotrophic factor (BDNF). Results from enzyme linked immunosorbent assay showed that the level of BDNF in astrocyte-conditioned medium was decreased in the astrocytes expressing htt552-100Q. Quantitative real-time polymerase chain reaction demonstrated that htt552-100Q reduced the transcripts of the BDNF III and IV, hence, repressed the transcription of BDNF. Furthermore, immunofluorescence showed that aggregates formed by htt552-100Q entrapped transcription factors cAMP-response element-binding protein and stimulatory protein 1, which might account for the reduction of BDNF transcription. These findings suggest that mhtt552 reduces BDNF transcription in astrocytes, which might contribute to the neuronal dysfunction in HD.
Expression of mutant N-terminal huntingtin fragment (htt552-100Q) in astrocytes suppresses the secretion of BDNF.
Wang Linhui,Lin Fang,Wang Jin,Wu Junchao,Han Rong,Zhu Lujia,Difiglia Marian,Qin Zhenghong
Huntington's disease (HD) is an inheritable neurological disorder caused by an abnormal expansion of the polyglutamine tract in the N-terminus of the protein huntingtin (htt). Mutant htt (mhtt) leads to selective neurodegeneration that preferentially affects striatal medium spiny neurons. Although mhtt is also expressed in astrocytes, whether and how astrocyte derived mhtt contributes to the neurodegeneration in HD remains largely unknown. In this study, a glia HD model, using an adenoviral vector to express wild-type and mutant N-terminal huntingtin fragment 1-552 aa (htt552) in rat primary cortical astrocytes, was generated. The influence of htt552 on the protein level of brain-derived neurotrophic factor (BDNF) in astrocytes was evaluated. Immunofluorescence showed that htt552-100Q formed aggregates in some astrocytes. These mhtt aggregates sequestered clathrin immunoreactivities and dispersed the Golgi complex. ELISA and immunofluorescence demonstrated an increase in BDNF levels in the astrocytes expressing htt552-100Q. Western blot analysis showed that there was an increase in pro-BDNF, but a decrease in mature BDNF in the astrocytes expressing htt552-100Q. Furthermore, medium collected from astrocytes expressing htt552-100Q showed a lower level of mature BDNF and less activity in supporting neurite development of primary cortical neurons. These results suggest that aggregates formed by mutant htt552 affect processing and secretion of the BDNF in astrocytes, which might contribute to the neuronal dysfunction and degeneration in HD.
TP53-induced glycolysis and apoptosis regulator (TIGAR) ameliorates lysosomal damage in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-mediated mouse model of Parkinson's disease.
Ge Jianbin,Lin Hongyan,Yang Jie,Li QiQi,Zhou Jingsi,Qin Zhenghong,Wu Feng
The progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) correlates with rupture of lysosome in Parkinson's disease (PD). It has been found that TP53-induced glycolysis and apoptosis regulator (TIGAR) has been attributed to the regulation of metabolic pathways and neuroprotective effect. In the present study, we showed in a mouse model that 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) caused lysosomal damage and DA neurons loss in the SNpc. MPTP only induced SP1-mediated TIGAR upregulation in the early stage of neurotoxin-induced pathology, and this compensatory mechanism was not enough to maintain normal lysosomal function. MPTP significantly decreased the levels of NADPH and GSH, and the effects were ameliorated by the expression of exogenous TIGAR but execerbated by knockdown of TIAGR. TIGAR or NADPH alleviated oxidative stress, rescued lysosomal dysfunction and attenuated DA neurons degeneration. Overexpression of TIGAR or NADPH supplement inhibited MPP-mediated reactive oxygen species (ROS), lysosomal membrane permeabilization (LMP) and autophagic flux impairment in PC12 cells. Together, these findings suggest that TIGAR reduces MPTP-mediated oxidative stress, lysosomal depletion and DA neuron damage.