Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autop...Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy.Methods:The primary hippocampal neurons,N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy,which was analysed by Student’s two-tailed t-test.The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1(mTORC1)activity and the vacuolar H+-ATPase(v-ATPase)activity,respectively,which were analysed by One-way ANOVA with post hoc tests.The Western blotting,co-immunoprecipitation and immunofuorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations,as analysed by Student’s two-tailed t-test or One-way ANOVA with post hoc tests.The autophagosome formation was detected by immunofuorescence staining and transmission electron microscopy.The amino acids(AA)levels were detected by high performance liquid chromatography(HPLC).Results:We observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits.Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1(PRD-TIA1)and this association significantly increased the intercellular level of amino acids(Leucine,P=0.0038;Glutamic acid,P=0.0348;Alanine,P=0.0037;Glycine,P=0.0104),with concordant upregulation of mTORC1 activity[phosphorylated eukaryotic translation initiation factor 4E-binding protein 1(p-4EBP1),P<0.0001;phosphorylated 70 kD ribosomal protein S6 kinase 1(p-p70S6K1),P=0.0001,phosphorylated unc-51-like autophagyactivating kinase 1(p-ULK1),P=0.0015]and inhibition of autophagosome formation[microtubuleassociated protein light chain 3 II(LC3 II),P=0.0073;LC3 puncta,P<0.0001].As expected,this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation.Importantly,we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1,downregulating the endogenous TIA1 expression by shRNA,or downregulating tau protein level by a small proteolysis targeting chimera(PROTAC)could remarkably attenuate tau-induced autophagy impairment.Conclusions:Our findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway,and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treat-ment and that of related tauopathies.展开更多
Autophagy is a conserved and programmed catabolic process that degrades damaged pro- teins and organelles. But the underlying mechanism and functions of autophagy in the ische- mia-reperfusion （IR）-induced injury ar...Autophagy is a conserved and programmed catabolic process that degrades damaged pro- teins and organelles. But the underlying mechanism and functions of autophagy in the ische- mia-reperfusion （IR）-induced injury are unknown. In this study, we employed simulated IR of N2a cells as an in vitro model of IR injury to the neurons and monitored autophagic processes. It was found that the levels of Beclin-1 （a key molecule of autophay complex, Beclin-1/elass III PI3K） and LC-3 II （an autophagy marker） were remarkably increased with time during the process of ischemia and the process of reperfusion after 90 min of ischemia, while the protein kinases pTOS6K and roTOR which are involved in autophagy regulation showed delayed inactivation after reperfusion. Admini- stration of 3-methyladenine （3MA）, an inhibitor of class III PI3K, abolished autophagy during reper- fusion, while employment of rapamycin, an inhibitor of mTORC1 （normally inducing autophagy）, surprisingly weakened the induction of autophagy during reperfusion. Analyses of mitochondria function by relative cell viability demonstrated that autophagy inhibition by 3-MA attenuated the de- cline of mitochondria function during reperfusion. Our data demonstrated that there were two distinct dynamic patterns of autophagy during IR-induced N2a injury, Beclin-1/class III PI3K com- plex-dependent and mTORCl-dependent. Inhibition of over-autophagy improved cell survival. These suggest that targeting autophagy therapy will be a novel strategy to control IR-induced neuronal damage.展开更多
The mechanistic target of rapamycin complex 1(mTORC1)controls cell growth and metabolism in response to various environmental inputs,especially amino acids.In fact,the activity of mTORC1 is highly sensitive to changes...The mechanistic target of rapamycin complex 1(mTORC1)controls cell growth and metabolism in response to various environmental inputs,especially amino acids.In fact,the activity of mTORC1 is highly sensitive to changes in amino acid levels.Over past decades,a variety of proteins have been identified as participating in the mTORC1 pathway regulated by amino acids.Classically,the Rag guanosine triphosphatases(GTPases),which reside on the lysosome,transmit amino acid availability to the mTORC1 pathway and recruit mTORC1 to the lysosome upon amino acid sufficiency.Recently,several sensors of leucine,arginine,and S-adenosylmethionine for the amino acidstimulated mTORC1 pathway have been coming to light.Characterization of these sensors is requisite for understanding how cells adjust amino acid sensing pathways to their different needs.In this review,we summarize recent advances in amino acid sensing mechanisms that regulate mTORC1 activity and highlight these identified sensors that accurately transmit specific amino acid signals to the mTORC1 pathway.展开更多
基金supported by grants from the Natural Science Foundation of China(91949205,31730035,81721005)the Science and Technology Committee of China(2016YFC1305800)+1 种基金the Special Project of Technological Innovation of Hubei Province(2018ACA142)Guangdong Provincial Key S&T Program(2018B030336001)。
文摘Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy.Methods:The primary hippocampal neurons,N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy,which was analysed by Student’s two-tailed t-test.The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1(mTORC1)activity and the vacuolar H+-ATPase(v-ATPase)activity,respectively,which were analysed by One-way ANOVA with post hoc tests.The Western blotting,co-immunoprecipitation and immunofuorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations,as analysed by Student’s two-tailed t-test or One-way ANOVA with post hoc tests.The autophagosome formation was detected by immunofuorescence staining and transmission electron microscopy.The amino acids(AA)levels were detected by high performance liquid chromatography(HPLC).Results:We observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits.Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1(PRD-TIA1)and this association significantly increased the intercellular level of amino acids(Leucine,P=0.0038;Glutamic acid,P=0.0348;Alanine,P=0.0037;Glycine,P=0.0104),with concordant upregulation of mTORC1 activity[phosphorylated eukaryotic translation initiation factor 4E-binding protein 1(p-4EBP1),P<0.0001;phosphorylated 70 kD ribosomal protein S6 kinase 1(p-p70S6K1),P=0.0001,phosphorylated unc-51-like autophagyactivating kinase 1(p-ULK1),P=0.0015]and inhibition of autophagosome formation[microtubuleassociated protein light chain 3 II(LC3 II),P=0.0073;LC3 puncta,P<0.0001].As expected,this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation.Importantly,we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1,downregulating the endogenous TIA1 expression by shRNA,or downregulating tau protein level by a small proteolysis targeting chimera(PROTAC)could remarkably attenuate tau-induced autophagy impairment.Conclusions:Our findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway,and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treat-ment and that of related tauopathies.
文摘目的·探究雷帕霉素靶蛋白复合物1(mechanistic target of rapamycin complex 1,mTORC1)对3型固有淋巴细胞(group 3 innate lymphoid cell,ILC3)功能的影响。方法·使用mTORC1信号通路的特异性抑制剂雷帕霉素体外刺激野生型C57BL/6小鼠肠道固有层淋巴细胞(lamina propria leukocyte,LPL),初步观察ILC3细胞数量及功能的变化;之后采用流式细胞技术分选出小鼠肠道ILC3,用白细胞介素23(interleukin 23,IL-23)刺激使其活化,再通过实时荧光定量PCR检测活化后ILC3中编码转录因子视黄酸受体相关的孤儿核受体(retinoic acid receptor related orphan receptor,RORγt)、IL-22及mTORC1关键组分Raptor(regulatory associated protein of mTOR)的基因mRNA水平;构建ILC3细胞特异性敲除Raptor的基因工程小鼠,使用苏木精-伊红染色、流式细胞技术和实时荧光定量PCR检测mTORC1功能缺失后对肠道结构、结肠ILC3细胞数量及功能的影响。结果·雷帕霉素刺激后LPL中ILC3数量无变化,但分泌IL-22减少;ILC3被IL-23激活后,检测到编码Raptor、RORγt和IL-22的基因mRNA表达水平同步上调;Raptor缺失的小鼠肠道结构无明显损伤,结肠ILC3数量和亚群比例与对照小鼠无明显差异,但ILC3活化后分泌细胞因子IL-22的水平显著下降。结论·mTORC1功能缺失显著抑制ILC3分泌IL-22的能力,对肠道结构及肠道ILC3的发育无影响,可见mTORC1信号对肠道ILC3功能具有正向调控作用。
基金supported by grants from the Graduate Programs Foundation of Ministry of Education of China(No.20070487101)Postdoctoral Foundation of China(No.20090450216)
文摘Autophagy is a conserved and programmed catabolic process that degrades damaged pro- teins and organelles. But the underlying mechanism and functions of autophagy in the ische- mia-reperfusion （IR）-induced injury are unknown. In this study, we employed simulated IR of N2a cells as an in vitro model of IR injury to the neurons and monitored autophagic processes. It was found that the levels of Beclin-1 （a key molecule of autophay complex, Beclin-1/elass III PI3K） and LC-3 II （an autophagy marker） were remarkably increased with time during the process of ischemia and the process of reperfusion after 90 min of ischemia, while the protein kinases pTOS6K and roTOR which are involved in autophagy regulation showed delayed inactivation after reperfusion. Admini- stration of 3-methyladenine （3MA）, an inhibitor of class III PI3K, abolished autophagy during reper- fusion, while employment of rapamycin, an inhibitor of mTORC1 （normally inducing autophagy）, surprisingly weakened the induction of autophagy during reperfusion. Analyses of mitochondria function by relative cell viability demonstrated that autophagy inhibition by 3-MA attenuated the de- cline of mitochondria function during reperfusion. Our data demonstrated that there were two distinct dynamic patterns of autophagy during IR-induced N2a injury, Beclin-1/class III PI3K com- plex-dependent and mTORCl-dependent. Inhibition of over-autophagy improved cell survival. These suggest that targeting autophagy therapy will be a novel strategy to control IR-induced neuronal damage.
基金National Natural Science Foundation of China(Nos.31520103915,31730090,and 31322053)the Hubei Provincial Natural Science Foundation of China(No.2018CFA020)
文摘The mechanistic target of rapamycin complex 1(mTORC1)controls cell growth and metabolism in response to various environmental inputs,especially amino acids.In fact,the activity of mTORC1 is highly sensitive to changes in amino acid levels.Over past decades,a variety of proteins have been identified as participating in the mTORC1 pathway regulated by amino acids.Classically,the Rag guanosine triphosphatases(GTPases),which reside on the lysosome,transmit amino acid availability to the mTORC1 pathway and recruit mTORC1 to the lysosome upon amino acid sufficiency.Recently,several sensors of leucine,arginine,and S-adenosylmethionine for the amino acidstimulated mTORC1 pathway have been coming to light.Characterization of these sensors is requisite for understanding how cells adjust amino acid sensing pathways to their different needs.In this review,we summarize recent advances in amino acid sensing mechanisms that regulate mTORC1 activity and highlight these identified sensors that accurately transmit specific amino acid signals to the mTORC1 pathway.
