Alanine-serine-cysteine transporter 2(ASCT2)is reported to participate in the progression of tumors and metabolic diseases.It is also considered to play a crucial role in the glutamate-glutamine shuttle of neuroglial ...Alanine-serine-cysteine transporter 2(ASCT2)is reported to participate in the progression of tumors and metabolic diseases.It is also considered to play a crucial role in the glutamate-glutamine shuttle of neuroglial network.However,it remains unclear the involvement of ASCT2 in neurological diseases such as Parkinson’s disease(PD).In this study,we demonstrated that high expression of ASCT2 in the plasma samples of PD patients and the midbrain of MPTP mouse models is positively correlated with dyskinesia.We further illustrated that ASCT2 expressed in astrocytes rather than neurons significantly upregulated in response to either MPP+or LPS/ATP challenge.Genetic ablation of astrocytic ASCT2 alleviated the neuroinflammation and rescued dopaminergic(DA)neuron damage in PD models in vitro and in vivo.Notably,the binding of ASCT2 to NLRP3 aggravates astrocytic inflammasometriggered neuroinflammation.Then a panel of 2513 FDA-approved drugs were performed via virtual molecular screening based on the target ASCT2 and we succeed in getting the drug talniflumate.It is validated talniflumate impedes astrocytic inflammation and prevents degeneration of DA neurons in PD models.Collectively,these findings reveal the role of astrocytic ASCT2 in the pathogenesis of PD,broaden the therapeutic strategy and provide a promising candidate drug for PD treatment.展开更多
Senescence of activated hepatic stellate cells(aHSCs) is a stable growth arrest that is implicated in liver fibrosis regression.Senescent cells often accompanied by a multi-faceted senescence-associated secretory phen...Senescence of activated hepatic stellate cells(aHSCs) is a stable growth arrest that is implicated in liver fibrosis regression.Senescent cells often accompanied by a multi-faceted senescence-associated secretory phenotype(SASP).But little is known about how alanine-serine-cysteine transporter type-2(ASCT2),a high affinity glutamine transporter,affects HSC senescence and SASP during liver fibrosis.Here,we identified ASCT2 is mainly elevated in aHSCs and positively correlated with liver fibrosis in human and mouse fibrotic livers.We first discovered ASCT2 inhibition induced HSCs to senescence in vitro and in vivo.The proinflammatory SASP were restricted by ASCT2 inhibition at senescence initiation to prevent paracrine migration.Mechanically,ASCT2 was a direct target of glutaminolysisdependent proinflammatory SASP,interfering IL-1α/NF-κB feedback loop via interacting with precursor IL-1α at Lys82.From a translational perspective,atractylenolide Ⅲ is identified as ASCT2 inhibitor through directly bound to Asn230 of ASCT2.The presence of -OH group in atractylenolide Ⅲ is suggested to be favorable for the inhibition of ASCT2.Importantly,atractylenolide Ⅲ could be utilized to treat liver fibrosis mice.Taken together,ASCT2 controlled HSC senescence while modifying the proinflammatory SASP.Targeting ASCT2 by atractylenolide Ⅲ could be a therapeutic candidate for liver fibrosis.展开更多
Background and Aims:Recognition of excessive activa-tion of hepatic stellate cells(HSCs)in liver fibrosis prompt-ed us to investigate the regulatory mechanisms of HSCs.We aimed to examine the role of O-GlcNAcylation m...Background and Aims:Recognition of excessive activa-tion of hepatic stellate cells(HSCs)in liver fibrosis prompt-ed us to investigate the regulatory mechanisms of HSCs.We aimed to examine the role of O-GlcNAcylation modifica-tion of alanine,serine,cysteine transporter 2(ASCT2)in HSCs and liver fibrosis.Methods:The expression of O-Glc-NAcylation modification in fibrotic mice livers and activated HSCs was analyzed by western blotting.Immunoprecipita-tion was used to assess the interaction of ASCT2 and O-Glc-NAc transferase(OGT).In addition,ASCT2 protein stability was assayed after cycloheximide(CHX)treatment.The O-GlcNAcylation site of ASCT2 was predicted and mutated by site-directed mutagenesis.Real-time PCR,immunofluores-cence,kit determinations and Seahorse assays were used to clarify the effect of ASCT2 O-GlcNAcylation on HSC glu-taminolysis and HSC activation.Western blotting,immuno-chemistry,and immunohistofluorescence were used to ana-lyze the effect of ASCT2 O-GlcNAcylation in vivo.Results:We observed significantly increased O-GlcNAcylation modi-fication of ASCT2.ASCT2 was found to interact with OGT to regulate ASCT2 stability.We predicted and confirmed that O-GlcNAcylation of ASCT2 at Thr122 site resulted in HSCs activation.We found Thr122 O-GlcNAcylation of ASCT2 me-diated membrane trafficking of glutamine transport and attenuated HSC glutaminolysis.Finally,we validated the expression and function of ASCT2 O-GlcNAcylation after in-jection of AAV8-ASCT2 shRNA in CCl4-induced liver fibrosis mice in vivo.Conclusions:Thr122 O-GlcNAcylation regu-lation of ASCT2 resulted in stability and membrane traf-ficking-mediated glutaminolysis in HSCs and liver fibrosis.Further studies are required to assess its role as a putative therapeutic target.展开更多
基金the support of the Experiment Center for Science and Technology,Nanjing University of Chinese Medicine(Nanjing,China)supported by grants from the National Key R&D Program of China(No.2021ZD0202903)the National Natural Science Foundation of China(Nos.81922066,82173797,82003725,81991523,and 82003722)。
文摘Alanine-serine-cysteine transporter 2(ASCT2)is reported to participate in the progression of tumors and metabolic diseases.It is also considered to play a crucial role in the glutamate-glutamine shuttle of neuroglial network.However,it remains unclear the involvement of ASCT2 in neurological diseases such as Parkinson’s disease(PD).In this study,we demonstrated that high expression of ASCT2 in the plasma samples of PD patients and the midbrain of MPTP mouse models is positively correlated with dyskinesia.We further illustrated that ASCT2 expressed in astrocytes rather than neurons significantly upregulated in response to either MPP+or LPS/ATP challenge.Genetic ablation of astrocytic ASCT2 alleviated the neuroinflammation and rescued dopaminergic(DA)neuron damage in PD models in vitro and in vivo.Notably,the binding of ASCT2 to NLRP3 aggravates astrocytic inflammasometriggered neuroinflammation.Then a panel of 2513 FDA-approved drugs were performed via virtual molecular screening based on the target ASCT2 and we succeed in getting the drug talniflumate.It is validated talniflumate impedes astrocytic inflammation and prevents degeneration of DA neurons in PD models.Collectively,these findings reveal the role of astrocytic ASCT2 in the pathogenesis of PD,broaden the therapeutic strategy and provide a promising candidate drug for PD treatment.
基金financially supported by the National Natural Science Foundation of China (81870423, 82173874 and 82073914)the Major Project of the Natural Science Research of Jiangsu Higher Education Institutions (19KJA310005, China)+3 种基金the Open Project of Chinese Materia Medica First-Class Discipline of Nanjing University of Chinese Medicine (2020YLXK022 and 2020YLXK023, China)the Open Project of Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica (JKLPSE202005, China)the Qing Lan Project of Jiangsu Higher Institutions (Young and Middle-Aged Academic Leader, China)the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX20_1493, China)
文摘Senescence of activated hepatic stellate cells(aHSCs) is a stable growth arrest that is implicated in liver fibrosis regression.Senescent cells often accompanied by a multi-faceted senescence-associated secretory phenotype(SASP).But little is known about how alanine-serine-cysteine transporter type-2(ASCT2),a high affinity glutamine transporter,affects HSC senescence and SASP during liver fibrosis.Here,we identified ASCT2 is mainly elevated in aHSCs and positively correlated with liver fibrosis in human and mouse fibrotic livers.We first discovered ASCT2 inhibition induced HSCs to senescence in vitro and in vivo.The proinflammatory SASP were restricted by ASCT2 inhibition at senescence initiation to prevent paracrine migration.Mechanically,ASCT2 was a direct target of glutaminolysisdependent proinflammatory SASP,interfering IL-1α/NF-κB feedback loop via interacting with precursor IL-1α at Lys82.From a translational perspective,atractylenolide Ⅲ is identified as ASCT2 inhibitor through directly bound to Asn230 of ASCT2.The presence of -OH group in atractylenolide Ⅲ is suggested to be favorable for the inhibition of ASCT2.Importantly,atractylenolide Ⅲ could be utilized to treat liver fibrosis mice.Taken together,ASCT2 controlled HSC senescence while modifying the proinflammatory SASP.Targeting ASCT2 by atractylenolide Ⅲ could be a therapeutic candidate for liver fibrosis.
基金the Leading Talent Project of Ji-angsu Province Traditional Chinese Medicine(SLJ0216)the National Natural Science Foundation of China(82073914,81870423)+2 种基金the Major Project of the Natural Science Research of Jiangsu Higher Education Institutions(19KJA310005)the Postgraduate Research&Practice Innovation Program of Jiangsu Province,Grant/Award Number:KYCX20_1493the Joint Project of Jiangsu Key Laboratory for Pharmacol-ogy and Safety Evaluation of Chinese Materia Medica and Yangtze River Pharmaceutical(JKLPSE202005)。
文摘Background and Aims:Recognition of excessive activa-tion of hepatic stellate cells(HSCs)in liver fibrosis prompt-ed us to investigate the regulatory mechanisms of HSCs.We aimed to examine the role of O-GlcNAcylation modifica-tion of alanine,serine,cysteine transporter 2(ASCT2)in HSCs and liver fibrosis.Methods:The expression of O-Glc-NAcylation modification in fibrotic mice livers and activated HSCs was analyzed by western blotting.Immunoprecipita-tion was used to assess the interaction of ASCT2 and O-Glc-NAc transferase(OGT).In addition,ASCT2 protein stability was assayed after cycloheximide(CHX)treatment.The O-GlcNAcylation site of ASCT2 was predicted and mutated by site-directed mutagenesis.Real-time PCR,immunofluores-cence,kit determinations and Seahorse assays were used to clarify the effect of ASCT2 O-GlcNAcylation on HSC glu-taminolysis and HSC activation.Western blotting,immuno-chemistry,and immunohistofluorescence were used to ana-lyze the effect of ASCT2 O-GlcNAcylation in vivo.Results:We observed significantly increased O-GlcNAcylation modi-fication of ASCT2.ASCT2 was found to interact with OGT to regulate ASCT2 stability.We predicted and confirmed that O-GlcNAcylation of ASCT2 at Thr122 site resulted in HSCs activation.We found Thr122 O-GlcNAcylation of ASCT2 me-diated membrane trafficking of glutamine transport and attenuated HSC glutaminolysis.Finally,we validated the expression and function of ASCT2 O-GlcNAcylation after in-jection of AAV8-ASCT2 shRNA in CCl4-induced liver fibrosis mice in vivo.Conclusions:Thr122 O-GlcNAcylation regu-lation of ASCT2 resulted in stability and membrane traf-ficking-mediated glutaminolysis in HSCs and liver fibrosis.Further studies are required to assess its role as a putative therapeutic target.
