In situ direct reprogramming technology can directly convert endogenous glial cells into functional neurons in vivo for central nervous system repair. Polypyrimidine tract-binding protein 1(PTB) knockdown has been sho...In situ direct reprogramming technology can directly convert endogenous glial cells into functional neurons in vivo for central nervous system repair. Polypyrimidine tract-binding protein 1(PTB) knockdown has been shown to reprogram astrocytes to functional neurons in situ. In this study, we used AAV-PHP.e B-GFAP-sh PTB to knockdown PTB in a mouse model of ischemic stroke induced by endothelin-1, and investigated the effects of GFAP-sh PTB-mediated direct reprogramming to neurons. Our results showed that in the mouse model of ischemic stroke, PTB knockdown effectively reprogrammed GFAP-positive cells to neurons in ischemic foci, restored neural tissue structure, reduced inflammatory response, and improved behavioral function. These findings validate the effectiveness of in situ transdifferentiation of astrocytes, and suggest that the approach may be a promising strategy for stroke treatment.展开更多
Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located...Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located in ZIKV 5'UTR and virus production.Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining,Subsequently,liquid chromatographytandem mass spectrometry(LC-MS/MS),bioinformatics analysis,and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production,respecitvely.Results tRSA RNA pull-down assay,LC-MS/MS,and Western blot analysis showed that polypyrimidine tractbinding protein(PTB)bound to the ZIKV 5'UTR.Furthermore,dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV(t=10.220,P<0.001),while PTB knockdown had the opposite effect(t=4.897,P<0.01).Additionally,virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer(t=6.400,P<0.01),whereas reducing PTB expression level weakened virus infectivity(t=5.055,P<0.01).Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.展开更多
After spinal cord injury(SCI),a fibroblast-and microglia-mediated fibrotic scar is formed in the lesion core,and a glial scar is formed around the fibrotic scar as a res ult of the activation and proliferation of astr...After spinal cord injury(SCI),a fibroblast-and microglia-mediated fibrotic scar is formed in the lesion core,and a glial scar is formed around the fibrotic scar as a res ult of the activation and proliferation of astrocytes.Simultaneously,a large number of neuro ns are lost in the injured area.Regulating the dense glial scar and re plenishing neurons in the injured area are essential for SCI repair.Polypyrimidine tra ct binding protein(PTB),known as an RNA-binding protein,plays a key role in neurogenesis.Here,we utilized short hairpin RNAs(shRNAs)and antisense oligonucleotides(ASOs)to knock down PTB expression.We found that reactive spinal astrocytes from mice were directly reprogrammed into motoneuron-like cells by PTB downregulation in vitro.In a mouse model of compressioninduced SCI,adeno-associated viral shRNA-mediated PTB knockdown replenished motoneuron-like cells around the injured area.Basso Mouse Scale scores and forced swim,inclined plate,cold allodynia,and hot plate tests showed that PTB knockdown promoted motor function recovery in mice but did not improve sensory perception after SCI.Furthermore,ASO-mediated PTB knockdown improved motor function resto ration by not only replenishing motoneuron-like cells around the injured area but also by modestly reducing the density of the glial scar without disrupting its overall structure.Together,these findings suggest that PTB knockdown may be a promising therapeutic strategy to promote motor function recovery during spinal cord repair.展开更多
Neuronal injury,aging,and cerebrovascular and neurodegenerative diseases such as cerebral infarction,Alzheimer’s disease,Parkinson’s disease,frontotemporal dementia,amyotrophic lateral sclerosis,and Huntington’s di...Neuronal injury,aging,and cerebrovascular and neurodegenerative diseases such as cerebral infarction,Alzheimer’s disease,Parkinson’s disease,frontotemporal dementia,amyotrophic lateral sclerosis,and Huntington’s disease are characte rized by significant neuronal loss.Unfo rtunately,the neurons of most mammals including humans do not possess the ability to self-regenerate.Replenishment of lost neurons becomes an appealing therapeutic strategy to reve rse the disease phenotype.Transplantation of pluripotent neural stem cells can supplement the missing neurons in the brain,but it carries the risk of causing gene mutation,tumorigenesis,severe inflammation,and obstructive hydrocephalus induced by brain edema.Conversion of neural or non-neural lineage cells into functional neurons is a promising strategy for the diseases involving neuron loss,which may overcome the above-mentioned disadvantages of neural stem cell therapy.Thus far,many strategies to transfo rm astrocytes,fibroblasts,microglia,Muller glia,NG2 cells,and other glial cells to mature and functional neurons,or for the conversion between neuronal subtypes have been developed thro ugh the regulation of transcription factors,polypyrimidine tra ct binding protein 1(PTBP1),and small chemical molecules or are based on a combination of several factors and the location in the central nervous system.However,some recent papers did not obtain expected results,and discrepancies exist.Therefore,in this review,we discuss the history of neuronal transdifferentiation,summarize the strategies for neuronal replenishment and conversion from glia,especially astrocytes,and point out that biosafety,new strategies,and the accurate origin of the truly co nverted neurons in vivo should be focused upon in future studies.It also arises the attention of replenishing the lost neurons from glia by gene therapies such as up-regulation of some transc ription factors or downregulation of PTBP1 or drug interfe rence therapies.展开更多
Objective To screen the proteins associated with four-and-a-half LIM domains 3(FHL3) 3' untranslated region(3'UTR) in glioma cells. Methods Western blot was adopted to detect the regulatory effect of poly(C)-b...Objective To screen the proteins associated with four-and-a-half LIM domains 3(FHL3) 3' untranslated region(3'UTR) in glioma cells. Methods Western blot was adopted to detect the regulatory effect of poly(C)-binding protein 2(PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3'UTR. Then liquid chromatography-tandem mass spectrometry(LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immunoprecipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1(PTBP1), a binding protein identified by LC-MS/MS. Results PCBP2 could bind to FHL3 mRNA 3'UTR-A and inhibited the expression of FHL3 in T98 G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3'UTR and interacted with PCBP2 protein. Conclusion PCBP2 and PTBP1 can both associate with FHL3 mRNA 3'UTR through forming a protein complex.展开更多
Background and Aims:Ras-related nuclear(RAN)protein is a small GTP-binding protein that is indispensable for the translocation of RNA and proteins through the nuclear pore complex.Recent studies have indicated that RA...Background and Aims:Ras-related nuclear(RAN)protein is a small GTP-binding protein that is indispensable for the translocation of RNA and proteins through the nuclear pore complex.Recent studies have indicated that RAN plays an important role in virus infection.However,the role of RAN in hepatitis C virus(HCV)infection is unclear.The objective of this study was to investigate the role and underlying mechanisms of RAN in HCV infection.Methods:Huh7.5.1 cells were infected with the JC1-Luc virus for 24 h and then were incubated with complete medium for an additional 48 h.HCV infection and RAN expression were determined using luciferase assay,quantitative reverse transcription-PCR and western blotting.Small interfering RNA was used to silence RAN.Western blotting and immunofluorescence were used to evaluate the cytoplasmic translocation of polypyrimidine tract-binding(PTB),and coimmunoprecipitation was used to examine the interaction between RAN and PTB.Results:HCV infection significantly induced RAN expression and cytoplasmic redistribution of PTB.Knockdown of RAN dramatically inhibited HCV infection and the cytoplasmic accumulation of PTB.Colocalization of RAN and PTB was determined by immunofluorescence,and a direct interaction of RAN with PTB was demonstrated by coimmunoprecipitation.Conclusions:PTB in the host cytoplasm is directly associated with HCV replication.These findings demonstrate that the involvement of RAN in HCV infection is mediated by influencing the cytoplasmic translocation of PTB.展开更多
Administration of human umbilical cord-derived mesenchymal stem cells(hUC-MSCs)is believed to be an effective method for treating neurodevelopmental disorde rs.In this study,we investigated the possibility of hUC-MSCs...Administration of human umbilical cord-derived mesenchymal stem cells(hUC-MSCs)is believed to be an effective method for treating neurodevelopmental disorde rs.In this study,we investigated the possibility of hUC-MSCs treatment of neonatal hypoxic/ischemic brain injury associated with maternal immune activation and the underlying mechanism.We established neonatal rat models of hypoxic/ischemic brain injury by exposing pregnant rats to lipopolysaccharide on day 16 or 17 of pregnancy.Rat offspring were intranasally administe red hUC-MSCs on postnatal day 14.We found that polypyrimidine tract-binding protein-1(PTBP-1)participated in the regulation of lipopolysaccharide-induced maternal immune activation,which led to neonatal hypoxic/ischemic brain injury.Intranasal delive ry of hUC-MSCs inhibited PTBP-1 expression,alleviated neonatal brain injury-related inflammation,and regulated the number and function of glial fibrillary acidic protein-positive astrocytes,there by promoting plastic regeneration of neurons and im p roving brain function.These findings suggest that hUC-MSCs can effectively promote the repair of neonatal hypoxic/ischemic brain injury related to maternal immune activation through inhibition of PTBP-1 expression and astrocyte activation.展开更多
目的:研究在非小细胞肺癌及癌旁组织中ceacam1通过选择性拼接而产生的两种转录产物的调控机制。方法:将用PCR方法获得ceacam1基因中从内含子5至外显子8长1 606 bp DNA片段插入到真核表达载体pCMV中,构建成ceacam1迷你基因模型并与ptb基...目的:研究在非小细胞肺癌及癌旁组织中ceacam1通过选择性拼接而产生的两种转录产物的调控机制。方法:将用PCR方法获得ceacam1基因中从内含子5至外显子8长1 606 bp DNA片段插入到真核表达载体pCMV中,构建成ceacam1迷你基因模型并与ptb基因共转染,用PCR法鉴定转染后的产物变化;根据外显子7序列设计的探针GAE(16-nt)及ACE(8-nt)进行凝胶阻滞分析实验。分离与探针结合的蛋白并进行质谱分析。结果:ptb3种cDNA与ceacam1迷你基因共转染后,CEACAM lL表达水平下降,其中ptb4对迷你基因的表达产物影响最大。仅转染迷你基因的细胞中ceacam1L在两条带中所占比例为76.7%,而与ptb3种重组质粒共转染后,比例分别下降至58.3%、64.8%和54.0%。凝胶阻滞实验表明,探针GAE能与核蛋白结合,而ACE基本不能与核蛋白结合,与GAE结合的蛋白经质谱分析为PTB。结论:PTB过表达与ceacam1低表达有明显的相关性,拼接因子PTB参与ceacam1的选择性拼接。展开更多
基金supported by the National Natural Science Foundation of China,No.82071418the Natural Science Foundation of Fujian Province,No.2020J01612 (both to EH)。
文摘In situ direct reprogramming technology can directly convert endogenous glial cells into functional neurons in vivo for central nervous system repair. Polypyrimidine tract-binding protein 1(PTB) knockdown has been shown to reprogram astrocytes to functional neurons in situ. In this study, we used AAV-PHP.e B-GFAP-sh PTB to knockdown PTB in a mouse model of ischemic stroke induced by endothelin-1, and investigated the effects of GFAP-sh PTB-mediated direct reprogramming to neurons. Our results showed that in the mouse model of ischemic stroke, PTB knockdown effectively reprogrammed GFAP-positive cells to neurons in ischemic foci, restored neural tissue structure, reduced inflammatory response, and improved behavioral function. These findings validate the effectiveness of in situ transdifferentiation of astrocytes, and suggest that the approach may be a promising strategy for stroke treatment.
文摘Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located in ZIKV 5'UTR and virus production.Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining,Subsequently,liquid chromatographytandem mass spectrometry(LC-MS/MS),bioinformatics analysis,and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production,respecitvely.Results tRSA RNA pull-down assay,LC-MS/MS,and Western blot analysis showed that polypyrimidine tractbinding protein(PTB)bound to the ZIKV 5'UTR.Furthermore,dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV(t=10.220,P<0.001),while PTB knockdown had the opposite effect(t=4.897,P<0.01).Additionally,virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer(t=6.400,P<0.01),whereas reducing PTB expression level weakened virus infectivity(t=5.055,P<0.01).Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.
基金supported by the National Natural Science Foundation of China,Nos.82101455(to RYY),31872773(to GC),82001168(to JYP)the Key Research and Development Program(Social Development)of Jiangsu Province,No.BE2020667(to GC)+3 种基金the Foundation of Jiangsu Province,333 Project High-level Talents",No.BRA2020076(to GC)the Nantong Civic Science and Technology Project of China,No.JC2020028(to RYY)the Natural Science Research of Jiangsu Higher Education Institutions of China,No.19KJB310012(to RYY)Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)。
文摘After spinal cord injury(SCI),a fibroblast-and microglia-mediated fibrotic scar is formed in the lesion core,and a glial scar is formed around the fibrotic scar as a res ult of the activation and proliferation of astrocytes.Simultaneously,a large number of neuro ns are lost in the injured area.Regulating the dense glial scar and re plenishing neurons in the injured area are essential for SCI repair.Polypyrimidine tra ct binding protein(PTB),known as an RNA-binding protein,plays a key role in neurogenesis.Here,we utilized short hairpin RNAs(shRNAs)and antisense oligonucleotides(ASOs)to knock down PTB expression.We found that reactive spinal astrocytes from mice were directly reprogrammed into motoneuron-like cells by PTB downregulation in vitro.In a mouse model of compressioninduced SCI,adeno-associated viral shRNA-mediated PTB knockdown replenished motoneuron-like cells around the injured area.Basso Mouse Scale scores and forced swim,inclined plate,cold allodynia,and hot plate tests showed that PTB knockdown promoted motor function recovery in mice but did not improve sensory perception after SCI.Furthermore,ASO-mediated PTB knockdown improved motor function resto ration by not only replenishing motoneuron-like cells around the injured area but also by modestly reducing the density of the glial scar without disrupting its overall structure.Together,these findings suggest that PTB knockdown may be a promising therapeutic strategy to promote motor function recovery during spinal cord repair.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences,No.XDB39050600(to RL)the National Natural Science Foundation of China,No.81971610(to RL)Beijing Rehabilitation Hospital Introduction of Talent Research Start-up Fund,No.2021R-008(to JZ)。
文摘Neuronal injury,aging,and cerebrovascular and neurodegenerative diseases such as cerebral infarction,Alzheimer’s disease,Parkinson’s disease,frontotemporal dementia,amyotrophic lateral sclerosis,and Huntington’s disease are characte rized by significant neuronal loss.Unfo rtunately,the neurons of most mammals including humans do not possess the ability to self-regenerate.Replenishment of lost neurons becomes an appealing therapeutic strategy to reve rse the disease phenotype.Transplantation of pluripotent neural stem cells can supplement the missing neurons in the brain,but it carries the risk of causing gene mutation,tumorigenesis,severe inflammation,and obstructive hydrocephalus induced by brain edema.Conversion of neural or non-neural lineage cells into functional neurons is a promising strategy for the diseases involving neuron loss,which may overcome the above-mentioned disadvantages of neural stem cell therapy.Thus far,many strategies to transfo rm astrocytes,fibroblasts,microglia,Muller glia,NG2 cells,and other glial cells to mature and functional neurons,or for the conversion between neuronal subtypes have been developed thro ugh the regulation of transcription factors,polypyrimidine tra ct binding protein 1(PTBP1),and small chemical molecules or are based on a combination of several factors and the location in the central nervous system.However,some recent papers did not obtain expected results,and discrepancies exist.Therefore,in this review,we discuss the history of neuronal transdifferentiation,summarize the strategies for neuronal replenishment and conversion from glia,especially astrocytes,and point out that biosafety,new strategies,and the accurate origin of the truly co nverted neurons in vivo should be focused upon in future studies.It also arises the attention of replenishing the lost neurons from glia by gene therapies such as up-regulation of some transc ription factors or downregulation of PTBP1 or drug interfe rence therapies.
基金Supported by Peking Union Medical College Youth Fundthe Fundamental Research Funds for the Central Universities(3332013052)
文摘Objective To screen the proteins associated with four-and-a-half LIM domains 3(FHL3) 3' untranslated region(3'UTR) in glioma cells. Methods Western blot was adopted to detect the regulatory effect of poly(C)-binding protein 2(PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3'UTR. Then liquid chromatography-tandem mass spectrometry(LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immunoprecipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1(PTBP1), a binding protein identified by LC-MS/MS. Results PCBP2 could bind to FHL3 mRNA 3'UTR-A and inhibited the expression of FHL3 in T98 G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3'UTR and interacted with PCBP2 protein. Conclusion PCBP2 and PTBP1 can both associate with FHL3 mRNA 3'UTR through forming a protein complex.
基金The work was supported by the Anhui Provincial Natural Science Foundation(1608085QH172)States S&T Projects of 13th Five Year(2018ZX10302206),Chinese Founda-tion for Hepatitis Prevention and Control(TQGB20200151,TQGB20180367)Anhui Key Program of Medical Scien-tific Research of China(#2010A010).
文摘Background and Aims:Ras-related nuclear(RAN)protein is a small GTP-binding protein that is indispensable for the translocation of RNA and proteins through the nuclear pore complex.Recent studies have indicated that RAN plays an important role in virus infection.However,the role of RAN in hepatitis C virus(HCV)infection is unclear.The objective of this study was to investigate the role and underlying mechanisms of RAN in HCV infection.Methods:Huh7.5.1 cells were infected with the JC1-Luc virus for 24 h and then were incubated with complete medium for an additional 48 h.HCV infection and RAN expression were determined using luciferase assay,quantitative reverse transcription-PCR and western blotting.Small interfering RNA was used to silence RAN.Western blotting and immunofluorescence were used to evaluate the cytoplasmic translocation of polypyrimidine tract-binding(PTB),and coimmunoprecipitation was used to examine the interaction between RAN and PTB.Results:HCV infection significantly induced RAN expression and cytoplasmic redistribution of PTB.Knockdown of RAN dramatically inhibited HCV infection and the cytoplasmic accumulation of PTB.Colocalization of RAN and PTB was determined by immunofluorescence,and a direct interaction of RAN with PTB was demonstrated by coimmunoprecipitation.Conclusions:PTB in the host cytoplasm is directly associated with HCV replication.These findings demonstrate that the involvement of RAN in HCV infection is mediated by influencing the cytoplasmic translocation of PTB.
基金the National Natural Science Foundation of China,No.81471308(to JL)Stem cell Clinical Research Registry Program,No.CMR-20161129-1003(to JL)+2 种基金Liaoning Province Excellent Talent Program Project of China,No.XLYC1902031(to JL)Dalian Innovation Fund of China,No.2018J11CY025(to JL)National Defense Science and Technology New Special Zone Contract,No.19-163-00-kx-003-001-01(to JL)。
文摘Administration of human umbilical cord-derived mesenchymal stem cells(hUC-MSCs)is believed to be an effective method for treating neurodevelopmental disorde rs.In this study,we investigated the possibility of hUC-MSCs treatment of neonatal hypoxic/ischemic brain injury associated with maternal immune activation and the underlying mechanism.We established neonatal rat models of hypoxic/ischemic brain injury by exposing pregnant rats to lipopolysaccharide on day 16 or 17 of pregnancy.Rat offspring were intranasally administe red hUC-MSCs on postnatal day 14.We found that polypyrimidine tract-binding protein-1(PTBP-1)participated in the regulation of lipopolysaccharide-induced maternal immune activation,which led to neonatal hypoxic/ischemic brain injury.Intranasal delive ry of hUC-MSCs inhibited PTBP-1 expression,alleviated neonatal brain injury-related inflammation,and regulated the number and function of glial fibrillary acidic protein-positive astrocytes,there by promoting plastic regeneration of neurons and im p roving brain function.These findings suggest that hUC-MSCs can effectively promote the repair of neonatal hypoxic/ischemic brain injury related to maternal immune activation through inhibition of PTBP-1 expression and astrocyte activation.
文摘目的:研究在非小细胞肺癌及癌旁组织中ceacam1通过选择性拼接而产生的两种转录产物的调控机制。方法:将用PCR方法获得ceacam1基因中从内含子5至外显子8长1 606 bp DNA片段插入到真核表达载体pCMV中,构建成ceacam1迷你基因模型并与ptb基因共转染,用PCR法鉴定转染后的产物变化;根据外显子7序列设计的探针GAE(16-nt)及ACE(8-nt)进行凝胶阻滞分析实验。分离与探针结合的蛋白并进行质谱分析。结果:ptb3种cDNA与ceacam1迷你基因共转染后,CEACAM lL表达水平下降,其中ptb4对迷你基因的表达产物影响最大。仅转染迷你基因的细胞中ceacam1L在两条带中所占比例为76.7%,而与ptb3种重组质粒共转染后,比例分别下降至58.3%、64.8%和54.0%。凝胶阻滞实验表明,探针GAE能与核蛋白结合,而ACE基本不能与核蛋白结合,与GAE结合的蛋白经质谱分析为PTB。结论:PTB过表达与ceacam1低表达有明显的相关性,拼接因子PTB参与ceacam1的选择性拼接。
