Direct in vivo conversion of astrocytes into functional new neurons induced by neural transcription factors has been recognized as a potential new therapeutic intervention for neural injury and degenerative disorders....Direct in vivo conversion of astrocytes into functional new neurons induced by neural transcription factors has been recognized as a potential new therapeutic intervention for neural injury and degenerative disorders. However, a few recent studies have claimed that neural transcription factors cannot convert astrocytes into neurons, attributing the converted neurons to pre-existing neurons mis-expressing transgenes. In this study, we overexpressed three distinct neural transcription factors––NeuroD1, Ascl1, and Dlx2––in reactive astrocytes in mouse cortices subjected to stab injury, resulting in a series of significant changes in astrocyte properties. Initially, the three neural transcription factors were exclusively expressed in the nuclei of astrocytes. Over time, however, these astrocytes gradually adopted neuronal morphology, and the neural transcription factors was gradually observed in the nuclei of neuron-like cells instead of astrocytes. Furthermore,we noted that transcription factor-infected astrocytes showed a progressive decrease in the expression of astrocytic markers AQP4(astrocyte endfeet signal), CX43(gap junction signal), and S100β. Importantly, none of these changes could be attributed to transgene leakage into preexisting neurons. Therefore, our findings suggest that neural transcription factors such as NeuroD1, Ascl1, and Dlx2 can effectively convert reactive astrocytes into neurons in the adult mammalian brain.展开更多
Mouse cortical radial glial cells(RGCs)are primary neural stem cells that give rise to cortical oligodendrocytes,astrocytes,and olfactory bulb(OB)GABAergic interneurons in late embryogenesis.There are fundamental gaps...Mouse cortical radial glial cells(RGCs)are primary neural stem cells that give rise to cortical oligodendrocytes,astrocytes,and olfactory bulb(OB)GABAergic interneurons in late embryogenesis.There are fundamental gaps in understanding how these diverse cell subtypes are generated.Here,by combining single-cell RNA-Seq with intersectional lineage analyses,we show that beginning at around E16.5,neocortical RGCs start to generate ASCL1^(+)EGFR^(+)apical multipotent intermediate progenitors(MIPCs),which then differentiate into basal MIPCs that express ASCL1,EGFR,OLIG2,and MKI67.These basal MIPCs undergo several rounds of divisions to generate most of the cortical oligodendrocytes and astrocytes and a subpopulation of OB interneurons.Finally,single-cell ATAC-Seq supported our model for the genetic logic underlying the specification and differentiation of cortical glial cells and OB interneurons.Taken together,this work reveals the process of cortical radial glial cell lineage progression and the developmental origins of cortical astrocytes and oligodendrocytes.展开更多
Objective:Glioblastoma(GBM)is the most prevalent and aggressive adult primary cancer in the central nervous system.Therapeutic approaches for GBM treatment are under intense investigation,including the use of emerging...Objective:Glioblastoma(GBM)is the most prevalent and aggressive adult primary cancer in the central nervous system.Therapeutic approaches for GBM treatment are under intense investigation,including the use of emerging immunotherapies.Here,we propose an alternative approach to treat GBM through reprogramming proliferative GBM cells into non-proliferative neurons.Methods:Retroviruses were used to target highly proliferative human GBM cells through overexpression of neural transcription factors.Immunostaining,electrophysiological recording,and bulk RNA-seq were performed to investigate the mechanisms underlying the neuronal conversion of human GBM cells.An in vivo intracranial xenograft mouse model was used to examine the neuronal conversion of human GBM cells.Results:We report efficient neuronal conversion from human GBM cells by overexpressing single neural transcription factor Neurogenic differentiation 1(Neuro D1),Neurogenin-2(Neurog2),or Achaete-scute homolog 1(Ascl1).Subtype characterization showed that the majority of Neurog2-and Neuro D1-converted neurons were glutamatergic,while Ascl1 favored GABAergic neuron generation.The GBM cell-converted neurons not only showed pan-neuronal markers but also exhibited neuron-specific electrophysiological activities.Transcriptome analyses revealed that neuronal genes were activated in glioma cells after overexpression of neural transcription factors,and different signaling pathways were activated by different neural transcription factors.Importantly,the neuronal conversion of GBM cells was accompanied by significant inhibition of GBM cell proliferation in both in vitro and in vivo models.Conclusions:These results suggest that GBM cells can be reprogrammed into different subtypes of neurons,leading to a potential alternative approach to treat brain tumors using in vivo cell conversion technology.展开更多
1文献来源研究一:Rudin CM,Poirier JT,Byers LA,et al.Molecular subtypes of small cell lung cancer:A synthesis of human and mouse model data[J].Nat Rev Cancer,2019,19(5):289-297.研究二:Owonikoko TK,Dwivedi B,Chen ZJ,et a...1文献来源研究一:Rudin CM,Poirier JT,Byers LA,et al.Molecular subtypes of small cell lung cancer:A synthesis of human and mouse model data[J].Nat Rev Cancer,2019,19(5):289-297.研究二:Owonikoko TK,Dwivedi B,Chen ZJ,et al.YAP1 positive small-cell lung cancer subtype is associated with the T-cell inflamed gene expression profile and confers good prognosis and long term survival[J].J Clin Oncol,2020,38(15S):Abstr 9019.展开更多
Differentiation of human fibroblasts into functional neurons depends on the introduction of viral-mediated transcription factors, which present risks of viral gene integration and tumorigenicity. In recent years, alth...Differentiation of human fibroblasts into functional neurons depends on the introduction of viral-mediated transcription factors, which present risks of viral gene integration and tumorigenicity. In recent years, although some studies have been successful in directly inducing neurons through sustained expression of small molecule compounds, they have only been shown to be effective on mouse-derived cells. Thus, herein we delivered vectors containing Epstein-Barr virus-derived oriP/Epstein-Barr nuclear antigen 1 encoding the neuronal transcription factor, Ascl1, the neuron-specific microRNA, miR124, and a small hairpin directed against p53, into human fibroblasts. Cells were incubated in a neuron-inducing culture medium. Immunofluorescence staining was used to detect Tuj-1, microtubule-associated protein 2, neuron-specific nucleoprotein NeuN and nerve cell adhesion molecules in the induced cells. The proportion of Tuj1-positive cells was up to 36.7% after induction for 11 days. From day 21, these induced neurons showed neuron-specific expression patterns of microtubule-associated protein 2, NeuN and neural cell adhesion molecule. Our approach is a simple, plasmid-based process that enables direct reprogramming of human fibroblasts into neurons, and provides alternative avenues for disease modeling and neurodegenerative medicine.展开更多
The retina of zebrafish can regenerate completely after injury.M ultiple studies have demonstrated that metabolic alte rations occur during retinal damage;however to date no study has identified a link between metabol...The retina of zebrafish can regenerate completely after injury.M ultiple studies have demonstrated that metabolic alte rations occur during retinal damage;however to date no study has identified a link between metabolites and retinal regeneration of zebrafish.Here,we performed an unbiased metabolome sequencing in the N-methyl-D-aspartic acid-damaged retinas of zebrafish to demonstrate the metabolomic mechanism of retinal regeneration.Among the differentially-ex pressed metabolites,we found a significant decrease in p-aminobenzoic acid in the N-methyl-D-aspartic acid-damaged retinas of zebrafish.Then,we investigated the role of p-aminobenzoic acid in retinal regeneration in adult zebrafish.Impo rtantly,p-aminobenzoic acid activated Achaetescute complex-like 1a expression,thereby promoting Müller glia reprogramming and division,as well as Müller glia-derived progenitor cell proliferation.Finally,we eliminated folic acid and inflammation as downstream effectors of PABA and demonstrated that PABA had little effect on Müller glia distribution.Taken together,these findings show that PABA contributes to retinal regeneration through activation of Achaetescute complex-like 1a expression in the N-methyl-Daspartic acid-damaged retinas of zebrafish.展开更多
基金supported by the Key Project of Guangzhou City,No.202206060002Science and Technology Project of Guangdong Province,No.2018B030332001Guangdong Provincial Pearl River Project,No.2021ZT09Y552 (all to GC)。
文摘Direct in vivo conversion of astrocytes into functional new neurons induced by neural transcription factors has been recognized as a potential new therapeutic intervention for neural injury and degenerative disorders. However, a few recent studies have claimed that neural transcription factors cannot convert astrocytes into neurons, attributing the converted neurons to pre-existing neurons mis-expressing transgenes. In this study, we overexpressed three distinct neural transcription factors––NeuroD1, Ascl1, and Dlx2––in reactive astrocytes in mouse cortices subjected to stab injury, resulting in a series of significant changes in astrocyte properties. Initially, the three neural transcription factors were exclusively expressed in the nuclei of astrocytes. Over time, however, these astrocytes gradually adopted neuronal morphology, and the neural transcription factors was gradually observed in the nuclei of neuron-like cells instead of astrocytes. Furthermore,we noted that transcription factor-infected astrocytes showed a progressive decrease in the expression of astrocytic markers AQP4(astrocyte endfeet signal), CX43(gap junction signal), and S100β. Importantly, none of these changes could be attributed to transgene leakage into preexisting neurons. Therefore, our findings suggest that neural transcription factors such as NeuroD1, Ascl1, and Dlx2 can effectively convert reactive astrocytes into neurons in the adult mammalian brain.
基金supported by grants from the National Key Research and Development Program of China(2018YFA0108000)the National Natural Science Foundation of China(31630032,31820103006,and 32070971)+1 种基金a Shanghai Municipal Science and Technology Major Project(2018SHZDZX01)ZJLab,and grants from NIH(R01MH094589 and R01NS089777)。
文摘Mouse cortical radial glial cells(RGCs)are primary neural stem cells that give rise to cortical oligodendrocytes,astrocytes,and olfactory bulb(OB)GABAergic interneurons in late embryogenesis.There are fundamental gaps in understanding how these diverse cell subtypes are generated.Here,by combining single-cell RNA-Seq with intersectional lineage analyses,we show that beginning at around E16.5,neocortical RGCs start to generate ASCL1^(+)EGFR^(+)apical multipotent intermediate progenitors(MIPCs),which then differentiate into basal MIPCs that express ASCL1,EGFR,OLIG2,and MKI67.These basal MIPCs undergo several rounds of divisions to generate most of the cortical oligodendrocytes and astrocytes and a subpopulation of OB interneurons.Finally,single-cell ATAC-Seq supported our model for the genetic logic underlying the specification and differentiation of cortical glial cells and OB interneurons.Taken together,this work reveals the process of cortical radial glial cell lineage progression and the developmental origins of cortical astrocytes and oligodendrocytes.
基金supported by the Charles H.“Skip”Smith Endowment Fund and the Verne M.Willaman Endowment Fund from the Pennsylvania State University to G.C。
文摘Objective:Glioblastoma(GBM)is the most prevalent and aggressive adult primary cancer in the central nervous system.Therapeutic approaches for GBM treatment are under intense investigation,including the use of emerging immunotherapies.Here,we propose an alternative approach to treat GBM through reprogramming proliferative GBM cells into non-proliferative neurons.Methods:Retroviruses were used to target highly proliferative human GBM cells through overexpression of neural transcription factors.Immunostaining,electrophysiological recording,and bulk RNA-seq were performed to investigate the mechanisms underlying the neuronal conversion of human GBM cells.An in vivo intracranial xenograft mouse model was used to examine the neuronal conversion of human GBM cells.Results:We report efficient neuronal conversion from human GBM cells by overexpressing single neural transcription factor Neurogenic differentiation 1(Neuro D1),Neurogenin-2(Neurog2),or Achaete-scute homolog 1(Ascl1).Subtype characterization showed that the majority of Neurog2-and Neuro D1-converted neurons were glutamatergic,while Ascl1 favored GABAergic neuron generation.The GBM cell-converted neurons not only showed pan-neuronal markers but also exhibited neuron-specific electrophysiological activities.Transcriptome analyses revealed that neuronal genes were activated in glioma cells after overexpression of neural transcription factors,and different signaling pathways were activated by different neural transcription factors.Importantly,the neuronal conversion of GBM cells was accompanied by significant inhibition of GBM cell proliferation in both in vitro and in vivo models.Conclusions:These results suggest that GBM cells can be reprogrammed into different subtypes of neurons,leading to a potential alternative approach to treat brain tumors using in vivo cell conversion technology.
文摘1文献来源研究一:Rudin CM,Poirier JT,Byers LA,et al.Molecular subtypes of small cell lung cancer:A synthesis of human and mouse model data[J].Nat Rev Cancer,2019,19(5):289-297.研究二:Owonikoko TK,Dwivedi B,Chen ZJ,et al.YAP1 positive small-cell lung cancer subtype is associated with the T-cell inflamed gene expression profile and confers good prognosis and long term survival[J].J Clin Oncol,2020,38(15S):Abstr 9019.
基金supported by the National Natural Science Foundation of China,No.81471126(to XZC)and 81771216(to XZC)the Natural Science Foundation of Zhejiang Province of China,No.LY17H090005(to JLP)a grant from the Medical Science and Technology Plan Project of Zhejiang Province of China,No.2016KYB119(to JLP)
文摘Differentiation of human fibroblasts into functional neurons depends on the introduction of viral-mediated transcription factors, which present risks of viral gene integration and tumorigenicity. In recent years, although some studies have been successful in directly inducing neurons through sustained expression of small molecule compounds, they have only been shown to be effective on mouse-derived cells. Thus, herein we delivered vectors containing Epstein-Barr virus-derived oriP/Epstein-Barr nuclear antigen 1 encoding the neuronal transcription factor, Ascl1, the neuron-specific microRNA, miR124, and a small hairpin directed against p53, into human fibroblasts. Cells were incubated in a neuron-inducing culture medium. Immunofluorescence staining was used to detect Tuj-1, microtubule-associated protein 2, neuron-specific nucleoprotein NeuN and nerve cell adhesion molecules in the induced cells. The proportion of Tuj1-positive cells was up to 36.7% after induction for 11 days. From day 21, these induced neurons showed neuron-specific expression patterns of microtubule-associated protein 2, NeuN and neural cell adhesion molecule. Our approach is a simple, plasmid-based process that enables direct reprogramming of human fibroblasts into neurons, and provides alternative avenues for disease modeling and neurodegenerative medicine.
基金supported by the National Natural Science Foundation of China,Nos.81974134(to XX)and 82000895(to HL)National Key Research and Development Program of China,Nos.2021YFA1101200&2021YFA1101202National Natural Science Foundation of Hunan Province,China,No.2022JJ30071(to HL)。
文摘The retina of zebrafish can regenerate completely after injury.M ultiple studies have demonstrated that metabolic alte rations occur during retinal damage;however to date no study has identified a link between metabolites and retinal regeneration of zebrafish.Here,we performed an unbiased metabolome sequencing in the N-methyl-D-aspartic acid-damaged retinas of zebrafish to demonstrate the metabolomic mechanism of retinal regeneration.Among the differentially-ex pressed metabolites,we found a significant decrease in p-aminobenzoic acid in the N-methyl-D-aspartic acid-damaged retinas of zebrafish.Then,we investigated the role of p-aminobenzoic acid in retinal regeneration in adult zebrafish.Impo rtantly,p-aminobenzoic acid activated Achaetescute complex-like 1a expression,thereby promoting Müller glia reprogramming and division,as well as Müller glia-derived progenitor cell proliferation.Finally,we eliminated folic acid and inflammation as downstream effectors of PABA and demonstrated that PABA had little effect on Müller glia distribution.Taken together,these findings show that PABA contributes to retinal regeneration through activation of Achaetescute complex-like 1a expression in the N-methyl-Daspartic acid-damaged retinas of zebrafish.
