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
The onset of retinal degenerative disease is often associated with neuronal loss. Therefore, how to regenerate new neurons to restore vision is an important issue. NeuroD1 is a neural transcription factor with the abi...The onset of retinal degenerative disease is often associated with neuronal loss. Therefore, how to regenerate new neurons to restore vision is an important issue. NeuroD1 is a neural transcription factor with the ability to reprogram brain astrocytes into neurons in vivo. Here, we demonstrate that in adult mice, NeuroD1 can reprogram Müller cells, the principal glial cell type in the retina, to become retinal neurons. Most strikingly, ectopic expression of NeuroD1 using two different viral vectors converted Müller cells into different cell types. Specifically, AAV7 m8 GFAP681::GFP-ND1 converted Müller cells into inner retinal neurons, including amacrine cells and ganglion cells. In contrast, AAV9 GFAP104::ND1-GFP converted Müller cells into outer retinal neurons such as photoreceptors and horizontal cells, with higher conversion efficiency. Furthermore, we demonstrate that Müller cell conversion induced by AAV9 GFAP104::ND1-GFP displayed clear dose-and time-dependence. These results indicate that Müller cells in adult mice are highly plastic and can be reprogrammed into various subtypes of retinal neurons.展开更多
目的·筛查青少年的成人起病型糖尿病(maturity-onset diabetes of the young,MODY)家系中NEUROD1基因突变,分析突变与中国人MODY6发病的相关性及其潜在的致病机制。方法·采用PCR-直接测序法对96例GCK/MODY2、HNF1A/MODY3、HNF...目的·筛查青少年的成人起病型糖尿病(maturity-onset diabetes of the young,MODY)家系中NEUROD1基因突变,分析突变与中国人MODY6发病的相关性及其潜在的致病机制。方法·采用PCR-直接测序法对96例GCK/MODY2、HNF1A/MODY3、HNF1B/MODY5突变阴性的中国MODY先证者进行NEUROD1突变筛查,同时比较96例MODY先证者与100例非糖尿病对照者NEUROD1基因变异的基因型频率。采用从头建模法构建NEUROD1蛋白野生型和突变体的3D结构,采用双荧光素酶报告基因系统检测野生型和突变体蛋白对胰岛素基因转录活性的影响。结果·在一个MODY家系中发现NEUROD1基因杂合错义突变Glu59Gln (NM_002500.5,c.175G>C)。3D结构分析发现,该突变将野生型中带负电荷的Glu59转化为突变中不带电荷的Gln59,导致两个盐桥键Glu59-Arg54和Glu59-Lys88缺失,并形成一个新的氢键Gln59-Arg54。与野生型相比,Glu59Gln突变体的胰岛素基因转录活性下降36.3%(P<0.05)。与非糖尿病对照相比,96例MODY先证者中Ala45Thr (G-A)变异的AA+GA基因型频率显著升高(P=0.002)。结论·Glu59Gln突变改变了NEUROD1蛋白N端的分子构象,导致其胰岛素基因转录活性显著下降,是该家系突变携带者胰岛素分泌缺陷的原因。Ala45Thr变异与MODY6先证者糖尿病发病年龄的提前有关。展开更多
A new technology called in vivo glia-to-neuron conversion has emerged in recent years as a promising next generation therapy for neural regeneration and repair. This is achieved through reprogramming endogenous glial ...A new technology called in vivo glia-to-neuron conversion has emerged in recent years as a promising next generation therapy for neural regeneration and repair. This is achieved through reprogramming endogenous glial cells into neurons in the central nervous system through ectopically expressing neural transcriptional factors in glial cells. Previous studies have been focusing on glial cells in the grey matter such as the cortex and striatum, but whether glial cells in the white matter can be reprogrammed or not is unknown. To address this fundamental question, we express NeuroD1 in the astrocytes of both grey matter(cortex and striatum) and white matter(corpus callosum) to investigate the conversion efficiency, neuronal subtypes, and electrophysiological features of the converted neurons. We discover that NeuroD1 can efficiently reprogram the astrocytes in the grey matter into functional neurons, but the astrocytes in the white matter are much resistant to neuronal reprogramming. The converted neurons from cortical and striatal astrocytes are composed of both glutamatergic and GABAergic neurons, capable of firing action potentials and having spontaneous synaptic activities. In contrast, the few astrocyte-converted neurons in the white matter are rather immature with rare synaptic events. These results provide novel insights into the differential reprogramming capability between the astrocytes in the grey matter versus the white matter, and highlight the impact of regional astrocytes as well as microenvironment on the outcome of glia-toneuron conversion. Since human brain has large volume of white matter, this study will provide important guidance for future development of in vivo glia-to-neuron conversion technology into potential clinical therapies. Experimental protocols in this study were approved by the Laboratory Animal Ethics Committee of Jinan University(approval No. IACUC-20180321-03) on March 21, 2018.展开更多
Accumulation of aberrant proteins and inclusion bodies are hallmarks in most neurodegenerative diseases. Consequently, these aggregates within neurons lead to toxic effects, overproduction of reactive oxygen species a...Accumulation of aberrant proteins and inclusion bodies are hallmarks in most neurodegenerative diseases. Consequently, these aggregates within neurons lead to toxic effects, overproduction of reactive oxygen species and oxidative stress. Autophagy is a significant intracellular mechanism that removes damaged organelles and misfolded proteins in order to maintain cell homeostasis. Excessive or insufficient autophagic activity in neurons leads to altered homeostasis and influences their survival rate, causing neurodegeneration. The review article provides an update of the role of autophagic process in representative chronic and acute neurodegenerative disorders.展开更多
AIM： To investigate the role of O-GIcNAcylation of nuclear factor-kappa B （NF-KB） in retinal ganglion cell （RGC） death and analysedthe effect of Aralia elata （AE） on neurodegen- eration in diabetic mice. METH...AIM： To investigate the role of O-GIcNAcylation of nuclear factor-kappa B （NF-KB） in retinal ganglion cell （RGC） death and analysedthe effect of Aralia elata （AE） on neurodegen- eration in diabetic mice. METHODS： C57BL/6mice with streptozotocin-induced diabetes were fed daily with AE extract or control （CTL） diet at the onset of diabetes mellitus （DM）. Two months af- tar injection of streptozotocin or saline, the degree of cell death and the expression of O-GIcNAc transferase （OGT）, N-acetyl-b-D-glucosaminidase （OGA）, O-GIcNAcylated pro- teins, and O-GIcNAcylation of NF-KB were examined. RESULTS： AE did not affect the metabolic status of diabetic mice. The decrease in the inner retinal thickness （P〈0.001 vs CTL, P〈0.01 vs DM） and increases in RGCs with terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling （P〈0.001 vs CTL, P〈0.0001 vs DM）, glial activation, and active caspase-3 （P〈0.0001 vs CTL, P〈0.0001 vs DM） were blocked in diabetic retinas of AE extract-fed mice. Expression levels of protein O-GIcNAcylation and OGT were increased in diabetic retinas （P〈0.0001 vs CTL）, and the level of O-GIcNAcylation of the NF-KB p65 subunit was higher in diabetic retinas than in controls （P〈0.0001 vs CTL）. AE extract downregulated O-GIcNAcylation of NF-KB and prevented neurodegeneration induced by hyperglycemia （P〈0.0001 vs DM）. CONCLUSION： O-GIcNAcylation of NF-KB is concerned in neuronal degeneration and that AE prevents diabetes-in- duced RGC apoptosis via downregulation of NF-KB O-GI- cNAcylation. Hence, O-GIcNAcylation may be a new object for the treatment of DR, and AE may have therapeutic pos- sibility to prevent diabetes-induced neurodegeneration.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 the Guangdong Grant Key Technologies for Treatment of Brain Disorders,China,No. 2018B030332001 (to GC)the Guangzhou Key Projects of Brain Science and Brain-Like Intelligence Technology,No. 20200730009 (to YX)the Guangdong Basic and Applied Basic Research Foundation,No. 2020A1515110898 (to WYC)。
文摘The onset of retinal degenerative disease is often associated with neuronal loss. Therefore, how to regenerate new neurons to restore vision is an important issue. NeuroD1 is a neural transcription factor with the ability to reprogram brain astrocytes into neurons in vivo. Here, we demonstrate that in adult mice, NeuroD1 can reprogram Müller cells, the principal glial cell type in the retina, to become retinal neurons. Most strikingly, ectopic expression of NeuroD1 using two different viral vectors converted Müller cells into different cell types. Specifically, AAV7 m8 GFAP681::GFP-ND1 converted Müller cells into inner retinal neurons, including amacrine cells and ganglion cells. In contrast, AAV9 GFAP104::ND1-GFP converted Müller cells into outer retinal neurons such as photoreceptors and horizontal cells, with higher conversion efficiency. Furthermore, we demonstrate that Müller cell conversion induced by AAV9 GFAP104::ND1-GFP displayed clear dose-and time-dependence. These results indicate that Müller cells in adult mice are highly plastic and can be reprogrammed into various subtypes of retinal neurons.
文摘目的·筛查青少年的成人起病型糖尿病(maturity-onset diabetes of the young,MODY)家系中NEUROD1基因突变,分析突变与中国人MODY6发病的相关性及其潜在的致病机制。方法·采用PCR-直接测序法对96例GCK/MODY2、HNF1A/MODY3、HNF1B/MODY5突变阴性的中国MODY先证者进行NEUROD1突变筛查,同时比较96例MODY先证者与100例非糖尿病对照者NEUROD1基因变异的基因型频率。采用从头建模法构建NEUROD1蛋白野生型和突变体的3D结构,采用双荧光素酶报告基因系统检测野生型和突变体蛋白对胰岛素基因转录活性的影响。结果·在一个MODY家系中发现NEUROD1基因杂合错义突变Glu59Gln (NM_002500.5,c.175G>C)。3D结构分析发现,该突变将野生型中带负电荷的Glu59转化为突变中不带电荷的Gln59,导致两个盐桥键Glu59-Arg54和Glu59-Lys88缺失,并形成一个新的氢键Gln59-Arg54。与野生型相比,Glu59Gln突变体的胰岛素基因转录活性下降36.3%(P<0.05)。与非糖尿病对照相比,96例MODY先证者中Ala45Thr (G-A)变异的AA+GA基因型频率显著升高(P=0.002)。结论·Glu59Gln突变改变了NEUROD1蛋白N端的分子构象,导致其胰岛素基因转录活性显著下降,是该家系突变携带者胰岛素分泌缺陷的原因。Ala45Thr变异与MODY6先证者糖尿病发病年龄的提前有关。
基金supported in part by the National Natural Science Foundation of China(Grant No.31701291 to WL,U1801681 to GC)the China Postdoctoral Science Foundation(Grant No.2016M602600 to WL)+1 种基金the Guangdong Grant ‘Key Technologies for Treatment of Brain Disorders’(Grant No.2018B030332001 to GC)the Internal Funding of Jinan University,China(Grant No.21616110 to GC)
文摘A new technology called in vivo glia-to-neuron conversion has emerged in recent years as a promising next generation therapy for neural regeneration and repair. This is achieved through reprogramming endogenous glial cells into neurons in the central nervous system through ectopically expressing neural transcriptional factors in glial cells. Previous studies have been focusing on glial cells in the grey matter such as the cortex and striatum, but whether glial cells in the white matter can be reprogrammed or not is unknown. To address this fundamental question, we express NeuroD1 in the astrocytes of both grey matter(cortex and striatum) and white matter(corpus callosum) to investigate the conversion efficiency, neuronal subtypes, and electrophysiological features of the converted neurons. We discover that NeuroD1 can efficiently reprogram the astrocytes in the grey matter into functional neurons, but the astrocytes in the white matter are much resistant to neuronal reprogramming. The converted neurons from cortical and striatal astrocytes are composed of both glutamatergic and GABAergic neurons, capable of firing action potentials and having spontaneous synaptic activities. In contrast, the few astrocyte-converted neurons in the white matter are rather immature with rare synaptic events. These results provide novel insights into the differential reprogramming capability between the astrocytes in the grey matter versus the white matter, and highlight the impact of regional astrocytes as well as microenvironment on the outcome of glia-toneuron conversion. Since human brain has large volume of white matter, this study will provide important guidance for future development of in vivo glia-to-neuron conversion technology into potential clinical therapies. Experimental protocols in this study were approved by the Laboratory Animal Ethics Committee of Jinan University(approval No. IACUC-20180321-03) on March 21, 2018.
文摘Accumulation of aberrant proteins and inclusion bodies are hallmarks in most neurodegenerative diseases. Consequently, these aggregates within neurons lead to toxic effects, overproduction of reactive oxygen species and oxidative stress. Autophagy is a significant intracellular mechanism that removes damaged organelles and misfolded proteins in order to maintain cell homeostasis. Excessive or insufficient autophagic activity in neurons leads to altered homeostasis and influences their survival rate, causing neurodegeneration. The review article provides an update of the role of autophagic process in representative chronic and acute neurodegenerative disorders.
基金Supported by the Basic Science Research Program Through the National Research Foundation(NRF)of Korea Funded by the Ministry of Science,ICT,and Future Planning 2014049413,NRF-2015R1A5A2008833 and NRF-2015R1C1A1A02037702
文摘AIM： To investigate the role of O-GIcNAcylation of nuclear factor-kappa B （NF-KB） in retinal ganglion cell （RGC） death and analysedthe effect of Aralia elata （AE） on neurodegen- eration in diabetic mice. METHODS： C57BL/6mice with streptozotocin-induced diabetes were fed daily with AE extract or control （CTL） diet at the onset of diabetes mellitus （DM）. Two months af- tar injection of streptozotocin or saline, the degree of cell death and the expression of O-GIcNAc transferase （OGT）, N-acetyl-b-D-glucosaminidase （OGA）, O-GIcNAcylated pro- teins, and O-GIcNAcylation of NF-KB were examined. RESULTS： AE did not affect the metabolic status of diabetic mice. The decrease in the inner retinal thickness （P〈0.001 vs CTL, P〈0.01 vs DM） and increases in RGCs with terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling （P〈0.001 vs CTL, P〈0.0001 vs DM）, glial activation, and active caspase-3 （P〈0.0001 vs CTL, P〈0.0001 vs DM） were blocked in diabetic retinas of AE extract-fed mice. Expression levels of protein O-GIcNAcylation and OGT were increased in diabetic retinas （P〈0.0001 vs CTL）, and the level of O-GIcNAcylation of the NF-KB p65 subunit was higher in diabetic retinas than in controls （P〈0.0001 vs CTL）. AE extract downregulated O-GIcNAcylation of NF-KB and prevented neurodegeneration induced by hyperglycemia （P〈0.0001 vs DM）. CONCLUSION： O-GIcNAcylation of NF-KB is concerned in neuronal degeneration and that AE prevents diabetes-in- duced RGC apoptosis via downregulation of NF-KB O-GI- cNAcylation. Hence, O-GIcNAcylation may be a new object for the treatment of DR, and AE may have therapeutic pos- sibility to prevent diabetes-induced neurodegeneration.
