Background:Generation of neurons is essential in cell replacement therapy for neurodegenerative disorders like Parkinson’s disease.Several studies have reported the generation of dopaminergic(DA)neurons from mouse an...Background:Generation of neurons is essential in cell replacement therapy for neurodegenerative disorders like Parkinson’s disease.Several studies have reported the generation of dopaminergic(DA)neurons from mouse and human fibroblasts by ectopic expression of transcription factors,in which genetic manipulation is associated with potential risks.Methods:The small molecules and protein factors were selected based on their function to directly induce human fetal lung IMR-90 fibroblasts into DA neuron-like cells.Microscopical,immunocytochemical,and RT-qPCR analyses were used to characterize the morphology,phenotype,and gene expression features of the induced cells.The wholecell patch-clamp recordings were exploited to measure the electrophysiological properties.Results:Human IMR-90 fibroblasts were rapidly converted into DA neuron-like cells after the chemical induction using small molecules and protein factors,with a yield of approximately 95%positive TUJ1-positive cells.The induced DA neuron-like cells were immunopositive for pan-neuronal markers MAP2,NEUN,and Synapsin 1 and DA markers TH,DDC,DAT,and NURR1.The chemical induction process did not involve a neural progenitor/stem cell intermediate stage.The induced neurons could fire single action potentials,which reflected partially the electrophysiological properties of neurons.Conclusions:We developed a chemical cocktail of small molecules and protein factors to convert human fibroblasts into DA neuron-like cells without passing through a neural progenitor/stem cell intermediate stage.The induced DA neuron-like cells from human fibroblasts might provide a cellular source for cell-based therapy of Parkinson’s disease in the future.展开更多
Objective: To investigate the mechanism of oxidative stress in rotenone neurotoxicity to dopaminergic neuron PC12. Methods: High differentiated PC12 cells as dopaminergic neurons were treated by different concentratio...Objective: To investigate the mechanism of oxidative stress in rotenone neurotoxicity to dopaminergic neuron PC12. Methods: High differentiated PC12 cells as dopaminergic neurons were treated by different concentrations of rotenone. The morphology was observed with inverted phase contrast microscope and transmission electron microscope. Cell viability and proliferation inhibition were assessed by MTT. SOD and MDA were detected with biochemical assay. And the specific fluorescent probe (DCF-DA) was used to examine ROS in PC12 cells. Results: After treated with rotenone for 24 h, most of the PC12 cells became smaller and rounder. The process of axon was reduced, shortened or broken in a time and concentration dependent manner. The mitochondrial structure and metabolism were changed. Endoplasmic reticulum expanded and the free ribosome increased. Compared with the control group, cell proliferation inhibition increased and cell viability decreased. SOD increased and MDA decreased. The intensity of fluorescence was more obvious in PC12 cells treated by rotenone compared with control group. Conclusion : Rotenone is neurotoxic to cultured dopaminergic neuron PC12. Rotenone might exert this effect through the metabolism of oxidative stress on the pathogenesis of the neuron.展开更多
Objective: To study the sequence and function of the glial cell line-derived neurotrophic factor (GDNF) transcript in subjects of Han nationality. Methods: The Han nationality GDNF transcript was amplified by RT-PCR a...Objective: To study the sequence and function of the glial cell line-derived neurotrophic factor (GDNF) transcript in subjects of Han nationality. Methods: The Han nationality GDNF transcript was amplified by RT-PCR and expressed by baculovirus expression system. Biological activity of the expressed product was measured by the primary culture of midbrain dopaminergic neurons. Results: There only existed the shorter GDNF transcript of 555 bp in the Han nationality. The secretory expression product of the shorter transcript in insect cells promoted the survival and differentiation of dopaminergic neurons. Conclusion: It is found that there is a 78 bp deletion in the Han nationality GDNF transcript compared with the reported 633 bp GDNF transcript. The 78 bp deletion does not affect the secretory expression and biological activity of GDNF mature protein.展开更多
Parkinson's disease(PD)is one of the most common neurodegenerative diseases.The loss of dopaminergic(DAergic)neurons in the substantia nigra and the decrease of dopamine(DA)levels accelerate the process of PD.L-Er...Parkinson's disease(PD)is one of the most common neurodegenerative diseases.The loss of dopaminergic(DAergic)neurons in the substantia nigra and the decrease of dopamine(DA)levels accelerate the process of PD.L-Ergothioneine(EGT)is a natural antioxidant derived from microorganisms,especially in edible mushrooms.EGT can penetrate blood-brain barrier and its levels are significantly decreased in the plasma of PD patients.Therefore,we speculated that EGT could ameliorate PD,and determined its effect on PD development by using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced PD mouse models and neurotoxin 1-methyl-4-phenylpyridinium(MPP^(+))-induced cell models.Our results show that EGT alleviated MPTP-induced behavioral dysfunction in mice.Mechanistically,we innovatively revealed that EGT was a key regulator of DJ-1.EGT restored DA levels by activating the DJ-1-nuclear receptor-related factor 1(Nurr1)axis.Furthermore,it reduced reactive oxygen species(ROS)levels by regulating the DJ-1-nuclear factor erythroid 2-related factor 2(Nrf2)pathway,which inhibited oxidative stress-induced DAergic neuronal apoptosis.Combined treatment with DJ-1-si RNA transfection revealed that blocking DJ-1 reversed EGT upregulated Nurr1 and Nrf2 expression in the nucleus,which significantly decreased the benefits of EGT.Taken together,our study suggests that EGT can ameliorate PD and be considered as a strategy for PD treatment.展开更多
基金supported in part by the National Nature Science Foundation of China(81830064,81721092)the National Key Research and Development Plan(2017YFC1103304)+1 种基金the CAMS Innovation Fund for Medical Sciences(CIFMS,2019-I2M-5-059)the Military Medical Research and Development Projects(AWS17J005,2019–126)。
文摘Background:Generation of neurons is essential in cell replacement therapy for neurodegenerative disorders like Parkinson’s disease.Several studies have reported the generation of dopaminergic(DA)neurons from mouse and human fibroblasts by ectopic expression of transcription factors,in which genetic manipulation is associated with potential risks.Methods:The small molecules and protein factors were selected based on their function to directly induce human fetal lung IMR-90 fibroblasts into DA neuron-like cells.Microscopical,immunocytochemical,and RT-qPCR analyses were used to characterize the morphology,phenotype,and gene expression features of the induced cells.The wholecell patch-clamp recordings were exploited to measure the electrophysiological properties.Results:Human IMR-90 fibroblasts were rapidly converted into DA neuron-like cells after the chemical induction using small molecules and protein factors,with a yield of approximately 95%positive TUJ1-positive cells.The induced DA neuron-like cells were immunopositive for pan-neuronal markers MAP2,NEUN,and Synapsin 1 and DA markers TH,DDC,DAT,and NURR1.The chemical induction process did not involve a neural progenitor/stem cell intermediate stage.The induced neurons could fire single action potentials,which reflected partially the electrophysiological properties of neurons.Conclusions:We developed a chemical cocktail of small molecules and protein factors to convert human fibroblasts into DA neuron-like cells without passing through a neural progenitor/stem cell intermediate stage.The induced DA neuron-like cells from human fibroblasts might provide a cellular source for cell-based therapy of Parkinson’s disease in the future.
基金Supported by the National Natural Science Foundation of China (No. 30400347)
文摘Objective: To investigate the mechanism of oxidative stress in rotenone neurotoxicity to dopaminergic neuron PC12. Methods: High differentiated PC12 cells as dopaminergic neurons were treated by different concentrations of rotenone. The morphology was observed with inverted phase contrast microscope and transmission electron microscope. Cell viability and proliferation inhibition were assessed by MTT. SOD and MDA were detected with biochemical assay. And the specific fluorescent probe (DCF-DA) was used to examine ROS in PC12 cells. Results: After treated with rotenone for 24 h, most of the PC12 cells became smaller and rounder. The process of axon was reduced, shortened or broken in a time and concentration dependent manner. The mitochondrial structure and metabolism were changed. Endoplasmic reticulum expanded and the free ribosome increased. Compared with the control group, cell proliferation inhibition increased and cell viability decreased. SOD increased and MDA decreased. The intensity of fluorescence was more obvious in PC12 cells treated by rotenone compared with control group. Conclusion : Rotenone is neurotoxic to cultured dopaminergic neuron PC12. Rotenone might exert this effect through the metabolism of oxidative stress on the pathogenesis of the neuron.
基金National Natural Science Foundation of China (No.30000048) National Basic Research Program of China (G1999054000)
文摘Objective: To study the sequence and function of the glial cell line-derived neurotrophic factor (GDNF) transcript in subjects of Han nationality. Methods: The Han nationality GDNF transcript was amplified by RT-PCR and expressed by baculovirus expression system. Biological activity of the expressed product was measured by the primary culture of midbrain dopaminergic neurons. Results: There only existed the shorter GDNF transcript of 555 bp in the Han nationality. The secretory expression product of the shorter transcript in insect cells promoted the survival and differentiation of dopaminergic neurons. Conclusion: It is found that there is a 78 bp deletion in the Han nationality GDNF transcript compared with the reported 633 bp GDNF transcript. The 78 bp deletion does not affect the secretory expression and biological activity of GDNF mature protein.
基金supported by the National Natural Science Foundation of China(U22A20272,82173807,82170497)。
文摘Parkinson's disease(PD)is one of the most common neurodegenerative diseases.The loss of dopaminergic(DAergic)neurons in the substantia nigra and the decrease of dopamine(DA)levels accelerate the process of PD.L-Ergothioneine(EGT)is a natural antioxidant derived from microorganisms,especially in edible mushrooms.EGT can penetrate blood-brain barrier and its levels are significantly decreased in the plasma of PD patients.Therefore,we speculated that EGT could ameliorate PD,and determined its effect on PD development by using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced PD mouse models and neurotoxin 1-methyl-4-phenylpyridinium(MPP^(+))-induced cell models.Our results show that EGT alleviated MPTP-induced behavioral dysfunction in mice.Mechanistically,we innovatively revealed that EGT was a key regulator of DJ-1.EGT restored DA levels by activating the DJ-1-nuclear receptor-related factor 1(Nurr1)axis.Furthermore,it reduced reactive oxygen species(ROS)levels by regulating the DJ-1-nuclear factor erythroid 2-related factor 2(Nrf2)pathway,which inhibited oxidative stress-induced DAergic neuronal apoptosis.Combined treatment with DJ-1-si RNA transfection revealed that blocking DJ-1 reversed EGT upregulated Nurr1 and Nrf2 expression in the nucleus,which significantly decreased the benefits of EGT.Taken together,our study suggests that EGT can ameliorate PD and be considered as a strategy for PD treatment.
