In the developing and adult brain, neurotrophic growth factors support the growth and protec tion of dopaminergic neuronal systems. Recently, links between impaired neurotrophin support of dopamine (DA) neurons has be...In the developing and adult brain, neurotrophic growth factors support the growth and protec tion of dopaminergic neuronal systems. Recently, links between impaired neurotrophin support of dopamine (DA) neurons has been described in Parkinson’s Disease (PD). Fibro- blast growth factor (FGF) has a unique association with DA neurons in that FGF signaling is vitally important for the development and protection of adult DA neurons. We assessed the role of substantia nigra (SN)-expressed FGFs in the nigrostriatal dopaminergic system using a transgenic mouse, th-fgfr1(tk-). In these mice, generated by expression of dominant negative FGFR1(TK-) from the tyrosine hydroxylase (TH) gene promoter, reduced FGF signaling results in smaller and less dense adult nigrostriatal DA neurons, similar to what is observed in PD. With unilateral 6-hydroxydopamine (6-OHDA) lesions, th-fgfr1(tk-) mice exhibited extensive unilateral nigrostriatal damage with robust spontaneous (non-drug induced) asymmetrical turning and a decreased latency to remain on the accelerating rotarod. L-DOPA remains the gold standard for PD therapy despite debilitating hyperkinetic and dyskinetic side effects. The nicotinic acetylcholine system has recently been targeted as an alternative system to combat PD motor symptoms. Nicotine effectively stimulates dopaminergic transmission in the nigrostriatal pathway and mediates movement. Using unilaterally lesioned th-fgfr1(tk-) mice, long term (11 day) oral administration of nicotine increased spontaneous bidirectional turning and increased the latency before falling from the accelerating rotarod. In a separate analysis, L-DOPA treatment reversed directionality of rotation and further deepened motor discoordination, suggesting activation of hypersensitive postsynaptic DA receptors in the denervated striata. These results in a transgenic model of PD provide insights into the cellular mechanisms underlying L-DOPA and nicotinic therapies and offer further evidence of nicotine’s capacity to facilitate movement and enhance motor coordination in PD.展开更多
Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases.They also represent a potential source of transplanted cells for therapeutic applications.In vi...Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases.They also represent a potential source of transplanted cells for therapeutic applications.In vitro differentiation of functional midbrain dopaminergic neurons provides an accessible platform to study midbrain neuronal dysfunction and can be used to examine obstacles to dopaminergic neuronal development.Emerging evidence and impressive advances in human induced pluripotent stem cells,with tuned neural induction and differentiation protocols,makes the production of induced pluripotent stem cell-derived dopaminergic neurons feasible.Using SB431542 and dorsomorphin dual inhibitor in an induced pluripotent stem cell-derived neural induction protocol,we obtained multiple subtypes of neurons,including 20%tyrosine hydroxylase-positive dopaminergic neurons.To obtain more dopaminergic neurons,we next added sonic hedgehog(SHH)and fibroblast growth factor 8(FGF8)on day 8 of induction.This increased the proportion of dopaminergic neurons,up to 75%tyrosine hydroxylase-positive neurons,with 15%tyrosine hydroxylase and forkhead box protein A2(FOXA2)co-expressing neurons.We further optimized the induction protocol by applying the small molecule inhibitor,CHIR99021(CHIR).This helped facilitate the generation of midbrain dopaminergic neurons,and we obtained 31-74%midbrain dopaminergic neurons based on tyrosine hydroxylase and FOXA2 staining.Thus,we have established three induction protocols for dopaminergic neurons.Based on tyrosine hydroxylase and FOXA2 immunostaining analysis,the CHIR,SHH,and FGF8 combined protocol produces a much higher proportion of midbrain dopaminergic neurons,which could be an ideal resource for tackling midbrain-related diseases.展开更多
文摘In the developing and adult brain, neurotrophic growth factors support the growth and protec tion of dopaminergic neuronal systems. Recently, links between impaired neurotrophin support of dopamine (DA) neurons has been described in Parkinson’s Disease (PD). Fibro- blast growth factor (FGF) has a unique association with DA neurons in that FGF signaling is vitally important for the development and protection of adult DA neurons. We assessed the role of substantia nigra (SN)-expressed FGFs in the nigrostriatal dopaminergic system using a transgenic mouse, th-fgfr1(tk-). In these mice, generated by expression of dominant negative FGFR1(TK-) from the tyrosine hydroxylase (TH) gene promoter, reduced FGF signaling results in smaller and less dense adult nigrostriatal DA neurons, similar to what is observed in PD. With unilateral 6-hydroxydopamine (6-OHDA) lesions, th-fgfr1(tk-) mice exhibited extensive unilateral nigrostriatal damage with robust spontaneous (non-drug induced) asymmetrical turning and a decreased latency to remain on the accelerating rotarod. L-DOPA remains the gold standard for PD therapy despite debilitating hyperkinetic and dyskinetic side effects. The nicotinic acetylcholine system has recently been targeted as an alternative system to combat PD motor symptoms. Nicotine effectively stimulates dopaminergic transmission in the nigrostriatal pathway and mediates movement. Using unilaterally lesioned th-fgfr1(tk-) mice, long term (11 day) oral administration of nicotine increased spontaneous bidirectional turning and increased the latency before falling from the accelerating rotarod. In a separate analysis, L-DOPA treatment reversed directionality of rotation and further deepened motor discoordination, suggesting activation of hypersensitive postsynaptic DA receptors in the denervated striata. These results in a transgenic model of PD provide insights into the cellular mechanisms underlying L-DOPA and nicotinic therapies and offer further evidence of nicotine’s capacity to facilitate movement and enhance motor coordination in PD.
基金supported by the National Natural Science Foundation of China,No.81771222(to LS)Guangzhou Key Research Program on Brain Science,Nos.202007030011,202206060001(to LS)the Program of Introducing Talents of Discipline to Universities of China,No.B14036(to KFS)。
文摘Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases.They also represent a potential source of transplanted cells for therapeutic applications.In vitro differentiation of functional midbrain dopaminergic neurons provides an accessible platform to study midbrain neuronal dysfunction and can be used to examine obstacles to dopaminergic neuronal development.Emerging evidence and impressive advances in human induced pluripotent stem cells,with tuned neural induction and differentiation protocols,makes the production of induced pluripotent stem cell-derived dopaminergic neurons feasible.Using SB431542 and dorsomorphin dual inhibitor in an induced pluripotent stem cell-derived neural induction protocol,we obtained multiple subtypes of neurons,including 20%tyrosine hydroxylase-positive dopaminergic neurons.To obtain more dopaminergic neurons,we next added sonic hedgehog(SHH)and fibroblast growth factor 8(FGF8)on day 8 of induction.This increased the proportion of dopaminergic neurons,up to 75%tyrosine hydroxylase-positive neurons,with 15%tyrosine hydroxylase and forkhead box protein A2(FOXA2)co-expressing neurons.We further optimized the induction protocol by applying the small molecule inhibitor,CHIR99021(CHIR).This helped facilitate the generation of midbrain dopaminergic neurons,and we obtained 31-74%midbrain dopaminergic neurons based on tyrosine hydroxylase and FOXA2 staining.Thus,we have established three induction protocols for dopaminergic neurons.Based on tyrosine hydroxylase and FOXA2 immunostaining analysis,the CHIR,SHH,and FGF8 combined protocol produces a much higher proportion of midbrain dopaminergic neurons,which could be an ideal resource for tackling midbrain-related diseases.
