Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons re...Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin（sh）RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 sh RNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region（GDNF-luciferase） were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-sh RNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-sh RNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.展开更多
Background: The authors recently identified three large genetically unrelated families with an identical 17 base pair duplication mutation in exon 4 of the PITX3 gene. Here, they report the detailed clinical phenotype...Background: The authors recently identified three large genetically unrelated families with an identical 17 base pair duplication mutation in exon 4 of the PITX3 gene. Here, they report the detailed clinical phenotype. Methods: Affected and unaffected individuals in the three families with autosomal dominant posterior polar cataract underwent full clinical examination and donated blood samples for DNA extraction and molecular genetic studies. Results: In all three families, an identical 17 base pair duplication mutation in PITX3 was identified which cosegregated with disease status in the family. All affected individuals had bilateral progressive posterior polar cataracts. In one family, posterior polar cataract was the only clinical abnormality but in the other two families, one of 10 affected individuals and four of 11 affected individuals also had anterior segment mesenchymal dysgenesis (ASMD). Conclusion: Mutations in the PITX3 gene in humans result in posterior polar cataract and variable ASMD. The gene encodes a transcription factor which has a key role in lens and anterior segment development. The mechanism by which the mutant protein gives rise to such a regional pattern of lens opacity remains to be elucidated.展开更多
BACKGROUND： Parkinson＇s disease （PD） is a common, age-dependent degenerative neurological disorder impairing motor control function and cognition. A key pathology of PD is a degeneration of the nigrostriatal dopam...BACKGROUND： Parkinson＇s disease （PD） is a common, age-dependent degenerative neurological disorder impairing motor control function and cognition. A key pathology of PD is a degeneration of the nigrostriatal dopamine system, leading to a severe dopamine denervation in the striatum and dynsfunction of the striatal neural circuits. OBJECTIVE： To better understand the pathophysiology of the nigrostriatal dopamine denervation and to discover better treatments, animal PD models are needed. METHODS： The authors＇ original research on the transcription factor Pitx3 null mutant mice and the relevant literature were reviewed. RESULTS： An important feature of an animal PD model is the severe, PD-like nigrostriatal dopamine denervation. This feature is provided in the transcription factor Pitx3 null mutant mice. These mice have a severe and bilateral nigral dopamine neuron loss and dopamine denervation in the dorsal striatum, while the dopamine neuron loss in the ventral tegmental area and dopamine denervation in the ventral striatum are moderate, creating a dorsal-ventral dopamine loss gradient and mimicking the dopamine denervation pattern in PD. Pitx3 null mice show motor function deficits in the balance beam and pole tests and these deficits are reversed by L-3,4-dihydroxyphenylalanine （L-dopa）. These mice also show impaired cognitive functions as indicated by reduced motor learning and avoidance memory. L-dopa, D 1 agonists and, to a lesser extent, D2 agonists, induce normal horizontal movements （walking） and also dyskinesia-like movements consisting of vertical body trunk movements and waving paw movements. CONCLUSIONS： The easy-to-maintain Pitx3 null mice with an autogenic, consistent and gradient dopamine denervation are a convenient and suitable mouse model to study the consequences of dopamine loss in PD and to test dopaminergic replacement therapies for PD.展开更多
基金supported by the National Natural Science Foundation of China,No.81372698
文摘Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin（sh）RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 sh RNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region（GDNF-luciferase） were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-sh RNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-sh RNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.
文摘Background: The authors recently identified three large genetically unrelated families with an identical 17 base pair duplication mutation in exon 4 of the PITX3 gene. Here, they report the detailed clinical phenotype. Methods: Affected and unaffected individuals in the three families with autosomal dominant posterior polar cataract underwent full clinical examination and donated blood samples for DNA extraction and molecular genetic studies. Results: In all three families, an identical 17 base pair duplication mutation in PITX3 was identified which cosegregated with disease status in the family. All affected individuals had bilateral progressive posterior polar cataracts. In one family, posterior polar cataract was the only clinical abnormality but in the other two families, one of 10 affected individuals and four of 11 affected individuals also had anterior segment mesenchymal dysgenesis (ASMD). Conclusion: Mutations in the PITX3 gene in humans result in posterior polar cataract and variable ASMD. The gene encodes a transcription factor which has a key role in lens and anterior segment development. The mechanism by which the mutant protein gives rise to such a regional pattern of lens opacity remains to be elucidated.
文摘BACKGROUND： Parkinson＇s disease （PD） is a common, age-dependent degenerative neurological disorder impairing motor control function and cognition. A key pathology of PD is a degeneration of the nigrostriatal dopamine system, leading to a severe dopamine denervation in the striatum and dynsfunction of the striatal neural circuits. OBJECTIVE： To better understand the pathophysiology of the nigrostriatal dopamine denervation and to discover better treatments, animal PD models are needed. METHODS： The authors＇ original research on the transcription factor Pitx3 null mutant mice and the relevant literature were reviewed. RESULTS： An important feature of an animal PD model is the severe, PD-like nigrostriatal dopamine denervation. This feature is provided in the transcription factor Pitx3 null mutant mice. These mice have a severe and bilateral nigral dopamine neuron loss and dopamine denervation in the dorsal striatum, while the dopamine neuron loss in the ventral tegmental area and dopamine denervation in the ventral striatum are moderate, creating a dorsal-ventral dopamine loss gradient and mimicking the dopamine denervation pattern in PD. Pitx3 null mice show motor function deficits in the balance beam and pole tests and these deficits are reversed by L-3,4-dihydroxyphenylalanine （L-dopa）. These mice also show impaired cognitive functions as indicated by reduced motor learning and avoidance memory. L-dopa, D 1 agonists and, to a lesser extent, D2 agonists, induce normal horizontal movements （walking） and also dyskinesia-like movements consisting of vertical body trunk movements and waving paw movements. CONCLUSIONS： The easy-to-maintain Pitx3 null mice with an autogenic, consistent and gradient dopamine denervation are a convenient and suitable mouse model to study the consequences of dopamine loss in PD and to test dopaminergic replacement therapies for PD.
