RESUMO
OBJECTIVE: Loss of function mutations in PINK1 typically lead to early onset Parkinson disease (PD). Zebrafish (Danio rerio) are emerging as a powerful new vertebrate model to study neurodegenerative diseases. We used a pink1 mutant (pink(-/-) ) zebrafish line with a premature stop mutation (Y431*) in the PINK1 kinase domain to identify molecular mechanisms leading to mitochondrial dysfunction and loss of dopaminergic neurons in PINK1 deficiency. METHODS: The effect of PINK1 deficiency on the number of dopaminergic neurons, mitochondrial function, and morphology was assessed in both zebrafish embryos and adults. Genome-wide gene expression studies were undertaken to identify novel pathogenic mechanisms. Functional experiments were carried out to further investigate the effect of PINK1 deficiency on early neurodevelopmental mechanisms and microglial activation. RESULTS: PINK1 deficiency results in loss of dopaminergic neurons as well as early impairment of mitochondrial function and morphology in Danio rerio. Expression of TigarB, the zebrafish orthologue of the human, TP53-induced glycolysis and apoptosis regulator TIGAR, was markedly increased in pink(-/-) larvae. Antisense-mediated inactivation of TigarB gave rise to complete normalization of mitochondrial function, with resulting rescue of dopaminergic neurons in pink(-/-) larvae. There was also marked microglial activation in pink(-/-) larvae, but depletion of microglia failed to rescue the dopaminergic neuron loss, arguing against microglial activation being a key factor in the pathogenesis. INTERPRETATION: Pink1(-/-) zebrafish are the first vertebrate model of PINK1 deficiency with loss of dopaminergic neurons. Our study also identifies TIGAR as a promising novel target for disease-modifying therapy in PINK1-related PD.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas Reguladoras de Apoptose/genética , Neurônios Dopaminérgicos/patologia , Larva/genética , Larva/metabolismo , Microglia/metabolismo , Doenças Mitocondriais/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Fibroblast growth factor 8 (FGF8), sonic hedgehog (SHH) and nodal signalling pathways play key roles in both development and survival of dopaminergic neurons. Both heterozygous mutations in autosomal recessively inherited Parkinson's disease (PD) genes such as parkin or PINK1 and exposure to exogenous toxins are thought to contribute to the pathogenesis of PD. The aim of our study was to investigate whether heterozygote mutations in fgf8, shh or oep lead to a reduced number of ascending dopaminergic neurons in zebrafish (Danio rerio) or confer increased susceptibility to the PD neurotoxin 1-methyl-4-phenyl-pyridinium (MPPâº). At 3 days post fertilization, heterozygous mutations in fgf8, shh or oep did not affect the number of ascending dopaminergic neurons, nor did heterozygous mutations in fgf8, shh or oep result in increased susceptibility to MPPâº. Further work is needed to determine whether haploinsufficiency in other neurodevelopmental genes might confer increased susceptibility to PD-related pathomechanisms.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Proteínas Hedgehog/genética , Proteínas de Homeodomínio/genética , Proteína Nodal/genética , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , 1-Metil-4-fenilpiridínio/toxicidade , Animais , Sobrevivência Celular , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Embrião não Mamífero , Larva , Mutação , Transdução de Sinais , Peixe-ZebraRESUMO
During pregnancy some maternal cells reach the fetal circulation. Microchimerism (Mc) refers to low levels of genetically disparate cells or DNA. Maternal Mc has recently been found in the peripheral blood of healthy adults. We asked whether healthy women have maternal Mc in T and B lymphocytes, monocyte/macrophages and NK cells and, if so, at what levels. Cellular subsets were isolated after fluorescence activated cell sorting. A panel of HLA-specific real-time quantitative PCR assays was employed targeting maternal-specific HLA sequences. Maternal Mc was expressed as the genome equivalent (gEq) number of microchimeric cells per 100,000 proband cells. Thirty-one healthy adult women probands were studied. Overall 39% (12/31) of probands had maternal Mc in at least one cellular subset. Maternal Mc was found in T lymphocytes in 25% (7/28) and B lymphocytes in 14% (3/21) of probands. Maternal Mc levels ranged from 0.9 to 25.6 and 0.9 to 25.3 gEq/100,000 in T and B lymphocytes, respectively. Monocyte/macrophages had maternal Mc in 16% (4/25) and NK cells in 28% (5/18) of probands with levels from 0.3 to 36 and 1.8 to 3.2 gEq/100,000, respectively. When compared to fetal Mc, as assessed by quantification of male DNA in women with sons, maternal Mc was substantially less prevalent in all cellular subsets; fetal Mc prevalence in T and B lymphocytes, monocyte/macrophages and NK cells was 58, 75, 50 and 62% (P=0.01, P=0.005, P=0.04, P=0.05) respectively. In summary, maternal Mc was identified among lymphoid and myeloid compartments of peripheral blood in healthy adult women. Maternal Mc was less frequent than fetal Mc in all cellular subsets tested. Studies are needed to investigate the immunological effects and function of maternal Mc and to explore whether maternal Mc in cellular subsets has biological effects on her progeny.
