Abstract
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. 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. 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. 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.
Highlights
It has previously been demonstrated that an insect orthologue, Tribolium castaneum PTEN-induced kinase 1 (PINK1) (TcPINK1), exhibits robust kinase activity in vitro as judged by phosphorylation of substrates including myelin basic protein (MBP) and the ubiquitinlike (Ubl) domain of parkin at serine 65.14,19 We modeled the zebrafish Y431X into TcPINK1 and determined the effect on kinase activity
We found that the Y419X mutation abolished TcPINK1 activity against the parkin Ubl domain and MBP (Supplementary Fig 4)
Genetic research has provided crucial new insight into the pathogenesis of Parkinson disease (PD), but the precise mechanisms leading to neuronal cell death remain to be elucidated.[33]
Summary
Loss of function mutations in PINK1 typically lead to early onset Parkinson disease (PD). Loss of function mutations in PTEN-induced kinase 1 (PINK1) typically lead to early onset Parkinson disease (EOPD).[1] The. PINK1 protein is expressed ubiquitously throughout the human brain.[2] Impaired mitochondrial function and morphology have been described in both human PINK1 mutant patient tissue and different PINK1-deficient in vitro or in vivo model systems.[3,4] PINK1 has been implicated in oxidative stress defense, mitophagy, and the regulation of mitochondrial calcium homeostasis.[3,4,5]. Genome-wide gene expression studies identified upregulation of TigarB, the zebrafish homologue of the TP53-induced glycolysis and apoptosis regulator (TIGAR).[13] Remarkably, TigarB knockdown resulted in normalization of mitochondrial function and complete rescue of ascending dopaminergic neurons. Modulation of TIGAR-related mechanisms may be a promising novel strategy to develop disease-modifying therapy for PINK1-related PD
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