Abstract
Two genes linked to early onset Parkinson's disease, PINK1 and Parkin, encode a protein kinase and a ubiquitin-ligase, respectively. Both enzymes have been suggested to support mitochondrial quality control. We have reported that Parkin is phosphorylated at Ser65 within the ubiquitin-like domain by PINK1 in mammalian cultured cells. However, it remains unclear whether Parkin phosphorylation is involved in mitochondrial maintenance and activity of dopaminergic neurons in vivo. Here, we examined the effects of Parkin phosphorylation in Drosophila, in which the phosphorylation residue is conserved at Ser94. Morphological changes of mitochondria caused by the ectopic expression of wild-type Parkin in muscle tissue and brain dopaminergic neurons disappeared in the absence of PINK1. In contrast, phosphomimetic Parkin accelerated mitochondrial fragmentation or aggregation and the degradation of mitochondrial proteins regardless of PINK1 activity, suggesting that the phosphorylation of Parkin boosts its ubiquitin-ligase activity. A non-phosphorylated form of Parkin fully rescued the muscular mitochondrial degeneration due to the loss of PINK1 activity, whereas the introduction of the non-phosphorylated Parkin mutant in Parkin-null flies led to the emergence of abnormally fused mitochondria in the muscle tissue. Manipulating the Parkin phosphorylation status affected spontaneous dopamine release in the nerve terminals of dopaminergic neurons, the survivability of dopaminergic neurons and flight activity. Our data reveal that Parkin phosphorylation regulates not only mitochondrial function but also the neuronal activity of dopaminergic neurons in vivo, suggesting that the appropriate regulation of Parkin phosphorylation is important for muscular and dopaminergic functions.
Highlights
Mutations of the Parkin and PINK1 genes cause selective degeneration of midbrain dopaminergic neurons in early-onset Parkinson’s disease (PD) [1,2]
We and Kondapalli et al have reported that Ser65 in the human Parkin Ubl domain is phosphorylated by PINK1, which is Parkinson’s disease is a neurodegenerative disorder caused by degeneration of the midbrain dopaminergic system in addition to other nervous systems
We previously found that Parkin is phosphorylated by PINK1 in mammalian cultured cells, the physiological significance of this interaction in vivo remained unclear
Summary
Mutations of the Parkin and PINK1 genes cause selective degeneration of midbrain dopaminergic neurons in early-onset Parkinson’s disease (PD) [1,2]. Our group and Wang et al have proposed a model in which PINK1 and Parkin prevent damaged mitochondria from moving to the nerve terminals by degrading Miro, an adaptor for microtubule-dependent mitochondrial transport [20,21] Supporting this notion, ectopic expression of WT Parkin in DA neurons suppressed mitochondrial distribution outside cell bodies, which was recovered in the PINK1-null genetic background. Regardless of the status of PINK1, SE Parkin enhanced the mitochondrial phenotypes observed when WT Parkin was expressed with endogenous PINK1 such that perinuclear accumulation of mitochondria was observed in the cell bodies These findings indicate that phosphorylation of Parkin by PINK1 boosts its E3 activity, regulating mitochondrial motility and morphology through degradation of mitochondrial proteins, such as Miro and Mfn
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