Original Contribution
Mutant PINK1 upregulates tyrosine hydroxylase and dopamine levels, leading to vulnerability of dopaminergic neurons

https://doi.org/10.1016/j.freeradbiomed.2013.12.015Get rights and content

Abstract

PINK1 mutations cause autosomal recessive forms of Parkinson disease (PD). Previous studies suggest that the neuroprotective function of wild-type (WT) PINK1 is related to mitochondrial homeostasis. PINK1 can also localize to the cytosol; however, the cytosolic function of PINK1 has not been fully elucidated. In this study we demonstrate that the extramitochondrial PINK1 can regulate tyrosine hydroxylase (TH) expression and dopamine (DA) content in dopaminergic neurons in a PINK1 kinase activity-dependent manner. We demonstrate that overexpression of full-length (FL) WT PINK1 can downregulate TH expression and DA content in dopaminergic neurons. In contrast, overexpression of PD-linked G309D, A339T, and E231G PINK1 mutations upregulates TH and DA levels in dopaminergic neurons and increases their vulnerability to oxidative stress. Furthermore transfection of FL WT PINK1 or PINK1 fragments with the PINK1 kinase domain can inhibit TH expression, whereas kinase-dead (KD) FL PINK1 or KD PINK1 fragments upregulate TH level. Our findings highlight a potential novel function of extramitochondrial PINK1 in dopaminergic neurons. Deregulation of these functions of PINK1 may contribute to PINK1 mutation-induced dopaminergic neuron degeneration. However, deleterious effects caused by PINK1 mutations may be alleviated by iron-chelating agents and antioxidant agents with DA quinone-conjugating capacity.

Section snippets

Plasmids and vectors

To create human stable PINK1 SHSY-5Y cell lines, WT full-length human PINK1 (PINK1-FL) cDNA was cloned into pcDNA3.1 (−) mammalian expression vectors (Invitrogen) and PINK1 A339T and E231G mutations were created by a PCR-based technique using a QuikChange site-directed mutagenesis kit (Stratagene) as previously reported [25]; or PINK1-FL cDNA was cloned into the EGFP-C1 vector and the PINK1 G309D mutation was created using a QuikChange site-directed mutagenesis kit (Stratagene). PINK1-FL or

PD-linked A339T and E231G PINK1 mutants upregulated TH and DA levels

We demonstrated the overexpression of WT and mutant PINK1 in our SHSY-5Y stable cell lines (Fig. 1A, C, and D). The expression levels of WT and mutant PINK1 in stable cells were at least 20 times higher than those of vector cells (Fig. 1A). Furthermore, we found that stable overexpression of WT PINK1 downregulated TH expression in SHSY-5Y cells (Fig. 1B, C, and E). In contrast, stable overexpression of A339T and E231G PINK1 mutants upregulated TH expression in SHSY-5Y cells (Fig. 1B, C, and E).

Discussion

Two homozygous PINK1 mutations (W437X and G309D) were initially identified and linked to hereditary early-onset PD in 2004 [28]. Subsequently, multiple novel PD-linked mutations or truncations in PINK1 have been reported [29], [30]. Previous studies suggest that single heterozygous PINK1 mutations may lead to the development of late-onset sporadic PD [31], [32], [33]. To date, at least 40 PINK1 heterozygous mutations have been reported and most of these mutants are missense substitutions

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