Author Affiliations: Sanford-Burnham Medical Research Institute, La Jolla, California (Drs Choo and Zhang and Mr Tang); and State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China (Dr Zhang and Mr Tang).
Mutations in parkin and phosphatase and tensin homolog–induced putative kinase 1 (PINK1) are individually associated with recessive familial forms of Parkinson disease (PD). Parkin is an E3 ligase that promotes ubiquitination of a number of substrates, including itself. In Drosophila, parkin plays essential roles in PINK1-mediated mitochondrial morphology and functions.1,2 We have recently shown that parkin, PINK1, and DJ-1 form an ubiquitin E3 ligase complex (the PPD complex) in which PINK1 increases parkin's ligase activity.3 Parkin levels are dependent inversely on PINK1 levels in cultured mammalian cells.3,4 PINK1 is required for parkin E3 ligase activity during stress conditions. However, regulation of parkin by PINK1 during physiological conditions in mammalian cells remains to be addressed. Here, we demonstrate that parkin levels are increased in PINK1-deficient mouse brains, suggesting that PINK1 plays a critical role in regulating parkin E3 ligase activity and, subsequently, its degradation in vivo.
The parkin-, and PINK1-deficient C57BL/6 mouse lines were generated previously.3,5 All animal procedures were approved by our Institutional Animal Care and Usage Committees. The mouse genotypes were determined by polymerase chain reaction.3,5 Age and sex-matched parkin-deficient (n = 5), wild-type (n = 7), and PINK1-deficient (n = 6) mice were sacrificed (FigureeFigure). Whole-brain lysates were prepared using 2% sodium dodecyl sulfate buffer.3 We separated 25 μg of lysate proteins on 4% to 20% Tris-Glycine gels and immunoblotted with anti-p38/JTV-1 (1:1000 dilution; Proteintech Group, Inc, Chicago, Illinois), anti-α synuclein (1:1000 dilution; Cell Signaling Technology, Inc, Danvers, Massachusetts), anti-parkin, and anti-actin (1:2000 dilution; Sigma-Aldrich, St Louis, Missouri) antibodies. The ratio of parkin or p38/JTV-1 to actin was quantified using ImageJ and analyzed with the unpaired t test with Welch's correction using GraphPad Prism 5 (GraphPad Software, Inc, La Jolla, California).
Parkin and p38/JTV-1 levels in parkin-deficient (parkin KO), wild-type (WT), and PINK1-deficient (PINK1 KO) mouse brains. A, Immunoblotting is shown. Unlike parkin or p38/JTV-1, α-synuclein, another Parkinson disease–related protein, does not show any notable difference in its level between parkin-deficient and wild-type mouse brains. B, Mice genotyping is shown. C, Quantification of normalized parkin level is shown (* P = .01, 2-tailed). D, Quantification of normalized p38/JTV-1 level is shown (* P = .03, 1-tailed).
To comprehend the in vivo function and interaction of mammalian parkin and PINK1, we examined parkin levels in the brains of wild-type, parkin-deficient, and PINK1-deficient mice. As shown in the Figure, A, parkin is undetectable in parkin-deficient mouse brains. In contrast, the steady-state level of parkin is significantly elevated in PINK1-deficient brains compared with those in wild-type brains (P = .01). The increased detection of parkin in PINK1-deficient mouse brains was not notably affected by age or sex (eFigure). The results suggest that PINK1 plays a critical role in regulating parkin levels in vivo. One potential explanation for the increased parkin protein in PINK1-deficient brains is that parkin's ubiquitin E3 ligase activity is reduced without PINK1.3 To address this hypothesis, we examined levels of p38/JTV-1, another parkin substrate, in these samples. Consistent with a previous article,6 our results revealed increased p38/JTV-1 levels in parkin-deficient mouse brains compared with wild-type mouse brains. In addition, p38/JTV-1 levels were shown to be greater in PINK1-deficient brains than in wild-type brains (P = .03, 1-tailed) (Figure, D) despite an increased level of parkin in PINK1-deficient brains. Quantitative real-time polymerase chain reaction indicates that parkin messenger RNA levels remain similar in brains of wild-type and PINK1-deficient mice (data not shown). The results suggest that PINK1 plays a critical role in the degradation of parkin substrates parkin and p38/JTV-1.
We have shown in this study that the steady-state level of parkin protein is regulated by PINK1 in vivo. The increased parkin level in PINK1-deficient mouse brains is likely caused by suppressed parkin E3 ligase activity in the absence of PINK1. Consistent with this notion, the level of p38/JTV-1 is also increased in brains of both parkin- and PINK1-deficient mice. Furthermore, the results of this study support our previous finding that parkin/PINK1/DJ-1 complex functions as an E3 ligase complex to promote degradation of parkin substrates and that PINK1 plays a crucial role in regulating parkin E3 ligase activity.3 p38/JTV-1 is a parkin substrate shown to accumulate in the ventral midbrain/hindbrain of parkin-deficient mice.6 By showing an increase in p38/JTV-1 levels in brains of an additional line of parkin-deficient mice, we provide further supporting evidence that p38/JTV-1 is an authentic parkin substrate. Additionally, the results suggest that parkin is the substrate of itself in vivo. Parkin and PINK1 interact in Drosophila and mammalian cells to either maintain mitochondrial function or promote degradation of unfolded proteins via the ubiquitin proteasomal pathway.1- 3 The results of this study offer in vivo evidence to support parkin/PINK1 E3 ligase function in mammalian cells.
Correspondence: Dr Zhang, Sanford-Burnham Medical Research Institute, 10901 N Torrey Pines Rd, La Jolla, CA 92037 (firstname.lastname@example.org).
Accepted for Publication: December 29, 2010.
Author Contributions:Study concept and design: Choo, Tang, and Zhang. Acquisition of data: Choo. Analysis and interpretation of data: Choo and Zhang. Drafting of the manuscript: Choo. Critical revision of the manuscript for important intellectual content: Tang and Zhang. Statistical analysis: Choo. Obtained funding: Choo and Zhang. Administrative, technical, and material support: Tang and Zhang. Study supervision: Zhang.
Financial Disclosure: None reported.
Funding/Support: This study was supported by National Institute of Health grants RO1 NS057289 and PO1 ES016738 (Dr Zhang); California Institute for Regenerative Medicine grants RL1-00682-1 (Dr Zhang); Chinese Natural Science Foundation grants (Dr Zhang); and the American Parkinson Disease Association (Dr Choo).
Additional Contributions: We thank Rena Baek, PhD, for editing the manuscript.
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