Biochemical and Biophysical Research Communications
Parkin protects dopaminergic neurons from excessive Wnt/β-catenin signaling
Introduction
The molecular mechanisms underlying the degeneration of dopaminergic (DA) neurons causing Parkinson’s disease (PD) remain elusive but increasing evidence points to the importance of cell cycle deregulation in post-mitotic neuronal cell death [1], [2]. Interestingly, cell cycle proteins such as cyclin-dependent kinase 5 and cyclin E have been found to accumulate in PD patient brains [3], [4]. To ensure tight temporal expression, the turnover of many cell cycle components is regulated through rapid ubiquitylation and subsequent proteasomal degradation events [5]. Dysfunction of the ubiquitin proteasome pathway has been linked to PD through the identification of mutations in key enzymes, like Parkin, in familial forms of the disease, and the presence of Lewy bodies (aggregation bodies of misfolded proteins). Parkin is an E3 ubiquitin protein ligase thought to regulate the turnover of misfolding- and aggregation-prone proteins [6], [7] and contains two RING finger motifs [8]. Like other RING proteins, Parkin has been reported to act within a multiprotein Skp1-Cullin1-F-box/WD repeat protein (SCF) ligase complex, together with the F-box/WD repeat protein Fbw7 [4]. Interestingly, Lewy bodies are often absent in Parkin-related PD, suggesting that Parkin may assist in maintenance of cell integrity by facilitating inclusion of dysfunctional proteins into aggregation bodies [9]. In the central nervous system, Parkin expression is primarily restricted to post-mitotic neurons [10] where it appears to protect post-mitotic neurons against proliferative signals and cell cycle re-entry attempts. Accumulation of cyclin E in stressed post-mitotic neurons can be counteracted by overexpression of Parkin, resulting in ubiquitylation and subsequent targeting of cyclin E for degradation [4]. Collectively, these reports suggest that proliferative signals and Parkin play opposing roles and negatively regulate each other.
The Wnt family of secreted glycoproteins has been reported to regulate patterning, cell fate determination, proliferation, and differentiation during CNS development [11]. The canonical Wnt signaling pathway acts through the stabilization of β-catenin. In the absence of Wnt, cytosolic β-catenin is constantly phosphorylated by a protein complex consisting of glycogen synthase kinase-3β (GSK-3β), axin, adenomatous polyposis coli, and casein kinase 1 [12]. The phosphorylation of β-catenin triggers a rapid ubiquitylation response by the SCF/β-TrCP ligase [13], resulting in proteasome-mediated degradation, as reported for the p53 inducible gene seven in absentia homolog 1 (Siah-1), thereby linking apoptosis signaling and cell cycle control [14].
In this report, we examined the role of Parkin and its effects on the Wnt/β-catenin pathway in post-mitotic dopaminergic neurons.
Section snippets
Material and methods
COS7 and MN9D cultures. COS7 cells were grown in D-MEM medium containing 10% fetal calf serum, 1% glutamine, and 0.8% gentamicin and transfected with Lipofectamine 2000 (all from Invitrogen) according to manufacturer’s recommendation. COS7 cells were treated with 50 ng/ml Wnt3a (R&D systems) for 9 h. Control cells were treated with 0.05‰ CHAPS.
MN9D cells were grown as described [15] and transfected using polyethylenimine (PEI) at 0.8 μg/ml in PBS (PEI, 25 kDa, Sigma–Aldrich). Briefly, PEI was mixed
Parkin interacts with endogenous β-catenin
To study the role of Parkin in DA cells, we transfected full length human parkin into proliferating MN9D cells, a dopaminergic cell line, and found that the Parkin protein is predominantly in the cytoplasm and overlaps with endogenous cytoplasmic β-catenin (Fig. 1A). To address if the two proteins interact, a glutathione-s-transferase (GST) pull-down assay was performed using Parkin protein N-terminally fused to GST (GST-Parkin), produced in E. coli[16]. Equal amounts of GST or GST-Parkin were
Discussion
Despite efforts to unravel the molecular mechanisms underlying the degenerative process in PD, many issues remain unresolved. Studies have illustrated that Parkin can influence events such as ubiquitylation and aggregation of proteins. However few of the identified Parkin substrates in vitro are elevated in parkin null mice [23], [24]. Our finding that β-catenin levels are consistently and specifically upregulated in the midbrains of parkin null mice thus suggests that Parkin indeed regulates
Acknowledgments
We thank Dr. C. Parish for fruitful discussions and assistance in adult tissue preparation, Dr. C. Hampe for advice and assistance in the biochemistry experiments, Drs. J. Staropoli and A. Abeliovich for experimental advice and materials, Drs. N. Dantuma and K.M. Sousa for critical reading of the manuscript, and K. Lundgren for PEI reagent. S37A β-catenin was a gift from Dr. S. Byers, the MN9D were from A. Heller and H. Simon. This work was supported by EuroStemCell (EU), VR, Swedish Foundation
References (30)
- et al.
Activation of cell-cycle-associated proteins in neuronal death: a mandatory or dispensable path?
Trends Neurosci.
(2001) - et al.
Parkin is a component of an SCF-like ubiquitin ligase complex and protects postmitotic neurons from kainate excitotoxicity
Neuron
(2003) - et al.
How the cyclin became a cyclin: regulated proteolysis in the cell cycle
Cell
(1999) - et al.
RING finger proteins: mediators of ubiquitin ligase activity
Cell
(2000) - et al.
Differential expression and tissue distribution of parkin isoforms during mouse development
Brain Res. Dev. Brain Res.
(2001) - et al.
Siah-1, SIP, and Ebi collaborate in a novel pathway for beta-catenin degradation linked to p53 responses
Mol. Cell
(2001) - et al.
Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion
Brain Res.
(1991) - et al.
Dynamic temporal and cell type-specific expression of Wnt signaling components in the developing midbrain
Exp. Cell Res.
(2006) - et al.
Serine phosphorylation-regulated ubiquitination and degradation of beta-catenin
J. Biol. Chem.
(1997) - et al.
Wnt signaling in lung cancer
Cancer Lett.
(2005)
Modulation of beta-catenin phosphorylation/degradation by cyclin-dependent kinase 2
J. Biol. Chem.
Kainic acid-induced apoptosis in cerebellar granule neurons: an attempt at cell cycle re-entry
Neuroreport
Cortical and brainstem-type Lewy bodies are immunoreactive for the cyclin-dependent kinase 5
Am. J. Pathol.
Parkin ubiquitinates the alpha-synuclein-interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease
Nat. Med.
Familial Parkinson disease gene product, parkin, is a ubiquitin–protein ligase
Nat. Genet.
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