Regular articleAdenosine monophosphate-activated protein kinase overactivation leads to accumulation of α-synuclein oligomers and decrease of neurites
Introduction
Aging and exposure to neurotoxins are considered the most prominent risk factors for development of idiopathic Parkinson disease (PD) (Andersen et al., 1999; Calne and Langston, 1983; Chu et al., 2002). Ample evidence supported that these 2 factors can cause mitochondrial dysfunction, which plays a central role in PD pathogenesis (Larsson, 2010; Wang et al., 2011; Zhu and Chu, 2010), and that mitochondria impairment can lead to elevated lactate levels (Dykens et al., 2008; Ross et al., 2010; Weissman et al., 2008). Previous studies have shown increased lactate levels in aging brain (Ross et al., 2010), in PD-affected subjects when compared with age-matched controls (Bowen et al., 1995), and in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) (Koga et al., 2006), which is a neurotoxin known to cause PD symptoms and mitochondria dysfunction (Przedborski et al., 2004). Moreover, it has been noted that α-synuclein (α-syn) levels increase in aging brain (Chu and Kordower, 2007; Jellinger, 2004; Li et al., 2004) and in MPTP-treated animals (Kowall et al., 2000; Vila et al., 2000). It remains unknown whether elevation of lactate levels has an effect on α-syn accumulation/aggregation.
Previous studies have demonstrated that lactic acid can upregulate the activity of adenosine monophosphate-activated protein kinase (AMPK) (Chen et al., 2010a). AMPK is a serine-threonine kinase protein complex and a central regulator of cellular energy homeostasis (Hardie, 2011). Its activity can also be regulated by physiological stimuli, independent of cell energy charge, including hormones, and nutrients (Chen and Downs, 2008; Towler and Hardie, 2007). AMPK consists of α, β, and γ subunits. Among them, the α subunit displays catalytic activity. Phosphorylation of Thr residue at amino acid 172 in α subunit is essential for AMPK activation to function as a protein kinase (Woods et al., 2000).
Recently, cell-based studies have demonstrated that incubation of cultured cells with lactic acid induces aberrant amyloid precursor protein processing to increase amyloid-β40 and 42 peptides (Xiang et al., 2010) and enhances AMPK activation (Chen et al., 2010a). Besides, H2O2 treatment of SH-SY5Y cells was shown to cause not only oxidative stress but also AMPK activation, and such oxidative stress was protected by decreasing AMPK level via gene knockdown (Chen et al., 2010b). Moreover, increased AMPK activation/levels were found in cultured cells treated with MPTP (Choi et al., 2010). In view of these findings and aging being a prominent risk factor for PD, we hereby investigated whether dysregulation of AMPK activation has a role in development of synucleinopathy.
To examine this issue we used experimental models such as differentiated dopaminergic neuroblastoma cells 3D5 (Takahashi et al., 2007) and mouse primary neurons which overexpress wild type human α-syn by the tetracycline-off (TetOff) inducible mechanism and viral infection, respectively. We upregulated AMPK activity in these cultured cells by exposing the cells to lactate or overexpressing active form of AMPK regarded as AT-AMPK, downregulated AMPK by overexpressing a dominant negative (DN) form of AMPK referred to as DN-AMPK (Za'tara et al., 2008), or expressing short hairpin RNA (shRNA) of AMPK α units (for knockdown) referred to as KD-AMPK. We also determined whether the pattern of activated AMPK distribution in cultured cells and human brain tissues can be affected by lactate treatment and synucleinopathy, respectively.
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Cell models
α-Syn transfectant 3D5 was derived from the human neuroblastoma BE2-M17D cell line (Takahashi et al., 2007). 3D5 cells express wild type human α-syn and display neuronal phenotypes on TetOff induction and incubation with retinoic acid (RA), respectively (Ko et al., 2008). Under certain experimental conditions, 3D5 cells are capable of accumulating oligomeric α-syn and form aggregates. Cultures of 3D5 were maintained in Dulbecco's modified Eagle medium (DMEM)/10% fetal bovine serum with 2 μg/mL
Exposure to lactate causes accumulation of α-syn in 3D5 cells in a time- and concentration-dependent manner
To determine the effects of lactate exposure on α-syn accumulation, we treated 3D5 cultures with 20 mM sodium lactate (referred to as lactate or LA) for 3 or 6 days or with different concentrations (0, 4, 8, 16, or 24 mM) of lactate for 3 days. Before the treatment, 3D5 cultures were differentiated with RA and induced to express α-syn for 4 days. Sibling cultures without lactate treatment were included as controls. As demonstrated in previous studies (Jiang et al., 2010; Ko et al., 2008),
Discussion
Previous genetic studies have shown that some patients with familial PD overexpress wild type α-syn because of multiplications of its gene SNCA and that the clinical phenotype correlates with such upregulation (Chartier-Harlin et al., 2004; Nishioka et al., 2006; Singleton and Gwinn-Hardy, 2004; Singleton et al., 2003). Moreover, the risk to develop PD increases with polymorphisms within the SNCA promoter (Chiba-Falek et al., 2005; Hadjigeorgiou et al., 2006; Pals et al., 2004). These findings,
Disclosure statement
All authors have no actual or potential conflicts of interest.
All animal work was approved by the Institutional IACUC.
Acknowledgements
The authors thank Dr Rachel Hertz and Dr Thomas C. Südhof for their generous gifts of lentiviral plasmids carrying AT-AMPK, DN-AMPK, and Myc tagged α-syn. This study was supported by the National Institute of Health (P50-NS40256), the Mayo Foundation (Yen) and the Mangurian's Foundation (Yen).
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