Research reportAccelerated α-synuclein aggregation after differentiation of SH-SY5Y neuroblastoma cells
Introduction
In Parkinson's disease (PD), α-synuclein (α-syn) has been implicated to play central roles in the pathogenesis, since missense mutations in the α-syn gene have recently been demonstrated to be the cause of rare autosomal dominant familial PD [24], [40], and α-syn has been confirmed to be a major constituent of pathological hallmarks including Lewy bodies (LBs) and Lewy neurites in PD [46]. Recent studies indicate that both genetic and environmental factors are important in the pathogenesis of PD [12], [20]. Among a variety of environmental factors potentially toxic to nigral cells, iron is supposed to play an important role in the pathogenesis of PD [18]. It was reported that the concentrations of circulating iron, ferritin and transferrin were significantly lower in PD than in controls [27], whereas iron and its binding protein lactotransferrin were increased in the mesencephalon of patients with PD [18], [26]. In vitro studies showed that ferrous iron not only induced oxidative stress [18] but also facilitated fibril formation of the α-syn [50]. Furthermore, enhanced vulnerability to the ferrous iron in neuroblastoma cells transfected with mutant α-syn suggested close relationships between iron and α-syn metabolism [29].
It was suggested that the processes of α-syn fibrillation and inclusion formation were associated with the neurodegeneration [51]. On the other hand, previous studies demonstrated that, in transgenic mice expressing wild-type or mutant human α-syn, LB-type inclusions was not developed in the nigrostriatal system [13], even though high levels of α-syn protein in dopaminergic cells were achieved [31]. These results suggest that overexpression of α-syn is neither sufficient to cause LB-like inclusions nor to initiate neurodegenerative changes.
To obtain a better understanding of the molecular pathogenesis underlying synucleinopathy, we established a novel cellular model, which can effectively reproduce intracytoplasmic inclusions, by using the combined methods of α-syn overexpression and cellular differentiation.
Section snippets
Expression plasmid construction
To construct α-syn expression plasmids, a set of primer pairs, 5′-TCGTGAGCGGAGAACTGGGAG-3′ and 5′-TCAAGAAACTGGGAGCAAAGAT-3′, was used to amplify the entire coding sequence of α-syn by polymerase chain reaction with template cDNA prepared from human peripheral lymphocytes. The resultant product was gel-purified and ligated into the pGEM-T vector (Promega, Madison, WI). After nucleotide sequencing, the insert wild-type (WT) α-syn cDNA was subcloned into pKF18K vector at SalI and SphI sites and
Overexpression of α-synuclein alone is not sufficient to cause intracytoplasmic inclusions in SH-SY5Y cells
Firstly, to study the effect of cellular differentiation on the formation of α-syn aggregates, we established polyclonal SH-SY5Y cell lines overexpressing WT and mutant human α-syn [32]. Then, these transfectants were sequentially treated with RA and BDNF [9], which induced cell differentiation accompanied by growth retardation and the outgrowth of neuritic processes in transfected cells. After these treatments, small and scattered aggregates were occasionally seen in the cytoplasm of
Discussion
The cellular models for the synucleinopathies postulated thus far can be classified into models using tumor-derived cells [3], [32], [37], [38] or primary-cultured cells [33], [52]. In the former model, cells are continuously dividing and are completely different from postmitotic neuronal cells. Moreover, the cellular populations might be altered during repeated subcultivation. In a primary culture system, it is hard to maintain transformed cells for a substantial period and to obtain
Acknowledgments
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the Nagao Memorial Fund. We also thank to Mr. Brent Bell for reading the manuscript.
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