Elsevier

Brain Research

Volume 1013, Issue 1, 2 July 2004, Pages 51-59
Brain Research

Research report
Accelerated α-synuclein aggregation after differentiation of SH-SY5Y neuroblastoma cells

https://doi.org/10.1016/j.brainres.2004.04.018Get rights and content

Abstract

α-Synuclein (α-syn) is a major component of inclusion bodies in Parkinson's disease (PD) and other synucleinopathies. To clarify the possible roles of α-syn in the molecular pathogenesis of neurodegenerative diseases, we have established a novel cellular model based on the differentiation of SH-SY5Y cells that overexpress α-syn. In the presence of ferrous iron, differentiation of the cells led to the formation of large perinuclear inclusion bodies, which developed from scattered small aggregates seen in undifferentiated cells. The iron-induced α-syn-positive inclusions co-localized largely with ubiquitin, and some of them were positive for nitrotyrosine, lipid, γ-tubulin and dynein. Notably, treatment with nocodazole, a microtubule depolymerizing agent, interrupted the aggregate formation but led to a concomitant increase of apoptotic cells. Therefore, it appears that an intracellular retrograde transport system via microtubules plays a crucial role in the aggregate formation and also that the aggregates may represent a cytoprotective response against noxious stimuli. This cellular model will enable better understanding of the molecular pathomechanisms of synucleinopathy.

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.

References (53)

  • G.E Meredith et al.

    Lysosomal malfunction accompanies alpha-synuclein aggregation in a progressive mouse model of Parkinson's disease

    Brain Res.

    (2002)
  • P.J Muchowski

    Protein misfolding, amyloid formation, and neurodegeneration: a critical role for molecular chaperones?

    Neuron

    (2002)
  • L Narhi et al.

    Both familial Parkinson's disease mutations accelerate alpha-synuclein aggregation

    J. Biol. Chem.

    (1999)
  • R.J Perrin et al.

    Exposure to long chain polyunsaturated fatty acids triggers rapid multimerization of synucleins

    J. Biol. Chem.

    (2001)
  • A.N Pronin et al.

    Synucleins are a novel class of substrates for G protein-coupled receptor kinases

    J. Biol. Chem.

    (2000)
  • E.K Richfield et al.

    Behavioral and neurochemical effects of wild-type and mutated human alpha-synuclein in transgenic mice

    Exp. Neurol.

    (2002)
  • M.A Smith et al.

    Amyloid-beta and tau serve antioxidant functions in the aging and Alzheimer brain

    Free Radic. Biol. Med.

    (2002)
  • M.M Tompkins et al.

    Contribution of somal Lewy bodies to neuronal death

    Brain Res.

    (1997)
  • V.N Uversky et al.

    Metal-triggered structural transformations, aggregation, and fibrillation of human alpha-synuclein. A possible molecular NK between Parkinson's disease and heavy metal exposure

    J. Biol. Chem.

    (2001)
  • W Zhou et al.

    Overexpression of human alpha-synuclein causes dopamine neuron death in primary human mesencephalic culture

    Brain Res.

    (2002)
  • M Zhu et al.

    The association of alpha-synuclein with membranes affects bilayer structure, stability and fibril formation

    J. Biol. Chem.

    (2003)
  • P.K Auluck et al.

    Chaperone suppression of alpha-synuclein toxicity in a Drosophila model for Parkinson's disease

    Science

    (2002)
  • N.F Bence et al.

    Impairment of the ubiquitin-proteasome system by protein aggregation

    Science

    (2001)
  • D.F Clayton et al.

    Synucleins in synaptic plasticity and neurodegenerative disorders

    J. Neurosci. Res.

    (1999)
  • K.A Conway et al.

    Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease

    Nat. Med.

    (1998)
  • W.A Den Hartog Jager

    Sphingomyelin in Lewy inclusion bodies in Parkinson's disease

    Arch. Neurol.

    (1969)
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