Neuroscience Letters 399 (2006) 27–32 Proteasomal inhibition hypersensitizes differentiated neuroblastoma cells to oxidative damage Nirit Lev ∗ , Eldad Melamed, Daniel Offen Department of Neurology and Laboratory of Neuroscience, FMRC, Rabin Medical Center, Tel Aviv University, Tel Aviv, Petah-Tiqva 49100, Israel Received 27 June 2005; received in revised form 5 September 2005; accepted 18 September 2005 Abstract Parkinson’s disease (PD) is a multifactorial disease caused by both genetic and environmental factors. Alpha-synuclein is of particular interest in PD since it is a major component of Lewy bodies and mutations in the alpha-synuclein gene were identified in familial PD. Oxidative stress and proteasomal dysfunction are implicated in the pathogenesis of PD but their interactions as well as their effect on aggregates formation are not yet clear. We therefore examined the roles of oxidative stress and proteasomal inhibition on protein aggregates induction in na¨ ıve and neuronally differentiated neuroblastoma SH-SY5Y cells. Neuroblastoma cells were stably transfected with wild type (WT) and A53T mutant alpha-synuclein. Na¨ ıve and transfected cells were exposed to oxidative stress induced by rotenone, SIN-I, FeCl 2, and to proteasomal inhibition by lactacystin. Proteasomal inhibition caused a dose-dependent decrease in viability and induced protein aggregates formation containing alpha-synuclein and ubiquitin. Proteasomal inhibition induced significantly increased alpha-synuclein aggregation in cells expressing mutant alpha-synuclein. Exposure to reactive oxygen species (ROS) combined with proteasomal inhibition increased aggregates formation. Inclusion body formation and cell death of differentiated neuroblastoma cells overexpressing alpha-synuclein can serve as a valuable model for elucidating the molecular components that cause neurodegeneration in PD as well as evaluating pharmacological interventions. © 2006 Published by Elsevier Ireland Ltd. Keywords: Parkinson’s disease; Alpha-synuclein; Ubiquitin–proteasome system; Protein aggregates Parkinson’s disease (PD) is the most common neurodegenera- tive movement disorder. The pathologic hallmark of PD is the appearance of cytoplasmic inclusion bodies, named Lewy bod- ies [4]. All forms of familial and idiopathic PD have Lewy bodies with the exception of the autosomal recessive juvenile-onset type [19]. Lewy bodies contain a heterogeneous mixture of insoluble, filamentous proteins and lipids, including alpha-synuclein, ubiq- uitin, neurofilaments, and oxidized/nitrated proteins [5,7,21,26]. Brain stem and cortical Lewy bodies have numerous features in common including the diameter of their filaments (7–12 nm) and a large proportion of their proteins are alpha-synuclein and ubiq- uitin [19]. Neuronal pathology in PD is associated with oxidative stress, mitochondrial dysfunction, and excitotoxicity but it is not clear how these events contribute to the neurodegenerative pro- cess [9]. ∗ Corresponding author. Tel.: +972 3 9376130; fax: +972 3 9376277. E-mail addresses: niritlev@post.tau.ac.il, niril@hotmail.com (N. Lev), doffen@post.tau.ac.il (D. Offen). Recent evidence suggests that failure of the ubiquitin– proteasome system (UPS) leading to protein accumulation, con- tributes to the degeneration of dopaminergic neurons and Lewy body formation in the substantia nigra pars compacta (SNc) in both familial and sporadic forms of PD [17]. The UPS is primar- ily responsible for degradation of damaged proteins in eukary- otic cells. Impairment of proteolytic activities of 26/20S protea- somes in the SNc of patients with sporadic PD was demonstrated in the post mortem examination of their midbrains [14,15]. In addition, various deletions and point mutations found in patients with familial PD are also related to the UPS: mutations in the parkin gene and ubiquitin C-terminal hydrolase L1 (UCH-L1) [11,24]. Alpha-synuclein is a major component of Lewy bodies in sporadic PD. In addition, in cases of autosomal dominant PD, missense mutations in the gene encoding alpha-synuclein (4q21–q23) have been shown to produce proteins that are prone to misfold and aggregate [6,22]. Mutant alpha-synuclein resists and inhibits proteolysis, and increases the sensitivity of cells to a variety of toxic insults [2,10]. In vitro experiments demonstrated 0304-3940/$ – see front matter © 2006 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2005.09.086