Amyloid formation and disaggregation of α-synuclein and its tandem repeat (α-TR)

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Abstract

The aggregation of α-synuclein is clearly related to the pathogenesis of Parkinson’s disease. Therefore, detailed understanding of the mechanism of fibril formation is highly valuable for the development of clinical treatment and also of the diagnostic tools. Here, we have investigated the interaction of α-synuclein with ionic liquids by using several biochemical techniques including Thioflavin T assays and transmission electron microscopy (TEM). Our data shows a rapid formation of α-synuclein amyloid fibrils was stimulated by 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [BIMbF3Im], and these fibrils could be disaggregated by polyphenols such as epigallocatechin gallate (EGCG) and baicalein. Furthermore, the effect of [BIMbF3Im] on the α-synuclein tandem repeat (α-TR) in the aggregation process was studied.

Research highlights

► Formation of the α-synuclein amyloid fibrils by [BIMbF3Im]. ► Disaggregation of amyloid fibrils by epigallocatechin gallate (EGCG) and baicalein. ► Amyloid formation of α-synuclein tandem repeat (α-TR).

Introduction

Parkinson’s disease (PD) is characterized by degeneration of dopaminergic neurons in the substantia nigra and by deposits of cytoplasmic inclusions (Lewy bodies). α-Synuclein, which is involved in neuronal plasticity, stabilization of lipid membranes, neurotransmitter release, and protein networks, is the major building block of the pathological fibrillar deposits within Lewy bodies [1], [2], [3]. α-Synuclein is a small (14 kDa) low-structured protein, possessing several imperfect KTKEGV repeats in the N-terminal portion (a.a. 1–95), and an highly acidic C-terminal portion (a.a. 96–140). Specifically, the central region (a.a. 61–95) is the core region that is mainly responsible for fibril formation. For example, the alterations of several hydrophobic amino acids in this region can significantly change the polymerization into amyloid [4], [5], [6]. Using several biophysical techniques, α-synuclein was shown to undergo conformational changes from its low-structured native state to the highly crossed β sheet-rich conformation of amyloid fibrils [7], [8], [9]. Additionally, the conversion of soluble α-synuclein into insoluble aggregates is believed to be a key event in the pathogenesis of PD and related diseases. Interestingly, in contrast to α-synuclein, β- and γ-synuclein, which are not related to neurodegenerative diseases, cannot readily form amyloid fibrils in vitro [6], [8]. Therefore, a comprehensive understanding of the mechanism of amyloid fibril formation at the molecular level is very important to elucidate the pathogenesis of PD and for the development of clinical treatment and diagnostic tools.

Although increasing evidences suggest that a prefibrillar intermediate, not amyloid fibril, is mainly responsible for neuronal cell death, the nature of the neurotoxic species and its mode of action still remain largely unknown. To date, therapeutic strategies are mainly focused on the inhibition of the amyloid formation process, as early as its initial stage, by using organic molecules, heat shock proteins and antibodies [10], [11], [12]. Here, we suggest alternative strategies which avoid cytotoxic intermediates by using ionic liquids (ILs) that could stabilize the formation of amyloid fibrils [13], [14]. As a first step, we have investigated the interaction of α-synuclein with ionic liquids, using several biochemical techniques, including Thioflavin T assays and transmission electron microscopy (TEM). We showed a rapid formation of α-synuclein amyloid fibrils stimulated by 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [BIMbF3Im]. Furthermore, the effect of [BIMbF3Im] on the α-synuclein tandem repeat (α-TR) in the aggregation process was studied. Our data shows that these fibrils could be disaggregated by the polyphenols such as epigallocatechin gallate (EGCG) and baicalein. Therefore, use of ionic liquids such as [BIMbF3Im] could represent an alternative strategy for attacking neurodegenerative diseases. Considering the enormous chemical variability of ionic liquids, this study will encourage further investigations on the effects of ionic liquids on the neurodegenerative diseases such as Parkinson’s disease (PD).

Section snippets

Expression and purification of α-synuclein and its related proteins

Wild type α-, β-, and γ-synuclein were expressed and purified as previously described [7]. γ-synuclein plasmid was a kind gift from Dr. J. Liu (Department of Veterans Affairs, Palo Alto Health Care System, USA.). For α-synuclein tandem repeat (α-TR), codon optimization of the complete ORF (840 bp) of the gene sequence was performed, which was then commercially synthesized (Genscript Corp., Piscataway, NJ). The synthesized α-TR gene was cloned into pET-21a using NdeI and HindIII restriction sites

Results

Abnormal aggregation of α-synuclein is clearly related to the etiology of Parkinson’s diseases (PD) and current studies are mainly focused on the suppression or inhibition of the formation of cytotoxic intermediates [2], [5], [8]. In a previous report, we have shown that ionic liquids could assist and stabilize the fibril formation process of α-synuclein [13]. Here, we examined the effects of two representative ionic liquids, 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [BIMbF3

Discussion

Amyloid deposits of α-synuclein are clearly linked to the pathogenesis of Parkinson’s disease (PD), but little information is known concerning the nucleation steps and cytotoxic forms of α-synuclein. Recent studies suggest that α-synuclein protofibrillar intermediates, not amyloid fibrils, are the pathogenic species in neuronal cell death and neurodegeneration [2], [3], [23], [24]. Therefore, inhibition or suppression of cytotoxic intermediates, as early as its nucleation steps, is highly

Acknowledgments

This work was supported by a Korean Research Foundation Grant funded by the Korean Government (KRF-2009-0089832) and by a 2008 Ajou University research fellowship to T.D.K. S.L. was supported through the Chronic Inflammatory Disease Research Center at Ajou University (KOSEF-R13-2003-019).

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    Present address: Science Education Division, Korea Institute for Curriculum and Evaluation.

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