A novel link between the conformations, exposure of specific epitopes, and subcellular localization of α-synuclein

https://doi.org/10.1016/j.bbagen.2015.09.006Get rights and content

Highlights

  • We identified the specific epitopes of α-Syn antibodies.

  • We classified the α-Syn conformations on the basis of the specific epitope.

  • We showed association between conformation and subcellular localization of α-Syn.

Abstract

Background

Genetic studies and the abundance of alpha-synuclein (α-Syn) in presynaptic terminals suggest that α-Syn plays a critical role in maintaining synaptic vesicle pools. However, there are still few experimental tools for elucidating its physiological roles.

Methods

Unexpectedly, we detected various cellular distribution patterns of endogenous α-Syn by immunofluorescence assays (IFAs). To provide new molecular insights into α-Syn research, we identified associations between epitopes, conformations, and subcellular localization of α-Syn and categorized them.

Results

The α-Syn exposing Y125 was found to coexist with F-actin at the edge of the cells, including the plasma membrane. α-Syn conformations exposing P128 or both F94 and K97 were partly localized to the mitochondria. These results indicate that various conformations of α-Syn are associated with specific subcellular localizations. Intriguingly, we demonstrate for the first time that the phosphorylated α-Syn at Ser129, also known as a Parkinson's disease (PD)-causing form, is targeted to the mitochondria.

Conclusions

Our study showed that different subcellular distribution patterns of α-Syn reflect the existence of various α-Syn conformations under normal conditions.

General significance

This study provides novel clues for deciphering the physiological function of α-Syn in connection with subcellular localization. Dissecting the specific α-Syn conformations may lead to useful strategies in PD therapy and diagnosis.

Introduction

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease [1], [2]. It is characterized by cell death of dopaminergic neurons and accumulation of intracellular protein aggregates, known as Lewy bodies (LBs) [3], [4], [5]. Alpha-synuclein (α-Syn) is one of the main causative proteins in PD and the main component of LBs [6], [7], [8], [9], [10]. Biochemical studies have shown that α-Syn normally adopts over 20 different unstructured conformations [11], [12], raising the possibility that conformational changes exposing hydrophobic or the C-term region are intimately associated with the progression of PD [13], [14], [15], [16]. Many in vitro biophysical and biochemical studies have shown that conformational states of α-Syn dynamically change depending on environmental conditions [17], [18], [19], [20]. These data suggest that the dynamic nature of α-Syn is due to its unique structure, which includes several amino acids with negative charges in the C-terminal acidic region and highly hydrophobic residues in the NAC region [21], [22], [23], [24]. However, the conformational diversity of α-Syn in in vivo systems and the roles of the various conformations of α-Syn have not yet been elucidated.

Several immunohistochemical studies using α-Syn antibodies (Abs) have reported different distribution patterns of endogenous α-Syn in the brain tissues of patients with PD and various cell lines [25], [26]. Consistent with previous studies, we have also observed different distribution patterns of α-Syn in cells immunostained with commercially available α-Syn Abs, although these Abs can recognize α-Syn as the same antigen (Ag) (Fig. 1). Immunostaining patterns rely on antibody (Ab)-recognition of a specific epitope on an antigen (Ag) [27], [28]. Because most proteins have only a few fixed conformations, a similar immunostaining pattern is detected for Abs recognizing different epitopes on the same target protein [29]. Taking these findings together, we suggest that immunofluorescence staining and immunological studies using α-Syn Abs could provide a clue to the association between various conformations of α-Syn and their cellular distribution patterns.

In the present study, using immunological and 3D structure image analysis, we classified the conformations of α-Syn following the exposure of different epitopes on the hydrophilic surface of α-Syn and identified distinct associations between conformations and subcellular localization patterns of α-Syn in cells. Interestingly, we found that phosphorylated α-Syn, which is closely associated with the pathogenesis of PD and carries additional negative charges in the C-terminal acidic region, was partly localized in the mitochondria. Collectively, our results imply that endogenous α-Syn displays conformational plasticity in a subcellular environment-dependent manner and that its subcellular fate is determined by its conformational dynamics. Our data provide clues for understanding the physiological roles of α-Syn and the molecular pathogenesis of α-Syn-associated PD as well as for diagnosis of PD.

Section snippets

Antibodies and chemicals

All chemicals were purchased from Sigma Chemical Co. unless otherwise stated. Immunological assays were performed with anti-α-Synuclein (211) monoclonal Ab (Syn125 mAb, Catalog No. sc-12767, Santa Cruz Biotechnology, Inc.), anti-α-Synuclein (C-20)-R polyclonal Ab (Syn128 pAb, Catalog No. sc-7011-R, Santa Cruz Biotechnology, Inc.), and anti-α-Synuclein monoclonal Ab (Syn97, Catalog No. 610786, BD Transduction Laboratories) (Supplementary Table S2). Anti-phosphorylated α-Synuclein monoclonal Ab

Accurate identification of epitopes of α-Syn Abs as the first step toward characterizing subcellular distribution patterns of α-Syn

To assess amino acid (aa) sequence similarity in the synuclein family consisting of α-Syn, β-Syn, and γ-Syn, we aligned three mammalian α-Syn orthologs and human β-Syn and γ-Syn with the ClustalW2 program (Supplementary Table S1). α-Syn was highly conserved between mammalian species including human, mouse, and rat, showing 95% aa sequence identity [31]. A comparison among the three human synuclein family members showed that the N terminus of α-Syn has 90% and 77% identity with that of β-Syn and

Acknowledgments

This work was supported by National Research Foundation of Korea (NRF) Grant funded by the Korean Government: [(MSIP) (NRF-2010-0027963)], [(MEST) (NRF-2014R1A1A2053568)], [(MSIP) (NRF-2015M2B2A9031726)], and the financial support of the Catholic Medical Center Research Foundation made in the program year of 2013 (CMCRF-2013). We thank Integrative Research Support Center of Catholic University for the excellent technical assistance in confocal image processing.

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    These authors contributed equally to this work.

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