Introduction

α-Synuclein (α-Syn) is a 140-amino acid protein, and the major component of the Lewy bodies found in the brains of patients with Parkinson’s disease and Dementia with Lewy bodies. α-Syn changes from a monomer to a soluble oligomer. An in vitro study reported that an α-Syn oligomer adopts an α-helical structure and exhibits neurotoxicity [1]. However, the structure of the α-Syn oligomer occurring in vivo is unclear. An in-cell NMR study demonstrated that α-Syn delivered into neuronal cells exists as a disordered monomer [2]. The goal of our study is to investigate the in vivo structure of the α-Syn oligomer, by introducing ¹³C-labeled α-Syn into the brains of mice and detecting the ¹³C signals of the α-Syn oligomer by a ¹³C CEST (Chemical exchange saturation transfer) experiment [3] with a ¹³C cryogenic probe [4]. To perform the CEST experiment, the in vivo detection of the ¹³C signals from the α-Syn monomer is required. The intracellular concentration of α-Syn in neurons is 5 to 50 μM [5, 6]. To generate a phantom that mimics a physiological environment, an agarose gel can be used [7]. The efficiency of the CEST phenomenon from the oligomer to the monomer depends on the exchange rate between them. Previous reports on the in vitro oligomerization of α-Syn in Gly-NaOH, sodium phosphate, and Tris-HCl buffers described differences in the resulting final products [1, 8, 9], suggesting their different oligomerization kinetics.

Purpose

We aimed to detect the ¹³C signals of α-Syn by an MRS experiment, using an agarose phantom that mimics a tissue environment. We also attempted the in vitro oligomerization of α-Syn in different buffer conditions, to examine whether the monomer-to-oligomer exchange of α-Syn can be controlled by changing the solvent conditions.

Methods and Materials

Uniformly ¹⁵N- and ¹³C/¹⁵N-labeled α-Syn samples were produced by an E. coli expression system. The agarose gel phantom was prepared by adding 10 mg agarose powder to 1.0 ml of 50 μM uniformly ¹³C/¹⁵N-labeled α-Syn solution, in 50 mM sodium phosphate buffer (pH 7.4) containing 0.02% sodium azide, dissolving it by heating to 100ºC, and then cooling and solidifying it in a 1.5 ml tube. The ¹³C MRS spectrum was acquired for the agarose phantom, using a 7.0T MRI scanner (Bruker BioSpec 70/20) equipped with a mouse brain ¹³C cryogenic probe (Bruker BioSpin) [4]. The ¹³C MRS measurements were performed without voxel selection (acquisition time: 163 msec, number of averages: 4,096, TR: 1 sec). The in vitro oligomerization of α-Syn was performed using 0.3 mM U-¹⁵N-labeled α-Syn dissolved in three different buffers: (i) 20 mM Gly-NaOH (pH 7.4), (ii) 50 mM sodium phosphate (pH 7.4), and (iii) 50 mM Tris-HCl, 150 mM KCl (pH 7.5). The three α-Syn solutions were incubated at 37°C, with shaking at 50 rpm. ¹H-¹⁵N HSQC spectra were recorded on the three samples at 0, 48 and 129 hr of incubation, using a 14.1T NMR machine (Bruker BioSpin).

Results

The ¹³C signals of α-Syn were successfully detected by MRS after 1 hr of accumulation, using the agarose phantom (Fig. 1). For a comparison, the ¹³C NMR spectrum was recorded on the uniformly ¹³C/¹⁵N labeled sample, which exhibited an almost identical spectral pattern to that of the MRS experiment (Fig. 2). The rates of the oligomerization were significantly different in the in vitro oligomerization experiments. At 48 hr of incubation, almost all signals of α-Syn were broadened beyond detection in the Tris-HCl buffer, and those in the sodium phosphate buffer were broadened to some extent. At 129 hr of incubation, the signals of the C-terminal ~30 residues were detectable in the Gly-NaOH buffer, and a smaller number of signals were detected in the sodium phosphate buffer (Fig. 3). The distinct spectral changes upon the incubations suggested that their oligomer-to-monomer kinetics were different.

Discussion

The intracellular half-life of α-Syn was reported to be ~50 hr [2]. As the ¹³C signals of α-Syn were detectable in the 1 hr experiment, the time-course of the α-Syn oligomerization can be monitored. The ¹³C signals derived from the α-Syn monomer serve as the target of observation in the ¹³C CEST experiment. These ¹³C signals can readily be assigned by a conventional solution NMR assignment method. However, many of the ¹³C signals of the protein overlap each other on the ¹³C 1D spectrum. To detect only the signals of interest, the use of a residue- and site-specific ¹³C labeling method is promising. As a future prospect, ¹³C-labeled α-Syn will be delivered into nerve cells, which will then be implanted into mouse brains for in vivo ¹³C MRS experiments.

Acknowledgements

This work was supported in part by the Grant-in-Aid for Challenging Exploratory Research (JP16K15110) (to H.T.) from the Japan Society for the Promotion of Science.