Intrtoduction

Intracellular proteins undergo numerous effects under physiological conditions. α-Synuclein (α-Syn) is the major component of Lewy bodies, found in the brains of patients with dementia,¹ and reportedly adopts a compact structure within cells.² a-Syn presumably experiences molecular crowding, interactions with other molecules, and oligomerization/fibrillization under physiological conditions (Fig. 1). We sought to deliver ¹³C-labeled proteins into the mouse brain, to investigate them by ¹³C MRS. We previously detected ¹³C-labeled α-Syn embedded into an agarose gel by ¹³C MRS,³ which confirmed that protein signals can be detected by MRS. Recent NMR studies have observed stable isotope-labeled proteins delivered into cultured cells.^2,4 We envisaged that cultured cells containing delivered target proteins can be used to test the feasibility of the MRS detection under more in vivo-like conditions. Therefore, we explored the delivery of isotope labeled proteins into cultured cells and the observations of the delivered proteins by NMR and MRS.

Materials and Methods

HeLa S3 cells were treated with a pore-forming toxin, streptolysin O⁵ (20 ng/ml), and suspended in a solution containing ¹⁵N-labeled α-Syn (1 mM). After resealing by calcium chloride, 2.0 × 10⁷ cells were suspended in 200 ml of Dulbecco's Modified Eagle's Medium, containing 25 mM HEPES (pH 7.5), 20% D₂O, and 35% Redigrad (GE Healthcare), and then transferred to an NMR tube. The ¹H-¹⁵N HSQC experiment was recorded at 10^oC, using a 14.1 T NMR spectrometer (Bruker Biospin). The ¹³C-labeled α-Syn was embedded at 50 mM in Mebiol gel (Mebiol), and the gel was placed within a 2.5 ml syringe (Terumo). The MRS experiment was performed using a 7.0 Tesla Bruker BioSpec 70/20 scanner and a ¹³C mouse brain cryogenic coil (Bruker BioSpin).The 2.5 ml syringe was directly attached to the coil. Non-selective ¹³C MRS was recorded. MRS was also recorded with a syringe filled with only the Mebiol gel, which was used to subtract background signals. The cell viability was assessed by trypan blue staining. HeLa S3 cells were embedded in an alginate gel.

Results

The ¹⁵N-labeled a-Syn was previously embedded in an agarose gel, but no significant changes were detected in the HSQC experiment, as compared to that in buffer (Fig. 2A).¹ In contrast, when ¹⁵N-labeled α-Syn was delivered into HeLa S3 cells, the resulting spectrum was quite different from that in the buffer (Fig. 2B). Some peaks exhibited significant chemical shift changes and line-broadening, and were totally different from those obtained in agarose (Fig. 2C), presumably reflecting physiological effects that cannot be reproduced in the agarose gel. In our previous study, we found that the ¹³C signals of α-Syn in buffer and an agarose gel could be detected by MRS, with comparable quality to NMR (Fig. 3A, B, C). However, cells contain lipids and small molecules that produce ¹³C background signals, making it difficult to detect the α-Syn signals. We thus tested whether the α-Syn signals can be detected as a difference spectrum. α-Syn was embedded in the Mebiol gel, a copolymer of hydrophobic and hydrophilic polymers, as a proxy for biomolecules. We placed the gel in a 2.5 ml syringe and performed the ¹³C MRS experiment. After subtracting the spectrum of the Mebiol gel in the absence of α-Syn from that in the presence of α-Syn, the ¹³C signals of a-Syn were successfully detected even in the Mebiol gel (Fig. 3D). Next, we constructed a system for observing cultured cells by MRI. In this system, the cells are placed into a tube at a high density (~10⁸ cells/ml), leading to nutrient depletion. Therefore, a bioreactor system,⁶ which perfuses the cells during the experiment, was constructed. In this system, the cells are embedded in an alginate gel and placed in a 2.5 ml syringe, with one end connected to a syringe pump and the other linked to a waste receptacle. To keep the cells in the syringe, the cells were entrapped within a gel, which was placed in the syringe (Fig. 4A). We constructed this system and confirmed that the viability of the cells was improved by the perfusion (Fig. 4B).

Diiscussion

α-Syn delivery into cultured cells allows it undergo numerous physiological phenomena that cannot be reproduced in an agarose gel. We plan to perform MRS experiments on cells containing α-Syn, delivered using the perfusion system. In the in-cell NMR and MRS experiments, the cell density in the sample space is on the order of 10⁸ cells/ml. Considering that the total number of neuronal cells in the human brain is on the order of 10¹¹ cells and the volume of the brain is 1,500 ml, the cell density in the NMR and MRS experiments matches that of the brain regions. Thus, the MRS detection of proteins delivered into cultured cells serves as a milestone for achieving the in vivo detection by MRS.

Acknowledgements

We thank Prof. Shimada and Dr. Nishida (Tokyo University) for advice regarding the in-cell NMR experiments.

References

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