Effect of magnetic modulation of mitochondrial voltage-dependent anion channel 2 against beta-amyloid induced neurotoxicity

Abstract

The magnetic capture of mitochondrial VDAC2 with bacterial magnetic particles (BMPs) conjugated VDAC2 antibody (BMPs–Ab) significantly decreased the expressed intracellular calcium levels and neurotoxicity induced by Aβ. This magnetic modulation of mitochondrial VDAC, playing a key role in various Ca²⁺ flux pathways, should provide attractive targets for the future development of AD treatments.

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Mitochondria are the governors of both cell life (e.g. energy generation) and cell death. A slight regulation of both of these functions occurs at the level of the outer membrane and results in the control of the flow of metabolites and the release of intermembrane space proteins into the cytosol. The VDAC proteins, which are pore-forming proteins predominantly found in the outer mitochondrial membrane, are major pathways for anions, cations, ATP, Ca²⁺ and metabolites that flux through the outer membrane.^1,2 These can be regulated in many ways, and the integration of these regulatory inputs allows mitochondrial metabolism to be adjusted to the changing cellular conditions.^3,4

Ca²⁺ is known to synchronize mitochondrial metabolism as well as intracellular Ca²⁺ signalling, which is fundamental to neuronal physiology and viability. Therefore, Ca²⁺ signalling has become a major focus of study in multifactorial neurodegenerative diseases such as Alzheimer's disease (AD).^5,6 In AD, neurotoxic mechanisms that are associated with β-amyloid (Aβ) include mitochondria dysfunctions, which cause disturbances in calcium homeostasis.⁷ This suggests that the control of intracellular calcium flux can prevent or inhibit fatal injury caused by Aβ-induced neurotoxicity.

Here, we demonstrate that the magnetic modulation of mitochondrial VDAC2, which is the only mammalian-specific isoform among VDAC isoforms, can contribute to protect the neurodegenerative disease, attenuating the changes in the intracellular calcium levels that were induced by beta-amyloid. In this study, BMPs originated from Magnetospirillum sp. AMB-1 directly conjugated with VDAC2 antibody using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) linker, which is used to couple carboxyl groups to primary amines (Fig. 1(a)).

Fig. 1 (a) Schematic of the BMP–VDAC2 Ab complex, (b) specific targeting of BMP–VDAC2 Ab complex to mitochondria inside SH-SY5Y cells; blue: nucleus, red: mitochondria, green: BMP; scale bar: 10 μm.

BMPs–VDAC2 antibody complexes (BMPs–Ab) are introduced into SH-SY5Y cells, human derived neuroblasts, which are often used as in vitro models of neuronal function and differentiation. As shown in Fig 1(b), most of the BMPs–Ab were successfully internalized into the SH-ST5Y cells and were observed with correlation to mitochondria (yellow color). In contrast, BMPs without VDAC-2 antibodies were randomly distributed inside cells, with no particular mitochondrial localization. Because of their numerous roles in energy production, cell-death regulation, and cell signalling transduction, mitochondria have been considered an important target for the therapeutic treatment of various diseases.^8,9 Thereby, the efficient internalization and specific mitochondrial VDAC2 targeting of the BMP–Ab is promising for the mitochondria specific manipulation of cell function.

Aβ, a hallmark of AD, destabilizes intracellular Ca²⁺ homeostasis, resulting in an elevation of intracellular free Ca²⁺ concentration.^6,10,11 We investigated the effect of magnetically modulated VDAC2 on the change of intracellular Ca²⁺ levels induced by Aβ (Fig. 2). SH-SY5Y cells were loaded with 5 μM Fluo-3 AM (Sigma-Aldrich Co., USA) for 30 min, and then the changes in the level of Ca²⁺ before and after treatment with Aβ were measured by a 488 nm laser source to excite Fluo-3. Fluorescence intensity reflecting intracellular Ca²⁺ concentration was measured by the Microplate Reader (Victor 3, Perkin Elmer, USA). As shown in Fig. 2, the intracellular calcium levels of VDAC2 targeted SH-SY5Y cells with BMPs–Ab moderately decreased compared to those of the untargeted cells, when the Aβ was not treated.

Fig. 2 Effect of the BMP–VDAC2 Ab complex on amyloid beta-induced Ca²⁺ influx in the SH-SY5Y cells. Control: VDAC2 untargeted, BMP–VDAC2 Ab: VDAC2 targeted cells, Control + 50 μM of Aβ: VDAC2 untargeted cells under Aβ treated condition, BMP–VDAC2 Ab + 50 μM of Aβ: VDAC2 targeted cells with BMPs–Ab under Aβ treated condition. (*p < 0.05 and **p < 0.01, n = 3) Calibration method for Fluo-3 fluorescence signals to [Ca²⁺] is described in the ESI (Materials and methods section and Fig. S5†).¹⁹

After the treatment of Aβ, however, the capture of VDAC2 with BMPs–Ab was found to more significantly decrease the expressed intracellular calcium levels when compared to those of the untargeted cells.

Ca²⁺ is an interesting second messenger, which can initiate both cellular life and death pathways in mitochondria. Mitochondria accumulate Ca²⁺ for cellular bioenergetics metabolism and suppression of mitochondrial motility within the cell. Excessive Ca²⁺ uptake into mitochondria often leads to mitochondrial membrane permeabilization and induction of apoptosis.

Interestingly, the magnetic modulation of VDAC2 considerably increases the proliferation of SH-ST5Y cells, up to 50% for 3 days culture (Fig. 3(a)). The increased growth can be ascribed to the ATP content of the VDAC2 targeted cells that improved about 30% higher compared with that of the VDAC2 untargeted cells (Fig. 3(b)). These results were reconfirmed with the change in cell viability in the presence of Aβ (25–35), which destabilizes intracellular Ca²⁺ homeostasis. As shown in Fig. 3(c) and (d), the Aβ treatment induced lethal cell death; however, the magnetic capture of VDAC2 with BMPs–Ab significantly reduced the Aβ-induced toxicity in SH-SY5Y cells.

Fig. 3 Effect of the BMP–VDAC2 Ab complex on cell growth by (a) MTS assay and (b) ATP level. Under the amyloid-beta induced neurotoxic condition, the effect of BMP–VDAC2 Ab complex on cell growth by (c) MTS assay and (d) ATP level. Control: VDAC2 untargeted cells, BMP–VDAC2 Ab: VDAC2 targeted cells with BMPs, Control + 50 μM of Aβ: amyloid-beta treated VDAC2 untargeted cells, and BMP–VDAC2 Ab + 50 μM of Aβ: amyloid-beta treated VDAC2 targeted cells. (*p < 0.05, **p < 0.01, and ***p < 0.001, n = 4).

Ca²⁺ signalling causes transient changes in the cytosolic Ca²⁺ concentration. Mitochondria rapidly take up Ca²⁺ when a physiological stimulus elicits an increase in cytosolic Ca²⁺ concentrations. This uptake machinery allows mitochondria to act as “Ca²⁺ buffers” to maintain the normal homeostasis. Aβ destabilizes intracellular Ca²⁺ homeostasis as well as localizes to mitochondrial membranes, blocks the transport of nuclear-encoded mitochondrial proteins to mitochondria, interact with mitochondrial proteins, disrupt the electron transport chain, increase reactive oxygen species (ROS) production, cause mitochondrial damage, and eventually induce the neurodegeneration or cell death (Fig. 4 right).^12–14 The magnetic modulation of VDAC2 should block the localization of Aβ to the mitochondria and promote the Ca²⁺ uptake into the mitochondria within the threshold. Consequently, mitochondria should be able to maintain its functions such as the ATP production and other energy-dependent functions (Fig. 4, left).

Fig. 4 Schematic map of mitochondrial Ca²⁺ transporters in the amyloid-beta induced toxicity. Aβ results in elevated cytosolic calcium levels and localizes to mitochondrial membranes. Aβ in mitochondria inhibits mitochondrial ATP production and other energy-dependent functions, and releases calcium stored in mitochondria, thereby further deregulating neuronal calcium signaling. Finally, a release of proapoptotic proteins from damaged mitochondria results in neuronal injury. However, the magnetic modulation of VDAC2 should block the localization of Aβ to the mitochondria, and promote the Ca²⁺ uptake into the mitochondria within the threshold. Mitochondria produce ATP and maintain other functions.

In the previous studies, we reported that magnetic stimulation can lead to changes in a wide range of cellular properties such as cell shape, cytoskeletal organization, and cell fate.^15,16 Moreover, some other groups have demonstrated that the activation of ion channels is possible using nanoscale magnetic particles.^17–19 The magnetic modulation of mitochondrial VDAC, playing a key role in various Ca²⁺ influx and efflux pathways, should provide attractive targets for the future development of AD treatments.

Conclusions

BMPs–VDAC2 antibody complexes (BMPs–Ab) introduced into SH-SY5Y cells were successfully internalized into the SH-SY5Y cells. The capture of VDAC2 with BMPs–Ab significantly decreased the expressed intracellular calcium levels induced by Aβ. This magnetic modulation of VDAC2 considerably increases the proliferation and reduces the Aβ-induced toxicity in SH-SY5Y. These results suggest that the magnetic modulation of VDAC-2 can protect the neurodegenerative disease, attenuating the changes in the intracellular calcium levels that were induced by Aβ.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (2014R1A1A2057209) and by the National Research Foundation of Korea (NRF) grant funded by the Korea goverment (Ministry of Science, ICT & Future Planning, grant no. NRF-2013R1A2A1A01015644/2012R1A1A3010079) and a Korea University Grant, and by the Center for Integrated Smart Sensors funded by the Ministry of Science, ICT & Future Planning as Global Frontier Project (CISS-2012M3A6A6054193).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental methods and additional proofs. See DOI: 10.1039/c4ra10755a

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