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Cite this: DOI: 10.1039/c5nr02390d Received 22nd April 2015, Accepted 28th April 2015 DOI: 10.1039/c5nr02390d www.rsc.org/nanoscale
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Real-time investigation of cytochrome c release profiles in living neuronal cells undergoing amyloid beta oligomer-induced apoptosis† Jae Young Lee,‡a Younggeun Park,‡b San Pun,c Sung Sik Lee,c Joe F. Loc and Luke P. Lee*b
Intracellular Cyt c release profiles in living human neuroblastoma undergoing amyloid β oligomer (AβO)-induced apoptosis, as a model Alzheimer’s disease-associated pathogenic molecule, were analysed in a real-time manner using plasmon resonance energy transfer (PRET)-based spectroscopy.
Alzheimer’s disease (AD) is the most common cause of senile dementia in the elderly population. This progressive neurodegenerative disorder contributes to cognitive impairment and neuronal loss in the brain.1–3 Amyloid β (Aβ) peptide species compose brain plaques in AD patients that have been used as one of the histopathological hallmarks of AD.4–6 These peptides, especially Aβ1–42, have been thought to cause dysfunction of neuronal cells in AD patients.7–9 Recent studies have shown that soluble oligomeric forms of the amyloid β peptide (AβO) play key roles in AD pathogenesis and exhibit higher toxicity to neuronal cells compared to the Aβ monomer or fibril forms.10–14 The exact mechanism of AβO neurotoxicity has not been fully understood; however, it has been proposed that AβO causes apoptosis through a series of steps, such as damage to membrane integrity, cation dyshomeostasis (e.g., increase in intracellular calcium), mitochondrial dysfunction, caspase activation, and cell death.15–18 Importantly, this amyloid β-mediated apoptosis involves mitochondrial dysfunction that causes mitochondrial membrane depolarization and subsequent Cytochrome c (Cyt c) release from mitochondria to the cytosol leading to an elevated cytosolic Cyt c level14–16 (Fig. 1a and b). The intracellular Cyt c binds with apoptotic protease activating factor 1 (Apaf-1), followed by activation of
a School of Materials Science and Engineering, Gwangju Institute of Science and Technology, Gwangju 500-715, Republic of Korea b Department of Bioengineering, Berkeley Sensor and Actuator Center University of California Berkeley, Berkeley, CA 94720-1762, USA. E-mail: [email protected]; Fax: +1-510-642-5835; Tel: +1-510-642-5855 c Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5nr02390d ‡ These authors contributed equally to this work.
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Fig. 1 Amyloid-beta oligomer (AβO)-induced apoptosis and plasmon resonance energy transfer (PRET)-based Cytochrome C (Cyt c) real-time detection in a living cell. (a) Illustration of the AβO-induced apoptosis mechanism, which involves Cyt c redistribution from mitochondria to the cytosol. (b) Roles of AβO and the nerve growth factor (NGF) in induction and prevention of intracellular Cyt c-mediated apoptosis. (c) Cytosolic Cyt c interacts with plasmonic nanoparticles in living cells. (d) Cyt c-plasmonic particle complexation forms a resonant energy state between them, leading to PRET. (e) Scattering spectra from the nanoparticles in the absence (red) or presence (blue) of Cyt c.
caspase. Hence, cytosolic Cyt c is a key component and an indicator of an apoptotic event.19,20 Thus, monitoring cytosolic Cyt c levels is essential for understanding the dynamics of pathological progress in many different neurodegenerative diseases, including AD. Nanoparticle biosensors of different sizes, shapes, and chemical components have been widely adapted for novel applications in biomedical imaging and biosensing for clinical diagnostics.21–23 Plasmonic nanoprobes with nanoantenna structures provide an efficient biosensing platform with spatiotemporal resolution because they enable direct and continuous acquirement of molecular spectral information on a target with high sensitivity;24–29 these unique properties of plasmonic nanoprobes can also address problems of conventional techniques, such as photobleaching or blinking in fluorescence-based detection.30 A plasmon resonance energy trans-
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fer (PRET)-based biosensing has been developed for molecular imaging of biomolecules for characterization and quantification of target molecules in a selective and sensitive manner.26,31–33 External light can induce the collective oscillations of conduction electrons at resonance wavelengths (called plasmon) on the surface of nanoplasmonic probes.34,35 When a target molecule binds to the surface of a nanoplasmonic probe, energy transfer occurs in the resonance state between the plasmonic energy of the probe and the absorption energy of the molecule (Fig. 1d). This energy transfer results in a spectral quenching dip in the scattering spectrum of the plasmonic probe (PRET probe) (Fig. 1e). This resulting quenching dip is matched with the absorption peak(s) of the target molecule with respect to its electronic states of absorption spectroscopy.26,31,36 Accordingly, spectral analysis of the location and depth of the quenching dips can be used to quantify analyte molecule information in a real-time manner. It should also be noted that the most PRET-based biosensing studies were performed for simple detection of molecules in the non-living systems, such as a cell-free solution. Thus, a study of PRET techniques in biologically relevant models (e.g., living cells) is still required to demonstrate the feasibility of the PRET-based biosensors. In our study, we employed the PRET technique to study apoptotic effects of AβO by directly monitoring the intracellular Cyt c level from individual living cells. For Cyt c detection using the PRET technique, we utilized mercaptopropionic acid (MPA)-functionalized 50 nm gold nanoparticles. The MPAfunctionalized nanoparticles have shown a fast and weak electrostatic force-based interaction with Cyt c (Ka = 2.5 × 10−4 M−1), which is suitable for real-time monitoring of intracellular Cyt c.26,37 These nanoparticles, as PRET probes, exhibit strong light scattering in the range between 450 and 600 nm. This range includes the absorption peaks of Cyt c (520–560 nm) which is the target molecule of interest in this study. PRET signals of Cyt c exhibit intensified quenching dips in the scattering spectra. In addition, the PRET technique allows the identification of both reduced and oxidized states of Cyt c. Different characteristic quenching dips are observed at their different characteristic absorption peak positions of reduced Cyt c (523 and 550 nm) and oxidized Cyt c (530 nm), respectively (Fig. S1†). We investigated the neuronal toxicity of AβO in neuroblastoma SH-SY5Y cells during cell incubation with AβO. The PRET was employed to study AβO-induced apoptosis, which involves increases in cytosolic Cyt c levels and PRET signals (Fig. 1 and Fig. S2†). Fig. 2a shows bright-field and dark-field images of living cells with the probes. After AβO (2.5 μM) treatment, scattering spectra were acquired using the individual PRET probes (marked with arrows) and their spectral changes were monitored using the probes in each cell (denoted as cell a and cell b, respectively). Cyt c signals, characteristic quenching dips of Cyt c at around 530 nm, from both the cells were observed using the multiple probes after AβO introduction. Both cell a and cell b eventually released Cyt c. However, the overall Cyt c signal profiles and intensities were different
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Fig. 2 Monitoring Cyt c release dynamics from living cells undergoing AβO-induced apoptosis using PRET analysis. (a) Bright-field (top) and dark-field (bottom) images of SH-SY5Y. Internalized plasmonic probes are shown as dots. Scattering spectra PRET probes (marked with arrows) in cell a and cell b were analyzed to study Cyt c analysis after the incubation with AβO. Scale bar = 20 μm. (b) Intensity change profiles of Cyt c from the probes in cell a and cell b. At least five probes in each cell were analyzed for the dip intensity from their scattering spectra. The average and the range of standard deviations were plotted as a solid line and green area.
between the cells (Fig. 2b). In cell a, significant Cyt c signals began to be detected with strong quenching dips, exhibiting a >15% intensity decrease, after 2 hours of incubation with AβO. Eventually, the overall signal intensity was saturated in the range of 0.2–0.3. On the other hand, Cyt c dips from the probes in cell b were detected within 30 min and their dip intensities remained relatively constant at around 0.1–0.15. These results imply that the scattering information within individual cells appears to be relatively homogeneous, whereas AβO-induced apoptotic progress is different among cells. These different profiles may be attributed to general heterogeneous cellular traits, such as metabolism, cell cycles, and gene expressions.38,39 Thus, it is practically important to examine multiple cells to investigate the overall cellular responses to AβO. For collective determination of AβO-associated toxicity and its dynamics, we analyzed Cyt c signals of multiple living cells (n > 15) from their PRET spectra (Fig. 3). AβO triggers apoptosis within a short time. Approximately 50% of the cells from the AβO-treated cells began to release Cyt c over 30 minutes after AβO introduction. Most of the cells (92%) showed quenching dips in the spectra for 1 hour, indicating fast induction of mitochondrial dysfunction and apoptosis. This acute action of AβO on mitochondrial dysfunction can be correlated with the findings of another study that prompt calcium dysregulation (elevation of intracellular calcium levels) occurred within 5 seconds of the AβO introduction, which in turn impaired mitochondrial function.13 Moreover, we found the larger portions of the cells with stronger Cyt c signals with increasing incubation time. To study the changes in Cyt c signal intensities in a semi-quantitative way, cells were classi-
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Fig. 3 Profiles of Cyt c releasing cells after AβO introduction. (a) Spectral analysis of multiple cells was performed to determine the presence and intensity of Cyt c with AβO treatment. (b) Effects of NGF pretreatment on AβO-induced apoptosis. Cells were classified into ΔI5% dip for ‘cells with weak dips’ and ΔI15% dip for ‘cells with strong dips’ when the quenching dip intensities at 530 nm are 5–15% and >15%, respectively. Total apoptotic cells were defined as the sum of the cells. (c) Immunostaining with FITC-Annexin V of cells cultured with the control (without AβO), AβO, or AβO + NGF. Cells were stained and imaged 3 hours after the treatment. Presented images were constructed by merging phase contrast images and green fluorescence. Scale bar = 100 µm.
fied into ΔI5% dip (5–15% intensity decrease), and ΔI15% dip (>15% intensity decrease), when the scattering intensities at 530 nm decreased by 5–10% and >10%, respectively. Cells with strong dips (ΔI15% dip) occupied 62% in 1 hour and this portion increased to 69% and 100% with 2 hour and 4 hour incubation, respectively. These results imply that AβO triggers cellular damage and mitochondrial dysfunction and AβOinduced apoptotic signals accumulate over the incubation. For untreated control cells, few probes exhibited weak Cyt c signals (data not shown). In addition, we tested the nerve growth factor (NGF), as a model anti-apoptotic molecule in neuronal cells, to investigate its effects on preventing AβO-induced Cyt c release in a realtime manner. NGF is a type of neurotrophin that plays important roles in neuronal differentiation and cell survival.40,41 A failure of NGF signaling is known to trigger AD onsets and induce neuronal cell death.42,43 An NGF signal cascade prevents proapoptotic Bax protein homodimerization and Cyt c extrusion. Neuroprotective mechanisms can also be accounted for by the anti-oxidative activity of NGF as is known to suppress activities of intracellular reactive oxygen species.44,45 Neuroblastoma cultured with NGF (50 ng mL−1) showed significantly smaller cell populations that presented Cyt c signals when AβO was treated, compared to the NGF-untreated controls (Fig. 3b), confirming NGF’s anti-apoptotic activity. In 0.5 and 1 hour, 7% and 17% of the cells exhibited Cyt c signals, respectively, all of
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which showed weak Cyt c quenching dips. Both cell populations with Cyt c signals and their signal intensities eventually increased over the incubation. After 3 hours, 60% out of the total analyzed cells displayed the Cyt c signals consisting of 33% cells with a weak dip (ΔI5% dip) and 27% with a strong dip (ΔI15% dip). PRET-based analysis of the AβO-induced apoptosis was well matched with the results obtained with conventional methods for apoptosis analysis, such as Annexin V-FITC (apoptotic cells). As shown in Fig. 3c, a significant increase in the number of Annexin V-positive cells was found in AβO-treated cells compared to the untreated control. In addition, JC-1 staining indicated that J-aggregates mostly disappeared after 3 hours of incubation with AβO due to the loss of the mitochondrial membrane potential, suggesting that AβO induces mitochondrial dysfunction (Fig. S3†). As mentioned earlier, NGF was tested as a model anti-apoptotic drug in this study to explore its protective effects on AβO-induced apoptosis. We found that NGF prevents AβO-induced Cyt c release. Interestingly, NGF does not completely block the AβO-induced apoptosis. Instead, NGF retards toxic effects of AβO on early apoptotic signaling (i.e., Cyt c release).
Conclusions In conclusion, dynamic apoptotic progression in response to AD-associated pathologic molecules (i.e., AβO) was observed in living cells in the manner of real time detection using the PRET technique. We successfully monitored the progression of AβO-induced apoptotic events in individual single living neuroblastoma by analyzing intracellular Cyt c levels. NGF pretreatment was found to attenuate AβO-induced Cyt c release, which results from anti-apoptotic effects of NGF. This PRET-based real-time spectroscopy will allow for studies on dynamic effects of apoptotic molecules in living cells and can potentially be employed in drug development for neurodegenerative diseases.
Acknowledgements This research was supported by the Global Research Lab. Program (2013-050616) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT (Information and Communication Technologies) and Future Planning. This work was also supported by the Converging Research Center Program (2010K000383) through the Ministry of Science. This work was also partially supported by and carried out in ETH Zurich, D-BSSE (LL).
Notes and references 1 C. P. Ferri, M. Prince, C. Brayne, H. Brodaty, L. Fratiglioni, M. Ganguli, K. Hall, K. Hasegawa, H. Hendrie, Y. Huang, A. Jorm, C. Mathers, P. R. Menezes, E. Rimmer and M. Scazufca, Lancet, 2005, 366, 2112. 2 D. J. Selkoe, Physiol. Rev., 2001, 81, 741.
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3 K. Ziegler-Graham, R. Brookmeyer, E. Johnson and H. M. Arrighi, Alzheimer’s Dementia, 2008, 4, 316. 4 D. J. Selkoe, Science, 1990, 248, 1058. 5 D. J. Selkoe, Nature, 2003, 426, 900. 6 M. Li, C. Zhao, X. Yang, J. Ren, C. Xu and X. Qu, Small, 2013, 9, 52. 7 L. Canevari, A. Y. Abramov and M. R. Duchen, Neurochem. Res., 2004, 29, 637. 8 W. E. Muller, G. P. Eckert, K. Scheuer, N. J. Cairns, A. Maras and W. F. Gattaz, Amyloid, 1998, 5, 10. 9 T. Luhrs, C. Ritter, M. Adrian, D. Riek-Loher, B. Bohrmann, H. Dobeli, D. Schubert and R. Riek, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 17342. 10 R. Kayed, E. Head, J. L. Thompson, T. M. McIntire, S. C. Milton, C. W. Cotman and C. G. Glabe, Science, 2003, 300, 486. 11 A. Laganowsky, C. Liu, M. R. Sawaya, J. P. Whitelegge, J. Park, M. Zhao, A. Pensalfini, A. B. Soriaga, M. Landau, P. K. Teng, D. Cascio, C. Glabe and D. Eisenberg, Science, 2012, 335, 1228. 12 C. Haass and D. J. Selkoe, Nat. Rev. Mol. Cell Biol., 2007, 8, 101. 13 A. Demuro, E. Mina, R. Kayed, S. C. Milton, I. Parker and C. G. Glabe, J. Biol. Chem., 2005, 280, 17294. 14 S. Sanz-Blasco, R. A. Valero, I. Rodriguez-Crespo, C. Villalobos and L. Nunez, PLoS One, 2008, 3, e2718. 15 M. Sakono and T. Zako, FEBS J., 2010, 277, 1348. 16 M. T. Lin and M. F. Beal, Nature, 2006, 443, 787. 17 P. J. Crouch, S. M. Harding, A. R. White, J. Camakaris, A. I. Bush and C. L. Masters, Int. J. Biochem. Cell Biol., 2008, 40, 181. 18 P. Calissano, C. Matrone and G. Amadoro, Commun. Integr. Biol., 2009, 2, 163. 19 M. O. Hengartner, Nature, 2000, 407, 770. 20 M. Okada, N. I. Smith, A. F. Palonpon, H. Endo, S. Kawata, M. Sodeoka and K. Fujita, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 28. 21 Y. Liu and H. Wang, Nat. Nanotechnol., 2007, 2, 20. 22 X. Chi, D. Huang, Z. Zhao, Z. Zhou, Z. Yin and J. Gao, Biomaterials, 2012, 33, 189.
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23 M. Mahmoudi, V. Serpooshan and S. Laurent, Nanoscale, 2011, 3, 3007. 24 H. W. Cheng, S. Y. Huan and R. Q. Yu, Analyst, 2012, 137, 3601. 25 G. Baffou and R. Quidant, Chem. Soc. Rev., 2014, 43, 3898. 26 Y. Choi, T. Kang and L. P. Lee, Nano Lett., 2009, 9, 85. 27 G. Konstantatos and E. H. Sargent, Nat. Nanotechnol., 2010, 5, 391. 28 K. E. Fong and L. Y. Yung, Nanoscale, 2013, 5, 12043. 29 A. Biswas, T. Wang and A. S. Biris, Nanoscale, 2010, 2, 1560. 30 B. Hötzer, I. L. Medintz and N. Hildebrandt, Small, 2012, 8, 2297. 31 G. L. Liu, Y. T. Long, Y. Choi, T. Kang and L. P. Lee, Nat. Methods, 2007, 4, 1015. 32 W. G. Qu, B. Deng, S. L. Zhong, H. Y. Shi, S. S. Wang and A. W. Xu, Chem. Commun., 2011, 47, 1237. 33 Y. Choi, Y. Park, T. Kang and L. P. Lee, Nat. Nanotechnol., 2009, 4, 742. 34 K. A. Willets and R. P. Van Duyne, Annu. Rev. Phys. Chem., 2007, 58, 267. 35 S. Eustis and M. A. El-Sayed, Chem. Soc. Rev., 2006, 35, 209. 36 I. Kim, S. L. Bender, J. Hranisavljevic, L. M. Utschig, L. Huang, G. P. Wiederrecht and D. M. Tiede, Nano Lett., 2012, 11, 3091. 37 S. M. Bonk and F. Lisdat, Biosens. Bioelectron., 2009, 25, 739. 38 A. Raza and N. Galili, Nat. Rev. Cancer, 2012, 12, 849. 39 A. Marusyk and K. Polyak, Biochim. Biophys. Acta, 2010, 1805, 105. 40 E. M. Shooter, Annu. Rev. Neurosci., 2001, 24, 601. 41 G. Ichim, S. Tauszig-Delamasure and P. Mehlen, Exp. Cell Res., 2010, 318, 1221. 42 S. Capsoni and A. Cattaneo, Cell Mol. Neurobiol., 2006, 26, 619. 43 S. Capsoni, S. Giannotta and A. Cattaneo, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 12432. 44 R. A. Kirkland, G. M. Saavedra and J. L. Franklin, J. Neurosci., 2007, 27, 11315. 45 J. L. Franklin, Antioxid. Redox Signaling, 2011, 14, 1437.
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Cite this: DOI: 10.1039/c5nr02390d Received 22nd April 2015, Accepted 28th April 2015 DOI: 10.1039/c5nr02390d www.rsc.org/nanoscale
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Real-time investigation of cytochrome c release profiles in living neuronal cells undergoing amyloid beta oligomer-induced apoptosis† Jae Young Lee,‡a Younggeun Park,‡b San Pun,c Sung Sik Lee,c Joe F. Loc and Luke P. Lee*b
Intracellular Cyt c release profiles in living human neuroblastoma undergoing amyloid β oligomer (AβO)-induced apoptosis, as a model Alzheimer’s disease-associated pathogenic molecule, were analysed in a real-time manner using plasmon resonance energy transfer (PRET)-based spectroscopy.
Alzheimer’s disease (AD) is the most common cause of senile dementia in the elderly population. This progressive neurodegenerative disorder contributes to cognitive impairment and neuronal loss in the brain.1–3 Amyloid β (Aβ) peptide species compose brain plaques in AD patients that have been used as one of the histopathological hallmarks of AD.4–6 These peptides, especially Aβ1–42, have been thought to cause dysfunction of neuronal cells in AD patients.7–9 Recent studies have shown that soluble oligomeric forms of the amyloid β peptide (AβO) play key roles in AD pathogenesis and exhibit higher toxicity to neuronal cells compared to the Aβ monomer or fibril forms.10–14 The exact mechanism of AβO neurotoxicity has not been fully understood; however, it has been proposed that AβO causes apoptosis through a series of steps, such as damage to membrane integrity, cation dyshomeostasis (e.g., increase in intracellular calcium), mitochondrial dysfunction, caspase activation, and cell death.15–18 Importantly, this amyloid β-mediated apoptosis involves mitochondrial dysfunction that causes mitochondrial membrane depolarization and subsequent Cytochrome c (Cyt c) release from mitochondria to the cytosol leading to an elevated cytosolic Cyt c level14–16 (Fig. 1a and b). The intracellular Cyt c binds with apoptotic protease activating factor 1 (Apaf-1), followed by activation of
a School of Materials Science and Engineering, Gwangju Institute of Science and Technology, Gwangju 500-715, Republic of Korea b Department of Bioengineering, Berkeley Sensor and Actuator Center University of California Berkeley, Berkeley, CA 94720-1762, USA. E-mail: [email protected]; Fax: +1-510-642-5835; Tel: +1-510-642-5855 c Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5nr02390d ‡ These authors contributed equally to this work.
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Fig. 1 Amyloid-beta oligomer (AβO)-induced apoptosis and plasmon resonance energy transfer (PRET)-based Cytochrome C (Cyt c) real-time detection in a living cell. (a) Illustration of the AβO-induced apoptosis mechanism, which involves Cyt c redistribution from mitochondria to the cytosol. (b) Roles of AβO and the nerve growth factor (NGF) in induction and prevention of intracellular Cyt c-mediated apoptosis. (c) Cytosolic Cyt c interacts with plasmonic nanoparticles in living cells. (d) Cyt c-plasmonic particle complexation forms a resonant energy state between them, leading to PRET. (e) Scattering spectra from the nanoparticles in the absence (red) or presence (blue) of Cyt c.
caspase. Hence, cytosolic Cyt c is a key component and an indicator of an apoptotic event.19,20 Thus, monitoring cytosolic Cyt c levels is essential for understanding the dynamics of pathological progress in many different neurodegenerative diseases, including AD. Nanoparticle biosensors of different sizes, shapes, and chemical components have been widely adapted for novel applications in biomedical imaging and biosensing for clinical diagnostics.21–23 Plasmonic nanoprobes with nanoantenna structures provide an efficient biosensing platform with spatiotemporal resolution because they enable direct and continuous acquirement of molecular spectral information on a target with high sensitivity;24–29 these unique properties of plasmonic nanoprobes can also address problems of conventional techniques, such as photobleaching or blinking in fluorescence-based detection.30 A plasmon resonance energy trans-
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fer (PRET)-based biosensing has been developed for molecular imaging of biomolecules for characterization and quantification of target molecules in a selective and sensitive manner.26,31–33 External light can induce the collective oscillations of conduction electrons at resonance wavelengths (called plasmon) on the surface of nanoplasmonic probes.34,35 When a target molecule binds to the surface of a nanoplasmonic probe, energy transfer occurs in the resonance state between the plasmonic energy of the probe and the absorption energy of the molecule (Fig. 1d). This energy transfer results in a spectral quenching dip in the scattering spectrum of the plasmonic probe (PRET probe) (Fig. 1e). This resulting quenching dip is matched with the absorption peak(s) of the target molecule with respect to its electronic states of absorption spectroscopy.26,31,36 Accordingly, spectral analysis of the location and depth of the quenching dips can be used to quantify analyte molecule information in a real-time manner. It should also be noted that the most PRET-based biosensing studies were performed for simple detection of molecules in the non-living systems, such as a cell-free solution. Thus, a study of PRET techniques in biologically relevant models (e.g., living cells) is still required to demonstrate the feasibility of the PRET-based biosensors. In our study, we employed the PRET technique to study apoptotic effects of AβO by directly monitoring the intracellular Cyt c level from individual living cells. For Cyt c detection using the PRET technique, we utilized mercaptopropionic acid (MPA)-functionalized 50 nm gold nanoparticles. The MPAfunctionalized nanoparticles have shown a fast and weak electrostatic force-based interaction with Cyt c (Ka = 2.5 × 10−4 M−1), which is suitable for real-time monitoring of intracellular Cyt c.26,37 These nanoparticles, as PRET probes, exhibit strong light scattering in the range between 450 and 600 nm. This range includes the absorption peaks of Cyt c (520–560 nm) which is the target molecule of interest in this study. PRET signals of Cyt c exhibit intensified quenching dips in the scattering spectra. In addition, the PRET technique allows the identification of both reduced and oxidized states of Cyt c. Different characteristic quenching dips are observed at their different characteristic absorption peak positions of reduced Cyt c (523 and 550 nm) and oxidized Cyt c (530 nm), respectively (Fig. S1†). We investigated the neuronal toxicity of AβO in neuroblastoma SH-SY5Y cells during cell incubation with AβO. The PRET was employed to study AβO-induced apoptosis, which involves increases in cytosolic Cyt c levels and PRET signals (Fig. 1 and Fig. S2†). Fig. 2a shows bright-field and dark-field images of living cells with the probes. After AβO (2.5 μM) treatment, scattering spectra were acquired using the individual PRET probes (marked with arrows) and their spectral changes were monitored using the probes in each cell (denoted as cell a and cell b, respectively). Cyt c signals, characteristic quenching dips of Cyt c at around 530 nm, from both the cells were observed using the multiple probes after AβO introduction. Both cell a and cell b eventually released Cyt c. However, the overall Cyt c signal profiles and intensities were different
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Fig. 2 Monitoring Cyt c release dynamics from living cells undergoing AβO-induced apoptosis using PRET analysis. (a) Bright-field (top) and dark-field (bottom) images of SH-SY5Y. Internalized plasmonic probes are shown as dots. Scattering spectra PRET probes (marked with arrows) in cell a and cell b were analyzed to study Cyt c analysis after the incubation with AβO. Scale bar = 20 μm. (b) Intensity change profiles of Cyt c from the probes in cell a and cell b. At least five probes in each cell were analyzed for the dip intensity from their scattering spectra. The average and the range of standard deviations were plotted as a solid line and green area.
between the cells (Fig. 2b). In cell a, significant Cyt c signals began to be detected with strong quenching dips, exhibiting a >15% intensity decrease, after 2 hours of incubation with AβO. Eventually, the overall signal intensity was saturated in the range of 0.2–0.3. On the other hand, Cyt c dips from the probes in cell b were detected within 30 min and their dip intensities remained relatively constant at around 0.1–0.15. These results imply that the scattering information within individual cells appears to be relatively homogeneous, whereas AβO-induced apoptotic progress is different among cells. These different profiles may be attributed to general heterogeneous cellular traits, such as metabolism, cell cycles, and gene expressions.38,39 Thus, it is practically important to examine multiple cells to investigate the overall cellular responses to AβO. For collective determination of AβO-associated toxicity and its dynamics, we analyzed Cyt c signals of multiple living cells (n > 15) from their PRET spectra (Fig. 3). AβO triggers apoptosis within a short time. Approximately 50% of the cells from the AβO-treated cells began to release Cyt c over 30 minutes after AβO introduction. Most of the cells (92%) showed quenching dips in the spectra for 1 hour, indicating fast induction of mitochondrial dysfunction and apoptosis. This acute action of AβO on mitochondrial dysfunction can be correlated with the findings of another study that prompt calcium dysregulation (elevation of intracellular calcium levels) occurred within 5 seconds of the AβO introduction, which in turn impaired mitochondrial function.13 Moreover, we found the larger portions of the cells with stronger Cyt c signals with increasing incubation time. To study the changes in Cyt c signal intensities in a semi-quantitative way, cells were classi-
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Fig. 3 Profiles of Cyt c releasing cells after AβO introduction. (a) Spectral analysis of multiple cells was performed to determine the presence and intensity of Cyt c with AβO treatment. (b) Effects of NGF pretreatment on AβO-induced apoptosis. Cells were classified into ΔI5% dip for ‘cells with weak dips’ and ΔI15% dip for ‘cells with strong dips’ when the quenching dip intensities at 530 nm are 5–15% and >15%, respectively. Total apoptotic cells were defined as the sum of the cells. (c) Immunostaining with FITC-Annexin V of cells cultured with the control (without AβO), AβO, or AβO + NGF. Cells were stained and imaged 3 hours after the treatment. Presented images were constructed by merging phase contrast images and green fluorescence. Scale bar = 100 µm.
fied into ΔI5% dip (5–15% intensity decrease), and ΔI15% dip (>15% intensity decrease), when the scattering intensities at 530 nm decreased by 5–10% and >10%, respectively. Cells with strong dips (ΔI15% dip) occupied 62% in 1 hour and this portion increased to 69% and 100% with 2 hour and 4 hour incubation, respectively. These results imply that AβO triggers cellular damage and mitochondrial dysfunction and AβOinduced apoptotic signals accumulate over the incubation. For untreated control cells, few probes exhibited weak Cyt c signals (data not shown). In addition, we tested the nerve growth factor (NGF), as a model anti-apoptotic molecule in neuronal cells, to investigate its effects on preventing AβO-induced Cyt c release in a realtime manner. NGF is a type of neurotrophin that plays important roles in neuronal differentiation and cell survival.40,41 A failure of NGF signaling is known to trigger AD onsets and induce neuronal cell death.42,43 An NGF signal cascade prevents proapoptotic Bax protein homodimerization and Cyt c extrusion. Neuroprotective mechanisms can also be accounted for by the anti-oxidative activity of NGF as is known to suppress activities of intracellular reactive oxygen species.44,45 Neuroblastoma cultured with NGF (50 ng mL−1) showed significantly smaller cell populations that presented Cyt c signals when AβO was treated, compared to the NGF-untreated controls (Fig. 3b), confirming NGF’s anti-apoptotic activity. In 0.5 and 1 hour, 7% and 17% of the cells exhibited Cyt c signals, respectively, all of
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which showed weak Cyt c quenching dips. Both cell populations with Cyt c signals and their signal intensities eventually increased over the incubation. After 3 hours, 60% out of the total analyzed cells displayed the Cyt c signals consisting of 33% cells with a weak dip (ΔI5% dip) and 27% with a strong dip (ΔI15% dip). PRET-based analysis of the AβO-induced apoptosis was well matched with the results obtained with conventional methods for apoptosis analysis, such as Annexin V-FITC (apoptotic cells). As shown in Fig. 3c, a significant increase in the number of Annexin V-positive cells was found in AβO-treated cells compared to the untreated control. In addition, JC-1 staining indicated that J-aggregates mostly disappeared after 3 hours of incubation with AβO due to the loss of the mitochondrial membrane potential, suggesting that AβO induces mitochondrial dysfunction (Fig. S3†). As mentioned earlier, NGF was tested as a model anti-apoptotic drug in this study to explore its protective effects on AβO-induced apoptosis. We found that NGF prevents AβO-induced Cyt c release. Interestingly, NGF does not completely block the AβO-induced apoptosis. Instead, NGF retards toxic effects of AβO on early apoptotic signaling (i.e., Cyt c release).
Conclusions In conclusion, dynamic apoptotic progression in response to AD-associated pathologic molecules (i.e., AβO) was observed in living cells in the manner of real time detection using the PRET technique. We successfully monitored the progression of AβO-induced apoptotic events in individual single living neuroblastoma by analyzing intracellular Cyt c levels. NGF pretreatment was found to attenuate AβO-induced Cyt c release, which results from anti-apoptotic effects of NGF. This PRET-based real-time spectroscopy will allow for studies on dynamic effects of apoptotic molecules in living cells and can potentially be employed in drug development for neurodegenerative diseases.
Acknowledgements This research was supported by the Global Research Lab. Program (2013-050616) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT (Information and Communication Technologies) and Future Planning. This work was also supported by the Converging Research Center Program (2010K000383) through the Ministry of Science. This work was also partially supported by and carried out in ETH Zurich, D-BSSE (LL).
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