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A novel method for the preparation of a neurotoxic complex
Analytical Biochemistry 435 (2013) 137–139
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
A novel method for the preparation of a neurotoxic complex Bum Han Ryu, Eunjin Jang, Hansol Ju, T. Doohun Kim ⇑ Department of Molecular Science and Technology, Graduate School of Interdisciplinary Programs, Ajou University, Suwon 443-749, South Korea
a r t i c l e
i n f o
Article history: Received 10 October 2012 Received in revised form 28 December 2012 Accepted 7 January 2013 Available online 16 January 2013
a b s t r a c t Neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) can be attributed to the specific degeneration of neuronal cells in the brain. However, the natures and action modes of toxic species remain largely unknown. Here, we present a simple and fast method for the preparation of neurotoxic complex with a-synuclein, which is implicated in PD. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Neurotoxicity Apoptosis FACS
Neurodegenerative diseases such as Parkinson’s disease (PD),1 Alzheimer’s disease (AD), and amyotrophic lateral sclerosis (ALS) are marked by the progressive degeneration of neurons [1–3]. In these diseases, the pathogenesis has been linked to conformational changes of proteins and peptides that inappropriately cause them to gain neurotoxic properties. Although these diseases are characterized by the formation of pathological protein aggregation, the natures of neurotoxic species as well as their modes of action remain to be elucidated. Currently, no effective chemical treatments can prevent, rescue, or slow down these neurodegenerative diseases. One of the reasons is clearly the lack of knowledge about the exact nature of the neurotoxic species, although recent studies have largely focused on characterization of oligomeric protein complexes with neurotoxic properties [4,5]. Recently, protein/lipid complexes have been reported to show toxic properties. For example, the neurotoxicity of prion protein correlates with its interaction with lipids [6]. Furthermore, disease-related properties of superoxide dismutase 1 (SOD 1) could be induced by unsaturated fatty acids [7]. In accordance, partially unfolded a-lactalbumin (a-LA) complexed with oleic acid (OA) acquires apoptotic properties such as cytochrome c release and activating cell death pathways [8]. Here, we report a novel method for the preparation of a neurotoxic complex with a-synuclein, which is implicated in PD. a-Synuclein is a small presynaptic protein with a length of 140 amino acids [9]. As shown in Fig. 1A, the N-terminal region (aa 1–60) contains an 11-amino-acid repeat sequence for interaction with lipid ⇑ Corresponding author. Fax: +82 31 219 2394. E-mail address: [email protected] (T.D. Kim). Abbreviations used: PD, Parkinson’s disease; a-LA, a-lactalbumin; OA, oleic acid; NAC, non-amyloid component; PBS, phosphate-buffered saline; MTT, methyl tetrazolium; LDH, lactate dehydrogenase; FBS, fetal bovine serum; calcein AM, calcein acetoxymethyl ester; Eth D-1, ethidium homodimer-I; a-syn TR, a-synuclein and its tandem repeat; AFM, atomic force microscopy. 1
0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.01.005
membranes, whereas the C-terminal region (aa 96–140) is enriched in acidic amino residues for modulation of amyloid aggregation. The central non-amyloid component (NAC, aa 61–95) region is composed of mainly hydrophobic residues. Based on the toxic properties of protein/lipid complexes observed in several proteins, we investigated the neurotoxic properties of a-synuclein/OA complex. Initially, we conducted turbidity analysis, which is frequently used to measure the formation of large aggregates or complexes, to investigate the specific interactions between a-synuclein and OA [10,11]. Fig. 1B shows the absorbance of solutions containing increasing amounts of OA in phosphate-buffered saline (PBS) at pH 7.4 in the absence or presence of a-synuclein. The addition of OA led to a gradual increase in absorbance with the formation of large OA aggregates. However, after the addition of a-synuclein, the turbid OA suspension became clear, indicating that a-synuclein was able to inhibit the formation of large aggregates. In the presence of a-synuclein (10 lM), no significant increase in turbidity was observed up to 500 lM OA (Fig. 1B, filled circles). The addition of a-synuclein to 2 mM OA suspension led to a large decrease in absorbance compared with the OA solution alone. Furthermore, intrinsic fluorescence analysis also led to the conclusion that effective complex formations occurred with the addition of a-synuclein to OA suspension (see Fig. S1 in supplementary material). Based on these results, a-synuclein was directly suspended in the OA solution and the mixture was heated to facilitate efficient formation of the complex [12]. After incubation for 45 min, the mixtures were cooled to room temperature. To investigate neurotoxic properties of a-synuclein/OA complex toward human neuroblastoma SH-SY5Y cells, we employed the methyl tetrazolium (MTT) reduction and lactate dehydrogenase (LDH) release assay [13]. SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium, supplemented with 10% (v/v) fetal bovine serum (FBS), in a 5% CO2 atmosphere
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Notes & Tips / Anal. Biochem. 435 (2013) 137–139
Fig.1. Characterization of the neurotoxic properties of a-synuclein/OA complex. (A) Schematic representation of a-synuclein and its tandem repeat (a-syn TR). (B) Turbidity analysis of OA solutions in the absence (s) or presence (d) of a-synuclein. Absorbance of samples containing up to 2 mM OA was measured at 405 nm. (C) Neurotoxic effects of a-synuclein/OA complex after heat treatments on SH-SY5Y cells were measured by the MTT assay. Cell viability is expressed as a percentage of controls. (D) LDH assay of SH-SY5Y cells treated with OA solutions in the absence or presence of a-synuclein (140 and 700 lM).
at 37 °C. Exponentially growing SH-SY5Y cells were seeded in 96well plates at a density of 5 104 cells/well. With approximately 80% confluence cells, a-synuclein (34.5 lM), OA (140 lM), or asynuclein/OA complex (1:20 molar ratio) was added to the culture medium. After incubation for 30 min at 37 °C, FBS was added to the culture. After 3 h of incubation, the SH-SY5Y cells were gently washed and incubated with 500 lg/ml MTT solution. The medium was then replaced with 100 ll of dimethyl sulfoxide (DMSO), and absorbance of blue formazan was measured at 570 nm using a microplate spectrophotometer (model 680, Bio-Rad, Hercules, CA, USA). Control cells were incubated with medium only, and their viability was considered to be 100%. The cell viability was expressed as a percentage of the absorbance compared with control cells. When cells were incubated with a-synuclein or OA alone, cell viability was comparable to that of the control, indicating that OA and a-synuclein possessed almost no cellular toxicities at these concentrations (Fig. 1C). However, the viability of SH-SY5Y cells treated with a-synuclein/OA complex (incubated at 37 °C) was reduced to approximately 20%. Higher temperature incubations (e.g., 50 or 60 °C) showed similar cellular toxic properties (Fig. 1C). LDH release, an indicator of cell membrane integrity, was evaluated by an LDH Cytotoxicity Detection Kit according to the manufacturer’s instructions (Takara Bio, Shiga, Japan). After incubation for 18 h in 96-well plates, a-synuclein (34.5 lM), OA (140 and 700 lM), and a-synuclein/OA complex in PBS were added to the culture medium. After 3.5 h, 100 ll of supernatants per well was harvested and transferred into a flat-bottomed 96-well plate. LDH reagent (100 ll) was added to each well, and the plates were incubated for 5 min at room temperature protected from light. The LDH activity was measured at 490/650 nm. In contrast to a-synuclein or OA alone, the addition of a-synuclein/OA resulted in significantly increased LDH activity. This increase was comparable to that induced
by 100 mM H2O2, that is, approximately 2.0-fold higher than basal levels (Fig. 1D). The morphological changes of SH-SY5Y cells after the addition of a-synuclein/OA complex were observed under a light microscope. Cells exposed to OA or a-synuclein alone were not visibly different from control cells (Fig. 2A). No significant cell deaths or cell growth retardation was observed, indicating the absence of cellular toxicity of a-synuclein or OA. However, after exposure to a-synuclein/OA complex, SH-SY5Y cells were severely damaged, suggesting that asynuclein/OA complex was strongly toxic. Similar to the addition of 100 mM H2O2, exposure to a-synuclein/OA complex results in apoptotic morphologies such as membrane rupture, cell shrinkage, loose cell attachment, and disruption of intercellular junctions (Fig. S1). To assess cell viability, live and/or dead cells were measured using a LIVE/DEAD Viability and Cytotoxicity Kit (Invitrogen, Carlsbad, CA, USA). SH-SY5Y cells, which were treated with a-synuclein, OA, and a-synuclein/OA complex for 3.5 h, were subjected to 2 lM calcein acetoxymethyl ester (calcein AM) and 4 lM ethidium homodimer-I (Eth D-1) staining. Calcein AM permeates all cells, leading to green fluorescence, whereas Eth D-1 gives a red fluorescence if cytoplasmic membrane integrity of cells is absent [14]. Approximately 40 min after staining, cells were examined using a fluorescence microscope (AE31, Motic, UK). Wavelengths for excitation/emission at 475/530 nm with a longpass filter of 542/ 620 nm were used. Representative images of the live (green-colored) and dead (red-colored) cells are shown in Fig. 2B. SH-SY5Y cells treated with a-synuclein or OA alone were mostly similar to control cells. However, red staining was seen in approximately 90% of cells treated with a-synuclein/OA complex, indicating that cell deaths had largely occurred. Furthermore, flow cytometry experiments using a FACSCalibur and CellQuest software (Becton Dickinson, San Jose, CA, USA) were also carried out to investigate
Notes & Tips / Anal. Biochem. 435 (2013) 137–139
139
Fig.2. Microscopic and flow cytometric analyses of the neurotoxicities of a-synuclein/OA complex. (A) Light microscopic images of SH-SY5Y cells treated with H2O2, OA, asynuclein, and a-synuclein/OA complex. (B) Calcein acetoxymethyl ester (calcein AM) and ethidium homodimer-I (Eth D-1) staining of SH-SY5Y cells treated with asynuclein, OA, and a-synuclein/OA complex. Live cells are stained with green fluorescent calcein AM. Damaged cells are stained with red fluorescent Eth D-1. (C) Flow cytometric analysis of cell death induced by a-synuclein/OA complex. SH-SY5Y cells treated with OA (blue), H2O2 (red), and a-synuclein/OA complex (purple) were analyzed using calcein AM and Eth D-1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the neurotoxic properties of a-synuclein/OA complex. Treatment with 100 mM H2O2 was used as a positive control. OA-treated cells resembled control cells, with a small decrease in fluorescence intensity of calcein AM (Fig. 2C). However, after a-synuclein/OA complex treatments, most cells were Eth D-1 positive, which was similar to the case of 100 mM H2O2 treatment. Previous studies have described the biochemical characterization and amyloid formation of a-synuclein and its tandem repeat (a-syn TR) [15]. In atomic force microscopy (AFM) studies, both a-synuclein and a-syn TR showed similar morphologies of amyloid fibrils (see Fig. S2 in supplementary material). In accordance with a-synuclein, SH-SY5Y cells treated with a-syn TR/OA complex suffered a considerable loss of viability and high LDH release through apoptosis-like death. By contrast, a-syn TR alone displayed negligible neurotoxicity. The extent of toxicity of a-syn TR/OA complex was comparable to that observed with a-synuclein/OA complex, indicating that length extension did not interfere with the neurotoxic properties. Interestingly, higher temperature incubations of a-syn TR with OA gave better neurotoxic activity, which was similar to the case of a-LA [12]. In summary, using MTT assay, LDH release, light microscopy, fluorescence microscopy, and flow cytometry, we observed neurotoxic properties of a-synuclein/OA complex. These a-synuclein/OA complexes could provide a valuable tool to study a-synuclein-related neurodegenerative mechanisms, although toxic species generated in vivo during the pathogenesis of PD remain to be identified. Acknowledgments This work was supported by a Korean Research Foundation grant funded by the Korean Government (KRF-2012R1A1A2000910). We acknowledge the help of Chan Bum Park (Korea Advanced Institute of Science and Technology) for AFM studies. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2013.01.005.
References [1] V.N. Uversky, D. Eliezer, Biophysics of Parkinson’s disease: structure and aggregation of a-synuclein, Curr. Protein Pept. Sci. 10 (2009) 483–499. [2] K.A. Jellinger, Recent advances in our understanding of neurodegeneration, J. Neural Transm. 116 (2009) 1111–1162. [3] E. Karran, M. Mercken, B. De Strooper, The amyloid cascade hypothesis for Alzheimer’s disease: an appraisal for the development of therapeutics, Nat. Rev. Drug Discov. 10 (2011) 698–712. [4] L. Crews, E. Masliah, Molecular mechanisms of neurodegeneration in Alzheimer’s disease, Hum. Mol. Genet. 19 (2010) R12–R20. [5] E.A. Waxman, B.I. Giasson, Molecular mechanisms of synuclein neurodegeneration, Biochim. Biophys. Acta 1792 (2009) 616–624. [6] X. Wang, F. Wang, L. Arterburn, R. Wollmann, J. Ma, The interaction between cytoplasmic prion protein and the hydrophobic lipid core of membrane correlates with neurotoxicity, J. Biol. Chem. 281 (2006) 13559–13565. [7] Y.J. Kim, R. Nakatomi, T. Akagi, T. Hashikawa, R. Takahashi, Unsaturated fatty acids induce cytotoxic aggregate formation of amyotrophic lateral sclerosislinked superoxide dismutase 1 mutants, J. Biol. Chem. 280 (2005) 21515– 21521. [8] J. Pettersson-Kastberg, A.K. Mossberg, M. Trulsson, Y.J. Yong, S. Min, Y. Lim, J.E. O’Brien, C. Svanborg, K.H. Mok, a-Lactalbumin, engineered to be nonnative and inactive, kills tumor cells when in complex with oleic acid: a new biological function resulting from partial unfolding, J. Mol. Biol. 394 (2009) 994– 1010. [9] H. Hwang, H. Choi, H.K. Kim, H. Jo, T.D. Kim, Ionic liquids promote amyloid formation from a-synuclein, Anal. Biochem. 386 (2009) 293–295. [10] M. Ahn, S. Kim, M. Kang, Y. Ryu, T.D. Kim, Chaperone-like activities of asynuclein: a-synuclein assists enzyme activities of esterases, Biochem. Biophys. Res. Commun. 346 (2006) 1142–1149. [11] S. Tolin, G. De Franceschi, B. Spolaore, E. Frare, M. Canton, P. Polverino de Laureto, A. Fontana, The oleic acid complexes of proteolytic fragments of alactalbumin display apoptotic activity, FEBS J. 277 (2010) 163–173. [12] T. Kamijima, A. Ohmura, T. Sato, K. Akimoto, M. Itabashi, M. Mizuguchi, M. Kamiya, T. Kikukawa, T. Aizawa, M. Takahashi, K. Kawano, M. Demura, Heattreatment method for producing fatty acid-bound a-lactalbumin that induces tumor cell death, Biochem. Biophys. Res. Commun. 376 (2008) 211–214. [13] G. Fotakis, J.A. Timbrell, In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT, and protein assay in hepatoma cell lines following exposure to cadmium chloride, Toxicol. Lett. 160 (2006) 171–177. [14] E.S. Kaneshiro, M.A. Wyder, Y. Wu, M.T. Cushio, Reliability of calcein acetoxy methyl ester and ethidium homodimer or propidium iodide for viability assessment of microbes, J. Microbiol. Methods 17 (1993) 1–16. [15] S.Y. Bae, S. Kim, H. Hwang, H.K. Kim, H.C. Yoon, J.H. Kim, S. Lee, T.D. Kim, Amyloid formation and disaggregation of a-synuclein and its tandem repeat (a-TR), Biochem. Biophys. Res. Commun. 400 (2010) 531–536.
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
A novel method for the preparation of a neurotoxic complex Bum Han Ryu, Eunjin Jang, Hansol Ju, T. Doohun Kim ⇑ Department of Molecular Science and Technology, Graduate School of Interdisciplinary Programs, Ajou University, Suwon 443-749, South Korea
a r t i c l e
i n f o
Article history: Received 10 October 2012 Received in revised form 28 December 2012 Accepted 7 January 2013 Available online 16 January 2013
a b s t r a c t Neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) can be attributed to the specific degeneration of neuronal cells in the brain. However, the natures and action modes of toxic species remain largely unknown. Here, we present a simple and fast method for the preparation of neurotoxic complex with a-synuclein, which is implicated in PD. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Neurotoxicity Apoptosis FACS
Neurodegenerative diseases such as Parkinson’s disease (PD),1 Alzheimer’s disease (AD), and amyotrophic lateral sclerosis (ALS) are marked by the progressive degeneration of neurons [1–3]. In these diseases, the pathogenesis has been linked to conformational changes of proteins and peptides that inappropriately cause them to gain neurotoxic properties. Although these diseases are characterized by the formation of pathological protein aggregation, the natures of neurotoxic species as well as their modes of action remain to be elucidated. Currently, no effective chemical treatments can prevent, rescue, or slow down these neurodegenerative diseases. One of the reasons is clearly the lack of knowledge about the exact nature of the neurotoxic species, although recent studies have largely focused on characterization of oligomeric protein complexes with neurotoxic properties [4,5]. Recently, protein/lipid complexes have been reported to show toxic properties. For example, the neurotoxicity of prion protein correlates with its interaction with lipids [6]. Furthermore, disease-related properties of superoxide dismutase 1 (SOD 1) could be induced by unsaturated fatty acids [7]. In accordance, partially unfolded a-lactalbumin (a-LA) complexed with oleic acid (OA) acquires apoptotic properties such as cytochrome c release and activating cell death pathways [8]. Here, we report a novel method for the preparation of a neurotoxic complex with a-synuclein, which is implicated in PD. a-Synuclein is a small presynaptic protein with a length of 140 amino acids [9]. As shown in Fig. 1A, the N-terminal region (aa 1–60) contains an 11-amino-acid repeat sequence for interaction with lipid ⇑ Corresponding author. Fax: +82 31 219 2394. E-mail address: [email protected] (T.D. Kim). Abbreviations used: PD, Parkinson’s disease; a-LA, a-lactalbumin; OA, oleic acid; NAC, non-amyloid component; PBS, phosphate-buffered saline; MTT, methyl tetrazolium; LDH, lactate dehydrogenase; FBS, fetal bovine serum; calcein AM, calcein acetoxymethyl ester; Eth D-1, ethidium homodimer-I; a-syn TR, a-synuclein and its tandem repeat; AFM, atomic force microscopy. 1
0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.01.005
membranes, whereas the C-terminal region (aa 96–140) is enriched in acidic amino residues for modulation of amyloid aggregation. The central non-amyloid component (NAC, aa 61–95) region is composed of mainly hydrophobic residues. Based on the toxic properties of protein/lipid complexes observed in several proteins, we investigated the neurotoxic properties of a-synuclein/OA complex. Initially, we conducted turbidity analysis, which is frequently used to measure the formation of large aggregates or complexes, to investigate the specific interactions between a-synuclein and OA [10,11]. Fig. 1B shows the absorbance of solutions containing increasing amounts of OA in phosphate-buffered saline (PBS) at pH 7.4 in the absence or presence of a-synuclein. The addition of OA led to a gradual increase in absorbance with the formation of large OA aggregates. However, after the addition of a-synuclein, the turbid OA suspension became clear, indicating that a-synuclein was able to inhibit the formation of large aggregates. In the presence of a-synuclein (10 lM), no significant increase in turbidity was observed up to 500 lM OA (Fig. 1B, filled circles). The addition of a-synuclein to 2 mM OA suspension led to a large decrease in absorbance compared with the OA solution alone. Furthermore, intrinsic fluorescence analysis also led to the conclusion that effective complex formations occurred with the addition of a-synuclein to OA suspension (see Fig. S1 in supplementary material). Based on these results, a-synuclein was directly suspended in the OA solution and the mixture was heated to facilitate efficient formation of the complex [12]. After incubation for 45 min, the mixtures were cooled to room temperature. To investigate neurotoxic properties of a-synuclein/OA complex toward human neuroblastoma SH-SY5Y cells, we employed the methyl tetrazolium (MTT) reduction and lactate dehydrogenase (LDH) release assay [13]. SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium, supplemented with 10% (v/v) fetal bovine serum (FBS), in a 5% CO2 atmosphere
138
Notes & Tips / Anal. Biochem. 435 (2013) 137–139
Fig.1. Characterization of the neurotoxic properties of a-synuclein/OA complex. (A) Schematic representation of a-synuclein and its tandem repeat (a-syn TR). (B) Turbidity analysis of OA solutions in the absence (s) or presence (d) of a-synuclein. Absorbance of samples containing up to 2 mM OA was measured at 405 nm. (C) Neurotoxic effects of a-synuclein/OA complex after heat treatments on SH-SY5Y cells were measured by the MTT assay. Cell viability is expressed as a percentage of controls. (D) LDH assay of SH-SY5Y cells treated with OA solutions in the absence or presence of a-synuclein (140 and 700 lM).
at 37 °C. Exponentially growing SH-SY5Y cells were seeded in 96well plates at a density of 5 104 cells/well. With approximately 80% confluence cells, a-synuclein (34.5 lM), OA (140 lM), or asynuclein/OA complex (1:20 molar ratio) was added to the culture medium. After incubation for 30 min at 37 °C, FBS was added to the culture. After 3 h of incubation, the SH-SY5Y cells were gently washed and incubated with 500 lg/ml MTT solution. The medium was then replaced with 100 ll of dimethyl sulfoxide (DMSO), and absorbance of blue formazan was measured at 570 nm using a microplate spectrophotometer (model 680, Bio-Rad, Hercules, CA, USA). Control cells were incubated with medium only, and their viability was considered to be 100%. The cell viability was expressed as a percentage of the absorbance compared with control cells. When cells were incubated with a-synuclein or OA alone, cell viability was comparable to that of the control, indicating that OA and a-synuclein possessed almost no cellular toxicities at these concentrations (Fig. 1C). However, the viability of SH-SY5Y cells treated with a-synuclein/OA complex (incubated at 37 °C) was reduced to approximately 20%. Higher temperature incubations (e.g., 50 or 60 °C) showed similar cellular toxic properties (Fig. 1C). LDH release, an indicator of cell membrane integrity, was evaluated by an LDH Cytotoxicity Detection Kit according to the manufacturer’s instructions (Takara Bio, Shiga, Japan). After incubation for 18 h in 96-well plates, a-synuclein (34.5 lM), OA (140 and 700 lM), and a-synuclein/OA complex in PBS were added to the culture medium. After 3.5 h, 100 ll of supernatants per well was harvested and transferred into a flat-bottomed 96-well plate. LDH reagent (100 ll) was added to each well, and the plates were incubated for 5 min at room temperature protected from light. The LDH activity was measured at 490/650 nm. In contrast to a-synuclein or OA alone, the addition of a-synuclein/OA resulted in significantly increased LDH activity. This increase was comparable to that induced
by 100 mM H2O2, that is, approximately 2.0-fold higher than basal levels (Fig. 1D). The morphological changes of SH-SY5Y cells after the addition of a-synuclein/OA complex were observed under a light microscope. Cells exposed to OA or a-synuclein alone were not visibly different from control cells (Fig. 2A). No significant cell deaths or cell growth retardation was observed, indicating the absence of cellular toxicity of a-synuclein or OA. However, after exposure to a-synuclein/OA complex, SH-SY5Y cells were severely damaged, suggesting that asynuclein/OA complex was strongly toxic. Similar to the addition of 100 mM H2O2, exposure to a-synuclein/OA complex results in apoptotic morphologies such as membrane rupture, cell shrinkage, loose cell attachment, and disruption of intercellular junctions (Fig. S1). To assess cell viability, live and/or dead cells were measured using a LIVE/DEAD Viability and Cytotoxicity Kit (Invitrogen, Carlsbad, CA, USA). SH-SY5Y cells, which were treated with a-synuclein, OA, and a-synuclein/OA complex for 3.5 h, were subjected to 2 lM calcein acetoxymethyl ester (calcein AM) and 4 lM ethidium homodimer-I (Eth D-1) staining. Calcein AM permeates all cells, leading to green fluorescence, whereas Eth D-1 gives a red fluorescence if cytoplasmic membrane integrity of cells is absent [14]. Approximately 40 min after staining, cells were examined using a fluorescence microscope (AE31, Motic, UK). Wavelengths for excitation/emission at 475/530 nm with a longpass filter of 542/ 620 nm were used. Representative images of the live (green-colored) and dead (red-colored) cells are shown in Fig. 2B. SH-SY5Y cells treated with a-synuclein or OA alone were mostly similar to control cells. However, red staining was seen in approximately 90% of cells treated with a-synuclein/OA complex, indicating that cell deaths had largely occurred. Furthermore, flow cytometry experiments using a FACSCalibur and CellQuest software (Becton Dickinson, San Jose, CA, USA) were also carried out to investigate
Notes & Tips / Anal. Biochem. 435 (2013) 137–139
139
Fig.2. Microscopic and flow cytometric analyses of the neurotoxicities of a-synuclein/OA complex. (A) Light microscopic images of SH-SY5Y cells treated with H2O2, OA, asynuclein, and a-synuclein/OA complex. (B) Calcein acetoxymethyl ester (calcein AM) and ethidium homodimer-I (Eth D-1) staining of SH-SY5Y cells treated with asynuclein, OA, and a-synuclein/OA complex. Live cells are stained with green fluorescent calcein AM. Damaged cells are stained with red fluorescent Eth D-1. (C) Flow cytometric analysis of cell death induced by a-synuclein/OA complex. SH-SY5Y cells treated with OA (blue), H2O2 (red), and a-synuclein/OA complex (purple) were analyzed using calcein AM and Eth D-1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the neurotoxic properties of a-synuclein/OA complex. Treatment with 100 mM H2O2 was used as a positive control. OA-treated cells resembled control cells, with a small decrease in fluorescence intensity of calcein AM (Fig. 2C). However, after a-synuclein/OA complex treatments, most cells were Eth D-1 positive, which was similar to the case of 100 mM H2O2 treatment. Previous studies have described the biochemical characterization and amyloid formation of a-synuclein and its tandem repeat (a-syn TR) [15]. In atomic force microscopy (AFM) studies, both a-synuclein and a-syn TR showed similar morphologies of amyloid fibrils (see Fig. S2 in supplementary material). In accordance with a-synuclein, SH-SY5Y cells treated with a-syn TR/OA complex suffered a considerable loss of viability and high LDH release through apoptosis-like death. By contrast, a-syn TR alone displayed negligible neurotoxicity. The extent of toxicity of a-syn TR/OA complex was comparable to that observed with a-synuclein/OA complex, indicating that length extension did not interfere with the neurotoxic properties. Interestingly, higher temperature incubations of a-syn TR with OA gave better neurotoxic activity, which was similar to the case of a-LA [12]. In summary, using MTT assay, LDH release, light microscopy, fluorescence microscopy, and flow cytometry, we observed neurotoxic properties of a-synuclein/OA complex. These a-synuclein/OA complexes could provide a valuable tool to study a-synuclein-related neurodegenerative mechanisms, although toxic species generated in vivo during the pathogenesis of PD remain to be identified. Acknowledgments This work was supported by a Korean Research Foundation grant funded by the Korean Government (KRF-2012R1A1A2000910). We acknowledge the help of Chan Bum Park (Korea Advanced Institute of Science and Technology) for AFM studies. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2013.01.005.
References [1] V.N. Uversky, D. Eliezer, Biophysics of Parkinson’s disease: structure and aggregation of a-synuclein, Curr. Protein Pept. Sci. 10 (2009) 483–499. [2] K.A. Jellinger, Recent advances in our understanding of neurodegeneration, J. Neural Transm. 116 (2009) 1111–1162. [3] E. Karran, M. Mercken, B. De Strooper, The amyloid cascade hypothesis for Alzheimer’s disease: an appraisal for the development of therapeutics, Nat. Rev. Drug Discov. 10 (2011) 698–712. [4] L. Crews, E. Masliah, Molecular mechanisms of neurodegeneration in Alzheimer’s disease, Hum. Mol. Genet. 19 (2010) R12–R20. [5] E.A. Waxman, B.I. Giasson, Molecular mechanisms of synuclein neurodegeneration, Biochim. Biophys. Acta 1792 (2009) 616–624. [6] X. Wang, F. Wang, L. Arterburn, R. Wollmann, J. Ma, The interaction between cytoplasmic prion protein and the hydrophobic lipid core of membrane correlates with neurotoxicity, J. Biol. Chem. 281 (2006) 13559–13565. [7] Y.J. Kim, R. Nakatomi, T. Akagi, T. Hashikawa, R. Takahashi, Unsaturated fatty acids induce cytotoxic aggregate formation of amyotrophic lateral sclerosislinked superoxide dismutase 1 mutants, J. Biol. Chem. 280 (2005) 21515– 21521. [8] J. Pettersson-Kastberg, A.K. Mossberg, M. Trulsson, Y.J. Yong, S. Min, Y. Lim, J.E. O’Brien, C. Svanborg, K.H. Mok, a-Lactalbumin, engineered to be nonnative and inactive, kills tumor cells when in complex with oleic acid: a new biological function resulting from partial unfolding, J. Mol. Biol. 394 (2009) 994– 1010. [9] H. Hwang, H. Choi, H.K. Kim, H. Jo, T.D. Kim, Ionic liquids promote amyloid formation from a-synuclein, Anal. Biochem. 386 (2009) 293–295. [10] M. Ahn, S. Kim, M. Kang, Y. Ryu, T.D. Kim, Chaperone-like activities of asynuclein: a-synuclein assists enzyme activities of esterases, Biochem. Biophys. Res. Commun. 346 (2006) 1142–1149. [11] S. Tolin, G. De Franceschi, B. Spolaore, E. Frare, M. Canton, P. Polverino de Laureto, A. Fontana, The oleic acid complexes of proteolytic fragments of alactalbumin display apoptotic activity, FEBS J. 277 (2010) 163–173. [12] T. Kamijima, A. Ohmura, T. Sato, K. Akimoto, M. Itabashi, M. Mizuguchi, M. Kamiya, T. Kikukawa, T. Aizawa, M. Takahashi, K. Kawano, M. Demura, Heattreatment method for producing fatty acid-bound a-lactalbumin that induces tumor cell death, Biochem. Biophys. Res. Commun. 376 (2008) 211–214. [13] G. Fotakis, J.A. Timbrell, In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT, and protein assay in hepatoma cell lines following exposure to cadmium chloride, Toxicol. Lett. 160 (2006) 171–177. [14] E.S. Kaneshiro, M.A. Wyder, Y. Wu, M.T. Cushio, Reliability of calcein acetoxy methyl ester and ethidium homodimer or propidium iodide for viability assessment of microbes, J. Microbiol. Methods 17 (1993) 1–16. [15] S.Y. Bae, S. Kim, H. Hwang, H.K. Kim, H.C. Yoon, J.H. Kim, S. Lee, T.D. Kim, Amyloid formation and disaggregation of a-synuclein and its tandem repeat (a-TR), Biochem. Biophys. Res. Commun. 400 (2010) 531–536.