Fluorescent Copper Probe Inhibiting Aβ1–16-Copper(II)-Catalyzed

Mar 20, 2017 - Synopsis. A fluorescent copper probe, OBEP-CS1, inhibits amyloid β (Aβ) peptide−copper-catalyzed intracellular reactive oxygen spec...
0 downloads 0 Views 972KB Size
Communication pubs.acs.org/IC

Fluorescent Copper Probe Inhibiting Aβ1−16-Copper(II)-Catalyzed Intracellular Reactive Oxygen Species Production Gulshan R. Walke,† Dnyanesh S. Ranade,† Shefali N. Ramteke,† Srikanth Rapole,‡ Cristina Satriano,§,⊥ Enrico Rizzarelli,§,∥,⊥ Gaetano A. Tomaselli,§ Giuseppe Trusso Sfrazzetto,§ and Prasad P. Kulkarni*,† †

Bioprospecting Group, Agharkar Research Institute, Pune, India Proteomics Laboratory, National Centre for Cell Science (NCCS), University of Pune Campus, Pune, India § Department of Chemical Sciences, University of Catania, Viale Andrea Doria 6, 95125 Catania, Italy ⊥ Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici, via Celso Ulpiani, 27, 70125 Bari, Italy ∥ Institute of Biostructures and Bioimages, National Council of Research, Via P. Gaifami 18, 95126 Catania, Italy ‡

S Supporting Information *

an area of interest for many researchers. The use of chelators specific for CuII and CuI is one of the approaches for the treatment of AD.9 For instance, a biocompatible phosphane 1,3,5-triaza-7-phosphadaamante ligand has been demonstrated to remove CuI from Aβ and prevent ROS production and oligomer formation.10 Recently, OBEP-CS1, (Scheme 1), a

ABSTRACT: A variety of fluorescent probes are proposed to monitor the intracellular copper content. So far, none of the probes have been evaluated for their potential to inhibit copper-associated intracellular oxidative stress. Herein, we studied the ability of a fluorescent copper probe, OBEP-CS1, to inhibit intracellular oxidative stress associated with an amyloid β (Aβ) peptide−copper complex. The data showed that OBEP-CS1 completely inhibits the copper-catalyzed oxidation as well as decarboxylation/deamination of Aβ1−16. Moreover, the cell imaging experiments confirmed that OBEP-CS1 can inhibit Aβ-CuII-catalyzed reactive oxygen species production in SH-SY5Y cells. We also demonstrated that Aβ1−16 peptide can bind intracellular copper and thereby exert oxidative stress.

Scheme 1. Structure of OBEP-CS1 (A) and Sequence of Aβ1− 16 Peptide (B)

fluorescent water-soluble CuI probe was developed, which demonstrated a good level of cellular uptake and no cytotoxicity up to relatively long incubation times.11,12 However, the potential of any fluorescent probe of copper against coppermediated intracellular oxidative stress has not been studied so far. Herein, we report on the ability of OBEP-CS1 to chelate CuI, avoiding copper-catalyzed oxidations of Aβ1−16 peptide. Aβ1− 16 peptide was used as a model for the full-length Aβ peptide because it contains binding sites for both CuII and CuI, and this truncated fragment does not aggregate easily.13 OBEP-CS1 is known to bind CuI ions in 1:1 stoichiometry, with an affinity binding constant K of 2.50 × 1013 M−1.11 Under reducing conditions, Aβ-CuII reduces to the Aβ-CuI complex, which displays a significantly lower binding constant of K = 1.9 × 107 M−1.14 Therefore, OBEP-CS1 is expected to bind CuI more tightly than monomeric Aβ in the competitive metal-binding process, thus chelating CuI ions from the Aβ1−16 environment and inhibiting copper-catalyzed oxidation of Aβ1−16. Thus, this study provides an excellent opportunity to correlate the change in the intracellular copper status and copper-catalyzed ROS generation in the presence of the Aβ1−16-CuII complex.

C

opper is present throughout the brain, and its dyshomeostasis is implicated directly or indirectly in the etiology of neurodegenerative diseases, including Alzheimer’s disease (AD), amyotrophic lateral sclerosis, Parkinson’s disease, and prion disease.1,2 Amyloid β (Aβ) peptide involved in AD is known to bind copper to form Aβ-CuII and Aβ-CuI complexes.3 Aβ peptide is a 40−42 amino acid chain and comprises a metal binding region (1−16) and an aggregation-prone region (17−40/42).3 CuII binds to His6 along with an N-terminal of peptide,4,5 while CuI binds to His13 and His14 in a linear fashion.6 In the presence of biological reductants such as ascorbate and glutathione, AβCuII reduces to Aβ-CuI. Further, in the presence of biological oxidants such as molecular oxygen, the Aβ-CuI complex is reoxidized to Aβ-CuII. The conversion of Aβ-CuI to Aβ-CuII initiates Fenton’s reaction to produce reactive oxygen species (ROS) and generates oxidative stress in the neuronal cell.7 The uninterrupted shuttling of copper bound to Aβ results in the generation of ROS extracellularly. In addition to this, ROS reacts with Aβ to produce toxic oxidation products such as dimer, trimer, etc.8 Copper is known to enhance peptide aggregation kinetics, leading to amyloid plaque formation in the brain, which is one of the hallmarks of AD.3 Thus, copper plays a crucial role in the neurodegeneration and progression of AD.1−3 An extracellular and intracellular copper-induced oxidative stress in AD is © XXXX American Chemical Society

Received: December 2, 2016

A

DOI: 10.1021/acs.inorgchem.6b02915 Inorg. Chem. XXXX, XXX, XXX−XXX

Communication

Inorganic Chemistry

OBEP-CS1, the mass spectrum of Aβ1−16-CuII showed the complete inhibition of oxidation peaks [m/z 1971, 1987, and 2003, decarboxylation (m/z 1925), and deamination (m/z 1941) peaks; Figure S1(B)]. Moreover, additional peaks were observed at m/z 1910 and 2018, the latter assigned to Aβ1−16-CuII adducts, not observed in the copper-catalyzed oxidation of Aβ1− 16 in the absence of OBEP-CS1. To investigate whether OBEP-CS1 is able to protect cells against ROS generated by Aβ1−16-CuII, we monitored ROS generation by using a CellROX green reagent in SH-SY5Y cells. CellROX green measures nuclear and cytosolic oxidative stresses in the cells.17 Neuroblastoma SH-SY5Y cells were treated with a preformed Aβ1−16-CuII complex in the absence and presence of OBEP-CS1 and imaged by laser confocal microscopy. The results in Figure 2, displaying only the fluorescence of a CellROX

To study the effect of OBEP-CS1 on the electron-transfer reaction of the Aβ1−16-CuII complex, we performed cyclic voltammetry and an ascorbate consumption experiment for the Aβ1−16-CuII complex in the absence and presence of OBEPCS1 (Figure 1). Thereafter, the copper-catalyzed oxidation

Figure 1. (A) Cyclic voltammograms of 250 μM Aβ1−16-CuII and 250 μM Aβ1−16-CuII in the presence of 250 μM OBEP-CS1. (B) Ascorbate consumption by 10 μM Aβ1−16-CuII and 10 μM Aβ1−16-CuII in the presence of 10 μM OBEP-CS1.

reaction of Aβ1−16 was carried out, and the products were analyzed using mass spectrometry. The voltammogram of the Aβ1−16-CuII complex shows a quasi-reversible redox wave with a E1/2 value of −0.0015 V, while in the presence of OBEP-CS1, a large positive shift is observed. The decrease in the cathodic and anodic current indicates OBEP-CS1 chelation of redox-active copper bound to Aβ1−16. As we previously reported, the positive shift in the redox wave suggests a decrease in H2O2 generation during the redox cycle.15,16 To study the formation of ROS by Aβ1−16-CuII, we carried out ascorbate consumption assay. An exactly similar rate of autooxidation of ascorbate was observed in all of the samples until 5 min had elapsed. In the following minutes, in the absence of OBEP-CS1, a rapid and complete consumption of ascorbate by Aβ1−16-CuII was obtained within 20 min. On the other hand, in the presence of OBEP-CS1, ascorbate consumption by Aβ1−16-CuII was significantly inhibited, with only a small amount of ascorbate consumed by Aβ1−16-CuII with a slower rate of ascorbate oxidation. The higher rate of ascorbate oxidation by Aβ1−16CuII corresponds to the greater ability to generate H2O2. The above findings suggest that OBEP-CS1 inhibits Aβ1−16CuII-catalyzed ROS generation. The ROS generated, such as hydroxyl radicals (•OH), can further react with Aβ, which leads to the formation of toxic oxidation products of Aβ and oligomeric species.8,15 The effects of OBEP-CS1 on the copper-catalyzed oxidation of Aβ1−16 and on the formation of oxidation products were analyzed using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS; see the Supporting Information for details). Aβ1−16-CuII was reduced to Aβ1−16-CuI species by ascorbic acid and then allowed to oxidize back to Aβ1−16-CuII in the presence of atmospheric oxygen. The generated ROS target the amino acid residues nearby the copperbinding site. OBEP-CS1 alone was found to behave as redoxinactive because it did not contribute to the generation of ROS. Thus, the extent of oxidation of Aβ1−16 was considered to be a measure of the effect of OBEP-CS1 on the redox reaction of Aβ1−16-CuII. In the absence of OBEP-CS1, the mass spectrum of Aβ1−16-CuII showed three oxidation peaks at m/z 1971, 1987 and 2003, respectively (Figure S1(A)). Additionally, three peaks at m/z 1941, 1925 and 1910 were also observed, assigned to deamination, decarboxylation, and decarboxylation/deamination of the Asp1 residue of Aβ1−16, respectively.15 In the presence of

Figure 2. Live confocal imaging of SH-SY5Y cells stained with 5 μM CellROX green upon 10 min of treatment (λex/em = 485/500−550 nm): (A) cells treated with 50 μM Aβ1−16-CuII (1:1) for 18 h; (B) cells treated with 50 μM Aβ1−16-CuII for 18 h, washed with phosphatebuffered saline, and treated with 5 μM OBEP-CS1 for 15 min; (C) normalized mean intensity of emission from quantitative analysis (by ImageJ software) of the green region in images A and B.

green probe, showed that cell treatment with Aβ1−16-CuII generates significant ROS production, as evidenced by the intense intracellular green fluorescence of CellROX compared to that of control cells (Figure S1 and S3). In the presence OBEPCS1, a significant decrease (Figure 2C) in the intensity of green fluorescence was observed. In Figure 3, the intracellular fluorescence of both OBEP-CS1 (in red) and CellROX (in green), together with the merged images, are shown. In the micrographs in Figure 3, the red fluorescence of OBEP-CS1 shows a decrease in intensity in response to the increase in the intracellular concentration of CuI ions. At the same time, the CellROX green fluorescence increase indicates an increase in ROS generation. This gives an excellent opportunity to track the correlation between intracellular copper and the levels of intracellular ROS generation. Untreated cells exhibit a diffuse red fluorescence, most intense in the extranuclear area, due to the intrinsic emission of the OBEPCS1 probe in basal conditions, as well as a green fluorescence, also spread in the whole cytoplasm but most intense in the nuclei. The merged image well displays the different preferential distribution of the two probes. It was earlier reported that Aβ peptide particularly increases mitochondrial oxidative stress.18 Indeed, after treatment with 50 μM Aβ1−16 peptide, the cells show a remarkable decrease of nuclear red fluorescence and an increase in red fluorescence along a pattern that likely displays the possible mitochondrial organelle distribution compared to the background diffuse fluorescence. Simultaneously, the green fluorescence increases in intensity both in the nuclei and in the cytoplasm, pointing to the increase in ROS formation, thus confirming that Aβ1−16 is able to compete with OBEP-CS1 to bind intracellular labile copper, which results in an increase in B

DOI: 10.1021/acs.inorgchem.6b02915 Inorg. Chem. XXXX, XXX, XXX−XXX

Communication

Inorganic Chemistry

ROS, leading to a decrease in the green fluorescence intensity. A comparison of the red fluorescence intensities in the presence of CuSO4 and a preformed Aβ1−16-CuII complex suggests that OBEP-CS1 is able to compete with Aβ for copper binding. Overall, our in vitro studies suggest that OBEP-CS1 is able to bind copper, decrease the formation of Aβ-CuII, and subsequently reduce intracellular oxidative stress. The fluorescent nature of OBEP-CS1 allows us to monitor the intracellular copper status and corresponding oxidative stress using a CellROX green reagent. We were also able to demonstrate for the first time that Aβ1−16 peptide can bind intracellular copper and thereby exert oxidative stress.20 In conclusion, OBEP-CS1, a water-soluble, fluorescent, nontoxic CuI sensor, was studied in this work in terms of its effect on Aβ-CuII-catalyzed ROS production. A nonstop shuttling of Aβ-CuII/CuI is responsible for ROS production, which subsequently results in oxidative stress in neuronal cells. We carried out cyclic voltammetry and ascorbate consumption experiments, which demonstrated that OBEP-CS1 can interfere in the redox cycling of the Aβ-CuII complex and thereby decrease Aβ-CuII-catalyzed ROS production. The •OH generated by AβCuII reacts with Aβ peptide itself. The mass spectrometry data confirmed that OBEP-CS1 completely inhibits the coppercatalyzed oxidation of Aβ1−16 peptide, with decarboxylation/ deamination processes also inhibited moderately. Moreover, the cell imaging experiments confirmed that OBEP-CS1 can inhibit Aβ-CuII-catalyzed ROS production in SH-SY5Y cells. OBEPCS1 can also be used in combination with CellROX green for simultaneous monitoring of Aβ peptide-associated intracellular copper redistribution and subsequent changes in ROS production in SH-SY5Y.



Figure 3. Confocal images of SH-SY5Y cells incubated for 18 h with 50 μM Aβ1−16-CuII (1:1), then further treated with 5 μM OBEP-CS1 for 15 min, washed with a phosphate-buffered saline (pH 7.4), and stained with 5 μM CellROX green (10 min of treatment): (A) OBEP fluorescence (λex/em = 543/560−600 nm); (B) CellROX green (λex/em = 485/500−550 nm); (C) merged images of A + B. (D) Comparison of the red and green regions in images A and B by using ImageJ analysis.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b02915.

intracellular oxidative stress. Similar results were obtained for full-length Aβ1−42 peptide (Figure S4 and S5). It is difficult to perform experiments using Aβ1−42 peptide in the presence of copper because the addition of copper to Aβ1−42 peptide results in rapid aggregation of the peptide. We have therefore continued using Aβ1−16 peptide for further experiments. In the presence of Aβ1−16, the intensities of red and green fluorescence were found to be highest among all of the samples, assigned as 100%. In control cells, the red and green fluorescence intensities were found to be 91% and 64%, respectively (Figure 3D). We and others have already demonstrated that the Aβ-CuII complex is toxic to cells and the mode of action is the formation of ROS.19 Therefore, we further treated cells with a preformed Aβ-CuII complex. In the presence of Aβ-CuII, the red and green fluorescence intensities were found to be 75% and 63%, respectively (Figure 3D). The cell treatment with 50 μM AβCuII shows an overall decrease in the red fluorescence in comparison to untreated cells, corresponding to OBEP-CS1 binding to the CuI ions, and a concomitant decrease in the green fluorescence, indicating decreased cellular oxidative stress. Finally, the treatment with 50 μM CuSO4 leads to a decrease in the intensities of both red and green fluorescence, 40% and 55%, respectively (Figure 3D). OBEP-CS1 forms a highly stable complex with CuI and, hence, may prevent the generation of



Experimental material and procedures for coppercatalyzed oxidation, MALDI-MS, cyclic voltammetry, and confocal images (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Cristina Satriano: 0000-0001-5348-5863 Gaetano A. Tomaselli: 0000-0002-6146-6934 Prasad P. Kulkarni: 0000-0002-3929-2990 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS

This work was funded by the Department of Science and Technology, India (EMR/2014/001235). S.N.R. thanks University Grants Commission, Government of India, for SRF. The authors thank V. Naik, NCCS, for help with mass spectrometry and Dr. K. M. Paknikar, Agharkar Research Institute, for use of their instrumental facility. C

DOI: 10.1021/acs.inorgchem.6b02915 Inorg. Chem. XXXX, XXX, XXX−XXX

Communication

Inorganic Chemistry



(18) Hu, H.; Tan, C. C.; Tan, L.; Yu, J. T. A mitocentric view of Alzheimer’s disease. Mol. Neurobiol. 2016, DOI: 10.1007/s12035-0160117-7. (19) (a) Walke, G. R.; Ranade, D. S.; Bapat, A. M.; Srikanth, R.; Kulkarni, P. P. Mn(III)-Salen protect against different ROS species generated by the Aβ16-Cu complex. ChemistrySelect 2016, 1, 3497− 3501. (b) Ramteke, S. N.; Walke, G. R.; Joshi, B. N.; Rapole, S.; Kulkarni, P. P. Effects of oxidation on redox and cytotoxic properties of copper complex of Aβ1−16 peptide. Free Radical Res. 2014, 48, 1417−25. (c) Huang, X.; Moir, R. D.; Tanzi, R. E.; Bush, A. I.; Rogers, J. T. Redoxactive metals, oxidative stress, and Alzheimer’s disease pathology. Ann. N. Y. Acad. Sci. 2004, 1012, 153−163. (20) White, A. R.; Barnham, K. J.; Bush, A. I. Metal homeostasis in Alzheimer’s disease. Expert Rev. Neurother. 2006, 6, 711−722.

REFERENCES

(1) Gaggelli, E.; Kozlowski, H.; Valensin, D.; Valensin, G. Copper homeostasis and neurodegenerative disorders (Alzheimer’s, prion, and Parkinson’s diseases and amyotrophic lateral sclerosis). Chem. Rev. 2006, 106, 1995−2044. (2) Pappalardo, G.; Milardi, D.; Rizzarelli, E.; Sovago, I. The inorganic side of Alzheimer’s disease. In Neurodegeneration: Metallostasis and Proteostasis; Milardi, D., Rizzarelli, E., Eds.; Royal Chemical Society, 2011; Chapter 6, pp 112−140. (3) (a) Kepp, K. P. Bioinorganic chemistry of Alzheimer’s disease. Chem. Rev. 2012, 112, 5193−5239. (b) Faller, P.; Hureau, C. Bioinorganic chemistry of copper and zinc ions coordinated to amyloid-beta peptide. Dalton Trans. 2009, 7, 1080−1094. (4) Ginotra, Y. P.; Ramteke, S. N.; Srikanth, R.; Kulkarni, P. P. Mass spectral studies reveal the structure of Aβ1−16−Cu2+ complex resembling ATCUN motif. Inorg. Chem. 2012, 51, 7960−7962. (5) Damante, C. A.; Osz, K.; Nagy, Z.; Pappalardo, G.; Grasso, G.; Impellizzeri, G.; Rizzarelli, E.; Sovago, I. The metal loading ability of beta-amyloid N-terminus: a combined potentiometric and spectroscopic study of copper(II) complexes with beta-amyloid(1−16), its short or mutated peptide fragments, and its polyethylene glycol (PEG)ylated analogue. Inorg. Chem. 2008, 47, 9669−9683. (6) Shearer, J.; Szalai, V. A. The amyloid-β peptide of Alzheimer’s disease binds Cu(I) in a linear bis-His coordination environment: Insight into a possible neuroprotective mechanism for the amyloid-β peptide. J. Am. Chem. Soc. 2008, 130, 17826−17835. (7) Huang, X.; Cuajungco, M. P.; Atwood, C. S.; Hartshorn, M. A.; Tyndall, J. D. A.; Hanson, G. R.; Stokes, K. C.; Leopold, M.; Multhaup, G.; Goldstein, L. E.; Scarpa, R. C.; Saunders, A. J.; Lim, J.; Moir, R. D.; Glabe, C.; Bowden, E. F.; Masters, C. L.; Fairlie, D. P.; Tanzi, R. E.; Bush, A. I. Cu(II) potentiation of Alzheimer Aβ neurotoxicity- Correlation with cell-free hydrogen peroxide production and metal reduction. J. Biol. Chem. 1999, 274, 37111−37116. (8) Ramteke, S. N.; Ginotra, Y. P.; Walke, G. R.; Joshi, B. N.; Kumbhar, A. S.; Rapole, S.; Kulkarni, P. P. Effects of oxidation on copper-binding properties of Aβ1−16 peptide: A pulse radiolysis study. Free Radical Res. 2013, 47, 1046−1053. (9) Rodriguez-Rodriguez, C.; Telpoukhovskaia, M.; Orvig, C. The art of building multifunctional metal-binding agents from basic molecular scaffolds for the potential application in neurodegenerative diseases. Coord. Chem. Rev. 2012, 256, 2308−2332. (10) Atrian-Blasco, E.; Cerrada, E.; Conte-Daban, A.; Testemale, D.; Faller, P.; Laguna, M.; Hureau, C. Copper(I) targeting in the Alzheimer’s disease context: a first example using the biocompatible PTA ligand. Metallomics 2015, 7, 1229−1232. (11) Giuffrida, M. L.; Rizzarelli, E.; Tomaselli, G. A.; Satriano, C.; Trusso Sfrazzetto, G. A novel fully water-soluble Cu(I) probe for fluorescence live cell imaging. Chem. Commun. 2014, 50, 9835−9838. (12) Trusso Sfrazzetto, G.; Satriano, C.; Tomaselli, G.; Rizzarelli, E. Synthetic fluorescent probes to map metallostasis and intracellular fate of zinc and copper. Coord. Chem. Rev. 2016, 311, 125−167. (13) Alies, B.; Renaglia, E.; Rozga, M.; Bal, W.; Faller, P.; Hureau, C. Cu(II) Affinity for the Alzheimers Peptide: Tyrosine fluorescence studies revisited. Anal. Chem. 2013, 85, 1501−1508. (14) Alies, B.; Badei, B.; Faller, P.; Hureau, C. Re-evaluation of copper(I) affinity for amyloid-β peptides by competition with ferrozineAn unusual copper(I) indicator. Chem. - Eur. J. 2012, 18, 1161−1167. (15) Walke, G. R.; Rapole, S.; Kulkarni, P. P. Cisplatin inhibits the formation of a reactive intermediate during copper-catalyzed oxidation of amyloid β peptide. Inorg. Chem. 2014, 53, 10003−10005. (16) Liu, L.; Jiang, D.; McDonald, A.; Hao, Y.; Millhauser, G. L.; Zhou, F. Copper redox cycling in the prion protein depends critically on binding mode. J. Am. Chem. Soc. 2011, 133, 12229−12237. (17) Luís, A.; Martins, J. D.; Silva, A.; Ferreira, I.; Cruz, M. T.; Neves, B. M. Oxidative stress-dependent activation of the eIF2α−ATFr unfolded protein response branch by skin sensitizer 1-fluoro-2,4-dinitrobenzene modulates dendritic-like cell maturation and inflammatory status in a biphasic manner. Free Radical Biol. Med. 2014, 77, 217−229. D

DOI: 10.1021/acs.inorgchem.6b02915 Inorg. Chem. XXXX, XXX, XXX−XXX