Copper brain protein protection against free radical-induced neuronal death: Survival ratio in SH-SY5Y neuroblastoma cell cultures

Journal of Trace Elements in Medicine and Biology 39 (2017) 50–53

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Copper brain protein protection against free radical-induced neuronal death: Survival ratio in SH-SY5Y neuroblastoma cell cultures Roger Deloncle a,∗ , Bernard Fauconneau b , Olivier Guillard c,1 , José Delaval d , Gérard Lesage e , Alain Pineau f,1 a

Université Franc¸ois Rabelais de Tours, Toxicology Laboratory Faculty of Pharmacy, 31 Avenue Monge, 37200 Tours, France GREVIC, EA 3808, Biology and Health Pole, 86022 Poitiers, France c University of Poitiers, Faculty of Medicine and Pharmacy, 86000 Poitiers, France d Touraine Laboratory, Indre et Loire General Council, BP 67357- 37073 Tours Cedex 02, France e Université Franc¸ois Rabelais de Tours, Virology-Immunology Laboratory Faculty of Pharmacy, 31 Avenue Monge, 37200 Tours, France f Université de Nantes, Toxicology Laboratory Faculty of Pharmacy, 9 Rue Bias, BP 53508- 44035 Nantes Cedex 1, France b

a r t i c l e

i n f o

Article history: Received 10 February 2016 Received in revised form 3 May 2016 Accepted 27 July 2016 Keywords: Copper Manganese Free radicals SH-SY5Y cells Neuronal death Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

a b s t r a c t In Creutzfeldt Jakob, Alzheimer and Parkinson diseases, copper metalloproteins such as prion, amyloid protein precursor and ␣-synuclein are able to protect against free radicals by reduction from cupric Cu+2 to cupreous Cu+ . In these pathologies, a regional copper (Cu) brain decrease correlated with an iron, zinc or manganese (Mn) increase has previously been observed, leading to local neuronal death and abnormal deposition of these metalloproteins in ␤-sheet structures. In this study we demonstrate the protective effect of Cu metalloproteins against deleterious free-radical effects. With neuroblastoma SH-SY5Y cell cultures, we show that bovine brain prion protein in Cu but not Mn form prevents free radical-induced neuronal death. The survival ratio of SH-SY5Y cells has been measured after UV irradiation (free radical production), when the incubating medium is supplemented with bovine brain homogenate in native, Cu or Mn forms. This ratio, about 28% without any addition or with bovine brain protein added in Mn form, increases by as much as 54.73% with addition to the culture medium of native bovine brain protein and by as much as 95.95% if the addition is carried out in cupric form. This protective effect of brain copper protein against free radical-induced neuronal death has been confirmed with Inductively Coupled Plasma Mass Spectrometry Mn and Cu measurement in bovine brain homogenates: respectively lower than detection limit and 9.01 ␮g/g dry weight for native form; lower than detection limit and 825.85 ␮g/g dry weight for Cu-supplemented form and 1.75 and 68.1 ␮g/g dry weight in Mn-supplemented brain homogenate. © 2016 Elsevier GmbH. All rights reserved.

1. Introduction In Creutzfeldt Jakob (CJD) and Alzheimer’s diseases (AD), it has been shown that copper (Cu) bound to prion protein or amyloid protein precursor (APP) is able to protect against the deleterious effects of free radicals by valence reduction from Cu+2 to Cu+ [1,2]. In Parkinson (PD) and Lewy Body (LBD) diseases, Cu can bind with ␣synuclein [3] even though this metal has been suspected in protein misfolding [4,5]. In these pathologies, a regional brain Cu decrease is observed with abnormal deposition of brain copper proteins in proteinase K-resistant ␤-sheet conformations. A manganese (Mn)

∗ Corresponding author. E-mail address: [email protected] (R. Deloncle). 1 SFERETE (Société Francophone d’Etude et de Recherche sur les Eléments traces Toxiques Essentiels) member. http://dx.doi.org/10.1016/j.jtemb.2016.07.006 0946-672X/© 2016 Elsevier GmbH. All rights reserved.

increase has been described in human or animal transmissible spongiform encephalopathies [6,7]. Subsequent to a local brain Cu substitution by bivalent cations (Fe++ , Zn++ , Mn++ ), we have recently proposed a free radical mechanism aimed at explaining CJD, AD, PD and LBD diseases [8]. Since they are in their lower oxidation degree +2, these bivalent cations cannot be further reduced. Free radicals, by accumulation in the cellular medium, will then induce local neuronal death and also, following a radical chain process, generate D-amino acid in metalloprotein sequences leading to their deposition as ␤-sheet structures. Deloncle et al. [9] have shown with bovine brain homogenates that in prion protein, Cu could be substituted for transition metals such as Mn in reductive medium, the substitution being reversible on return to oxidative conditions. In order to demonstrate the protective effect of Cu metalloproteins against free radicals’ deleterious effects, we wish to show

R. Deloncle et al. / Journal of Trace Elements in Medicine and Biology 39 (2017) 50–53

with neuroblastoma SH-SY 5Y cell cultures that bovine brain prion protein in Cu but not Mn form prevents free radical-induced neuronal death. The survival ratio of SH-SY 5Y cells is measured after UV-induced free radical irradiation when the incubating medium is supplemented with bovine brain homogenate in native form or with Cu or Mn.

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2. Materials and methods

was withdrawn from the eight flasks and replaced with 4 ml nutritive medium. The cell cultures were then supplemented with the different Obex suspensions containing 120 ␮g protein/ml for a total 5 ml volume. Flasks 1 and 2 were supplemented with 1 ml nutritive medium more; flasks 3 and 4 with 1 ml diluted native bovine brain homogenate; flasks 5 and 6 with 1 ml diluted copper bovine brain homogenate; flasks 7 and 8 with 1 ml diluted manganese bovine brain homogenate.

2.1. Chemicals

2.5. Irradiation process

Nitric acid suprapure® and chemicals of analytical grade for preparation of bovine brain homogenates were purchased from Merck. All chemicals for Cu and Mn (ICP-MS) measurements and Triton X100 were of suprapure quality and purchased from Fisher Scientific (Illkirch, France), Rhodium from SCP Sciences (Villebon sur Yvette, France) For cell cultures, chemicals were of analytical grade and the quantity of total proteins was measured with the Quant-it® protein assay, all purchased from Gibco Invitrogen laboratories.

Flasks with even numbers were submitted under slight stirring to a 5 min UV irradiation with a SOL2 apparatus under 14 mW/cm2 . Odd-numbered flasks were not submitted to irradiation. After UVA irradiation, the nutritive mediums of all the flasks were removed and rinsed twice with 5 ml PBS. They were then filled with 15 ml of nutritive medium supplemented with retinoic acid (10−5 M in sterile DMSO) and allowing for 24 more hours of cell growth before numeration of the surviving cells. Numeration of the surviving cells was carried on as follows: The medium was withdrawn and the flask rinsed twice with 5 ml PBS before addition of 2 ml trypsin EDTA and 8 ml nutritive culture medium. Centrifugation was then carried out at +4 ◦ C for 5 min at 1500 rpm. The supernatant was removed and replaced with 1 ml of nutritive medium. The resulting cell suspension in nutritive medium was diluted 1:10 with 0.2% PBS Blue Trypan for numeration with a Malassez cell.

2.2. Instrumentation UVA irradiation was performed at 372 nm with a SOL2 apparatus (Hönle, Plannegg, Germany). ICP-MS: Instrument parameters using a NexION® 3OOX ICPMS spectrometer (PerkinElmer® , Inc., Shelton, CT, USA), are in agreement with Li et al. [10]. 2.3. Sample preparation of bovine brain obex homogenate Bovine brain homogenates were prepared, from 300 bovine brain obex negative to Prionics test [11]. Protein purification was carried out by trichloroacetic defecation before division into three identical fractions. The first fraction of non-treated bovine obex homogenate was submitted to dialysis at +4 ◦ C in phosphate buffer saline (pH 7.2) on an Amicon ultrafiltration apparatus equipped with a Millipore cellulose regenerated membrane allowing filtration retention for molecules over 3000 kDa. Filtration was carried on until Cu or Mn was found in the dialysate under the limit of detection (LOD) by ICP MS. The second fraction was set up under oxidative conditions by addition of 5 mM sodium persulfate and supplemented with 10−4 M Cu sulfate. This persulfate concentration was verified as oxidative in the homogenate by precipitating copper as blue cupric hydroxide. This second fraction was dialysed as described above. The third fraction was set up under reductive conditions by addition of 5 mM hydrogen peroxide. These conditions were verified as reductive in the homogenate by precipitating Cu as brown cuprous hydroxide. This third fraction was then supplemented by manganese sulfate (10−4 M), and submitted to dialysis like the first fraction. In order to obtain fluid mixtures suitable for further uses, all three resulting dialysed bovine brain homogenates were diluted up to 80% PBS and pH adjusted to 7.00.

2.6. ICP-MS copper and manganese analysis Samples of 1 ml of the diluted bovine brain homogenates native, or subjected to Cu or Mn supplementation, were digested according to the method of van Ginkel et al. [12] as modified for dry-weight samples. After homogenization, mineralized samples were diluted 1/100 in a mixed solution of nitric acid (1%) and Rh (100 ␮g/L). Standards and reference material were prepared following the same procedure. Aqueous standard solutions prepared daily from mono-element standard of 1 g/L were used for calibration. As the differences of the calibration slopes of samples and acid-based standards were less than 10%, the external calibration mode (linear thru zero) was used. Concentrations of the elements were as follows: blank, 5, 10, 20, 50 and 100 ␮g/L for Cu and blank, 2, 5, 10, 20 and 50 ␮g/L for Mn. To compensate for possible instrumental drift and matrix effects, all samples and solutions contained of Rh and 0.5% of Triton X100 (1% in ultrapure water). Peak area mode and 2 s data acquisition time and three replicates were used for measurement. 2.7. Quality assurance and quality control For assessment of the accuracy and precision of the concentration of Cu and Mn determined in mineralized samples, a certified reference material NIST 1640a (National Institute of Standards & Technology, USA) was used.

2.4. SH-SY 5Y neuroblastoma cell culture

2.8. Statistical analysis

The human SH-SY5Y neuroblastoma cell line(ATCC® ) was cultured in a Memmert incubator (+37 ◦ C, 5% CO2 , hygrometry 80%) and was initially allowed to grow in a single 75 cm2 flask (NUNCTM ) before seeding evenly into eight 25 cm2 flasks (NUNCTM ). They were then allowed to grow until cell confluence (3 × 106 cells/flask) before differentiation with retinoic acid (10−5 M in sterile DMSO). The culture change was carried on every two days. In order to avoid a possible effect of retinoic acid with regard to free radicals on bovine brain obex homogenate, whole nutritive medium

Results were analyzed using GraphPad Prism® software. Comparisons between groups were performed by ANOVA followed by Newman-Keuls’ test. The level of significance was set at p < 0.05. 3. Results and discussion In Table 1, the survival percentages of neuroblastoma cells after 5 min of UVA irradiation are indicated. The cell suspensions supplemented with 20% diluted Obex-Cu in cupric form present a

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R. Deloncle et al. / Journal of Trace Elements in Medicine and Biology 39 (2017) 50–53

Table 1 Determination of the survival rate (%) of SH-SY5Y cells after 5 min UVA irradiation. n = 10

Cell culture without addition (control)

Cell culture + Native Obex 20%

Cell culture + Obex-Cu 20%

Cell culture + Obex-Mn 20%

Meana (±SD)

27.92 ± 0.07

54.73 ± 2.55***

95.95 ± 2.28*** , †††

28.06 ± 1.52‡‡‡ , †††

a *** ‡‡‡ †††

Triplicate analysis for each sample. Significantly different from control (p < 0.001). Significantly different from Obex-Cu 20% (p < 0.001). Significantly different from Native Obex 20% (p < 0.001).

Table 2 Copper and manganese measurement in 20% diluted bovine brain homogenates. n = 13

b

Mean (±SD) a b *** ‡‡‡

Native Obex 20%

Obex-Cu 20%

Obex-Mn 20%

Cua

Mna

Cua

Mna

9.01 ± 0.50

≤L.O.D.

825.85 ± 26.14

***

≤L.O.D.

Cua

Mna ‡‡‡ , ***

1.75 ± 0.27

68.10 ± 2.46

␮g/g dry weight. Triplicate analysis for each sample; L.O.D.: Limit of Detection. Significantly different from Cu in Native Obex 20% (p < 0.001). Significantly different from Cu in Obex-Cu 20% (p < 0.001).

survival rate significantly higher (p < 0.001) than that of the control without any added Obex. Following addition of a 20% diluted Obex-Mn suspension, the survival rate was essentially the same as that observed in the control, without supplementation, [28.06% and 27.92% respectively (NS)], but was very significantly different from that observed after addition of Obex-Cu (p < 0.001). It should also be noted that addition of 20% diluted native Obex translated into a significantly increased survival ratio (p < 0.001). In order to explain the differing survival ratios in Obex-Cu and Obex-Mn, it seemed indispensable to measure the Cu and Mn concentrations in the different additions of Obex to the SH-SY 5Y cell suspensions. Assay of these metals was performed by ICP-MS after mineralization of the different Obex homogenates. This ICP-MS method has been validated in our laboratory. All sample preparations were carried out and analyzed in a clean laboratory environment, equipped with a laminar flow bench and fume cupboards. Details of procedures have been described by Pineau et al. [13]. After mineralization of certified reference samples, mean values for Cu and Mn were 82.12 ± 2.32 ␮g/L and 40.16 ± 1.20 ␮g/L (n = 6) vs 85.75 ± 0.51 ␮g/L and 40.39 ± 0.36 ␮g/Lrespectively. Their stability was tested daily (within-run, n = 6) and over longer periods of 14 days (betweenrun, n = 14). The CVs (%) were 0.67% and 0.90% for Cu and Mn respectively as regards within-run precision and 0.54% and 1.56% for Cu and Mn as regards between-run precision (all measurements in three replicates). The results showed low variability and high stability. In addition, it bears mentioning that detection limits (LOD: ␮g/L) and quantification limits (LOQ: ␮g/L) were calculated as 3 times and 10 times the standard deviation of replicate measurement of the blank reagent respectively. For LOD, the results were 1.0 ␮g/L and 0.5 ␮g/L and for LOQ they were 3.0 ␮g/L and 1.0 ␮g/L for Cu and Mn respectively. In light of these analyses, we consider this method as being adapted to detection of low Cu and Mn concentrations. In agreement with this validation, Cu and Mn concentrations in various diluted brain homogenates are given in Table 2. These results show that 20% diluted native-Obex brain homogenates contain little Cu and that measured Mn concentration is lower than the LOD. Preparation of the Obex-Cu suspension indicates that in the native-Obex samples, the maximum capacity of Cu fixation was far from having been reached: in an oxidizing environment, Cu fixation can be close to one hundred times greater (825.85 vs. 9.01 ␮g/g dry wt; p < 0.001). As for preparation of the Obex-Mn suspensions in a reducing medium, it seems that on all the sites where Cu atoms have been fixed, they cannot be totally exchanged for Mn atoms, since Mn fixation at saturation is lower than that observed for Cu

(68.10 vs. 825.85 ␮g/g dry wt). It also bears mentioning that the Obex-Mn homogenate continues to contain a residual concentration of Cu (1.75 ± 0.27 ␮g/g dry wt), a finding tending to prove that in the homogenates, fixation sites for Cu and Mn are indeed not altogether interchangeable. Given the results shown in Table 1, subsequent to addition of an Obex-Cu suspension we have observed a heightened survival rate, which would appear to indicate that with regard to free radicals, copper fulfills a protective function. Analogous to the protection afforded by superoxide dismutase (SOD), this function has been demonstrated in prion protein [1]. In a previous study [9], we noted that in metalloproteins, substitution of Mn for Cu resulted in inhibition of superoxide dismutase-like protective properties against free radicals and it appears (Table 1) that cell survival rate following addition of Obex Mn (28.06%) is practically identical to the cell survival rate observed in the absence of any supplementation (control: 27.92%). This result justifies the affirmation that Obex Mn supplementation affords no protection against free radicals. Conversely, even when copper concentration is pronouncedly lower (9.01 ␮g/g dry wt) than that of Obex-Cu (825.85 ␮g/g dry wt), the addition of native Obex tangibly enhances protection (54.73%). To sum up, the fact that added Obex-Cu is more protective than added native Obex appears congruent with the fact that Cu concentration is higher in Obex-Cu. In light of these observations, preventive efforts aimed at combating the deleterious effects of free radicals could be reinforced by the protective effects of Cu metalloproteins. In neurodegenerative diseases, these kinds of deleterious effects can be observed subsequent to a brain copper deficiency as has been shown in Parkinson and Lewy body [14,15], Alzheimer [16] and Creutzfeldt Jakob [17], and a number of controversial hypotheses have been put forward concerning the role of this metal in these pathologies. Exley et al. [18] showed copper diminution in 60 human brains to be correlated with higher deposition of ␤-amyloid in brain tissue and concluded that copper might fulfill a protective function in AD and related disorders. Conversely, Singh et al. [19] demonstrated in mouse that Cu’s effect on brain A␤ homeostasis depends on whether it is accumulated in capillaries or in parenchyma and concluded that faulty A␤ clearance across the BBB due to increased Cu levels in the aging brain vessels may lead to accumulation of neurotoxic A␤ in brains. These different hypotheses with regard to AD are by no means incompatible. Indeed, using a meta-analytic strategy, Squitti et al. [20] have shown copper indices in general circulation to be significantly higher in AD subjects compared to healthy controls and concluded that systemic copper

R. Deloncle et al. / Journal of Trace Elements in Medicine and Biology 39 (2017) 50–53

dysfunction characterizes AD. This is supported by the increased labile copper levels observed within the brain [21]. 4. Conclusion Our results suggest that copper associated with bovine brain proteins is able to provide protection against the neuronal death induced by free radicals. Indeed, when copper in proteins is replaced by manganese, this potential protective effect disappears. Substitution in copper proteins is possible in a reductive medium when copper is at its lower oxidative degree. Metal substitution in complexes is closely associated with the metal’s ionic potential (ratio of ionic charge to ionic ratio): the greater the ratio, the more stable the complex [22]. To sum up, these different findings suggest that an impairment in brain copper homeostasis leading to oxidative abnormalities may be involved in neurodegenerative diseases. Conflict of interest The authors declare that there is no conflict of interest. References [1] D.R. Brown, B.S. Wong, F. Hafiz, C. Clive, S.J. Haswell, I.M. Jones, Normal prion protein has an activity like that of superoxide dismutase, Biochem. J. 344 (1999) 1–5. [2] C.J. Lin, H.C. Huang, Z.F. Jiang, Cu(II) interaction with amyloid-beta peptide: a review of neuroactive mechanisms in AD brains, Brain Res. Bull. 82 (5–6) (2010) 235–242. [3] A. Ahmad, C.S. Burns, A.L. Fink, V.N. Uversky, Peculiarities of copper binding to alpha-synuclein, Biomol. Struct. Dyn. 29 (4) (2012) 825–842. [4] F. Rose, M. Hodak, J. Bernholc, Mechanism of copper(II)-induced misfolding of Parkinson’s disease protein, Sci. Rep. 1 (2011) 11. [5] F. Arnesano, S. Scintilla, V. Calo, E. Bonfrate, C. Ingrosso, M. Losacco, T. Pellegrino, E. Rizzarelli, G. Natile, Copper-triggered aggregation of ubiquitin, PLoS One 4 (9) (2009), e7052. [6] S. Hesketh, J. Sassoon, R. Knight, J. Hopkins, D.R. Brown, Elevated manganese levels in blood and central nervous system occur before onset of clinical signs in scrapie and bovine spongiform encephalopathy, J. Anim. Sci. 85 (6) (2007) 1596–1609. [7] S. Hesketh, J. Sassoon, R. Knight, D.R. Brown, Elevated manganese levels in blood and CNS in human prion disease, Mol. Cell. Neurosci. 37 (2008) 590–598.

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