Cellular prion protein protects against reactive-oxygen-species-induced DNA damage

Free Radical Biology & Medicine 43 (2007) 959 – 967 www.elsevier.com/locate/freeradbiomed

Original Contribution

Cellular prion protein protects against reactive-oxygen-species-induced DNA damage Nicole T. Watt a , Michael N. Routledge b , Christopher P. Wild b , Nigel M. Hooper a,⁎ a

b

Proteolysis Research Group, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences and Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, UK Molecular Epidemiology Unit, Leeds Institute of Genetics, Health and Therapeutics, Faculty of Medicine and Health, University of Leeds, Leeds LS2 9JT, UK Received 3 January 2007; revised 26 April 2007; accepted 5 June 2007 Available online 13 June 2007

Abstract Although the cellular form of the prion protein (PrPC) is critical for the development of prion disease through its conformational conversion into the infectious form (PrPSc), the physiological role of PrPC is less clear. Using alkaline single-cell gel electrophoresis (the Comet assay), we show that expression of PrPC protects human neuroblastoma SH-SY5Y cells against DNA damage under basal conditions and following exposure to reactive oxygen species, either hydroxyl radicals following exposure to Cu2+ or Fe2+ or singlet oxygen following exposure to the photosensitizer methylene blue and white light. Cells expressing either PrPΔoct which lacks the octapeptide repeats or the prion-diseaseassociated mutants A116V or PG14 had increased levels of DNA damage compared to cells expressing PrPC. In PrPSc-infected mouse ScN2a cells there was a significant increase in DNA damage over noninfected N2a cells (median tail DNA 2.87 and 7.33%, respectively). Together, these data indicate that PrPC has a critical role to play in protecting cells against reactive-oxygen-species-mediated DNA damage; a function which requires the octapeptide repeats in the protein, is lost in disease-associated mutants of the protein or upon conversion to PrPSc, and thus provide further support for the neuroprotective role for PrPC. © 2007 Published by Elsevier Inc. Keywords: Prion; DNA damage; Reactive oxygen species; Comet assay; Copper

Introduction The transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative disorders which include scrapie in sheep and goats, bovine spongiform encephalopathy in cattle, Creutzfeldt–Jakob disease (CJD) and Gerstmann–Sträussler– Scheinker (GSS) syndrome in humans, and chronic wasting disease in cervids [1]. TSEs or prion diseases are due to the conformational conversion of the normal cellular form of the prion protein (PrPC) to the infectious form of the protein (PrPSc), which is resistant to protease digestion. While PrPC is an Abbreviations: CJD, Creutzfeldt–Jakob disease; FPG, formamidopyrimidine DNA glycosylase; GSS, Gerstmann–-Sträussler–Scheinker; 8-OHG, 8hydroxyguanosine; PBS, phosphate-buffered saline; PrP, prion protein; PrPC, cellular form of PrP; PrPSc, infectious, protease-resistant form of PrP; ROS, reactive oxygen species; TSE, transmissible spongiform encephalopathy; DCFDA, dihydrodicholorofluorescein diacetate; IQR, interquartile range. ⁎ Corresponding author. Fax: +44 113 343 3167. E-mail address: [email protected] (N.M. Hooper). 0891-5849/$ - see front matter © 2007 Published by Elsevier Inc. doi:10.1016/j.freeradbiomed.2007.06.004

absolute prerequisite in the development of prion disease by providing the substrate for conversion [2,3], the normal physiological role of PrP C remains unclear, raising the possibility that prion diseases may, in part, be due to the loss of a normal neuroprotective function of PrPC [4]. In support of this, animals at the terminal stage of illness have a marked decrease in the amount of PrPC which can be detected in their brains [5]. The brain encounters high levels of oxidative stress as it consumes approximately 20% of the inhaled oxygen and possesses low levels of antioxidant enzymes. PrPC appears to play a critical role in the cellular resistance to oxidative stress [reviewed in 6,7]. In the brains of PrPC knockout mice there were marked elevations in protein oxidation and lipid peroxidation [8], and cells deficient in PrPC were less viable in culture and more susceptible to oxidative damage and toxicity caused by reactive oxygen species (ROS) compared with cells which expressed PrPC [9–12]. Furthermore, a cell line that was selected to be more resistant to oxidative stress had a higher level of PrPC expression

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[13]. Exposure of cells to ROS causes cell surface PrPC to undergo β-cleavage within or adjacent to the Cu2+-binding octapeptide repeats in the N-terminal half of the protein [14,15]. This ROSmediated β-cleavage is an early and critical event necessary for cells to respond appropriately to oxidative insult [15]. Direct damage to cellular components, including DNA, can occur following oxidative stress. The attack on deoxyribose by ROS results in single-strand breaks and cells employ several mechanisms to repair such breaks [16,17]. Defects in the repair of, or response to, DNA damage have been associated with many human disorders, including neurodegenerative diseases [18]. In this context, increased levels of 8-hydroxyguanosine (8-OHG; a modified base formed when DNA is attacked by hydroxyl radicals) were detected in hippocampal and temporal cortical neurons from patients with GSS [19], 8-OHG immunoreactivity correlated with PrPSc deposition in CJD patients [20], and increased oxidative damage to nucleic acids was detected in the brains of CJD patients [21]. Furthermore, in experimental rodent models of prion disease increased oxidative stress has been observed [22,23]. Using the sensitive alkaline single-cell gel electrophoresis assay (Comet assay), we have investigated the role of PrPC in protecting against DNA damage in neuronal cells (human SHSY5Y and mouse N2a). PrPC reduced both basal DNA damage and that detectable following exposure of the cells to ROS produced either by divalent metal ions or by the photosensitizer methylene blue. Cells transfected with a mutant form of PrPC in which the octarepeat sequence had been deleted, PrPΔoct, exhibited elevated levels of DNA damage consistent with a role for the Cu2+-binding octapeptide repeat region in this protective effect of PrPC. Elevated levels of DNA damage were observed both in cells expressing disease-associated mutants of PrPC and in cells infected with PrPSc. These data indicate that PrPC has a critical role to play in protecting cells against ROS-mediated DNA damage, a function which is lost upon mutation of the protein or upon conversion to PrPSc, and provide further support for the neuroprotective role for PrPC. Materials and methods All reagents were obtained from Sigma (Poole, Dorset, UK) unless otherwise stated. cDNA constructs and cell culture The construction of murine PrPC, encoding the epitopes for antibody 3F4, PrPΔoct, PG14, and A116V in pIRESneo, has been described previously [15,24]. Human neuroblastoma SHSY5Y cells were cultured and transfected by electroporation, and pooled stable cell lines were obtained by antibiotic selection as described previously [25]. Murine N2a and ScN2a cells (obtained from S. Lehmann, Montpellier, France) were cultured in Dulbecco's minimal essential medium with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (0.1 mg/ml). All metals were routinely administered to the cells in the presence of fetal calf serum to provide both histidine and albumin as physiological ligands to enable uptake into the cells. Cells were

harvested either with trypsin or by scraping into phosphatebuffered saline (PBS; 1.5 mM KH2PO4, 2.7 mM Na2HPO4, 150 mM NaCl, pH 7.4), pelleted by centrifugation at 1000g for 5 min, and resuspended in either Opti-MEM or lysis buffer (10 mM Tris/HCl, 0.5% (w/v) sodium deoxycholate, 0.5% (v/v) Nonidet P-40, 100 mM NaCl, 10 mM EDTA, pH 7.8, supplemented with complete protease inhibitor cocktail (P8340, Sigma)). The protein content of the lysates was determined using bicinchoninic acid in a microtiter plate assay with bovine serum albumin as standard [26]. SDS–PAGE and Western blot analysis Samples (containing 30 μg of protein) were resolved by electrophoresis through 14.5% polyacrylamide gels. Where indicated, samples were digested with 5 μg/ml proteinase K for 1 h at 37 °C prior to SDS–PAGE. For Western blot detection, resolved proteins were transferred to Hybond-P polyvinylidene difluoride membrane (GE Healthcare, Little Chalfont, UK). The membrane was blocked by incubation for 1 h with PBS containing 0.1% (v/v) Tween 20 and 5% (w/v) dried milk powder. Incubation with antibody 3F4 (Signet Laboratories, Inc., Dedham, MA, USA) or antibody SAF32 (Cayman Chemical, Ann Arbour, MI, USA) and peroxidase-conjugated secondary antibodies was performed for 1 h in the same buffer. Bound peroxidase conjugates were visualized using an enhanced chemiluminescence detection system (Pierce, Cramlington, UK). Measurement of DNA damage by alkaline single-cell gel electrophoresis Cells were cultured in 25-cm3 tissue culture flasks or six-well plates. Once they had attained confluence, the cell monolayer was washed twice with Opti-MEM before incubating with the relevant compounds. Exposure to either Cu2+, Zn2+, Fe2+, or Mn2+ involved a fetal-calf-serum-containing stock which was diluted into Opti-MEM just prior to use and the cells were left for 2 h at 37 °C. Experiments with methylene blue (diluted in Dulbecco's PBS) were carried out with the cell monolayer being coated with 50 μl of 0.01% methylene blue for 1 min in the presence of focused white light (Schott Fibreoptics, Mainz, Germany) positioned 40 cm above the six-well plate. The monolayers were harvested following two washes in PBS using a 1:10 dilution of trypsin/EDTA. The cells were pelleted by centrifugation at 1000g for 5 min and resuspended in Opti-MEM to a concentration of 1 × 106 cells/ml. A 60-μl aliquot of this suspension was mixed with 300 μl of 1% low-melting-point agarose in PBS, and 162 μl was placed onto duplicate microscope slides precoated with 1% agarose and left to solidify on ice. The slides were then treated with a detergent lysis solution (2.5 M NaCl, 1 mM EDTA, 10 mM Tris, 10% dimethyl sulfoxide, 1% Triton X-100, pH 10) overnight at 4 °C. This treatment solubilized the cell membrane to leave the nuclear DNA contained within the cytoskeletal scaffold. At the end of the lysis period, the slides were placed directly into an electrophoresis tank containing alkaline electrophoresis buffer

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(0.3 M NaOH, 1 mM EDTA, pH N 13) to allow the nuclear DNA to unwind for 40 min. Alternatively, after treatment of the slides with the lysis solution, they were washed 3× with buffer (40 mM Hepes, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin, pH 8) prior to incubation with formamidopyrimidine DNA glycosylase (FPG; diluted in buffer, obtained from A. Collins, Oslo, Norway) for 30 min at 37 °C in the dark. At the end of the incubation period, the slides were placed in the electrophoresis tank to allow the DNA to unwind as described above. A current of 23 V was passed through the tank for 20 min. After rinsing with neutralization buffer (0.4 M Tris/HCl, pH 7.5) for 5 min, the nuclear DNA was stained with ethidium bromide (25 μg/ml). The slides were viewed with a Zeiss fluorescent microscope and digitally analyzed using the Komet 5.5 software. Measurement of intracellular oxidative activity

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DNA fragments caused by the induction of DNA strand breaks are drawn out from a nucleoid created following lysis of whole cells embedded in agarose. The damaged DNA forms a “comet tail” and the amount of DNA contained within the tail is linearly related to the DNA strand break frequency [27,28]. Analysis of untransfected SH-SY5Y cells, which lack detectable PrPC expression (Fig. 1A) [24], showed that DNA could be detected in a comet tail (Figs. 1B–1D), indicating that even under routine culturing conditions a degree of DNA damage occurred within the cells. However, upon stable expression of PrPC within these cells (Fig. 1A), there was a significant reduction (p b 0.05) in the amount of DNA which could be detected within the comet tail (Figs. 1C and 1D); the median value for tail DNA content (IQR) reduced from 4.58 (1.70–9.31)% in the untransfected cells to 1.68 (0.68–3.40)% in the cells expressing PrPC. This indicated that there was a reduction in the amount of DNA damage in the

The level of intracellular free radicals was determined following exposure of the cells to Cu2+ (0–200 μM), Zn2+, Mn2+, or Fe2+ (all 100 μM), or 0.01% methylene blue in 5% fetal-calf-serum-containing medium for 6 h using 100 μM dihydrodichlorofluorescein diacetate (DCF-DA) as described previously [15]. At the end of the 2-h kinetic read, the cells were fixed in 70% ethanol at room temperature for 5 min and the adherent cell monolayers stained with the DNA-binding fluorochrome Hoescht 33342 (8.8 μM). The DCF-DA fluorescence was corrected for DNA content within each well of the 96well plate. Once dry, the fluorescence of each well was measured on a Synergy HT (Bio-Tek) (350 nm excitation and 450 nm emission wavelengths) to determine the cell number in each well. Data analysis In each experiment, the tail DNA (%) from 100 comets (50 per slide from duplicate slides) was analyzed and scored per condition. All experiments were performed independently at least three times. The amount of tail DNA in the PrPCexpressing cells was found to be consistently below 5%, therefore all cells were grouped according to the amount of tail DNA with b 5% as the lowest banding. The remaining cells were grouped into 5.01–10%, 10.01–20%, and N 20% tail DNA. The mean value from a minimum of three replicates is shown for each group as a graphical representation of the data. A median value for the percentage tail DNA was determined for the total number of cells examined per condition and tabulated together with the first and third quartiles (interquartile range; IQR). As the data were not normally distributed, nonparametric statistical analysis was performed with significance being inferred if p b 0.05. Results PrPC reduces basal DNA damage Alkaline single-cell gel electrophoresis (comet assay) was used to assess the amount of DNA damage. In this assay, cellular

Fig. 1. PrPC reduces basal DNA damage. (A) Western blot comparing the expression of PrPC in the untransfected (Un) SH-SY5Y cells with that in the cells stably expressing PrPC. (B) Examples of comets with differing amounts of DNA in the tail from untransfected SH-SY5Y cells. (C and D)Tail DNA (%) as a measurement of DNA damage in untransfected SH-SY5Y cells compared with those stably expressing PrPC. (C) Untransfected SH-SY5Y cells (white bars) and PrPC-expressing SH-SY5Y cells (black bars) grouped according to the amount of comet tail DNA (%) as a marker of DNA damage. Data shown as the mean of the median tail DNA (%) ± SE (n = 3). (D) Mean distribution of tail DNA (%) taken from three independent experiments. Those with b5% tail DNA are shown as white sections, 5.01–10% tail DNA as dotted regions, 10.01–20% tail DNA as striped areas, and N20% tail DNA as black (n = 3).

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Fig. 2. PrPC is protective against Cu2+-induced DNA damage. (A) The generation of ROS was measured using the dye DCF-DA in untransfected SH-SY5Y cells (white bars) or SH-SY5Y cells expressing PrPC (black bars) treated with Cu2+ at the concentrations indicated for 2 h. Data are expressed as absolute fluorescence units of DCF-DA corrected for DNA content within each well for each individual condition (n ≥ 8). Statistical significance inferred whether test values were higher than the respective control values *p b 0.05, #p b 0.05. Untransfected SH-SY5Y cells (B) or SH-SY5Y cells expressing PrPC (C) were exposed to the indicated concentrations of Cu2+ for 2 h. Cells were grouped according to the amount of comet tail DNA (%) as a marker of DNA damage. Those with b 5% tail DNA are shown as white sections, 5.01–10% tail DNA as dotted regions, 10.01–20% tail DNA as striped areas, and N 20% tail DNA as black (n = 3).

presence of PrPC. No significant difference was found in the amount of DNA contained in the comet tail of the untransfected SH-SY5Y cells compared with those which had been mock transfected with the empty pIRESneo vector (data not shown). PrPC protects against hydroxyl-radical-induced DNA damage PrPC is capable of binding Cu2+ ions at the octapeptide repeats in the N-terminal half of the protein and may be involved in copper homeostasis within the brain [29]. The ability of Cu2+ ions to participate in Fenton chemistry results in the formation of

the highly reactive hydroxyl radical that is capable of causing damage to cellular macromolecules. Following exposure of untransfected SH-SY5Y cells to elevated concentrations of Cu2+ in the culture medium, significant dose-dependent increases (p b 0.05) in DCF-DA fluorescence was seen at all Cu2+ concentrations investigated (Fig. 2A, white bars). Though colorless in its native state, cleavage of the DCF-DA by intracellular esterases and contact with ROS causes oxidation and fluorescence to be emitted. Increases in fluorescence, therefore, correlate with elevated intracellular ROS levels. Concomitant increases in the amount of DNA found within the comet tail were observed at 100 μM Cu2+ and above (Fig. 2B, Table 1). However, in the cells expressing PrPC, exposure to Cu2+ did not result in a change in the amount of DNA within the comet tail (Fig. 2C, Table 1). While an increase in DCF-DA fluorescence was measurable in the cells expressing PrPC, the levels were not as significant as those found in the untransfected SH-SY5Y cells (Fig. 2A, black bars). These data indicate that, while there was an increase in ROS generation, as determined by DCF-DA fluorescence, and associated DNA damage in the untransfected SH-SY5Y cells upon exposure to Cu2+, the presence of PrPC in the cells protected against this Cu2+-induced DNA damage. The doses of Cu2+ used are within the range of the reported extracellular concentration of Cu2+ in the brain [30,31] and are known to enhance the generation of radical species but were associated with no significant change in viability of the cells over the period of exposure [32]. Like Cu2+, Fe2+ also participates in Fenton chemistry. Although neither Zn2+ nor Mn2+ participate in Fenton chemistry [33], both these metals are involved in PrPC biology [34,35]. Thus we examined whether these other metal ions caused DNA damage that could be protected against by PrPC. Untransfected SH-SY5Y cells exposed to 100 μM Zn2+ showed no significant increase in either DCF-DA fluorescence or tail DNA over untreated cells (Figs. 3A and 3B, Table 2). Attempts to measure ROS generation using DCF-DA in untransfected SH-SY5Y cells treated with 100 μM Mn2+ led to an artificially high background level of fluorescence, however, no change in tail DNA was measurable in the cells under these conditions. Exposure of the untransfected SH-SY5Y cells to 100 μM Fe2+ resulted in a significant increase (p b 0.05) in both DCF-DA fluorescence and tail DNA (Figs. 3A and 3B, Table 2). The amount of DNA damage induced by 100 μM Fe2+ was comparable to that induced by exposure of the cells to 100 μM Cu2+ (Fig. 3B, Table Table 1 PrPC is protective against Cu2+-induced DNA damage Cu2+ (μM)

0 50 100 200

% Median tail DNA (IQR) Untransfected

PrPC expressing

3.72 (1.28–7.53) 3.53 (1.37–8.10) 4.38 (1.79–9.57) ⁎ 5.11 (2.18–9.46) ⁎

2.34 (0.80–4.22) 2.70 (0.86–4.90) 2.04 (0.60–4.40) 2.43 (0.85–5.28)

Median tail DNA (%) and IQR as a measurement of DNA damage in untransfected SH-SY5Y cells compared with those stably expressing PrPC upon exposure to Cu2+ (n = 300–400). ⁎ p b 0.05.

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PrPC protects against singlet-oxygen-induced DNA damage To provide an alternative model of oxidative DNA damage, cells were exposed to the photosensitizer methylene blue in the presence of white light which produces singlet oxygen that is capable of reacting with and damaging cellular DNA [36]. Exposure of untransfected SH-SY5Y cells to white light alone resulted in a nonsignificant increase in the amount of DNA found in the comet tail compared with cells not exposed to white light (Table 3). Following exposure of the untransfected SH-SY5Y cells to white light and 0.01% methylene blue there was a substantial increase in DCF-DA fluorescence (p b 0.05, data not shown) with an additional significant increase (p b 0.05) in the amount of DNA found in the comet tail (Fig. 4, Table 3). Exposure of the SH-SY5Y cells expressing PrPC to 0.01% methylene blue and white light resulted in a slight increase in DNA damage, however, this increase was significantly less than that seen in the untransfected cells (Fig. 4, Table 3). Cells expressing PrPC which were exposed to white light alone did not display any increased DNA damage. These data indicate that PrPC can protect cellular DNA against singlet-oxygeninduced damage. Inclusion of FPG demonstrates the presence of oxidative DNA damage

Fig. 3. PrPC is protective against divalent metal-ion-induced DNA damage. (A) The generation of ROS was measured using the dye DCF-DA in untransfected SH-SY5Y cells (white bars) or SH-SY5Y cells expressing PrPC (black bars) treated with Cu2+, Fe2+, Zn2+, or Mn2+ at 100 μM for 2 h. Data are expressed as absolute fluorescence units of DCF-DA corrected for DNA content within each well for each individual condition (n ≥ 8). Statistical significance inferred whether test values were higher than the respective control values *p b 0.05, # p b 0.05.; n.d., not determined. Untransfected SH-SY5Y cells (B) or SH-SY5Y cells expressing PrPC (C) were exposed to the various metals for 2 h. Cells were grouped according to the amount of comet tail DNA (%) as a marker of DNA damage. Those with b 5% tail DNA are shown as white sections, 5.01–10% tail DNA as dotted regions, 10.01–20% tail DNA as striped areas, and N 20% tail DNA as black (n = 3).

To further explore whether the DNA damage that had been induced was caused by oxidative mechanisms, an incubation with the enzyme FPG was included in the comet assay protocol after the cells had been lysed and before alkali incubation. FPG excises the oxidatively damaged base 8-OHG and nicks the DNA backbone to leave a single-strand break that is detected as additional DNA migration in the comet tail [37,38]. Treatment of cells expressing PrPC with FPG showed that, even under routine growth conditions or following exposure to white light, significant increases (p b 0.05) in tail DNA could be determined (Table 4). Cells which had been exposed to 100 μM Cu2+ or 0.01% methylene blue similarly displayed further significant increases in tail DNA following incubation with FPG (p b 0.05). These data confirm that exposure to 100 μM Cu2+ and 0.01% methylene blue resulted in oxidative damage occurring within the nuclear DNA. Table 2 PrPC is protective against divalent metal ion-induced DNA damage Metal ion

C

2). SH-SY5Y cells expressing PrP showed no significant increase in tail DNA when exposed to either Zn2+ or Mn2+ compared with untreated cells (Fig. 3C, Table 2). Although there was a significant (p b 0.05) increase in both DCF-DA fluorescence (Fig. 3A) and tail DNA in the SH-SY5Y cells expressing PrPC upon exposure to 100 μM Fe2+ (Fig. 3C, Table 2), this increase was lower than that observed in the untransfected SHSY5Y cells, indicating that PrPC can provide some protection against Fe2+-induced DNA damage.

None Cu2+ Fe2+ Zn2+ Mn2+

% Median tail DNA (IQR) Untransfected

PrPC expressing

3.57 (1.61–7.08) 4.44 (1.33–8.97) ⁎ 4.61 (1.87–10.82) ⁎ 3.75 (1.65–7.00) 3.25 (1.23–5.98)

1.67 (0.72–3.57) 1.58 (0.59–4.46) 2.84 (1.00–6.20) ⁎ 1.46 (0.43–3.60) 1.83 (0.82–3.76)

Median tail DNA (%) and IQR as a measurement of DNA damage in untransfected SH-SY5Y cells compared with those stably expressing PrPC upon exposure to 100 μM Cu2+, Fe2+, Zn2+, or Mn2+ (n = 400). ⁎ p b 0.05.

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Table 3 PrPC protects against singlet-oxygen-induced DNA damage Methylene blue

Table 4 Oxidative DNA damage in cells expressing PrPC

% Median tail DNA (IQR)

0 0.01%

Treatment

Untransfected

PrPC expressing

6.02 (2.99–9.49) 14.18 (8.61–20.54) ⁎

1.97 (0.62–4.17) 3.75 (1.37–7.52)*

Median tail DNA (%) and IQR as a measurement of DNA damage in untransfected SH-SY5Y cells compared with those stably expressing PrPC upon exposure to white light alone or white light and 0.01% methylene blue (n = 300). ⁎ p b 0.05.

The octapeptide repeat region of PrPC is required to protect against DNA damage 2+

C

As the Cu -binding octapeptide repeat region of PrP is required to protect cells against ROS [15], we investigated whether this region of the protein was required also to mediate the protection against DNA damage. Cells expressing a construct, PrPΔoct, in which the octarepeat regions (residues 51–90) have been removed (Fig. 5A), were analyzed using the comet assay. PrPΔoct was expressed at a level similar to that of PrPC in the SHSY5Y cells (Fig. 5B, lanes 1 and 2). The cells expressing PrPΔoct showed a significant increase (p b 0.05) in the amount of DNA damage compared with the cells expressing PrPC and the amount of DNA damage was comparable to that found in the untransfected cells (Fig. 5C, Table 5). Similarly there was a significant increase in DNA damage upon exposure of the cells expressing PrPΔoct to 100 μM Cu2+ (4.66% median tail DNA in the untreated cells compared with 9.30% in the Cu2+-treated cells). These data clearly indicate that the octapeptide repeat region is required for the protective effect of PrPC against DNA damage.

None Cu2+ White light White light + methylene blue

% Median tail DNA (IQR) − FPG

+FPG

1.83 (0.69–3.55) 2.29 (1.01–4.21) 1.97 (0.62–4.17) 3.75 (1.37–7.52)

2.21 (0.895–4.18) ⁎ 6.41 (1.98–9.56) ⁎ 3.10 (1.25–6.84) ⁎ 15.97 (15.32–26.87) ⁎

Median tail DNA (%) and IQR as a measurement of DNA damage in cells expressing PrPC following exposure to 100 μM Cu2+ or 0.01% methylene blue in the presence of white light. Cells were pretreated with FPG prior to the Comet assay which allowed the measurement of oxidized base damage (n = 300). ⁎ p b 0.05.

ing, equivalent to Ala117 in human PrP) is mutated to Val, is associated with GSS [41] (Fig. 5A). Both of these constructs were expressed in the SH-SY5Y cells at levels comparable to those of PrPC (Fig. 5B, lanes 3 and 4). Cells expressing A116V showed considerable amounts of DNA damage which were significantly greater (p b 0.05) than those found in the cells expressing PrPC but that were comparable to those found in the untransfected SH-SY5Y cells (Fig. 5C, Table 5). Similarly there was a significant increase in DNA damage upon exposure of the cells expressing A116V to 100 μM Cu2+ (4.33% median tail DNA in the untreated cells compared with 6.67% in the Cu2+treated cells). The cells expressing PG14 displayed levels of DNA damage which were significantly greater (p b 0.01) than those observed in either the cells expressing PrPC or the untransfected cells (Fig. 5C, Table 5). These data indicate that these two disease-associated mutations in PrPC result in the loss of the protein's protective effect against DNA damage. Cells infected with PrPSc have increased DNA damage

Disease-associated mutant forms of PrPC do not protect against DNA damage Previously we have shown [5] that two disease-associated mutants of PrP, PG14 and A116V, fail to protect cells against oxidative stress. PG14 contains an extra nine copies of the octapeptide repeat and is associated with familial human prion disease [39,40]. A116V, in which Ala116 (murine PrP number-

Fig. 4. PrPC protects against singlet-oxygen-induced DNA damage. Mean tail DNA (%) as a measurement of DNA damage in untransfected SH-SY5Y cells compared with those stably expressing PrPC upon exposure to 0.01% methylene blue (MB) (n = 3). Cells were grouped according to the amount of comet tail DNA (%) as a marker of DNA damage. Those with b5% tail DNA are shown as white sections, 5.01–10% tail DNA as dotted regions, 10.01–20% tail DNA as striped areas, and N20% tail DNA as black (n = 3).

Finally we used the comet assay to compare the amount of DNA damage in noninfected and prion-infected N2a cells to determine whether the protective effect of PrPC is compromised upon its conversion to PrPSc. The mouse neuroblastoma N2a cell line is infected with the Chandler mouse-adapted scrapie strain and produces PrPSc molecules constitutively (Fig. 6A) [42]. In the noninfected N2a cells, whose endogenous level of PrPC is similar to that in the SH-SY5Y cells overexpressing PrPC, there was only a small amount of DNA in the comet tail (Fig. 6B), similar to that in the SH-SY5Y cells stably expressing PrPC. However, the infected ScN2a cells had a significantly greater (p b 0.01) amount of DNA contained within the comet tail than the noninfected N2a cells (Fig. 6B); the median value for tail DNA content (IQR) increased from 2.87 (1.30–6.06)% in the noninfected N2a cells to 7.33 (2.86–12.93)% in the infected ScN2a cells, indicating that there was a significantly higher degree of DNA damage in the infected cells. This would suggest that, upon prion infection and the formation of PrPSc, the cells are less well protected against DNA damage. Discussion The brain is prone to oxidative stress and has limited antioxidant defence systems to prevent both ongoing oxidative

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Table 5 Removal of the octapeptide repeats or disease-associated mutations in PrPC negate its protective effect against DNA damage Cell Type

% Median tail DNA (IQR)

Untransfected PrPC PrPΔoct PG14 A116V

5.08 (2.53–10.08) ⁎ 2.69 (1.24–4.42) 4.39 (1.87–9.57) ⁎ 9.31 (4.86–15.57)⁎⁎ 5.06 (1.99–9.06) ⁎

Median tail DNA (%) and IQR as a measurement of DNA damage in untransfected SH-SY5Y cells and cells expressing either PrPC, PrPΔOct, PG14, or A116V (n = 300). ⁎ p b 0.05.

and gene expression and cause replication errors and genomic instability [44]. Alkaline single-cell gel electrophoresis (the Comet assay) is a sensitive analytical method used to detect DNA damage. Using this technique, we have shown that PrPC protects against both basal and ROS-mediated DNA damage, providing further evidence for the neuroprotective function of PrPC. The SH-SY5Y cells, which do not express detectable PrPC, had a basal level of DNA damage which was significantly reduced upon expression of PrPC, implying that under normal culture conditions there is ongoing intracellular damage occurring which PrPC protects against. Exposure to Cu2+ brought about a dose-dependent increase in ROS being generated within

Fig. 5. Removal of the octapeptide repeats or disease-associated mutations in PrPC negate its protective effect against DNA damage. (A) Schematic diagram of the PrP mutants used. PrPC is shown as the mature, full-length protein with its C-terminal GPI anchor, two N-linked glycosylation sites (residues 180 and 196, lollipops), and the octapeptide repeat region (shaded). PrPΔoct lacks the entire octapeptide region, PG14 has an additional nine octapeptide repeats, and A116V has a single point mutation Ala → Val at position 116. (B) Western blot of lysates from SH-SH5Y cells expressing PrPC (lane 1), PrPΔoct (lane 2), PG14 (lane 3), or A116V (lane 4). (C) Cells were grouped according to the amount of comet tail DNA (%) as a marker of DNA damage. Those with b5% tail DNA are shown as white sections, 5.01–10% tail DNA as dotted regions, 10.01–20% tail DNA as striped areas, and N20% tail DNA as black (n = 3).

damage and that imposed by neurodegenerative diseases [43]. Cellular DNA is a sensitive target of damage following oxidative stress. This damage can include chemical and structural modifications to purine and pyrimidine bases and 2′-deoxyribose and the formation of single- and double-strand breaks. Strand breaks within DNA can occur either directly due to damage from ROS exposure or indirectly due to cleavage of the DNA backbone during DNA base excision repair [17]. Continued oxidative damage to DNA can alter signaling cascades

Fig. 6. Cells infected with PrPSc have increased DNA damage. (A) Lysates from noninfected (lanes 1 and 2) and infected (lanes 3 and 4) N2a cells were incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 20 μg/ml proteinase K for 30 min at 37 °C prior to SDS–PAGE and Western blot analysis with antibody 6H4. The blot shown was overexposed to detect the low amount of proteinase-K-resistant PrPSc in the infected ScN2a cells. (B) Cells were grouped according to the amount of comet tail DNA (%) as a marker of DNA damage. Those with b5% tail DNA are shown as white sections, 5.01–10% tail DNA as dotted regions, 10.01–20% tail DNA as striped areas, and N20% tail DNA as black (n = 3).

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the cells and a concomitant increase in DNA damage in the untransfected SH-SY5Y cells, consistent with the generation via Fenton chemistry of the highly reactive hydroxyl radical that then attacks the nuclear DNA. Although PrPC binds Cu2+ both at the octapeptide repeats and at other sites in the protein [29,45], this effect was not Cu2+ specific as exposure to Fe2+, which also undergoes Fenton chemistry, similarly caused increased damage to the nuclear DNA that could be protected against by PrPC. Neither Zn2+ nor Mn2+, which do not participate in Fenton chemistry but which have been implicated in prion biology [34,35], showed measurable ROS generation or DNA damage. This confirms that the DNA-damaging action of Cu2+ and Fe2+ was due to the generation of ROS, independent of their binding to PrPC. These data are in agreement with data from a previous study [46] which reported that PrPC protects rabbit kidney epithelial cells against DNA damage induced by hydroxyl radical generated from paraquat. The observation that cells expressing PrPC were also protected against singlet oxygen radicals, following exposure to the photosensitizer methylene blue and white light, provides evidence that PrPC has a wider role in protecting against multiple ROS and not just hydroxyl radicals. Confirmation of an oxidative mechanism causing the DNA damage following exposure to methylene blue or Cu2+ was shown by treatment of the cells with FPG. This enzyme reveals oxidative DNA damage due to its ability to excise the 8-OHG lesion from nuclear DNA [37,38]. The enzyme also nicks the DNA at the resulting abasic site, which is visualized in the comet assay as further migration of the tail DNA. Our observation that cells expressing the disease-associated mutants of PrPC, A116V and PG14, had a substantial increase in basal DNA damage compared with cells expressing PrPC are consistent with the observed increases in oxidative damage in the brains from individuals with CJD [19–21]. Previously we have shown [15] that cells expressing PrPΔoct, A116V, or PG14 had a reduced viability and glutathione peroxidase activity and increased intracellular free radical generation compared to cells expressing PrPC. The observation that cells expressing PrPΔoct, PG14, or A116V have increased levels of DNA damage compared to cells expressing PrPC adds further experimental evidence to the observation that such cells are less resistant to oxidative stress than cells expressing PrPC and is consistent with our previous work indicating a correlation between the ability of PrP to undergo β-cleavage, which these three mutants fail to do, and the cellular resistance to oxidative stress [15]. As PrP can interact directly with nuclear DNA and form large nucleoprotein complexes [47–49], we cannot rule out an alternative mechanism whereby PrPC, or fragments of the protein, protect DNA against oxidative damage by binding to it. Previously it was reported that prion-infected hypothalamic neuronal GT1 cells had increased lipid peroxidation and signs of apoptosis associated with a dramatic reduction in the activities of the glutathione-dependent and superoxide dismutase antioxidant systems compared to noninfected cells [50]. Our observation that, in another well-characterized model of prion disease, infected ScN2a cells there was a significant amount of DNA damage compared with the noninfected N2a cells adds further experimental evidence to the conclusion that

prion infection impairs the cellular resistance to oxidative stress [50]. Although we favor the hypothesis that this impaired cellular resistance to oxidative stress upon prion infection is due to a loss of the normal protective function of PrPC, we cannot rule out an additional gain of toxic function contributed by the accumulation of PrPSc in the cells. In cells transfected with Parkinson's-disease-associated mutants of α-synuclein, Fe2+-induced DNA damage was exacerbated compared with cells expressing the wild-type αsynuclein [51]. These authors suggested that a possible interpretation for the effects of the mutations in α-synuclein was that, in the presence of Fe2+, the mutant forms of the protein aggregate into toxic oligomers more rapidly than the wild-type α-synuclein [51]. These oligomers then induce oxidative damage to the cells via the direct formation of ROS. Unlike the situation with PrPC, expression of wild-type α-synuclein in the human dopaminergic BE(2)-M17 neuroblastoma cells, which do not express significant levels of endogenous α-synuclein, did not afford any protection against Fe2+-induced DNA damage [51]. Thus, in contrast to the gain of toxic function afforded by the mutants of α-synuclein, our data are consistent with PrPC having a normal function in protecting cells against oxidative damage to DNA and that either in cells expressing disease-associated mutants of PrPC or in prion-infected cells there is a loss of this normal function. Acknowledgments The financial support of the Medical Research Council of Great Britain is gratefully acknowledged. We thank J. Olliver and R. Sturmey for assistance with the comet analysis. References [1] Aguzzi, A. Prion diseases of humans and farm animals: epidemiology, genetics, and pathogenesis. J. Neurochem. 97:1726–1739; 2006. [2] Bueler, H.; Aguzzi, A.; Sailer, A.; Greiner, R. A.; Autenried, P.; Aguet, M.; Weissmann, C. Mice devoid of PrP are resistant to scrapie. Cell 73:1339–1347; 1993. [3] Prusiner, S. B.; Groth, D.; Serban, A.; Koehler, R.; Foster, D.; Torchia, M.; Burton, D.; Yang, S. L.; DeArmond, S. J. Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. Proc. Natl. Acad. Sci. USA 90:10608–10612; 1993. [4] Hetz, C.; Maundrell, K.; Soto, C. Is loss of function of the prion protein the cause of prion disorders? Trends Mol. Med. 9:237–243; 2003. [5] Yokoyama, T.; Kimura, K. M.; Ushiki, Y.; Yamada, S.; Morooka, A.; Nakashiba, T.; Sassa, T.; Itohara, S. In vivo conversion of cellular prion protein to pathogenic isoforms, as monitored by conformation-specific antibodies. J. Biol. Chem. 276:11265–11271; 2001. [6] Vassallo, N.; Herms, J. Cellular prion protein function in copper homeostasis and redox signalling at the synapse. J. Neurochem. 86:538–544; 2003. [7] Roucou, X.; Gains, M.; LeBlanc, A. C. Neuroprotective functions of prion protein. J. Neurosci. Res. 75:153–161; 2004. [8] Wong, B. S.; Liu, T.; Li, R.; Pan, T.; Petersen, R. B.; Smith, M. A.; Gambetti, P.; Perry, G.; Manson, J. C.; Brown, D. R.; Sy, M. S. Increased levels of oxidative stress markers detected in the brains of mice devoid of prion protein. J. Neurochem. 76:565–572; 2001. [9] Brown, D. R.; Schulz-Schaeffer, W. J.; Schmidt, B.; Kretzschmar, H. A. Prion protein-deficient cells show altered response to oxidative stress due to decreased SOD-1 activity. Exp. Neurol. 146:104–112; 1997.

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