Prion protein protects against zinc-mediated cytotoxicity by modifying intracellular exchangeable zinc and inducing metallothionein expression

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Journal of Trace Elements in Medicine and Biology 23 (2009) 214–223 www.elsevier.de/jtemb

PATHOBIOCHEMISTRY

Prion protein protects against zinc-mediated cytotoxicity by modifying intracellular exchangeable zinc and inducing metallothionein expression Walid Rachidia,, Fabrice Chimientib, M’hammed Aouffena, Abderrahmane Senatorc, Pascale Guiraudc, Michel Seved, Alain Faviera a

INAC/SCIB/LAN, CEA de Grenoble, 17 rue des Martyrs, 38054 Grenoble, France MELLITECH SAS, INAC/SCIB, 17 rue des Martyrs, 38054 Grenoble, France c ORSOX, Faculte´ de Pharmacie, Domaine de la Merci, 38706 La Tronche-Grenoble Cedex 9, France d Institut Albert Bonniot, Centre de Recherche INSERM/UJF U823, Centre d’Innovation en Biologie, CHU Grenoble, Domaine de la Merci, 38700 La Tronche, France b

Received 13 June 2008; accepted 12 February 2009

Abstract PrPC contains several octapeptide repeats sequences toward the N-terminus which have binding affinity for divalent metals such as copper, zinc, nickel and manganese. However, the link between PrPC expression and zinc metabolism remains elusive. Here we studied the relationship between PrPC and zinc ions intracellular homeostasis using a cell line expressing a doxycycline-inducible PrPC gene. No significant difference in 65Zn2+ uptake was observed in cells expressing PrPC when compared with control cells. However, PrPC-expressing cells were more resistant to zinc-induced toxicity, suggesting an adaptative mechanism induced by PrPC. Using zinquin-ethyl-ester, a specific fluorophore for vesicular free zinc, we observed a significant re-localization of intracellular exchangeable zinc in vesicles after PrPC expression. Finally, we demonstrated that PrPC expression induces metallothionein (MT) expression, a zincupregulated zinc-binding protein. Taken together, these results suggest that PrPC modifies the intracellular localization of zinc rather than the cellular content and induces MT upregulation. These findings are of major importance since zinc deregulation is implicated in several neurodegenerative disorders. It is postulated that in prion diseases the conversion of PrPC to PrPSc may deregulate zinc homeostasis mediated by metallothionein. Crown Copyright r 2009 Published by Elsevier GmbH. All rights reserved. Keywords: Prion protein; Zinc ions; Metallothionein; Neurodegeneration

Introduction

Abbreviations: AD, Alzheimer disease; Dox, doxycycline; MT, metallothionein; PBS, phosphate buffer saline; PD, Parkinson disease; PrPC, cellular isoform of prion protein; PrPSc, scrapie isoform of prion protein; S.D., standard deviation. Corresponding author. Tel.: +33 4 38 78 50 11; fax: +33 4 38 78 50 90. E-mail address: [email protected] (W. Rachidi).

Prion diseases are fatal neurodegenerative disorders that affect humans and some animals [1]. All these diseases are characterized by the accumulation of an abnormally folded isoform of the normal prion protein PrPC, denoted PrPSc, which represents the major component of infectious prion diseases. The normal prion protein physiologically binds copper, through a repeated five octapeptide domain at N-terminus [2].

0946-672X/$ - see front matter Crown Copyright r 2009 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2009.02.007

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Several studies have reported that transition metals including copper, zinc, manganese and nickel present a binding capacity to the prion protein [3,4]. However the physiological relevancy of such a metal binding capacity remains questionable. Recently, using a murine PrP-transfected rabbit kidney cell model (RK13), we have shown a copper binding activity of the PrPC, for which a correlation between copper binding and PrPC expression was established [5]. In addition, other groups [6] and us (unpublished data) have found that PrPC can bind zinc as well as copper, and undergoes endocytosis in response to extracellular zinc. The relationship between the prion protein and zinc ion is not clear. It has been hypothesized that PrPC could be involved in zinc uptake into cells. PrPC might also act as a zinc sensor [7]. In the present work we used radioactive zinc (65Zn), absorption spectrometry and fluorescent detection to investigate the impact of PrPC expression on zinc binding, zinc uptake and subcellular zinc ions localization in A74 cells, a murine cell line in which PrPC expression is dose-dependently inducible by doxycycline treatment. We demonstrate that PrPC expression leads to intracellular zinc re-localization, which in turn leads to metallothionein (MT) induction and protection against zinc toxicity.

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supplemented with 10% heat-inactivated fetal calf serum and were usually split at one-fourth dilution each week.

Cellular

65

Zn uptake assay

Cells were cultured in 35-mm Petri dishes. Culture medium was replaced by 2 mL of fresh complete medium in the absence or in the presence of 500 ng/mL of doxycycline (to stimulate PrPC expression). After 24 h, stimulated and unstimulated cells were exposed to 0.25 mM 65Zn (CIS Biointernational, Gif-surYvette, France, specific activity 20 mCi/mg) to evaluate zinc binding to cells as a function of PrPC expression. Cells were incubated at 37 1C under 5% CO2. The radioactive medium was removed after 0, 2, 8, 12, 24 and 30 h. Cells were rinsed twice with 2 mL of diluted Puck’s saline A solution (Invitrogen, France), and harvested after addition of 1 mL of 0.25% trypsin solution. After harvesting, each dish was rinsed with 1 mL of Puck’s saline A. The final 2 mL obtained for each dish were counted for 2 min using a Packard Cobra III, monowell gamma counter (Packard Instrument Company, CT, USA). Protein content was assayed with the BCA protein assay reagent kit (Pierce, France). Data were normalized to obtain results as mCi of 65Zn incorporated or retained per mg of protein.

Materials and methods

Cellular copper and zinc determination

Cell culture and treatment

For intracellular copper and zinc determination, stimulated (500 ng/mL Dox for 24 h) or unstimulated A74 cell, were trypsinized, washed three times in Ca/Mg-free phosphate-buffered saline, and lysed by three cycles of freeze thawing. Lysates (total extract) were then centrifuged at 13,000 rpm for 10 min to obtain the soluble fraction. Copper and zinc concentrations were determined by electrothermal atomic absorption spectrophotometry (PerkinElmer Life Sciences, France). Their levels were normalized to the protein content, measured with a protein assay kit.

The A74 cells derive from an heterologous epithelial cell line (RK13) in which the expression of murine PrPC was made adjustable in a dose-dependent-manner by doxycycline (Dox) treatment [8,9]. Doxycycline (98% purity) was purchased from Sigma. Generation of A74 cells was described previously [5]. Briefly, the murine PrPC was cloned in the pTRE plasmid (Clontech, France), and the resulting plasmid was transfected by the Lipofectamine method (Invitrogen, France) into rabbit kidney epithelial cells (RK13). Stable transfectants, containing the pTRE plasmid bearing a puromycin resistance gene, were selected in the presence of puromycin (1 mg/mL), which inhibits protein synthesis at translation by prematurely terminating a peptide chain in both prokaryotic and eukaryotic cells [10]. One clone (A74) was amplified for further study. Expression of PrPC was related to doxycycline concentration in the culture medium [5]. Therefore, in the following experiments doxycycline was used at 500 ng/mL, a concentration responding to a maximum expression of PrPC and already used in other studies [5]. RK13 and A74 cells were grown at 37 1C in a 5% CO2-enriched atmosphere in minimal essential medium

Flow cytometry analysis of exchangeable zinc Flow analysis cell sorter (FACS) was used to measure exchangeable zinc in unstimulated and doxycyclinestimulated A74 cells. Cells were cultured in 35-mm Petri dishes at 37 1C under 5% CO2 in complete medium. To induce PrPC expression, cells were challenged with 500 ng/mL doxycycline for 24 h. Unstimulated and PrPC-expressing cells were exposed to 100 mmol/L ZnSO4, in OptiMEM medium (Invitrogen, France) for 1 h at 37 1C under 5% CO2. Cells were washed three times in PBS with calcium magnesium (PBS-CM) and loaded with 10 mmol/L zinpyr-1 in PBS-CM for 30 min

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[11]. Then cells were washed three times in PBS-CM and harvested by treatment with 0.25% trypsin-EDTA solution in PBS for 5 min at 37 1C under 5% CO2. Finally cells were washed three times in PBS and analyzed by a FACSCaliburTM Flow Cytometry System (Becton Dickinson, France) using an excitation wavelength 488 nm and an emission wavelength 520 nm. Untreated cells were used as negative control to adjust the FACS parameters for the granularity and auto fluorescence parameters.

Determination of exchangeable zinc by zinquin Cells were grown in Lab-tek II chambered cover glass systems (Nunc, France). Twenty-four hours later, culture medium was replaced by 2 mL of fresh complete medium containing doxycycline (0–500 ng/mL) or not to induce murine PrPC expression in A74 cells, and incubated for another 24 h. Then, cells were washed three times in PBS and loaded with a micromolar solution of zinquin-ethyl-ester in PBS at 371 for 30 min [12]. Cells were then washed three times in PBS and observed under an inverted fluorescence microscope (excitation wavelength 365 nm, emission wavelength 485 nm, Axiovert 200, Zeiss). Cells were photographed with a cooled digital camera ORCA-100 (Hamamatsu, Japan) and fluorescence was quantified using Scion Image software.

Cell viability assay Cell viability was determined by a modified 3-(4,5dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. 3000 cells per well were plated in 96-well microtiter plates in 100 mL complete medium. The next day, the medium was changed, and the cells were challenged for 24 h with 100 mL complete medium without (unstimulated cells) or with 500 ng/mL doxycycline to induce the PrPC expression. Unstimulated and doxycycline-treated cells were exposed to various concentrations of zinc (0–400 mmol/L) in complete medium for 24 h at 37 1C. Cells were washed three times in complete medium and then incubated for an additional 24 h without drugs. For the MTT assay, 10 mL of MTT (5 mg/mL stock in PBS) were added to each well for 3 h at 37 1C. The medium was removed and 100 mL of dimethyl sulfoxide (99.5% purity) were added to dissolve the formazan crystals. After shaking the plates to ensure adequate solubilization, the absorbance readings for each well were performed at 570 nm using Multiscan Ascent microtiter plate reader (Labsystems, France). The absorbance is proportional to viable cell number, and survival was calculated as the percentage of the staining values of untreated cultures. The percentage viability was calculated as follows: % specific

viability [(AB)/(CB)]/100 where A ¼ OD570 of the treated sample, B ¼ OD570 of the medium and C ¼ OD570 of the control (phosphate buffer saline-treated cells).

Metallothionein reporter gene assay RK13 cells and A74 cells were transfected with MT promoter-driven luciferase reporter (MT-Luc) using Fugen transfection reagent (Roche, France). Exponentially growing cells were seeded in 24-well plates at a density of 4  104 cells per well in 1 mL of complete medium and growing for 24 h prior to transfection. One microgram plasmid in 100 mL serum-free MEM was mixed with 20 mL of the transfection reagent for 30 min. The mix was added to the cell layer together with 400 mL serum-free MEM and left for 5 h at 371C in a CO2 incubator. In all, 500 mL 20% serum-containing MEM was then added to each well and left for 24 h. To induce the expression of PrP, doxycycline was added at various concentrations in complete medium and left for another 24 h at 37 1C in a CO2 incubator. The luciferase assay was performed accordingly to the manufacturer (Promega, France). Luciferase activity, expressed in arbitrary light units, was normalized for protein concentration obtained by the BCA micro method (Pierce, France) using bovine serum albumin as standard.

Immunofluorescence analysis Immunofluorescence analysis of untreated and treated cells was performed at 4 1C, with anti-metallothionein mouse polyclonal antibody (Stressgen, USA). Cyanine3conjugated IgG (Santa Cruz Biotechnology, USA) was used as a second antibody. Imaging was performed with a Leica microscope equipped by Kamassoki camera and controlled by a twain software.

Statistical analysis The Student’s t-test was applied to the data and a p-valueo0.05 was considered statistically significant. The results were normally distributed.

Results PrPC expression increases resistance to zinc toxicity Unstimulated and doxycycline-treated cells were exposed to zinc at various concentrations in complete medium for 24 h at 37 1C (Fig. 1). Assessment of cell viability reveals a drastic increase in cell death in the presence of high concentrations of zinc. However,

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Fig. 1. Effect of PrPC expression on resistance to zinc cytotoxicity. Cell lines were incubated with the indicated concentration of zinc for 24 h, and viability was then measured. PrPC-expressing A74 cells were much more resistant toward the high zinc levels compared to control cells. Results are expressed as mean of three experiments percentage 7S.D. of viable cells, assuming 100% viability for zinc-untreated A74 cells. *, po0.005; ], po0.01.

PrPC-expressing A74 cells were much more resistant toward the high zinc levels compared to control cells. Interestingly, treatment of PrPC-expressing A74 cells with 50 or 100 mmol/L zinc actually increased cell viability compared with untreated controls. Moreover, this protection was specific to PrPC expression, since treatment of RK13 cells (non-transfected cells used as control) with doxycyline (500 ng/mL) did not offer any protection against zinc cytotoxicity (data not shown).

Effect of PrPC on cellular

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Zinc uptake

To explore the impact of the prion protein on zinc uptake in A74 cells, cells were exposed to 65Zn2+ (0.25 mCi/mL) in complete medium in the absence (0 Dox) or in the presence of 500 ng/mL doxycycline (500 Dox) over 30 h of incubation time at 37 1C. Fig. 2 shows a kinetic curve of zinc uptake for unstimulated and PrPC-expressing A74 cells, which is time dependent. PrPC expression did not enhance zinc uptake. These results were confirmed by measuring total cellular zinc content by electrothermal atomic absorption spectrometry (Table 1). No difference in zinc content was observed between control and Dox-stimulated cells. Since doxycycline induces PrPC synthesis in A74 cells in a time- and concentration-dependent manner [5], the expression levels as well as the induction time of PrPC may explain the absence of the impact of this protein on zinc uptake in such conditions. To verify this hypothesis, PrPC expression was induced in A74 cells 24 h prior to the zinc uptake kinetic assay but no difference was observed (data not shown). We also carried out these experiments in both PBS and MEM medium. In this case, a significant difference in zinc uptake was observed between cells incubated in MEM medium compared to

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Fig. 2. Kinetic curve of zinc uptake after 0, 2, 8, 12, 24 and 30 h incubation in radioactive media for unstimulated and PrPC-expressing A74 cells. PrPC expression did not enhance zinc uptake. Zinc binding was quantified and normalized to the protein content of cell lysates. Data represent the mean of three experiments 7S.D. Table 1. Determination of copper and zinc concentrations in whole cells lysate. Cells

Cu (mg Cu/g prot)

Zn (mg Zn/g prot)

0 Dox 500 Dox

8.371.4 15.572.9

309.1745.3 318.2735.7

Copper and zinc concentrations have been determined in whole cells lysate supernatants by electrothermal atomic absorption spectrophotometry. Values are expressed as copper or zinc concentration normalized to protein content. The standard errors are the standard deviations of the means from several measurements (n ¼ 3).  po0.05 versus 0 Dox.

those incubated in PBS (data not shown). This result suggests that amino acids present in the culture medium have an important effect on the zinc uptake. However, both unstimulated and PrPC-expressing A74 cells showed again, either in PBS or complete medium, a similar kinetic curve in zinc uptake.

Exchangeable zinc levels and localization studies The intracellular free zinc concentration and localization were then studied by flow cytometry and cell imaging, using zinpyr-1 as a probe. Flow cytometry analysis of both unstimulated and doxycycline-treated A74 cells is presented in Fig. 3A. Unstimulated and Dox-treated cells displayed an important fluorescence intensity when they were loaded with 10 mmol/L zinpyr1, compared to the negative control cells. However, the expression of PrPC did not significantly modify the fluorescence intensity in doxycycline-stimulated cells. The impact of PrPC expression on exchangeable zinc has also been investigated after extracellular zincinduced excessive zinc uptake in A74 cells. Unstimulated and stimulated A74 cells were exposed to various concentrations of zinc (0–100 mmol/L) in OptiMEM medium for 1 h at 37 1C and then analyzed by FACS as

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Fig. 4. Effect of PrPC expression on the intracellular free zinc analyzed by using zinquin-ethyl-ester. Unstimulated or Doxstimulated cells were incubated with a micromolar solution of zinquin-ethyl-ester in PBS at 37 1C for 30 min. Then, cells observed under an inverted fluorescence microscope (excitation wavelength 365 nm, emission wavelength 420 nm). Cells were photographed with a cooled digital camera ORCA-100 (Hamamatsu, Japan) and fluorescence was quantified using the Scion Image software. Zinc fluorescence was drastically increased in Dox-stimulated cells compared to control *, po0.0001 versus 0 Dox; 1, po0.05 versus 10 Dox.

Fig. 3. (A) Effect of PrPC expression on exchangeable zinc analyzed by flow cytometry after zinpyr-1 staining. Unstimulated and Dox-treated cells displayed important fluorescence intensity when they were loaded with 10 mmol/L zinpyr-1, compared to the negative control cells, which were used as negative control to adjust the cell sorter parameters for the granularity and auto fluorescence parameters. However, PrPc expression after Dox treatment did not modify fluorescence intensity of zinpr-1-loaded cells. (B) Effect of PrPC expression on exchangeable zinc analyzed by fluorescence microscopy after zinpyr-1 staining. Unstimulated A74 cells mainly present a strong perinuclear fluorescence signal (upper left). Almost all fluorescence was abolished when cells were incubated with TPEN (upper right). PrPC-expressing A74 cells displayed a strong cytoplasmic signal (lower left). This signal could not be completely abolished by TPEN treatment (lower right).

described above. A remarkable increase in fluorescence intensity was observed in both conditions (data not shown). Therefore, addition of zinc ions to the culture medium, but not PrPC expression, strongly increased intracellular accumulation of free zinc ion in a concentration-dependent manner. As shown in Fig. 3B, unstimulated A74 cells mainly presented a strong perinuclear fluorescence signal, with a very slight, diffuse cytoplasmic fluorescence. Almost

all fluorescence was abolished when cells were incubated with TPEN, a strong membrane-permeant zinc-specific chelator. Contrary to control cells, PrPC-expressing A74 cells displayed a quite different staining: in these cells a perinuclear, Golgi-like region was still observed, but a strong cytoplasmic, punctuate signal appeared after doxycycline treatment. Moreover, this vesicular zinc observed with PrPC expression could not be completely abolished by TPEN treatment, suggesting that zinc is highly concentrated in these vesicular organelles. To confirm that PrPc expression induced zinc re-localization in zinc-rich vesicles/lysosome-like structures, we stained the cells with the membrane-permeant UVexcitable Zn-specific fluorophore zinquin-ethyl-ester, which stains vesicular zinc. In these conditions, zinc fluorescence was drastically increased in Dox-stimulated cells compared to control (Fig. 4).

PrPC expression increases metallothionein gene expression and protein levels Because zinc is mainly stored inside cells as bound to metallothioneins, we examined whether these proteins were expressed differently in the various cell lines. MT gene transcription was measured with a luciferase reporter

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MT promoter transcriptional activity was strongly activated with PrPC expression. Metallothionein proteins were revealed by immunocytochemistry on fixed cells. Exogenous zinc ions induced MT expression at the protein level in both cell types, as expected (Fig. 6). Dox treatment alone had no effect on metallothioneins expression, since Dox-treated RK13 cells have the same MT levels as untreated RK13 cells. Contrasting with RK13 cells, treatment of A74 cells with doxycycline obviously increased MT protein expression level (Fig. 6). These data suggest that PrPC expression is sufficient to induce MT upregulation at the protein level, consistent with data obtained with the gene reporter assay.

Discussion Fig. 5. Effect of PrPC expression on metallothionein promoter activity in RK13 cells and A74 cells. Control RK13 cells displayed basal levels of MT promoter activity. However, Dox-stimulated cells exhibited a stronger promoter activation of transcription. Luciferase assay was performed on cell lysate and results were expressed as arbitrary light units after correction for protein concentration. Results are expressed as mean from several measurements (n ¼ 3) 7S.D. Zinc-treated cells were used as a positive control for metallothionein promoter activation (*, po0.01).

Fig. 6. Effect of PrPC expression on metallothionein expression in RK13 and A74 cells. Cells were cultivated and treated as indicated in ‘Material and methods’ and immunostained for the metallothionein protein and zinc-treated cells were used as a positive control for metallothionein immunofluorescence detection. Treatment of A74 cells with doxycycline (contrary to RK 13 control cells) increased MT protein expression level.

gene assay driven by the metallothionein I promoter, including the region 727 to 13 from the transcription start site. Results are shown in Fig. 5. Control RK13 cells displayed basal levels of MT promoter activity. In contrast, Dox-stimulated cells exhibited a stronger promoter activation of transcription. Supplementation of both control and Dox-stimulated cells with 100 mmol/L ZnSO4 for 24 h similarly activated MT promoter. Thus,

In this study, we have investigated in detail the relationship between the prion protein and zinc ions using a cellular model in which the expression of murine PrPC was adjustable in a dose-dependent manner by a doxycycline treatment. Cell death determination revealed decreased zinc-mediated toxicity in Dox-stimulated compared to unstimulated cells. We have shown previously that PrPC presents a powerful protective effect against copper-mediated cytotoxicity in A74 cells [5]. However, expression of PrPC in A74 cells was unable to offer a protection against manganese or cadmiummediated cytoxicity in these cells. This specific protective effect of PrPC on copper and zinc toxicity may be due to the chelating or buffering effect of PrPC on the neuron surface. The brain has one of the highest zinc content with respect to other organs. The average of total brain zinc concentration was estimated to be approximately 150 mmol/L [13]; 5–15% of brain zinc is present up to millimolar concentrations in presynaptic vesicles of certain glutamatergic terminals [14,15]. Upon excitation, these vesicles fuse with the presynaptic membrane and release their zinc into the synaptic cleft where it can reach concentrations in the 100–300 mmol/L range [16]. At present, it is known that zinc excess causes apoptosis or necrotic cell death [17]. Application of 150–300 mmol/L Zn for 30 min generated toxic-free radicals and caused necrotic death in cortical cell cultures [18], and programmed cell death in cultured C6 rat glioma cells [19]. In some cases, zinc can even act as a pro-oxidant metal, by enhancing production of reactive oxygen species [20]. PrPC could protect neurons by either sequestering zinc excess or by being involved in a re-uptake mechanism as described by Pauly and Harris [6]. Regarding the literature data, controversy has emerged concerning the binding capacity of PrPC to zinc ions. Indeed, using a recombinant PrPC or a synthetic peptide corresponding to the PrPC protein,

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some studies suggested that PrPC has a binding capacity to zinc ions, while, the other group reported that PrPC binds copper but not zinc ions (for review see [21]). Such controversy may be explained by the fact that most studies have used an in vitro assay to investigate the relationship between PrPC and zinc ions, which do not reflect the reality in cellular event. In addition, no data is available yet about the effect of the prion protein on the cellular zinc uptake. To clarify the role of PrPC in zinc metabolism, we used the radioactive zinc ions (65Zn). A remarkable 65Zn2+ uptake was observed in both Doxstimulated and unstimulated cells, which is more pronounced in the presence of complete medium than PBS, suggesting a role of amino acid in such process. Indeed, it has been reported that amino acids, especially histidine, enhanced considerably the cellular zinc uptake in rat erythrocytes [22]. A specific effect of histidine upon Zn(II) uptake has been observed previously in a wide range of species and tissues [23]. Surprisingly, PrPC expression does not affect zinc uptake by A74 cells, supporting a weaker or undetectable interaction of this protein with zinc ions [24]. This was also confirmed by flow cytometry analysis, using the zinc-specific probe zinpyr-1, which shows no significant global difference in their intracellular exchangeable zinc content between the Dox-stimulated and unstimulated cells. Furthermore, spectrofluorometry quantification of total zinc corroborates the absence of PrPC impact on the zinc accumulation. Therefore, it was puzzling that PrPC-expressing cells were more resistant to zinc. Because intracellular localization of zinc ions has been proved important for zinc signaling [25–27], especially zinc binding and release from proteins, or zinc exchange between intracellular organelles, we aimed to assess whether zinc ions localization was affected by PrPC expression. Using zinc-specific fluorophores, especially zinquin-ethyl-ester, a specific fluorophore for vesicular free zinc [28], we observed a significant re-localization of intracellular exchangeable zinc in zinc-rich vesicles, socalled ‘zincosomes’ [15], after PrPC expression. Then, we studied the effect of PrPC expression on metallothioneins (MT), the main intracellular free zinc-buffering proteins, and observed an increase in both transcription and expression of MT in PrPC-expressing cells. MT display a high zinc-binding affinity (KZn ¼ 3.2  1013 at pH 7.4) and bind seven zinc ions due to a particular coordination of the metal with their cysteine sulfur ligands [29]. MT play a role in maintaining the homeostasis of essential trace metals in the brain [30]. They may also play some yet unidentified role in the response to prion infection. For example it has been suggested that the expression of MT in astrocytes is regulated differentially in different human prion diseases and modified locally by abnormal prion protein deposition [31]. Immunoreactivity for both MT-I/II and MT-III in the astrocytes of Creutzfeldt–Jakob disease patients with

a relatively long disease course was significantly reduced. Another study showed that the concentration of MT-I/II was greater in the medulla oblongata of cattle with BSE than in healthy control animals [32]. However, Vidal et al. [33] demonstrated very recently a lack of differences in prion disease progression and lesion between the MT-I/II knockout and wild-type mice, which could be due to the presence of compensatory mechanisms such as upregulation of MT-III or HSP25 [33]. MT are able to sequester zinc immediately after its uptake by the cell forming Zn-MT complex, thus preventing an excessive increase of free cytoplasmic zinc and avoiding toxic effects of the metal [34]. Since PrPC expression by itself did not stimulate zinc uptake, we explain the PrPC-induced MT induction by the increased uptake of copper due to PrPC expression. Copper, for which MT have greater affinity than zinc ions, then replaces zinc from zinc bound MT. Free zinc released from MT then stimulates MT gene expression through the zinc-sensing transcription factor MTF1 [35]. The relationship between MT and PrPC is very important and can explain many mysterious points, especially the link between PrPC, resistance to oxidative stress and metal toxicity. It has been reported that PrPC presents an antioxidant activity, which was revealed in enhancing the survival of cells in culture exposed to oxidative stress conditions [36]. Moreover, the protective effect against oxidative stress injury of PrPC was demonstrated in several studies in both cellular [5,36,37] and animal [36] models. We have shown that PrPC presents a powerful protective effect against coppermediated cytotoxicity in A74 cells [5] and prion-infected hypothalamic neuronal GT1 cells were more sensitive to induced oxidative stress than uninfected cells and showed decreased GPX and glutathione reductase activities together with altered SOD metabolism [38]. The functions of PrPC can be explained by the fact that MT is involved in metal detoxification, such as zinc and copper. Furthermore, MT appear to be regulated by oxidative stress [39,40] and have antioxidant function [41,42]. MT can protect cells from oxidative stress damage to lipid and DNA because of their free radical scavenging activities due to their high thiol content [43,44]. In a zinc-resistant cell model, MT mediate the main cellular protective effect of zinc against oxidative injury [45]. Recently, it has been shown that MT may provide neuro-protection by decreasing peroxinitrite-induced oxidative stress in Parkinson disease [46]. The link between zinc, MT and PrPC may explain the mechanism of neurodegeneration in prion diseases because zinc and other transition metals play an important role in the neuropathology of neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. In Alzheimer disease the amyloid precursor protein (APP) is proteolytically cleaved by the b- and g-secretase to form the

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amyloidogenic and neurotoxic amyloid-b (Ab) peptide, which binds both copper and zinc [47,48]. Interestingly, a decrease in Ab formation in the brains of transgenic mice lacking the synaptic ZnT3 zinc transporter [49], which controls the total amount of elemental zinc in synaptic vesicles [50], has been reported. Studies using mice have shown promising results using clioquinol, an antibiotic that selectively chelates zinc and copper. Clioquinol treatment for only 9 weeks was capable to inhibit 49% of Ab deposition [51]. Zinc homeostasis deregulation may also play an important role in Parkinson disease (PD). It has been shown that zinc deficiency is present in PD [52]. Other studies in PD brains found a significantly decreased levels of zinc in the cerebrospinal fluid [53]. Similarly, the involvement of MT has been hypothesized in PD: increased MT-I and MT-II levels have been found in PD brains [54]. MT isoforms seem capable of attenuating oxidative stress by scavenging free radicals and free metals, thus providing neuro-protection in PD [54]. In conclusion, we have shown that expression of PrPC confers high resistance to zinc toxicity, induces re-localization of intracellular exchangeable zinc. Moreover, these results demonstrate for the first time a relationship between PrPC and MT expression through zinc ions signaling. However, we could not exclude that MT induction could be also dependent on other mechanisms, and therefore further studies are required to verify that such mechanisms would be applicable in vivo. These findings are of major importance since zinc deregulation is implicated in several neurodegenerative disorders. We can postulate that in prion diseases the conversion of PrPC to PrPSc may deregulate zinc homeostasis mediated by MT.

Acknowledgement This work was supported by a Grant from European Community QLRT-2000-02353, molecular basis of neurodegeneration. FC was supported in part by the ‘‘Programme de Toxicologie Nucle´aire Environnementale’’ (http://www.toxnuc-e.org). WR is supported by Fondation Recherche Medicale (FRM). We thank Dr. Josiane Arnaud for trace element measurement by atomic spectrophotometry and Jocelyne Chantegrel for technical assistance.

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