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NP7 protects from cell death induced by oxidative stress in neuronal and glial midbrain cultures from parkin null mice
FEBS Letters 583 (2009) 168–174
journal homepage: www.FEBSLetters.org
NP7 protects from cell death induced by oxidative stress in neuronal and glial midbrain cultures from parkin null mice M.A. Mena a,*, M.J. Casarejos a, R. Solano a, J.A. Rodríguez-Navarro a, A. Gómez a, I. Rodal b, M. Medina c, J.G. de Yebenes b a
Departmento de Neurobiología-Investigación, CIBERned, Hospital Ramón y Cajal, Carretera de Colmenar, km 9, Madrid 28034, Spain Department of Neurobiology, CIBERned, Hospital Ramon y Cajal, 28034 Madrid, Spain c Noscira, Tres Cantos, Madrid, Spain b
a r t i c l e
i n f o
Article history: Received 20 October 2008 Revised 19 November 2008 Accepted 27 November 2008 Available online 10 December 2008 Edited by Jesus Avila Keywords: Parkinson’s disease Parkin knockout mice Dopamine neurons Glia Oxidative stress Apoptosis p-ERK and p-AKT signaling pathways Aged glia
a b s t r a c t Parkin mutations produce Parkinson’s disease (PD) in humans and nigrostriatal dopamine lesions related to increased free radicals in mice. We examined the effects of NP7, a synthetic, marine derived, free radical scavenger which enters the brain, on H2O2 toxicity in cultured neurons and glia from wild-type (WT) and parkin null mice (PK-KO). NP7, 5–10 lM, prevented the H2O2 induced apoptosis and necrosis of midbrain neuronal and glial cultures from WT and PK-KO mice. NP7 suppressed microglial activation and the H2O2 induced drop-out of dopamine neurons. Furthermore, NP7 prevented the increased phosphorylation of ERK and AKT induced by H2O2. NP7 may be a promising neuroprotector against oxidative stress in PD. Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Parkinson’s disease (PD) is a neurodegenerative disorder of multifactorial origin [1–3]. The majority of the cases are sporadic and of unknown origin [4] and in most cases it is considered that the drop-out of nigrostriatal dopamine neurons, one of the land marks of the disease, could be mediated by multiple mechanisms including genetic abnormalities of several proteins, aging, accumulation of free radicals, mitochondrial dysfunction and others [4–6]. Mutations of Park-2, a gene which codes for parkin, a protein with ubiquitin ligase function, are the most common cause of autosomal recessive parkinsonism in humans world wide [7–9]. The pathogenic mechanism of parkin related PD is considered, at least partly, to be due to excessive free radical formation in the intracellular mono amino oxidase (MAO) mediated metabolism of dopamine (DA) [10,11]. The suppression of parkin impairs the release of dopamine and shifts its metabolism to dihydroxy-phenyl-acetic
* Corresponding author. Fax: +34 91 3369016. E-mail address: [email protected] (M.A. Mena).
acid (DOPAC), producing a molecule of H2O2 per molecule of DA [10–12]. Parkin null mice, while young, are able to compensate the excessive free radical production, due to their abnormal metabolism of DA, with an increased production of glutathione (GSH) [10] and, therefore, are more resistant than wild-type (WT) to L-DOPA toxicity [12], which is also mediated by increased free radical production through the metabolism of catecholamines, or to other free radical producing neurotoxins such as nitric oxide [13]. But parkin null cells are more susceptible to additional insults such as treatment with rotenone, a mitochondrial toxin [14], calcium channel antagonists, such as cinnarizine [15] or to the effects of aging, when the supportive function of glia is reduced and the increased production of GSH collapses [16]. Parkin enhances the survival of the dopamine neurons [14,17– 21]. Parkin dysfunction impairs astrocytic function and contributes to the pathogenesis of parkin-linked AR-PD [22]. Furthermore, a reduction in parkin levels renders the rat glial-like cell line RG-6 more susceptible to dopamine (DA)- and H2O2-induced cell death [23]. In PK-KO aging midbrain glia, low H2O2 concentration induced cell death [16].
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.11.051
M.A. Mena et al. / FEBS Letters 583 (2009) 168–174
We have hypothesized that, if the high production of free radicals is the key pathogenic mechanism for the death of the nigrostriatal dopamine neurons in parkin related PD and that if the increased production of GSH is able to compensate such increase in free radicals as long as it lasts, the treatment of parkin null nervous cells with free radical scavengers will reduce the cell death of DA neurons. Here we report the neuroprotective effects of NP7 in parkin null dopamine neurons and glial cells challenged with H2O2. NP7 is a new antioxidant, marine derived, with a different chemical molecular structure that the classic phytochemicals, obtained by lead optimization system from the Streptomyces species, with free radical scavenger properties in the sub lM–nM range and which crosses the blood brain barrier in mammals [24].
2. Materials and methods 2.1. Materials Drugs, culture media, chemical reagents and antibodies. NP7 was provided by Noscira from a group of synthetic, marine derived antioxidants. Culture media and antibodies were used as previously described [12,16] and are detailed in Supplementary material. 2.2. Cell cultures Neuronal-enriched and glial mesencephalic cultures were obtained from 129SV/C57BL6 WT or parkin mutant mice (PK-KO) as previously described [10,12,16]. In accordance with the European
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Union Directives, all efforts were made to minimize the number of animals used and their suffering. Neuronal-enriched mesencephalic and glial cultures were obtained and prepared as previously described [12,16,25]. Culture medium from glial cultures was refreshed after 6–7 days and every 7 days thereafter. After 18 days in culture, positive staining with anti-GFAP antibody identified the astrocytes in these cultures and contained at least 80–90% of total cells. 2.3. Cell treatments WT and PK-KO neuronal-enriched midbrain cultures, maintained for 7 days in vitro (DIV), were seeded with a density of 250.000 cells in P24 multiwells or 200.000 cells in cover-slides, for immunocytochemistry (ICQ) studies. For the curve dose– response experiments, the cultures were treated for 3 h with increasing concentrations of NP7. In another set of experiments, WT and PK-KO cultures were treated with NP7 (5–10 lM) 60 min before the treatment with H2O2 (50 lM). The growth medium was changed to EMEM plus D-glucose (6 mg/ml), the most appropriate medium for H2O2 stability, for the experiments with hydrogen peroxide. Glial cell treatments. After 15–20 DIV in the growth medium (DMEM-FCS), glial cultures had reached confluent; at this time, the cells were mildly trypsinized and reseeded for the experiments at a density of 1.3 104 cells/cm2 in 24- 12-well plates cultures and maintained in growth medium for 6–7 additional days. In the experiments, the cultures were treated 90 min before H2O2 (100 lM) treatment with NP7 at the different concentrations tested. In the experiments with H2O2, the growth medium was
Fig. 1. Effects of NP7 on hydrogen peroxide-induced toxicity in neuronal-enriched mesencephalic cultures from WT mice. After 7 days in vitro, the cells were pretreated for 1 h with 5 lM NP7 in EMEM + D-glucose medium and then was added 50 lM H2O2 for two additional hours. (A) Mitochondrial activity measured by MTT assay. (B) Percentage of apoptotic cells with respect to the total number. (C) Number of DA neurons expressed as percentage of TH+ cells (TH+ cells in control, 450 ± 10). (D) Optical intensity of the immunoreactivity of astroglial (GFAP+) cells. Values are the mean ± S.E.M. of three–four independent cultures with six replicates each. Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls multiple comparison test. ***P < 0.001 H2O2 treated cultures vs. controls, DDDP < 0.001 H2O2 treatment plus NP7 vs. the corresponding H2O2 treatments alone.
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changed to EMEM plus D-glucose (6 mg/ml). In another set of experiments, we used aged glial cultures maintained in culture for 5–7 months, changing the growth medium (DMEM-FCS) every week. 2.4. Cellular characterization and viability, anti-oxidants and pro-oxidants DA neurons were characterized by immunostaining with a mouse anti-TH antibody (1:500) and astrocytes with a rabbit anti-GFAP antibody (1:500); microglial cells were identified with isolectin B4 peroxidase-labelled (12.5 lg/ml) [26,27]. The number of immunoreactive cells was counted in one-seventh of the total area of the cover-slides. The cells were counted in predefined parallel strips using a counting reticule inserted into the ocular. Cell viability was analyzed with the mitochondrial activity assay which measures the metabolism of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Apoptosis was measured by light microscopy, DNA staining with bis-benzimide (Hoechst 33342) and the TUNEL assay [16,28,29]. Necrosis was measured according to lactate dehydrogenase activity [30] in the culture medium and by trypan blue dye exclusion in cells [29]. Total glutathione, reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured by the methods of Tietze [31] and Griffith [32], respectively. The concentration of H2O2 was estimated with a colorimetric assay; 3,30 -dimethoxybenzidine, which is colourless in its reduced form, is oxidized in the presence of H2O2 and peroxidased into a red-coloured product [16]. The concentration of intracellular iron in midbrain glial cultures was estimated by the colorimetric ferrozine assay [33].
2.5. Western blot analysis The levels of proteins involved in signaling of survival responses, pERK and IP3/AKT pathways, were measured by Western blot according to previously described techniques. We used a mouse anti-phospho-ERK-1/2 and rabbit anti-ERK-1/2 antibodies from Sigma (Madrid, Spain) diluted 1:5000 and 1:10 000, respectively. Rabbit anti-phosphorylated AKT and rabbit polyclonal anti AKT antibodies from Cell-Signaling (Boston, MA, USA) at 1/400 each [16,25,34]. 2.6. Statistical analysis The statistical evaluation was performed by two-way analysis of variance followed by the Bonferroni test, or by one-way ANOVA followed by Newman–Keuls test, using the GraphPad PRISM 4 software. Differences were considered significant when P < 0.05.
3. Results and discussion 3.1. Effects of NP7 on H2O2 toxicity in midbrain neuronal cultures from WT and PK-KO mice The curve dose–response at concentrations of NP7 from 0.5 to 10 lM did not show any toxicity in the MTT assay (data not shown). NP7, 5 lM, 60 min before the treatment with H2O2 (50 lM), prevented partially the effects of H2O2 on mitochondrial activity (H2O2-treated groups had a 81% decreased vs. WT and
Fig. 2. Effects of NP7 on cell viability and H2O2 detoxification in neuronal-enriched mesencephalic cultures from WT and PK-KO mice. After 7 days in vitro, WT and PK-KO cells, 60 min before the treatment with H2O2 (50 lM) for 2 h, received 10 lM of NP7. (A) Mitochondrial activity (MTT assay). (B) LDH activity. (C) H2O2 levels in the medium 10 min post-treatment with H2O2, 50 lM. (D) Percentage of apoptosis, condensated and fragment cells staining with bis-benzimide, in WT and PK-KO mesencephalic cultures treated with solvent, H2O2 (50 lM) or H2O2 plus NP7 (10 lM). (E) Photomicrographs of total nuclei stained with bis-benzimide and positive co-localization of apoptoticfragmented cells obtained by the TUNEL assay corresponding to the same field. Scale bar = 30 lm. Amplified inset shows that chromatin-condensed nuclei are not comarked by TUNEL assay. (F) Percentage of apoptotic-fragmented cells by TUNEL assay. Values are the mean ± S.E.M. of two–three independent cultures with six replicates each. Statistical analysis was performed by two-way ANOVA (the interaction between genotype and treatment was P < 0.01) followed by Bonferroni post-test. *P < 0.05; **P < 0.01; *** P < 0.001 H2O2 treated cultures vs. controls, ++P < 0.01; +++P < 0.001 PK-KO vs. WT cultures; DP < 0.05; DDP < 0.01; DDDP < 0.001 H2O2 treatment plus NP7 vs. the corresponding H2O2 treatments alone.
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Fig. 3. Effects of NP7 on dopamine neurons and microglial cells in mesencephalic cultures from WT and PK-KO mice. After 7 days in vitro, the cells were treated with H2O2 (50 lM) for 2 h; NP7, 10 lM, was added to pre-established groups 60 min before the peroxide. (A) Photomicrographs of DA neurons (TH+ cells), scale bar = 30 lm. (B) DA neurons expressed as percentage of TH+ (TH+ cells in WT control, 427 ± 11; TH+ cells in PK-KO control, 411 ± 10). (C) Photomicrographs showing microglial (Isolectin B4+) cells, scale bar = 30 lm. (D) Percentage of microglial cells present in WT and PK-KO midbrain cultures (Isolectin B4+ cells in WT and PK-KO controls were 448 ± 8 and 749 ± 48, respectively). Values are the mean ± S.E.M. of two independent cultures with six replicates each. Statistical analysis was performed by one-way ANOVA followed by Newman Keuls multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001 H2O2 treated cultures vs. controls, + P < 0.05 PK-KO vs. WT cultures; DP < 0.05; DDP < 0.01; DDDP < 0.001 H2O2 treatment plus NP7 vs. the corresponding H2O2 treatments alone.
Fig. 4. NP7 protects WT and PK-KO midbrain glial cultures from cell death induced by H2O2. Glial cultures maintained during 20–30 DIV in DMEM + 15% FCS were used for these experiments; 6–7 days after reseeding, the cells were treated with 100 lM of H2O2 for 2 h in EMEM + D-glucose; 90 min before treatment pre-established groups were treated with NP7 (0.5 or 5 lM). (A) Mitochondrial and LDH (B) activities in WT glial cultures pre-exposed to 0.5 and 5 lM of NP7 previous to H2O2 100 lM treatment. (C) Photomicrographs showing trypan blue dye exclusion and (D) number of necrotic cells in WT glial cultures. (E) LDH activity in WT and PK-KO glial cells treated with H2O2 (100 lM), solvent or H2O2 (100 lM) plus NP7 (5 lM). (F) Intracellular glutathione. (G) Levels of H2O2 in the medium. Values are the mean ± S.E.M. of two independent cultures with six replicates each. Statistical analysis was performed by one- or two-way ANOVA (the interaction between genotype and treatment was P < 0.01) followed by Newman–Keuls multiple comparison test and Bonferroni post-test, respectively. *P < 0.05; **P < 0.01; ***P < 0.001 H2O2 treated cultures vs. controls, +P < 0.05; ++P < 0.01; +++ P < 0.001 PK-KO vs. WT cultures; DP < 0.05, DDP < 0.01, DDDP < 0.001 H2O2 treatment plus NP7 vs. the corresponding H2O2 treatments alone.
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NP7 + H2O2 prevented to a 64% decreased vs. WT) (Fig. 1A) and on apoptosis (H2O2-treated groups had a 52%, NP7 + H2O2 prevented to a 15% increased vs. the 5.9% of WT control) (Fig. 1B). However, NP7, 5 lM, prevented totally the drop-out of dopamine (TH+) neurons (Fig. 1C) and astroglia activation (Fig. 1D). Therefore, NP7, when administered before H2O2 has a potent neuroprotective effect in WT midbrain neuronal-enriched cultures. We have previously shown that WT and PK-KO neuronal-enriched cultures grown in B27/NBL serum-free defined medium for 7–8 days in vitro are formed by 80% neurons (MAP-2+), of which around 1–2% are DAergic (TH+), and 10–15% glia, mostly astrocytes (GFAP+), glial progenitors (A2B5), microglia (isolectin B4) and oligodendrocytes (O1+); the rest are nestin+ neuronal progenitors [12]. Now, we have compared the neuroprotective effects of NP7 on WT and PK-KO midbrain neurons. In both types of neurons, H2O2 altered and NP7, 10 lM, prevented the decreased MTT metabolism, from 40% to 70% in H2O2 and NP7 + H2O2 treated groups vs. controls, respectively (Fig. 2A). LDH increased activity-induced by H2O2 was totally prevented with NP7, 10 lM (Fig. 2B), as well as the remaining H2O2 levels in the medium (Fig. 2C) and the percentage of apoptotic cells (Fig. 2D–F). As expected, after 2 h of H2O2 treatment, we observed a higher increase in early condensatedapoptotic cells, measured by bis-benzimide staining (Fig. 2D and E) than in fragmented-apoptotic cells valued by the TUNEL assay (Fig. 2E and F). The percentage of TH+ neurons loss (Fig. 3A and B) and the activation of microglial cells were totally prevented with NP7, 10 lM (Fig. 3C and D). In general, the effects and counter effects of H2O2 and NP7, respectively, on these parameters were similar in both WT and PK-KO mice, but there were some very interesting exceptions worthy of discussion. The levels of H2O2 were significantly lower in H2O2 treated PK-KO neurons than in H2O2 treated WT and, accordingly, the drop-out of TH+ neurons after H2O2 treatment was less severe in PK-KO than in WT cultures. We have described that PK-KO mice have greater levels of GSH in brain than their WT controls, perhaps as a mechanism of compensation of the chronic increased production of free radicals which they have. These increased GSH levels could work as a scavenging mechanism for H2O2, reducing its levels and its toxicity.
in PK-KO mice increase with age [20,21,35]. Therefore, we have investigated the effects of H2O2 and NP7 on midbrain glia maintained in growth medium (DMEM-FCS) for 5–7 months, changing the growth medium (DMEM-FCS) every week. Aged PK-KO glial cells are more sensitive than aged WT to H2O2 [16], so we treated glial cultures with lower concentrations of H2O2, 50 lM. At this concentration of H2O2, WT glia does not produce elevation of LDH (Fig. 5A). The levels of LDH, which are greater in untreated aged PK-KO glia than in untreated aged WT, greatly increase with the treatment with H2O2, 50 lM, an effect which is almost completely prevented by NP7, 5 lM (from a 204% increased in H2O2 to a 129% in NP7 + H2O2) (Fig. 5A). As expected, the treatment with H2O2, 50 lM, increased the levels of H2O2 in WT and much more in PK-KO aged glia (Fig. 5 B). Baseline H2O2 levels were also greater in PK-KO than in WT glia (Fig. 5B). In summary, our data suggest that aged PK-KO glia has difficulties in handling the endogenous production of H2O2 and becomes very susceptible to relatively small concentrations of this oxidating agent which appear to be well tolerated by WT aged glia.
3.2. NP7 protects WT and PK-KO midbrain glial cultures from cell death induced by H2O2 In this work, we found that NP7 protects astroglial cells from WT and PK-KO neuronal-enriched cultures from the H2O2 toxicity. In WT glial cultures treated with H2O2, NP7 restores MTT levels from concentrations of 0.5 lM (Fig. 4A) and has a dose-dependent effect on the reversal of the elevation of LDH levels from 0.5 to 5 lM (Fig. 4B). At a concentration of 5 lM, NP7 completely blocks necrosis in H2O2 treated WT glia (Fig. 4C–E). As in the case of neuronal cultures, H2O2 toxicity is less marked in PK-KO than in WT glial cultures. The role of GSH in the differences between WT and PK-KO cells is shown in Fig. 4F. PK-KO cells have a 128% increased GSH levels vs. WT, and NP7 did not protected from the H2O2-induced decreased in GSH (Fig. 4F). The treatment with H2O2 and NP7 does not alter the levels of iron in WT or PK-KO glia (data not shown). As expected, the treatment with H2O2 increases the H2O2 levels in WT and PK-KO glia but the co-treatment with NP7 slightly reduced them, from 375% to 275% in WT and from 180% to 130% en PK-KO cultures (Fig. 4G). 3.3. Effect of NP7 on H2O2 toxicity in aged glial cultures PD and other neurodegenerative disorders affect mature or senile patients and we have observed that neurochemical deficits
Fig. 5. Effect of NP7 on H2O2-induced toxicity in aged WT and PK-KO glial cultures. Glia cultures maintained for 5–7 months in DMEM + 15% FCS were used for these experiments. (A) LDH activity in WT and PK-KO aged cultures treated for 2 h in EMEM + D-glucose with H2O2 (50 lM); 90 min before the peroxide treatment, preestablished groups received NP7 (5 lM). (B) H2O2 remnant in the medium after 10 min post-treatment with H2O2 (50 lM and 100 lM). Values are the mean ± S.E.M. of two independent cultures with six replicates each. Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls multiple comparison test. **P < 0.01; ***P < 0.001 H2O2 treated cultures vs. controls, +P < 0.05; +++P < 0.001 PK-KO vs. WT cultures, DDP < 0.01; DDDP < 0.001 H2O2 treatment plus NP7 vs. the corresponding H2O2 treatments alone.
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3.4. Effects of oxidative stress and NP7 in signaling pathways H2O2 and other free radical generating agents induce a strong activation of ERKs, JNKs and p38-MAPK on many cellular signaling systems [34,36,37]. Parkin and DJ-1 lead to impairments of oxidative stress response and PI3K/AKT signaling [38,39]. In young (18– 30 DIV) WT glial cultures, treatment with H2O2 produced a rapid and progressive ERK phosphorylation, as measured by the ratio p-ERK/ERK, from 15 to 60 min after treatment (data not shown). NP7, 5 lM protected from p-ERK activation in young glial cultures from WT (465% increased with H2O2 and 300% with NP7 + H2O2) and PK-KO mice (438% increased with H2O2 and 305% with NP7 + H2O2) (Fig. 6A) and from the H2O2 induced p-AKT activation (424% increased with H2O2 and 285% with NP7 + H2O2 in WT and 322% increased with H2O2 and 222% with NP7 + H2O2 in PK-KO mice) (Fig. 6B). Aged PK-KO glia is more sensitive to oxidative stress than WT, and H2O2 induced a higher p-ERK (254% increased with H2O2 and 106% with NP7 + H2O2 in WT and 629% increased with H2O2 and
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139% with NP7 + H2O2 in PK-KO mice) (Fig. 6C) and p-AKT activation (385% increased with H2O2 and 213% with NP7 + H2O2 in WT and 1246% increased with H2O2 and 526% with NP7 + H2O2 in PK-KO mice) (Fig. 6D). NP7 protected very effectively in aged glial cultures from the p-ERK (Fig. 6C) and from the p-AKT activation (Fig. 6D). The special sensitivity of aged PK-KO glia to oxidative stress while there is strong resistance in young PK-KO cells, suggest that these cells are exposed to a greater oxidative challenge and that a compensatory GSH synthesis is increased for some time until it collapses with senescence and renders the cells more susceptible [16]. Furthermore, in the differential oxidative stress response between WT and PK-KO cultures, parkin as an E3 ligase, which participates in the ubiquitin–proteasome system, protein aggregation and protein posttranslational modifications, may play a very important role [40–43]. In summary, NP7 has a strong protective effect in free radical related stress in neurons and glia from WT and PK-KO mice and, may therefore be a promising candidate for neuroprotection against oxidative stress in PD.
Fig. 6. Effects of H2O2 and NP7 on p-ERK and p-AKT signaling pathways. Young and aged glial cultures maintained in DMEM + 15% FCS were used for these experiments; 6–7 days after reseeding, the cells were treated with 100 lM of H2O2 for 30 min in EMEM + D-glucose; 90 min before treatment, pre-established groups were treated with NP7 (5 lM). (A) p-ERK signaling pathway in young glia cultures. (B) p-AKT signaling pathway in young glia cultures. (C) p-ERK signaling pathway in aged glia cultures. (D) p-AKT signaling pathway in aged glia cultures. Values are the mean ± S.E.M. of three independent cultures with six replicates each. Statistical analysis was performed by one- or twoway ANOVA (the interaction between aged glia genotype and treatment was P < 0.01) followed by Newman–Keuls multiple comparison test and Bonferroni post-test, respectively. ***P < 0.001 H2O2 treated cultures vs. controls, +++P < 0.001 PK-KO vs. WT cultures, DP < 0.05; DDP < 0.01; DDDP < 0.001 H2O2 treatment plus NP7 vs. the corresponding H2O2 treatments alone.
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Acknowledgements Supported in part by grants from the Spanish Ministry of Health, FIS 2007/PI07037, NDG 07-4, CAM 0202/2006 and CIBER 2006/05/0059. The authors thank M. Serrano for excellent technical assistance; Mrs. Claire Marsden for editorial help. Appendix A. Supplementary data Materials (Culture media, Antibodies and Chemicals) and Methods (Cell cultures; Immunocytochemistry; Cell viability assays; Determination of GSH, H2O2 and iron levels; and Western blot). Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2008.11.051. References [1] Clarke, C.E. (2007) Parkinson’s disease. BMJ 335, 441–445. [2] Klein, C. and Schlossmacher, M.G. (2007) Parkinson disease, 10 years after its genetic revolution: multiple clues to a complex disorder. Neurology 69, 2093– 2104. [3] Levy, G. (2007) The relationship of Parkinson disease with aging. Arch. Neurol. 64, 1242–1246. [4] Mouradian, M.M. (2002) Recent advances in the genetics and pathogenesis of Parkinson disease. Neurology 58, 179–185. [5] Sadrzadeh, S.M. and Saffari, Y. (2004) Iron and brain disorders. Am. J. Clin. Pathol. 121 (Suppl), S64–S70. [6] Sulzer, D. (2007) Multiple hit hypotheses for dopamine neuron loss in Parkinson’s disease. Trends Neurosci. 30, 244–250. [7] Hardy, J. (2000) Pathways to primary neurodegenerative disease. Ann. NY Acad. Sci. 924, 29–34. [8] Hattori, N., Shimura, H., Kubo, S., Wang, M., Shimizu, N., Tanaka, K. and Mizuno, Y. (2000) Importance of familial Parkinson’s disease and parkinsonism to the understanding of nigral degeneration in sporadic Parkinson’s disease. J. Neural. Transm. Suppl., 101–116. [9] Polymeropoulos, M.H. (2000) Genetics of Parkinson’s disease. Ann. NY Acad. Sci. 920, 28–32. [10] Itier, J.M., Ibanez, P., Mena, M.A., et al. (2003) Parkin gene inactivation alters behaviour and dopamine neurotransmission in the mouse. Hum. Mol. Genet. 12, 2277–2291. [11] Menendez, J., Rodriguez-Navarro, J.A., Solano, R.M., Casarejos, M.J., Rodal, I., Guerrero, R., Sanchez, M.P., Avila, J., Mena, M.A. and de Yebenes, J.G. (2006) Suppression of Parkin enhances nigrostriatal and motor neuron lesion in mice over-expressing human-mutated tau protein. Hum. Mol. Genet. 15, 2045– 2058. [12] Casarejos, M.J., Solano, R.M., Menendez, J., Rodriguez-Navarro, J.A., Correa, C., Garcia de Yebenes, J. and Mena, M.A. (2005) Differential effects of l-DOPA on monoamine metabolism, cell survival and glutathione production in midbrain neuronal-enriched cultures from parkin knockout and wild-type mice. J. Neurochem. 94, 1005–1014. [13] Solano, R.M., Menendez, J., Casarejos, M.J., Rodriguez-Navarro, J.A., Garcia de Yebenes, J. and Mena, M.A. (2006) Midbrain neuronal cultures from parkin mutant mice are resistant to nitric oxide-induced toxicity. Neuropharmacology 51, 327–340. [14] Casarejos, M.J., Menendez, J., Solano, R.M., Rodriguez-Navarro, J.A., Garcia de Yebenes, J. and Mena, M.A. (2006) Susceptibility to rotenone is increased in neurons from parkin null mice and is reduced by minocycline. J. Neurochem. 97, 934–946. [15] Serrano, A., Menendez, J., Casarejos, M.J., Solano, R.M., Gallego, E., Sanchez, M., Mena, M.A. and Garcia de Yebenes, J. (2005) Effects of cinnarizine, a calcium antagonist that produces human parkinsonism, in parkin knock out mice. Neuropharmacology 49, 208–219. [16] Solano, R.M., Casarejos, M.J., Menendez-Cuervo, J., Rodriguez-Navarro, J.A., Garcia de Yebenes, J. and Mena, M.A. (2008) Glial dysfunction in parkin null mice: effects of aging. J. Neurosci. 28, 598–611. [17] Kitada, T., Asakawa, S., Hattori, N., Matsumine, H., Yamamura, Y., Minoshima, S., Yokochi, M., Mizuno, Y. and Shimizu, N. (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392, 605–608. [18] Abbas, N., Lucking, C.B., Ricard, S., Durr, A., Bonifati, V., De Michele, G., Bouley, S., Vaughan, J.R., Gasser, T., Marconi, R., Broussolle, E., Brefel-Courbon, C., Harhangi, B.S., Oostra, B.A., Fabrizio, E., Bohme, G.A., Pradier, L., Wood, N.W., Filla, A., Meco, G., Denefle, P., Agid, Y. and Brice, A. (1999) A wide variety of mutations in the parkin gene are responsible for autosomal recessive parkinsonism in Europe. French Parkinson’s disease genetics study group and the European consortium on genetic susceptibility in Parkinson’s Disease.. Hum. Mol. Genet. 8, 567–574. [19] Lucking, C.B., Durr, A., Bonifati, V., Vaughan, J., De Michele, G., Gasser, T., Harhangi, B.S., Meco, G., Denefle, P., Wood, N.W., Agid, Y. and Brice, A. (2000) Association between early-onset Parkinson’s disease and mutations in the parkin gene. N. Engl. J. Med. 342, 1560–1567.
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journal homepage: www.FEBSLetters.org
NP7 protects from cell death induced by oxidative stress in neuronal and glial midbrain cultures from parkin null mice M.A. Mena a,*, M.J. Casarejos a, R. Solano a, J.A. Rodríguez-Navarro a, A. Gómez a, I. Rodal b, M. Medina c, J.G. de Yebenes b a
Departmento de Neurobiología-Investigación, CIBERned, Hospital Ramón y Cajal, Carretera de Colmenar, km 9, Madrid 28034, Spain Department of Neurobiology, CIBERned, Hospital Ramon y Cajal, 28034 Madrid, Spain c Noscira, Tres Cantos, Madrid, Spain b
a r t i c l e
i n f o
Article history: Received 20 October 2008 Revised 19 November 2008 Accepted 27 November 2008 Available online 10 December 2008 Edited by Jesus Avila Keywords: Parkinson’s disease Parkin knockout mice Dopamine neurons Glia Oxidative stress Apoptosis p-ERK and p-AKT signaling pathways Aged glia
a b s t r a c t Parkin mutations produce Parkinson’s disease (PD) in humans and nigrostriatal dopamine lesions related to increased free radicals in mice. We examined the effects of NP7, a synthetic, marine derived, free radical scavenger which enters the brain, on H2O2 toxicity in cultured neurons and glia from wild-type (WT) and parkin null mice (PK-KO). NP7, 5–10 lM, prevented the H2O2 induced apoptosis and necrosis of midbrain neuronal and glial cultures from WT and PK-KO mice. NP7 suppressed microglial activation and the H2O2 induced drop-out of dopamine neurons. Furthermore, NP7 prevented the increased phosphorylation of ERK and AKT induced by H2O2. NP7 may be a promising neuroprotector against oxidative stress in PD. Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Parkinson’s disease (PD) is a neurodegenerative disorder of multifactorial origin [1–3]. The majority of the cases are sporadic and of unknown origin [4] and in most cases it is considered that the drop-out of nigrostriatal dopamine neurons, one of the land marks of the disease, could be mediated by multiple mechanisms including genetic abnormalities of several proteins, aging, accumulation of free radicals, mitochondrial dysfunction and others [4–6]. Mutations of Park-2, a gene which codes for parkin, a protein with ubiquitin ligase function, are the most common cause of autosomal recessive parkinsonism in humans world wide [7–9]. The pathogenic mechanism of parkin related PD is considered, at least partly, to be due to excessive free radical formation in the intracellular mono amino oxidase (MAO) mediated metabolism of dopamine (DA) [10,11]. The suppression of parkin impairs the release of dopamine and shifts its metabolism to dihydroxy-phenyl-acetic
* Corresponding author. Fax: +34 91 3369016. E-mail address: [email protected] (M.A. Mena).
acid (DOPAC), producing a molecule of H2O2 per molecule of DA [10–12]. Parkin null mice, while young, are able to compensate the excessive free radical production, due to their abnormal metabolism of DA, with an increased production of glutathione (GSH) [10] and, therefore, are more resistant than wild-type (WT) to L-DOPA toxicity [12], which is also mediated by increased free radical production through the metabolism of catecholamines, or to other free radical producing neurotoxins such as nitric oxide [13]. But parkin null cells are more susceptible to additional insults such as treatment with rotenone, a mitochondrial toxin [14], calcium channel antagonists, such as cinnarizine [15] or to the effects of aging, when the supportive function of glia is reduced and the increased production of GSH collapses [16]. Parkin enhances the survival of the dopamine neurons [14,17– 21]. Parkin dysfunction impairs astrocytic function and contributes to the pathogenesis of parkin-linked AR-PD [22]. Furthermore, a reduction in parkin levels renders the rat glial-like cell line RG-6 more susceptible to dopamine (DA)- and H2O2-induced cell death [23]. In PK-KO aging midbrain glia, low H2O2 concentration induced cell death [16].
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.11.051
M.A. Mena et al. / FEBS Letters 583 (2009) 168–174
We have hypothesized that, if the high production of free radicals is the key pathogenic mechanism for the death of the nigrostriatal dopamine neurons in parkin related PD and that if the increased production of GSH is able to compensate such increase in free radicals as long as it lasts, the treatment of parkin null nervous cells with free radical scavengers will reduce the cell death of DA neurons. Here we report the neuroprotective effects of NP7 in parkin null dopamine neurons and glial cells challenged with H2O2. NP7 is a new antioxidant, marine derived, with a different chemical molecular structure that the classic phytochemicals, obtained by lead optimization system from the Streptomyces species, with free radical scavenger properties in the sub lM–nM range and which crosses the blood brain barrier in mammals [24].
2. Materials and methods 2.1. Materials Drugs, culture media, chemical reagents and antibodies. NP7 was provided by Noscira from a group of synthetic, marine derived antioxidants. Culture media and antibodies were used as previously described [12,16] and are detailed in Supplementary material. 2.2. Cell cultures Neuronal-enriched and glial mesencephalic cultures were obtained from 129SV/C57BL6 WT or parkin mutant mice (PK-KO) as previously described [10,12,16]. In accordance with the European
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Union Directives, all efforts were made to minimize the number of animals used and their suffering. Neuronal-enriched mesencephalic and glial cultures were obtained and prepared as previously described [12,16,25]. Culture medium from glial cultures was refreshed after 6–7 days and every 7 days thereafter. After 18 days in culture, positive staining with anti-GFAP antibody identified the astrocytes in these cultures and contained at least 80–90% of total cells. 2.3. Cell treatments WT and PK-KO neuronal-enriched midbrain cultures, maintained for 7 days in vitro (DIV), were seeded with a density of 250.000 cells in P24 multiwells or 200.000 cells in cover-slides, for immunocytochemistry (ICQ) studies. For the curve dose– response experiments, the cultures were treated for 3 h with increasing concentrations of NP7. In another set of experiments, WT and PK-KO cultures were treated with NP7 (5–10 lM) 60 min before the treatment with H2O2 (50 lM). The growth medium was changed to EMEM plus D-glucose (6 mg/ml), the most appropriate medium for H2O2 stability, for the experiments with hydrogen peroxide. Glial cell treatments. After 15–20 DIV in the growth medium (DMEM-FCS), glial cultures had reached confluent; at this time, the cells were mildly trypsinized and reseeded for the experiments at a density of 1.3 104 cells/cm2 in 24- 12-well plates cultures and maintained in growth medium for 6–7 additional days. In the experiments, the cultures were treated 90 min before H2O2 (100 lM) treatment with NP7 at the different concentrations tested. In the experiments with H2O2, the growth medium was
Fig. 1. Effects of NP7 on hydrogen peroxide-induced toxicity in neuronal-enriched mesencephalic cultures from WT mice. After 7 days in vitro, the cells were pretreated for 1 h with 5 lM NP7 in EMEM + D-glucose medium and then was added 50 lM H2O2 for two additional hours. (A) Mitochondrial activity measured by MTT assay. (B) Percentage of apoptotic cells with respect to the total number. (C) Number of DA neurons expressed as percentage of TH+ cells (TH+ cells in control, 450 ± 10). (D) Optical intensity of the immunoreactivity of astroglial (GFAP+) cells. Values are the mean ± S.E.M. of three–four independent cultures with six replicates each. Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls multiple comparison test. ***P < 0.001 H2O2 treated cultures vs. controls, DDDP < 0.001 H2O2 treatment plus NP7 vs. the corresponding H2O2 treatments alone.
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changed to EMEM plus D-glucose (6 mg/ml). In another set of experiments, we used aged glial cultures maintained in culture for 5–7 months, changing the growth medium (DMEM-FCS) every week. 2.4. Cellular characterization and viability, anti-oxidants and pro-oxidants DA neurons were characterized by immunostaining with a mouse anti-TH antibody (1:500) and astrocytes with a rabbit anti-GFAP antibody (1:500); microglial cells were identified with isolectin B4 peroxidase-labelled (12.5 lg/ml) [26,27]. The number of immunoreactive cells was counted in one-seventh of the total area of the cover-slides. The cells were counted in predefined parallel strips using a counting reticule inserted into the ocular. Cell viability was analyzed with the mitochondrial activity assay which measures the metabolism of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Apoptosis was measured by light microscopy, DNA staining with bis-benzimide (Hoechst 33342) and the TUNEL assay [16,28,29]. Necrosis was measured according to lactate dehydrogenase activity [30] in the culture medium and by trypan blue dye exclusion in cells [29]. Total glutathione, reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured by the methods of Tietze [31] and Griffith [32], respectively. The concentration of H2O2 was estimated with a colorimetric assay; 3,30 -dimethoxybenzidine, which is colourless in its reduced form, is oxidized in the presence of H2O2 and peroxidased into a red-coloured product [16]. The concentration of intracellular iron in midbrain glial cultures was estimated by the colorimetric ferrozine assay [33].
2.5. Western blot analysis The levels of proteins involved in signaling of survival responses, pERK and IP3/AKT pathways, were measured by Western blot according to previously described techniques. We used a mouse anti-phospho-ERK-1/2 and rabbit anti-ERK-1/2 antibodies from Sigma (Madrid, Spain) diluted 1:5000 and 1:10 000, respectively. Rabbit anti-phosphorylated AKT and rabbit polyclonal anti AKT antibodies from Cell-Signaling (Boston, MA, USA) at 1/400 each [16,25,34]. 2.6. Statistical analysis The statistical evaluation was performed by two-way analysis of variance followed by the Bonferroni test, or by one-way ANOVA followed by Newman–Keuls test, using the GraphPad PRISM 4 software. Differences were considered significant when P < 0.05.
3. Results and discussion 3.1. Effects of NP7 on H2O2 toxicity in midbrain neuronal cultures from WT and PK-KO mice The curve dose–response at concentrations of NP7 from 0.5 to 10 lM did not show any toxicity in the MTT assay (data not shown). NP7, 5 lM, 60 min before the treatment with H2O2 (50 lM), prevented partially the effects of H2O2 on mitochondrial activity (H2O2-treated groups had a 81% decreased vs. WT and
Fig. 2. Effects of NP7 on cell viability and H2O2 detoxification in neuronal-enriched mesencephalic cultures from WT and PK-KO mice. After 7 days in vitro, WT and PK-KO cells, 60 min before the treatment with H2O2 (50 lM) for 2 h, received 10 lM of NP7. (A) Mitochondrial activity (MTT assay). (B) LDH activity. (C) H2O2 levels in the medium 10 min post-treatment with H2O2, 50 lM. (D) Percentage of apoptosis, condensated and fragment cells staining with bis-benzimide, in WT and PK-KO mesencephalic cultures treated with solvent, H2O2 (50 lM) or H2O2 plus NP7 (10 lM). (E) Photomicrographs of total nuclei stained with bis-benzimide and positive co-localization of apoptoticfragmented cells obtained by the TUNEL assay corresponding to the same field. Scale bar = 30 lm. Amplified inset shows that chromatin-condensed nuclei are not comarked by TUNEL assay. (F) Percentage of apoptotic-fragmented cells by TUNEL assay. Values are the mean ± S.E.M. of two–three independent cultures with six replicates each. Statistical analysis was performed by two-way ANOVA (the interaction between genotype and treatment was P < 0.01) followed by Bonferroni post-test. *P < 0.05; **P < 0.01; *** P < 0.001 H2O2 treated cultures vs. controls, ++P < 0.01; +++P < 0.001 PK-KO vs. WT cultures; DP < 0.05; DDP < 0.01; DDDP < 0.001 H2O2 treatment plus NP7 vs. the corresponding H2O2 treatments alone.
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Fig. 3. Effects of NP7 on dopamine neurons and microglial cells in mesencephalic cultures from WT and PK-KO mice. After 7 days in vitro, the cells were treated with H2O2 (50 lM) for 2 h; NP7, 10 lM, was added to pre-established groups 60 min before the peroxide. (A) Photomicrographs of DA neurons (TH+ cells), scale bar = 30 lm. (B) DA neurons expressed as percentage of TH+ (TH+ cells in WT control, 427 ± 11; TH+ cells in PK-KO control, 411 ± 10). (C) Photomicrographs showing microglial (Isolectin B4+) cells, scale bar = 30 lm. (D) Percentage of microglial cells present in WT and PK-KO midbrain cultures (Isolectin B4+ cells in WT and PK-KO controls were 448 ± 8 and 749 ± 48, respectively). Values are the mean ± S.E.M. of two independent cultures with six replicates each. Statistical analysis was performed by one-way ANOVA followed by Newman Keuls multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001 H2O2 treated cultures vs. controls, + P < 0.05 PK-KO vs. WT cultures; DP < 0.05; DDP < 0.01; DDDP < 0.001 H2O2 treatment plus NP7 vs. the corresponding H2O2 treatments alone.
Fig. 4. NP7 protects WT and PK-KO midbrain glial cultures from cell death induced by H2O2. Glial cultures maintained during 20–30 DIV in DMEM + 15% FCS were used for these experiments; 6–7 days after reseeding, the cells were treated with 100 lM of H2O2 for 2 h in EMEM + D-glucose; 90 min before treatment pre-established groups were treated with NP7 (0.5 or 5 lM). (A) Mitochondrial and LDH (B) activities in WT glial cultures pre-exposed to 0.5 and 5 lM of NP7 previous to H2O2 100 lM treatment. (C) Photomicrographs showing trypan blue dye exclusion and (D) number of necrotic cells in WT glial cultures. (E) LDH activity in WT and PK-KO glial cells treated with H2O2 (100 lM), solvent or H2O2 (100 lM) plus NP7 (5 lM). (F) Intracellular glutathione. (G) Levels of H2O2 in the medium. Values are the mean ± S.E.M. of two independent cultures with six replicates each. Statistical analysis was performed by one- or two-way ANOVA (the interaction between genotype and treatment was P < 0.01) followed by Newman–Keuls multiple comparison test and Bonferroni post-test, respectively. *P < 0.05; **P < 0.01; ***P < 0.001 H2O2 treated cultures vs. controls, +P < 0.05; ++P < 0.01; +++ P < 0.001 PK-KO vs. WT cultures; DP < 0.05, DDP < 0.01, DDDP < 0.001 H2O2 treatment plus NP7 vs. the corresponding H2O2 treatments alone.
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NP7 + H2O2 prevented to a 64% decreased vs. WT) (Fig. 1A) and on apoptosis (H2O2-treated groups had a 52%, NP7 + H2O2 prevented to a 15% increased vs. the 5.9% of WT control) (Fig. 1B). However, NP7, 5 lM, prevented totally the drop-out of dopamine (TH+) neurons (Fig. 1C) and astroglia activation (Fig. 1D). Therefore, NP7, when administered before H2O2 has a potent neuroprotective effect in WT midbrain neuronal-enriched cultures. We have previously shown that WT and PK-KO neuronal-enriched cultures grown in B27/NBL serum-free defined medium for 7–8 days in vitro are formed by 80% neurons (MAP-2+), of which around 1–2% are DAergic (TH+), and 10–15% glia, mostly astrocytes (GFAP+), glial progenitors (A2B5), microglia (isolectin B4) and oligodendrocytes (O1+); the rest are nestin+ neuronal progenitors [12]. Now, we have compared the neuroprotective effects of NP7 on WT and PK-KO midbrain neurons. In both types of neurons, H2O2 altered and NP7, 10 lM, prevented the decreased MTT metabolism, from 40% to 70% in H2O2 and NP7 + H2O2 treated groups vs. controls, respectively (Fig. 2A). LDH increased activity-induced by H2O2 was totally prevented with NP7, 10 lM (Fig. 2B), as well as the remaining H2O2 levels in the medium (Fig. 2C) and the percentage of apoptotic cells (Fig. 2D–F). As expected, after 2 h of H2O2 treatment, we observed a higher increase in early condensatedapoptotic cells, measured by bis-benzimide staining (Fig. 2D and E) than in fragmented-apoptotic cells valued by the TUNEL assay (Fig. 2E and F). The percentage of TH+ neurons loss (Fig. 3A and B) and the activation of microglial cells were totally prevented with NP7, 10 lM (Fig. 3C and D). In general, the effects and counter effects of H2O2 and NP7, respectively, on these parameters were similar in both WT and PK-KO mice, but there were some very interesting exceptions worthy of discussion. The levels of H2O2 were significantly lower in H2O2 treated PK-KO neurons than in H2O2 treated WT and, accordingly, the drop-out of TH+ neurons after H2O2 treatment was less severe in PK-KO than in WT cultures. We have described that PK-KO mice have greater levels of GSH in brain than their WT controls, perhaps as a mechanism of compensation of the chronic increased production of free radicals which they have. These increased GSH levels could work as a scavenging mechanism for H2O2, reducing its levels and its toxicity.
in PK-KO mice increase with age [20,21,35]. Therefore, we have investigated the effects of H2O2 and NP7 on midbrain glia maintained in growth medium (DMEM-FCS) for 5–7 months, changing the growth medium (DMEM-FCS) every week. Aged PK-KO glial cells are more sensitive than aged WT to H2O2 [16], so we treated glial cultures with lower concentrations of H2O2, 50 lM. At this concentration of H2O2, WT glia does not produce elevation of LDH (Fig. 5A). The levels of LDH, which are greater in untreated aged PK-KO glia than in untreated aged WT, greatly increase with the treatment with H2O2, 50 lM, an effect which is almost completely prevented by NP7, 5 lM (from a 204% increased in H2O2 to a 129% in NP7 + H2O2) (Fig. 5A). As expected, the treatment with H2O2, 50 lM, increased the levels of H2O2 in WT and much more in PK-KO aged glia (Fig. 5 B). Baseline H2O2 levels were also greater in PK-KO than in WT glia (Fig. 5B). In summary, our data suggest that aged PK-KO glia has difficulties in handling the endogenous production of H2O2 and becomes very susceptible to relatively small concentrations of this oxidating agent which appear to be well tolerated by WT aged glia.
3.2. NP7 protects WT and PK-KO midbrain glial cultures from cell death induced by H2O2 In this work, we found that NP7 protects astroglial cells from WT and PK-KO neuronal-enriched cultures from the H2O2 toxicity. In WT glial cultures treated with H2O2, NP7 restores MTT levels from concentrations of 0.5 lM (Fig. 4A) and has a dose-dependent effect on the reversal of the elevation of LDH levels from 0.5 to 5 lM (Fig. 4B). At a concentration of 5 lM, NP7 completely blocks necrosis in H2O2 treated WT glia (Fig. 4C–E). As in the case of neuronal cultures, H2O2 toxicity is less marked in PK-KO than in WT glial cultures. The role of GSH in the differences between WT and PK-KO cells is shown in Fig. 4F. PK-KO cells have a 128% increased GSH levels vs. WT, and NP7 did not protected from the H2O2-induced decreased in GSH (Fig. 4F). The treatment with H2O2 and NP7 does not alter the levels of iron in WT or PK-KO glia (data not shown). As expected, the treatment with H2O2 increases the H2O2 levels in WT and PK-KO glia but the co-treatment with NP7 slightly reduced them, from 375% to 275% in WT and from 180% to 130% en PK-KO cultures (Fig. 4G). 3.3. Effect of NP7 on H2O2 toxicity in aged glial cultures PD and other neurodegenerative disorders affect mature or senile patients and we have observed that neurochemical deficits
Fig. 5. Effect of NP7 on H2O2-induced toxicity in aged WT and PK-KO glial cultures. Glia cultures maintained for 5–7 months in DMEM + 15% FCS were used for these experiments. (A) LDH activity in WT and PK-KO aged cultures treated for 2 h in EMEM + D-glucose with H2O2 (50 lM); 90 min before the peroxide treatment, preestablished groups received NP7 (5 lM). (B) H2O2 remnant in the medium after 10 min post-treatment with H2O2 (50 lM and 100 lM). Values are the mean ± S.E.M. of two independent cultures with six replicates each. Statistical analysis was performed by one-way ANOVA followed by Newman–Keuls multiple comparison test. **P < 0.01; ***P < 0.001 H2O2 treated cultures vs. controls, +P < 0.05; +++P < 0.001 PK-KO vs. WT cultures, DDP < 0.01; DDDP < 0.001 H2O2 treatment plus NP7 vs. the corresponding H2O2 treatments alone.
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3.4. Effects of oxidative stress and NP7 in signaling pathways H2O2 and other free radical generating agents induce a strong activation of ERKs, JNKs and p38-MAPK on many cellular signaling systems [34,36,37]. Parkin and DJ-1 lead to impairments of oxidative stress response and PI3K/AKT signaling [38,39]. In young (18– 30 DIV) WT glial cultures, treatment with H2O2 produced a rapid and progressive ERK phosphorylation, as measured by the ratio p-ERK/ERK, from 15 to 60 min after treatment (data not shown). NP7, 5 lM protected from p-ERK activation in young glial cultures from WT (465% increased with H2O2 and 300% with NP7 + H2O2) and PK-KO mice (438% increased with H2O2 and 305% with NP7 + H2O2) (Fig. 6A) and from the H2O2 induced p-AKT activation (424% increased with H2O2 and 285% with NP7 + H2O2 in WT and 322% increased with H2O2 and 222% with NP7 + H2O2 in PK-KO mice) (Fig. 6B). Aged PK-KO glia is more sensitive to oxidative stress than WT, and H2O2 induced a higher p-ERK (254% increased with H2O2 and 106% with NP7 + H2O2 in WT and 629% increased with H2O2 and
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139% with NP7 + H2O2 in PK-KO mice) (Fig. 6C) and p-AKT activation (385% increased with H2O2 and 213% with NP7 + H2O2 in WT and 1246% increased with H2O2 and 526% with NP7 + H2O2 in PK-KO mice) (Fig. 6D). NP7 protected very effectively in aged glial cultures from the p-ERK (Fig. 6C) and from the p-AKT activation (Fig. 6D). The special sensitivity of aged PK-KO glia to oxidative stress while there is strong resistance in young PK-KO cells, suggest that these cells are exposed to a greater oxidative challenge and that a compensatory GSH synthesis is increased for some time until it collapses with senescence and renders the cells more susceptible [16]. Furthermore, in the differential oxidative stress response between WT and PK-KO cultures, parkin as an E3 ligase, which participates in the ubiquitin–proteasome system, protein aggregation and protein posttranslational modifications, may play a very important role [40–43]. In summary, NP7 has a strong protective effect in free radical related stress in neurons and glia from WT and PK-KO mice and, may therefore be a promising candidate for neuroprotection against oxidative stress in PD.
Fig. 6. Effects of H2O2 and NP7 on p-ERK and p-AKT signaling pathways. Young and aged glial cultures maintained in DMEM + 15% FCS were used for these experiments; 6–7 days after reseeding, the cells were treated with 100 lM of H2O2 for 30 min in EMEM + D-glucose; 90 min before treatment, pre-established groups were treated with NP7 (5 lM). (A) p-ERK signaling pathway in young glia cultures. (B) p-AKT signaling pathway in young glia cultures. (C) p-ERK signaling pathway in aged glia cultures. (D) p-AKT signaling pathway in aged glia cultures. Values are the mean ± S.E.M. of three independent cultures with six replicates each. Statistical analysis was performed by one- or twoway ANOVA (the interaction between aged glia genotype and treatment was P < 0.01) followed by Newman–Keuls multiple comparison test and Bonferroni post-test, respectively. ***P < 0.001 H2O2 treated cultures vs. controls, +++P < 0.001 PK-KO vs. WT cultures, DP < 0.05; DDP < 0.01; DDDP < 0.001 H2O2 treatment plus NP7 vs. the corresponding H2O2 treatments alone.
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Acknowledgements Supported in part by grants from the Spanish Ministry of Health, FIS 2007/PI07037, NDG 07-4, CAM 0202/2006 and CIBER 2006/05/0059. The authors thank M. Serrano for excellent technical assistance; Mrs. Claire Marsden for editorial help. Appendix A. Supplementary data Materials (Culture media, Antibodies and Chemicals) and Methods (Cell cultures; Immunocytochemistry; Cell viability assays; Determination of GSH, H2O2 and iron levels; and Western blot). Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2008.11.051. References [1] Clarke, C.E. (2007) Parkinson’s disease. BMJ 335, 441–445. [2] Klein, C. and Schlossmacher, M.G. (2007) Parkinson disease, 10 years after its genetic revolution: multiple clues to a complex disorder. Neurology 69, 2093– 2104. [3] Levy, G. (2007) The relationship of Parkinson disease with aging. Arch. Neurol. 64, 1242–1246. [4] Mouradian, M.M. (2002) Recent advances in the genetics and pathogenesis of Parkinson disease. Neurology 58, 179–185. [5] Sadrzadeh, S.M. and Saffari, Y. (2004) Iron and brain disorders. Am. J. Clin. Pathol. 121 (Suppl), S64–S70. [6] Sulzer, D. (2007) Multiple hit hypotheses for dopamine neuron loss in Parkinson’s disease. Trends Neurosci. 30, 244–250. [7] Hardy, J. (2000) Pathways to primary neurodegenerative disease. Ann. NY Acad. Sci. 924, 29–34. [8] Hattori, N., Shimura, H., Kubo, S., Wang, M., Shimizu, N., Tanaka, K. and Mizuno, Y. (2000) Importance of familial Parkinson’s disease and parkinsonism to the understanding of nigral degeneration in sporadic Parkinson’s disease. J. Neural. Transm. Suppl., 101–116. [9] Polymeropoulos, M.H. (2000) Genetics of Parkinson’s disease. Ann. NY Acad. Sci. 920, 28–32. [10] Itier, J.M., Ibanez, P., Mena, M.A., et al. (2003) Parkin gene inactivation alters behaviour and dopamine neurotransmission in the mouse. Hum. Mol. Genet. 12, 2277–2291. [11] Menendez, J., Rodriguez-Navarro, J.A., Solano, R.M., Casarejos, M.J., Rodal, I., Guerrero, R., Sanchez, M.P., Avila, J., Mena, M.A. and de Yebenes, J.G. (2006) Suppression of Parkin enhances nigrostriatal and motor neuron lesion in mice over-expressing human-mutated tau protein. Hum. Mol. Genet. 15, 2045– 2058. [12] Casarejos, M.J., Solano, R.M., Menendez, J., Rodriguez-Navarro, J.A., Correa, C., Garcia de Yebenes, J. and Mena, M.A. (2005) Differential effects of l-DOPA on monoamine metabolism, cell survival and glutathione production in midbrain neuronal-enriched cultures from parkin knockout and wild-type mice. J. Neurochem. 94, 1005–1014. [13] Solano, R.M., Menendez, J., Casarejos, M.J., Rodriguez-Navarro, J.A., Garcia de Yebenes, J. and Mena, M.A. (2006) Midbrain neuronal cultures from parkin mutant mice are resistant to nitric oxide-induced toxicity. Neuropharmacology 51, 327–340. [14] Casarejos, M.J., Menendez, J., Solano, R.M., Rodriguez-Navarro, J.A., Garcia de Yebenes, J. and Mena, M.A. (2006) Susceptibility to rotenone is increased in neurons from parkin null mice and is reduced by minocycline. J. Neurochem. 97, 934–946. [15] Serrano, A., Menendez, J., Casarejos, M.J., Solano, R.M., Gallego, E., Sanchez, M., Mena, M.A. and Garcia de Yebenes, J. (2005) Effects of cinnarizine, a calcium antagonist that produces human parkinsonism, in parkin knock out mice. Neuropharmacology 49, 208–219. [16] Solano, R.M., Casarejos, M.J., Menendez-Cuervo, J., Rodriguez-Navarro, J.A., Garcia de Yebenes, J. and Mena, M.A. (2008) Glial dysfunction in parkin null mice: effects of aging. J. Neurosci. 28, 598–611. [17] Kitada, T., Asakawa, S., Hattori, N., Matsumine, H., Yamamura, Y., Minoshima, S., Yokochi, M., Mizuno, Y. and Shimizu, N. (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392, 605–608. [18] Abbas, N., Lucking, C.B., Ricard, S., Durr, A., Bonifati, V., De Michele, G., Bouley, S., Vaughan, J.R., Gasser, T., Marconi, R., Broussolle, E., Brefel-Courbon, C., Harhangi, B.S., Oostra, B.A., Fabrizio, E., Bohme, G.A., Pradier, L., Wood, N.W., Filla, A., Meco, G., Denefle, P., Agid, Y. and Brice, A. (1999) A wide variety of mutations in the parkin gene are responsible for autosomal recessive parkinsonism in Europe. French Parkinson’s disease genetics study group and the European consortium on genetic susceptibility in Parkinson’s Disease.. Hum. Mol. Genet. 8, 567–574. [19] Lucking, C.B., Durr, A., Bonifati, V., Vaughan, J., De Michele, G., Gasser, T., Harhangi, B.S., Meco, G., Denefle, P., Wood, N.W., Agid, Y. and Brice, A. (2000) Association between early-onset Parkinson’s disease and mutations in the parkin gene. N. Engl. J. Med. 342, 1560–1567.
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