Susceptibility to rotenone is increased in ... - Wiley Online Library

Journal of Neurochemistry, 2006, 97, 934–946

doi:10.1111/j.1471-4159.2006.03777.x

Susceptibility to rotenone is increased in neurons from parkin null mice and is reduced by minocycline M. J. Casarejos,*,1 J. Meneńdez,*,1 R. M. Solano,* J. A. Rodrı´guez-Navarro,* J. Garcıá de Ye´benes and M. A. Mena* *Department of Neurobiology and Department of Neurology, Hospital ‘Ramoń y Cajal’, Madrid, Spain

Abstract Parkinson’s disease is a neurodegenerative disorder which is in most cases of unknown etiology. Mutations of the Park-2 gene are the most frequent cause of familial parkinsonism and parkin knockout (PK-KO) mice have abnormalities that resemble the clinical syndrome. We investigated the interaction of genetic and environmental factors, treating midbrain neuronal cultures from PK-KO and wild-type (WT) mice with rotenone (ROT). ROT (0.025–0.1 lM) produced a dosedependent selective reduction of tyrosine hydroxylase-immunoreactive cells and of other neurons, as shown by the immunoreactivity to microtubule-associated protein 2 in PK-KO cultures, suggesting that the toxic effect of ROT involved dopamine and other types of neurons. Neuronal death was mainly apoptotic and suppressible by the caspase inhibitor t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone (Boc-D-FMK). PK-KO cultures were more susceptible to apoptosis induced by low doses of ROT than those from WT. ROT increased the proportion of astroglia and microglia more in PK-KO than in WT cultures. Indomethacin, a cyclo-oxygenase inhibitor, worsened the effects of ROT on tyrosine hydroxylase cells, apoptosis and astroglial (glial fibrillary acidic protein) cells. N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, increased ROT-induced apoptosis but did not change tyrosine hydroxylase-immunoreactive or

glial fibrillary acidic protein area. Neither indomethacin nor N-nitro-L-arginine methyl ester had any effect on the reduction by ROT of the mitochondrial potential as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Microglial NADPH oxidase inhibition, however, protected against ROT. The roles of p38 MAPK and extracellular signalregulated kinase signaling pathways were tested by treatment with SB20358 and PD98059, respectively. These compounds were inactive in ROT-naive cultures but PD98059 slightly increased cellular necrosis, as measured by lactate dehydrogenase levels, caused by ROT, without changing mitochondrial activity. SB20358 increased the mitochondrial failure and lactate dehydrogenase elevation induced by ROT. Minocycline, an inhibitor of microglia, prevented the dropout of tyrosine hydroxylase and apoptosis by ROT; the addition of microglia from PK-KO to WT neuronal cultures increased the sensitivity of dopaminergic neurons to ROT. PK-KO mice were more susceptible than WT to ROT and the combined effects of Park-2 suppression and ROT reproduced the cellular events observed in Parkinson’s disease. These events were prevented by minocycline. Keywords: dopamine neurons, extracellular signal-regulated kinase, microglial cells, NADPH oxidase, Park-2 gene, Parkinson’s disease. J. Neurochem. (2006) 97, 934–946.

The cause of Parkinson’s disease (PD), the second most prevalent neurodegenerative disorder after Alzheimer’s disease with an estimated prevalence of 0.3% of individuals in the developed world (Olanow and Tatton 1999), is unknown in the majority of cases and has been a topic for debate for the last half century. PD is characterized by akinesia, rigidity, postural instability and tremor at rest, and perceptive and cognitive deficits after years of progression. The pathology is consistent with remarkable degeneration of nigrostriatal dopaminergic neurons and other brain nuclei and the presence of neuronal cytoplasmic inclusions containing

Received December 19, 2005; revised manuscript received January 4, 2006; accepted January 4, 2006. Address correspondence and reprint requests to Dr M. A. Mena, Departamento Investigacioń, Hospital Ramoń y Cajal, Carretera de Colmenar, Km. 9, Madrid 28034, Spain. E-mail: [email protected] 1 These authors contributed equally to this article. Abbreviations used: COX, cyclo-oxygenase; DA, dopamine; DIV, days in vitro; GFAP, glial fibrillary acidic protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PD, Parkinson’s disease; PK-KO, parkin knockout; ROT, rotenone; TH, tyrosine hydroxylase; TUNEL, TdT-mediated dUTP Nick-end labelling; WT, wild-type.

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2006 The Authors Journal Compilation 2006 International Society for Neurochemistry, J. Neurochem. (2006) 97, 934–946

Effects of rotenone on PK-KO mice 935

ubiquitinated proteins in these structures. The characteristic symptoms of the disease patently appear when > 70% of the nigrostriatal dopaminergic neurons are lost (Lees 1992). PD appears with familial aggregation in 10–25% of patients and, so far, more than 10 responsible genes or loci have been reported (Hardy 2003). In some sporadic cases environmental factors, including exposure to toxins that impair mitochondrial function or increase free radicals, drugs with different pharmacological profiles and infectious agents that may damage the nervous system and other external agents, have been reported (Allam et al. 2005). In the majority of the sporadic patients no clear pathogenic mechanisms are known and there is still debate about the putative role of the combination of the genetic risk factor with common environmental agents. The most frequent cause of familial PD is related to deletions or point mutations of Park-2, a gene that codifies for parkin, a 465-amino-acid protein with ubiquitine ligase function. We have investigated the pathogenic mechanisms of disease production in parkin null mice and found that, in addition to alterations of processing of proteins, these animals have an abnormal release and metabolism of dopamine (DA) associated with increased free radical production that is compensated, at least in part, by overproduction of glutathione (Itier et al. 2003; Casarejos et al. 2005; Serrano et al. 2005). Among the neurotoxins that interfere with mitochondrial function and produce PD the best studied is 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine. An epidemic of 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism appeared in the early 1980s when several hundred young drug addicts were intoxicated by a batch of 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine, a precursor of the 1-methyl-4-phenylpyridinium ion, a compound that is selectively taken up by the DA transporter and selectively inhibits complex I of the mitochondrial respiratory chain (Nicklas et al. 1985; Ramsay et al. 1986a,b). This is of interest because post-mortem studies have shown mitochondrial abnormalities in patients with idiopathic PD (Schapira et al. 1990) and epidemiological studies (Di Monte 2003) have suggested an association with environmental toxins, mainly mitochondrial complex I inhibitors such as the widely used pesticide rotenone (ROT). Rotenone is a lipophilic compound that freely crosses cell membranes and accesses cytoplasm and mitochondria. In vitro, it produces cell apoptosis, caspase 3 activation, change of mitochondrial membrane potential, accumulation and aggregation of alpha-synuclein and ubiquitin, oxidative damage, and endoplasmic reticulum stress (Ryu et al. 2002; Sherer et al. 2002, 2003c; Ahmadi et al. 2003; Tada-Oikawa et al. 2003; Diaz-Corrales et al. 2005; Moon et al. 2005; Zoccarato et al. 2005), i.e. many of the findings observed in the brains of patients with PD. Chronic systemic administration of ROT in vivo reproduces an animal model of PD in

Drosophila melanogaster (Coulom and Birman 2004) and in rodents (Betarbet et al. 2000; Alam and Schmidt 2002; Hoglinger et al. 2003; Sherer et al. 2003b; Bashkatova et al. 2004; Lapointe et al. 2004). It is important to notice that, in addition to abnormalities of protein processing, mitochondrial function and free radical balance that cause a severe nigrostriatal DA cell loss, the neuropathology of PD is characterized by a robust glial reaction in the substantia nigra pars compacta which has also been observed in some experimental models of PD (Hirsch and Hunot 2000; Du et al. 2001; Vila et al. 2001; Wu et al. 2002). The role of gliosis is controversial as it is sometimes associated with neuroprotective effects (Mena et al. 2002) but in other studies it appears to be deleterious (Hirsch and Hunot 2000; Du et al. 2001; Vila et al. 2001; Wu et al. 2002). Nigrostriatal cell death, as a result of microglial activation, could result from the multifunctional nature of activated microglia that encompasses the up-regulation of cytotoxic molecules, including reactive oxygen species, nitric oxide and a variety of proinflammatory cytokines such as interleukin-1b (Banati et al. 1993). It is of great interest to know whether subjects with a genetic susceptibility for PD are more susceptible to putative environmental neurotoxic agents. In this study we have found that exposure to ROT is more toxic for DA neurons from parkin null than wild-type (WT) mice. In addition, we have investigated the differential effects of ROT on cellular phenotype, microglial activation, cell survival and tyrosine hydroxylase (TH) expression in neuronal-enriched midbrain cultures from Park-2 null and WT mice. We found that inhibition of microglial activation by minocycline protects against the toxic effects of ROT.

Materials and methods Materials The culture reagents B27 supplement, neurobasal TM medium, fetal calf serum, penicillin-streptomycin and L-glutamine were obtained from Gibco-Life Technologies (Paisley, Scotland, UK). ROT was purchased from Sigma (Madrid, Spain). All other agents were of the highest purity commercially available from Merck (Darmstadt, Germany) or Sigma. Antibodies Anti-TH and the molecular marker of mature neurons (neuronal nuclei) antibodies, made in mouse, were obtained from Chemicon (Temecula, CA, USA); polyclonal anti-glial fibrillary acidic protein (GFAP) antibody, raised in rabbit, was from DAKO (Glostrup, Denmark); isolectin B4 from Bandeiraea simplicifolia peroxidaselabeled and anti-microtubule-associated protein 2a + 2b antibody were purchased from Sigma; anti-rabbit IgG conjugated with tetramethylrhodamine and anti-mouse IgG fluorescein were from Jackson (West Grove, PA, USA); and anti-mouse IgG Alexa Fluor 568 was obtained from Molecular Probes (Eugene, OR, USA).


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Fig. 1 Dose-dependent differential neurotoxic effects of rotenone (ROT) on midbrain neuronal-enriched cultures from wild-type (WT) and parkin knockout (PK-KO) mice. After 8 days in vitro, the cells were treated with ROT (0.025, 0.05 and 0.1 lM) for 5 h. (a) Photomicrographs of dopamine (DA) neurons [tyrosine hydroxylase-immunoreactive (TH+) cells] from cultures treated with ROT or solvent. Scale bar, 30 lm. (b) Number of DA neurons. (c) Chromatin condensed and fragmented nuclei were counted and expressed as a percentage of

apoptotic cells with respect to the total cell number. Values are expressed as the mean ± SEM from three independent experiments with six replicates each. Statistical analysis was performed by two-way ANOVA (the interaction between genotype and treatment was p < 0.001) followed by Bonferroni post-test. *p < 0.05, **p < 0.01, ***p < 0.001 ROT-treated cultures versus controls; ++p < 0.01, +++p < 0.001 PK-KO versus WT cultures.

Chemicals Trypan blue, bovine serum albumin, poly-D-lysine, p-phenylenediamine, bis-benzimide, N-nitro-L-arginine methyl ester and minocycline were from Sigma. Laminin was from Roche (Barcelona, Spain); the cytotoxicity detection kit for lactate dehydrogenase and cell proliferation kit I [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] were from Boehringer-Mannheim (Barcelona, Spain). The apoptosis TdT-mediated dUTP Nick-end labelling (TUNEL) detection kit was obtained from Promega (Madison, WI, USA); SB20358 and PD98059 were from Alexis (Carlsbad, CA, USA); and indomethacin, apocynin and the caspase inhibitor III t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone (Boc-D-FMK) were from Calbiochem (Darmstadt, Germany). The bicinchoninic acid protein assay kit was from Pierce (Rockford, IL, USA). All other reagents were of the highest purity commercially available from Merck or Sigma.

by western blot analysis of parkin in the cultures (Casarejos et al. 2005). The ventral mesencephalon was removed from embryonic tissue (13th day of gestation) and incubated with 0.36 mg/mL papain in phosphate-buffered saline/D-glucose (6 mg/mL)/1% bovine serum albumin buffer for 15 min at 37C and mechanically dissociated in the presence of 10 mg/mL Dnase-I. The cells were seeded in B27/ neurobasal TM medium with 15% (v/v) heat-inactivated fetal calf serum supplemented with glutamine (4 mM), penicillin (100 U/mL) and streptomycin (100 lg/mL) at a density of 2.5 · 105 cells/cm2 in multiwells or 2 · 105 cells/cm2 in glass cover-slides pre-coated with poly-D-lysine (4.5 lg/cm2) in 0.1 M borate buffer, pH 8.4, and laminin (3 lg/mL). The cultures were kept in a humidified chamber at 37C in a 5% CO2 atmosphere for 7–8 days in vitro (DIV). At 24 h after plating, the cells were changed to serum-free defined medium (B27/neurobasal TM medium). Microglia-enriched cultures were prepared from sibling cells kept in culture from 15 days to 3 months in Dulbecco’s modified Eagle’s medium with 15% fetal calf serum as described previously (de Bernardo et al. 2003). After reaching confluence (15 d), microglia were separated from astrocytes by shaking the flasks for 5 h at 150 r.p.m. The enriched microglia were > 95% pure, as determined by immunostaining with antibodies against GFAP and staining with isolectin B4.

Cell culture Transgenic animals were obtained from 129SV/C57BL6 WT (+/+) or parkin null (–/–) mice (Itier et al. 2003). Neuronalenriched mesencephalic primary cultures were derived from littermate –/– and +/+ embryos obtained from homozygous colonies previously generated by heterozygous parkin –/+ intercross. The genotype was confirmed by PCR analysis of tail tissue and



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Fig. 2 Rotenone (ROT) induces apoptosis in neuronal parkin knockout (PK-KO) cultures. (a) Effects of ROT (0.05 lM · 24 h) at 7 days in vitro (DIV) on microtubule-associated protein 2a + 2b (MAP-2)+ cells from wild-type (WT) and PK-KO mice. The interaction between genotype and treatment was p < 0.001. (b) Photomicrographs of colocalization of neurons with apoptotic cells, nuclei stained with bisbenzimide and neuronal nuclei stained with anti-neuronal nuclei (NeuN). (c) TUNEL+ cells in ROT-treated PK-KO neuronal cultures. The broad-spectrum caspase inhibitor (Casp I) Boc-D-FMK (30 lM) was added 30 min before ROT treatment (0.05 lM · 24 h) at 7 DIV of PK-KO midbrain cultures. (d) Photomicrographs of double-stained cells with TUNEL assay and bis-benzimide from the same field. Insets

show that peripheral chromatin-condensed nuclei are not comarked by TUNEL assay and that fragmented cells are TUNEL+. Scale bar, 30 lm. The caspase inhibitor protects partially from total apoptotic cells (e) and did not recover to normal mitochondrial activity (f) or lactate dehydrogenase activity in the medium of ROT-treated cultures (g). Statistical analysis was performed by ANOVA followed by Newman Keuls multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 ROT-treated cultures versus controls; +++p < 0.001 PK-KO versus WT cultures; p < 0.05, p < 0001 ROT plus caspase inhibitortreated cultures versus ROT-treated cultures. MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Experimental treatments Wild-type and parkin knockout (PK-KO) neuronal-enriched midbrain cultures were seeded with a density of 200000 cells/coverslides and maintained in culture for 7–8 DIV for neuronal and glial phenotype characterization. For genotype characterization of parkin protein, the cells were grown for 7–8 DIV, in 12-well culture plates, at a density of 400 000 cells/well. The time- and dose-dependent effects of ROT on DA cell survival were studied in cultures from WT and PK-KO mice grown in coverslides at 7–8 DIV and treated with ROT (0.025–0.1 lM) for 6 and 24 h.

The roles of p38 MAPK and extracellular signal-regulated kinase signaling pathways in the ROT effects in PK-KO cultures were investigated using the mitogen extracellular signal regulated kinase (MEK) inhibitor PD98059 (5, 10 and 20 lM) and the specific p38 MAPK inhibitor SB20358 (10 and 20 lM) 30 min before the ROT treatment (0.05 lM for 24 h) at 7 DIV. In order to study whether NADPH oxidase, cyclo-oxygenase (COX) and nitric oxide synthase inhibition block the ROT-induced cell death in PK-KO cultures, after 7 DIV, the cultures were pretreated with apocynin (0.5 mM), indomethacin (5, 10 and 15 lM) and N-nitro-L-arginine methyl ester (1 mM) 30 min before the



Fig. 3 Effects of rotenone (ROT) on midbrain glial population. After 8 days in vitro, the cells were treated with ROT (0.05 and 0.1 lM for 5 h). (a) Photomicrographs show microglial (isolectin B4+) cells in cultures from wild-type (WT) (left panel) and parkin knockout (PK-KO) (right panel) mice treated with ROT (0.05 and 0.1 lM) or solvent (control). (b) Number of microglial cells (isolectin B4+). (c) Photomicrographs of astroglial cells [glial fibrillary acidic protein (GFAP)+]. (d)

Expression of GFAP area. Scale bar, 30 lm. Values are expressed as the mean ± SEM from three independent experiments 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 < 0.01, ***p < 0.001 ROT-treated cultures versus controls; ++p < 0.01, +++p < 0.001 PK-KO versus WT cultures.

addition of ROT (0.05 lM for 24 h). The broad-spectrum caspase inhibitor Boc-D-FMK (30 lM) and minocycline (20 lM), a secondgeneration tetracycline, were added 30 min before ROT treatment in order to prevent the induced cell death. To investigate the role of microglial cells in ROT-induced neuronal cell death, 12 000 PK-KO microglial cells were added to WT midbrain neuronal-enriched cultures at 5 DIV. After 24 h, ROT or solvent treatment was performed in neuronal-enriched versus neuron/microglia cultures.

37C, 100 lL was transferred into 96-well microtiter plates and the absorption value at 540 nm was measured in an automatic microtiter reader (Spectra Fluor, Tecan, Salzburg, Austria). Chromatin condensation and fragmentation were assessed by DNA staining with bis-benzimide (Hoechst 33342). Cells growing on cover-slides were fixed in 4% paraformaldehyde and nuclei were stained with bis-benzimide added in the antifading solution (3 · 10)6 M final concentration) (Hilwig and Gropp 1975; Pardo et al. 1997). The apoptosis TUNEL detection system measures the fragmented DNA of apoptotic cells by incorporating fluorescein-12dUTP* at the 3¢-OH ends of the DNA using the enzyme terminal deoxynucleotidyl transferase (Kerr et al. 1972; Gavrieli et al. 1992). For this assay, the cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. The fluorescein12-dUTP-labeled DNA of apoptotic cells was visualized by fluorescence microscopy (positive cells with green fluorescence). The number of apoptotic cells was counted in one of 14 fields of the cover-slide area. Cells were counted in pre-defined parallel strips using a counting reticule inserted into the ocular. Cells

Cell viability measurements Mitochondrial activity was measured with the MTT assay. Cells were grown on 24-well culture plates with 500 lL defined medium and treated with various reagents according to the experimental design. The MTT assay measures the ability of cells to metabolize MTT. At the end of the treatment period, 300 lL of culture medium was removed from each well and 20 lL of MTT solution (5 mg/mL) was added and incubated for 2 h. At this time, 200 lL of solubilization solution (10% sodium dodecyl sulfate in HCl 0.01 M) was added to the wells and, after 24 h of incubation at



Fig. 4 Cyclo-oxygenase (COX) and nitric oxide synthase (NOS) inhibitors did not protect against rotenone (ROT)-induced cell death in parkin knockout (PK-KO) midbrain cultures. Indomethacin (Ind) (15 lM) and N-nitro-L-arginine methyl ester (L-NAME) (1 mM) were added 30 min before ROT treatment (0.05 lM · 24 h) at 7 days in vitro of PK-KO midbrain cultures. Effects on dopamine neurons (a), apoptotic cells (b), mitochondrial activity (c), astroglial cells (d) and

microglial cells (e). Statistical analysis was performed by ANOVA followed by Newman Keuls multiple comparison test. **p < 0.01, ***p < 0.001 ROT-treated cultures versus controls; p < 0.05, p < 0.001 ROT plus COX or NOS inhibitor-treated cultures versus ROT-treated cultures. GFAP, glial fibrillary acidic protein; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TH, tyrosine hydroxylase.

incubated with buffer in the absence of terminal deoxynucleotidyl transferase enzyme were used as negative controls (RodriguezMartin et al. 2000). For necrotic cell death determination, lactate dehydrogenase activity was measured in the culture medium by using a cytotoxicity detection kit (Decker and Lohmann-Matthes 1988). In neuronalenriched cultures, lactate dehydrogenase release to the culture medium correlates with cell death measured by trypan blue dye exclusion assay (Mena et al. 1997; Canals et al. 2001).

positive cells under fluorescent microscopy. For microglial cell identification, cultures were fixed with 4% paraformaldehyde + 1% glutaraldehyde and incubated at 4C for 24 h with isolectin B4 peroxidase-labeled diluted in Tris-buffered saline/ 0.1% triton X-100; the positive cells were developed with a 3,3¢diamino benzidine (DAB) system (LSAB2 system, DAKO) and visualized under optical microscopy. 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.

Immunocytochemistry Dopamine neurons were characterized by immunostaining with a mouse anti-TH antibody (1 : 500) and astrocytes with a rabbit antiGFAP antibody (1 : 500). Microglial cells were identified with isolectin B4 peroxidase-labeled (12.5 lg/mL) (Streit and Kreutzberg 1987; Ashwell 1991). To detect all neurons in the culture, a mouse anti-neuronal nuclei antibody (1 : 10) and mouse monoclonal antimicrotubule-associated protein 2a + 2b antibody (1 : 250) were used. The cell cultures were fixed with 4% paraformaldehyde, washed in 0.1 M phosphate-buffered saline, pH 7.4, permeabilized with ethanol/acetic acid (19 : 1) and incubated at 4C for 24 h with primary antibodies diluted in phosphate-buffered saline containing 10% fetal calf serum. Fluorescein- and rhodamineconjugated secondary antibodies were employed to visualize

Uptake studies H-Dopamine uptake was measured after incubation of the cells with 3H-DA (10)8 M, 70 Ci/mmol) in the presence of pargyline (10)5 M) and ascorbic acid (10)3 M) at 37C for 30 min. Nonspecific uptake/binding was calculated in the presence of 10)5 M mazindol and represented £ 5% (Canals et al. 2001). 3

Statistical analysis The results were statistically evaluated for significance with a twoway ANOVA followed by the Bonferroni test as a post-hoc evaluation or by a one-way ANOVA followed by Newman Keuls multiple comparison test. Differences were considered statistically significant when p < 0.05.



Fig. 5 NADPH oxidase inactivation protected dopamine (DA) neurons against rotenone (ROT)-induced toxicity in parkin knockout (PK-KO) midbrain cultures. The effect of microglial NADPH oxidase activity inhibition [apocynin (Apo) 0.5 mM 30 min before ROT treatment] on tyrosine hydroxylase-immunoreactive (TH+) cells (a), 3H-DA uptake (b), microglia (c), apoptosis (d) and mitochondrial activity (e) in PK-KO

neuro-enriched cultures treated with 0.05 lM ROT for 5 h. Statistical analysis was performed by ANOVA followed by Newman Keuls multiple comparison test. *p < 0.05, ***p < 0.001 ROT-treated cultures versus controls; p < 0.05, p < 0.01, p < 0.001 ROT plus NADPH oxidase inhibitor-treated cultures versus ROT-treated cultures. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Results

2a + 2b-positive cells) were decreased after ROT treatment in PK-KO but not in WT cultures (Fig. 2a); there was a highly significant interaction between the genotype and the treatment. Neuronal death induced by a ROT concentration of 0.05 lM involved mainly, but not exclusively, apoptosis shown by colocalization of neuronal nuclei-positive immunocytochemistry and Hoechst staining (Fig. 2b). Apoptosis is characterized by an early event with shrunken cells and rounded cells with chromatin condensation without DNA fragmentation (negative for TUNEL stain) and by a later phase with chromatin-fragmentized cells that are TUNEL+ (Figs 2c, d and d inset). After 5 h of treatment with a ROT concentration of 0.05 lM, the cell death is characterized mainly by apoptotic peripheral chromatin-condensed cells (TUNEL+). Signs of necrosis (Fig. 2g) and apoptotic (TUNEL+) caspase-dependent cell death (Figs 2c and d) were detected after 24 h of treatment. Caspase inhibition abolished the increase in TUNEL+ cells induced by ROT in PK-KO cultures. However, the caspase

Differential dose-dependent effects of rotenone in neuronal-enriched midbrain cultures from wild-type and parkin knockout mice Dopamine neurons [TH-immunoreactive (TH+)] from PK-KO mice are dose- and time-dependently more susceptible to ROT-induced cell death than those from WT animals (Figs 1a and b). Although significant cell death was only observed in WT cultures at a ROT concentration of 0.1 lM, it was present in PK-KO cultures treated with ROT concentrations of 0.025 and 0.05 lM (Fig. 1c). Cell death had the characteristics of both apoptosis and necrosis (Fig. 2). There was a significant interaction between the genotype (WT or PK-KO) and the treatment (ROT or solvent). Pattern of cell death and phenotype affected by rotenone in midbrain cultures Low doses of ROT induced neuronal cell death and glial dysfunction in PK-KO midbrain cultures but not in WT. The total midbrain neurons (microtubule-associated protein



inhibitor partially protected against apoptosis (Fig. 2e) and did not recover to normal mitochondrial activity or lactate dehydrogenase levels (Figs 2f and g). Rotenone induced a dose-dependent increase in microglia (Figs 3a and b). The number of microglial cells increased in 0.05 lM ROT-treated cultures from PK-KO mice but not in WT cultures. Midbrain cultures from PK-KO mice had a greater number of microglia than those of WT, with and without ROT treatment. ROT (0.05 lM) further increased the levels of microglia in PK-KO but not in WT cultures (Fig. 3b). Rotenone (0.05 lM) increased GFAP+ cells in PK-KO cultures but did not affect astroglial cells in WT cultures (Figs 3c and d). Cyclo-oxygenase and nitric oxide synthase activation are not involved in the rotenone effects in parkin knockout cultures In order to test if nitric oxide synthase and COX expression was up-regulated in our model with microglial activation, we used a nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester, 1 mM) and a COX inhibitor (indomethacin, 5 and 15 lM) 30 min before ROT (0.05 lM for 24 h) treatment. Both inhibitors failed to protect against DA neuron loss or against ROT-induced cell death in PK-KO cultures (Fig. 4). Furthermore, indomethacin exacerbated astrogliosis and the DA cell death induced by ROT (Figs 4a and c). Both inhibitors increased ROTinduced apoptosis (Fig. 4b) and did not protect against decreased mitochondrial activity induced by ROT (Fig. 4d). Furthermore, neither inhibitor protected against the microglial activation induced by ROT in PK-KO midbrain cultures (Fig. 4e). NADPH oxidase inactivation protected dopaminergic neurons against rotenone-induced toxicity in parkin knockout midbrain cultures Apocynin (0.5 mM) protected against ROT-induced microglia activation (Fig. 5c), cell death (Figs 5d and e) and DA neurodegeneration (Figs 5a and b). ROT at 0.05 lM for 5 h, a dose that did not alter WT neuronal-enriched midbrain cultures, stimulated PK-KO microglia activation and PK-KO neuronal cell death that was attenuated by pre-treatment with inhibitors of NADPH oxidase. Involvement of cell death signaling pathways in the rotenone-induced cell death In models of neurotoxicity with microglial activation, the involvement of p38 MAPK and p-extracellular signalregulated kinase MAPK has been postulated. To confirm the involvement of these signaling pathways we used the specific p38 MAPK inhibitor SB20358 (10 and 20 lM) and the MEK inhibitor PD98059 (5, 15 and 20 lM) 30 min before ROT (0.05 lM for 24 h) treatment. Both inhibitors

Fig. 6 Involvement of cell death signaling pathways in the rotenone (ROT)-induced cell death in parkin knockout (PK-KO) midbrain cultures. p38 MAPK inhibitor [SB20358 (SB), 20 lM] and the MEK 1/2 inhibitor [PD98059 (PD), 20 lM] were added 30 min before ROT treatment (0.05 lM · 24 h) at 7 days in vitro of PK-KO midbrain cultures. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (a) and lactate dehydrogenase (LDH) assays (b). Statistical analysis was performed by ANOVA followed by Newman Keuls multiple comparison test. ***p < 0.001 ROT-treated cultures versus controls; p < 0.05, p < 0.01, p < 0.001 ROT plus inhibitor-treated cultures versus ROT-treated cultures.

failed to protect against ROT-induced cell death in PK-KO cultures and further increased the induced cell death (Fig. 6). However, the broad-spectrum caspase inhibitor protected against neuronal-induced cell death in PK-KO cultures (Fig. 2). Minocycline protected dopaminergic neurons and prevented the rotenone-induced cell death Minocycline completely prevented the ROT-induced loss of TH+ cells and total number of neurons in mesencephalic cultures from PK-KO mice (Figs 7a and c). Minocycline also blocked ROT-increased microglial expression (Fig. 7f) and apoptotic-induced cell death (Figs 7b and d) but failed to reduce GFAP expression in PK-KO cultures (Fig. 7e). Therefore, minocycline appears to be a very effective



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Fig. 7 Minocycline (Min) prevented the rotenone (ROT)-induced cell death in parkin knockout (PK-KO) midbrain cultures. After 8 days in vitro, the cells were treated with ROT (0.05 lM) for 5 h. Minocycline (20 lM) was added 30 min before the treatment with ROT. (a) Photomicrographs of dopamine (DA) cells [tyrosine hydroxylase-immunoreactive (TH+)]. (b) Photomicrographs of total nuclei stained with bis-benzimide. Scale bar, 30 lm. (c) Number of DA neurons expressed as number of TH+ cells/well. (d) Percentage of apoptotic

cells with respect to the total number. (e) Glial fibrillary acidic protein (GFAP) expression and (f) microglial cells. Statistical analysis was performed by two-way ANOVA (the interaction between genotype and treatment was p < 0.001) followed by Bonferroni post-test. ***p < 0.001 ROT-treated cultures versus controls; +p < 0.05, +++p < 0.001 PK-KO versus wild-type (WT) cultures; p < 0.05, p < 0.001 ROT plus minocycline-treated cultures versus ROTtreated cultures.

neuroprotective agent in ROT-induced DA cell death in parkin null mice.

Neurodegeneration of DA neurons was evaluated by H-DA uptake (Fig. 8b) and the number of TH+ cells with processes (Fig. 8a).

Addition of parkin knockout microglia to wild-type neuron-enriched cultures significantly increases the sensitivity of dopamine neurons to rotenone-induced neurotoxicity Rotenone at 0.05 lM, a concentration that is non-toxic for DA neurons in WT neuron-enriched cultures, in the presence of PK-KO microglia induces significant and selective degeneration of DA neurons (Fig. 8). Microglial cells play a central role in ROT-induced toxicity in midbrain neuronalenriched cultures. The addition of 1.2 · 104 PK-KO microglial cells to WT neuron-enriched cultures significantly increases the sensitivity of dopaminergic neurons to ROTinduced neurotoxicity.

3

Discussion

In this study, we have shown that ROT, a respiratory chain complex I inhibitor, induces cell death of fetal midbrain neurons, including DA cells, in PK-KO mice. Treatment with low doses of ROT and for short periods of time induces cell death and increases microglial activation in these parkindeficient but not in WT animals. Taking in consideration the limitations of translating results from rodents to humans, these results suggest that individuals with lack of parkin function are more susceptible to the toxic effects of environmental agents such as ROT and



Fig. 8 The effect of addition of microglia to PK-KO/WT neuron-enriched cultures on rotenone (ROT)-induced degeneration of dopamine neurons. Wild-type neuron-enriched mesencephalic cultures were supplemented with 1.2 · 104 PK-KO microglial cells/well. At 24 h later the cultures were treated with vehicle or 0.05 lM ROT and the number of tyrosine hydroxylase-immunoreactive (TH+) cells with processes (a) and 3H-dopamine (DA) uptake (b) were determined 5 h after the ROT treatment. Statistical analysis was performed by ANOVA followed by Newman Keuls multiple comparison test. **p < 0.01, ***p < 0.001 ROT-treated cultures in presence of parkin knockout (PK-KO) microglia versus controls.

other complex I inhibitors used as pesticides or herbicides. We have therefore established a link between genetic and environmental factors that may contribute to the pathogenesis of PD. As our results were obtained in mice with homozygote deletions of the Park-2 gene, we do not know whether the same conclusions apply to individuals with partial parkin function due to heterozygote mutations of even polymorphisms. Homozygotes or heterozygotes with combined mutations of the two genes that code for parkin usually present with early onset autosomal recessive PD. Heterozygotes or individuals with certain polymorphisms of parkin do

not usually show clinical symptoms of PD. Patients with partial parkin disfunction may show the combined effect of a genetic trait and an environmental agent and they are likely to present typical symptoms of sporadic or even drug-induced PD (Ampuero et al. manuscript in preparation). Little is known about the precise mechanisms of ROT action that can lead to neurodegeneration. Previous studies suggested that pesticides and herbicides could act by aggregating alpha-synuclein (Uversky et al. 2001; Manning-Bog et al. 2002). As synuclein and parkin interact with each other, it is possible that such a mechanism could be relevant. The effect of ROT could be greater in DA neurons from PK-KO mice as they have high levels of reactive oxygen related to the metabolism of DA (Palacino et al. 2004; Periquet et al. 2005). Signs of oxidative damage have been detected frequently in DA neurons from PD patients, suggesting an involvement of oxidative stress in this disease (Beal 2001; Dauer and Przedborski 2003; Jenner 2003). The primary action of ROT is to promote extracellular O2– release via activation of NADPH oxidase in the microglia (Gao et al. 2003; Zoccarato et al. 2005). In ROT-dependent parkinsonism, NADPH oxidase-derived superoxide triggers the extracellular oxidation of DA to quinones, and these species enter into the neurons to initiate the peroxide production, supported by the increase of intramitochondrial Ca2+ (Zoccarato et al. 2005). Furthermore, PK-KO mice have increased monoamine oxidase-B activity (Casarejos et al. 2005) and are more susceptible to cinnarizine, a calcium antagonist that produces human parkinsonism (Serrano et al. 2005). Neuroinflammation, as an integral component of the progressive neurodegenerative process, has been increasingly implicated in the pathogenesis of PD. Microglial activation, a hallmark of neuroinflammation, has been detected frequently in PD patients and experimental models. Our recent work demonstrated increased microglial cells in midbrain PK-KO cultures (Casarejos et al. 2005). The presence of microglia markedly enhanced ROT neurotoxicity and the ROT-stimulated microglial activation preceded dopaminergic neurodegeneration (Gao et al. 2003). Similar results have been reported in experiments in vivo (Sherer et al. 2003a). Our present findings, that microglia from PK-KO mice enhance DA toxicity in WT cultures and that inhibition of microglial NADPH oxidase protects against ROT-induced cell death, further clarify the cytotoxic role of microglia-induced neuroinflammation in PD. Nitric oxide synthase and COX inhibitors are known to modulate microglial function and microglial production of cytokines (Pyo et al. 1999; Choi et al. 2003). In our model nitric oxide synthase and COX inhibitors did not reduce the DA cell loss. In contrast, the prevention of both cell death and DA degeneration in PK-KO midbrain cultures by minocycline and partially by the wide-spectrum caspase inhibitor, as well as the NADPH oxidase inhibitor, suggests



that ROT-induced cell death results from a complex process which is both dependent and independent of microglia and caspase activation. Minocycline, a second-generation tetracycline that effectively crosses the blood–brain barrier (Zhu et al. 2002; Domercq and Matute 2004) and blocks the proliferation of microglia, has marked neuroprotective properties in models of cerebral ischemia (Yrjanheikki et al. 1998), amyotrophic lateral sclerosis (Zhang et al. 2003; McGeer and McGeer 2005), Huntington’s disease (Chen et al. 2000; Bantubungi et al. 2005) and PD (Du et al. 2001; Wu et al. 2002; Thomas and Le 2004). Relevant studies have further demonstrated that minocycline-mediated neuroprotection is possibly associated with inhibition of caspase 1, caspase 3 and COX-2 (McGeer and McGeer 2005). Inducible nitric oxide synthase transcriptional up-regulation and activation (Tikka and Koistinaho 2001; Tikka et al. 2001) and p38 MAPK pathway (Lin et al. 2001; Zhu et al. 2002; Pi et al. 2004) may also be involved in minocycline-mediated neuroprotection. Minocycline may have additional neuroprotective effects through microglia-independent and caspase 3-dependent mechanisms (Hughes et al. 2004). Minocycline prevents glutamate-induced apoptosis of cerebellar granule neurons by differential regulation of p38 and Akt pathways (Pi et al. 2004), and inhibits caspase activation (Du et al. 2001) and cytochrome c release from mitochondria (Chen et al. 2000). We show that in PK-KO midbrain cultures minocycline neuroprotection is related to inactivation of microglial cells and protection against neuronal apoptotic cell death. Minocycline has been proposed as a putative neuroprotective agent for a number of neurodegenerative disorders including PD, Huntington’s disease and amyotrophic lateral sclerosis (Blum et al. 2004). Our study adds further support to this idea at least in patients with PD and abnormal parkin function. This study also demonstrates that microgliaenhanced degeneration of DA neurons induced by ROT was mediated by activation of microglia and by consequent increased NADPH oxidase activity. In conclusion, exposure of PK-KO midbrain cultures to ROT represents a model of interaction of genetic and environmental factors involved in the pathogenesis of PD and provides an excellent model to analyse the effects of putative neuroprotective agents for PD. Minocycline appears to be the most interesting of the compounds tested in this study. Acknowledgements Supported in part by grants FIS 2002/020265, FIS 2004/040360, FIS CP04/0180 and red CIEN 03/06. JM and JAR-N had FIS predoctoral fellowships. The authors thank R Villaverde for excellent technical assistance.

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