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Dopaminergic neurons are preferentially sensitive to long-term rotenone toxicity in primary cell culture
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Toxicology in Vitro 22 (2008) 68–74 www.elsevier.com/locate/toxinvit
Dopaminergic neurons are preferentially sensitive to long-term rotenone toxicity in primary cell culture Khaled Radad
a,*,1
, Gabriele Gille
b,1
, Wolf-Dieter Rausch
c
a
c
Pathology Department, Faculty of Veterinary Medicine, Assiut University, Assiut 71526, Egypt b Technical University, Department of Neurology, Fetscherstr, 74, 01307 Dresden, Germany Institute for Medical Chemistry, University of Veterinary Medicine, Veterina¨rplatz 1, 1210 Vienna, Austria Received 11 December 2006; accepted 8 August 2007 Available online 1 September 2007
Abstract Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by the death of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the subsequent decrease of dopamine levels in the striatum. Epidemiological studies indicate environmental pollutants as a causative factor of sporadic PD. Experimental cell culture models have the inherent problem to mimick longlasting neurodegeneration and to tackle its time–concentration relationship. The present study was designed to investigate the sensitivity of primary dopaminergic neurons to long-term rotenone exposure relevant to PD. Primary cultures prepared from embryonic mouse mesencephala were treated with nanomolar concentrations of rotenone (1, 3, 5, 10 nM) on the 6th day in vitro (DIV) for 2, 4 and 6 days. The number of tyrosine hydroxylase immunoreactive (TH+) neurons and total hematoxylin-stained nuclei were counted. Astrocyte density was qualitatively evaluated by anti-glial fibrillary acidic protein (anti-GFAP) immunocytochemistry. It was found that dopaminergic neurons were highly sensitive to long-term rotenone treatment. Rotenone in a concentration- and time-dependent manner decreased the number of TH+ neurons and led to degenerative changes of their morphology. Counting of the total cell number revealed a significant deleterious effect on the overall culture after 6 days of rotenone exposure. However, our study demonstrates a higher sensitivity of dopaminergic neurons to long-term exposure to nanomolar concentrations of rotenone. Other cells in the culture including non-dopaminergic neurons and glia cells appeared less affected compared to dopaminergic neurons. 2007 Elsevier Ltd. All rights reserved. Keywords: Dopaminergic; Neurodegeneration; Parkinson’s; Pesticides; Rotenone
1. Introduction Parkinson’s disease (PD) represents the most common neurodegenerative disorder of the aging brain next to Alzheimer’s dementia (Bove et al., 2005). The disease is charAbbreviations: DIV, day in vitro; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulphoxide; DPBS, Dulbecco’s phosphate buffered saline; FCS, fetal calf serum; GFAP, glial fibrillary acidic protein; H2O2, hydrogen peroxide; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD, Parkinson’s disease; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase. * Corresponding author. Tel.: +20 882 333938; fax: +20 882 366503. E-mail address: [email protected] (K. Radad). 1 Authors contributed equally to the publication. 0887-2333/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.08.015
acterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and subsequent deficit in striatal dopamine levels leading to the clinical manifestation of the disease (Dauer and Przedborski, 2003). Only a small proportion of PD cases are caused by major gene mutations and therefore more than 90% of the cases are considered sporadic (de Lau and Breteler, 2006). Environmental conditions such as farming, rural living and well water consumption have a strong correlation with an increased incidence of non-familial PD (Kitazawa et al., 2001; Gao et al., 2002; Wang et al., 2002; Di Monte, 2003; Shimizu et al., 2003; Sherer et al., 2003b). Chemical pollutants, such as pesticides, are thought to participate in the pathogenesis of PD (Li et al., 2005; de Lau and
K. Radad et al. / Toxicology in Vitro 22 (2008) 68–74
Breteler, 2006). Post-mortem findings showed increased levels of pesticide residues in the SN of individuals with PD (Jenner, 2001). Rotenone, a naturally occurring compound derived from the roots of Derris and Lonchocarpus plant species, is used worldwide as a natural pesticide and insecticide (Jenner, 2001). As the active ingredient of hundreds of pesticide products it is widely used as a household insecticide and a tool to eradicate nuisance fish populations in lakes and reservoirs (Betarbet et al., 2000). Due to its extreme lipophilicity, rotenone easily crosses biological membranes and does not depend on membrane transporters (Greenamyre et al., 2001). Inside nerve cells, rotenone acts with high affinity as a specific inhibitor of mitochondrial NADH dehydrogenase within complex I of the respiratory chain (Thiffault et al., 2000). Rotenone is used to elucidate the mechanisms underlying the degeneration of dopaminergic neurons in PD both in in vitro and in vivo models. Dukes et al. (2005) reported that rotenone selectively damaged dopaminergic neurons in experimental animals and cell cultures. Dopaminergic cell death due to rotenone treatment is attributed to oxidative stress resulting from the release of free radicals and superoxide following complex I inhibition (Sherer et al., 2003a). Further downstream mitochondrial damage by rotenone leads to caspase-3-dependent apoptosis in primary dopaminergic neurons (Moon et al., 2005). Oxidative stress is also a typical feature of PD brains (Dexter et al., 1989; Pearce et al., 1997; Alam et al., 1997) and the levels of apoptosis-related factors such as Bcl-2, soluble FAS, p55, caspase 1 and caspase 3 are increased (Mogi et al., 2000). To date primary cell cultures have the inherent problem to mimick long-lasting neurodegeneration and most of the data presented from rotenone-exposed primary dopaminergic cell cultures were obtained after short-term exposure to relatively high concentrations. The present study was designed to investigate the effect of the long-term exposure of primary mesencephalic culture to nanomolar concentrations of rotenone, a pesticide with relevance to PD. 2. Materials and methods
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careful removal of the meninges, tissues were mechanically cut into small pieces in DPBS and transferred into a sterile test tube containing 2 ml of 0.1% trypsin (Invitrogen, Germany) and 2 ml 0.02% DNase I (Roche, Germany) in DPBS. The tube was incubated in a water bath at 37 C for 7 min. Then, 2 ml of trypsin inhibitor (0.125 mg/ml in DPBS) (Invitrogen, Germany) were added, the tissue was centrifuged (Hettich, ROTIXA/AP) at 100g for 4 min and the supernatant was aspirated. The tissue pellet was triturated with a fire-polished Pasteur pipette in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, Germany) containing 0.02% DNase I. Dissociated cells were collected in DMEM supplemented with Hepes buffer (25 mM), glucose (30 mM), glutamine (2 mM), penicillin–streptomycin (10 U/ml and 0.1 mg/ml, respectively) and heat inactivated fetal calf serum (FCS, 10%) (all from Sigma, Germany). The cell suspension was plated into four-well multidishes (Nunclon, Germany) pre-coated with poly-D-lysine (50 lg/ml) (Sigma, Germany). Cultures were grown at 37 C in an atmosphere of 5% CO2/95% air and 100% relative humidity. The medium was exchanged on the 1st day in vitro (DIV) and on the 3rd DIV. On the 5th DIV half of the medium was replaced with serum-free DMEM containing 0.02 ml B-27/ml (Invitrogen, Germany). Serum-free supplemented DMEM was used for feeding from the 6th DIV and subsequently replaced every 2nd day. 2.2. Treatment with rotenone A stock solution of 1 mM rotenone (Sigma, Germany) was prepared in dimethyl sulphoxide (DMSO) and then diluted in DMEM to the final concentrations. The DMSO concentration in the culture medium did not exceed 0.01%. On the 10th DIV, cultures were treated with 20 nM rotenone for 48 h as a short-time exposure. For long-term exposure, cultures were treated with 1, 3, 5 and 10 nM rotenone on the 6th DIV for 2, 4 and 6 days. Parallel concentrations of DMSO without rotenone were added to control cultures. Culture medium was replaced every 2nd day with the same rotenone concentrations. The treatment was stopped by aspiration of the culture medium and fixation with 4% paraformaldehyde.
2.1. Preparation of primary mesencephalic dopaminergic cell culture
2.3. Identification of TH+ neurons
Pregnant mice (OF1/SPF, Himberg, Austria) were cared and handled in accordance with the guidelines of the European Union Council (86/609/EU) for the use of laboratory animals. At gestation day 14, uterine horns were dissected after abdominal laparotomy and transferred to a Petri dish containing sterile Dulbecco’s phosphate buffered saline (DPBS, Invitrogen, Germany). Embryos were carefully removed under aseptic conditions and collected in DPBS at room temperature. Under a stereoscope (Nikon SMZ1B, 100 · magnification), the brains were dissected, the ventral mesencephala excised and primary cultures were prepared according to Radad et al. (2006). Briefly, after
Cultured cells were rinsed carefully with PBS (pH 7.2) and fixed in 4% paraformaldehyde for 45 min at 4 C. After washing with PBS (pH 7.2), cells were permeabilized with 0.4% Triton X-100 for 30 min at room temperature. Cultures were washed three times with PBS (pH 7.2) and incubated with 5% horse serum (Vectastain ABC Kit, Vector Laboratories, USA) for 90 min to block non-specific binding sites. Cells were sequentially incubated with anti-tyrosine hydroxylase (anti-TH) antibody (Chemicon, USA) overnight at 4 C, biotinylated secondary antibody (Vectastain) and avidin–biotin–horseradish peroxidase complex (Vectastain) for 90 min at room temperature and washed
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with PBS (pH 7.2) between incubation stages. The reaction product was developed in a solution of diaminobenzidine (1.4 mM) in PBS (pH 7.2) containing 3.3 mM hydrogen peroxide (H2O2) (Sigma, Germany). Total TH+ cell numbers were counted in 10 randomly selected fields (1.13 mm2/field) at 100· magnification with a Nikon inverted microscope. 2.4. Anti-glial fibrillary acidic protein (anti-GFAP) immunostaining Cultured cells were stained immunocytochemically using anti-GFAP antibody (Chemicon, USA) for visualizing the astrocyte population in the primary mesencephalic cell cultures. The same staining procedures were carried out as described above for anti-TH staining except that the antiTH antibody was replaced with the anti-GFAP antibody. 2.5. Counting of total cell number Following immunocytochemical staining, the nuclei of the cultured cells were stained with hematoxylin to count the total number of cells. Total numbers of hematoxylin-stained nuclei were counted in 10 randomly selected fields at 100· magnification with a Nikon inverted microscope.
by v2-test. p < 0.05 was considered as statistically significant. Calculations were performed using Statview software. 3. Results 3.1. Dopaminergic neurons are preferentially sensitive to long-term rotenone toxicity When primary dopaminergic cell cultures were exposed to long-term rotenone treatment, they showed a progressive concentration- and time-dependent loss of dopaminergic neurons. Treatment of primary mesencephalic culture with nanomolar concentrations of rotenone (1, 3, 5 and 10 nM) on the 6th DIV for 6 consecutive days significantly reduced the total number of TH+ neurons up to 93% (10 nM, 12th DIV) compared to controls (Fig. 1). Moreover, it was found that treatment with 1 nM rotenone on the 6th DIV for 6 consecutive days (Fig. 1) reduced TH+ neurons by 60%, approaching the reduction produced by 20 nM when added on the 10th DIV for 48 h (Fig. 2). Morphological changes of dopaminergic neurons varied considerably between 1 nM and 10 nM and exhibited a progressive deterioration of neurites and cell bodies during the treatment period (Fig. 3).
2.6. Statistical analysis
3.2. Effect of rotenone on the total number of ventral mesencephalic cells
Each experiment was run in triplicate with four wells in each experiment. Data were expressed as median ± standard error of the median of 12 values. Statistical differences were calculated with the Kruskal–Wallis (H) test followed
Counting of hematoxylin-stained nuclei revealed a significant decrease in the total cell number on the 12th DIV after 6 days of rotenone exposure (Fig. 4B). Moreover, there was an increase in the number of pyknotic
120
100
***
TH+ cells [%]
80
***
******
60
40
*** ***
***
***
20
***
0 8 DIV
0
10 DIV
1 nM
3 nM
12 DIV
5 nM
10 nM
Fig. 1. Effect of rotenone on the survival of TH+ neurons in primary mesencephalic cell culture. Control cultures were not treated with rotenone. The average number of TH+ cells in control cultures on the 8th, 10th and 12th DIV were 34, 34 and 30 cells/field, respectively. Values represent the median ± standard error of the median of three independent experiments with four wells in each treatment. In each well 10 randomly selected fields were counted for TH immunocytochemistry (***p < 0.0001 as compared to controls).
K. Radad et al. / Toxicology in Vitro 22 (2008) 68–74 120
TH+ cells [%]
100 80
***
60
71
nuclei (dark-stained nuclei) indicating cell death (Fig. 4A). However, on the 8th and 10th DIV (2 and 4 days of rotenone exposure, respectively) there were no significant changes in the total cell number indicating their lower sensitivity to short- and medium-term rotenone treatment (data not shown). 3.3. Rotenone altered the astrocyte population in primary mesencephalic culture
40 20 0 control
20 nM Rotenone
Fig. 2. Treatment of primary dopaminergic cell culture with 20 nM of rotenone on the 10th DIV for 48 h. Control cultures were not treated with rotenone. The average number of TH+ cells in control cultures on the 12th DIV was 10 cells/field. Values represent the median ± standard error of the median of three independent experiments with four wells in each treatment. In each well 10 randomly selected fields were counted for TH immunocytochemistry (***p < 0.0001 as compared to controls).
Immunocytochemical staining using anti-GFAP antibody revealed a marked decrease in the density of the astrocyte population in primary mesencephalic cell culture particularly after 6 days of rotenone treatment at all tested concentrations. When cultures were investigated on the 12th DIV after 6 days of treatment with 10 nM rotenone, astrocytes showed shrinkage, vacuolation and loss of their processes (Fig. 5).
Fig. 3. Representative micrographs of TH+ neurons. Control cultures (A–C) showing intact dopaminergic neurons with long and branched processes compared to cultures treated with 1 nM (D–F) and 10 nM (G–I) rotenone. The morphology of surviving cells progressively altered according to the concentrations and times of rotenone exposure. TH+ neurons were stained by immunocytochemistry.
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A
120
B total cells [%]
100
**
80
**
**
**
5 nM
10 nM
60 40 20 0 0
1 nM
3 nM
12 DIV Fig. 4. Effect of rotenone on the overall cells in primary mesencephalic culture on the 12th DIV after treatment with rotenone for 6 consecutive days. (A) Representative micrographs for hematoxylin-stained nuclei. Note the loss of a significant number of hematoxylin-stained nuclei and the presence of a considerable number of pyknotic ones (arrow) in 1 nM (b) and 10 nM (c) rotenone-treated cultures compared to control culture (a). (B) Rotenone significantly decreased the total number of cells in primary mesencephalic culture. Control cultures were not treated with rotenone. The average number of total cells in control cultures on the 8th, 10th and 12th DIV were 95, 131 and 156 cell/field, respectively. Values represent the median ± standard error of the median of three independent experiments with four wells in each treatment. In each well 10 randomly selected fields were counted for hematoxylinstained nuclei (**p = 0.0071).
Fig. 5. Representative micrographs of GFAP-positive cells (astrocytes). Control cultures show a dense population of astrocytes with many entangled cellular processes. Astrocyte density was decreased and astrocytes showed shrinkage and vacuolation in rotenone-exposed cultures (10 nM rotenone, 10– 12th DIV).
4. Discussion The pathogenesis of sporadic PD is still elusive. Epidemiological studies have suggested that several risk factors
including environmental toxins and aging are suspected of playing a major role in developing sporadic PD (Le Couteur et al., 2002). This suggestion and the similarities between the pathology produced by some environmental
K. Radad et al. / Toxicology in Vitro 22 (2008) 68–74
toxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), paraquat and rotenone, and the clinical findings in PD brains prompted researchers to use these toxins to model PD either in vivo or in vitro. In the present study, we investigated the effect of long-term treatment with rotenone on primary mesencephalic cell culture, because so far most of the data presented from primary cell cultures were obtained after short-term exposure to neurotoxins and little exists about long-term treatment except in cell line models. It was found that long-term (6 days) treatment of primary mesencephalic culture starting on the 6th DIV with nanomolar concentrations of rotenone destroyed TH+ neurons in a concentration- and time-dependent manner. In consistency, we previously reported that short-term treatment with 5, 10 and 20 nM of rotenone on the 10th DIV for 24 and 48 h also decreased the total number of TH+ neurons in a concentration- and time-dependent manner (Radad et al., 2006). Also, Moon et al. (2005) found that TH+ neurons in primary dopaminergic cell culture were gradually lost by increasing the concentration and exposure time of rotenone. These findings were also observed in dopaminergic tumor cell lines and organotypic slice cultures. For instance, Seaton et al. (1997) and Sherer et al. (2003b) reported that rotenone caused a concentration- and time-dependent decrease in cell survival in both PC12 and SK-N-MC neuroblastoma cells, respectively. Testa et al. (2005) showed a concentration- and timedependent destruction of SNpc neuronal processes, neuronal loss and decreased TH protein levels after chronic complex I inhibition over weeks by low concentrations (10–50 nM) of rotenone in rodent post-natal midbrain organotypic slices. When our cultures were treated with 1, 3, 5 and 10 nM rotenone concentrations on the 6th DIV for 2 days they exhibited a concentration-dependent progressive and selective loss of TH+ neurons demonstrating a sensitivity of TH+ neurons towards nanomolar concentrations of rotenone. In contrast, other cells in the culture appeared only significantly affected on the 12th DIV after 6 days rotenone exposure (time-dependent). This decrease in the total number of cells was not concentration-dependent as different rotenone concentrations (1, 3, 5 and 10 nM) produced a similar loss of overall cells. This is in agreement with Ahmadi et al. (2003) who found similar levels of apoptosis in primary rat mesencephalic cultures treated with 1–30 nM rotenone. Moon et al. (2005) also showed that death of the overall neuronal cells in primary mesencephalic cell culture was nearly equal for 1, 5 and 10 nM of rotenone as the levels of the neuronal marker protein MAP2 was equally reduced when cultures were treated with these concentrations for 24 h. The decrease in the total number of ventral mesencephalic cells might result from the loss in non-dopaminergic neurons and glia cells. Staining of our culture against anti-GFAP antibody showed shrinkage, vacuolation and loss of some astrocytes. The neurites of dopaminergic neurons were also concentrationand time-dependently deteriorated.
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Taken together, our data show that dopaminergic neurons in primary mesencephalic culture are more sensitive and selectively lost after prolonged treatment with rotenone at low doses. This is consistent with the results of Kweon et al. (2004) who reported in a cell line model that rotenone killed more dopaminergic MN9D cells than nondopaminergic MN9X cells. In parallel, Ahmadi et al. (2003) and Ren et al. (2005) reported that rotenone exhibited much higher toxicity to dopaminergic neurons in midbrain neuronal cultures compared to the total ventral mesencephalic cell population. Moreover, Dukes et al. (2005) reported that rotenone produced selective dopaminergic cell death both in in vivo and in vitro models including dopaminergic cell culture. On the other hand, Zhu et al. (2004) concluded that the specificity of rotenone toxicity for dopaminergic neurons remains controversial and Nakamura et al. (2000) showed that nanomolar rotenone administration in primary dopaminergic cell culture did not produce selective damage to TH+ neurons. However, we found that long-term treatment of primary dopaminergic cell culture with 1 nM of rotenone on the 6th DIV for 6 consecutive days significantly reduced the total number of TH+ neurons by 60%. This loss was approaching the value achieved when cultures were treated with 20 nM of rotenone on the 10th DIV for only 2 days (51%, Fig. 2). This indicates that even low concentrations of rotenone, when applied for a longer treatment period, result in the same degree of damage like higher concentrations applied as short-term treatment. Progressive loss of TH+ neurons induced by nanomolar concentrations of rotenone, particularly at 1 and 3 nM, over a relatively long period regarding the age of primary culture, appears to better simulate the degeneration process in Parkinson’s disease. The higher vulnerability of dopaminergic neurons to rotenone exposure in primary dopaminergic cell culture was attributed to their higher sensitivity to oxidative stress accompanying complex I inhibition, metabolism and autooxidation of dopamine (Giasson and Lee, 2000). 5. Conclusions The present study showed progressive and selective death of dopaminergic neurons in primary mesencephalic cell culture in response to rotenone toxicity at low nanomolar concentrations over a considerable period regarding the age of the primary culture. Other cells in the culture appeared less sensitive and were only affected on the 12th DIV after 6 days of rotenone treatment. Primary mesencephalic cell culture exposed to nanomolar concentrations of rotenone over a long period seem to better reflect the slow progressive neuronal degeneration in PD. Conflict of interest statement The authors state that there are no conflicts of interest with the publication of this manuscript.
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Acknowledgement This work was supported by the University of Veterinary Medicine, Vienna, Austria. References Ahmadi, F.A., Linseman, D.A., Grammatopoulos, T.N., Jones, S.M., Bouchard, R.J., Freed, C.R., Heidenreich, K.A., Zawada, W.M., 2003. The pesticide rotenone induces caspase-3-mediated apoptosis in ventral mesencephalic dopaminergic neurons. J. Neurochem. 87, 914–921. Alam, Z.I., Jenner, A., Daniel, S.E., Lees, A.J., Cairns, N., Marsden, C.D., Jenner, P., Halliwell, B., 1997. Oxidative DNA damage in the parkinsonian brain: an apparent selective increase in 8-hydroxyguanine levels in substantia nigra. J. Neurochem. 69, 1196–1203. Betarbet, R., Sherer, T.B., MacKenzie, G., Garcia-Osuna, M., Panov, A.V., Greenamyre, J.T., 2000. Chronic systemic pesticide exposure reproduces features of Parkinson’s disease. Nat. Neurosci. 3, 1301– 1306. Bove, J., Prou, D., Perier, C., Przedborski, S., 2005. Toxin-induced models of Parkinson’s disease. NeuroRx 2, 484–494. Dauer, W., Przedborski, S., 2003. Parkinson’s disease: mechanisms and models. Neuron 39, 889–909. de Lau, L.M., Breteler, M.M., 2006. Epidemiology of Parkinson’s disease. Lancet Neurol. 5, 525–535. Dexter, D.T., Carter, C.J., Wells, F.R., Javoy-Agid, F., Agid, Y., Lees, A., Jenner, P., Marsden, C.D., 1989. Basal lipid peroxidation in substantia nigra is increased in Parkinson’s disease. J. Neurochem. 52, 381–389. Di Monte, D.A., 2003. The environment and Parkinson’s disease: is the nigrostriatal system preferentially targeted by neurotoxins? Lancet Neurol. 2, 531–538. Dukes, A.A., Korwek, K.M., Hastings, T.G., 2005. The effect of endogenous dopamine in rotenone-induced toxicity in PC12 cells. Antioxid. Redox. Signal. 7, 630–638. Gao, H.M., Hong, J.S., Zhang, W., Liu, B., 2002. Distinct role for microglia in rotenone-induced degeneration of dopaminergic neurons. J. Neurosci. 22, 782–790. Giasson, B.I., Lee, V.M., 2000. A new link between pesticides and Parkinson’s disease. Nat. Neurosci. 3, 1227–1228. Greenamyre, J.T., Betarbet, R., Sherer, T., Panov, A., 2001. Response: Parkinson’s disease, pesticides and mitochondrial dysfunction. Trends Neurosci. 24, 247. Jenner, P., 2001. Parkinson’s disease, pesticides and mitochondrial dysfunction. Trends Neurosci. 24, 245–247. Kitazawa, M., Anantharam, V., Kanthasamy, A.G., 2001. Dieldrininduced oxidative stress and neurochemical changes contribute to apoptopic cell death in dopaminergic cells. Free Radic. Biol. Med. 31, 1473–1485. Kweon, G.R., Marks, J.D., Krencik, R., Leung, E.H., Schumacker, P.T., Hyland, K., Kang, U.J., 2004. Distinct mechanisms of neurodegeneration induced by chronic complex I inhibition in dopaminergic and non-dopaminergic cells. J. Biol. Chem. 279, 51783–51792.
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Toxicology in Vitro 22 (2008) 68–74 www.elsevier.com/locate/toxinvit
Dopaminergic neurons are preferentially sensitive to long-term rotenone toxicity in primary cell culture Khaled Radad
a,*,1
, Gabriele Gille
b,1
, Wolf-Dieter Rausch
c
a
c
Pathology Department, Faculty of Veterinary Medicine, Assiut University, Assiut 71526, Egypt b Technical University, Department of Neurology, Fetscherstr, 74, 01307 Dresden, Germany Institute for Medical Chemistry, University of Veterinary Medicine, Veterina¨rplatz 1, 1210 Vienna, Austria Received 11 December 2006; accepted 8 August 2007 Available online 1 September 2007
Abstract Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by the death of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the subsequent decrease of dopamine levels in the striatum. Epidemiological studies indicate environmental pollutants as a causative factor of sporadic PD. Experimental cell culture models have the inherent problem to mimick longlasting neurodegeneration and to tackle its time–concentration relationship. The present study was designed to investigate the sensitivity of primary dopaminergic neurons to long-term rotenone exposure relevant to PD. Primary cultures prepared from embryonic mouse mesencephala were treated with nanomolar concentrations of rotenone (1, 3, 5, 10 nM) on the 6th day in vitro (DIV) for 2, 4 and 6 days. The number of tyrosine hydroxylase immunoreactive (TH+) neurons and total hematoxylin-stained nuclei were counted. Astrocyte density was qualitatively evaluated by anti-glial fibrillary acidic protein (anti-GFAP) immunocytochemistry. It was found that dopaminergic neurons were highly sensitive to long-term rotenone treatment. Rotenone in a concentration- and time-dependent manner decreased the number of TH+ neurons and led to degenerative changes of their morphology. Counting of the total cell number revealed a significant deleterious effect on the overall culture after 6 days of rotenone exposure. However, our study demonstrates a higher sensitivity of dopaminergic neurons to long-term exposure to nanomolar concentrations of rotenone. Other cells in the culture including non-dopaminergic neurons and glia cells appeared less affected compared to dopaminergic neurons. 2007 Elsevier Ltd. All rights reserved. Keywords: Dopaminergic; Neurodegeneration; Parkinson’s; Pesticides; Rotenone
1. Introduction Parkinson’s disease (PD) represents the most common neurodegenerative disorder of the aging brain next to Alzheimer’s dementia (Bove et al., 2005). The disease is charAbbreviations: DIV, day in vitro; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulphoxide; DPBS, Dulbecco’s phosphate buffered saline; FCS, fetal calf serum; GFAP, glial fibrillary acidic protein; H2O2, hydrogen peroxide; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD, Parkinson’s disease; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase. * Corresponding author. Tel.: +20 882 333938; fax: +20 882 366503. E-mail address: [email protected] (K. Radad). 1 Authors contributed equally to the publication. 0887-2333/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.08.015
acterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and subsequent deficit in striatal dopamine levels leading to the clinical manifestation of the disease (Dauer and Przedborski, 2003). Only a small proportion of PD cases are caused by major gene mutations and therefore more than 90% of the cases are considered sporadic (de Lau and Breteler, 2006). Environmental conditions such as farming, rural living and well water consumption have a strong correlation with an increased incidence of non-familial PD (Kitazawa et al., 2001; Gao et al., 2002; Wang et al., 2002; Di Monte, 2003; Shimizu et al., 2003; Sherer et al., 2003b). Chemical pollutants, such as pesticides, are thought to participate in the pathogenesis of PD (Li et al., 2005; de Lau and
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Breteler, 2006). Post-mortem findings showed increased levels of pesticide residues in the SN of individuals with PD (Jenner, 2001). Rotenone, a naturally occurring compound derived from the roots of Derris and Lonchocarpus plant species, is used worldwide as a natural pesticide and insecticide (Jenner, 2001). As the active ingredient of hundreds of pesticide products it is widely used as a household insecticide and a tool to eradicate nuisance fish populations in lakes and reservoirs (Betarbet et al., 2000). Due to its extreme lipophilicity, rotenone easily crosses biological membranes and does not depend on membrane transporters (Greenamyre et al., 2001). Inside nerve cells, rotenone acts with high affinity as a specific inhibitor of mitochondrial NADH dehydrogenase within complex I of the respiratory chain (Thiffault et al., 2000). Rotenone is used to elucidate the mechanisms underlying the degeneration of dopaminergic neurons in PD both in in vitro and in vivo models. Dukes et al. (2005) reported that rotenone selectively damaged dopaminergic neurons in experimental animals and cell cultures. Dopaminergic cell death due to rotenone treatment is attributed to oxidative stress resulting from the release of free radicals and superoxide following complex I inhibition (Sherer et al., 2003a). Further downstream mitochondrial damage by rotenone leads to caspase-3-dependent apoptosis in primary dopaminergic neurons (Moon et al., 2005). Oxidative stress is also a typical feature of PD brains (Dexter et al., 1989; Pearce et al., 1997; Alam et al., 1997) and the levels of apoptosis-related factors such as Bcl-2, soluble FAS, p55, caspase 1 and caspase 3 are increased (Mogi et al., 2000). To date primary cell cultures have the inherent problem to mimick long-lasting neurodegeneration and most of the data presented from rotenone-exposed primary dopaminergic cell cultures were obtained after short-term exposure to relatively high concentrations. The present study was designed to investigate the effect of the long-term exposure of primary mesencephalic culture to nanomolar concentrations of rotenone, a pesticide with relevance to PD. 2. Materials and methods
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careful removal of the meninges, tissues were mechanically cut into small pieces in DPBS and transferred into a sterile test tube containing 2 ml of 0.1% trypsin (Invitrogen, Germany) and 2 ml 0.02% DNase I (Roche, Germany) in DPBS. The tube was incubated in a water bath at 37 C for 7 min. Then, 2 ml of trypsin inhibitor (0.125 mg/ml in DPBS) (Invitrogen, Germany) were added, the tissue was centrifuged (Hettich, ROTIXA/AP) at 100g for 4 min and the supernatant was aspirated. The tissue pellet was triturated with a fire-polished Pasteur pipette in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, Germany) containing 0.02% DNase I. Dissociated cells were collected in DMEM supplemented with Hepes buffer (25 mM), glucose (30 mM), glutamine (2 mM), penicillin–streptomycin (10 U/ml and 0.1 mg/ml, respectively) and heat inactivated fetal calf serum (FCS, 10%) (all from Sigma, Germany). The cell suspension was plated into four-well multidishes (Nunclon, Germany) pre-coated with poly-D-lysine (50 lg/ml) (Sigma, Germany). Cultures were grown at 37 C in an atmosphere of 5% CO2/95% air and 100% relative humidity. The medium was exchanged on the 1st day in vitro (DIV) and on the 3rd DIV. On the 5th DIV half of the medium was replaced with serum-free DMEM containing 0.02 ml B-27/ml (Invitrogen, Germany). Serum-free supplemented DMEM was used for feeding from the 6th DIV and subsequently replaced every 2nd day. 2.2. Treatment with rotenone A stock solution of 1 mM rotenone (Sigma, Germany) was prepared in dimethyl sulphoxide (DMSO) and then diluted in DMEM to the final concentrations. The DMSO concentration in the culture medium did not exceed 0.01%. On the 10th DIV, cultures were treated with 20 nM rotenone for 48 h as a short-time exposure. For long-term exposure, cultures were treated with 1, 3, 5 and 10 nM rotenone on the 6th DIV for 2, 4 and 6 days. Parallel concentrations of DMSO without rotenone were added to control cultures. Culture medium was replaced every 2nd day with the same rotenone concentrations. The treatment was stopped by aspiration of the culture medium and fixation with 4% paraformaldehyde.
2.1. Preparation of primary mesencephalic dopaminergic cell culture
2.3. Identification of TH+ neurons
Pregnant mice (OF1/SPF, Himberg, Austria) were cared and handled in accordance with the guidelines of the European Union Council (86/609/EU) for the use of laboratory animals. At gestation day 14, uterine horns were dissected after abdominal laparotomy and transferred to a Petri dish containing sterile Dulbecco’s phosphate buffered saline (DPBS, Invitrogen, Germany). Embryos were carefully removed under aseptic conditions and collected in DPBS at room temperature. Under a stereoscope (Nikon SMZ1B, 100 · magnification), the brains were dissected, the ventral mesencephala excised and primary cultures were prepared according to Radad et al. (2006). Briefly, after
Cultured cells were rinsed carefully with PBS (pH 7.2) and fixed in 4% paraformaldehyde for 45 min at 4 C. After washing with PBS (pH 7.2), cells were permeabilized with 0.4% Triton X-100 for 30 min at room temperature. Cultures were washed three times with PBS (pH 7.2) and incubated with 5% horse serum (Vectastain ABC Kit, Vector Laboratories, USA) for 90 min to block non-specific binding sites. Cells were sequentially incubated with anti-tyrosine hydroxylase (anti-TH) antibody (Chemicon, USA) overnight at 4 C, biotinylated secondary antibody (Vectastain) and avidin–biotin–horseradish peroxidase complex (Vectastain) for 90 min at room temperature and washed
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with PBS (pH 7.2) between incubation stages. The reaction product was developed in a solution of diaminobenzidine (1.4 mM) in PBS (pH 7.2) containing 3.3 mM hydrogen peroxide (H2O2) (Sigma, Germany). Total TH+ cell numbers were counted in 10 randomly selected fields (1.13 mm2/field) at 100· magnification with a Nikon inverted microscope. 2.4. Anti-glial fibrillary acidic protein (anti-GFAP) immunostaining Cultured cells were stained immunocytochemically using anti-GFAP antibody (Chemicon, USA) for visualizing the astrocyte population in the primary mesencephalic cell cultures. The same staining procedures were carried out as described above for anti-TH staining except that the antiTH antibody was replaced with the anti-GFAP antibody. 2.5. Counting of total cell number Following immunocytochemical staining, the nuclei of the cultured cells were stained with hematoxylin to count the total number of cells. Total numbers of hematoxylin-stained nuclei were counted in 10 randomly selected fields at 100· magnification with a Nikon inverted microscope.
by v2-test. p < 0.05 was considered as statistically significant. Calculations were performed using Statview software. 3. Results 3.1. Dopaminergic neurons are preferentially sensitive to long-term rotenone toxicity When primary dopaminergic cell cultures were exposed to long-term rotenone treatment, they showed a progressive concentration- and time-dependent loss of dopaminergic neurons. Treatment of primary mesencephalic culture with nanomolar concentrations of rotenone (1, 3, 5 and 10 nM) on the 6th DIV for 6 consecutive days significantly reduced the total number of TH+ neurons up to 93% (10 nM, 12th DIV) compared to controls (Fig. 1). Moreover, it was found that treatment with 1 nM rotenone on the 6th DIV for 6 consecutive days (Fig. 1) reduced TH+ neurons by 60%, approaching the reduction produced by 20 nM when added on the 10th DIV for 48 h (Fig. 2). Morphological changes of dopaminergic neurons varied considerably between 1 nM and 10 nM and exhibited a progressive deterioration of neurites and cell bodies during the treatment period (Fig. 3).
2.6. Statistical analysis
3.2. Effect of rotenone on the total number of ventral mesencephalic cells
Each experiment was run in triplicate with four wells in each experiment. Data were expressed as median ± standard error of the median of 12 values. Statistical differences were calculated with the Kruskal–Wallis (H) test followed
Counting of hematoxylin-stained nuclei revealed a significant decrease in the total cell number on the 12th DIV after 6 days of rotenone exposure (Fig. 4B). Moreover, there was an increase in the number of pyknotic
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TH+ cells [%]
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Fig. 1. Effect of rotenone on the survival of TH+ neurons in primary mesencephalic cell culture. Control cultures were not treated with rotenone. The average number of TH+ cells in control cultures on the 8th, 10th and 12th DIV were 34, 34 and 30 cells/field, respectively. Values represent the median ± standard error of the median of three independent experiments with four wells in each treatment. In each well 10 randomly selected fields were counted for TH immunocytochemistry (***p < 0.0001 as compared to controls).
K. Radad et al. / Toxicology in Vitro 22 (2008) 68–74 120
TH+ cells [%]
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nuclei (dark-stained nuclei) indicating cell death (Fig. 4A). However, on the 8th and 10th DIV (2 and 4 days of rotenone exposure, respectively) there were no significant changes in the total cell number indicating their lower sensitivity to short- and medium-term rotenone treatment (data not shown). 3.3. Rotenone altered the astrocyte population in primary mesencephalic culture
40 20 0 control
20 nM Rotenone
Fig. 2. Treatment of primary dopaminergic cell culture with 20 nM of rotenone on the 10th DIV for 48 h. Control cultures were not treated with rotenone. The average number of TH+ cells in control cultures on the 12th DIV was 10 cells/field. Values represent the median ± standard error of the median of three independent experiments with four wells in each treatment. In each well 10 randomly selected fields were counted for TH immunocytochemistry (***p < 0.0001 as compared to controls).
Immunocytochemical staining using anti-GFAP antibody revealed a marked decrease in the density of the astrocyte population in primary mesencephalic cell culture particularly after 6 days of rotenone treatment at all tested concentrations. When cultures were investigated on the 12th DIV after 6 days of treatment with 10 nM rotenone, astrocytes showed shrinkage, vacuolation and loss of their processes (Fig. 5).
Fig. 3. Representative micrographs of TH+ neurons. Control cultures (A–C) showing intact dopaminergic neurons with long and branched processes compared to cultures treated with 1 nM (D–F) and 10 nM (G–I) rotenone. The morphology of surviving cells progressively altered according to the concentrations and times of rotenone exposure. TH+ neurons were stained by immunocytochemistry.
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A
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B total cells [%]
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12 DIV Fig. 4. Effect of rotenone on the overall cells in primary mesencephalic culture on the 12th DIV after treatment with rotenone for 6 consecutive days. (A) Representative micrographs for hematoxylin-stained nuclei. Note the loss of a significant number of hematoxylin-stained nuclei and the presence of a considerable number of pyknotic ones (arrow) in 1 nM (b) and 10 nM (c) rotenone-treated cultures compared to control culture (a). (B) Rotenone significantly decreased the total number of cells in primary mesencephalic culture. Control cultures were not treated with rotenone. The average number of total cells in control cultures on the 8th, 10th and 12th DIV were 95, 131 and 156 cell/field, respectively. Values represent the median ± standard error of the median of three independent experiments with four wells in each treatment. In each well 10 randomly selected fields were counted for hematoxylinstained nuclei (**p = 0.0071).
Fig. 5. Representative micrographs of GFAP-positive cells (astrocytes). Control cultures show a dense population of astrocytes with many entangled cellular processes. Astrocyte density was decreased and astrocytes showed shrinkage and vacuolation in rotenone-exposed cultures (10 nM rotenone, 10– 12th DIV).
4. Discussion The pathogenesis of sporadic PD is still elusive. Epidemiological studies have suggested that several risk factors
including environmental toxins and aging are suspected of playing a major role in developing sporadic PD (Le Couteur et al., 2002). This suggestion and the similarities between the pathology produced by some environmental
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toxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), paraquat and rotenone, and the clinical findings in PD brains prompted researchers to use these toxins to model PD either in vivo or in vitro. In the present study, we investigated the effect of long-term treatment with rotenone on primary mesencephalic cell culture, because so far most of the data presented from primary cell cultures were obtained after short-term exposure to neurotoxins and little exists about long-term treatment except in cell line models. It was found that long-term (6 days) treatment of primary mesencephalic culture starting on the 6th DIV with nanomolar concentrations of rotenone destroyed TH+ neurons in a concentration- and time-dependent manner. In consistency, we previously reported that short-term treatment with 5, 10 and 20 nM of rotenone on the 10th DIV for 24 and 48 h also decreased the total number of TH+ neurons in a concentration- and time-dependent manner (Radad et al., 2006). Also, Moon et al. (2005) found that TH+ neurons in primary dopaminergic cell culture were gradually lost by increasing the concentration and exposure time of rotenone. These findings were also observed in dopaminergic tumor cell lines and organotypic slice cultures. For instance, Seaton et al. (1997) and Sherer et al. (2003b) reported that rotenone caused a concentration- and time-dependent decrease in cell survival in both PC12 and SK-N-MC neuroblastoma cells, respectively. Testa et al. (2005) showed a concentration- and timedependent destruction of SNpc neuronal processes, neuronal loss and decreased TH protein levels after chronic complex I inhibition over weeks by low concentrations (10–50 nM) of rotenone in rodent post-natal midbrain organotypic slices. When our cultures were treated with 1, 3, 5 and 10 nM rotenone concentrations on the 6th DIV for 2 days they exhibited a concentration-dependent progressive and selective loss of TH+ neurons demonstrating a sensitivity of TH+ neurons towards nanomolar concentrations of rotenone. In contrast, other cells in the culture appeared only significantly affected on the 12th DIV after 6 days rotenone exposure (time-dependent). This decrease in the total number of cells was not concentration-dependent as different rotenone concentrations (1, 3, 5 and 10 nM) produced a similar loss of overall cells. This is in agreement with Ahmadi et al. (2003) who found similar levels of apoptosis in primary rat mesencephalic cultures treated with 1–30 nM rotenone. Moon et al. (2005) also showed that death of the overall neuronal cells in primary mesencephalic cell culture was nearly equal for 1, 5 and 10 nM of rotenone as the levels of the neuronal marker protein MAP2 was equally reduced when cultures were treated with these concentrations for 24 h. The decrease in the total number of ventral mesencephalic cells might result from the loss in non-dopaminergic neurons and glia cells. Staining of our culture against anti-GFAP antibody showed shrinkage, vacuolation and loss of some astrocytes. The neurites of dopaminergic neurons were also concentrationand time-dependently deteriorated.
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Taken together, our data show that dopaminergic neurons in primary mesencephalic culture are more sensitive and selectively lost after prolonged treatment with rotenone at low doses. This is consistent with the results of Kweon et al. (2004) who reported in a cell line model that rotenone killed more dopaminergic MN9D cells than nondopaminergic MN9X cells. In parallel, Ahmadi et al. (2003) and Ren et al. (2005) reported that rotenone exhibited much higher toxicity to dopaminergic neurons in midbrain neuronal cultures compared to the total ventral mesencephalic cell population. Moreover, Dukes et al. (2005) reported that rotenone produced selective dopaminergic cell death both in in vivo and in vitro models including dopaminergic cell culture. On the other hand, Zhu et al. (2004) concluded that the specificity of rotenone toxicity for dopaminergic neurons remains controversial and Nakamura et al. (2000) showed that nanomolar rotenone administration in primary dopaminergic cell culture did not produce selective damage to TH+ neurons. However, we found that long-term treatment of primary dopaminergic cell culture with 1 nM of rotenone on the 6th DIV for 6 consecutive days significantly reduced the total number of TH+ neurons by 60%. This loss was approaching the value achieved when cultures were treated with 20 nM of rotenone on the 10th DIV for only 2 days (51%, Fig. 2). This indicates that even low concentrations of rotenone, when applied for a longer treatment period, result in the same degree of damage like higher concentrations applied as short-term treatment. Progressive loss of TH+ neurons induced by nanomolar concentrations of rotenone, particularly at 1 and 3 nM, over a relatively long period regarding the age of primary culture, appears to better simulate the degeneration process in Parkinson’s disease. The higher vulnerability of dopaminergic neurons to rotenone exposure in primary dopaminergic cell culture was attributed to their higher sensitivity to oxidative stress accompanying complex I inhibition, metabolism and autooxidation of dopamine (Giasson and Lee, 2000). 5. Conclusions The present study showed progressive and selective death of dopaminergic neurons in primary mesencephalic cell culture in response to rotenone toxicity at low nanomolar concentrations over a considerable period regarding the age of the primary culture. Other cells in the culture appeared less sensitive and were only affected on the 12th DIV after 6 days of rotenone treatment. Primary mesencephalic cell culture exposed to nanomolar concentrations of rotenone over a long period seem to better reflect the slow progressive neuronal degeneration in PD. Conflict of interest statement The authors state that there are no conflicts of interest with the publication of this manuscript.
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