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Slow N-acetyltransferase 2 status leads to enhanced intrastriatal dopamine depletion in 6-hydroxydopamine-lesioned rats
Experimental Neurology 187 (2004) 199 – 202 www.elsevier.com/locate/yexnr
Brief Communication
Slow N-acetyltransferase 2 status leads to enhanced intrastriatal dopamine depletion in 6-hydroxydopamine-lesioned rats M. Grundmann, C.D. Earl, J. Sautter, C. Henze, W.H. Oertel, and O. Bandmann * Department of Neurology, Philipps-University, Marburg, Germany Received 14 July 2003; revised 8 December 2003; accepted 5 January 2004
Abstract We previously reported an association between the N-acetyltransferase 2 (NAT2) slow acetylator status and Parkinson’s disease (PD). We have now investigated the possible functional relevance of this association by treating Fischer 344 (F344) rapid and Wistar – Kyoto (WKY) slow NAT2 acetylator rat strains with the neurotoxin 6-hydroxydopamine (6-OHDA). Intrastriatal treatment with either 10 or 20 Ag of 6-OHDA lead to a significantly greater reduction of striatal dopamine concentrations in the WKY slow acetylator rat strain than in the F344 rapid acetylator rat strain ( P < 0.004), reflecting a more marked degree of dopaminergic denervation. Nigral dopaminergic cell counts were also lower in the WKY rats, but this difference failed to reach statistical significance, suggesting that slow acetylation is especially deleterious at the level of striatal nerve endings. D 2004 Elsevier Inc. All rights reserved. Keywords: N-Acetyltransferase 2; 6-Hydroxydopamine; Wistar – Kyoto
There is growing evidence for an important role of genetic factors in the pathogenesis of Parkinson’s disease (PD), and the identification of alpha-synuclein, parkin, and other monogenically inherited, comparatively rare PD genes has significantly contributed to our understanding of the pathogenesis of this disorder (Warner and Schapira, 2003). However, genetic susceptibility factors remain to be identified in most patients with PD who are either sporadic or only have a small number of affected relatives. Epidemiological studies have consistently identified both a positive family history and exposure to toxins as a risk factor for PD (Warner and Schapira, 2003). We previously investigated the gene encoding the detoxification enzyme N-acetyltransferase 2 (NAT2) for association with PD and identified a strong association of the slow NAT2 acetylator status with familial PD (Bandmann et al., 1997). NAT2 is capable of Nacetylation, O-acetylation, and N,O-acetylation and is implicated in the activation and detoxification of known carcinogens and neurotoxins (Grant et al., 2000). Subse-
* Corresponding author. Department of Neurology, Academic Neurology Unit, Medical School, University of Sheffield, E Floor Beech Hill Road, Sheffield S10 2RX, UK. E-mail address: [email protected] (O. Bandmann). 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.01.001
quent association studies in PD have produced conflicting results. Most recently, a strong association between the NAT2 slow acetylator genotype and PD with an odds ratio of 5.53 was reported in Hong Kong Chinese (Chan et al., 2003), but a large study conducted in the US failed to find evidence for an association of NAT2 with PD (Walt et al., 2003). Thus, NAT2 acetylator status may only confer increased susceptibility in particular populations, depending on the genetic background and toxin exposure. The aim of this study was to investigate in vivo whether NAT2 slow acetylator status alters susceptibility to neurotoxins. Intrastriatal injection of 6-hydroxydopamine (6OHDA) in rats results in progressive dopaminergic nigral cell loss and constitutes a well-established animal model for PD (Glinka et al., 1997). We now demonstrate that intrastriatal 6-OHDA treatment of the NAT2 slow acetylator rat strain Wistar – Kyoto (WKY) leads to a considerably greater decrease of striatal dopamine (DA) levels compared to identical treatment of NAT2 rapid-acetylator Fischer 344 (F344) rats. In addition, there was more pronounced nigral dopaminergic cell loss in the WKY rats, but this difference failed to reach statistical significance, implying a predominantly synaptic action of acetylation on cell functionability. Michaelis –Menten kinetic constants of NAT2 for different rat strains have previously been characterized (Hein
200
Brief Communication / Experimental Neurology 187 (2004) 199–202
et al., 1997). Male 12- to 14-week-old WKY rats were utilized as NAT2 slow acetylators (Km: 98.2 F 6.1 AM; Vmax: 6.97 F 0.31 nmol/min/mg) and male F344 rats of identical age were utilized as NAT2 rapid acetylators (Km: 631 F 34 AM; Vmax: 431 F 13 nmol/min/mg). The rats were kept on a 12/12-h dark/light cycle with free access to food and water. Both the bilateral intrastriatal injection of fluorogold (FG) and the unilateral intrastriatal 6-OHDAinjection were performed on deeply anaesthetized (Ketavet and Xylocain i.m.) rats using a stereotactic frame (David Kopf Instruments, USA) as previously described (Sauer and Oertel, 1994). The experimental protocol consisted of three groups of each strain including two different concentrations (either 20 Ag 6-OHDA or 10 Ag 6-OHDA) and saline as control. The animals were analyzed after 2 weeks (n = 12 per group) and 4 weeks (n = 12 per group). The striatal tissue samples included the area of the injection site and were wrapped separately in aluminum foil, immediately frozen in liquid nitrogen, and then stored at 80jC until the biochemical analysis of DA with highpressure liquid chromatography (HPLC). The midbrain was immersion fixed in 4% paraformaldehyde and four series of coronal sections were cut on a cryostat at 35 Am. The frozen tissue samples were analyzed for DA using HPLC with electrochemical detection. FG fluorescence microscopy and tyrosine hydroxylase (TH) immunostaining and microscopic analysis were undertaken as previously described (Sauer and Oertel, 1994). Three sections from every animal were chosen and cell counts of labeled/stained SN cells were determined ipsi- and contralateral to the striatal 6-OHDA lesion and expressed as the mean of the three sections analyzed. TH-immunoreactive neurons were counted under bright-field illumination in adjacent sections according to the same criteria. For analysis of the intrastriatal dopamine content, FGlabeled cells and TH-stained cells, a two-way analysis of
variance (ANOVA), followed by post-hoc Scheffe test, was made. In all tests, significance was assigned when P < 0.05. In comparison to the unlesioned side, the DA concentrations were significantly decreased ipsilateral to the 6OHDA lesion (20 and 10 Ag 6-OHDA) at 2 and 4 weeks ( P < 0.001, two tailed Student’s test). Furthermore, onefactor analysis of variance (ANOVA), followed by a post hoc Scheffe test, revealed significant differences in dopamine concentrations in the different NAT2 acetylator rat strains at the 2- and 4-week time point after injection of the different doses of the 6-OHDA (see Fig. 1 and Table 1). Treatment with 20 Ag 6-OHDA of the F344 rapid acetylator rat strain resulted in a reduction of the DA content on the 6-OHDA injected side to 55.6% after 2 weeks compared to the saline injected side (SE 5.7; n = 12), but to 29.2% (SE 2.96; n = 12) in the WKY slow acetylator rat strain. The difference between the F344 rapid acetylator strain and the WKY slow acetylator strain was highly significant ( P = 0.004). After 4 weeks, the DA content was reduced to 59.2% in F344 and to 40.0% in WKY in the 20-Ag 6-OHDA-injected side compared to the saline injected side. The difference between F344 and WKY remained significant ( P = 0.033). Treatment with 10 Ag 6-OHDA resulted in a significant loss of DA with 65.7% (F344) and 38.2% (WKY) after 2 weeks ( P = 0.01), but not after 4 weeks ( P = 0.05). The number of FG-labeled nigral neurons was significantly decreased ipsilateral to the lesion at 2 and 4 weeks with 6-OHDA doses of 20 Ag (F344 P < 0.0001; WKY P < 0.0001) and 10 Ag (F344 P < 0.0001, WKY P < 0.0001). The cell loss was more marked in the WKY slow acetylator rats than the F344 rapid acetylator rats at both concentrations and time points (see Table 1), but none of these differences were significant ( P > 0.5). The number of THIR nigral cells was also significantly decreased ipsilateral to the lesion at 2 and 4 weeks with 6-OHDA doses of 20 Ag ( P < 0.01 for both F344 WKY) and 10 Ag 6-OHDA ( P < 0.05 for F344 and P < 0.01 for WKY) on the ipsilateral side in
Fig. 1. Striatal dopamine concentration 2 and 4 weeks after treatment with saline, 10 Ag or 20 Ag 6-hydroxydopamine (6-OHDA). The striatal dopamine concentrations on the lesioned side are given in percentage compared to the values measured on the unlesioned side. White bars: F344 rapid NAT2 acetylator rat strain; black bars: WKY slow NAT2 acetylator rat strain. ***P < 0.005, *P < 0.05.
Brief Communication / Experimental Neurology 187 (2004) 199–202
201
Table 1 Striatal dopamine concentration, fluorogold-labeled and tyrosine hydroxylase stained cell counts 2 and 4 weeks after injection with saline, 10 Ag and 20 Ag 6hydroxydopamine for both F344 rapid NAT2 acetylator and WKY slow acetylator rat strain Two weeks after injection
Two weeks after injection
F344
F344
WKY
Placebo (saline) Striatal dopamine concentration 97.18% (SE = 4.59) 94.25% (SE = 5.52)
Fluorogold-labeled cells 96.44% (SE = 2.55) 100.27% (SE = 3.70)
Tyrosine hydroxylase-labeled cells 94.33% (SE = 5.54) 96.17% (SE = 4.32)
Four weeks after injection WKY
20 Ag 6-OHDA
F344
WKY
20 Ag 6-OHDA
55.7% (SE = 5.7) P = 0.004 10 Ag 6-OHDA
29.17% (SE = 2.96)
59.17% (SE = 4.99) P = 0.033 10 Ag 6-OHDA
40.07% (SE = 3.31)
65.67% (SE = 6.05) P = 0.01
38.2% (SE = 4.49)
56.92% (SE = 3.85) P > 0.05
38.55% (SE = 4.28)
52.08% (SE = 3.66) 10 Ag 6-OHDA
44.33% (SE = 2.32)
29.1% (SE = 3.55) 10 Ag 6-OHDA
26.06% (SE = 2.59)
52.92% (SE = 4.52)
44.29% (SE = 3.0)
32.54% (SE = 2.42)
23.68% (SE = 1.8)
78.17% (SE = 7.04) 10 Ag 6-OHDA
65.5% (SE = 4.23)
74.91% (SE = 2.89) 10 Ag 6-OHDA
72.68% (SE = 3.93)
79.93% (SE = 2.01)
70.64% (SE = 1.99)
77.19% (SE = 2.44)
63.75% (SE = 2.75)
both strains compared with the number of nigral TH-IR cells on the contralateral side. Similar to the results of the FG staining, the loss of TH-labeled cells was more marked in the WKY slow acetylator rats than the F344 rapid acetylator rats at both concentrations and time points (see Table 1), but none of these differences were significant ( P > 0.5). The striatal DA concentration is a direct measure of the degree of dopaminergic denervation after 6-OHDA lesions in rats (Altar et al., 1987) Thus, the marked reduction of the DA levels in the WKY slow acetylator rat strain is likely to reflect a more pronounced distal damage of the dopaminergic neurons. The concentrations of the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were decreased in the same manner as DA itself in the F344 rapid and WKY slow acetylator rat strains (data not shown). The literature on genetics and PD is inundated with positive and negative candidate gene studies (Warner and Schapira, 2003). However, few studies have investigated the possible functional relevance of a particular genetic variant in vivo. We have now demonstrated a marked effect of NAT2 acetylator status on striatal DA levels in 6-OHDAtreated rats. We cannot exclude the possibility of other genetically encoded differences in the activity of other enzymes, transporter proteins, etc., between the two rat strains studied, which might also explain the observed variation in susceptibility to 6-OHDA rather than the postulated effect of NAT2 activity. However, such additional differences beyond the observed difference in NAT2 activity remain hypothetical. Intrastriatal injection of 6OHDA results in progressive degeneration of nigrostriatal neurons and thus resembles the natural time course of PD in humans closely. Terminal or axonal damage is an important aspect of 6-OHDA-induced cell death (Carman et al., 1991) and is compatible with our observation of a marked differ-
ence in DA levels (reflecting distal axonal damage) without significant difference in SN cell count between the F344 rapid acetylator strain and the WKY slow acetylator strain. 6-OHDA forms free radicals and is a potent inhibitor of the mitochondrial respiratory chain complexes I and IV, but there is also more recent evidence for an activation of extracellular, signal-regulated kinases and apoptotic pathways (Blum et al., 2001; Glinka et al., 1997; Kulich and Chu, 2001). NAT2 is widely expressed in adult rat brain tissue but not in glial cells or blood vessels (Ogawa, 1999). The precise mechanism as to how slow NAT2 acetylation might confer increased susceptibility to neurotoxins remains to be elucidated. 6-OHDA is only an experimental neurotoxin, and exposure to 6-OHDA has not been implicated in the pathogenesis of PD in humans. Both the MPTP and the rotenone model resemble PD more closely than the 6OHDA model (Betarbet et al., 2000; Przedborski and Jackson-Lewis, 1998). Further studies should therefore investigate whether NAT2 slow acetylator rodents are also more susceptible to other neurotoxins such as MPTP or rotenone. Only then can firm conclusions be drawn regarding the effect of NAT2 acetylator status on susceptibility to nigral neurotoxins. MPTP was not used in this study since marked differences in NAT activity between different mice strains were only reported after this study was completed (Boukouvala et al., 2002). Furthermore, this study was initiated before the effect of rotenone on the nigrostriatal system was reported (Betarbet et al., 2000). It should also be noted that at least one subsequent study showed a more widespread neurotoxic effect of rotenone (Ho¨glinger et al., 2003). The comparatively mild effect of NAT2 status on susceptibility to 6-OHDA and possibly also to other neurotoxins might at least partially explain why the different genetic association studies of NAT2 in PD have resulted in conflicting results.
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Brief Communication / Experimental Neurology 187 (2004) 199–202
Acknowledgments Financial support from the Fritz Thyssen Stiftung fu¨r Wissenschaftsfo¨rderung, Cologne, Germany, is gratefully acknowledged. We would also like to thank Edith Sim, David Hein, Etienne Hirsch, Inke Ko¨nig, and in particular Andreas Hartmann for helpful discussions.
References Altar, C.A., Marien, M.R., Marshall, J.F., 1987. Time course of adaptations in dopamine biosynthesis, metabolism, and release following nigrostriatal lesions: implications for behavioral recovery from brain injury. J. Neurochem. 48, 390 – 399. Bandmann, O., Vaughan, J., Holmans, P., Marsden, C.D., Wood, N.W., 1997. Association of slow acetylator genotype for N-acetyltransferase 2 with familial Parkinson’s disease. Lancet 350, 1136 – 1139. 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. Blum, D., Torch, S., Lambeng, N., et al., 2001. Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson’s disease. Prog. Neurobiol. 65, 135 – 172. Boukouvala, S., Price, N., Sim, E., 2002. Identification and functional characterization of novel polymorphisms associated with the genes for arylamine N-acetyltransferases in mice. Pharmacogenetics 12, 385 – 394. Carman, L.S., Gage, F.H., Shults, C.W., 1991. Partial lesion of the sub-
stantia nigra: relation between extent of lesion and rotational behavior. Brain Res. 553, 275 – 283. Chan, D.K.Y., Lam, M.K.P., Wong, R., Hung, W.T., Wilcken, D.E.L., 2003. Strong association between N-acetyltransferase 2 genotype and PD in Hong Kong Chinese. Neurology 60, 1002 – 1005. Glinka, Y., Gassen, M., Youdim, M.B., 1997. Mechanism of 6-hydroxydopamine neurotoxicity. J. Neural Transm., Suppl. 50, 55 – 66. Grant, D.M., Goodfellow, G.H., Sugamori, K.M., Durette, K., 2000. Pharmacogenetics of the human arylamine N-acetyltransferases. Pharmacology 61, 204 – 211. Hein, D.W., Doll, M.A., Fretland, A.J., et al., 1997. Rodent models of the human acetylation polymorphism: comparisons of recombinant acetyltransferases. Mutat. Res. 376, 101 – 106. Ho¨glinger, G.U., Feger, J., Prigent, A., et al., 2003. Chronic systemic complex I inhibition induces a hypokinetic multisystem degeneration in rats. J. Neurochem. 84, 491 – 502. Kulich, S.M., Chu, C.T., 2001. Sustained extracellular signal-regulated kinase activation by 6-hydroxydopamine: implications for Parkinson’s disease. J. Neurochem. 77, 1058 – 1066. Ogawa, M., 1999. Biochemical, molecular genetic and ecogenetic studies of polymorphic arylamine N-acetyltransferase (NAT2) in the brain. Fukuoka Igaku Zasshi 90, 118 – 131. Przedborski, S., Jackson-Lewis, V., 1998. Mechanisms of MPTP toxicity. Mov. Disord. 13 (Suppl. 1), 35 – 38. Sauer, H., Oertel, W.H., 1994. Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6hydroxydopamine: a combined retrograde tracing and immunocytochemical study in the rat. Neuroscience 59, 401 – 415. Walt, J.M., Martin, E.R., Scott, W.K., et al., 2003. Genetic polymorphisms of the N-acetyltransferase genes and risk of Parkinson’s disease. Neurology 60, 1189 – 1191. Warner, T.T., Schapira, A.H., 2003. Genetic and environmental factors in the cause of Parkinson’s disease. Ann. Neurol. 53 (Suppl. 3), S16 – S23.
Brief Communication
Slow N-acetyltransferase 2 status leads to enhanced intrastriatal dopamine depletion in 6-hydroxydopamine-lesioned rats M. Grundmann, C.D. Earl, J. Sautter, C. Henze, W.H. Oertel, and O. Bandmann * Department of Neurology, Philipps-University, Marburg, Germany Received 14 July 2003; revised 8 December 2003; accepted 5 January 2004
Abstract We previously reported an association between the N-acetyltransferase 2 (NAT2) slow acetylator status and Parkinson’s disease (PD). We have now investigated the possible functional relevance of this association by treating Fischer 344 (F344) rapid and Wistar – Kyoto (WKY) slow NAT2 acetylator rat strains with the neurotoxin 6-hydroxydopamine (6-OHDA). Intrastriatal treatment with either 10 or 20 Ag of 6-OHDA lead to a significantly greater reduction of striatal dopamine concentrations in the WKY slow acetylator rat strain than in the F344 rapid acetylator rat strain ( P < 0.004), reflecting a more marked degree of dopaminergic denervation. Nigral dopaminergic cell counts were also lower in the WKY rats, but this difference failed to reach statistical significance, suggesting that slow acetylation is especially deleterious at the level of striatal nerve endings. D 2004 Elsevier Inc. All rights reserved. Keywords: N-Acetyltransferase 2; 6-Hydroxydopamine; Wistar – Kyoto
There is growing evidence for an important role of genetic factors in the pathogenesis of Parkinson’s disease (PD), and the identification of alpha-synuclein, parkin, and other monogenically inherited, comparatively rare PD genes has significantly contributed to our understanding of the pathogenesis of this disorder (Warner and Schapira, 2003). However, genetic susceptibility factors remain to be identified in most patients with PD who are either sporadic or only have a small number of affected relatives. Epidemiological studies have consistently identified both a positive family history and exposure to toxins as a risk factor for PD (Warner and Schapira, 2003). We previously investigated the gene encoding the detoxification enzyme N-acetyltransferase 2 (NAT2) for association with PD and identified a strong association of the slow NAT2 acetylator status with familial PD (Bandmann et al., 1997). NAT2 is capable of Nacetylation, O-acetylation, and N,O-acetylation and is implicated in the activation and detoxification of known carcinogens and neurotoxins (Grant et al., 2000). Subse-
* Corresponding author. Department of Neurology, Academic Neurology Unit, Medical School, University of Sheffield, E Floor Beech Hill Road, Sheffield S10 2RX, UK. E-mail address: [email protected] (O. Bandmann). 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.01.001
quent association studies in PD have produced conflicting results. Most recently, a strong association between the NAT2 slow acetylator genotype and PD with an odds ratio of 5.53 was reported in Hong Kong Chinese (Chan et al., 2003), but a large study conducted in the US failed to find evidence for an association of NAT2 with PD (Walt et al., 2003). Thus, NAT2 acetylator status may only confer increased susceptibility in particular populations, depending on the genetic background and toxin exposure. The aim of this study was to investigate in vivo whether NAT2 slow acetylator status alters susceptibility to neurotoxins. Intrastriatal injection of 6-hydroxydopamine (6OHDA) in rats results in progressive dopaminergic nigral cell loss and constitutes a well-established animal model for PD (Glinka et al., 1997). We now demonstrate that intrastriatal 6-OHDA treatment of the NAT2 slow acetylator rat strain Wistar – Kyoto (WKY) leads to a considerably greater decrease of striatal dopamine (DA) levels compared to identical treatment of NAT2 rapid-acetylator Fischer 344 (F344) rats. In addition, there was more pronounced nigral dopaminergic cell loss in the WKY rats, but this difference failed to reach statistical significance, implying a predominantly synaptic action of acetylation on cell functionability. Michaelis –Menten kinetic constants of NAT2 for different rat strains have previously been characterized (Hein
200
Brief Communication / Experimental Neurology 187 (2004) 199–202
et al., 1997). Male 12- to 14-week-old WKY rats were utilized as NAT2 slow acetylators (Km: 98.2 F 6.1 AM; Vmax: 6.97 F 0.31 nmol/min/mg) and male F344 rats of identical age were utilized as NAT2 rapid acetylators (Km: 631 F 34 AM; Vmax: 431 F 13 nmol/min/mg). The rats were kept on a 12/12-h dark/light cycle with free access to food and water. Both the bilateral intrastriatal injection of fluorogold (FG) and the unilateral intrastriatal 6-OHDAinjection were performed on deeply anaesthetized (Ketavet and Xylocain i.m.) rats using a stereotactic frame (David Kopf Instruments, USA) as previously described (Sauer and Oertel, 1994). The experimental protocol consisted of three groups of each strain including two different concentrations (either 20 Ag 6-OHDA or 10 Ag 6-OHDA) and saline as control. The animals were analyzed after 2 weeks (n = 12 per group) and 4 weeks (n = 12 per group). The striatal tissue samples included the area of the injection site and were wrapped separately in aluminum foil, immediately frozen in liquid nitrogen, and then stored at 80jC until the biochemical analysis of DA with highpressure liquid chromatography (HPLC). The midbrain was immersion fixed in 4% paraformaldehyde and four series of coronal sections were cut on a cryostat at 35 Am. The frozen tissue samples were analyzed for DA using HPLC with electrochemical detection. FG fluorescence microscopy and tyrosine hydroxylase (TH) immunostaining and microscopic analysis were undertaken as previously described (Sauer and Oertel, 1994). Three sections from every animal were chosen and cell counts of labeled/stained SN cells were determined ipsi- and contralateral to the striatal 6-OHDA lesion and expressed as the mean of the three sections analyzed. TH-immunoreactive neurons were counted under bright-field illumination in adjacent sections according to the same criteria. For analysis of the intrastriatal dopamine content, FGlabeled cells and TH-stained cells, a two-way analysis of
variance (ANOVA), followed by post-hoc Scheffe test, was made. In all tests, significance was assigned when P < 0.05. In comparison to the unlesioned side, the DA concentrations were significantly decreased ipsilateral to the 6OHDA lesion (20 and 10 Ag 6-OHDA) at 2 and 4 weeks ( P < 0.001, two tailed Student’s test). Furthermore, onefactor analysis of variance (ANOVA), followed by a post hoc Scheffe test, revealed significant differences in dopamine concentrations in the different NAT2 acetylator rat strains at the 2- and 4-week time point after injection of the different doses of the 6-OHDA (see Fig. 1 and Table 1). Treatment with 20 Ag 6-OHDA of the F344 rapid acetylator rat strain resulted in a reduction of the DA content on the 6-OHDA injected side to 55.6% after 2 weeks compared to the saline injected side (SE 5.7; n = 12), but to 29.2% (SE 2.96; n = 12) in the WKY slow acetylator rat strain. The difference between the F344 rapid acetylator strain and the WKY slow acetylator strain was highly significant ( P = 0.004). After 4 weeks, the DA content was reduced to 59.2% in F344 and to 40.0% in WKY in the 20-Ag 6-OHDA-injected side compared to the saline injected side. The difference between F344 and WKY remained significant ( P = 0.033). Treatment with 10 Ag 6-OHDA resulted in a significant loss of DA with 65.7% (F344) and 38.2% (WKY) after 2 weeks ( P = 0.01), but not after 4 weeks ( P = 0.05). The number of FG-labeled nigral neurons was significantly decreased ipsilateral to the lesion at 2 and 4 weeks with 6-OHDA doses of 20 Ag (F344 P < 0.0001; WKY P < 0.0001) and 10 Ag (F344 P < 0.0001, WKY P < 0.0001). The cell loss was more marked in the WKY slow acetylator rats than the F344 rapid acetylator rats at both concentrations and time points (see Table 1), but none of these differences were significant ( P > 0.5). The number of THIR nigral cells was also significantly decreased ipsilateral to the lesion at 2 and 4 weeks with 6-OHDA doses of 20 Ag ( P < 0.01 for both F344 WKY) and 10 Ag 6-OHDA ( P < 0.05 for F344 and P < 0.01 for WKY) on the ipsilateral side in
Fig. 1. Striatal dopamine concentration 2 and 4 weeks after treatment with saline, 10 Ag or 20 Ag 6-hydroxydopamine (6-OHDA). The striatal dopamine concentrations on the lesioned side are given in percentage compared to the values measured on the unlesioned side. White bars: F344 rapid NAT2 acetylator rat strain; black bars: WKY slow NAT2 acetylator rat strain. ***P < 0.005, *P < 0.05.
Brief Communication / Experimental Neurology 187 (2004) 199–202
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Table 1 Striatal dopamine concentration, fluorogold-labeled and tyrosine hydroxylase stained cell counts 2 and 4 weeks after injection with saline, 10 Ag and 20 Ag 6hydroxydopamine for both F344 rapid NAT2 acetylator and WKY slow acetylator rat strain Two weeks after injection
Two weeks after injection
F344
F344
WKY
Placebo (saline) Striatal dopamine concentration 97.18% (SE = 4.59) 94.25% (SE = 5.52)
Fluorogold-labeled cells 96.44% (SE = 2.55) 100.27% (SE = 3.70)
Tyrosine hydroxylase-labeled cells 94.33% (SE = 5.54) 96.17% (SE = 4.32)
Four weeks after injection WKY
20 Ag 6-OHDA
F344
WKY
20 Ag 6-OHDA
55.7% (SE = 5.7) P = 0.004 10 Ag 6-OHDA
29.17% (SE = 2.96)
59.17% (SE = 4.99) P = 0.033 10 Ag 6-OHDA
40.07% (SE = 3.31)
65.67% (SE = 6.05) P = 0.01
38.2% (SE = 4.49)
56.92% (SE = 3.85) P > 0.05
38.55% (SE = 4.28)
52.08% (SE = 3.66) 10 Ag 6-OHDA
44.33% (SE = 2.32)
29.1% (SE = 3.55) 10 Ag 6-OHDA
26.06% (SE = 2.59)
52.92% (SE = 4.52)
44.29% (SE = 3.0)
32.54% (SE = 2.42)
23.68% (SE = 1.8)
78.17% (SE = 7.04) 10 Ag 6-OHDA
65.5% (SE = 4.23)
74.91% (SE = 2.89) 10 Ag 6-OHDA
72.68% (SE = 3.93)
79.93% (SE = 2.01)
70.64% (SE = 1.99)
77.19% (SE = 2.44)
63.75% (SE = 2.75)
both strains compared with the number of nigral TH-IR cells on the contralateral side. Similar to the results of the FG staining, the loss of TH-labeled cells was more marked in the WKY slow acetylator rats than the F344 rapid acetylator rats at both concentrations and time points (see Table 1), but none of these differences were significant ( P > 0.5). The striatal DA concentration is a direct measure of the degree of dopaminergic denervation after 6-OHDA lesions in rats (Altar et al., 1987) Thus, the marked reduction of the DA levels in the WKY slow acetylator rat strain is likely to reflect a more pronounced distal damage of the dopaminergic neurons. The concentrations of the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were decreased in the same manner as DA itself in the F344 rapid and WKY slow acetylator rat strains (data not shown). The literature on genetics and PD is inundated with positive and negative candidate gene studies (Warner and Schapira, 2003). However, few studies have investigated the possible functional relevance of a particular genetic variant in vivo. We have now demonstrated a marked effect of NAT2 acetylator status on striatal DA levels in 6-OHDAtreated rats. We cannot exclude the possibility of other genetically encoded differences in the activity of other enzymes, transporter proteins, etc., between the two rat strains studied, which might also explain the observed variation in susceptibility to 6-OHDA rather than the postulated effect of NAT2 activity. However, such additional differences beyond the observed difference in NAT2 activity remain hypothetical. Intrastriatal injection of 6OHDA results in progressive degeneration of nigrostriatal neurons and thus resembles the natural time course of PD in humans closely. Terminal or axonal damage is an important aspect of 6-OHDA-induced cell death (Carman et al., 1991) and is compatible with our observation of a marked differ-
ence in DA levels (reflecting distal axonal damage) without significant difference in SN cell count between the F344 rapid acetylator strain and the WKY slow acetylator strain. 6-OHDA forms free radicals and is a potent inhibitor of the mitochondrial respiratory chain complexes I and IV, but there is also more recent evidence for an activation of extracellular, signal-regulated kinases and apoptotic pathways (Blum et al., 2001; Glinka et al., 1997; Kulich and Chu, 2001). NAT2 is widely expressed in adult rat brain tissue but not in glial cells or blood vessels (Ogawa, 1999). The precise mechanism as to how slow NAT2 acetylation might confer increased susceptibility to neurotoxins remains to be elucidated. 6-OHDA is only an experimental neurotoxin, and exposure to 6-OHDA has not been implicated in the pathogenesis of PD in humans. Both the MPTP and the rotenone model resemble PD more closely than the 6OHDA model (Betarbet et al., 2000; Przedborski and Jackson-Lewis, 1998). Further studies should therefore investigate whether NAT2 slow acetylator rodents are also more susceptible to other neurotoxins such as MPTP or rotenone. Only then can firm conclusions be drawn regarding the effect of NAT2 acetylator status on susceptibility to nigral neurotoxins. MPTP was not used in this study since marked differences in NAT activity between different mice strains were only reported after this study was completed (Boukouvala et al., 2002). Furthermore, this study was initiated before the effect of rotenone on the nigrostriatal system was reported (Betarbet et al., 2000). It should also be noted that at least one subsequent study showed a more widespread neurotoxic effect of rotenone (Ho¨glinger et al., 2003). The comparatively mild effect of NAT2 status on susceptibility to 6-OHDA and possibly also to other neurotoxins might at least partially explain why the different genetic association studies of NAT2 in PD have resulted in conflicting results.
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Brief Communication / Experimental Neurology 187 (2004) 199–202
Acknowledgments Financial support from the Fritz Thyssen Stiftung fu¨r Wissenschaftsfo¨rderung, Cologne, Germany, is gratefully acknowledged. We would also like to thank Edith Sim, David Hein, Etienne Hirsch, Inke Ko¨nig, and in particular Andreas Hartmann for helpful discussions.
References Altar, C.A., Marien, M.R., Marshall, J.F., 1987. Time course of adaptations in dopamine biosynthesis, metabolism, and release following nigrostriatal lesions: implications for behavioral recovery from brain injury. J. Neurochem. 48, 390 – 399. Bandmann, O., Vaughan, J., Holmans, P., Marsden, C.D., Wood, N.W., 1997. Association of slow acetylator genotype for N-acetyltransferase 2 with familial Parkinson’s disease. Lancet 350, 1136 – 1139. 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. Blum, D., Torch, S., Lambeng, N., et al., 2001. Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson’s disease. Prog. Neurobiol. 65, 135 – 172. Boukouvala, S., Price, N., Sim, E., 2002. Identification and functional characterization of novel polymorphisms associated with the genes for arylamine N-acetyltransferases in mice. Pharmacogenetics 12, 385 – 394. Carman, L.S., Gage, F.H., Shults, C.W., 1991. Partial lesion of the sub-
stantia nigra: relation between extent of lesion and rotational behavior. Brain Res. 553, 275 – 283. Chan, D.K.Y., Lam, M.K.P., Wong, R., Hung, W.T., Wilcken, D.E.L., 2003. Strong association between N-acetyltransferase 2 genotype and PD in Hong Kong Chinese. Neurology 60, 1002 – 1005. Glinka, Y., Gassen, M., Youdim, M.B., 1997. Mechanism of 6-hydroxydopamine neurotoxicity. J. Neural Transm., Suppl. 50, 55 – 66. Grant, D.M., Goodfellow, G.H., Sugamori, K.M., Durette, K., 2000. Pharmacogenetics of the human arylamine N-acetyltransferases. Pharmacology 61, 204 – 211. Hein, D.W., Doll, M.A., Fretland, A.J., et al., 1997. Rodent models of the human acetylation polymorphism: comparisons of recombinant acetyltransferases. Mutat. Res. 376, 101 – 106. Ho¨glinger, G.U., Feger, J., Prigent, A., et al., 2003. Chronic systemic complex I inhibition induces a hypokinetic multisystem degeneration in rats. J. Neurochem. 84, 491 – 502. Kulich, S.M., Chu, C.T., 2001. Sustained extracellular signal-regulated kinase activation by 6-hydroxydopamine: implications for Parkinson’s disease. J. Neurochem. 77, 1058 – 1066. Ogawa, M., 1999. Biochemical, molecular genetic and ecogenetic studies of polymorphic arylamine N-acetyltransferase (NAT2) in the brain. Fukuoka Igaku Zasshi 90, 118 – 131. Przedborski, S., Jackson-Lewis, V., 1998. Mechanisms of MPTP toxicity. Mov. Disord. 13 (Suppl. 1), 35 – 38. Sauer, H., Oertel, W.H., 1994. Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6hydroxydopamine: a combined retrograde tracing and immunocytochemical study in the rat. Neuroscience 59, 401 – 415. Walt, J.M., Martin, E.R., Scott, W.K., et al., 2003. Genetic polymorphisms of the N-acetyltransferase genes and risk of Parkinson’s disease. Neurology 60, 1189 – 1191. Warner, T.T., Schapira, A.H., 2003. Genetic and environmental factors in the cause of Parkinson’s disease. Ann. Neurol. 53 (Suppl. 3), S16 – S23.