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- Materials and methods
The present studies were done to investigate the effect of long-term nicotine treatment against nigrostriatal damage in non-human primates. Monkeys were administered nicotine in drinking water for 6 months to provide chronic but intermittent delivery as with smoking. Plasma nicotine levels ranged from 10 to 15 ng/mL, which were within the range in cigarette smokers. Animals were then lesioned with low doses of the dopaminergic neurotoxin MPTP for several months while nicotine was continued. The results showed that levels of striatal tyrosine hydroxylase, dopamine transporter, vesicular monoamine transporter, dopamine and nicotinic receptors were greater in nicotine-treated MPTP-lesioned primates than in lesioned animals not receiving nicotine. Nicotine had no effect in unlesioned animals. Monoamine oxidase activity was similar in unlesioned and lesioned animals treated with or without nicotine, suggesting that nicotine did not exert its effects through changes in MPTP or dopamine metabolism. MPTP-induced cell loss in the substantia nigra was unaffected by nicotine treatment, indicating that nicotine acts at the striatal level to restore/maintain dopaminergic function. These data further support the possibility that nicotine contributes to the lower incidence of Parkinson's disease in smokers.
Tobacco use is generally known for its detrimental health-related effects. However, a large series of epidemiological studies over the past 50 years have shown an inverse relationship between smoking and Parkinson's disease, a progressive neurodegenerative movement disorder for which only symptomatic therapy is currently available (Lang and Obeso 2004; Olanow 2004; Samii et al. 2004). This decreased incidence (∼ 50%) of Parkinson's disease with tobacco use is reproducible, dose related and does not appear to be explained by selective mortality (Morens et al. 1995; Checkoway and Nelson 1999; Allam et al. 2004). The critical question is what agent in smoke is responsible, as its identification may provide a means to protect against the dopaminergic nigrostriatal damage that characterizes Parkinson's disease. However, tobacco contains numerous chemicals (∼ 4000), any one of which has the potential to alter biological processes and yield an apparent neuroprotective effect. One possibility, which is the focus of considerable research, is that nicotine in tobacco products may play a role.
Extensive evidence shows that nicotine exposure protects against toxic insults in cell culture systems including dopaminergic nigral neurons (O'Neill et al. 2002; Quik 2004). In vivo work to evaluate nicotine's protective potential against nigrostriatal damage has, however, yielded conflicting results, with protection observed in some studies (Janson et al. 1988, 1992; Carr and Rowell 1990; Shahi et al. 1991; Costa et al. 2001; Parain et al. 2001, 2003; Ryan et al. 2001), but not others (Behmand and Harik 1992; Janson et al. 1992; Hadjiconstantinou et al. 1994). These inconsistencies may be explained by inadequate nicotine dosing (too low or too high) and/or inappropriate nicotine treatment regimens, with more reproducible protection observed with frequent, intermittent rather than single dosing (Costa et al. 2001; Parain et al. 2001, 2003). Lesion size may also be an important factor, with the most robust protection observed with small chronic lesions (Costa et al. 2001). Coincidently, the factors that maximize the neuroprotective effects of nicotine in rodent studies resemble those encountered in humans who smoke and do not have clinically overt Parkinson's disease, that is frequent nicotine dosing during the day (as with smoking) and a slow progressive lesion.
Because of the importance of determining whether there is a link between nicotine and the reduced incidence of Parkinson's disease, we recently performed a study in non-human primates with nigrostriatal damage. This model offers the advantage of similar behavioral deficits to those in Parkinson's disease (Langston et al. 2000; Quik 2004). Moreover, nicotine is metabolized with a similar time course to that in humans (Schoedel et al. 2003; Hukkanen et al. 2005). Nicotine was administered over a 6-month period before MPTP treatment, and the animals were subsequently lesioned with MPTP over several months while nicotine treatment was continued. Nicotine administration significantly normalized aberrant striatal dopaminergic activity, including nicotine- and potassium-evoked dopamine release, dopamine turnover and synaptic plasticity in MPTP-treated non-human primates (Quik et al. 2006).
The present experiments were done to understand the molecular changes associated with the functional improvements described above. To approach this, we measured dopaminergic nerve terminal markers, including tyrosine hydroxylase (TH), the dopamine transporter, vesicular monoamine transporter, dopamine levels and nicotinic receptors, in striatum of unlesioned and lesioned monkeys treated with and without nicotine.
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- Materials and methods
The present data show that nicotine treatment to lesioned primates results in greater levels of several key dopaminergic nerve terminal measures in striatum. Striatal TH expression was shown to be higher in nicotine-treated lesioned animals using western analyses and immunocytochemistry. Similar results were obtained for the dopamine transporter, a membrane protein critical for dopamine reuptake. Importantly, the level of vesicular monoamine transporter was also increased in MPTP-lesioned animals treated with nicotine. This latter transporter is reported to be a very reliable marker of striatal dopaminergic nerve terminal integrity because it does not appear to be affected by drug treatments, unlike TH and the dopamine transporter (Vander Borght et al. 1995; Wilson and Kish 1996; Kemmerer et al. 2003). Striatal dopamine levels were also increased in MPTP-lesioned animals treated with nicotine. These results are consistent with the possibility that nicotine promotes the survival and/or regeneration of striatal dopaminergic terminals in animals with no overt parkinsonism. Studies to determine whether similar improvements are evident in monkeys with more severe nigrostriatal damage and consequent behavioral deficits remain to be done.
Such an interpretation is also supported by the results of recent studies (Quik et al. 2006), which show that chronic nicotine treatment of monkeys with nigrostriatal damage normalizes excessive dopaminergic activity in the striatum. This includes both nicotine- and potassium-evoked dopamine release, as well as dopamine turnover. In addition, chronic nicotine dosing to MPTP-lesioned monkeys restores corticostriatal synaptic plasticity, as measured using long-term depression, that arises with nigrostriatal damage (Quik et al. 2006).
The magnitude of the increase in dopaminergic markers in striatum of lesioned primates receiving nicotine is in agreement with that observed in other experimental models (O'Neill et al. 2002; Quik 2004). Work in cultured mesencephalic cells showed that nicotine exposure resulted in ∼ 20% protection against MPTP-induced toxicity. Nicotine treatment resulted in a similar protective effect against nigrostriatal damage in rodents, in which protection has been observed. Because Parkinson's disease develops only when there is a 70–80% deficit in the striatal dopaminergic system (Olanow 2004; Samii et al. 2004), a 20% reversal may delay the onset of symptoms by many years.
Nicotine administration and smoking are well known to increase CNS nicotinic receptors in rodents (Wonnacott et al. 1990; Wonnacott 1997; Gentry and Lukas 2002) and humans (Benwell et al. 1988; Breese et al. 1997; Perry et al. 1999). Various modes of administration are available in experimental animals, including its inclusion in the drinking water, a method that has been successfully used in rodents. This approach has the advantage that administration is long term and yet pulsatile because the animals drink intermittently during the course of the day, a mode that resembles smoking behavior. The present data demonstrating enhanced CNS nicotinic receptors with this method of nicotine treatment indicate that we were achieving biologically active levels of nicotine in monkey brain. Robust increases (∼ 50%) were observed in both cortex and striatum in control animals administered nicotine. Although MPTP lesioning decreased nicotinic receptors in striatum, concomitant nicotine treatment yielded nicotinic receptor levels similar to control values. Because nicotinic receptor stimulation modulates dopaminergic transmission (Wonnacott 1997), control receptor levels may allow for a more efficient regulation of striatal function.
To determine whether nicotine treatment also altered MPTP-induced decreases in substantia nigra neurons, dopaminergic cells were counted using stereological techniques (McCormack et al. 2004). No differences were observed in the number of dopaminergic neurons in lesioned animals treated without or with nicotine treatment. These data were somewhat unexpected because nicotine treatment partially reversed the dopaminergic nerve terminal deficits, as assessed by enhanced protein levels of striatal TH, dopamine transporter and vesicular monoamine transporter. These findings may suggest that nicotine treatment specifically affects the capacity of striatal dopamine terminals to respond to injury, perhaps through sprouting. Another hypothesis is that the remaining dopaminergic neurons in the substantia nigra of nicotine-treated lesioned animals are more functional than those in lesioned-only animals. Studies that include measurement of dopaminergic enzymes and transport processes in substantia nigra are necessary to evaluate this possibility.
The present finding of an improvement in striatal dopaminergic markers with no change in nigral dopaminergic cell number resembles that following treatment of MPTP-treated rhesus monkeys with glial cell line-derived neurotrophic factor (GDNF), which is being tested in patients with Parkinson's disease (Nutt et al. 2003; Harvey et al. 2005; Patel et al. 2005). Intranigral, intracaudate and intracerebroventricular infusion of GDNF led to ∼ 20% improvements in dopaminergic measures, including TH and dopamine levels on the lesioned side, in hemi-parkinsonian monkeys, but no significant increase in nigral dopamine neurons (Gash et al. 1996). This preferential effect at terminals is of interest because terminals are also damaged to a much greater extent. In animal models and also in Parkinson's disease, there is an ∼ 80% decline in striatal dopamine levels but only ∼ 50% loss in nigral dopaminergic neurons (Lang and Obeso 2004; Samii et al. 2004). Nicotine therefore appears to be most effective in the region that is most vulnerable.
Mechanisms whereby nicotine may exerts its protective effect include stimulation of trophic factors, which are increased in rodents after chronic treatment (Belluardo et al. 2000). Another possibility is that nicotine administration leads to receptor desensitization with a resulting reduction in dopamine release and a decrease in toxic metabolites arising from dopamine auto-oxidation (Rosenberg 1988; Masserano et al. 1996; McLaughlin et al. 1998; Berman and Hastings 1999; Quik et al. 2006). Alternatively or in addition, nicotine-stimulated dopamine release may have competed with MPTP for the dopamine transporter, although such a mechanism would also have applied to the substantia nigra, in which the dopaminergic cell number was not changed. Moreover, the lack of effect of nicotine on nigral cell number suggests that striatal changes are unlikely to be a mere consequence of nicotine affecting MPTP biodisposition. This is a relevant point as previous studies had shown that MPTP is metabolized to its active metabolite MPP+ by monoamine amine oxidase B (Langston 1996). Moreover, monoamine oxidase activity is decreased in the brains of smokers (Fowler et al. 1996a,b). However, no change was seen in enzymic activity in unlesioned or lesioned animals treated with nicotine.
As mentioned earlier, the conflicting data concerning the neuroprotective potential of nicotine against nigrostriatal damage in rodents may be explained by methodological differences between studies (O'Neill et al. 2002; Quik 2004). In addition, the variable degree of protection may relate to inherent species differences in nicotine pharmacokinetics and pharmacodynamics. Rodents metabolize nicotine somewhat differently, and there is a more rapid time course in rodents than in primates (Schoedel et al. 2003; Hukkanen et al. 2005). There are also differences in nicotinic receptor subtype distribution between species. The nicotinic α6* subtype forms only 15–20% of the total nicotinic receptor population in rodent striatum, but a much larger proportion (∼ 50%) in monkey striatum (Quik 2004). Although other nicotinic receptors are also present in striatum, the α6* subtype may be particularly relevant to Parkinson's disease because of its more restricted localization to the basal ganglia and selective vulnerability to nigrostriatal damage (Quik et al. 2001). Although it is uncertain at present which receptor subtypes mediate neuroprotection against nigrostriatal damage, evidence suggests that α2*–α6* subtypes may be involved (O'Neill et al. 2002; Quik 2004). It is possible that the larger population of α6* receptors in primate striatum may be linked to a more robust neuroprotective effect compared with that in rodents.
In summary, the present results show that nicotine treatment to lesioned primates results in greater levels of a wide range of dopaminergic nerve terminal measures in striatum. These molecular changes may be responsible, at least in part, for the normalization of aberrant activity in striatum of nicotine-treated lesioned animals compared with lesioned animals not receiving nicotine (Quik et al. 2006). Although the present results have not allowed us to evaluate whether nicotine is neuroprotective or neurorestorative, they do support the idea that the reduced incidence of Parkinson's disease in smokers may relate to the nicotine in tobacco. The present findings also have implications for the proposed use of nicotine as a therapeutic agent for Parkinson's disease, because nicotine or selective neuronal nicotinic receptor agonists (Lloyd and Williams 2000) may counteract disease progression particularly if given during the early stages.