The FASEB Journal express article 10.0983/fj.03-0983fje. Published online January 20, 2004. Neuroprotective effect of dextromethorphan in the MPTP Parkinson�s disease model: role of NADPH oxidase Wei Zhang,*,� Tongguang Wang,* Liya Qin,* Hui-Ming Gao,* Belinda Wilson,* Syed F. Ali,§ Wanqin Zhang,� Jau-Shyong Hong,* and Bin Liu¶

*Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA; �Department of Neurology, First Clinical Hospital, and �Department of Physiology, Dalian Medical University, Dalian, China; §Neurochemistry Laboratory, National Center for Toxicological Research/FDA, Jefferson, Arkansas, USA; ¶Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, Florida, USA

Corresponding author: Bin Liu, Department of Pharmacodynamics, College of Pharmacy, Box 100487 HSC, University of Florida, Gainesville, FL 32610, USA. E-mail: liu@cop.ufl.edu

ABSTRACT

Parkinson�s disease (PD) is a neurodegenerative movement disorder characterized by a progressive loss of dopaminergic neurons in the substantia nigra and depletion of the neurotransmitter dopamine in the striatum. Progress in the search for effective therapeutic strategies that can halt this degenerative process remains limited. Mechanistic studies using animal systems such as the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) rodent PD model have revealed the involvement of the brain�s immune cells and free radical-generating processes. We recently reported that dextromethorphan (DM), a widely used anti-tussive agent, attenuated endotoxin-induced dopaminergic neurodegeneration in vitro. In the current study, we investigated the potential neuroprotective effect of DM and the underlying mechanism of action in the MPTP rodent PD model. Mice (C57BL/6J) that received daily MPTP injections (15 mg free base/kg body weight, s.c.) for 6 consecutive days exhibited significant degeneration of the nigrostriatal dopaminergic pathway. However, the MPTP-induced loss of nigral dopaminergic neurons was significantly attenuated in those mice receiving DM (10 mg/kg body weight, s.c.). In mesencephalic neuron-glia cultures, DM significantly reduced the MPTP-induced production of both extracellular superoxide free radicals and intracellular reactive oxygen species (ROS). Because NADPH oxidase is the primary source of extracellular superoxide and intracellular ROS, we investigated the involvement of NADPH oxidase in the neuroprotective effect of DM. Indeed, the neuroprotective effect of DM was only observed in the wild-type but not in the NADPH oxidase-deficient mice, indicating that NADPH oxidase is a critical mediator of the neuroprotective activity of DM. More importantly, due to its proven safety record of long-term clinical use in humans, DM may be a promising agent for the treatment of degenerative neurological disorders such as PD.

Key words: reactive oxygen species � microglia � dopamine � substantia nigra � morphinan

arkinson�s disease (PD) is a degenerative neurological disorder characterized by a progressive loss of dopaminergic neurons in the substantia nigra (SN) pars compacta (SNpc) and dopamine-releasing fibers in the striatum, leading to movement impairment

that includes slow movement, resting tremor, rigidity, and gait disturbance (1). Despite the significant advances in recent decades, the etiology and the underlying mechanism responsible for the progressive neurodegeneration remain poorly understood. Furthermore, current therapies are primarily symptomatic and are largely ineffective in halting the progressive neurodegenerative process.

Administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively destroys the nigrostriatal dopaminergic pathway, resulting in Parkinsonian-like syndromes in humans, primates, and mice (2, 3). MPTP is converted by monoamine oxidase B in astroglia to 1-methyl-4-phenylpyridinium (MPP+), which enters dopaminergic neurons through the dopamine transporter. It is believed that MPP+ exerts its dopaminergic neurotoxicity through the inhibition of complex I of the mitochondrial electron transport chain, resulting in a depletion of ATP, collapse of the membrane potential, and, eventually, neuronal death. Recent studies, however, have demonstrated that glia, especially microglia, play an active role in mediating the MPTP-induced neurotoxicity. Damaged dopaminergic neurons induce a reactive response in glia, which are readily activated. Through activation of enzyme pathways that generate free radicals and up-regulate cytokine expression and release, activated glia, especially microglia, are thought to exacerbate the neurodegenerative process (4�7).

Dopaminergic neurons are characteristically vulnerable to oxidative damage due to their reduced antioxidant capacity and high content of oxidation-prone dopamine, lipids, and iron (8). In the SN region, the primary source of extracellular oxidative insults is most likely the NADPH oxidase-mediated release of superoxide free radicals by activated microglia. Originally identified as phagocyte oxidase (PHOX), NADPH oxidase is a multi-subunit oxidase. Upon activation, the cytosolic subunits (p40, p47, and p67) translocate to and become associated with the membrane-bound heterodimer p22-gp91. Small G proteins such as rac-1 are also part of the complex. The fully assembled enzyme catalyzes the electron transfer from NADPH to molecular oxygen to form superoxide (9). In the peripheral system, it is highly expressed in a variety of immune cells such as neutrophils and macrophages. In the CNS, however, microglia, the resident immune cells in the brain, may be the main cell type using this enzyme to produce superoxide (10).

Activation of microglia is an integral part of the neuro-inflammatory process that has been increasingly associated with the pathogenesis of degenerative neurological disorders, including PD (11, 12). Activated microglia release a variety of neurotoxic factors that include superoxide, nitric oxide (NO), cytokines, and eicosanoids. Because the SN area is especially rich in microglia (13, 14), NADPH oxidase-mediated generation of superoxide in microglia may play a key role in the induction of dopaminergic neurodegeneration. Using the acute MPTP rodent PD model, Wu et al. (15) have recently demonstrated that NADPH oxidase-deficient mice are less sensitive to the MPTP-induced neurotoxicity. In addition, in midbrain neuron-glia cultures, lack of NADPH oxidase renders dopaminergic neurons less vulnerable to degeneration induced by rotenone (16). These results suggest that NADPH oxidase may be a highly valuable target for drug intervention for PD.

P

Dextromethorphan (DM) is the active ingredient in a variety of widely used cough remedies. It was initially synthesized as a dextrorotatory morphinan to replace codeine as a non-opioid anti-tussive agent (17). Although the detailed mechanism of action responsible for its anti-tussive activity remains unclear, several groups have reported its neuroprotective activity using animal models of cerebral ischemia and hypoglycemic neural injuries (18�23). We recently reported that DM protected dopaminergic neurons in rat mesencephalic neuron-glia cultures against inflammation-mediated degeneration (24). The neuroprotective effect of DM did not appear to involve NMDA receptors but seemed to be related to its ability to inhibit microglial release of superoxide, nitric oxide (NO), and tumor necrosis factor α (TNF-α). Among those microglia-derived factors, DM was most effective in inhibiting the production of superoxide. The long-standing safety record in clinical usage and its potential non-NMDA-related but anti-inflammation-related neuroprotective activity make DM a seemingly excellent candidate for further investigation for its usefulness in the treatment of PD.

In this study, we examined the effect of DM on the MPTP-induced dopaminergic neurodegeneration in a subchronic MPTP PD mouse model (25). The potential mechanism of action was analyzed using both the primary mouse neuron-glia cultures and NADPH oxidase-deficient mice. We report here that DM afforded significant neuroprotection of SNpc dopaminergic neurons in the MPTP PD model and that the neuroprotective activity of DM seemed to be mediated through its inhibition of the activity of NADPH oxidase.

MATERIALS AND METHODS

Reagents

DM and MPTP were purchased from Sigma-Aldrich (St. Louis, MO). Cell culture ingredients were obtained from Invitrogen (Carlsbad, CA). [3H]Dopamine (DA, 30 Ci/mmol) was purchased from Perkin Elmer Life Sciences (Boston, MA). The polyclonal anti-tyrosine hydroxylase (TH) antibody was a generous gift from Dr. John Reinhard of GlaxoSmithKline (Research Triangle Park, NC). The Vectastain ABC kit and biotinylated secondary antibodies were purchased from Vector Laboratories (Burlingame, CA). The fluorescence probe H2DCFDA was obtained from Calbiochem (San Diego, CA)

Animal studies

Eight-week-old NADPH oxidase null (26) and matching wild-type (C57BL/6J) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Animals were kept on a 12-h light/dark cycle with ad libitum access to food and water and acclimated to their environment for 2�4 wk before use. Mice (25�30 g) received daily s.c. injections of vehicle saline or MPTP (15 mg free base/kg body weight) for 6 consecutive days. To study the effect of DM on MPTP-induced neurotoxicity, we gave mice twice-daily injections of vehicle saline or DM (10 mg/kg body weight) for the first 6 days and once-daily injections for the remainder of the study. Six or 21 days after the last MPTP injection, the animals were killed. Striatal tissues were rapidly dissected, immediately frozen on dry ice, and stored at �70°C until use. The rest of the brain was fixed in 3.7% paraformaldehyde followed by cryoprotection in 30% sucrose before use.

Immunohistochemistry and cell counting

Coronal sections (35 µm) covering the entire SN and striatum were obtained. Immunostaining for nigral dopaminergic neurons and striatal dopaminergic fibers was performed with anti-TH antiserum, as described previously (25, 27). In brief, after blocking, the sections were incubated overnight at 4°C with the anti-TH antiserum (1:10,000) followed by biotinylated secondary antibody and Vectastain ABC reagents. The bound complex was visualized using 3,3′-diaminobenzidine.

The number of TH-immunoreactive (TH-ir) neurons in the SN pars compacta region was visually counted under a microscope (200×), as described previously (25, 27). The boundary between the SNpc and the adjacent ventral tagmental area was defined following the mouse brain atlas (28). To ensure the accuracy of the count, a normal distribution (rostral to caudal) of SNpc TH-ir neurons was first established in the control mice. In brief, we determined the number of SNpc TH-ir neurons for each of the 24 consecutive coronal sections that encompass the entire SN. Counts from 4 animals for each group were averaged to create a normal distribution curve. No apparent differences were observed in the distribution patterns for SNpc TH-ir neurons between wild-type and PHOX−/− mice. To count the number of SNpc TH-ir neurons in the saline- or MPTP-injected mice, we used the first (rostral) and every forth section of the 24 sections of each brain (i.e., 8 sections/brain) for the counting, which was performed by three to four individuals who were blind to the treatment. The distribution of the SNpc TH-ir neurons in each set of brain sections was compared with that of its respective sham-control to correct for potential �frame-shifting� errors resulting from brain slicing and assignment of the first of the 24 sections. A mean value for the number of SNpc TH-ir neurons was then deduced by averaging the counts of the 8 sections for each animal; the results were expressed as the average number of TH-ir neurons per SNpc.

Analysis of striatal catecholamine content

The levels of dopamine (DA) and its metabolites [3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)] were determined by HPLC, coupled with electrochemical detection as described (29). Briefly, striatal tissues were sonicated in 0.2 M perchloric acid (20% W/V) containing the internal standard 3,4-dihydroxybenzylamine (100 ng/ml). After centrifugation, 150 µl of the supernatant was passed through a 0.2 µm Nylon-66 filter, and 25 µl of the filtrate representing 2.5 mg of striatal tissue was injected. The concentrations of DA, DOPAC, and HVA were calculated using standard curves that were generated by determining, in triplicate, the ratios between three known amounts of the internal standard.

Mouse mesencephalic neuron-glia cultures

Primary mesencephalic neuron-glia cultures were prepared from the brains of embryonic day 12/13 C57BL/6J mice (Jackson Laboratory), following our previously described protocol (30) with subsequent modifications (31). In brief, the ventral mesencephalic tissues were removed and dissociated by a mild mechanical trituration. Cells were seeded at 1 x 105/well on 96-well culture plates precoated with poly-D-lysine (20 µg/ml) and maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air in maintenance medium (0.2 ml/well). The medium consisted of minimum essential medium (MEM) containing 10% heat-inactivated fetal bovine

serum (FBS) and 10% heat-inactivated horse serum (HS), 1 g/l glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µM non-essential amino acids, 50 U/ml penicillin, and 50 µg/ml streptomycin. Three days after the initial seeding, 0.1 ml of fresh maintenance medium was added to each well. Seven-day-old cultures were used for treatment. The composition of the cultures at the time of treatment was ~48% astrocytes, 11% microglia, 40% neurons, and ~0.5% TH-ir neurons.

Assay of superoxide release in neuron-glia cultures

The production of superoxide was determined by measuring the superoxide dismutase (SOD)-inhibitable reduction of the tetrazolium salt WST-1, originally reported by Tan and Berridge (32) and Peskin and Winterbourn (33) and adapted for neural cell cultures by Liu et al. (34). To measure the reactive microgliosis-related release of superoxide, we pretreated neuron-glia cultures in 96-well culture plates for 30 min with vehicle or DM (1 µM) before treatment with 1 µM MPTP in the treatment medium (phenol red-free MEM containing 2% FBS, 2% HS, 100 µl/well) following the protocol described by Gao et al. (7). Two and 4 days after MPTP treatment, DM (1 µM final concentration) was added again to the DM-treated cultures. On day 6, 75 µl of WST-1 in treatment medium with and without SOD (50 U/ml final concentration) were added to each well (1 mM final concentration). Absorbance at 450 nm was read with a SpectraMax Plus microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). The difference in absorbance observed in the absence and presence of SOD was considered to be the amount of superoxide produced. Results were expressed as percent of vehicle-treated control cultures.

Determination of the levels of intracellular reactive oxygen species (ROS) in neuron-glia cultures

The levels of intracellular ROS in the neuron-glia cultures were measured using the fluorescent probe H2DCFDA as described previously with modifications (35). In brief, 6 days after treatment, cultures were treated with 10 µM H2DCFDA diluted in phenol red-free Hanks balanced salt solution (HBSS) containing 2% FBS and 2% HS. After an additional 30 min incubation at 37°C, fluorescence intensity was measured at 485 nm for excitation and 530 nm for emission using a SpectraMax Gemini XS fluorescence microplate reader (Molecular Devices).

Nitrite assay

The production of NO was determined 6 days after MPTP treatment by measuring the accumulated levels of nitrite in the supernatant with Griess reagent, as described previously (30).

Statistical analysis

Statistical significance of the differences was assessed with ANOVA followed by Bonferroni�s t test using the StatView program (Abacus Concepts, Berkeley, CA). A value of P < 0.05 was considered significant.

RESULTS

DM protects SNpc TH-ir neurons against MPTP-induced degeneration

In this study, we used a subchronic dosing regiment of MPTP injections to induce lesions in the nigrostriatal dopaminergic pathway. C57BL/6J mice received daily subcutaneous injections of vehicle or MPTP (15 mg free base/kg body weight) for 6 consecutive days. Six days later, the mice were killed. The loss of SNpc TH-ir neurons and the decrease in striatal dopamine content were determined. To determine the effect of DM on MPTP-induced dopaminergic neuronal lesions, mice were given twice-daily s.c. injections of DM (10 mg/kg body weight) during the first 6 days and once-daily DM injections thereafter. Mice were killed 6 days after the last MPTP injection. Compared with the saline-injected control mice, MPTP induced a significant decrease in the TH immunoreactivity in both the neuronal cell bodies and neuronal fibers in the SNpc (Fig. 1A). Quantitative analysis of the SNpc TH-ir neurons indicated a 30% loss of TH-ir neurons in the MPTP-injected mice compared with the vehicle (saline)-injected control mice (P<0.001, Fig. 1B). This is consistent with our previous findings using the same MPTP dosing regiment (25). Administration of DM, however, significantly reduced the MPTP-induced reduction of TH immunoreactivity and the loss of SNpc TH-ir neurons (Fig. 1). No significant difference was observed in either the TH immunoreactivity or the number of SNpc TH-ir neurons between mice that received saline injections and those that received injections of DM alone (Fig. 1). These results demonstrated that SNpc TH-ir neurons in mice that received DM injections were resistant to MPTP-induced degeneration.

DM inhibits MPTP-induced production of ROS in neuron-glia cultures

We have recently reported that DM is capable of inhibiting bacterial endotoxin lipopolysaccharide-induced activation of cultured rat microglia and the production of superoxide free radicals (24). In addition, MPTP has recently been shown to induce a rapid reactive microgliosis in mice (4�6). MPTP-induced reactive microgliosis is closely associated with an increase in microglial release of superoxide and a rise in the intracellular levels of ROS (7). Therefore, it was necessary to determine whether DM had any effect on the MPTP-induced ROS generation in mouse neuron-glia cultures. First, we tested the effect of DM on superoxide production. Mouse mesencephalic neuron-glia cultures were pretreated for 30 min with DM (1 µM) before the addition of MPTP (1 µM). DM (1 µM final concentration) was again added at 2 and 4 days after MPTP treatment. On day 6, release of superoxide from activated microglia was determined. As shown in Figure 2A, treatment of cultures with 1 µM DM almost completely inhibited the MPTP (1 µM)-induced superoxide production. Second, the effect of DM on the levels of intracellular ROS was determined. Cultures were treated with DM and/or MPTP in the same manner as described above, and the levels of intracellular ROS were determined 6 days after MPTP treatment. DM treatment greatly reduced the MPTP-induced increase in the level of intracellular ROS (Fig. 2B). Hence, these results demonstrated that DM was capable of reducing the MPTP-induced reactive activation of microglia and, most notably, their production of ROS.

Role of NADPH oxidase in the neuroprotective effect of DM

The observation that DM effectively inhibited the MPTP-induced production of ROS prompted us to speculate that inhibition of ROS production was the major underlying mechanism of action

for the observed neuroprotective activity of DM. Because NADPH oxidase (also called phagocyte oxidase, PHOX) is the primary enzyme for the production of ROS, especially superoxide, in a variety of cell types, including macrophages, neutrophils, and microglia, we hypothesized that the inhibition of NADPH oxidase was vital to the neuroprotective effect of DM. As an important control, we first compared the effect of MPTP in wild-type (PHOX+/+) and NADPH oxidase null (PHOX−/−) mice. Both groups of mice received daily injections of MPTP (15 mg free base/kg body weight, s.c.) for 6 days. Six or 21 days after the last MPTP injection, mice were killed. As shown in Figure 3, in mice killed 21 days after the last MPTP injection, a significant loss (32%) of TH-ir neurons was observed in the PHOX+/+ mice that received MPTP injections, compared with the saline-injected animals (P<0.001). In contrast, in PHOX−/− mice, compared with saline-injected animals, the MPTP-induced loss of nigral TH-ir neurons was only 14%, which is significantly less than that observed in the corresponding MPTP-injected PHOX+/+ mice (32%, P<0.05, Fig. 3B). The number of TH-ir neurons in the saline-injected PHOX+/+ mice did not differ significantly from that of saline-injected PHOX−/− mice (Fig. 3B). The reduced sensitivity of SNpc TH-ir neurons in PHOX−/− mice to the MPTP-induced neurotoxicity was also found in mice that were killed 6 days after the last MPTP injection (data not shown). These results demonstrated that in mice subchronically receiving MPTP injections, NADPH oxidase at least partially mediated the MPTP-induced loss of SNpc TH-ir neurons, consistent with the observation reported by Wu et al. (15) in mice receiving the acute MPTP injection (16 mg/kg body weight, 4 i.p. injections with 2 h intervals).

To determine the role of NADPH oxidase in the neuroprotective effect of DM, wild-type or PHOX−/− mice were given once-daily s.c. injections of saline or MPTP (15 mg free base/kg body weight) and twice-daily injections of saline or DM (10 mg/kg body weight) for the first 6 days. The DM dosing (10 mg/kg) was subsequently reduced to once-daily for the remainder of the experiment. Six days after the last MPTP injection, mice were killed. As shown in Figure 4, DM was totally ineffective in protecting the SNpc TH-ir neurons in PHOX−/− mice against the MPTP-induced degeneration. These results demonstrated that the neuroprotective effect of DM depended on the normal function of NADPH oxidase. Thus, NADPH oxidase appeared to be a critical mediator of DM�s neuroprotective activity.

In addition to counting the SNpc TH-ir neurons, striatal catecholamine content was also determined by HPLC analysis. As shown in Table 1, MPTP induced similar degrees of depletion in the content of DA (85%) and DOPAC in both the PHOX+/+ and PHOX−/− mice that were killed 6 days after the last MPTP injection. No difference in the HVA/DA ratio was observed between PHOX+/+ and PHOX−/− mice that received either saline or MPTP injections (Table 1). The basal levels of DA, DOPAC, and HVA in saline-injected PHOX+/+ and PHOX−/− mice did not differ significantly from each other (Table 1). Administration of DM did not affect either the basal or MPTP-induced changes (Table 1). Besides the 6-day observation period, striatal catecholamine content was also determined in mice killed 21 days after the last MPTP injection, the same time point used for the SNpc TH-ir neuron counts presented in Figure 3B. As shown in Table 2, 21 days after the last MPTP injection, striatal DA levels in the MPTP-injected mice remained at 20% of that of saline-injected mice in both the PHOX+/+ and PHOX−/− mice.

Effect of DM on MPTP-induced nitrite production

Recent studies have indicated that MPTP-induced neurotoxicity also involves the participation of the inducible nitric oxide synthase (iNOS) (4). To test the role of iNOS in the neuroprotective effect of DM in the MPTP PD model, mouse mesencephalic neuron-glia cultures were pretreated for 30 min with DM (1 µM) before treatment with MPTP (1 µM). At 2 and 4 days after MPTP treatment, DM (1 µM final concentration) was added again to the DM-containing treatment medium. The accumulated levels of nitrite, an indicator of NO production, were measure at 6 days after MPTP treatment. As shown in Figure 5, the levels of nitrite in cultures that received DM and MPTP were significant lower than those that received MPTP only, demonstrating that DM at least partially suppressed the MPTP-induced nitrite production.

DISCUSSION

In this study, we demonstrated that DM afforded significant protection of nigral dopaminergic neurons in the mouse MPTP PD model. The protective effect of DM observed in this study appeared to be related to the ability of DM to inhibit microglial production of ROS. Supporting this conclusion was the observation that DM markedly reduced the MPTP-induced production of superoxide free radicals and, to a lesser degree, the elevation of intracellular ROS. Furthermore, the neuroprotective effect of DM appeared to be closely related to NADPH oxidase, the primary enzymatic system in microglia for the generation of ROS, because the neuroprotective effect of DM was observed only in the wild-type but not the NADPH oxidase-deficient mice.

PD is a devastating movement disorder characterized by progressive degeneration of the nigrostriatal dopaminergic pathway. Despite decades of research, the etiology and underlying mechanism of the progressive neurodegenerative process remain poorly understood. Lack of such knowledge has hampered efforts to develop effective treatment strategies to halt the neurodegenerative process. Recently, several groups have reported that the MPTP-induced dopaminergic neurodegeneration involves the active participation of the resident immune cells in the brain, the microglia. For example, in mice that received acute administrations of MPTP, a rapid activation of microglia preceded astroglial activation in the SN and striatum (4). The glial response either preceded or coincided with the onset of the MPTP-induced neuronal injuries, suggesting that reactive gliosis is a crucial element of MPTP-induced neurodegeneration (4). In neuron-glia cultures, reactive microglial activation exacerbated the MPTP-induced neurotoxicity (7). Furthermore, mice lacking both TNF-α receptors were resistant to the MPTP-induced neurotoxicity (36).

Of the multiple factors such as cytokines, free radicals, and fatty acid metabolites produced from activated glial cells that are associated with neurodegeneration (37, 38), ROS may be the key mediator of glia-facilitated MPTP neurotoxicity. Dopaminergic neurons in the nigra are known to be particularly vulnerable to oxidative stress, presumably due to their lower antioxidant capacity, increased accumulation of ion and oxidation-prone DA, and possible defects in mitochondria (39). The midbrain region that encompasses the substantia nigra is particularly enriched with microglia (13, 14) that are known to be the primary contributor to ROS production through the activity of NADPH oxidase (7, 10, 15, 16, 40). Hence, inhibition of microglial activity and their production of proinflammatory and neurotoxic factors would be an effective strategy to slow down or possibly even stop the neurodegenerative process.

DM was initially synthesized as a dextrorotatory morphinan and has been widely used as a non-opioid anti-tussive agent with minimal side effects. In addition to its cough-suppressing activity, several groups have reported its neuroprotective effects in several model systems (18�22, 41�45). Although results from some studies appeared to associate DM�s neuroprotective effect with its possible antagonistic activity toward the NMDA receptors (41, 44), other studies did not seem to fully support this hypothesis (42, 43, 45). We recently reported that DM afforded significant protection of dopaminergic neurons in rat neuron-glia cultures against endotoxin-induced microglial activation-mediated degeneration (24). Results from that study appeared to suggest a novel mechanism for the observed neuroprotective effect of DM: inhibition of microglial production of NO, secretion of cytokines such as TNF-α, and release of superoxide free radicals (24). In this study, DM was found to provide significant protection of SNpc TH-ir neurons in the MPTP mouse PD model (Fig. 1). More importantly, the neuroprotection offered by DM relied upon a functional NADPH oxidase because no neuroprotection was observed in NADPH oxidase-deficient mice (Fig. 4). The in vivo observations were in line with in vitro results in which DM effectively inhibited the MPTP-induced release of superoxide and elevation of intracellular ROS (Fig. 2). Hence, NADPH oxidase appears to be a primary mediator of DM neuroprotective activity, although the exact relationship between DM and NADPH oxidase remains to be determined.

In addition to inhibiting the MPTP-induced ROS production, DM appeared to be able to partially reduce MPTP-induced NO production, consistent with its ability to reduce NO production in endotoxin-activated microglia (24). It has been proposed that NO and superoxide are able to form a more toxic intermediate, peroxynitrite (46). Therefore, it is advantageous that DM is capable of inhibiting the production of both ROS and NO. Delineating the precise mechanism of action by which DM interferes with these pathways will be crucial to the understanding of DM�s therapeutic efficacy.

Note that in this study, administration of DM protected against the MPTP-induced loss of SNpc TH-ir neurons (Fig. 1 and 4) but not the MPTP-induced depletion of striatal DA (Table 1). This finding appears to be analogous to the well-documented differential effects of MPTP on the loss of dopaminergic neurons in the SN and the depletion of dopamine in the striatum (47). MPTP-induced depletion of striatal dopamine occurs much more rapidly than the degeneration of nigral dopaminergic neurons. Dopamine depletion appears to involve an acute release of dopamine from storage vesicles in the nerve terminals. In contrast, degeneration of nigral dopaminergic neurons is a relatively delayed process and appears to require the participation of glial cells. Initial neuronal injuries trigger a reactive microglial activation (microgliosis) that facilitates the degeneration of dopamine neurons in the nigra, as well as degeneration of dopamine terminals in the striatum, resulting in more dramatic depletion of dopamine. Therefore, the depletion of striatal dopamine is always far more severe than the degeneration of nigral dopaminergic neurons. Furthermore, the replenishment of striatal dopamine appears to be a slow-occurring process. Chiueh et al. (48) reported a minimum of a two-month lag before a partial recovery of striatal dopamine following MPTP treatment. We postulate that DM protects dopaminergic neurons by reducing the oxidative insults as a consequence of its suppression of the MPTP-induced reactive microgliosis. In contrast, DM may not be capable of preventing the MPTP-induced fast and severe depletion of striatal dopamine. It remains to be determined whether DM will have any effect on restoring the striatal dopamine content at a longer time interval (e.g., 2�3 months) following MPTP administrations in the MPTP mouse PD model.

One of the important considerations in developing drug therapies for PD patients is the potential side-effect after long-term administrations. Widely used as an over-the-counter anti-cough agent in the last several decades, DM has a very impressive clinical safety record. In addition, the neuroprotective effect of DM is not limited to MPTP-induced neurotoxicity. DM is also effective in protecting dopaminergic neurons against damage induced by other agents such as the bacterial endotoxin lipopolysaccharide (24). More importantly, contrary to current drugs such as L-DOPA that can provide only symptomatic relief for a limited period of time, DM, which is capable of inhibiting microglial activation that appears to serve as a driving force to the progressive neurodegenerative process in PD, stands a great chance to be a prototype drug that can effectively slow down the progressive neurodegeneration. In light of an extremely limited inventory of effective drugs, further studies on DM as a potential drug for the treatment of PD are certainly warranted.

ACKNOWLEDGMENTS

We are grateful to C. Paras and R. Gentry for help with cell counting and B. Robinson for the HPLC analysis. This work is supported in part by the New Investigator Award (to B.L.) from the College of Pharmacy of the University of Florida.

REFERENCES

1. Olanow, C. W., and Tatton, W. G. (1999) Etiology and pathogenesis of Parkinson�s disease. Annu. Rev. Neurosci. 22, 123�144

2. Langston, J. W., Ballard, P., Tetrud, J. W., and Irwin, I. (1983) Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science 219, 979�980

3. Uhl, G. R., Javitch, J. A., and Snyder, S. H. (1985) Normal MPTP binding in parkinsonian substantia nigra: evidence for extraneuronal toxin conversion in human brain. Lancet 8435, 956�957

4. Liberatore, G. T., Jackson-Lewis, V., Vukosavic, S., Mandir, A. S., Vila, M., McAuliffe, W. G., Dawson, V. L., Dawson, T. M., and Przedborski, S. (1999) Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease. Nat. Med. 5, 1403�1409

5. Du, Y., Ma, Z., Lin, S., Dodel, R. C., Gao, F., Bales, K. R., Triarhou, L. C., Chernet, E., Perry, K. W., Nelson, D. L., et al. (2001) Minocycline prevents nigrostriatal dopaminergic neurodegeneration in the MPTP model of Parkinson's disease. Proc. Natl. Acad. Sci. USA 98, 14669�14674

6. Wu, D. C., Jackson-Lewis, V., Vila, M., Tieu, K., Teismann, P., Vadseth, C., Choi, D. K., Ischiropoulos, H., and Przedborski, S. (2002) Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine mouse model of Parkinson disease. J. Neurosci. 22, 1763�1771

7. Gao, H. M., Liu, B., Zhang, W., and Hong, J. S. (2003a) Critical role of microglial NADPH oxidase-derived free radicals in the in vitro MPTP model of Parkinson's disease. FASEB J. 17, 1954�1956

8. Greenamyre, J. T., MacKenzie, G. M., Peng, T.-I., and Stephans, S. E. (1999) Mitochondrial dysfunction in Parkinsons�s disease. Biochem. Soc. Symp. 66, 85�97

9. Babior, B. M. (1999) NADPH oxidase: an update. Blood 93, 1464�1476

10. Green, S. P., Cairns, B., Rae, J., Errett-Baroncini, C., Hongo, J. A., Erickson, R. W., and Curnutte, J. T. (2001) Induction of gp91-phox, a component of the phagocyte NADPH oxidase, in microglial cells during central nervous system inflammation. J. Cereb. Blood Flow Metab. 21, 374�384

11. McGeer, P. L., Yasojima, K., and McGeer, E. G. (2001) Inflammation in Parkinson's disease. Adv. Neurol. 86, 83�89

12. Liu, B., and Hong, J.-S. (2003) Role of microglia in inflammation-mediated neurodegenerative diseases: study of mechanism and strategies for therapeutic intervention. J. Pharmacol. Exp. Ther. 304, 1�7

13. Lawson, L. J., Perry, V. H., Dri, P., and Gordon, S. (1990) Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain. Neuroscience 39, 151�170

14. Kim, W. G., Mohney, R. P., Wilson, B., Jeohn, G. H., Liu, B., and Hong, J. S. (2000) Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia. J. Neurosci. 20, 6309�6316

15. Wu, D.C., Teismann, P., Tieu, K., Vila, M., Jackson-Lewis, V., Ischiropoulos, H., and Przedborski, S. (2003) NADPH oxidase mediates oxidative stress in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson�s disease. Proc. Natl. Acad. Sci. USA. 100, 6145�6150

16. Gao, H. M., Liu, B., and Hong, J. S. (2003b) Critical role for microglial NADPH oxidase in rotenone-induced degeneration of dopaminergic neurons. J. Neurosci. 23, 6181�6187

17. Tortella, F. C., Pellicano, M., and Bowery, N. G. (1989) Dextromethorphan and neuromodulation: old drug coughs up new activities. Trends Pharmacol. Sci. 10, 501�507

18. Steinberg, G.K., George, C.P., DeLaPaz, R., Shibata, D.K., and Gross, T. (1988) Dextromethorphan protects against cerebral injury following transient focal ischemia in rabbits. Stroke 19, 1112�1118

19. George, C. P., Goldberg, M. P., Choi, D. W., and Steinberg, G. K. (1988) Dextromethorphan reduces neocortical ischemic neuronal damage in vivo. Brain Res. 440, 375�379

20. Prince, D. A., and Feeser, H. R. (1988) Dextromethorphan protects against cerebral infarction in a rat model of hypoxia-ischemia. Neurosci. Lett. 85, 291�296

21. Monyer, H., and Choi, D. W. (1988) Morphinans attenuate cortical neuronal injury induced by glucose deprivation in vitro. Brain Res. 446, 144�148

22. Britton, P., Lu, X. C., Laskosky, M. S., and Tortella, F. C. (1997) Dextromethorphan protects against cerebral injury following transient, but not permanent, focal ischemia in rats. Life Sci. 60, 1729�1740

23. Tortella, F. C., Britton, P., Williams, A., Lu, X. C., and Newman, A. H. (1999) Neuroprotection (focal ischemia) and neurotoxicity (electroencephalographic) studies in rats with AHN649, a 3-amino analog of dextromethorphan and low-affinity N-methyl-D-aspartate antagonist. J. Pharmacol. Exp. Ther. 291, 399�408

24. Liu, Y. X., Qin, L., Li, G., Zhang, W., An, L., Liu, B., and Hong, J. S. (2003) Dextromethorphan protects dopaminergic neurons against inflammation-mediated degeneration through inhibition of microglial activation. J. Pharmacol. Exp. Ther. 305, 212�218

25. Feng, Z.H., Wang, T.G., Li, D-D., Wilson, B.C., Liu, B., Ali, S.F., Langenbach, R., and Hong, J-S. (2002) Cyclooxygenase-2 deficient mice are resistant to 1-methyl-4-phenyl 1, 2, 3, 6-tetrahydropyridine-induced damage of dopaminergic neurons in the substantia nigra. Neurosci. Lett. 329, 354�358

26. Pollock, J. D., Williams, D. A., Gifford, M. A., Li, L. L., Du, X., Fisherman, J., Orkin, S. H., Doerschuk, C. M., and Dinauer, M. C. (1995) Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat. Genet. 9, 202�209

27. Liu, B., Jiang, J. W., Wilson, B., Du, L., Yang, S. N., Wang, J. Y., Wu, G. C., Cao, X. D., and Hong, J. S. (2000b) Systemic infusion of naloxone reduces degeneration of rat substantia nigral dopaminergic neurons induced by intranigral injection of lipopolysaccharide. J. Pharmacol. Exp. Ther. 295, 125�132

28. Paxinos, G., and Franklin, K. B. J. (2001) The Mouse Brain in Stereotaxic Coordinates, 2nd ed., Academic Press, San Diego, CA

29. Ali, S. F., Newport, G. D., Holson, R. R., Slikker, W., Jr., and Bowyer, J. F. (1994) Low environmental temperatures or pharmacologic agents that produce hypothermia decrease methamphetamine neurotoxicity in mice. Brain Res. 658, 33�38

30. Liu, B., Du, L., and Hong, J. S. (2000a) Naloxone protects rat dopaminergic neurons against inflammatory damage through inhibition of microglia activation and superoxide generation. J. Pharmacol. Exp. Ther. 293, 607�617

31. Gao, H. M., Zhang, W., Hong, J. S., and Liu, B. (2002) Synergistic dopaminergic neurotoxicity of the pesticide rotenone and inflammogen lipopolysaccharide: relevance to the etiology of Parkinson�s disease. J. Neurosci. 23, 1285�1297

32. Tan, A. S., and Berridge, M. V. (2000) Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents. J. Immunol. Methods 238, 59�68

33. Peskin, A. V., and Winterbourn, C. C. (2000) A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1). Clin. Chim. Acta 293, 157�166

34. Liu, Y. X., Qin, L., Wilson, B. C., An, L., Hong, J. S., and Liu, B. (2002) Inhibition by naloxone stereoisomers of beta-amyloid peptide (1-42)-induced superoxide production in microglia and degeneration of cortical and mesencephalic neurons. J. Pharmacol. Exp. Ther. 302, 1212�1219

35. Rosenkranz, A. R., Schmaldienst, S., Stuhlmeier, K. M., Chen, W., Knapp, W., and Zlabinger, G. J. (1992) A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate. J. Immunol. Methods 156, 39�45

36. Sriram, K., Matheson, J. M., Benkovic, S. A., Miller, D. B., Luster, M. I., and O'Callaghan, J. P. (2002) Mice deficient in TNF receptors are protected against dopaminergic neurotoxicity: implications for Parkinson's disease. FASEB J. 16, 1474�1476

37. Chao, C. C., Hu, S., and Peterson, P. K. (1996) Glia: the not so innocent bystanders. J. Neurovirol. 2, 234�239

38. Gayle, D. A., Ling, Z., Tong, C., Landers, T., Lipton, J. W., and Carvey, P. M. (2002) Lipopolysaccharide (LPS)-induced dopamine cell loss in culture: roles of tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide. Brain Res. Dev. Brain Res. 133, 27�35

39. Jenner, P., and Olanow, C. W. (1998) Understanding cell death in Parkinson's disease. Ann. Neurol. 44, S72�S84

40. Bianca, V. D., Dusi, S., Bianchini, E., Dal Pra, I., and Rossi, F. (1999) beta-amyloid activates the O-2 forming NADPH oxidase in microglia, monocytes, and neutrophils: A possible inflammatory mechanism of neuronal damage in Alzheimer's disease. J. Biol. Chem. 274, 15493�15499

41. Choi, D. W. (1987) Dextrorphan and dextromethorphan attenuate glutamate neurotoxicity. Brain Res. 403, 333�336

42. DeCoster, M. A., Klette, K. L., Knight, E. S., and Tortella, F. C. (1995) Sigma receptor-mediated neuroprotection against glutamate toxicity in primary rat neuronal cultures. Brain Res. 671, 45�53

43. Lesage, A. S., De Loore, K. L., Peeters, L., and Leysen, J. E. (1995) Neuroprotective sigma ligands interfere with the glutamate-activated NOS pathway in hippocampal cell culture. Synapse 20, 156�164

44. Berman, F. W., and Murray, T. F. (1996) Characterization of [3H]MK-801 binding to N-methyl-D-aspartate receptors in cultured rat cerebellar granule neurons and involvement in glutamate-mediated toxicity. J. Biochem. Toxicol. 11, 217�226

45. Haberecht, M. F., Mitchell, C. K., Lo, G. J., and Redburn, D. A. (1997) N-methyl-D-aspartate-mediated glutamate toxicity in the developing rabbit retina. J. Neurosci. Res. 47, 416�426

46. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA 87, 1620�1624

47. Miyake, H., and Chiueh, C. C. (1989) Effects of MPP+ on the release of serotonin and 5-hydroxindoleacetic acid from rat striatum in vivo. Eur. J. Pharmacol. 166, 49�55

48. Chiueh, C. C., Johannessen, J. N., Sun, J. L., Bacon, J. P., and Markey, S. P. Reversible neurotoxicity of MPTP in the nigrostriatal dopaminergic system of mice (1986). In MPTP: A Neurotoxin Producing a Parkinsonian Syndrome (Markey, S. P., et al., eds) pp. 473�479 Academic Press, New York

Received September 16, 2003; accepted December 3, 2003.

Table 1 Striatal levels of catecholamine at 6 days after the last MPTP injectiona DA DOPAC HVA/DA PHOX+/+-Saline 1045 ± 46 68 ± 6 0.09 ± 0.03 PHOX+/+-MPTP 159 ± 20b 24 ± 2b 0.33 ± 0.03b PHOX+/+-DM/MPTP 174 ± 17b 28 ± 6b 0.37 ± 0.03b PHOX+/+-DM 1007 ± 22 66 ± 4 0.09 ± 0.02 PHOX–/–-Saline 1044 ± 55 74 ± 3 0.09 ± 0.01 PHOX–/–-MPTP 129 ± 11b 31 ± 2b 0.37 ± 0.02b PHOX–/–-DM/MPTP 149 ± 15b 40 ± 8b 0.37 ± 0.03b PHOX–/–-DM 996 ± 19 64 ± 8 0.10 ± 0.03

aWild-type (PHOX+/+) or NADPH oxidase-deficient (PHOX–/–) mice were injected with vehicle (saline), MPTP, DM, or DM plus MPTP as described in Materials and Methods. Six days after the last MPTP injection, mice were killed and striatal tissues were removed. The levels of catecholamine were determined with HPLC and expressed as ng/100 mg of wet tissue. bP < 0.05 compared with corresponding saline-injected control mice (n=5).

Table 2 Striatal levels of catecholamine at 21 days after the last MPTP injectiona DA DOPAC HVA/DA PHOX+/+ - Saline 895 ± 10 72 ± 11 0.11 ± 0.01 PHOX+/+ - MPTP 182 ± 30b 27 ± 9c 0.24 ± 0.02c PHOX–/– - Saline 882 ± 2 79 ± 12 0.12 ± 0.01 PHOX–/– - MPTP 180 ± 27b 28 ± 6c 0.26 ± 0.02c

aWild-type (PHOX+/+) or NADPH oxidase-null (PHOX–/–) mice received daily injection (s.c.) of saline or 15 mg/kg MPTP for 6 consecutive days. Twenty-one days after the last MPTP injection, mice were killed and striatal tissues were removed. The levels of catecholamine were determined with HPLC and expressed as ng/100 mg of wet tissue. bP < 0.005 compared with corresponding saline-injected control mice (n=8–10). cP < 0.05 compared with corresponding saline-injected control mice (n=8–10).

Fig. 1

Figure 1. Effect of DM on the MPTP-induced loss of SNpc TH-ir neurons. C57BL/6J mice were injected s.c. once daily with vehicle (saline) or MPTP (15 mg free base/kg body weight) for 6 consecutive days. To test the effect of DM, we gave mice twice-daily s.c. injections of saline or DM (10 mg/kg body weight) for the first 6 days, and once-daily thereafter. Mice were killed 6 days after the last MPTP injection, and their brains were removed; coronal brains sections were obtained and immunostained for TH immunoreactivity. Nine to 10 mice were used in each group. The differences were analyzed using a multifactorial ANOVA; a difference of P < 0.05 was considered significant.

Fig. 2

Figure 2. Effect of DM on the MPTP-induced ROS production. Primary mesencephalic neuron-glia cultures were prepared from embryonic E12/13 brain tissues of C57BL/6J mice. Cultures were pretreated for 30 min with vehicle (saline) or DM (1 µM) before treatment with 1 µM (A) or 0.5 µM (B) of MPTP. Two and 4 days after MPTP treatment, DM (1 µM final concentration) was added again to the DM-treated cultures. On day 6, the release of superoxide and the levels of intracellular ROS were determined as described in Materials and Methods. Superoxide production was expressed as a percentage of the vehicle-treated control cultures and as the mean ± SE of 3 experiments performed in triplicate. The levels of intracellular ROS were expressed as arbitrary fluorescent units and are given as the mean ± SE of 3 experiments performed in triplicate. *P < 0.05 compared with the control cultures; +P < 0.05 compared with the MPTP-treated cultures.

Fig. 3

Figure 3. SNpc TH-ir neurons of mice lacking NADPH oxidase activity were less sensitive to the MPTP-induced degeneration. Wild-type (PHOX+/+) or NADPH oxidase-null (PHOX–/–) mice were injected s.c. for 6 consecutive days with saline or 15 mg free base/kg body weight of MPTP. Twenty-one days after the last MPTP injection, mice were killed and their brains were removed. Brain sections were immunostained for TH and SNpc TH-ir neurons were counted as described in Materials and Methods. The number of PHOX+/+ or PHOX–/– mice used for the saline injections was 7, and that for MPTP injections 10. Results are expressed as the average number of TH-ir neurons in each SNpc region.

Fig. 4

Figure 4. Lack of neuroprotective effect of DM in NADPH oxidase-null mice. Wild-type or NADPH oxidase-deficient mice received injections of saline, DM (10 mg/kg body weight), and/or MPTP (15 mg free base/kg body weight) as described in Figure 1. Six days after the last MPTP injection, mice were killed and brain sections were stained for TH-ir neurons. Eight mice were used for each group. The differences were analyzed using a multifactorial ANOVA; a difference of P < 0.05 was considered significant.

Fig. 5

Figure 5. Effect of DM on the MPTP-induced nitrite production. Primary mesencephalic neuron-glia cultures were pretreated for 30 min with vehicle (saline) or DM (1 µM) before treatment with MPTP (1 µM). Two and 4 days after MPTP treatment, DM (1 µM final concentration) was added again to the DM-containing cultures. On day 6, the levels of nitrite in the supernatants were determined. Results are mean ± SE of 3 experiments performed in triplicate. *P < 0.05 compared with the control cultures; +P < 0.05 compared with the MPTP-treated cultures.