Abbreviations used: DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid; iNOS and nNOS, inducible and neuronal nitric oxide synthase, respectively; NO[UNK], nitric oxide; NOS, nitric oxide synthase; PBS, phosphate-buffered saline; PD, Parkinson's disease; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase.
Address correspondence and reprint requests to Dr. J. B. Schulz at Department of Neurology, Hoppe-Seyler-Strasse 3, 72076 Tübingen, Germany. E-mail: firstname.lastname@example.org
Abstract: MPTP produces clinical, biochemical, and neuropathologic changes reminiscent of those that occur in idiopathic Parkinson's disease (PD). In the present study we show that MPTP treatment led to activation of microglia in the substantia nigra pars compacta (SNpc), which was associated and colocalized with an increase in inducible nitric oxide synthase (iNOS) expression. In iNOS-deficient mice the increase of iNOS expression but not the activation of microglia was blocked. Dopaminergic SNpc neurons of iNOS-deficient mice were almost completely protected from MPTP toxicity in a chronic paradigm of MPTP toxicity. Because the MPTP-induced decrease in striatal concentrations of dopamine and its metabolites did not differ between iNOS-deficient mice and their wild-type littermates, this protection was not associated with a preservation of nigrostriatal terminals. Our results suggest that iNOS-derived nitric oxide produced in microglia plays an important role in the death of dopaminergic neurons but that other mechanisms contribute to the loss of dopaminergic terminals in MPTP neurotoxicity. We conclude that inhibition of iNOS may be a promising target for the treatment of PD.
Pathologically, the hallmark of idiopathic Parkinson's disease (PD) is loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), leading to the major clinical and pharmacological abnormalities that characterize the disease. The cause of neuronal loss in the substantia nigra is not known. However, recent advances have been made in defining morphological and biochemical events in the pathogenesis of the disease. Inhibition of oxidative phosphorylation, excitotoxicity, and generation of reactive oxygen species are considered important mediators of neuronal death in PD (Beal, 1995).
Insights into the pathogenesis of PD have been achieved experimentally by using the neurotoxin MPTP. MPTP produces irreversible clinical, biochemical, and neuropathological effects that closely mimic those observed in idiopathic PD (Bloem et al., 1990). This meperidine analogue is metabolized to MPP+ by the enzyme monoamine oxidase B. MPP+ is subsequently selectively taken up by dopaminergic terminals and concentrated in neuronal mitochondria in the substantia nigra. MPP+ binds to and inhibits complex I of the electron transport chain (Tipton and Singer, 1993), thereby producing the same biochemical defect as detected in SNpc of PD patients.
We and others have previously shown that the toxicity of MPTP may be mediated by nitric oxide (NO[UNK]) (Schulz et al., 1995b; Hantraye et al., 1996; Przedborski et al., 1996; Matthews et al., 1997). Three isoforms of NO[UNK] synthase (NOS) have been identified so far: constitutive neuronal (nNOS; NOS1) and endothelial (eNOS, NOS3) isoforms and an inducible isoform (iNOS; NOS2) originally isolated from macrophages. Pharmacological inhibition of NOS in MPTP studies was achieved by 7-nitroindazole and S-methylthiocitrulline, which are considered to be rather selective inhibitors of nNOS. As 7-nitroindazole provides complete but nNOS-deficient mice only partial protection, its selectivity was questioned for the dose used in these studies. In fact, it was shown that, in addition to nNOS, 7-nitroindazole may inhibit monoamine oxidase B (Di Monte et al., 1997) and, at least in vitro, may inhibit iNOS as well (Moore et al., 1993). Based on these findings and as iNOS expression in glia cells of SNpc has already been suggested to play a role in the pathogenesis of PD (Hunot et al., 1996, 1999; Hirsch et al., 1998), we studied whether (a) MPTP treatment leads to the induction of microglia and iNOS expression in the striatum and substantia nigra of mice and (b) MPTP toxicity is attenuated in iNOS-deficient mice.
MATERIALS AND METHODS
Eight-week-old male iNOS-deficient mice (Laubach et al., 1995) (C57/bl-NOS2tm 1 Lau; Jackson Laboratories, Bar Harbor, ME, U.S.A.) and their wild-type littermates were treated with either normal saline or MPTP hydrochloride (Research Biochemicals, Cologne, Germany). MPTP was administered in 0.1 ml of phosphate-buffered saline (PBS) at a dose of 30 mg/kg i.p. at 24-h intervals for five doses (Tatton and Kish, 1997). Ten animals were used in each group. Animals were killed 1 week after the last MPTP injection. The two striata were rapidly dissected, rapidly frozen, and stored at -80°C until analysis. On the day of the assay, tissue samples were sonicated in 20 μl of 0.1 M perchloric acid/mg of striatal tissue. After centrifugation (15,000 g, 10 min, 4°C), 20 μl of supernatant was injected onto a C18 reverse-phase HR-80 catecholamine column (ESA, Bedford, MA, U.S.A.). Dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were quantified by HPLC with electrochemical detection. The mobile phase (pH = 2.9) consisted of 90% 75 mM sodium phosphate, 275 mg/L octane sulfonic acid solution, and 10% methanol. Flow rate was 1 ml/min. Peaks were detected by an ESA Coulochem II with a model 5010 detector (E1 = 50 mV, E2 = 400 mV). Data were collected and processed using the Chromeleon computer system (Gynkotek, Gemering, Germany).
Tyrosine hydroxylase (TH) immunohistochemistry was performed on 10-μm paraffin-embedded frontal brain sections. After dewaxing (3 × 10 min xylol, 2 × 5 min 100%, 2 × 5 min 96%, and 2 × 5 min 70% isopropanol) slices were washed in PBS (3 × 5 min) and treated with normal goat serum (10% in PBS with 0.3% Triton X-100) for 10 min. Afterward slices were incubated at 4°C overnight with primary antibodies diluted in PBS (containing 0.3% Triton X-100 and 1% normal goat serum). For detection of TH immunoreactivity a monoclonal antibody (dilution 1:1,000; Diasorin, Stillwater, MN, U.S.A.) was used. Activated microglia was identified by isolectin-B4 staining (BSI-B4, fluorescein isothiocyanate-labeled, dilution 1:20; Sigma, Deisenhofen, Germany); iNOS was stained by a polyclonal antibody (anti-mouse iNOS rabbit polyclonal antibody, dilution 1:500; Upstate Biotechnology, Lake Placid, NY, U.S.A.). After washing with PBS (3 × 5 min) the sections were incubated at room temperature for 2 h with respective secondary antibodies: goat anti-rabbit IgG, carbocyanine 3-labeled, and goat anti-mouse IgG, carbocyanine 2-labeled (Biotrend, Cologne; 1:200). Sections were washed, mounted on coverslips, and then analyzed by confocal laser scanning microscopy (LSM 510; Carl Zeiss, Jena, Germany). The specificity of secondary antibodies was tested by omitting the primary antisera and monoclonal antibodies.
SNpc neuronal counts were performed manually by workers blinded to the treatment schedule and the genetic phenotype of the mice. Nucleated, process-bearing, TH-positive SNpc cells were bilaterally counted on at least three TH-immunostained mesencephalic sections at the widest dimension of the SNpc at AP -3.16 (Franklin and Paxinos, 1996) lateral to the roots of the third cranial nerve separating medial and lateral SNpc using a confocal microscope (LSM 510; Zeiss). To rule out a possible change of SNpc volume as an influencing factor, the results are expressed as a ratio of TH-positive cells per area of SNpc.
MPTP treatment induced a robust increase in the number of activated microglia in the striatum and the SNpc as detected by BSI-B4 isolectin histochemistry at 12 h after the last (fifth) MPTP injection (Fig. 1A-C). Several but not all BSI-B4-positive cells in the striatum and in the SNpc stained positive for iNOS (Fig. 1D). As all iNOS-positive cells showed colocalization with BSI-B4-positive cells (Fig. 1E) but not with markers for neurons (NeuN; data not shown) or astrocytes (glial fibrillary acidic protein; data not shown), iNOS expression appears to be restricted to activated microglia. We characterized the specificity of the iNOS antibody by western blot of lysates from tumor necrosis factor-α- and lipopolysaccharide-treated differentiated PC12 cells, as shown earlier (Heneka et al., 1998). Although the glial response after MPTP treatment was not altered in iNOS-deficient mice (Fig. 1B), the induction of iNOS was not detectable in iNOS-deficient mice compared with littermates.
Using iNOS-deficient mice we investigated the role of iNOS expression on MPTP toxicity. TH-positive cells in SNpc were almost completely protected against MPTP toxicity in iNOS-deficient mice compared with littermate controls (Figs. 2 and 3). MPTP treatment of littermate controls led to 39% survival of TH-positive cells compared with vehicle-treated mice, whereas survival was increased to 96% in iNOS-deficient mice. However, the extent of MPTP-induced decrease of striatal dopamine, DOPAC, or HVA concentrations was not significantly different between iNOS-deficient mice and their littermates (Table 1).
Table 1. Striatal concentrations of catecholamines
iNOS-deficient mice (iNOS-/-) are not protected from MPTP-induced decrease of striatal catecholamine concentrations at 7 days after the last MPTP injection. Data are mean ± SEM values, in pmol/mg of tissue.
ANOVA followed by Tukey's post hoc test: ap < 0.01 versus saline-treated wild-type mice; bp < 0.001, cp < 0.01, dp < 0.05 versus saline-treated iNOS-/- mice.
Increasing evidence suggests that free radical-induced oxidative stress is involved in the mechanism of nerve cell death in PD. Among several toxic oxidative species, a major role has been proposed for NO[UNK] (Schulz et al., 1995a). Based on reports of increased density of microglia (Banati et al., 1998) expressing iNOS in the SNpc of PD patients (Hunot et al., 1996), we investigated the role of iNOS in MPTP toxicity. We show here that MPTP treatment of mice leads to a robust increase of activated microglia and iNOS expression in SNpc. Although microglia may have beneficial effects under certain circumstances (Gonzalez-Scarano and Baltuch, 1999), our data strongly indicate that iNOS expression in microglia contributes to cell death of dopaminergic neurons in SNpc and is neither beneficial nor an epiphenomenon. We show that in iNOS-deficient mice MPTP-induced cell loss of dopaminergic SNpc neurons is almost completely prevented, although the microglia activation still occurs.
NO[UNK] is only a weak oxidant and may not directly lead to deleterious effects on cells. However, it may react with superoxide to form peroxynitrite. 3-Nitrotyrosine, a marker for peroxynitrite-induced damage, is increased in MPTP toxicity (Schulz et al., 1995b; Pennathur et al., 1999) and in PD brains (Good et al., 1998). Once formed, peroxynitrite can diffuse over several cell diameters where it can oxidize lipids, proteins, and DNA. It also can produce nitronium ions, which then nitrate tyrosine residues (Schulz et al., 1995a).
It is surprising that the protective effects did not extend to the dopaminergic terminal markers in the striatum. The concentrations of dopamine and its metabolites were similarly decreased in wild-type animals and iNOS-deficient mice. In contrast, treatment with 7-nitroindazole or S-methylthiocitrulline, inhibitors of nNOS, or deficiency of nNOS protects against the decrease of dopaminergic terminal markers and against loss of dopaminergic neurons in SNpc (Schulz et al., 1995b; Hantraye et al., 1996; Przedborski et al., 1996; Matthews et al., 1997). The striatum contains a rich density of nNOS-positive neurons and fibers (Bredt et al., 1991). Therefore, dopamine nerve terminals may be the primary target of nNOS-mediated MPTP neurotoxicity followed by a slower and secondary death of the SNpc dopamine cell bodies mediated in part by NO[UNK] derived from activated microglia expressing iNOS. The block of glia activation in the SNpc after treatment with 7-nitroindazole is consistent with this hypothesis (Hantraye et al., 1996). As nNOS expression also occurs in melanized neurons of SNpc (Hunot et al., 1996), inhibitors of nNOS may also act at the level of dopaminergic soma in SNpc in addition to its protective effects on nerve terminals in the striatum.
While this article was in preparation Liberatore et al. (1999) reported very similar results, confirming a protection of dopaminergic SNpc cells in iNOS-deficient mice without preservation of catecholamine concentrations in the striatum. They also showed that the conversion of MPTP to its active metabolite MPP+ in the striatum was not altered in iNOS-deficient mice, ruling out that reduced striatal MPP+ concentrations were the reason for the protective effects observed. In their study the degree of protection against dopaminergic cell death in the SNpc was 50%, whereas we observed ∼90% protection. This discrepancy is likely due to a different MPTP treatment paradigm: These authors treated acutely with four doses of 20 mg/kg MPTP at 2-h intervals, whereas we chose a more chronic paradigm with five doses of 30 mg/kg MPTP at 24-h intervals, in which the contribution of the inflammatory response to dopaminergic cell death may be more substantial. Further support for a slowly progressive degeneration of dopaminergic SNpc neurons after MPTP treatment comes from a recent neuropathological study of patients who developed parkinsonism after MPTP poisoning (Langston et al., 1999). This study revealed active ongoing degeneration and inflammation in SNpc at 3-16 years after MPTP exposure.
Our results further support the pathophysiological significance of NO[UNK] in MPTP neurotoxicity. They provide evidence that NO[UNK] derived from iNOS-expressing activated microglia contributes to the death of dopaminergic neurons in MPTP toxicity and in PD. Treatment strategies that block the inflammatory response or iNOS activity may halt the progression of PD. However, as terminal markers are not preserved—at least in this mouse model of MPTP toxicity—inhibition of iNOS may not result in functional benefit. A combination with a therapy that leads to outgrowth of fibers, e.g., growth factors, may overcome this disadvantage. Furthermore, a combination of nNOS and iNOS inhibitors may provide additive effects.