The role of TWEAK/Fn14 signaling in the MPTP-model of Parkinson's disease.

The tumor necrosis factor like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor-inducible 14 (Fn14), mediate inflammation and neuronal apoptosis in cerebral edema, ischemic stroke and multiple sclerosis. The downstream effectors and pathways linked to TWEAK-Fn14 signaling are strongly implicated in the pathology of Parkinson's disease (PD), thus indicating a putative role for TWEAK/Fn14 signaling in PD neurodegeneration. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model, we aimed to determine whether genetic ablation or pharmacologic mitigation of the TWEAK protein and its Fn14 receptor affected substantia nigra and striatum Parkinsonian pathology. Changes in endogenous TWEAK protein expression were also quantified in tissue from both MPTP-treated mice and PD human samples. TWEAK protein expression was transiently increased in the striatal tissue but remained unaltered in substantia nigra tissue of MPTP-treated mice. There was also no change of TWEAK protein levels in the substantia nigra or the striatum of human PD patients as compared to matched control subjects. Mitigating the effects of endogenous TWEAK protein using neutralizing antibody did affect MPTP-mediated neurotoxicity in the substantia nigra using the sub-acute model of MPTP (30mg/kg i.p. over five consecutive days). Neither TWEAK nor Fn14 genetic ablation led to attenuation of MPTP-toxicity in the acute model. These findings suggest that TWEAK signaling might be an aspect of MPTP-mediated neuropathology and be involved in the overall neurodegenerative pathology of PD.

The effect of neuronal conditional knock-out of peroxisome proliferator-activated receptors in the MPTP mouse model of Parkinson's disease.

Activation of peroxisome proliferator-activated receptors (PPARs), namely PPARγ and PPARδ, has been shown to provide neuroprotection in a number of neurodegenerative disorders, such as Alzheimer's and Parkinson's disease (PD). The observed neuroprotective effects in experimental models of PD have been linked to anti-oxidant and anti-inflammatory actions. This study aimed to analyze the full influence of these receptors in neuroprotection by generating a nerve cell-specific conditional knock-out of these receptors and subjecting these genetically modified mice to the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin to model dopaminergic degeneration. Mice null for both receptors show the lowest levels of tyrosine hydroxylase (TH)-positive cell bodies following MPTP administration. Presence of one or both these receptors show a trend toward protection against this degeneration, as higher dopaminergic cell immunoreactivity and striatal monoamine levels are evident. These data supplement recent studies that have elected to use agonists of the receptors to regulate immune responses. The results place further importance on the activation of PPARs and the neuroprotective roles these have in inflammatory processes linked to neurodegenerative processes.

[Myeloperoxidase in the neurodegenerative process of Parkinson's disease]. / Neurodegenerativer Prozess bei Morbus Parkinson: Rolle der Myeloperoxidase.

Myeloperoxidase (MPO) is a hemoprotein which is involved in the unspecific immune response. In this process hypochloric acid is released. Hypochloric acid can react with lipids and proteins and thus lead to cell damage. We were able to show that MPO, as well as a biomarker for MPO - 3-Chlorotyrosine - are upregulated in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-model of Parkinson's disease as well as in the disease itself. Ablation of MPO resulted in a neuroprotective effect in the MPTP-model. Herein we describe the different function of MPO, and how these can lead to the cellular demise as seen in Parkinson's disease.

A peroxisome proliferator-activated receptor-δ agonist provides neuroprotection in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease.

Peroxisome proliferator-activated receptor (PPAR)-γ and PPARα have shown neuroprotective effects in models of Parkinson's disease (PD). The role of the third, more ubiquitous isoform PPARδ has not been fully explored. This study investigated the role of PPARδ in PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to model the dopaminergic neurodegeneration of PD. In vitro administration of the PPARδ antagonist GSK0660 (1 µM) increased the detrimental effect of 1-methyl-4-phenylpyridinium iodide (MPP⁺) on cell viability, which was reversed by co-treatment with agonist GW0742 (1 µM). GW0742 alone did not affect MPP⁺ toxicity. PPARδ was expressed in the nucleus of dopaminergic neurons and in astrocytes. Striatal PPARδ levels were increased (over two-fold) immediately after MPTP treatment (30 mg/kg for 5 consecutive days) compared to saline-treated mice. PPARδ heterozygous mice were not protected against MPTP toxicity. Intra-striatal infusion of GW0742 (84 µg/day) reduced the MPTP-induced loss of dopaminergic neurons (5036±195) when compared to vehicle-infused mice (3953±460). These results indicate that agonism of PPARδ provides protection against MPTP toxicity, in agreement with the effects of other PPAR agonists.

Mitochondrial dysfunction triggered by loss of HtrA2 results in the activation of a brain-specific transcriptional stress response.

Cellular stress responses can be activated following functional defects in organelles such as mitochondria and the endoplasmic reticulum. Mitochondrial dysfunction caused by loss of the serine protease HtrA2 leads to a progressive movement disorder in mice and has been linked to parkinsonian neurodegeneration in humans. Here, we demonstrate that loss of HtrA2 results in transcriptional upregulation of nuclear genes characteristic of the integrated stress response, including the transcription factor CHOP, selectively in the brain. We also show that loss of HtrA2 results in the accumulation of unfolded proteins in the mitochondria, defective mitochondrial respiration and enhanced production of reactive oxygen species that contribute to the induction of CHOP expression and to neuronal cell death. CHOP expression is also significantly increased in Parkinson's disease patients' brain tissue. We therefore propose that this brain-specific transcriptional response to stress may be important in the advance of neurodegenerative diseases.

Overexpression of human wildtype torsinA and human DeltaGAG torsinA in a transgenic mouse model causes phenotypic abnormalities.

Primary torsion dystonia is an autosomal-dominant inherited movement disorder. Most cases are caused by an in-frame deletion (GAG) of the DYT1 gene encoding torsinA. Reduced penetrance and phenotypic variability suggest that alteration of torsinA amino acid sequence is necessary but not sufficient for development of clinical symptoms and that additional factors must contribute to the factual manifestation of the disease. We generated 4 independent transgenic mouse lines, two overexpressing human mutant torsinA and two overexpressing human wildtype torsinA using a strong murine prion protein promoter. Our data provide for the first time in vivo evidence that not only mutant torsinA is detrimental to neuronal cells but that also wildtype torsinA can lead to neuronal dysfunction when overexpressed at high levels. This hypothesis is supported by (i) neuropathological findings, (ii) neurochemistry, (iii) behavioral abnormalities and (iv) DTI-MRI analysis.

COX-2 and neurodegeneration in Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Recent observations link cyclooxygenase type-2 (COX-2) to the progression of the disease. Consistent with this notion, studies with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) show that inhibition and ablation of COX-2 markedly reduce the deleterious effects of this toxin on the nigrostriatal pathway. The similarity between this experimental model and PD strongly supports the possibility that COX-2 expression is also pathogenic in PD.

Increased levels of the pro-inflammatory prostaglandin PGE2 in CSF from ALS patients.

Levels of the potent pro-inflammatory prostaglandin E(2) (PGE(2)) are elevated in postmortem spinal cords from patients with ALS, and inhibition of a key PGE(2)-synthesizing enzyme, cylcooxygenase-2, is neuroprotective in an in vitro model of ALS. The authors report that 82% of the patients with ALS studied had 2 to 10 times higher PGE(2) levels in CSF compared with normal control subjects. That affected areas of the CNS are inflamed in ALS supports this. CSF PGE(2) measurement may be useful in monitoring treatment for ALS.

Dopamine mediates striatal malonate toxicity via dopamine transporter-dependent generation of reactive oxygen species and D2 but not D1 receptor activation.

Intrastriatal injection of the reversible succinate dehydrogenase inhibitor malonate results in both chemically induced hypoxia and striatal lesions that are similar to those seen in Huntington's disease and cerebral ischaemia. The mechanisms leading to neuronal death involve secondary excitotoxicity, the release of dopamine from nigrostriatal fibres and the generation of reactive oxygen species (ROS) including nitric oxide (NO) and hydroxyl radicals. Here, we further investigated the contribution and mechanism of dopamine on malonate-induced striatal lesions. Prior lesions of the nigrostriatal pathway with 6-OHDA or the depletion of striatal dopamine stores by pretreatment with reserpine, an inhibitor or the vesicular monoamine transporter type-2 (VMAT2), in combination with alpha-methyl-p-tyrosine resulted in a significant reduction of malonate-induced striatal lesion volumes. This was paralleled by block or reduction of the malonate-induced generation of ROS, as measured by the conversions of salicylate to 2,3-dihydroxybenzoic acid (2,3-DHBA) using microdialysis. Systemic or intrastriatal application of L-DOPA or dopamine, respectively, reconstituted malonate toxicity and the generation of ROS in 6-OHDA-lesioned rats. Block of the dopamine transporter by GBR12909 did not result in a reduction of malonate-induced dopamine release, but significantly reduced the generation of hydroxyl radicals. The D2 receptor agonist lisuride and the mixed D1 and D2 receptor agonist apomorphine, but not the D1 receptor agonist SKF38393, partially restored malonate toxicity in 6-OHDA-lesioned rats without increasing the generation of ROS. In line with these results sulpiride, an inhibitor of D2 receptors, reduced the malonate-induced lesion volume, whereas SCH23390, an inhbitor of D1 receptors, was ineffective. Our data suggest that malonate-induced dopamine toxicity to energetically impaired neurons is mediated by two independent pathways: (i) dopamine transporter uptake-dependent, dopamine receptor-independent generation of ROS, and (ii) excessive stimulation of D2 receptors.

Comparison of two independent aromatic hydroxylation assays in combination with intracerebral microdialysis to determine hydroxyl free radicals.

The phenylalanine- and salicylate assay were compared to investigate the production of hydroxyl free radicals. In vitro experiment: Phenylalanine (100 micromol/l) or salicylic acid (100 micropmol/l) were incubated in a hydroxyl radical generating in vitro Fenton system with increasing concentrations (1.25--40 micromol/l) of equimolar hydrogen peroxide and ferrous ions. Both, phenylalanine and salicylic acid were able to trap hydroxyl radicals in a reliable way indicated by the linear relationship between the concentration of the Fenton reagents and either the phenylalanine derived products (ortho-, meta-, para-tyrosine) or the salicylic acid-derived products (2,3- and 2,5-dihydroxybenzoic acid (DHBA)). In vivo experiment: Wistar rats were implanted with microdialysis probes and striatal perfusion with either 5 mmol/l phenylalanine or 5 mmol/l salicylic acid was performed. Addition of the dopaminergic neurotoxin 6-hydroxydopamine (100 micromol/l, flow rate 2 microl/min, 60 min) to the perfusion fluid significantly increased the concentrations of ortho- and meta-tyrosine or 2,3-DHBA in comparison to control animals. All increases determined were rapidly reversible after changing back to pre-stimulation conditions. The results demonstrate that aromatic hydroxylation of phenylalanine or salicylic acid is a useful technique to investigate hydroxyl free radical formation in vitro and in vivo. Advantages and disadvantages of both methods are discussed.

The role of glial cells in Parkinson's disease.

Parkinson's disease is a common neurodegenerative disorder characterized by the progressive loss of the dopaminergic neurons in the substantia nigra pars compacta. The loss of these neurons is associated with a glial response composed mainly of activated microglial cells and, to a lesser extent, of reactive astrocytes. This glial response may be the source of trophic factors and can protect against reactive oxygen species and glutamate. Aside from these beneficial effects, the glial response can mediate a variety of deleterious events related to the production of reactive species, and pro-inflammatory prostaglandin and cytokines. This article reviews the potential protective and deleterious effects of glial cells in the substantia nigra pars compacta of Parkinson's disease.

Nuclear factor-kappaB activation is not involved in a MPTP model of Parkinson's disease.

In the present study the involvement of hydroxyl free radicals and nuclear factor-kappaB (NF-kappaB) activation was investigated in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease. MPTP (30 mg/kg, s.c.) produced a significant 2-fold increase in hydroxyl free radicals in the striatum of C57BL/6 mice determined by microdialysis in combination with the salicylate hydroxylation assay. Electrophoretic mobility shift assays did not detect NF-kappaB activation after MPTP treatment. Furthermore, p50-deficient mice showed only minor differences in striatal dopamine and metabolite levels as well as tyrosine hydroxylase immunoreactivity after MPTP administration in comparison to wildtype mice. We postulate that, although hydroxyl radical production was enhanced, NF-kappaB plays only a minor role in the MPTP model because neither neurochemical nor immunocytochemical parameters were altered in p50-deficient mice in comparison to controls.

Inhibition of the cyclooxygenase isoenzymes COX-1 and COX-2 provide neuroprotection in the MPTP-mouse model of Parkinson's disease.

To study the possible role of the isoenzymes of cyclooxygenase COX-1 and COX-2 in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson's disease we used acetylsalicylic acid, a COX-1/COX-2 inhibitor, in comparison with meloxicam, a preferential COX-2 inhibitor. As markers of protection we determined the effects on MPTP-induced striatal dopamine depletion, locomotor activity, cell loss, and tyrosine hydroxylase immunoreactivity (TH-IR) in the substantia nigra pars compacta. Male C57BL/6 mice (n = 82) were treated with a single dose of acetylsalicylic acid (10, 50, 100 mg/kg i.p.) or meloxicam (2, 7.5, 50 mg/kg i.p.) immediately prior to administration of MPTP (30 mg/kg s.c.) or saline. After 7 days the mice were sacrificed to analyze striatal dopamine and metabolite levels. Nigral sections were processed for Nissl-staining and TH-IR. In the saline-treated MPTP control group striatal dopamine levels were reduced to 15.9% of control values. Dopamine depletion was significantly attenuated to values of 37.1 and 38.6% of saline control values by acetylsalicylic acid (50 and 100 mg/kg) and to values of 36 and 40% by meloxicam (7.5 and 50 mg/kg), respectively. MPTP-induced decrease of locomotor activity was significantly attenuated by acetylsalicylic acid and meloxicam. Remarkably, the MPTP-induced decrease of TH-IR as well as the loss of nigral neurons was nearly completely prevented by acetylsalicylic acid (100 mg/kg) and meloxicam (7.5 and 50 mg/kg). In conclusion, the inhibition of either COX-1/COX-2 by acetylsalicylic acid or preferentially COX-2 by meloxicam provided a clear neuroprotection against MPTP-toxicity on the striatal and nigral levels.

Increased expression of the pro-inflammatory enzyme cyclooxygenase-2 in amyotrophic lateral sclerosis.

Mutations in the copper/zinc superoxide dismutase (mSOD1) gene are associated with a familial form of amyotrophic lateral sclerosis (ALS), and their expression in transgenic mice produces an ALS-like syndrome. Recent observations suggest a role for inflammatory-related events in the progression and propagation of the neurodegenerative process in ALS. Consistent with this view, the present study demonstrates that, during the course of the disease, the expression of cyclooxygenase type 2 (Cox-2), a key enzyme in the synthesis of prostanoids, which are potent mediators of inflammation, is dramatically increased. In both early symptomatic and end-stage transgenic mSOD1 mice, neurons and, to a lesser extent, glial cells in the anterior horn of the spinal cord exhibit robust Cox-2 immunoreactivity. Cox-2 mRNA and protein levels and catalytic activity are also significantly increased in the spinal cord of the transgenic mSOD1 mice. The time course of the spinal cord Cox-2 upregulation parallels that of motor neuronal loss in transgenic mSOD1 mice. We also show that Cox-2 activity is dramatically increased in postmortem spinal cord samples from sporadic ALS patients. We speculate that Cox-2 upregulation, through its pivotal role in inflammation, is instrumental in the ALS neurodegenerative process and that Cox-2 inhibition may be a valuable therapeutic avenue for the treatment of ALS.

The dopamine agonist pramipexole scavenges hydroxyl free radicals induced by striatal application of 6-hydroxydopamine in rats: an in vivo microdialysis study.

Hydroxyl free radical production seems to play an important role in the pathogenesis of Parkinson's disease. In the present study, we investigated the dopamine agonists pramipexole and pergolide as well as the nitrone compound S-PBN (N-tert-butyl-alpha-(2-sulfophenyl)nitrone) to reduce hydroxyl radical formation. Microdialysis experiments were carried out in non-anaesthetized Wistar rats. Salicylate was incorporated into the perfusion fluid to measure indirectly hydroxyl radicals indicated by 2,3-dihydroxybenzoic acid (2,3-DHBA). Local perfusion with 0.2 or 2 nmol/2 microl/min 6-hydroxydopamine (6-OHDA) via the microdialysis probe significantly increased 2,3-DHBA levels 14-fold and 47-fold, respectively. Systemic application of either pergolide (0.05 mg/kg) or pramipexole (1 mg/kg) failed to significantly reduce 6-OHDA-induced hydroxyl radical production. In contrast, a 40 min pretreatment with pramipexole (2 and 10 nmol/2 microl/min via the probe) before onset of 6-OHDA perfusion, significantly attenuated 2, 3-DHBA levels compared with vehicle controls. S-PBN pretreatment (2 nmol/2 microl/min) was not effective to reduce 2,3-DHBA levels. In conclusion, pramipexole was able to reduce hydroxyl radical levels induced by 6-OHDA in vivo after local application. This property of pramipexole may be beneficial under conditions of enhanced hydroxyl radical formation in parkinsonian brains and may add to its well known dopamine D(2)-like receptor agonistic effects.

Effects of ensaculin on dopamine metabolite levels and K(+)-induced glutamate release.

In vivo microdialysis with the new antidementia compound ensaculin was performed in freely moving rats to study the alterations in dopaminergic and glutamatergic neurotransmission. Ensaculin (0.1 and 1 mg/kg i.p.) significantly increased extracellular levels of the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Furthermore, ensaculin (1 mg/kg i.p.) showed a non-significant tendency to reduce the K(+)-induced glutamate release. The data suggest that ensaculin may have moderate D(2) antagonistic properties. Thus, besides its possible role as a cognitive enhancer, ensaculin may also have moderate antipsychotic properties.

In vivo effects of the putative cognitive enhancer KA-672.HCl in comparison with 8-hydroxy-2-(di-N-propylamino) tetralin and haloperidol on dopamine, 3,4-dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid levels in striatal and cortical brain regions.

1. KA-672.HCl (7-methoxy-6-[3-[4-(2-methoxyphenyl)piperazin-1-yl]propoxy]-3,4-di methyl-2H-1-benzopyran-2-one hydrochloride), designed as a cognitive enhancer, has been investigated through behavioural and binding studies. However, little is known about its biochemical effects on the dopaminergic and serotoninergic system in vivo. 2. In the present study the authors investigated the effects of KA-672.HCl (0.1 mg/kg and 1 mg/kg), 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) (1 mg/kg), haloperidol (0.1 mg/kg) and a mixture of haloperidol and 8-OH-DPAT on dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels, in striatum and cerebral cortex of rats. 3. Male Wistar rats received an intraperitoneal injection of the drugs or vehicle 1 hour before striatal and cortical brain tissues were dissected out for neurochemical analysis. 4. KA-672.HCl, 8-OH-DPAT and haloperidol significantly reduced striatal DA levels, whereas only KA-672.HCl significantly reduced cortical DA levels. 8-OH-DPAT and haloperidol induced a significant increase in cortical DOPAC levels but only haloperidol significantly elevated the striatal DOPAC content. In contrast, only the higher dose of KA-672.HCl elevated striatal DOPAC levels. Furthermore, KA-672.HCl significantly reduced striatal 5-HT levels and slightly elevated striatal 5-HIAA concentrations. 8-OH-DPAT significantly decreased striatal 5-HIAA levels. All substances were able to enhance the cortical and striatal DA turnover. 5. The cortical and striatal 5-HT turnover was significantly decreased following 8-OH-DPAT treatment and significantly increased in the striatum after haloperidol and KA-672.HCl treatment. 6. The data suggest that KA-672.HCl possesses D2 antagonistic as well as 5-HT1A agonistic properties. However, additional mechanisms of actions by interaction with other neurotransmitter systems such as acetylcholine, excitatory or inhibitory amino acids need to be determined.

The salicylate hydroxylation assay to measure hydroxyl free radicals induced by local application of glutamate in vivo or induced by the Fenton reaction in vitro.

The direct measurement of hydroxyl radicals in vivo is extremely difficult. Therefore, the indirect determination of hydroxyl radicals using salicylate (2-hydroxybenzoate) is widely accepted. Reverse microdialysis with glutamate led to a dose-dependent production of hydroxyl free radicals indicated by the hydroxylation adduct of salicylate, namely 2,3-dihydroxybenzoic acid. The local stimulation of hydroxyl free radical formation seems to be suitable to investigate a radical-scavenging property of potential neuroprotective drugs. In vitro experiments using the Fenton reaction may be a helpful tool to assess whether or not a substance is able to act as a radical scavenger in a cell free environment, which is easy to handle and a simple screening method before in vivo experiments were performed. In the present study we present an in vivo approach using local application of glutamate into the striatum and an in vitro screening using the Fenton reaction to induce hydroxyl radical formation. The main goal is to reliable measure hydroxyl free radicals, which are the most reactive oxygen radicals in biology and medicine.

Comparison of the novel drug Ensaculin with MK-801 on the reduction of hydroxyl radical production in rat striatum after local application of glutamate.

Ensaculin interacts with various neurotransmitter systems (e.g., dopaminergic, serotoninergic, glutamatergic) and was originally designed for the treatment of dementia. In the present study Ensaculin was tested for its possible reduction of glutamate-induced hydroxyl free radical formation in vivo. The microdialysis experiment was carried out in non-anaesthetized Wistar rats, which were implanted with a microdialysis probe into the striatum. Salicylate (10 nmol/2 microl/min) was incorporated into the perfusion fluid to measure indirectly hydroxyl radicals indicated by 2,3-dihydroxybenzoic acid (DHBA) formation. After baseline recording, glutamate (100 or 500 nmol/2 microl/min) was perfused through the microdialysis probe (CMA 12, 4 mm, flow rate 2 microl/min). Ensaculin (0.1, 1 and 10 mg/kg), MK-801 (1 mg/kg) or saline was injected i.p. 20 min after the onset of glutamate perfusion (500 nmol/2 microl/min). Glutamate (100 nmol/2 microl/min) and (500 nmol/2 microl/min) perfusion produced a 2.6- and 17-fold increase of 2,3-DHBA, respectively. Treatment with Ensaculin (1 and 10 mg/kg i.p. ) significantly antagonized the formation of 2,3-DHBA, to values of 60.5% and 56.7% of control levels, respectively. In comparison, MK-801 attenuated 2,3-DHBA levels, to values of 65.8% compared to control values. Ensaculin may be useful in the treatment of neurodegenerative disorders associated with elevated hydroxyl free radicals and excitotoxicity.

The protective effects of PBN against MPTP toxicity are independent of hydroxyl radical trapping.

To study the mechanism of the protective effect of the spin-trapping agent alpha-phenyl-N-tert-butyl nitrone (PBN) against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) toxicity hydroxyl radicals and functional parameters of neuroprotection were determined. C57BL/6 mice received PBN (100 mg/kg IP) over a time period of 15 days and on day 8 MPTP (40 mg/kg SC). On day 15 striatal levels of dopamine, serotonin, and metabolites were analyzed. For radical determination mice received a single injection of salicylic acid (SA) (100 mg/kg IP) in the time period of 0.5 h before to 72 h after MPTP injection. In vivo maximum hydroxyl radical levels indicated by 2,3-dihydroxybenzoic acid/SA ratios were obtained 4 h after MPTP injection, and were not affected by PBN treatment. However, the MPTP-induced mortality, reduction of locomotor activity, continuous loss of body weight, and striatal dopamine depletion were significantly less pronounced in PBN-treated animals. These results elucidate the time course of hydroxyl free radical formation in MPTP toxicity. PBN improved the functional parameters of neuroprotection against MPTP toxicity, but there is no evidence for hydroxyl radical scavenging properties to this effect.