Effect of human interleukin-10 on the expressionof nitric oxide synthases in the MPTP-basedmodel of Parkinson’s disease

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Effect of human interleukin-10 on the expression of nitric oxide synthases in the MPTP-based

model of Parkinson’s disease

Joanna Schwenkgrub¹, Ilona Joniec-Maciejak¹, Anna Sznejder-Pacho³ek¹, Adriana Wawer¹, Agnieszka Ciesielska³, Krzysztof Bankiewicz³,

Anna Cz³onkowska^1,2, Andrzej Cz³onkowski¹

1Department of Experimental and Clinical Pharmacology, Medical University of Warsaw, Krakowskie Przedmieœcie 26/28, PL 00-927, Warszawa, Poland

2Second Department of Neurology, Institute of Psychiatry and Neurology, Sobieskiego 1/9, PL 02-957 Warszawa, Poland

3Department of Neurosurgery, University of California at San Francisco, 1855 Folsom Street, San Francisco, CA 94103-0555, USA

Correspondence: Ilona Joniec-Maciejak, e-mail: ilona.joniec@wum.edu.pl

Abstract:

Background: Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder. The degeneration of the nigro-striatal pathway has been linked with the inflammatory process accompanied by the robust up-regulation of the nitric oxide synthase (NOS) and production of the neurotoxic level of nitric oxide (NO). One of the therapeutic strategies of PD is based on the reduction of the detrimental neuroinflammatory markers in the lesioned nigro-striatal pathway.

In this study we have investigated the neuroprotective effect of the cerebral infusion of recombinant adeno-associated viral vector, expressing the gene for human interleukin-10 (AAV2-hIL-10) in a mouse model of PD induced by 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). It is known that IL-10 is a potent anti-inflammatory cytokine that limits the inducible nitric oxide synthase (iNOS) gene expression.

Methods: The striatal iNOS, neuronal nitric oxide synthase (nNOS) and tyrosine hydroxylase (TH) protein expression was evalu- ated by immunoblot analysis.

Results: The intracerebral injection of the AAV2-hIL-10, before the lesion, induced the upregulation of the striatal TH protein, depleted by MPTP intoxication. This AAV2-hIL-10-induced increase of TH level was associated with the suppression of iNOS protein expression in the lesioned striatum.

Conclusion: The results revealed protective properties of AAV2-hIL-10.

Key words:

Parkinson’s disease, MPTP, nitric oxide synthase, nNOS, iNOS, viral vector, IL-10

Abbreviations: AAV2-hIL-10 – adeno-associated viral type-2 vector containing the complementary DNA for human interleukin-10, DA – dopamine, IL-10 – interleukin-10, MPTP – 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, iNOS – in-

ducible nitric oxide synthase, NF-kB – nuclear factor-kB, nNOS – neuronal nitric oxide synthase, NO – nitric oxide, ONOO– peroxynitrite, PD – Parkinson’s disease, SNpc – substantia nigra pars compacta, TH – tyrosine hydroxylase

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Introduction

Parkinson’s disease (PD) is a progressive degenerative disorder, for which the etiology and pathogenesis re- mains unknown. Its symptomatic phase begins after a massive loss of up to 50% of the dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) and an 80% depletion in striatal DA [15, 27]. PD af- fects about 1.5% of the population [16] and is charac- terized by slowness of movement, rigidity and tremors.

It is postulated that nitric oxide (NO) is involved in the neuronal degeneration process in the pathogenesis of PD [25, 28]. NO is a free radical synthesized in the conversion of L-arginine to L-citrulline, catalyzed by nitric oxide synthases (NOSs). There are three isoforms of NOS: – neuronal (nNOS) – constitutively expressed in nerve cells, skeletal muscle and heart muscle; – endothelial (eNOS) – constitutively expressed in endothelial cells; – inducible (iNOS) – regulated tran- scriptionally and induced in glia, macrophages and neurons by inflammation [13, 17, 21].

The physiological NO levels (putatively nM) are produced by eNOS or nNOS. By contrast, during the neuroinflammatory process caused by pathogens, in- jury or hypoxia, iNOS is induced by pro-inflam- matory cytokines (TNF-a, IL-1b, and IFN-g) in acti- vated microglia or astrocytes [25, 29] and, once expressed, produces high, toxic level of NO (putatively µM) [14, 20, 21]. Under pathological conditions, NO can easily react with superoxide (O₂) to form peroxynitrite (ONOO), which is a strong oxidant. This compound is responsible for the nitration of proteins, predominantly on tyrosine residues, thiols and lipid oxidation, as well as DNA strand breaks and the inhibition of the respiratory chain [8, 9, 19].

There is a cross-talk between nNOS and iNOS. In- ducible NOS expression is under the transcriptional control of nuclear factor-kB (NF-kB), which is inhib- ited by physiological levels of NO. An arachidonic acid-dependent inhibition of constitutive NOS activity at the early phases of inflammation response leads to the activation of NF-kB, and thus iNOS expression [13, 20]. It is also indicated that anti-inflammatory cytokine interleukin-10 (IL-10) has an influence on iNOS expression via the suppression of NF-kB activ- ity [2]. This study examined the influence of the bilat- eral infusion of AAV2-hIL-10 into the striata on tyrosine hydroxylase (TH, the rate-limiting enzyme in dopamine biosynthesis), nNOS and iNOS expression in

the mouse model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP treatment leads to glia activation and the degeneration of nigrostriatal DA neurons, resulting in a decrease in TH expression and dopamine concentration [12].

Materials and Methods

Animals

One year-old male C57Bl mice were used in this study (body weight 35–40 g). The animals were housed in plastic cages with free access to food and water, ex- posed to 12:12 h light-dark cycle. Ambient temperature was maintained at 25 ± 2°C. There were 4–6 animals in each time point in each experimental group. Experi- ments were conducted according to the National Insti- tutes of Health Guide for the Care and Use of Labora- tory Animals and approved by the 2^nd Local Ethic Committee of Medical University of Warsaw.

Experimental protocol

AAV2-hIL-10 vector administration (stereotactic surgery)

Under surgical anesthesia [ketamine : xylazine (1:1);

2 ml/kg], the mouse was placed in a stereotactic frame (Stoelting). An incision was made in the skin overlying the skull and in the next step a hole with a needle just above the infusion site was made. Four weeks prior to MPTP intoxication, 2 µl of AAV2-hIL-10 (1 × 10¹³ vector genomes/ml) was infused bilaterally into the striatum by programmable microsyringe pump (World Precision Instruments; flow rate 0.5 µl/min; AP: +0.6, ML: ±1.75 relative to bregma and DV: -3.5 mm relative to dura) [24]. After AAV2-hIL-10 administration, the scalp was closed with sutures.

AAV2-hIL-10 treatment caused hIL-10 expression in the striata detected by ELISA test [11].

Vector genomes were obtained from prof. Krzysztof Bankiewicz, University of California, San Francisco.

MPTP intoxication

MPTP hydrochloride (MPTP-HCl; Sigma) was dis- solved in 0.9% sterile saline and was injected to the

Effect of IL-10 on iNOS and nNOS in MPTP model of PD

Joanna Schwenkgrub et al.

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equal volume of 0.9% NaCl. Animals were sacrificed by spinal cords dislocation at the different time inter- vals: 1, 7 and 21 days after MPTP injection. The in- tact brain was rapidly and completely removed from the skull, put it on the ice-cold glass plate. Striata (both right and left) were dissected using laboratory microscope, weighed, immediately placed in dry ice and stored at –80°C until further use. The following parameters were measured in all experimental groups:

TH, iNOS, nNOS proteins expression by western blot method (WB).

Western blot analysis

Samples were prepared from the striatum tissue according to the method described previously [4]. Sixty five µg of protein from each sample were separated on 7% (or 15% for TH) SDS polyacrylamide gel using Mini-PROTEAN Tetra Cell (Bio-Rad). Proteins were electrophoretically transferred (transfer time:

7 min; voltage: 23 V) to nitrocellulose membranes using iBlot Gel Transfer Device (Invitrogen) with iBlot Transfer Stacks (Invitrogen) for semi-dry blot- ting method.

The WB detection was performed using ONE- HOUR Western Kit (GenScript), in accordance with the manufacturer’s instructions.

antibodies (Santa Cruz Biotechnology, Inc.) that react with neuronal and inducible isoform of NOS, respec- tively.

• TH (1:1600), a rabbit polyclonal antibody (Milli- pore) that recognizes TH.

Each gel contained lanes from control group and each experimental group. The optical density of samples was expressed in terms of their ratio to control lanes, determined by gel analysis software program (Zero-descan, Scanalitics Inc.).

Statistical analysis

All values are expressed as the mean ± SEM. The differences between the groups were analyzed by one-way analysis of variance (ANOVA) followed by Mann- Whitney U-test; p < 0.05 was considered significant.

Results

The effect of pre-treatment with AAV2-hIL-10 on the TH protein expression in MPTP-treated mice is shown in Figure 1. Western blot analysis revealed significant reductions in the striatal TH protein level between 7 and 21 days after MPTP injections, whereas

Fig. 1. Expression of tyrosine hydroxylase (TH) in mice striatum. Data are presented as the means ± SEM of 4–6 mice per group. Groups of ani- mals: control – with saline injections; M (1, 7 or 21) – MPTP-treated and sacrificed at 1, 7 or 21 days after MPTP intoxication; W (28 + 1, 28 + 7, 28+ 21) – AAV2-hIL-10-treated and sacrificed at 29, 35 or 49 days following the vector’s administration; W28M (1, 7 or 21) – AAV2-hIL-10 vector was bilaterally administrated 28 days prior to MPTP intoxication, animals were sacrificed at 1, 7 or 21 days following MPTP injections

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in the group additionally pretreated with AAV2-hIL- 10, the MPTP intoxication was not able to significantly reduce the striatal TH expression.

There was no influence of AAV2-hIL-10 injection on the nNOS expression in the striatum after MPTP intoxication. The significant depletion in nNOS protein expression compared to the control group was obtained at 7 days after MPTP intoxication, as well as at 7 days in the MPTP group pretreated with AAV2- hIL-10 (Fig. 2). At all time points following AAV2- hIL-10 administration alone, nNOS protein expression was significantly decreased.

In case of iNOS expression, we observed some increase of iNOS protein expression after MPTP intoxication (up to

228% at 21 days compared to the control group), however, this increase did not reach the statistical sig- nificance compared with control (Fig. 3). The pretreatment with AAV2-hIL-10 suppresses the MPTP- induced tendency to increase iNOS protein expression in the striatum.

Discussion

The current study examined the impact of AAV2- hIL-10 pretreatment in the MPTP-based model of PD.

0 50 100 150 200 250 300 350

control M1 M7 M21 W28 + 1 W28 + 7 W28 + 21 W28M1 W28M7 W28M21

%ofcontrol

Fig. 3. Expression of inducible nitric oxide synthase (iNOS) in mice striatum. Data are presented as the means ± SEM of 5–6 mice per group.

Groups of animals: control – with saline injections; M (1, 7 or 21) – MPTP-treated and sacrificed at 1, 7 or 21 days after MPTP intoxication;

W (28 + 1, 28 + 7, 28 + 21) – AAV2-hIL-10-treated and sacrificed at 29, 35 or 49 days following the vector’s administration; W28M (1, 7 or 21) – AAV2-hIL-10 vector was bilaterally administrated 28 days prior to MPTP intoxication, animals were sacrificed at 1, 7 or 21 days following MPTP injections

Fig. 2. Expression of neuronal nitric oxide synthase (nNOS) in mice striatum. Data are presented as the means ± SEM of 4–6 mice per group.

Groups of animals: control – with saline injections; M (1, 7 or 21) – MPTP-treated and sacrificed at 1, 7 or 21 days after MPTP intoxication;

W (28 + 1, 28 + 7, 28 + 21) – AAV2-hIL-10-treated and sacrificed at 29, 35 or 49 days following the vector’s administration; W28M (1, 7 or 21) – AAV2-hIL-10 vector was bilaterally administrated 28 days prior to MPTP intoxication, animals were sacrificed at 1, 7 or 21 days following MPTP injections

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tem. In the group of animals pretreated with AAV2- hIL-10, the striatal TH protein level is almost un- changed by MPTP intoxication. First, we measured the effect of MPTP on striatal dopaminergic neurons terminals by alteration in TH protein levels. Not sur- prisingly, immunoblot analysis revealed significant reductions in TH protein expression in the striatum at 7 and 21 days after MPTP injections, whereas reversal of this effect was observed in the group additionally treated with AAV2-hIL-10. It is known that MPTP intoxication induces the glial activation and inflammatory reaction that precedes the degeneration of dopaminergic neurons [7, 10]. Inflammation in the lesioned brain is accompanied by significant nitric oxide release produced by iNOS [6]. This may be neuroprotective, if it is a short-term action, leading to the neutralization of pathogens [3]. However, PD pa- tients suffer from chronic inflammation that probably precedes neurodegeneration [18, 26]. Experiments on iNOS gene-deficient mice have provided evidence that the absence of this gene is associated with the partial dopaminergic neuron protection from MPTP- induced degeneration [5]. In our study, the pretreatment with AAV2-hIL-10 suppresses the MPTP- induced tendency to increase iNOS protein expression in the striatum. It was also reported that pretreatment of cultured astrocytes with IL-10 decreased the neuro- tropic Theiler’s virus-dependent iNOS expression and NF-kB binding activity [22]. The promoter region of the iNOS and nNOS gene contains several binding sites for transcriptional factors, including NF-kB. The activation of NF-kB requires the presence of cytoki- nes, oxidative stress, bacterial or viral products, and indirectly, the inhibition of nNOS activity [1, 13, 20, 25, 29]. NF-kB activity may be limited by NO. Ex- cessive NO formation due to the induction of iNOS and the presence of pro-inflammatory cytokines down-regulates nNOS gene expression [13]. We observed a significant decrease in nNOS protein expression at 7 days after MPTP intoxication. Muramatsu et al. [23] indicated that at 3 and 7 days after MPTP administration there were less nNOS-immunopositive cells in SN compared to the control. What is more, they did not notice any changes in immunoreactivity in the striatum of MPTP-treated mice. In this study, a significant decrease in nNOS protein levels was also observed after AAV2-hIL-10 administration at all

ministration on the nNOS protein concentration after MPTP intoxication.

In conclusion, human anti-inflammatory cytokine IL-10 delivered by an AAV2 vector attenuates the deleterious effect of MPTP on nigro-striatal system.

Our results might confirm the relevant role of the inflammatory reaction in the neurodegeneration process and indicate that the reduction of some inflammatory processes, might be a promising therapeutic strategy in PD.

Acknowledgment:

This study was supported by Grant No. N N401 0364 33 from the Ministry of Science and Higher Education, Warszawa, Poland.

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Received: January 9, 2012; in the revised form: August 23, 2012;

accepted: September 27, 2012.