Parkinsonism and Related Disorders xxx (2014) 1e6
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The NK1 receptor antagonist N-acetyl-L-tryptophan reduces dyskinesia in a hemi-parkinsonian rodent model Emma Thornton*, Mark Macquarie Hassall, Frances Corrigan, Robert Vink The Adelaide Centre for Neuroscience Research, School of Medical Sciences, The University of Adelaide, South Australia, Australia
a r t i c l e i n f o
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Article history: Received 3 October 2013 Received in revised form 7 January 2014 Accepted 8 February 2014
Background: Dyskinesia or abnormal involuntary movements (AIMs) are a disabling effect of chronic L-DOPA administration and consequent pulsatile stimulation of dopamine receptors. This abnormal activation causes maladaptive changes including upregulation of FosB expression in dynorphin containing striatal cells. Substance P (SP) is co-localized within dynorphin positive cells and is increased within the substantia nigra by L-DOPA (L-3,4-dihydroxyphenylalanine) treatment. Accordingly, we determined if treatment with a SP NK1 receptor antagonist reduced the onset of L-DOPA induced dyskinesia (LID) in the hemi-parkinsonian rodent model. Methods: Adult male SpragueeDawley rats underwent unilateral 6-OHDA (6-hydroxydopamine-hydrobromide) lesions of the medial forebrain bundle. At day 21, daily administration commenced of either L-DOPA (6 mg/kg plus 15 mg/kg of benseraside), L-DOPA with the NK1 antagonist N-acetyl-L-tryptophan (NAT) or equal volume of saline. Animals were tested with the rodent AIM scale assessing axial, contralateral forelimb and orolingual AIMs. Assessment of L-DOPA induced turning was undertaken, and motor function determined using the accelerating rotarod and adjusting step test. Dopaminergic neuronal counts and immunoreactivity for SP and FosB were undertaken. Results: All animals treated with L-DOPA alone developed dyskinesia, whereas combined administration of NAT with L-DOPA significantly reduced onset of AIMs and prevented mild to moderate dyskinesia. In non-dyskinetic NAT treated animals, similar numbers of FosBþ striatal cells were recorded as in saline treated animals. Importantly NAT treatment did not interfere with the anti-parkinsonian effect of L-DOPA. Conclusion: Daily administration of a SP NK1 receptor antagonist may represent a novel treatment regime that reduces the onset of LID whilst conserving motor function. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Substance P Tachykinin NK1 receptor L-DOPA Dyskinesia FosB Motor function
1. Introduction Since its inception over 50 years ago, L-DOPA (L-3,4dihydroxyphenylalanine), the precursor to dopamine, has remained the “gold standard” treatment for improving the motor deficits associated with Parkinson’s disease (PD). In early PD, L-DOPA treatment alleviates motor symptoms, however, its symptomatic relief becomes variable as the disease progresses with patients experiencing “wearing off” effects [1]. Along with this lessening in efficacy, chronic L-DOPA induces dyskinesia, or involuntary choreiform and dystonic movements of the trunk, face and limbs [2]. Unfortunately, almost all patients taking L-DOPA or * Corresponding author. School of Medical Sciences, The University of Adelaide, Level 4, Medical School South, Frome Rd, Adelaide, South Australia 5005, Australia. Tel.: þ61 8 313 3114. E-mail address:
[email protected] (E. Thornton).
dopamine agonists will become dyskinetic during their disease [3] and it is these abnormal involuntary movements (AIMs) that PD patients report reduce their quality of life [4]. To date, treatment for dyskinesia is extremely limited, with many patients seeking relief through invasive surgery like deep brain stimulation or pallidotomy. The onset of dyskinesia in patients has been linked to the pulsatile/oscillating activation of dopamine receptors by multiple daily doses of L-DOPA or dopamine agonists [5]. This abnormal activation of dopamine receptors results in maladaptive plasticity changes in striatal signaling and the expression of post-synaptic molecular markers including FosB, phosphorylation of DARP-32 (dopamine cAMP-regulated phosphoprotein of 32 kDa) at threonine 34 and activation of extracellular signal-related kinases 1 and 2 and protein kinase A [6,7]. Enhanced expression of prodynorphin (PDyn) mRNA has been strongly correlated to the onset and severity of dyskinesia [8e10]. Moreover, upregulation of DFosB, a truncated splice variant of FosB, within dynorphin containing striatal
http://dx.doi.org/10.1016/j.parkreldis.2014.02.008 1353-8020/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Thornton E, et al., The NK1 receptor antagonist N-acetyl-L-tryptophan reduces dyskinesia in a hemiparkinsonian rodent model, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.02.008
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E. Thornton et al. / Parkinsonism and Related Disorders xxx (2014) 1e6
projection neurons has been shown to play a vital role in the development of dyskinesia [11]. The neuropeptide substance P (SP) is co-localized with dynorphin in striatal projection neurons [9]. SP is located in primary sensory neurons throughout the brain, and found in particularly high levels in the substantia nigra (SN) and the basal ganglia [12]. Here, SP and DA are regulated through a positive feedback mechanism, whereby SP binding to its tachykinin NK1 receptor on dopamine neurons causes striatal DA release, and DA by binding to its D1 receptor on striatal projection neurons potentiates the release of SP within the SN [13]. Accordingly, a loss of nigral SP and striatal DA has been reported in the late stages of PD in both the 6-OHDA model [14] and clinical PD [15]. In this regard, L-DOPA treatment is also able to restore nigral SP content [14]. To date, the potential for SP to contribute to the onset of dyskinesia has not been established despite the known link between the onset of dyskinesia and the induction of DFosB in dynorphin/SP containing striatal projections neurons. Therefore we sought to determine if blockade of the SP NK1 receptor may reduce the onset of L-DOPA induced dyskinesia (LID). 2. Materials and methods All experiments were carried out under the guidelines established by the National Health and Medical Research Council and were approved by the University of Adelaide animal ethics committee. In total, 43 young adult male SpragueeDawley rats weighing 230e270 g were group housed under a 12 h lightedark cycle with access to food and water ad libitum.
together to determine a cumulative AIM score for the entire assessment period. If animals recorded a cumulative score of 5 turns/ min included in the study [17]. This rotational criterion excluded 3 animals. Furthermore, a significant association between dyskinetic behavior and 80% nigral TH cell loss was assessed by Fishers exact test (p ¼ 0.0045), thus animals with less than 80% TH cell loss were also excluded from the study. This criteria excluded a further 8 animals. Final group numbers were as follows: saline n ¼ 6; NAT alone n ¼ 2; L-DOPA alone n ¼ 9; L-DOPA þ NAT: n ¼ 11. 2.2. Drug administration Animals were randomly assigned to receive daily subcutaneous injections of either L-DOPA (6 mg/kg plus 15 mg/kg benseraside), N-acetyl-L-tryptophan 3,5-bis (trifluoromethyl)benzyl ester (NAT, Sigma, 0.5 mg/kg) or L-DOPA with NAT (0.5 and 5 mg/kg; these groups were pooled as no significant differences were observed for any tested parameter. NAT was administered immediately prior to L-DOPA) or equal volume vehicle (0.9% saline) for 21 days starting 3 weeks following 6-OHDA lesion. An L-DOPA dose of 6 mg/kg is clinically relevant and efficacious in experimental models of PD [10]. 2.3. AIMs rating L-DOPA induced AIMs were assessed according to a rat dyskinesia scale, which has been described elsewhere [10]. Briefly, animals were individually placed into transparent spherical bowls and observed for 1 min every 20 min for a total of 180 min following L-DOPA administration. Four subtypes of AIMs (locomotive, axial, forelimb and orolingual) were observed and scored between 0 (no movement observed) and 4 (movement continuous and uninterruptable). Locomotive AIMs were not included in the final AIM score as this subtype relates to the antiparkinsonian effect of L-DOPA [18]. Animals were assessed on days 3, 7, 10, 14, 17 and 21 following commencement of L-DOPA treatment. The AIM scores from each category were summed for each assessment day, with each daily AIM score added
Fig. 1. (A) All 6-OHDA hemi parkinsonian animals had similar TH immunoreactive cell loss in the ipsilateral substantia nigra regardless of treatment. F(4,32) ¼ 0.6444, p ¼ 0.6349. (B) Treatment with L-DOPA reverses the 6-OHDA induced loss of substance P in the SNc as assessed by color deconvolution F(3,21) ¼ 8.075, p ¼ 0.0009 (*-**p < 0.05e0.001 compared to saline treated animals; þþþp < 0.001 compared to sham).
Please cite this article in press as: Thornton E, et al., The NK1 receptor antagonist N-acetyl-L-tryptophan reduces dyskinesia in a hemiparkinsonian rodent model, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.02.008
E. Thornton et al. / Parkinsonism and Related Disorders xxx (2014) 1e6 specific primary antibody. Sections were incubated with biotinylated secondary immunoglobulin for 30 min at RT (Vector Laboratories, 1:250) then incubated with streptavidinehorseradish peroxidase for 60 min at RT with PBS washes between. To visualize antigen, sections were reacted with the chromagen, diaminobenzidine (DAB) containing 0.01% H2O2 for 7 min then counterstained in hematoxylin for 1 min. 2.6. Assessment of 6-OHDA induced loss of dopaminergic neurons Under light microscopy, TH immunoreactive neurons within the SN pars compacta (SNc) were manually counted at 20 magnification. The level that contained the interpeduncular fossa at approximately 5.0 mm posteriorly from bregma was assessed [16]. However to negate effects of slightly differing levels, TH immunoreactive neurons within the ipsilateral SNc were compared to neurons within the contralateral SNc (internal control) to determine percentage of ipsilateral cell loss. 2.7. Assessment of SP content within the ipsilateral SN For analysis of SP content within the ipsilateral SNc, slides were scanned (Nanozoomer, Hamamatsu) and viewed with the associated proprietary viewing software. A section of the SNc was isolated using 20 magnification and exported as a jpeg (virtual dissection). Color deconvolution as described elsewhere was used to determine the percentage of DAB contained in the image [22]. 2.8. Assessment of FosB/DFosB expression within the ipsilateral striatum Slides were scanned and viewed as described for SP. Two levels of ipsilateral striatum approximately 2 mm apart were assessed, with the area of the striatal sections determined at 1.25 magnification using the associated software.
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Sequential images of striatum were exported at 10 magnification. The number of FosB positive (þ) cells in these images were counted using ImageJ, with total number of þ cells expressed per 10 mm2 of striatum. 2.9. Statistical analysis All statistical tests were undertaken using PrismÒ (GraphPad). Rotarod and LDOPA turning were analyzed via two-way repeated measures ANOVA with Bonferroni post-test. Paired t-tests were used to analyze changes in the number of L-DOPA induced contralateral turns and adjusting steps from the first to the last assessment day, with baseline adjusting steps analyzed via unpaired t-tests with Welch’s correction. SP and TH immunoreactivity and FosBþ cells were analyzed with a oneway ANOVA with NeumaneKeuls post-test. All aforementioned data is expressed as mean SEM. Cumulative and daily AIM scores were analyzed with a KruskaleWallis ANOVA and Freidman test with Dunn’s post-hoc test, respectively. AIMs displayed as median and individual AIM rankings (cumulative) and median range (daily). The effect of NK1 antagonist treatment on the onset of dyskinesia was analyzed using the Fishers exact test.
3. Results The unilateral MFB 6-OHDA lesion resulted in near total loss of TH immunoreactive cells in the ipsilateral SNc, with all treatment groups recording similar nigral cell loss (Fig. 1A). At 6 weeks following 6-OHDA MFB injections, the saline treated group had a significant loss in SP content within the SNc compared to sham (p < 0.001; Fig. 1B). NAT treated animals resembled the saline group
Fig. 2. (A) Daily administration of NAT significantly reduced the onset of LID (Fishers exact test; *p ¼ 0.014). (B) When incorporating the entire assessment period, cumulative AIM scores assessing axial, forelimb and orolingual categories showed a large spread of AIM scores in the L-DOPA treated animals, whereas the NAT treated animals were predominantly severely dyskinetic (>350) or non-dyskinetic (
