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Keywords:

  • Parkinson;
  • cabergoline;
  • NMDA antagonist;
  • basal ganglia;
  • dyskinesia;
  • BDNF

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. Author Roles:
  8. REFERENCES

Levodopa-induced dyskinesias (LIDs) are frequent in parkinsonian patients and may result from an aberrant plasticity. Brain-derived neurotrophic factor (BDNF) represents a likely candidate to subserve neuroadaptive processes encountered in LIDs. We compared striatal BDNF levels measured by ELISA in levodopa-treated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys having developed LIDs compared with animals where LIDs were prevented by the addition of CI-1041 (NR1A/2B NMDA receptor antagonist) or low doses of cabergoline (dopamine D2 receptor agonist). We observed reduced striatal BDNF concentrations in levodopa-treated MPTP monkeys with or without LIDs, suggesting that levodopa treatment is associated with reduced striatal BDNF levels and is independent of dyskinesias. © 2009 Movement Disorder Society

The development of abnormal involuntary movements in response to prolonged administration of levodopa (L-dopa) significantly affects the quality of life of parkinsonian patients. The mechanism of L-dopa-induced dyskinesias (LIDs) remains poorly understood but is generally thought to result from an aberrant form of plasticity.1

Brain-derived neurotrophic factor (BDNF) was shown to be related to neuronal plasticity and synaptic transmission in the adult central nervous system.2 Despite the lack of striatal BDNF mRNA,3, 4 immunohistochemical studies in adult rhesus monkey brains,4 in accordance with studies on rodent brains,5 detected intense labeling of BDNF protein in the caudate nucleus and moderate labeling of few neurons and numerous fibers in the lateral putamen. It was shown that anterograde but not retrograde axonal transport is the source of BDNF in the striatum because striatal targets, the globus pallidus and substantia nigra pars reticulata, do not display BDNF mRNA, whereas the cell bodies of cortical, substantia nigra pars compacta, amygdala, and thalamic neurons that project to the striatum contain high level of BDNF mRNA.3–6 This study investigated whether BDNF concentration is a factor associated with the development of LIDs.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. Author Roles:
  8. REFERENCES

Animals and Treatments

Experiments were performed with 20 middle-aged ovariectomized female cynomolgus monkeys (Macaca fascicularis) weighing 3.0–4.3 kg. Handling of the primates was performed in accordance to the National Institute of Health Guide for the Care and Use of Laboratory Animals. All procedures, including means to minimize discomfort, were reviewed and approved by the Institutional Animal Care Committee of Laval University. Four drug-naive monkeys were used as normal healthy controls. Sixteen other monkeys were exposed to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Sigma-Aldrich Canada, Oakville, Ontario) dissolved in sterile saline and continuously administered using subcutaneous Alzet minipumps at a dose of 0.5 mg/24 hr, until a bilateral stable parkinsonian syndrome developed for at least 1 month (i.e., unchanged parkinsonian score of 8 or more). A detailed description of the behavioral response of these monkeys was previously reported.7, 8 The total dose and time necessary to obtain sustained parkinsonian features were in average 16.2 mg and 3.6 months, respectively. The MPTP monkeys were then divided into 4 groups (n = 4 in each group) to have the total dose of MPTP, time after MPTP exposure, and start of chronic treatment as well as basal parkinsonian score the same in each group.7, 8 One group injected with saline served as untreated MPTP monkeys. Three other groups were exposed during 4 weeks to the following drug treatments. The L-dopa group was chronically treated with a daily oral dose of 100 mg L-dopa and 25 mg benserazide (Prolopa®: Hoffmann-La Roche, Mississauga, ON) (termed “L-dopa” thereafter). The L-dopa + CI-1041 group was given simultaneously L-dopa (same dosage) and CI-1041 (Pfizer, Ann Arbor, MI) at the dose of 10 mg/kg (per os). CI-1041 (PD196860), a substituted piperidine related to ifenprodil, is a selective NR1A/2B receptor antagonist.9 The L-dopa + Cabergoline group was administered simultaneously L-dopa (same dosage) plus cabergoline [a long-acting dopamine (DA) D2 receptor agonist] at a dose of 0.015–0.035 mg/kg (s.c.). A cabergoline dose was chosen for each animal based on the behavioral subthreshold concentration that by itself did not induce a significant antiparkinsonian response.

Tissue Preparation

Monkeys were administered an overdose of sodium pentobarbital and killed 24 hours after the last drug treatment. The brains were removed and placed in isopentane for less than 30 seconds (−40°C), then kept frozen at −80°C until used. Brain tissue samples for BDNF assays were collected from brain slices cut on a cryostat (−18°C) and dissected from the anterior (levels A18–A22) and posterior (A15–A18) caudate nucleus (9 and 12 pieces of 12 μm, respectively) and putamen (12 pieces of 12 μm) according to the atlas of Szabo and Cowan.10

BDNF Assay

BDNF striatal levels of monkeys were measured by ELISA (Quantikine®, Human BDNF Immunoassay R&D systems, Minneapolis, MN) according to the manufacturer's specifications. Brain tissue samples were homogenized in 130 μL of radioimmunoprecipitation assay buffer [50 mM Tris-HCl; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.5% SDS; 2 mM EDTA; 1% PMSF 100 mM; 1% protease inhibitor cocktail; 1% phosphatase inhibitor cocktail (Sigma St. Louis, MO)]. Homogenates were put on ice for 15–20 minutes and then centrifuged for 20 minutes at 10,000g at 4°C. Supernatants were collected for protein assay and BDNF measurement. The protein assay for each sample (dilution 1:50) and standards was performed in duplicate using Micro BCA™ protein assay kit (Pierce, Rockford, IL). The optical density for protein assay was detected in a microplate reader set at 562 nm, and protein concentration of each sample was calculated from the standard curve. For BDNF assay, the samples and standards were also added in duplicate, and optical densities were detected in a microplate reader set at 450 nm. BDNF concentrations of each sample were calculated from the standard curve, and then concentrations determined from the assay kit (pg/mL) were divided by the protein content of each sample (μg/μL) to express BDNF levels in pg/mg of proteins. Not enough tissue was available for the posterior caudate of 1 vehicle-treated MPTP monkey, and 1 control monkey had BDNF values about half of those for the other controls suggesting degradation. Data from these two monkeys were omitted from the statistical analyses.

Data Analysis and Statistics

Statistical comparisons of data were performed by an analysis of variance, followed by post hoc pairwise comparisons with Fisher's probability of least significant difference test. Pearson Correlation analyses were performed using the Statview software. A P-value ≤0.05 was required for the results to be considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. Author Roles:
  8. REFERENCES

Behavioral Evaluation

The detailed behavioral evaluation of these monkeys was previously reported.7, 8 All MPTP monkeys treated with L-dopa alone developed dyskinesias, whereas CI-1041 and cabergoline completely prevented the induction of dyskinesias in 3 monkeys in each group. Only 1 monkey in the two latter groups developed mild dyskinesias. Antiparkinsonian effects of L-dopa were similar in all monkeys. All the MPTP monkeys displayed similar extensive reduction of DA concentrations (94–98% decrease) and prominent decrease of DA transporter-specific binding in the caudate and putamen of all treated MPTP monkeys.11, 12

BDNF Concentrations

Anterior striatum BDNF concentrations of monkeys are shown in Figure 1. Overall comparisons of BDNF concentrations in the anterior caudate and putamen did not show statistical significance when separated by treatment groups. No difference between saline-treated MPTP and saline-treated intact monkeys was observed. Chronic L-dopa treatment alone (dyskinetic) or combined with CI-1041 or cabergoline (nondyskinetic) tended to reduce striatal BDNF concentrations, and this was statistically significant when L-dopa-treated monkeys were analyzed together (Fig. 1). Hence, no difference of BDNF concentrations was observed in anterior caudate and putamen between dyskinetic and nondyskinetic monkeys, and this was also shown with the lack of correlation between individual dyskinetic scores of MPTP monkeys and anterior striatal BDNF levels (Fig. 1).

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Figure 1. Anterior striatum BDNF concentrations of intact and MPTP monkeys treated with saline, L-dopa, L-dopa + CI-1041, or L-dopa + cabergoline (CAB) shown for individual treatment groups (caudate: F4,13 = 2.45, P = 0.10; putamen: F4,13 = 2.03, P = 0.15). L-dopa-treated monkeys combined according to the presence (n = 6, Dysk) or absence of dyskinesias (n = 6, Non-dysk) were not different, and together L-dopa-treated MPTP monkeys were different from vehicle-treated MPTP monkeys and/or controls (caudate: F2,15 = 4.34, P = 0.03; putamen: F2,15 = 4.41, P = 0.03). The lower panels show no correlation between individual dyskinetic scores of MPTP monkeys and their anterior striatal BDNF concentrations.

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Overall comparisons of BDNF concentrations showed statistical significance when separated by treatment groups in the posterior caudate and were unchanged in the posterior putamen. In the posterior caudate, a significant reduction of BDNF concentrations was measured between saline-treated MPTP and saline-treated intact monkeys (Fig. 2). L-dopa treated and L-dopa + cabergoline-treated MPTP monkeys had significantly reduced levels of BDNF compared with controls in the caudate, and a tendency for decrease was observed in the L-dopa + CI-1041 group. L-dopa-treated monkeys were then analyzed according to the presence (n = 6) or absence of dyskinesias (n = 6); they were not different, and together L-DOPA-treated MPTP monkeys were different from controls in the caudate. No significant change of BDNF concentrations was measured in the posterior putamen with MPTP lesion and treatments. Individual dyskinetic scores of MPTP monkeys did not correlate with their posterior striatal BDNF concentrations (Fig. 2).

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Figure 2. Posterior striatum BDNF concentrations of intact and MPTP monkeys treated with saline, L-dopa, L-dopa + CI-1041, or L-dopa + cabergoline (CAB) shown for individual treatment groups (caudate: F4,13 = 3.08, P = 0.05; putamen F4,13 = 1.74, P = 0.20). L-dopa-treated monkeys combined according to presence (n = 6, Dysk) or absence of dyskinesias (n = 6, Non-dysk) were not different, and together L-dopa-treated MPTP monkeys were different from controls in the caudate: (F2,15 = 4.40, P = 0.03) but not in the putamen (F2,15 = 2.14, P = 0.15). The lower panels show no correlation between individual dyskinetic scores of MPTP monkeys and their posterior striatal BDNF concentrations.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. Author Roles:
  8. REFERENCES

Our results demonstrate a limited effect of MPTP lesion on striatal BDNF levels with a decrease only in the posterior caudate of these monkeys. Chronic L-dopa therapy was associated with a reduction of BDNF concentrations in the anterior and posterior caudate nucleus as well as in the anterior putamen of MPTP monkeys. However, no difference of BDNF concentrations was observed between dyskinetic and nondyskinetic monkeys. LIDs were prevented here with the addition of CI-1041, a NMDA receptor antagonist, glutamatergic overactivity being proposed to be associated with motor complications.7, 13 Cabergoline, a long-acting D2 receptor agonist, was used to prevent LID; it provides a more constant stimulation of DA receptors because their fluctuating activation is associated with dyskinesias.8 Moreover, the small doses of cabergoline used could also act presynaptically to decrease corticostriatal glutamate release.8 Preventing LID with these drugs by different mechanisms of action did not change striatal BDNF levels, supporting that this was not related to their specific activity.

Unilaterally, MPTP-lesioned female rhesus monkeys were shown to have a decrease of BDNF levels in the lesioned striata of young- and middle-aged animals.14 Old monkeys had decreased striatal BDNF levels in the intact side that did not decrease further with the MPTP lesion.14 This is in overall agreement with our findings with middle-aged ovariectomized female MPTP cynomolgus monkeys, where we measured a decrease of BDNF only in the posterior caudate.

A link is documented between L-dopa increasing BDNF and the induction of D3 DA receptors leading to behavioral sensitization in unilaterally 6-hydroxydopamine-lesioned rats.15 Moreover, higher D3 DA receptor binding was shown in the putamen and internal globus pallidus of MPTP monkeys having developed LIDs compared with nondyskinetic monkeys treated with a partial D3 receptor agonist, suggesting a role of D3 DA receptors in LIDs.16 We did see a correlation in the present group of monkeys between levels of D3 DA receptor-specific binding and the maximum dyskinesia scores (unpublished data), which is consistent with our previous results.17 However, our data do not support a similar relationship with BDNF levels. Indeed, MPTP monkeys with LID had similarly decreased BDNF levels as L-dopa-treated MPTP monkeys without dyskinesias compared with intact monkeys. Species, toxin, and duration of treatment differences may account for this difference.

According to recent studies, endogenously produced BDNF can increase the susceptibility of motor neurons to excitotoxic insult18–20; therefore, it could be suggested that the reduction of BDNF levels is a consequence of L-dopa therapy and acts as a compensatory mechanism to protect against DA toxicity in the striatum with reduced DA buffering capacity. A recent study showed that PD patients with a val66met allele polymorphism of BDNF, associated with lower activity-dependent secretion of BDNF, were at higher risk of developing dyskinesias earlier in the course of treatment with dopaminergic agents.21 Hence, the mechanism underlying L-dopa-mediated signals modulating BDNF expression associated with pathophysiological conditions involving nigrostriatal degeneration is still not well understood and requires further investigation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. Author Roles:
  8. REFERENCES

This work was supported by a grant from the Canadian Institute of Health Research (CIHR) (to T.D.P.). P. S. held a postdoctoral fellowship from CIHR-Rx&D.

Author Roles:

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. Author Roles:
  8. REFERENCES

Pershia Samadi participated in the conception of the experiments, performed the assays, did the statistical analyses, and wrote the first draft of the manuscript. Marc Morissette organized and setup the experiments. Daniel Levesque was a key participant in the conception of this study, interpretation of data, and revision of the manuscript. Thérèse Di Paolo supervised the conception, the organization, and execution of the project as well as reviewed, critique, and finalized the manuscript.

Financial Disclosure: During the past year, Dr. Di Paolo had contracts from Novartis Switzerland and Merck Serono Switzerland and Germany related to the present study. All other authors have no disclosures to report.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. Author Roles:
  8. REFERENCES