Striatal mRNA expression patterns underlying peak dose l-DOPA-induced dyskinesia in the 6-OHDA hemiparkinsonian rat
Introduction
Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder worldwide, estimated to affect 0.3% of the general population (Tanner and Aston, 2000). At the onset of motor symptoms (resting tremor, bradykinesia, rigidity, and postural instability) more than half of dopaminergic neurons have degenerated (Barbeau, 1960, Ehringer and Hornykiewicz, 1960, Barbeau et al., 1961), resulting in a loss of 80% of the dopamine in downstream nuclei, including the striatum (Ehringer and Hornykiewicz, 1960). The primary drug treatment for PD is replacement of endogenous dopamine with the precursor substance l-DOPA (Kaplan and Tarsy, 2013), in order to restore striatal function (Nicola et al., 2000).
l-DOPA treatment provides relief from the motor symptoms of PD, however the half-life of l-DOPA is only 50–90 min (Fabbrini et al., 1988, Olanow et al., 2000, Kang et al., 2010), and storage of dopamine is altered in the parkinsonian brain (Lotharius and Brundin, 2002). Thus PD patients require multiple doses of l-DOPA during the course of a day, which can lead to a number of side effects, including dyskinesia. l-DOPA-induced dyskinesia are additional, unwanted, and often abnormal movements that develop with ongoing l-DOPA treatment. Dyskinesia may affect all areas of the body, including the head and neck, limbs, and torso; interfering with the process of everyday tasks (Barbeau, 1969).
Three major risk factors for development of l-DOPA-induced dyskinesia are: age at diagnosis of PD, initial l-DOPA dose, and length of treatment. A diagnosis before the age of 50 is associated with a 1.5-fold increase in dyskinesia incidence, and an initial dose of l-DOPA of more than 600 mg increases the risk of developing dyskinesia 1.4-fold (Grandas et al., 1999). Treatment with l-DOPA for a period of five years or more will almost always result in development of dyskinesia (Ahlskog and Muenter, 2001). Given the prevalence and impact of l-DOPA-induced dyskinesia, it is critical to understand their etiology, in order to develop effective treatments to minimize dyskinesia and, in the long term, prevent them from developing altogether.
In animal models of PD, the development of dyskinesia is associated with an alteration in synaptic plasticity mechanisms in neurons in the striatum (Picconi et al., 2003). In a healthy animal, excitatory cortical axons that synapse onto striatal projection neurons can induce both an increase in synaptic transmission (long-term potentiation, LTP) and a decrease in synaptic transmission (long-term depression, LTD) following particular patterns of afferent stimulation (Reynolds and Wickens, 2000, Reynolds et al., 2001). Parkinsonian animals are unable to induce corticostriatal LTP without dopamine present (Picconi et al., 2003), however, only non-dyskinetic animals are able to induce de-potentiation, suggesting that the presence of dyskinesia is associated with an inability to reverse previously reinforced motor patterns. We hypothesize that changes in signaling will be accompanied with changes in gene expression.
The development of transcriptomic technologies (e.g., microarrays, RNA-seq) has facilitated large-scale investigation of the pattern of gene expression changes in the brain following treatments. Indeed several studies have investigated the changes associated with dyskinesia using this approach (Konradi et al., 2004, El Atifi-Borel et al., 2009, Heiman et al., 2014). These studies, however, only investigated gene expression in response to a large-dose challenge (El Atifi-Borel et al., 2009), or after brain levels of l-DOPA had markedly reduced (e.g., 18 h after the last dose; Konradi et al., 2004).
Here, for the first time, we use RNA-seq technology to examine global changes in striatal gene expression between dyskinetic and non-dyskinetic animals, at a time point consistent with peak-dose dyskinesia (Kang et al., 2010). We use a model of l-DOPA-induced dyskinesia where all treated animals received the same l-DOPA dose but only half displayed dyskinesia at three weeks of treatment (Picconi et al., 2011). This provides a control group (non-dyskinetic) that has been subjected to exactly the same chronic treatment regimen as the experimental group (dyskinetic). Using these animals we examined the gene expression changes at the peak of l-DOPA levels in the blood, 60 min after the last l-DOPA injection (Kang et al., 2010), with the aim of identifying genes associated with peak-dose dyskinesia that could act as potential drug targets in the future.
Section snippets
Animals
Eighteen male Wistar rats were obtained and housed in the Hercus Taieri Research Unit at the University of Otago. All rats were used in each aspect of the study. Rats were housed on a 12-h reverse light–dark cycle with food and water ad libitum. All procedures were approved by the University of Otago Animal Ethics Committee.
Lesioning and behavioral testing
At 7-weeks of age rats received a unilateral lesion of the medial forebrain bundle using 6-hydroxydopamine (6-OHDA). Rats were anesthetized with ketamine (7.5 mg/kg s.c.,
Analysis of lesion extent
The extent of the dopaminergic lesion was estimated pre-mortem and post-mortem (Fig. 1). Pre-mortem estimates used the adjusting step test and forelimb use asymmetry test (Olsson et al., 1995, Schallert et al., 2000, Tseng et al., 2005, Parr-Brownlie et al., 2007). Successfully lesioned animals used the affected right paw for less than 5% of movements in both the step test (Fig. 1A; p < 0.001, F = 259) and the asymmetry test (Fig. 1B; p < 0.001, F = 899). Following all experimental procedures, lesion
Discussion
l-DOPA remains the gold standard treatment for alleviating symptoms in PD patients, however the majority of patients will develop dyskinesia with prolonged l-DOPA treatment. The experiments presented here examined the gene expression profile associated with l-DOPA-induced dyskinesia at the peak of l-DOPA dose with the aim of finding patterns of gene expression changes related to the most common form of dyskinesia in humans, so-called peak-dose dyskinesia. This is a unique timepoint; previous
Acknowledgments
The authors acknowledge the technical assistance of Mr. Jason Gray, Dr. Melony Black, and Dr. Jennifer Davies. Funding was received from a University of Otago Research Grant (to JNJR, PKD and EJD) and for sequencing costs from Parkinson’s NZ (The King Bequest, to JNJR). LMS received a W & B Miller PhD Scholarship from the Neurological Foundation of New Zealand and JNJR received a Rutherford Discovery Fellowship from the Royal Society of NZ.
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