Elsevier

Neurobiology of Disease

Volume 17, Issue 2, November 2004, Pages 219-236
Neurobiology of Disease

Transcriptome analysis in a rat model of l-DOPA-induced dyskinesia

https://doi.org/10.1016/j.nbd.2004.07.005Get rights and content

We have examined the pattern of striatal messenger RNA expression of over 8000 genes in a rat model of levodopa (l-DOPA)-induced dyskinesia and Parkinson disease (PD). 6-Hydroxydopamine (6-OHDA)-lesioned rats were treated with l-DOPA or physiological saline for 22 days and repeatedly tested for antiakinetic response to l-DOPA and the development of abnormal involuntary movements (AIMs). In a comparison of rats that developed a dyskinetic motor response to rats that did not, we found striking differences in gene expression patterns. In rats that developed dyskinesia, GABA neurons had an increased transcriptional activity, and genes involved in Ca2+ homeostasis, in Ca2+-dependent signaling, and in structural and synaptic plasticity were upregulated. The gene expression patterns implied that the dyskinetic striatum had increased transcriptional, as well as synaptic activity, and decreased capacity for energy production. Some basic maintenance chores such as ribosome protein biosynthesis were downregulated, possibly a response to expended ATP levels.

Introduction

Parkinson disease (PD) is a progressive neurological disorder characterized by a degeneration of dopamine (DA) neurons in the substantia nigra (Dauer and Przedborski, 2003). DA deficiency in areas innervated by nigral efferent neurons causes the motor symptoms of PD, that is, bradykinesia, rigidity, tremor, and postural instability (Gelb et al., 1999). The etiology of the disease is largely unknown. l-Dihydroxyphenylalanine (levodopa, l-DOPA) is used to treat the symptoms of PD, but it leads to the development of abnormal involuntary movements (AIMs; dyskinesia) complicating long-term treatment (Bezard et al., 2001, Nutt, 2001, Rascol et al., 2003).

We have developed a model of l-DOPA-induced dyskinesia in the rat (Cenci et al., 1998, Lee et al., 2000, Lundblad et al., 2002, Winkler et al., 2002). Rats are lesioned unilaterally with 6-hydroxydopamine (6-OHDA) and subsequently treated with relatively low doses of l- DOPA for a few weeks, during which a majority of the animals develop abnormal involuntary movements (AIMs), while some rats are resistant. This model allows us to study the biochemical and molecular factors involved in dyskinesia in a pharmacologically and genetically controlled environment.

One of the main target areas of nigral DA neurons is the striatum, a structure critically involved in the pathophysiology of parkinsonian motor symptoms (Sian et al., 1999). The striatum is also a prominent site of maladaptive molecular and synaptic plasticity in l-DOPA-induced dyskinesia (Andersson et al., 1999, Andersson et al., 2001, Cenci et al., 1998, Johansson et al., 2001, Picconi et al., 2003, Westin et al., 2001). Most of the neurons in the striatum are projection neurons that use GABA as their neurotransmitter. These neurons can be further classified based on their expression of dopamine receptor subtypes and the neuropeptides enkephalin, dynorphin, and substance P (Gerfen, 1992a, Gerfen, 1992b, Le Moine et al., 1991). The striatum has, in addition, interneurons expressing either acetylcholine, parvalbumin, or somatostatin (Kubota and Kawaguchi, 2000, Parent and Hazrati, 1995).

The aim of this study was to define changes in striatal gene expression that are associated with l-DOPA-induced dyskinesia in the rat model. Gene array technology was used to study the expression of over 8000 genes and expressed sequence tags (ESTs) in the striatum. Approximately 3000 of the genes examined were of known or inferred function and were expressed above background levels in at least 20% of our samples. These genes were the focus of our investigation. We present the major differences in striatal gene expression patterns between rats that develop l-DOPA-induced dyskinesia and rats that do not develop dyskinetic side effects in response to l-DOPA, although they show motor improvement. Some of the most interesting genes were further subjected to in situ hybridization histochemistry (ISHH) in the dyskinesia model.

Section snippets

Subjects

The study was performed in Sprague–Dawley rats (BK Labs, Sweden) weighing 225 g at the beginning of the experiments. Rats were housed three per cage under a 12-h light–dark cycle, with ad libitum access to food and water. Rats from different experimental groups were randomly distributed in the cages. To conform to the procedures used in our previous studies, the experimental subjects were female. Estrus cycle phase was checked once a week in each rat by vaginal mucus analysis, and the different

Results

Approximately 4500 mRNAs (3000 known sequences plus ESTs) were above detection limit in at least 20% of the samples and were used for further analysis. At a probability level below 0.05 (P < 0.05), 12% of these mRNAs were expressed at altered levels in the dyskinetic striata compared to saline controls, whereas only 5% of all mRNAs of nondyskinetic striata had altered levels (Fig. 1). Samples from dyskinetic rats differed from the nondyskinetic cases in over 6% of all mRNA species. Of all

Discussion

Microarray analysis enables an examination of large numbers of mRNAs expressed in different biological systems, and it provides a valuable tool in a discovery process intended to lead to novel hypotheses. Although transcript changes might not be translated into protein levels in every instance, there is generally a good correlation between increases in mRNA and increased protein levels, albeit with some variation in magnitude (Pongrac et al., 2002).

In the present study, microarray analysis was

Acknowledgments

This work was supported by DA07134 (CK) and by grants from the Elsa and Thorsten Segerfalk Foundation, the Johan and Greta Kock Foundations, the Swedish Association of the Neurologically Disabled, the Swedish Foundation for Parkinson´s Research, and the Swedish National Research Council (MAC). We would like to thank the Gemzeus Foundation, The Marie Curie Host Fellowship Training Program, and the Socrates/Erasmus Program for their support to JEW, MC, and KK, respectively. We thank Hanna

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