Striatal inhibition of PKA prevents levodopa-induced behavioural and molecular changes in the hemiparkinsonian rat

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Abstract

l-3,4-dihydroxyphenylalanine methyl ester hydrochloride (l-DOPA) is the gold standard for symptomatic treatment of Parkinson's disease (PD), but long-term therapy is associated with the emergence of abnormal involuntary movements (AIMS) known as l-DOPA-induced dyskinesias (LID). The molecular changes underlying LID are not completely understood. Using the 6-hydroxydopamine-lesioned rat model of PD, we showed that l-DOPA elicits profound alterations in the activity of three LID molecular markers, namely ΔFosB, dopamine, cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), as well as in phosphorylation levels of the cytoskeletal-associated protein tau. These modifications are triggered by protein kinase A (PKA) activation and intermittent stimulation of dopamine receptors as they are totally prevented by intrastriatal injections of Rp-cAMPS, a PKA inhibitor, or by continuous administration of l-DOPA via subcutaneous mini-pump. Importantly, Rp-cAMPS does not modulate the positive effect of l-DOPA on locomotor deficits and significantly attenuates the emergence of AIMS in 6-hydroxydopamine hydrobromide-lesioned rats. Even if decreased PKA signalling in the striatum may represent a clinical challenge, these data provide novel evidence that PKA activation, through modification of striatal signalling and alterations of cytoskeletal constituents, plays a key role in the manifestation of LID.

Introduction

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta with subsequent loss of dopaminergic input to the striatum. Treatment strategies, consisting of dopamine replacement and l-3,4-dihydroxyphenylalanine methyl ester hydrochloride (l-DOPA), are still the most effective and commonly used in PD. With disease progression, the therapeutic window for l-DOPA narrows, and doses that reduce Parkinsonism also evoke abnormal involuntary movements (AIMS) known as l-DOPA-induced dyskinesias (LID) (Nutt, 2000, Ahlskog and Muenter, 2001). The majority of PD patients under l-DOPA therapy develop motor complications within 5–10 years of treatment (Rascol, 2000, Bezard, 2001). These complications impair fine motor control and constitute a challenge in the management of PD (Obeso et al., 2000). The pathogenesis of LID is not completely understood. Animal models have demonstrated that LID are paralleled by increased signal transduction and significant changes in striatal protein expression (Doucet, 1996, Brotchie, 1998, Cenci, 2002, Gerfen, 2002, Aubert, 2005). Basically, the most recognized molecular markers of LID are elevation of ΔFosB, phosphorylation of dopamine, cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) at threonine34 and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (Andersson, 1999, Cenci, 2002, Gerfen, 2002, Winkler, 2002, Picconi, 2003, Pavon, 2006, Santini, 2007, Westin, 2007). Interestingly, each of these signalling molecules can be physiologically activated by protein kinase A (PKA). However, the link between PKA activity, these molecular marker expression levels and the emergence of LID is unknown. We recently uncovered a novel molecular chain of events connecting PKA activation, through D1 dopamine receptor stimulation, and abnormal phosphorylation of the cytoskeletal-associated protein tau (Lebel et al., 2009). These data are interesting in that abnormal tau phosphorylation negatively regulates microtubule-binding and leads to destabilization of the microtubule network, cytoskeletal dysfunction and modification of synaptic plasticity (Iqbal and Grundke-Iqbal, 2006, Mazanetz and Fischer, 2007). Whether tau is another player in the molecular reorganization associated with LID is still not clear.

In the present study, we focused on a widely-known rat model of AIMS to investigate the role of PKA in striatal molecular reorganization and the emergence of motor complications in conjunction with the pulsatile administration of l-DOPA. In parallel, we evaluated whether continuous delivery of l-DOPA, by subcutaneous mini-pump in 6-hydroxydopamine hydrobromide (6-OHDA)-lesioned rats, affects the occurrence of AIMS. We demonstrated that the latter mode of l-DOPA delivery is not linked with either the apparition of AIMS or changes in striatal protein expression in response to intermittent dopamine receptor stimulation. Interestingly, intrastriatal inhibition of PKA entirely prevents increased levels of ΔFosB and phosphorylated ERK1/2 and DARPP-32 levels at threonine34 induced by pulsatile l-DOPA treatment. While tau phosphorylation levels were not affected by 6-OHDA lesion, they increased with pulsatile l-DOPA treatment in association with AIMS, and intrastriatal inhibition of PKA was able to prevent this phenomenon. Thus, inhibition of PKA remarkably restrains the expression and activation of these signalling molecules that are associated with the emergence of AIMS. Importantly, however, PKA inhibition only partially prevents AIMS evoked by l-DOPA in this rat model of Parkinsonism. We provide the first evidence that PKA inhibition, through modifications in the intracellular signalling of striatal neurons, plays a central part in the advent of motor complications deriving from chronically-administered l-DOPA.

Section snippets

Chemical reagents

Male Sprague–Dawley rats were obtained from Charles River Laboratories (St-Constant, QC, Canada). 6-OHDA, desipramine hydrochloride, l-DOPA, benserazide hydrochloride and R(−) apomorphine hydrochloride hemihydrate were procured from Sigma-Aldrich (Oakville, ON, Canada). Osmotic mini-pump (Models 2ML4 and 2004) and brain infusion kit 2 were purchased from ALZET (Cupertino, CA, USA). Rp-cAMPS was from BioMol (Plymouth, PA, USA). Phosphatase and protease inhibitor cocktails were sourced from Roche

Validation of 6-OHDA-induced nigrostriatal dopamine denervation

85 rats were unilaterally injected with 6-OHDA in the MFB. Fig. 1 describes the experimental design. The severity of depletion was evaluated 10 days after surgery by measuring the contraversive rotation response to apomorphine (1 mg/kg s.c.). Rotations were scored for 30 min, and only complete (360°) turns were considered. Apomorphine did not produce any circling behaviour in sham rats, whereas 6-OHDA-lesioned animals presented low (1 turn/min) to severe (8 turns/min) contralateral turning (

Discussion

It is now well-established that brain dopamine depletion results in the enhanced responsiveness of striatal neurons of the direct striatonigral pathway after intermittent l-DOPA administration. For instance, several studies have demonstrated that LID may be produced by persistent hyperactivation of cAMP signalling in these neurons (see Santini et al., 2008 for review). The ability of dopamine to stimulate adenylyl cyclase and PKA via activation of D1 receptors is enhanced in Parkinsonism

Acknowledgments

We thank Dominic Bastien for his invaluable help with the surgical procedures. This work was supported by the Parkinson Society of Canada (MC) and the Canada Research Chair in Molecular Neuropharmacology (MC).

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