M. A. Cenci

Striatal fosB expression is causally linked with l-DOPA-induced abnormal involuntary movements and the associated upregulation of striatal prodynorphin mRNA in a rat model of Parkinson's disease.

Rats with unilateral dopamine-denervating lesions sustained a 3-week treatment with a daily l-DOPA dose that is in the therapeutic range for Parkinsons disease. In most of the treated animals, chronic l-DOPA administration gradually induced abnormal involuntary movements affecting cranial, trunk, and limb muscles on the side of the body contralateral to the lesion. This effect was paralleled by an induction of FosB-like immunoreactive proteins in striatal subregions somatotopically related to the types of movements that had been elicited by l-DOPA. The induced proteins showed both regional and cellular colocalization with prodynorphin mRNA. Intrastriatal infusion of fosB antisense inhibited the development of dyskinetic movements that were related to the striatal subregion targeted and produced a local specific downregulation of prodynorphin mRNA. These data provide compelling evidence of a causal role for striatal fosB induction in the development of l-DOPA-induced dyskinesia in the rat and of a positive regulation of prodynorphin gene expression by FosB-related transcription factors.

A model of l-DOPA-induced dyskinesia in 6-hydroxydopamine lesioned mice: relation to motor and cellular parameters of nigrostriatal function ☆

L-DOPA-induced dyskinesia is a major complication of L-DOPA pharmacotherapy in Parkinsons disease, and is thought to depend on abnormal cell signaling in the basal ganglia. In this study, we have addressed the possibility to model L-DOPA-induced dyskinesia in the mouse at both the behavioral and the molecular level. C57BL/6 mice sustained unilateral injections of 6-hydroxydopamine (6-OHDA) either in the medial forebrain bundle (MFB) or in the sensorimotor part of the striatum. Both types of lesion produced a similar degree of forelimb akinesia on the contralateral side of the body. The lowest dose of L-DOPA that could significantly relieve this akinetic deficit (i.e., 6 mg/kg) did not differ between MFB and intrastriatal lesions. The L-DOPA threshold dose for the induction of dyskinesia did however differ between the two lesion types. A daily dose of 6 mg/kg L-DOPA caused MFB lesioned mice to develop abnormal movements affecting orofacial, trunk, and forelimb muscles on the side contralateral to the lesion, whereas a daily dose of 18 mg/kg was required to produce comparable dyskinetic effects in the intrastriatally lesioned animals. The development of abnormal movements was accompanied by a striatal induction of DeltaFosB-like proteins and prodynorphin mRNA, that is, molecular markers that are associated with L-DOPA-induced dyskinesia in both rats and nonhuman primates. We conclude that 6-OHDA lesioned mice exhibit behavioral and cellular features of akinesia and L-DOPA-induced dyskinesia that are similar to those previously characterized in rats. The mouse model of L-DOPA-induced dyskinesia will provide a useful tool to study the molecular determinants of this movement disorder in transgenic mice strains.

Role of striatal l-DOPA in the production of dyskinesia in 6-hydroxydopamine lesioned rats.

We explored possible differences in the peripheral and central pharmacokinetics of l‐DOPA as a basis for individual variation in the liability to dyskinesia. Unilaterally, 6‐hydroxydopamine (6‐OHDA) lesioned rats were treated chronically with l‐DOPA for an induction and monitoring of abnormal involuntary movements (AIMs). Comparisons between dyskinetic and non‐dyskinetic cases were then carried out with regard to plasma and striatal l‐DOPA concentrations, tissue levels of dopamine (DA), DA metabolites, and serotonin. After a single intraperitoneal injection of l‐DOPA, plasma l‐DOPA concentrations did not differ between dyskinetic and non‐dyskinetic animals, whereas peak levels of l‐DOPA in the striatal extracellular fluid were about fivefold larger in the former compared with the latter group. Interestingly, the time course of the AIMs paralleled the surge in striatal l‐DOPA levels. Intrastriatal infusion of l‐DOPA by reverse dialysis concentration dependently induced AIMs in all 6‐OHDA lesioned rats, regardless of a previous priming for dyskinesia. Steady‐state levels of DA and its metabolites in striatal and cortical tissue did not differ between dyskinetic and non‐dyskinetic animals, indicating that the observed difference in motor response to l‐DOPA did not depend on the extent of lesion‐induced DA depletion. These results show that an elevation of l‐DOPA levels in the striatal extracellular fluid is necessary and sufficient for the occurrence of dyskinesia. Individual differences in the central bioavailability of l‐DOPA may provide a clue to the varying susceptibility to dyskinesia in Parkinsons disease.

Persistent changes in striatal gene expression induced by long-term L-DOPA treatment in a rat model of Parkinson's disease

Current knowledge of the molecular changes induced by dopamine denervation and subsequent treatment with l‐DOPA is based on studies performed on relatively acute and young animal models of parkinsonism. It is highly warranted to ask how well these models simulate the state of chronic denervation and sustained l‐DOPA pharmacotherapy which are typical of advanced Parkinsons disease. This study investigates the effects of time postdenervation and l‐dopa treatment duration on the striatal expression of opioid precursor mRNAs and FosB/ΔFosB‐related proteins. Unilaterally 6‐hydroxydopamine‐lesioned rats were treated with therapeutical doses of l‐DOPA for one year (long‐term group) or a few weeks (short‐term group). Age‐matched lesioned rats received injections of vehicle or bromocriptine, an antiparkinsonian compound which does not produce dyskinesia when administered de novo. The lesion‐induced up‐regulation of preproenkephalin mRNA expression persisted at more than one year postlesion, and was unaffected by the pharmacological treatments applied. l‐DOPA, but not bromocriptine, induced high striatal levels of FosB/ΔFosB immunoreactivity and prodynorphin mRNA, and these did not differ between short‐term and long‐term l‐DOPA‐treated rats. The present data provide the first demonstration that l‐DOPA maintains high striatal levels of fosB and prodynorphin gene expression during a prolonged course of treatment, which simulates the clinical practice in Parkinsons disease more closely than the short‐treatment paradigms studied thus far.

The impact of graft size on the development of dyskinesia following intrastriatal grafting of embryonic dopamine neurons in the rat

Intrastriatal transplants of embryonic ventral mesencephalon can cause dyskinesia in patients with Parkinsons disease (PD). We assessed the impact of transplant size on the development of graft-induced dyskinesia. Rats with unilateral 6-hydroxydopamine lesions were primed to exhibit L-DOPA-induced dyskinesia. They were then intrastriatally grafted with different quantities of embryonic ventral mesencephalic tissue to give small and large grafts. Without drug treatment, discrete dyskinetic-like movements were observed in most rats with large grafts 2-6 weeks after transplantation, but disappeared later. Amphetamine evoked severe abnormal involuntary movements (AIMs) in grafted animals, which were more striking with large grafts. The AIMs coincided with contralateral rotation, but displayed a different temporal profile and pharmacological properties. Thus, selective dopamine uptake blockade elicited rotational behavior, whereas coadministration of both dopamine and serotonin uptake blockers was required to evoke significant orolingual and limb AIMs. In conclusion, robust and reproducible AIMs were evoked in rats with large grafts by blockade of monoamine reuptake. These AIMs may provide a new tool for assessing dyskinetic effects of neural grafting.

Time course of striatal DeltaFosB-like immunoreactivity and prodynorphin mRNA levels after discontinuation of chronic dopaminomimetic treatment.

ΔFosB‐like proteins are particularly stable transcription factors that accumulate in the brain in response to chronic perturbations. In this study we have compared the time‐course of striatal FosB/ΔFosB‐like immunoreactivity and prodynorphin mRNA expression after discontinuation of chronic cocaine treatment to intact rats and chronic L‐DOPA treatment to unilaterally 6‐hydroxydopamine (6‐OHDA) lesioned rats. The animals were killed between 3 h and 16 days after the last drug injection. In both treatment paradigms, the drug‐induced FosB/ΔFosB immunoreactivity remained significantly elevated in the caudate putamen even at the longest withdrawal period examined. The concomitant upregulation of prodynorphin mRNA, a target of ΔFosB, paralleled the time‐course of ΔFosB‐like immunoreactivity in the 6‐OHDA‐lesion/L‐DOPA model, but was more transient in animals treated with cocaine. These results suggest that ΔFosB‐like proteins have exceptional in vivo stability. In the dopamine‐denervated striatum, these proteins may exert sustained effects on the expression of their target genes long after discontinuation of L‐DOPA pharmacotherapy.

Proteomic analysis of striatal proteins in the rat model of L-DOPA-induced dyskinesia.

l‐DOPA‐induced dyskinesia (LID) is among the motor complications that arise in Parkinson’s disease (PD) patients after a prolonged treatment with l‐DOPA. To this day, transcriptome analysis has been performed in a rat model of LID [Neurobiol. Dis., 17 (2004), 219] but information regarding the proteome is still lacking. In the present study, we investigated the changes occurring at the protein level in striatal samples obtained from the unilaterally 6‐hydroxydopamine‐lesion rat model of PD treated with saline, l‐DOPA or bromocriptine using two‐dimensional difference gel electrophoresis and mass spectrometry (MS). Rats treated with l‐DOPA were allocated to two groups based on the presence or absence of LID. Among the 2000 spots compared for statistical difference, 67 spots were significantly changed in abundance and identified using matrix‐assisted laser desorption/ionization time‐of‐flight MS, atmospheric pressure matrix‐assisted laser desorption/ionization and HPLC coupled tandem MS (LC/MS/MS). Out of these 67 proteins, LID significantly changed the expression level of five proteins: αβ‐crystalin, gamma‐enolase, guanidoacetate methyltransferase, vinculin, and proteasome α‐2 subunit. Complementary techniques such as western immunoblotting and immunohistochemistry were performed to investigate the validity of the data obtained using the proteomic approach. In conclusion, this study provides new insights into the protein changes occurring in LID.

Priming for L-DOPA-induced abnormal involuntary movements increases the severity of amphetamine-induced dyskinesia in grafted rats.

In some patients, graft-induced dyskinesia develops following intrastriatal transplantation of embryonic neural tissue for the treatment of Parkinsons disease. The mechanisms underlying these involuntary movements need to be clarified before this approach to clinical cell therapy can be developed further. We previously found that rats with 6-OHDA lesions, primed with L-DOPA treatment and that have subsequently undergone intrastriatal graft surgery exhibit involuntary movements when subjected to amphetamine. This model of amphetamine-induced AIMs reflects a pattern of post-graft behaviours that in the absence of robust spontaneous GID in the rat is the closest approximation that we currently have available. We now show that they are associated with the chronic administration of L-DOPA prior to the transplantation surgery. We also demonstrate that neither changes in c-fos nor FosB/DeltaFosB expression in the lateral striatum are associated with the expression of these behaviours. Taken together, these data reveal that the severity of abnormal movements elicited by amphetamine in grafted animals may relate to previous L-DOPA exposure and dyskinesia development, but they develop through mechanisms that are independent of FosB/DeltaFosB upregulation.

Neuroinflammation in the generation of post-transplantation dyskinesia in Parkinson's disease

The observation that neural grafts can induce dyskinesias has severely hindered the development of a transplantation therapy for Parkinsons disease (PD). We addressed the hypothesis that inflammatory responses within and around an intrastriatal graft containing dopamine neurons can trigger dyskinetic behaviors. We subjected rats to unilateral nigrostriatal lesions with 6-hydroxydopamine (6-OHDA) and treated them with L-DOPA for 21 days in order to induce abnormal involuntary movements (AIMs). Subsequently, we grafted the rats with allogeneic embryonic ventral mesencephalic tissue in the dopamine-denervated striatum. In agreement with earlier studies, the grafted rats developed dyskinesia-like AIMs in response to amphetamine. We then used two experimental approaches to induce an inflammatory response and examined if the amphetamine-induced AIMs worsened or if spontaneous AIMs developed. In one experiment, we challenged the neural graft hosts immunologically with an orthotopic skin allograft of the same genetic origin as the intracerebral neural allograft. In another experiment, we infused the pro-inflammatory cytokine interleukin 2 (IL-2) adjacent to the intrastriatal grafts using osmotic minipumps. The skin allograft induced rapid rejection of the mesencephalic allografts, leading to disappearance of the amphetamine-induced AIMs. Contrary to our hypothesis, the rejection process itself did not elicit AIMs. Likewise, the IL-2 infusion did not induce spontaneous AIMs, nor did it alter L-DOPA-induced AIMs. The IL-2 infusions did, however, elicit the predicted marked striatal inflammation, as evidenced by the presence of activated microglia and IL2Ralpha-positive cells. These results indicate that an inflammatory response in and around grafted dopaminergic neurons is not sufficient to evoke dyskinetic behaviors in experimental models of PD.

The locus coeruleus Is Directly Implicated in L-DOPA-Induced Dyskinesia in Parkinsonian Rats: An Electrophysiological and Behavioural Study

Despite being the most effective treatment for Parkinson’s disease, L-DOPA causes a development of dyskinetic movements in the majority of treated patients. L-DOPA-induced dyskinesia is attributed to a dysregulated dopamine transmission within the basal ganglia, but serotonergic and noradrenergic systems are believed to play an important modulatory role. In this study, we have addressed the role of the locus coeruleus nucleus (LC) in a rat model of L-DOPA-induced dyskinesia. Single-unit extracellular recordings in vivo and behavioural and immunohistochemical approaches were applied in rats rendered dyskinetic by the destruction of the nigrostriatal dopamine neurons followed by chronic treatment with L-DOPA. The results showed that L-DOPA treatment reversed the change induced by 6-hydroxydopamine lesions on LC neuronal activity. The severity of the abnormal involuntary movements induced by L-DOPA correlated with the basal firing parameters of LC neuronal activity. Systemic administration of the LC-selective noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine did not modify axial, limb, and orolingual dyskinesia, whereas chemical destruction of the LC with ibotenic acid significantly increased the abnormal involuntary movement scores. These results are the first to demonstrate altered LC neuronal activity in 6-OHDA lesioned rats treated with L-DOPA, and indicate that an intact noradrenergic system may limit the severity of this movement disorder.