Hanna Lindgren

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.

L-DOPA-induced dopamine efflux in the striatum and the substantia nigra in a rat model of Parkinson's disease: temporal and quantitative relationship to the expression of dyskinesia.

J. Neurochem. (2010) 112, 1465–1476.

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.

Endothelial Proliferation and Increased Blood–Brain Barrier Permeability in the Basal Ganglia in a Rat Model of 3,4-Dihydroxyphenyl-l-Alanine-Induced Dyskinesia

3,4-Dihydroxyphenyl-l-alanine (l-DOPA)-induced dyskinesia is associated with molecular and synaptic plasticity in the basal ganglia, but the occurrence of structural remodeling through cell genesis has not been explored. In this study, rats with 6-hydroxydopamine lesions received injections of the thymidine analog 5-bromo-2′-deoxyuridine (BrdU) concomitantly with l-DOPA for 2 weeks. A large number of BrdU-positive cells were found in the striatum and its output structures (globus pallidus, entopeduncular nucleus, and substantia nigra pars reticulata) in l-DOPA-treated rats that had developed dyskinesia. The vast majority (60–80%) of the newborn cells stained positively for endothelial markers. This endothelial proliferation was associated with an upregulation of immature endothelial markers (nestin) and a downregulation of endothelial barrier antigen on blood vessel walls. In addition, dyskinetic rats exhibited a significant increase in total blood vessel length and a visible extravasation of serum albumin in the two structures in which endothelial proliferation was most pronounced (substantia nigra pars reticulata and entopeduncular nucleus). The present study provides the first evidence of angiogenesis and blood–brain barrier dysfunction in an experimental model of l-DOPA-induced dyskinesia. These microvascular changes are likely to affect the kinetics of l-DOPA entry into the brain, favoring the occurrence of motor complications.

The "motor complication syndrome" in rats with 6-OHDA lesions treated chronically with l-DOPA: Relation to dose and route of administration

L-DOPA-induced motor complications can be modelled in rats with 6-hydroxydopamine (6-OHDA) lesions by chronic injections of L-DOPA. We have compared the sensitisation and duration of rotational responses, and the occurrence of dose-failure episodes and abnormal involuntary movements (AIMs) in 6-OHDA-lesioned rats with regard to the dose and route of administration of L-DOPA. Rats were treated with either low (6mg/kg) or high (25mg/kg) doses of L-DOPA twice daily for 21 days whereas control animals received injections of either saline or bromocriptine (2.5mg/kg). A dose-dependent and gradual development of AIMs and contralateral turning was observed in rats treated chronically with l-DOPA. Rats treated with bromocriptine exhibited rotational sensitisation but no AIMs. A shortening of motor response duration was not seen in any of the drug-treated groups. In contrast, dose-failure episodes occurred frequently in both L-DOPA- and bromocriptine-treated animals. Changing the route of L-DOPA administration from intraperitoneal to subcutaneous completely abolished failures in motor response without affecting the development of dyskinesia. Based on the hypothesis that higher doses of L-DOPA may be toxic to dopaminoceptive structures, we compared the total number of neurons and the levels of activated microglia in the striatum. No signs of neurodegenerative changes could be seen in any of the treatment groups. In conclusion, both body AIMs and rotations were dose-dependently evoked by L-DOPA. Only AIMs, however, provided a specific measure of dyskinesia since rotations also were induced by bromocriptine, a drug with low dyskinesiogenic potential. Dose-failure episodes were not specific to L-DOPA treatment and could be attributed to erratic drug absorption from the peritoneal route.

l-DOPA dosage is critically involved in dyskinesia via loss of synaptic depotentiation

The emergence of levodopa (l-DOPA)-induced dyskinesia and motor fluctuations represents a major clinical problem in Parkinsons disease (PD). While it has been suggested that the daily dose of l-DOPA can play a critical role, the mechanisms linking l-DOPA dosage to the occurrence of motor complications have not yet been explored. Using an experimental model of PD we have recently demonstrated that long-term l-DOPA treatment leading to the induction of abnormal involuntary movements (AIMs) alters corticostriatal bidirectional synaptic plasticity. Dyskinetic animals, in fact, lack the ability to reverse previously induced long-term potentiation (LTP). This lack of depotentiation has been associated to a defect in erasing unessential motor information. Here chronic l-DOPA treatment was administered at two different doses to hemiparkinsonian rats, and electrophysiological recordings were subsequently performed from striatal spiny neurons. Both low and high doses of l-DOPA restored normal LTP, which was disrupted following dopamine (DA) denervation. By the end of the chronic treatment, however, while the low l-DOPA dose induced AIMs only in half of the rats, the high dose caused motor complications in all the treated animals. Interestingly, the dose-related expression of motor complications was associated with a lack of synaptic depotentiation. Our study provides further experimental evidence to support a direct correlation between the daily dosage of l-DOPA and the induction of motor complications and establishes a critical pathophysiological link between the lack of synaptic depotentiation and the expression of AIMs.

Vascular endothelial growth factor is upregulated by L-dopa in the parkinsonian brain: implications for the development of dyskinesia.

Angiogenesis and increased permeability of the blood-brain barrier have been reported to occur in animal models of Parkinsons disease and l-dopa-induced dyskinesia, but the significance of these phenomena has remained unclear. Using a validated rat model of l-dopa-induced dyskinesia, this study demonstrates that chronic treatment with l-dopa dose dependently induces the expression of vascular endothelial growth factor in the basal ganglia nuclei. Vascular endothelial growth factor was abundantly expressed in astrocytes and astrocytic processes in the proximity of blood vessels. When co-administered with l-dopa, a small molecule inhibitor of vascular endothelial growth factor signalling significantly attenuated the development of dyskinesia and completely blocked the angiogenic response and associated increase in blood-brain barrier permeability induced by the treatment. The occurrence of angiogenesis and vascular endothelial growth factor upregulation was verified in post-mortem basal ganglia tissue from patients with Parkinsons disease with a history of dyskinesia, who exhibited increased microvascular density, microvascular nestin expression and an upregulation of vascular endothelial growth factor messenger ribonucleic acid. These congruent findings in the rat model and human patients indicate that vascular endothelial growth factor is implicated in the pathophysiology of l-dopa-induced dyskinesia and emphasize an involvement of the microvascular compartment in the adverse effects of l-dopa pharmacotherapy in Parkinsons disease.

Differential Involvement of D1 and D2 Dopamine Receptors in L-DOPA-Induced Angiogenic Activity in a Rat Model of Parkinson's Disease.

Angiogenesis occurs in the brains of Parkinsons disease patients, but the effects of dopamine replacement therapy on this process have not been examined. Using rats with 6-hydroxydopamine lesions, we have compared angiogenic responses induced in the basal ganglia by chronic treatment with either L-DOPA, or bromocriptine, or a selective D1 receptor agonist (SKF38393). Moreover, we have asked whether L-DOPA-induced angiogenesis can be blocked by co-treatment with either a D1- or a D2 receptor antagonist (SCH23390 and eticlopride, respectively), or by an inhibitor of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (SL327). L-DOPA, but not bromocriptine, induced dyskinesia, which was associated with endothelial proliferation, upregulation of immature endothelial markers (nestin) and downregulation of endothelial barrier antigen in the striatum and its output structures. At a dose inducing dyskinesia (1.5 mg/kg/day), SKF38393 elicited angiogenic changes similar to L-DOPA. Antagonism of D1- but not D2 class receptors completely suppressed both the development of dyskinesia and the upregulation of angiogenesis markers. In fact, L-DOPA-induced endothelial proliferation was markedly exacerbated by low-dose D2 antagonism (0.01 mg/kg eticlopride). Inhibition of ERK1/2 by SL327 attenuated L-DOPA-induced dyskinesia and completely inhibited all markers of angiogenesis. These results highlight the specific link between treatment-induced dyskinesias and microvascular remodeling in the dopamine-denervated brain. L-DOPA-induced angiogenesis requires stimulation of D1 receptors and activation of ERK1/2, whereas the stimulation of D2 receptors seems to oppose this response.

Regulation of Transcriptional Activity of the Murine CD40 Ligand Promoter in Response to Signals Through TCR and the Costimulatory Molecules CD28 and CD2

We have analyzed the murine CD40 ligand promoter with regard to stimulation of transcriptional activity in Jurkat T cells after signaling via the TCR and the costimulatory molecules CD28 and CD2. TCR engagement was necessary for the induction of transcriptional activity from the CD40 ligand promoter, and costimulation through either CD28 or CD2 further increased the activity. Analysis of promoter deletants showed that the DNA elements needed for transcriptional activity induced by costimulatory molecules were located within two regions containing previously identified transcription factor NFAT sites. Further studies of the proximal NFAT site showed that it was not dependent on AP-1 binding for transcriptional activity induced by costimulation through CD28. Instead, a region between the TATA box and the proximal NFAT site was shown to bind proteins of the early growth response family and to contribute to NFAT-mediated transcriptional activation.

N-substituted benzamides inhibit nuclear factor-κB and nuclear factor of activated T cells activity while inducing activator protein 1 activity in T lymphocytes

N-substituted benzamides are compounds that have recently been reported to inhibit nuclear factor-kappaB (NF-kappaB) activity and induce apoptosis in a pre-B cell line. In this study, we focused on the effects of N-substituted benzamides on transcriptional regulation in Jurkat T cells. We used a model system where the cells can be stimulated either through TCR/CD28 or by treatment of the cells with PMA and ionomycin to induce transcription factors typical for T lymphocyte activation. Treatment of the Jurkat cells with procainamide did not influence the transcription factor profile of stimulated cells, while treatment with a derivative having an acetyl group in position 4 of the aromatic ring inhibited NF-kappaB and nuclear factor of activated T cells (NFAT) activity. Declopramide, which contains a chloride in position 3 of the aromatic ring, was inactive in this system, whereas also the acetylated derivative of this compound inhibited NF-kappaB and NFAT activity. In contrast, the transcriptional activity and nuclear expression of activator protein 1 induced by TCR/CD28 stimulation or PMA and ionomycin treatment was enhanced by the acetylated variants of the N-substituted benzamides. Finally, we investigated the effect of N-substituted benzamides on intact promoters for two genes central in immune regulation; the CD40 ligand (CD40L) and IL-2 promoters. The transcriptional activity of the CD40L promoter as well as surface expression of the CD40L induced by signaling through TCR/CD28 was inhibited by addition of acetylated N-substituted benzamides, while the transcriptional activity of the IL-2 promoter was enhanced. Taken together, these data indicate that derivatives of N-substituted benzamides are potential drug candidates for quantitative as well as qualitative modulation of immune functions.