1. Considering the pre-clinical and clinical evidence for continuous dopaminergic stimulation (CDS) This educational material has been supported by Abbott

2. Considering the pre-clinical and clinical evidence for continuous dopaminergic stimulation (CDS) Motor fluctuations and dyskinesias: what have we learned from the laboratory? >

3. Learning objectives At the end of this section you will have: An understanding of the animal models that have been developed to study motor complications in Parkinson’s disease Greater knowledge of the mechanisms underlying motor fluctuations and dyskinesias in Parkinson’s disease

4. Animal models of motor complications in Parkinson’s disease ModelPhysiological effectBehavioural effects Rodent 6-OHDA lesion (unilateral intracerebral injection) Selective permanent dopaminergic depletion of the nigrostriatal pathway Unilateral dopamine denervation causes motor deficits in the contralateral limbs and postural asymmetry, mimicking Parkinson’s disease Levodopa treatment improves these deficits but also causes AIMs mimicking dyskinesia. In most (but not all) rodent models, levodopa- induced AIMs are accompanied by contralateral rotation Non-human primate MPTP-treated Metabolite (MPP+) is taken up by the dopamine transporter and inhibits complex I of the mitochondrial respiratory chain Bradykinesia, rigidity, and postural deficits similar to Parkinson’s disease Levodopa improves motor performance and reduces disability Chronic intermittent levodopa treatment leads to AIMs (dyskinesia) AIMs = abnormal involuntary movements 6-OHDA = 6-hydroxydopamine MPTP = 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPP = 1-methyl-4-phenylpyridinium Adapted from: Chapter 1: The continuous dopaminergic stimulation concept and evidence to date. M Maral Mouradian. Managing Advanced Parkinson’s Disease: The role of continuous dopaminergic stimulation. Aquilonius and Lees (Ed). 2007.

5. StudyFinding Papa et al, 1994Rats with 95% dopamine neuron loss due to 6-OHDA lesions manifest ‘wearing-off’, while rats with less severe lesions do not Di Monte et al, 2000Moderate nigrostriatal denervation (60-70% striatal dopamine depletion) in MPTP treated primates led to levodopa-induced dyskinesias; the severity of the lesions’ enhanced sensitivity to levodopa Winkler et al, 2002Intrastriatal 6-OHDA-lesioned rats exhibited a lower predisposition to levodopa-induced dyskinesia than rats with complete bundle lesions Selective and partial denervation in the sensorimotor part of the striatum can confer cellular and behavioural supersensitivity to levodopa Paillie et al, 2007Levodopa given twice daily caused dyskinesias in bilateral 6-OHDA lesioned rats Dyskinesia severity correlated with extent of dopamine neuron loss increasing abruptly when neuron loss approached 75% Papa SM, et al. Brain Res 1994;662(1-2):69-74. Di Monte DA, et al. Mov Disord 2000;15(3):459-66. Winkler C, et al. Neurobiol Dis. 2002;10(2):165-86. Paillie V, et al. Mov Disord. 2007;22(4):533-9. Pre-clinical studies of motor complications Dopamine denervation is necessary for the occurrence of motor complications

6. StudyFinding Juncos et al, 198930-day intermittent levodopa, but not continuous levodopa treatment, produced behavioural sensitisation in 6-OHDA rats Bibbiani et al, 2005Continuous apomorphine infusion improved motor function in primates for up to 6 months without dyskinesias Intermittent apomorphine produced dyskinesias within 7-10 days of treatment Blanchet et al, 2001 Dyskinesias <10 days of intermittent dopaminergic treatment in MPTP-treated primates Only 3 of 6 primates developed dyskinesias with continuous dopaminergic stimulation, but complications diminished in intensity Schmidt et al, 2008 In unilateral 6-OHDA lesioned rats, pulsatile levodopa injections (1-2/day) caused contraversive rotations and AIMs Pulsatile (1-2/day) injections of the dopamine agonist rotigotine resulted in more contraversive rotations than a slow release formulation Stockwell et al, 2009 Continuous rotigotine delivery (via osmotic minipump) produces less dyskinesias than pulsatile rotigotine administration (twice daily) in MPTP-treated primates Stockwell et al, 2010 Switching from pulsatile levodopa or rotigotine administration to continuous rotigotine infusion reduced the severity and duration of dyskinesias in primates Juncos JL, et al. Ann Neurol 1989;25(5):473-8. Bibbiani F, et al. Exp Neurol 2005;192(1):73-8. Blanchet PJ, et al. Adv Neurol 2001;86:337-44. Schmidt WJ, et al. J Neural Transmiss 2008;115(10):1385-92. Stockwell KA, et al. Exp Neurol 2009;219(2):533-42. Stockwell KA, et al. Exp Neurol 2010;221(1):79-85. Pulsatile levodopa treatment is a cause of motor complications Pre-clinical studies of motor complications

7. A primate model of motor complications Reproduced from Exp Neurol 2005;192(1) Bibbiani F, et al. Continuous dopaminergic stimulation reduces risk of motor complications in parkinsonian primates. p 73-8, Copyright (2005) with permission from Elsevier. Effect of continuous versus intermittent dopaminergic treatment

8. The development of pulsatile levodopa-induced dyskinesias Study background Unilateral 6-OHDA in the nigrostriatal fibre bundle Severe (> 90%) striatal dopamine denervation Daily levodopa injections for 2-3 weeks Peak-dose abnormal involuntary movements (AIMs) Non-dyskinetic [movie] Dyskinetic [movie] Permission kindly granted by Dr Angela Cenci-Nilsson.

9. Potential mechanisms underlying pulsatile levodopa-induced dyskinesias Large peaks-and-troughs of extracellular dopamine: Rise and decline of levodopa and dopamine levels in the striatal extracellular fluid correlate with the timing of AIMs Larger increases in striatal levodopa and dopamine in dyskinetic 6-OHDA lesioned rats than in non-dyskinetic animals Chronic levodopa treatment may alter dopamine regulation and metabolism in striatum and may lead to treatment-related adaptations Serotonergic system: Treatment with serotonin autoreceptor agonists blunts the peak in extracellular dopamine levels in dyskinetic rats Removal of serotonin afferents blocks levodopa-induced dyskinesias in 6-OHDA lesioned rats Thus, dysregulated dopamine release from serotonergic neurons likely play a key role in dyskinesia development Cenci MA, Lundblad M. J Neurochem 2006;99(2):381-92. Lindgren H et al. J. Neurochem 2010;112(6):1465-76. Carta M, et al. Brain 2007;130(7):1819-33.

10. Serotonergic involvement in pulsatile levodopa-induced motor complications Levodopa-induced rat AIMs are abolished by dampening of serotonin neuron activity † Carta M, et al. Dopamine released from 5-HT terminals is the cause of L-DOPA-induced dyskinesia in parkinsonian rats. Brain 2007;130(Part 7):1819-33, reproduced with permission of Oxford University Press. † Munoz A, et al. Combined 5-HT1A and 5-HT1B receptor agonists for the treatment of L-DOPA-induced dyskinesia. Brain 2008;131(2):3380-94. † Similar study carried out in primates by Munoz et al * = significant versus sham/vehicle

11. Non-dyskinetic ratDyskinetic rat Prodynorphin mRNA (3 h to >2 weeks post-dosing) Phospho-ERK1/2 (15 min to 2 h post-dosing) ∆FosB (2 h to >2 weeks post-dosing) Abnormal molecular responses to levodopa in striatal neurons in dyskinetic animals Cenci MA, et al. Eur. J. Neurosci 1998;10(8):2694-2706. Andersson M, et al. J. Neurosci 2001;21(24):9930-9943. Andersson M, et al. Eur. J. Neurosci 2003;17(3): 661-666. Westin JE et al. (2007) Biol. Psychiatry 62: 800-810. Aubert I, et al. Biol Psychiatry 2007;61:836-44. Santini E, et al. Sci Signal 2009;21(2):ra36. (Images developed under the guidance of Dr Angela Cenci Nilsson). ERK1/2: extracellular signal- regulated kinases 1 and 2. Master regulator of neuronal plasticity. Become active when phosphorylated on specific amino acid. Causes changes in gene expression (via transcription factors and histone kinases) and changes in protein translation (via the mTOR pathway) ∆FosB: nuclear transcription factor Prodynorphin: effector gene (codes a neurotransmitter precursor) ERK 1/2 PDyn ∆FosB MSK-1Elk1

12. Enhanced response to dopamine Pulsatile dopaminergic stimulation leads to downstream changes in striatal dopamine neurones

13. Aetiology of dyskinesias: what we have learned from the laboratory Adapted from Thanvi B, et al. Postgrad Med J 2007;83:384-88

14. Nigrostriatal pathology Reduced dopamine storage capacity ‘Wearing-off’ (also postsynaptic changes can be important) early: several days effect late: only hours effect of oral levodopa Aetiology of ‘wearing off’: what we have learned from the laboratory

15. Mechanisms of levodopa-induced dyskinesia and clues to treatment Levodopa treatment Pulsatile drug delivery Microvascular remodeling Dopaminergic denervation Loss of nigrostriatal dopamine storage capacity Dopamine release from serotonin terminals Large intermittent surges of extracellular dopamine Abnormal signaling in striatal D1R-rich GABA+, Dyn+ neurons Altered firing patterns in GPi/SNr Drugs acting on post- synaptic receptors and/or signaling pathways (e.g. mGluR5 antagonists) New levodopa delivery methods (duodenal delivery; gene therapy) Deep brain stimulation 5HT1A and 1B agonists Dopamine-R supersensitivity Adapted from Cenci MA. Parkinsonism Relat Disord 2007;13 (Suppl 3):S263-7

16. Summary Pre-clinical studies have identified the potential mechanisms involved in the development of motor complications observed in patients with Parkinson’s disease In rodents and primates, the ‘wearing off’ phenomenon and dyskinesias are apparent with intermittent dopaminergic administration Basal ganglia undergo plastic changes following denervation and long-term pulsatile dopaminergic exposure These changes are at least partly reversible upon switching from pulsatile to continuous drug delivery