Martin Lundblad

Pharmacological validation of behavioural measures of akinesia and dyskinesia in a rat model of Parkinson's disease

In an attempt to define clinically relevant models of akinesia and dyskinesia in 6‐hydroxydopamine (6‐OHDA)‐lesioned rats, we have examined the effects of drugs with high (l‐DOPA) vs. low (bromocriptine) dyskinesiogenic potential in Parkinsons disease on three types of motor performance, namely: (i) abnormal involuntary movements (AIMs) (ii) rotational behaviour, and (iii) spontaneous forelimb use (cylinder test). Rats with unilateral 6‐OHDA lesions received single daily i.p. injections of l‐DOPA or bromocriptine at therapeutic doses. During 3 weeks of treatment, l‐DOPA but not bromocriptine induced increasingly severe AIMs affecting the limb, trunk and orofacial region. Rotational behaviour was induced to a much higher extent by bromocriptine than l‐DOPA. In the cylinder test, the two drugs initially improved the performance of the parkinsonian limb to a similar extent. However, l‐DOPA‐treated animals showed declining levels of performance in this test because the drug‐induced AIMs interfered with physiological limb use, and gradually replaced all normal motor activities. l‐DOPA‐induced axial, limb and orolingual AIM scores were significantly reduced by the acute administration of compounds that have antidyskinetic efficacy in parkinsonian patients and/or nonhuman primates (−91%, yohimbine 10 mg/kg; −19%, naloxone 4–8 mg/kg; −37%, 5‐methoxy 5‐N,N‐dimethyl‐tryptamine 2 mg/kg; −30%, clozapine 8 mg/kg; −50%, amantadine 40 mg/kg). l‐DOPA‐induced rotation was, however, not affected. The present results demonstrate that 6‐OHDA‐lesioned rats do exhibit motor deficits that share essential functional similarities with parkinsonian akinesia or dyskinesia. Such deficits can be quantified using novel and relatively simple testing procedures, whereas rotometry cannot discriminate between dyskinetic and antiakinetic effects of antiparkinsonian treatments.

Generation of Regionally Specified Neural Progenitors and Functional Neurons from Human Embryonic Stem Cells under Defined Conditions.

To model human neural-cell-fate specification and to provide cells for regenerative therapies, we have developed a method to generate human neural progenitors and neurons from human embryonic stem cells, which recapitulates human fetal brain development. Through the addition of a small molecule that activates canonical WNT signaling, we induced rapid and efficient dose-dependent specification of regionally defined neural progenitors ranging from telencephalic forebrain to posterior hindbrain fates. Ten days after initiation of differentiation, the progenitors could be transplanted to the adult rat striatum, where they formed neuron-rich and tumor-free grafts with maintained regional specification. Cells patterned toward a ventral midbrain (VM) identity generated a high proportion of authentic dopaminergic neurons after transplantation. The dopamine neurons showed morphology, projection pattern, and protein expression identical to that of human fetal VM cells grafted in parallel. VM-patterned but not forebrain-patterned neurons released dopamine and reversed motor deficits in an animal model of Parkinsons disease.

TFEB-mediated autophagy rescues midbrain dopamine neurons from α-synuclein toxicity.

Significance This study shows that neurodegenerative changes induced by α-synuclein in midbrain dopamine neurons in vivo can be blocked through activation of the autophagy-lysosome pathway. Using an adeno-associated virus model of Parkinson disease to overexpress α-synuclein in the substantia nigra, we show that genetic [transcription factor EB (TFEB) and Beclin-1 overexpression] or pharmacological (rapalog) manipulations that enhance autophagy protect nigral neurons from α-synuclein toxicity, but inhibiting autophagy exacerbates α-synuclein toxicity. The results provide a mechanistic link between α-synuclein toxicity and impaired TFEB function, and identify TFEB as a target for therapies aimed at neuroprotection and disease modification in Parkinson disease. The aggregation of α-synuclein plays a major role in Parkinson disease (PD) pathogenesis. Recent evidence suggests that defects in the autophagy-mediated clearance of α-synuclein contribute to the progressive loss of nigral dopamine neurons. Using an in vivo model of α-synuclein toxicity, we show that the PD-like neurodegenerative changes induced by excess cellular levels of α-synuclein in nigral dopamine neurons are closely linked to a progressive decline in markers of lysosome function, accompanied by cytoplasmic retention of transcription factor EB (TFEB), a major transcriptional regulator of the autophagy-lysosome pathway. The changes in lysosomal function, observed in the rat model as well as in human PD midbrain, were reversed by overexpression of TFEB, which afforded robust neuroprotection via the clearance of α-synuclein oligomers, and were aggravated by microRNA-128–mediated repression of TFEB in both A9 and A10 dopamine neurons. Delayed activation of TFEB function through inhibition of mammalian target of rapamycin blocked α-synuclein induced neurodegeneration and further disease progression. The results provide a mechanistic link between α-synuclein toxicity and impaired TFEB function, and highlight TFEB as a key player in the induction of α-synuclein–induced toxicity and PD pathogenesis, thus identifying TFEB as a promising target for therapies aimed at neuroprotection and disease modification in PD.

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.

Modulation of l-DOPA-induced abnormal involuntary movements by clinically tested compounds: Further validation of the rat dyskinesia model.

L-DOPA-induced dyskinesia (LID) is a major complication of the pharmacotherapy of Parkinsons Disease. A model of LID has recently been described in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. In the present study, the model was used in order to compare the efficacies of some clinically available compounds that have shown antidyskinetic effects in nonhuman primate models of LID and/or in patients, namely, amantadine (20 and 40 mg/kg), buspirone (1, 2 and 4 mg/kg), clonidine (0.01, 0.1 and 1 mg/kg), clozapine (4 and 8 mg/kg), fluoxetine (2.5 and 5 mg/kg), propranolol (5, 10 and 20mg/kg), riluzole (2 and 4 mg/kg), and yohimbine (2 and 10 mg/kg). Rats were treated for 3 weeks with L-DOPA for an induction and monitoring of abnormal involuntary movements (AIMs) prior to the drug screening experiments. The antidyskinetic drugs or their vehicles were administered together with L-DOPA, and their effects were evaluated according to a randomized cross-over design both on the AIM rating scale and on the rotarod test. Most of the compounds under investigation attenuated the L-DOPA-induced axial, limb and orolingual AIM scores. However, the highest doses of many of these substances (but for amantadine and riluzole) had also detrimental motor effects, producing a reduction in rotarod performance and locomotor scores. Since the present results correspond well to existing clinical and experimental data, this study indicates that axial, limb and orolingual AIMs possess predictive validity for the preclinical screening of novel antidyskinetic treatments. Combining tests of general motor performance with AIMs ratings in the same experiment allows for selecting drugs that specifically reduce dyskinesia without diminishing the anti-akinetic effect of L-DOPA.

Post- versus presynaptic plasticity in L-DOPA-induced dyskinesia

L‐3,4‐dihydroxyphenylalanine (L‐DOPA) remains the most efficacious drug for the treatment of Parkinsons disease (PD), but causes adverse effects that limit its utility. L‐DOPA‐induced dyskinesia (abnormal involuntary movements) is a significant clinical problem that attracts growing scientific interest. Current notions attribute the development of dyskinesia to two main factors, viz. the loss of nigrostriatal dopamine (DA) projections and the maladaptive changes produced by L‐DOPA at sites postsynaptic to the nigrostriatal neuron. Basic research in the past 15 years has placed a lot of emphasis on the postsynaptic plasticity associated with dyskinesia, but recent experimental work shows that also some presynaptic factors, involving the regulation of L‐DOPA/DA release and metabolism in the brain, may show plasticity during treatment. This review summarizes significant studies of L‐DOPA‐induced dyskinesia in patients and animal models, and outlines directions for future experiments addressing mechanisms of presynaptic plasticity. These investigations may uncover clues to the varying susceptibility to L‐DOPA‐induced dyskinesia among PD patients, paving the way for tailor‐made treatments.

Ratings of L‐DOPA‐Induced Dyskinesia in the Unilateral 6‐OHDA Lesion Model of Parkinson's Disease in Rats and Mice

This unit provides detailed protocols for establishing rodent models of L‐DOPA‐induced dyskinesia. The 6‐hydroxydopamine (6‐OHDA) lesion procedure is described in more detail for mice than for rats since the lesioning procedure in rats has been described extensively in previous work and is less difficult to perform. Unlike primate models, rodent models of L‐DOPA‐induced dyskinesia are relatively simple and fast to set up, thus being affordable to most laboratories. These models allow for studying the dyskinetic complications of L‐DOPA treatment on large groups of animals under strictly controlled experimental conditions. Along with information and structured protocols for the practical execution of the test, this unit provides a detailed description of the rating scale and the phenomenology of rodent abnormal involuntary movements, and suggestions for beginners. Curr. Protoc. Neurosci. 41:9.25.1‐9.25.23.

Impaired neurotransmission caused by overexpression of α-synuclein in nigral dopamine neurons

We used in vivo amperometry to monitor changes in synaptic dopamine (DA) release in the striatum induced by overexpression of human wild-type α-synuclein in nigral DA neurons, induced by injection of an adeno-associated virus type 6 (AAV6)–α-synuclein vector unilaterally into the substantia nigra in adult rats. Impairments in DA release evolved in parallel with the development of degenerative changes in the nigrostriatal axons and terminals. The earliest change, seen 10 d after vector injection, was a marked, ≈50%, reduction in DA reuptake, consistent with an early dysfunction of the DA transporter that developed before any overt signs of axonal damage. At 3 wk, when the first signs of axonal damage were observed, the amount of DA released after a KCl pulse was reduced by 70–80%, and peak DA concentration was delayed, indicating an impaired release mechanism. At later time points, 8–16 wk, overall striatal innervation density was reduced by 60–80% and accompanied by abundant signs of axonal damage in the form of α-synuclein aggregates, axonal swellings, and dystrophic axonal profiles. At this stage DA release and reuptake were profoundly reduced, by 80–90%. The early changes in synaptic DA release induced by overexpression of human α-synuclein support the idea that early predegenerative changes in the handling of DA may initiate, and drive, a progressive degenerative process that hits the axons and terminals first. Synaptic dysfunction and axonopathy would thus be the hallmark of presymptomatic and early-stage Parkinson disease, followed by neuronal degeneration and cell loss, characteristic of more advanced stages of the disease.

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.

Pharmacological validation of a mouse model of l-DOPA-induced dyskinesia.

Dyskinesia (abnormal involuntary movements) is a common complication of l-DOPA pharmacotherapy in Parkinsons disease, and is thought to depend on abnormal cell signaling in the basal ganglia. Dopamine (DA) denervated mice can exhibit behavioral and cellular signs of dyskinesia when they are treated with l-DOPA, but the clinical relevance of this animal model remains to be established. In this study, we have examined the pharmacological profile of l-DOPA-induced abnormal involuntary movements (AIMs) in the mouse. C57BL/6 mice sustained unilateral injections of 6-hydroxydopamine (6-OHDA) in the striatum. The animals were treated chronically with daily doses of l-DOPA that were sufficient to ameliorate akinetic features without inducing overt signs of dyskinesia upon their first administration. In parallel, other groups of mice were treated with antiparkinsonian agents that do not induce dyskinesia when administered de novo, that is, the D2/D3 agonist ropinirole, and the adenosine A2a antagonist KW-6002. During 3 weeks of treatment, l-DOPA-treated mice developed AIMs affecting the head, trunk and forelimb on the side contralateral to the lesion. These movements were not expressed by animals treated with ropinirole or KW-6002 at doses that improved forelimb akinesia. The severity of l-DOPA-induced rodent AIMs was significantly reduced by the acute administration of compounds that have been shown to alleviate l-DOPA-induced dyskinesia both in parkinsonian patients and in rat and monkey models of Parkinsons disease (amantadine, -47%; buspirone, -46%; riluzole, -33%). The present data indicate that the mouse AIMs are indeed a functional equivalent of l-DOPA-induced dyskinesia.