Modeling Parkinson's disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain.

Although a causative role of α-synuclein (α-syn) is well established in Parkinson's disease pathogenesis, available animal models of synucleinopathy do not replicate the full range of cellular and behavioral changes characteristic of the human disease. This study was designed to generate a more faithful model of Parkinson's disease by injecting human α-syn fibril seeds into the rat substantia nigra (SN), in combination with adenoassociated virus (AAV)-mediated overexpression of human α-syn, at levels that, by themselves, are unable to induce acute dopamine (DA) neurodegeneration. We show that the ability of human α-syn fibrils to trigger Lewy-like α-synuclein pathology in the affected DA neurons is dramatically enhanced in the presence of elevated levels of human α-syn. This synucleinopathy was fully developed already 10 days after fibril injection, accompanied by progressive degeneration of dopaminergic neurons in SN, neuritic swelling, reduced striatal DA release, and impaired motor behavior. Moreover, a prominent inflammatory response involving both activation of resident microglia and infiltration of CD4+ and CD8+ T lymphocytes was observed. Hypertrophic microglia were found to enclose or engulf cells and processes containing Lewy-like α-syn aggregates. α-Syn aggregates were also observed inside these cells, suggesting transfer of phosphorylated α-syn from the affected nigral neurons. The nigral pathology triggered by fibrils in combination with AAV-mediated overexpression of α-syn reproduced many of the cardinal features of the human disease. The short time span and the distinct sequence of pathological and degenerative changes make this combined approach attractive as an experimental model for the assessment of neuroprotective and disease-modifying strategies.

DREADD Modulation of Transplanted DA Neurons Reveals a Novel Parkinsonian Dyskinesia Mechanism Mediated by the Serotonin 5-HT6 Receptor.

Transplantation of DA neurons is actively pursued as a restorative therapy in Parkinson's disease (PD). Pioneering clinical trials using transplants of fetal DA neuroblasts have given promising results, although a number of patients have developed graft-induced dyskinesias (GIDs), and the mechanism underlying this troublesome side effect is still unknown. Here we have used a new model where the activity of the transplanted DA neurons can be selectively modulated using a bimodal chemogenetic (DREADD) approach, allowing either enhancement or reduction of the therapeutic effect. We show that exclusive activation of a cAMP-linked (Gs-coupled) DREADD or serotonin 5-HT6 receptor, located on the grafted DA neurons, is sufficient to induce GIDs. These findings establish a mechanistic link between the 5-HT6 receptor, intracellular cAMP, and GIDs in transplanted PD patients. This effect is thought to be mediated through counteraction of the D2 autoreceptor feedback inhibition, resulting in a dysplastic DA release from the transplant.

Progressive nigrostriatal terminal dysfunction and degeneration in the engrailed1 heterozygous mouse model of Parkinson's disease.

Current research on Parkinson's disease (PD) pathogenesis requires relevant animal models that mimic the gradual and progressive development of neuronal dysfunction and degeneration that characterizes the disease. Polymorphisms in engrailed 1 (En1), a homeobox transcription factor that is crucial for both the development and survival of mesencephalic dopaminergic neurons, are associated with sporadic PD. This suggests that En1 mutant mice might be a promising candidate PD model. Indeed, a mouse that lacks one En1 allele exhibits decreased mitochondrial complex I activity and progressive midbrain dopamine neuron degeneration in adulthood, both features associated with PD. We aimed to further characterize the disease-like phenotype of these En1(+/-) mice with a focus on early neurodegenerative changes that can be utilized to score efficacy of future disease modifying studies. We observed early terminal defects in the dopaminergic nigrostriatal pathway in En1(+/-) mice. Several weeks before a significant loss of dopaminergic neurons in the substantia nigra could be detected, we found that striatal terminals expressing high levels of dopaminergic neuron markers TH, VMAT2, and DAT were dystrophic and swollen. Using transmission electron microscopy, we identified electron dense bodies consistent with abnormal autophagic vacuoles in these terminal swellings. In line with these findings, we detected an up-regulation of the mTOR pathway, concurrent with a downregulation of the autophagic marker LC3B, in ventral midbrain and nigral dopaminergic neurons of the En1(+/-) mice. This supports the notion that autophagic protein degradation is reduced in the absence of one En1 allele. We imaged the nigrostriatal pathway using the CLARITY technique and observed many fragmented axons in the medial forebrain bundle of the En1(+/-) mice, consistent with axonal maintenance failure. Using in vivo electrochemistry, we found that nigrostriatal terminals in the dorsal striatum were severely deficient in dopamine release and reuptake. Our findings support a progressive retrograde degeneration of En1(+/-) nigrostriatal neurons, akin to what is suggested to occur in PD. We suggest that using the En1(+/-) mice as a model will provide further key insights into PD pathogenesis, and propose that axon terminal integrity and function can be utilized to estimate dopaminergic neuron health and efficacy of experimental PD therapies.

Highly efficient generation of induced neurons from human fibroblasts that survive transplantation into the adult rat brain.

Induced neurons (iNs) offer a novel source of human neurons that can be explored for applications of disease modelling, diagnostics, drug screening and cell replacement therapy. Here we present a protocol for highly efficient generation of functional iNs from fetal human fibroblasts, and also demonstrate the ability of these converted human iNs (hiNs) to survive transplantation and maintain their phenotype in the adult rat brain. The protocol encompasses a delay in transgene activation after viral transduction that resulted in a significant increase in conversion efficiency. Combining this approach with treatment of small molecules that inhibit SMAD signalling and activate WNT signalling provides a further increase in the conversion efficiency and neuronal purity, resulting in a protocol that provides a highly efficient method for the generation of large numbers of functional and transplantable iNs from human fibroblasts without the use of a selection step. When transplanting the converted neurons from different stages of in vitro culture into the brain of adult rats, we observed robust survival and maintenance of neuronal identity four weeks post-transplantation. Interestingly, the positive effect of small molecule treatment observed in vitro did not result in a higher yield of iNs surviving transplantation.

Dynamic changes in dopamine neuron function after DNSP-11 treatment: effects in vivo and increased ERK 1/2 phosphorylation in vitro.

Glial cell-line derived neurotrophic factor (GDNF) has demonstrated robust effects on dopamine (DA) neuron function and survival. A post-translational processing model of the human GDNF proprotein theorizes the formation of smaller, amidated peptide(s) from the proregion that exhibit neurobiological function, including an 11-amino-acid peptide named dopamine neuron stimulating peptide-11 (DNSP-11). A single treatment of DNSP-11 was delivered to the substantia nigra in the rat to investigate effects on DA-neuron function. Four weeks after treatment, potassium (K+) and D-amphetamine evoked DA release were studied in the striatum using microdialysis. There were no significant changes in DA-release after DNSP-11 treatment determined by microdialysis. Dopamine release was further examined in discrete regions of the striatum using high-speed chronoamperometry at 1-, 2-, and 4-weeks after DNSP-11 treatment. Two weeks after DNSP-11 treatment, potassium-evoked DA release was increased in specific subregions of the striatum. However, spontaneous locomotor activity was unchanged by DNSP-11 treatment. In addition, we show that a single treatment of DNSP-11 in the MN9D dopaminergic neuronal cell line results in phosphorylation of ERK1/2, which suggests a novel cellular mechanism responsible for increases in DA function.

A novel α-synuclein-GFP mouse model displays progressive motor impairment, olfactory dysfunction and accumulation of α-synuclein-GFP.

Compelling evidence suggests that accumulation and aggregation of alpha-synuclein (α-syn) contribute to the pathogenesis of Parkinson's disease (PD). Here, we describe a novel Bacterial Artificial Chromosome (BAC) transgenic model, in which we have expressed wild-type human α-syn fused to green fluorescent protein (GFP), under control of the mouse α-syn promoter. We observed a widespread and high expression of α-syn-GFP in multiple brain regions, including the dopaminergic neurons of the substantia nigra pars compacta (SNpc) and the ventral tegmental area, the olfactory bulb as well as in neocortical neurons. With increasing age, transgenic mice exhibited reductions in amphetamine-induced locomotor activity in the open field, impaired rotarod performance and a reduced striatal dopamine release, as measured by amperometry. In addition, they progressively developed deficits in an odor discrimination test. Western blot analysis revealed that α-syn-GFP and phospho-α-syn levels increased in multiple brain regions, as the mice grew older. Further, we observed, by immunohistochemical staining for phospho-α-syn and in vivo by two-photon microscopy, the formation of α-syn aggregates as the mice aged. The latter illustrates that the model can be used to track α-syn aggregation in vivo. In summary, this novel BAC α-syn-GFP model mimics a unique set of aspects of PD progression combined with the possibility of tracking α-syn aggregation in neocortex of living mice. Therefore, this α-syn-GFP-mouse model can provide a powerful tool that will facilitate the study of α-syn biology and its involvement in PD pathogenesis.

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

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.

Striatal glutamate release in L-DOPA-induced dyskinetic animals.

L-DOPA-induced dyskinesia is a common side effect developed after chronic treatment with 3,4-dihydroxyphenyl-l-alanine (l-DOPA) in Parkinson's disease. The biological mechanisms behind this side effect are not fully comprehended although involvement of dopaminergic, serotonergic, and glutamatergic systems has been suggested. The present study utilizes in vivo amperometry to investigate the impact from unilateral 6-hydroxydopamine lesions and l-DOPA (4 mg/kg, including benserazide 15 mg/kg) -induced dyskinetic behavior on striatal basal extracellular glutamate concentration and potassium-evoked glutamate release in urethane-anesthetized rats. Recordings were performed before and after local L-DOPA application in the striatum. In addition, effects from the 5-HT(1A) receptor agonist (2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OHDPAT; 1 mg/kg) was assessed on glutamate release and on dyskinetic behavior. The results revealed a bilateral ≈ 30% reduction of basal extracellular glutamate concentration and attenuated potassium-evoked glutamate release after a unilateral dopamine-depletion in L-DOPA naïve animals. In dyskinetic subjects, basal glutamate concentration was comparable to normal controls, although potassium-evoked glutamate release was reduced to similar levels as in drug naïve dopamine-lesioned animals. Furthermore, acute striatal L-DOPA administration attenuated glutamate release in all groups, except in the dopamine-lesioned striatum of dyskinetic animals. Co-administration of 8-OHDPAT and L-DOPA decreased dyskinesia in dopamine-lesioned animals, but did not affect potassium-evoked glutamate release, which was seen in normal animals. These findings indicate altered glutamate transmission upon dopamine-depletion and dyskinesia.

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 Parkinson's disease.

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.

Dopamine release from serotonergic nerve fibers is reduced in L-DOPA-induced dyskinesia.

L-DOPA is the most commonly used treatment for symptomatic control in patients with Parkinson's disease. Unfortunately, most patients develop severe side-effects, such as dyskinesia, upon chronic l-DOPA treatment. The patophysiology of dyskinesia is unclear; however, involvement of serotonergic nerve fibers in converting l-DOPA to dopamine has been suggested. Therefore, potassium-evoked dopamine release was studied after local application of l-DOPA in the striata of normal, dopamine- and dopamine/serotonin-lesioned l-DOPA naïve, and dopamine-denervated chronically l-DOPA-treated dyskinetic rats using in vivo chronoamperometry. The results revealed that local l-DOPA administration into normal and intact hemisphere of dopamine-lesioned l-DOPA naïve animals significantly increased the potassium-evoked dopamine release. l-DOPA application also increased the dopamine peak amplitude in the dopamine-depleted l-DOPA naïve striatum, although these dopamine levels were several-folds lower than in the normal striatum, whereas no increased dopamine release was found in the dopamine/serotonin-denervated striatum. In dyskinetic animals, local l-DOPA application did not affect the dopamine release, resulting in significantly attenuated dopamine levels compared with those measured in l-DOPA naïve dopamine-denervated striatum. To conclude, l-DOPA is most likely converted to dopamine in serotonergic nerve fibers in the dopamine-depleted striatum, but the dopamine release is several-fold lower than in normal striatum. Furthermore, l-DOPA loading does not increase the dopamine release in dyskinetic animals as found in l-DOPA naïve animals, despite similar density of serotonergic innervation. Thus, the dopamine overflow produced from the serotonergic nerve fibers appears not to be the major cause of dyskinetic behavior.

VGLUT2 in dopamine neurons is required for psychostimulant-induced behavioral activation.

The "One neuron-one neurotransmitter" concept has been challenged frequently during the last three decades, and the coexistence of neurotransmitters in individual neurons is now regarded as a common phenomenon. The functional significance of neurotransmitter coexistence is, however, less well understood. Several studies have shown that a subpopulation of dopamine (DA) neurons in the ventral tegmental area (VTA) expresses the vesicular glutamate transporter 2 (VGLUT2) and has been suggested to use glutamate as a cotransmitter. The VTA dopamine neurons project to limbic structures including the nucleus accumbens, and are involved in mediating the motivational and locomotor activating effects of psychostimulants. To determine the functional role of glutamate cotransmission by these neurons, we deleted VGLUT2 in DA neurons by using a conditional gene-targeting approach in mice. A DAT-Cre/Vglut2Lox mouse line (Vglut2(f/f;DAT-Cre) mice) was produced and analyzed by in vivo amperometry as well as by several behavioral paradigms. Although basal motor function was normal in the Vglut2(f/f;DAT-Cre) mice, their risk-taking behavior was altered. Interestingly, in both home-cage and novel environments, the gene targeted mice showed a greatly blunted locomotor response to the psychostimulant amphetamine, which acts via the midbrain DA system. Our results show that VGLUT2 expression in DA neurons is required for normal emotional reactivity as well as for psychostimulant-mediated behavioral activation.

Chronic intermittent L-DOPA treatment induces changes in dopamine release.

3,4-Dihydroxyphenyl-l-alanine (l-DOPA)-induced dyskinesia often develops as a side effect of chronic l-DOPA therapy. This study was undertaken to investigate dopamine (DA) release upon l-DOPA treatment. Chronoamperometric measurements were performed in unilaterally DA-depleted rats, chronically treated with l-DOPA, resulting in dyskinetic and non-dyskinetic animals. Normal and lesioned l-DOPA naïve animals were used as controls. Potassium-evoked DA releases were significantly reduced in intact sides of animals undertaken chronic l-DOPA treatment, independent on dyskinetic behavior. Acute l-DOPA further attenuated the amplitude of the DA release in the control sides. In DA-depleted striata, no difference was found in potassium-evoked DA releases, and acute l-DOPA did not affect the amplitude. While immunoreactivity to serotonin uptake transporter was higher in lesioned striata of animals displaying dyskinetic behavior, no correlation could be documented between serotonin transporter-positive nerve fiber density and the amplitude of released DA. In conclusions, the amplitude of potassium-evoked DA release is attenuated in intact striatum after chronic intermittent l-DOPA treatment. No change in amplitude was found in DA-denervated sides of either dyskinetic or non-dyskinetic animals, while release kinetics were changed. This indicates the importance of studying DA release dynamics for the understanding of both beneficial and adverse effects of l-DOPA replacement therapy.

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: alphabeta-crystalin, gamma-enolase, guanidoacetate methyltransferase, vinculin, and proteasome alpha-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.

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 Parkinson's 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.

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.

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.

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

L-3,4-dihydroxyphenylalanine (L-DOPA) remains the most efficacious drug for the treatment of Parkinson's 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.

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 Parkinson's disease.

Graft placement and uneven pattern of reinnervation in the striatum is important for development of graft-induced dyskinesia.

In two recent double-blind clinical trials of fetal ventral mesencephalic cell transplants into the striatum in patients with Parkinson's disease (PD), a significant proportion of the grafted patients developed dyskinetic side effects, which were not seen in the sham operated patients. Comparison between dyskinetic and non-dyskinetic grafted patients in one of the trials suggested that an uneven pattern of striatal reinnervation might be the leading cause of the dyskinesias. Here, we studied the importance of graft placement for the development of dyskinesias in parkinsonian rats. Abnormal involuntary movements resembling peak-dose dyskinesias seen in PD patients were induced by daily injections of L-DOPA for 6 weeks. The dyskinetic animals received about 130.000 fetal ventral mesencephalic cells as single grafts placement in the rostral or the caudal aspect of the head of striatum. The results show that grafts placed in the caudal, but not the rostral, part are effective in reducing the L-DOPA-induced limb and orolingual dyskinesia, predominantly seen as hyperkinesia. The same grafts, however, also induced a new type of dyskinetic behavior after activation with amphetamine, which were not seen in non-grafted lesion controls. The severity of these abnormal involuntary movements was significantly correlated with a higher graft-derived dopaminergic reinnervation in the caudal aspect of the head of striatum relative to the rostral part. The results indicate that graft-induced dyskinesias in PD patients may be linked to single, small graft deposits that provide an uneven, patchy reinnervation of the putamen.