Striatal overexpression of DeltaJunD resets L-DOPA-induced dyskinesia in a primate model of Parkinson disease.

BACKGROUND: Involuntary movements, or dyskinesia, represent a debilitating complication of dopamine replacement therapy for Parkinson disease (PD). The transcription factor DeltaFosB accumulates in the denervated striatum and dimerizes primarily with JunD upon repeated L-3,4-dihydroxyphenylalanine (L-DOPA) administration. Previous studies in rodents have shown that striatal DeltaFosB levels accurately predict dyskinesia severity and indicate that this transcription factor may play a causal role in the dyskinesia sensitization process. METHODS: We asked whether the correlation previously established in rodents extends to the best nonhuman primate model of PD, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned macaque. We used western blotting and quantitative polymerase chain reaction (PCR) to compare DeltaFosB protein and messenger RNA (mRNA) levels across two subpopulations of macaques with differential dyskinesia severity. Second, we tested the causal implication of DeltaFosB in this primate model. Serotype 2 adeno-associated virus (AAV2) vectors were used to overexpress, within the motor striatum, either DeltaFosB or DeltaJunD, a truncated variant of JunD lacking a transactivation domain and therefore acting as a dominant negative inhibitor of DeltaFosB. RESULTS: A linear relationship was observed between endogenous striatal levels of DeltaFosB and the severity of dyskinesia in Parkinsonian macaques treated with L-DOPA. Viral overexpression of DeltaFosB did not alter dyskinesia severity in animals previously rendered dyskinetic, whereas the overexpression of DeltaJunD dramatically dropped the severity of this side effect of L-DOPA without altering the antiparkinsonian activity of the treatment. CONCLUSIONS: These results establish a mechanism of dyskinesia induction and maintenance by L-DOPA and validate a strategy, with strong translational potential, to deprime the L-DOPA-treated brain.

Dopamine-deprived striatal GABAergic interneurons burst and generate repetitive gigantic IPSCs in medium spiny neurons.

Striatal GABAergic microcircuits modulate cortical responses and movement execution in part by controlling the activity of medium spiny neurons (MSNs). How this is altered by chronic dopamine depletion, such as in Parkinson's disease, is not presently understood. We now report that, in dopamine-depleted slices of the striatum, MSNs generate giant spontaneous postsynaptic GABAergic currents (single or in bursts at 60 Hz) interspersed with silent episodes, rather than the continuous, low-frequency GABAergic drive (5 Hz) observed in control MSNs. This shift was observed in one-half of the MSN population, including both "D(1)-negative" and "D(1)-positive" MSNs. Single GABA and NMDA channel recordings revealed that the resting membrane potential and reversal potential of GABA were similar in control and dopamine-depleted MSNs, and depolarizing, but not excitatory, actions of GABA were observed. Glutamatergic and cholinergic antagonists did not block the GABAergic oscillations, suggesting that they were generated by GABAergic neurons. In support of this, cell-attached recordings revealed that a subpopulation of intrastriatal GABAergic interneurons generated bursts of spikes in dopamine-deprived conditions. This subpopulation included low-threshold spike interneurons but not fast-spiking interneurons, cholinergic interneurons, or MSNs. Therefore, a population of local GABAergic interneurons shifts from tonic to oscillatory mode when dopamine deprived and gives rise to spontaneous repetitive giant GABAergic currents in one-half the MSNs. We suggest that this may in turn alter integration of cortical signals by MSNs.

Involvement of canonical and non-canonical D1 dopamine receptor signalling pathways in L-dopa-induced dyskinesia.

Chronic L-3,4-dihydroxyphenylalanine (L-dopa) treatment of Parkinson Disease (PD) often leads to debilitating involuntary movements, termed L-dopa-induced dyskinesia (LID). The past few years have seen an unprecedented increase in understanding the neural mechanisms underlying LID manifestation in PD associating them mostly with D1 dopamine (DA) receptor sensitisation and deregulated homologous desensitisation as well as hyperactivity of both canonical and non-canonical DA signalling pathways. We here review these recent findings and demonstrate that decreasing DA receptor-mediated signalling (i) by increasing D1 receptor internalization and (ii) by inhibiting the Ras-Extracellular signal-Regulated Kinase 1/2 non-canonical DA signalling cascade, might reduced LID severity. Strategy (i) uses the lentivirus-mediated over-expression of the G protein-coupled receptor kinase 6 that control the desensitisation of DA receptors. Strategy (ii) proposes to use statins that, besides being specific inhibitors of the rate-limiting enzyme in cholesterol biosynthesis, can also inhibit Ras isoprenylation and activity and subsequently the phosphorylation of ERK1/2. Experiments were performed in both the rodent and primate models of LID. Those results strongly suggest that different strategies might represent a treatment option for managing LID in PD.

Striatal proteomic analysis suggests that first L-dopa dose equates to chronic exposure.

L-3,4-dihydroxypheylalanine (L-dopa)-induced dyskinesia represent a debilitating complication of therapy for Parkinson's disease (PD) that result from a progressive sensitization through repeated L-dopa exposures. The MPTP macaque model was used to study the proteome in dopamine-depleted striatum with and without subsequent acute and chronic L-dopa treatment using two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry. The present data suggest that the dopamine-depleted striatum is so sensitive to de novo L-dopa treatment that the first ever administration alone would be able (i) to induce rapid post-translational modification-based proteomic changes that are specific to this first exposure and (ii), possibly, lead to irreversible protein level changes that would be not further modified by chronic L-dopa treatment. The apparent equivalence between first and chronic L-dopa administration suggests that priming would be the direct consequence of dopamine loss, the first L-dopa administrations only exacerbating the sensitization process but not inducing it.

Impact of chronic subthalamic high-frequency stimulation on metabolic basal ganglia activity: a 2-deoxyglucose uptake and cytochrome oxidase mRNA study in a macaque model of Parkinson's disease.

The mechanisms of action of high-frequency stimulation (HFS) of the subthalamic nucleus (STN) remain only partially understood. Hitherto, experimental studies have suggested that STN-HFS reduces the activity of STN neurons. However, some recent reports have challenged this view, showing that STN-HFS might also increase the activity of globus pallidus internalis (GPi) neurons that are under strong excitatory drive of the STN. In addition, most results emanate from studies applying acute STN-HFS, while parkinsonian patients receive chronic stimulation. Thus, the present study was designed to assess the effect of chronic (10 days) STN-HFS in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated nonhuman primate. For this purpose, 2-deoxyglucose (2-DG) uptake, a measure of global synaptic activity, was assessed in the basal ganglia and the motor thalamus after chronic unilateral STN-HFS. Cytochrome oxidase subunit 1 (COI) mRNA expression, a marker of efferent metabolic activity, was additionally assessed in the globus pallidus. Chronic STN-HFS (i) reversed abnormally decreased 2-DG uptake in the STN of parkinsonian nonhuman primates, (ii) reversed abnormally increased 2-DG accumulation in the GPi while COI mRNA expression was increased, suggesting global activation of GPi neurons, and (iii) reversed abnormally increased 2-DG uptake in the ventrolateral motor thalamus nucleus. The simultaneous decrease in 2-DG uptake and increase in COI mRNA expression are difficult to reconcile with the current model of basal ganglia function and suggest that the mechanisms by which STN-HFS exerts its clinical benefits are more complex than a simple reversal of abnormal activity in the STN and its targets.

Altered D(1) dopamine receptor trafficking in parkinsonian and dyskinetic non-human primates.

Dyskinesias represent a debilitating complication of levodopa therapy for Parkinson's disease (PD). While we recently demonstrated that levodopa-induced dyskinesia results from increased dopamine D(1) receptor-mediated transmission, we also questioned the possible role of subcellular localization of D(1) and D(2) receptors in mediating these effects as we previously showed that D(1) receptors undergo differential trafficking in striatal neurons of non-dyskinetic PD patients. Taking advantage of a monkey brain bank, we here report changes affecting the cellular and subcellular distribution of D(1) and D(2) dopamine receptors within the striatum of three experimental groups: normal, parkinsonian and dyskinetic L-dopa-treated parkinsonian animals. Our studies at both light and electron microscopy levels show a recruitment of D(1) receptor at the plasma membrane of striatal neurons in the parkinsonian animals and a strong increase of D(1) expression both at the membrane and in cytoplasm of dyskinetic animals, whereas D(2) receptor distribution is only modestly affected in all conditions. Our results rule out the hypothesis of a pathological overinternalization of dopamine receptors in levodopa-induced dyskinesia but raise the possibility for involvement of D(1) receptors in the priming phenomenon through massive and sudden internalization in response to the first ever administration of L-dopa and for an altered homologous desensitization mechanism in dyskinesia leading to an increased availability of D(1) receptors at membrane. Further experiments including parkinsonian monkeys chronically treated with L-dopa that show no dyskinesia and parkinsonian monkeys treated only once with L-dopa are now necessary to confirm our hypothesis.

Enhanced preproenkephalin-B-derived opioid transmission in striatum and subthalamic nucleus converges upon globus pallidus internalis in L-3,4-dihydroxyphenylalanine-induced dyskinesia.

BACKGROUND: A role for enhanced opioid peptide transmission has been suggested in the genesis of levodopa-induced dyskinesia. However, basal ganglia nuclei other than the striatum have not been regarded as potential sources, and the opioid precursors have never been quantified simultaneously with the levels of opioid receptors at the peak of dyskinesia severity. METHODS: The levels of messenger RNA (mRNA) encoding the opioid precursors preproenkephalin-A and preproenkephalin-B in the striatum and the subthalamic nucleus and the levels of mu, delta, and kappa opioid receptors were measured within the basal ganglia of four groups of nonhuman primates killed at the peak of effect: normal, parkinsonian, parkinsonian chronically-treated with levodopa without exhibiting dyskinesia, and parkinsonian chronically-treated with levodopa showing overt dyskinesia. RESULTS: Dyskinesia are associated with reduction in opioid receptor binding and specifically of kappa and mu receptor binding in the globus pallidus internalis (GPi), the main output structure of the basal ganglia. This decrease was correlated with enhancement of the expression of preproenkephalin-B mRNA but not that of preproenkephalin-A in the striatum and the subthalamic nucleus. CONCLUSIONS: Abnormal transmission of preproenkephalin-B-derived opioid coming from the striatum and the subthalamic nucleus converges upon GPi at the peak of dose to induce levodopa-induced dyskinesia.

Rotigotine treatment partially protects from MPTP toxicity in a progressive macaque model of Parkinson's disease.

Clinical DA agonist monotherapy trials, which used in vivo imaging of the DA transporter (DAT) to assess the rate of progression of nigrostriatal degeneration, have failed to demonstrate consistent evidence for neuroprotection. The present study aims at reconciling these experimental and clinical data by testing the protective property of the continuously delivered D3/D2/D1 dopamine receptor agonist rotigotine. Using a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned (MPTP) macaque model that mimics the progression of Parkinson's disease in vivo ([99mTc]-TRODAT-1 single photon emission computed tomography (SPECT)) and ex vivo ([125I]-nortropane DAT labelling) endpoints were evaluated. After 38 days of treatment followed by two weeks of washout, rotigotine-treated animals were significantly less parkinsonian than the vehicle-treated ones. Such behavioural difference is the consequence of a partial protection of the DA terminals as could be confirmed by ex vivo DAT labelling. However, the protection of nerve terminals was not detected using SPECT. The data suggest that rotigotine exerts partial protection but that conventional imaging would not be able to identify such protection.

Phenotype of striatofugal medium spiny neurons in parkinsonian and dyskinetic nonhuman primates: a call for a reappraisal of the functional organization of the basal ganglia.

The classic view of anatomofunctional organization of the basal ganglia is that striatopallidal neurons of the "indirect" pathway express D2 dopamine receptors and corelease enkephalin with GABA, whereas striatopallidal neurons of the "direct" pathway bear D1 dopamine receptors and corelease dynorphin and substance P with GABA. Although many studies have investigated the pathophysiology of the basal ganglia after dopamine denervation and subsequent chronic levodopa (L-dopa) treatment, none has ever considered the possibility of plastic changes leading to profound reorganization and/or biochemical phenotype modifications of medium spiny neurons. Therefore, we studied the phenotype of striatal neurons in four groups of nonhuman primates, including the following: normal, parkinsonian, parkinsonian chronically treated with L-dopa without exhibiting dyskinesia, and parkinsonian chronically treated with L-dopa exhibiting overt dyskinesia. To identify striatal cells projecting to external (indirect) or internal (direct) segments of the globus pallidus, the retrograde tracer cholera toxin subunit B (CTb) was injected stereotaxically into the terminal areas. Using immunohistochemistry techniques, brain sections were double labeled for CTb and dopamine receptors, opioid peptides, or the substance P receptor (NK1). We also used HPLC-RIA to assess opioid levels throughout structures of the basal ganglia. Our results suggest that medium spiny neurons retain their phenotype because no variations were observed in any experimental condition. Therefore, it appears unlikely that dyskinesia is related to a phenotype modification of the striatal neurons. However, this study supports the concept of axonal collateralization of striatofugal cells that project to both globus pallidus pars externa and globus pallidus pars interna. Striatofugal pathways are not as segregated in the primate as previously considered.

Levodopa-induced dyskinesia in MPTP-treated macaques is not dependent on the extent and pattern of nigrostrial lesioning.

The extent of nigrostriatal denervation is presumed to play a role in the genesis of levodopa-induced dyskinesia. Yet some parkinsonian patients who have been treated over a long period do not develop dyskinesia, raising the possibility that the pattern of denervation is as important as the extent of lesioning as a risk factor. Here we study the extent and pattern of nigrostriatal denervation in a homogeneous population of parkinsonian macaque monkeys chronically treated with levodopa. Based on the characteristics of the lesioning, non-dyskinetic animals could not be differentiated from those with dyskinesia. Indeed, the number of tyrosine-hydroxylase (TH)-immunopositive neurons in the substantia nigra pars compacta, striatal dopamine transporter (DAT) binding and TH immunostaining, as well as the overall TH striatal content measured by Western blotting were identical. Moreover, the patterns of lesioning assessed by a detailed analysis of the TH- and DAT-immunopositive striatal fibers were comparable in all functional quadrants and at all rostro-caudal levels considered. These data indicate that neither the extent nor the pattern of nigrostriatal lesioning are sufficient to explain the occurrence of levodopa-induced dyskinesia.

Involvement of sensorimotor, limbic, and associative basal ganglia domains in L-3,4-dihydroxyphenylalanine-induced dyskinesia.

Dyskinesia represents a debilitating complication of L-3,4-dihydroxyphenylalanine (L-dopa) therapy for Parkinson's disease. Such motor manifestations are attributed to pathological activity in the motor parts of basal ganglia. However, because consistent funneling of information takes place between the sensorimotor, limbic, and associative basal ganglia domains, we hypothesized that nonmotor domains play a role in these manifestations. Here we report the changes in 2-deoxyglucose (2-DG) accumulation in the sensorimotor, limbic, and associative domains of basal ganglia and thalamic nuclei of four groups of nonhuman primates: normal, parkinsonian, parkinsonian chronically treated with L-dopa without exhibiting dyskinesia, and parkinsonian chronically treated with L-dopa and exhibiting overt dyskinesia. Although nondyskinetic animals display a rather normalized metabolic activity, dyskinetic animals are distinguished by significant changes in 2-DG accumulation in limbic- and associative-related structures and not simply in sensorimotor-related ones, suggesting that dyskinesia is linked to a pathological processing of limbic and cognitive information. We propose that these metabolic changes reflect the underlying neural mechanisms of not simply motor dyskinesias but also affective, motivational, and cognitive disorders associated with long-term exposure to L-dopa.

Increased D1 dopamine receptor signaling in levodopa-induced dyskinesia.

Involuntary movements, or dyskinesia, represent a debilitating complication of levodopa therapy for Parkinson's disease. Although changes affecting D(1) and D(2) dopamine receptors have been studied in association with this condition, no causal relationship has yet been established. Taking advantage of a monkey brain bank constituted to study levodopa-induced dyskinesia, we report changes affecting D(1) and D(2) dopamine receptors within the striatum of normal, parkinsonian, nondyskinetic levodopa-treated parkinsonian, and dyskinetic levodopa-treated parkinsonian animals. Whereas D(1) receptor expression itself is not related to dyskinesia, D(1) sensitivity per D(1) receptor measured by D(1) agonist-induced [(35)S]GTPgammaS binding is linearly related to dyskinesia. Moreover, the striata of dyskinetic animals show higher levels of cyclin-dependent kinase 5 (Cdk5) and of the dopamine- and cAMP-regulated phosphoprotein of 32kDa (DARPP-32). Our data suggest that levodopa-induced dyskinesia results from increased dopamine D(1) receptor-mediated transmission at the level of the direct pathway.

Rabies virus is not cytolytic for rat spinal motoneurons in vitro.

Cultures of purified rat embryonic spinal cord motoneurons were used to investigate the capacity of the neurons to survive rabies virus infection in vitro. In crude primary spinal cord cultures, neurons did not survive more than 2 days after rabies virus infection with the fixed strain Challenge Virus Standard. In contrast, virus-infected purified motoneurons resisted cytolysis for at least 7 days, as also did infected motoneurons treated with conditioned medium sampled from rabies virus-infected crude spinal cord cultures. This survival rate was also observed when motoneurons were grown in the presence of astrocytes or fibroblasts and it was not dependent on the presence of growth factors in the culture medium. Moreover, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling experiments showed that only 30% of infected motoneurons were apoptotic after 7 days of infection. In vivo, despite the massive infection of the spinal cord in infected rat neonates, the moderate number of apoptotic cells in the ventral horn suggests that only a few motoneurons were affected by this mechanism of cell death. Morphometric analyses showed that motoneurons' axon elongated at a comparable rate in virus-infected and noninfected cultures, a sign of high metabolic activity maintained in rabies virus-infected motoneurons. In contrast, hippocampus neurons were susceptible to rabies virus infection, because 70% of infected neurons were destroyed within 3 days, a large proportion of them being apoptotic. These experiments suggest that spinal cord motoneurons consist in a neuronal population that survive rabies virus infection because the viral induction of apoptosis is delayed in these neurons. They suggest also that paralyses frequently observed in rabid animals could be the consequence of dysfunctions of the locomotor network or of the spinal cord motoneurons themselves, whose parameters could be studied in vitro.