Incarnation Aubert

Increased D1 dopamine receptor signaling in levodopa-induced dyskinesia

Involuntary movements, or dyskinesia, represent a debilitating complication of levodopa therapy for Parkinsons disease. Although changes affecting D1 and D2 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 D1 and D2 dopamine receptors within the striatum of normal, parkinsonian, nondyskinetic levodopa‐treated parkinsonian, and dyskinetic levodopa‐treated parkinsonian animals. Whereas D1 receptor expression itself is not related to dyskinesia, D1 sensitivity per D1 receptor measured by D1 agonist‐induced [35S]GTPγS 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 D1 receptor–mediated transmission at the level of the direct pathway. Ann Neurol 2004

Phenotypical characterization of the neurons expressing the D1 and D2 dopamine receptors in the monkey striatum

The striatum is regulated by dopaminergic inputs from the substantia nigra. Several anatomical studies using in situ hybridization have demonstrated that in rodents, dopamine D1 and D2 receptors are segregated into distinct striatal efferent populations: dopamine D1 receptor into γ‐aminobutyric acid (GABA)/substance P striatonigral neurons, and dopamine D2 receptor into GABA/enkephalin striatopallidal neurons. The existence of such a segregation has not been investigated in primates. Therefore, to quantify the efferent striatal GABAergic neurons in the adult Cynomolgus monkey, we detected GAD67 mRNA expression while considering that only a minority of the GABAergic population is composed of interneurons. To characterize the peptidergic phenotype of the neurons expressing dopamine D1 or D2 receptors, we examined the mRNA coding for these receptors in the striatum at the cellular level using single‐ and double in situ hybridization with digoxigenin and 35S ribonucleotide probes. Double in situ hybridization demonstrated a high coexpression of dopamine D1 receptor and substance P mRNAs (91–99%) as well as dopamine D2 receptor and preproenkephalin A mRNAs (96–99%) in medium‐sized neurons throughout the nucleus caudatus, putamen, and nucleus accumbens. Only a small subpopulation (2–5%) of the neurons that contained dopamine D1 receptor mRNA also expressed dopamine D2 receptor mRNA in all regions. Large‐sized neurons known to be cholinergic expressed D2R mRNA. However, within the nucleus basalis of Meynert, the large cholinergic neurons expressed D2R mRNA, but the neurons producing enkephalin expressed neither D1R nor D2R mRNA. These results demonstrate that the striatal organizational pattern of D1 and D2 receptor segregation in distinct neuronal populations described in rodent also exists in primate. J. Comp. Neurol. 418:22–32, 2000.

Lentiviral Overexpression of GRK6 Alleviates l-Dopa–Induced Dyskinesia in Experimental Parkinson’s Disease

G protein–coupled receptor kinase 6, which promotes desensitization of the dopamine receptor, alleviates dyskinesia without compromising the antiparkinsonian effect of l-dopa. Treatment for Tremors Without Side Effects As neurodegenerative diseases go, Parkinson’s disease is fairly treatable. Oral doses of l-dopa can still the tremors and normalize a patient’s movements—for a time. Eventually, however, most patients develop involuntary aimless gestures call dyskinesias, thought to be a result of oversensitive dopamine responses in the brain, caused by years of taking l-dopa. Now, Bezard and his colleagues have taken aim at a regulator of the dopamine receptor, G protein–coupled receptor kinase 6 (GRK6), to combat these disturbing side effects. The dopamine receptor, like others in its family, will desensitize after use. In this state, the receptor can no longer be activated and is taken up by the cell. The first step in desensitization is the phosphorylation of the receptor by GRK6. After many years of l-dopa, the amount of GRK in the brain starts to decline and the machinery that desensitizes the receptor does not work properly, leading, it is believed, to the uncontrolled movements of dyskinesia. The authors reinstated GRKs with gene therapy in mice that had an induced parkinsonian syndrome and showed that the dyskinesia-like movements of the mice were much reduced and, as expected, desensitization of the dopamine receptor was normalized. Repeating this experiment in macaque monkeys, in which a Parkinson-like disease had been artificially induced by a toxic agent, gave similar results: Increasing GRK6 expression in the brain could markedly improve the dyskinesia-like side effects of long-term l-dopa treatment, likely by correcting the desensitization of dopamine receptors. Notably, correction of GRK6 did not interfere with the therapeutic effects of l-dopa—an important attribute for the eventual application of such a therapy. These authors have identified a signaling pathway that seems to be responsible for the worst side effect of the standard treatment for Parkinson’s disease. Manipulation of one of its members, GRK6, or other components of dopamine receptor sensitization may prove to be an effective treatment for these side effects without hindering the efficacy of one of the most useful drugs in the neurologist’s armamentarium. Parkinson’s disease is caused primarily by degeneration of brain dopaminergic neurons in the substantia nigra and the consequent deficit of dopamine in the striatum. Dopamine replacement therapy with the dopamine precursor l-dopa is the mainstay of current treatment. After several years, however, the patients develop l-dopa–induced dyskinesia, or abnormal involuntary movements, thought to be due to excessive signaling via dopamine receptors. G protein–coupled receptor kinases (GRKs) control desensitization of dopamine receptors. We found that dyskinesia is attenuated by lentivirus-mediated overexpression of GRK6 in the striatum in rodent and primate models of Parkinson’s disease. Conversely, reduction of GRK6 concentration by microRNA delivered with lentiviral vector exacerbated dyskinesia in parkinsonian rats. GRK6 suppressed dyskinesia in monkeys without compromising the antiparkinsonian effects of l-dopa and even prolonged the antiparkinsonian effect of a lower dose of l-dopa. Our finding that increased availability of GRK6 ameliorates dyskinesia and increases duration of the antiparkinsonian action of l-dopa suggests a promising approach for controlling both dyskinesia and motor fluctuations in Parkinson’s disease.

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 Parkinsons 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.

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.

Cellular distribution of adenosine A2A receptor mrna in the primate striatum

The cellular expression of adenosine A2A receptor mRNA in the adult monkey and human striatum was examined by using single and double in situ hybridization with ribonucleotide probes. Analysis on adjacent sections demonstrated a homogeneous overlapping expression of adenosine A2A receptor and preproenkephalin A mRNAs throughout nucleus caudatus, putamen, and nucleus accumbens. By contrast, high expression of preproenkephalin A mRNA but no expression of adenosine A2A receptor mRNA was found in the nucleus basalis of Meynert. Double in situ hybridization demonstrated an extensive colocalization of adenosine A2A receptor and preproenkephalin A mRNAs in approximately 50% of the medium‐sized spiny neurons of the monkey nucleus caudatus, putamen, and nucleus accumbens. A small number of neurons (4–12%) that contained adenosine A2A receptor mRNA but not preproenkephalin A mRNA was found along the ventral borders of the striatum. Virtually all adenosine A2A receptor mRNA‐containing neurons co‐expressed dopamine D2 receptor mRNA, whereas only very few adenosine A2A receptor mRNA containing neurons co‐expressed dopamine D1 receptor or substance P mRNAs. In addition, a sub‐population of adenosine A2A receptor mRNA‐expressing neurons that also contained preproenkephalin A mRNA was found in the septum in monkeys. These results demonstrate that there is a high expression of adenosine A2A receptor mRNA in the primate striatum that is extensively co‐localized with dopamine D2 receptor and preproenkephalin A mRNAs. It is concluded that adenosine A2A receptors are likely to be important for the parallel organization of primate striatal neurotransmission and that these receptors could be a target for drug therapy in Parkinsons disease. J. Comp. Neurol. 399:229–240, 1998.

Striatal overexpression of ΔJunD 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.

Antagonizing L-type Ca2+ Channel Reduces Development of Abnormal Involuntary Movement in the Rat Model of L-3,4-Dihydroxyphenylalanine-Induced Dyskinesia

BACKGROUND Chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment of Parkinsons disease (PD) leads to debilitating involuntary movements, termed L-DOPA-induced dyskinesia. Striatofugal medium spiny neurons (MSN) lose their dendritic spines and cortico-striatal glutamatergic synapses in PD and in experimental models of DA depletion. This loss of connectivity is triggered by a dysregulation of intraspine Cav1.3 L-type Ca2+ channels. Here we address the possible implication of DA denervation-induced spine pruning in the development of L-DOPA-induced dyskinesia. METHODS The L-type Ca2+ antagonist, isradipine was subcutaneously delivered to rats at the doses of .05, .1, or .2 mg/kg/day, for 4 weeks, starting the day after a unilateral nigrostriatal 6-hydroxydopamine (6-OHDA) lesion. Fourteen days later, L-DOPA treatment was initiated. RESULTS Isradipine-treated animals displayed a dose-dependent reduction in L-DOPA-induced rotational behavior and abnormal involuntary movements. Dendritic spine counting at electron microscopy level showed that isradipine (.2 mg/kg/day) prevented the 6-OHDA-induced spine loss and normalized preproenkephalin-A messenger RNA expression. Involuntary movements were not reduced when isradipine treatment was started concomitantly with L-DOPA. CONCLUSIONS These results indicate that isradipine, at a therapeutically relevant dose, might represent a treatment option for preventing L-DOPA-induced dyskinesia in PD.

Neuroanatomical Study of the A11 Diencephalospinal Pathway in the Non-Human Primate

Background The A11 diencephalospinal pathway is crucial for sensorimotor integration and pain control at the spinal cord level. When disrupted, it is thought to be involved in numerous painful conditions such as restless legs syndrome and migraine. Its anatomical organization, however, remains largely unknown in the non-human primate (NHP). We therefore characterized the anatomy of this pathway in the NHP. Methods and Findings In situ hybridization of spinal dopamine receptors showed that D1 receptor mRNA is absent while D2 and D5 receptor mRNAs are mainly expressed in the dorsal horn and D3 receptor mRNA in both the dorsal and ventral horns. Unilateral injections of the retrograde tracer Fluoro-Gold (FG) into the cervical spinal enlargement labeled A11 hypothalamic neurons quasi-exclusively among dopamine areas. Detailed immunohistochemical analysis suggested that these FG-labeled A11 neurons are tyrosine hydroxylase-positive but dopa-decarboxylase and dopamine transporter-negative, suggestive of a L-DOPAergic nucleus. Stereological cell count of A11 neurons revealed that this group is composed by 4002±501 neurons per side. A 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) intoxication with subsequent development of a parkinsonian syndrome produced a 50% neuronal cell loss in the A11 group. Conclusion The diencephalic A11 area could be the major source of L-DOPA in the NHP spinal cord, where it may play a role in the modulation of sensorimotor integration through D2 and D3 receptors either directly or indirectly via dopamine formation in spinal dopa-decarboxylase-positives cells.