Michel Cyr

Striatal inhibition of PKA prevents levodopa-induced behavioural and molecular changes in the hemiparkinsonian rat

l-3,4-dihydroxyphenylalanine methyl ester hydrochloride (l-DOPA) is the gold standard for symptomatic treatment of Parkinsons disease (PD), but long-term therapy is associated with the emergence of abnormal involuntary movements (AIMS) known as l-DOPA-induced dyskinesias (LID). The molecular changes underlying LID are not completely understood. Using the 6-hydroxydopamine-lesioned rat model of PD, we showed that l-DOPA elicits profound alterations in the activity of three LID molecular markers, namely DeltaFosB, dopamine, cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), as well as in phosphorylation levels of the cytoskeletal-associated protein tau. These modifications are triggered by protein kinase A (PKA) activation and intermittent stimulation of dopamine receptors as they are totally prevented by intrastriatal injections of Rp-cAMPS, a PKA inhibitor, or by continuous administration of l-DOPA via subcutaneous mini-pump. Importantly, Rp-cAMPS does not modulate the positive effect of l-DOPA on locomotor deficits and significantly attenuates the emergence of AIMS in 6-hydroxydopamine hydrobromide-lesioned rats. Even if decreased PKA signalling in the striatum may represent a clinical challenge, these data provide novel evidence that PKA activation, through modification of striatal signalling and alterations of cytoskeletal constituents, plays a key role in the manifestation of LID.

Dopamine D1 receptor activation induces tau phosphorylation via cdk5 and GSK3 signaling pathways.

Increasing evidence is demonstrating that drugs affecting dopamine levels in the brain induce cytoskeletal modifications. These evolving changes may impact neuronal synaptic plasticity as cytoskeletal constituents are involved in the maintenance of dendritic processes, and any alterations in their stability could influence major cellular compartments of neurons, such as dendrites, spines and synapses. Here, we describe a molecular chain of events that links dopamine D1 receptor activation to hyperphosphorylation of the microtubule-associated protein tau, which is normally involved in microtubules stabilization. We show, in SK-N-MC cells and rat striatal sections, that phosphorylation of tau at serines 199-202 and 214 appears to be mediated through activation of calcium-dependent intracellular mechanism, subsequent to D1 receptor-induced cAMP-dependent protein kinase A (PKA). We demonstrate, using pharmacological tools, that PKA activation causes increase of calcium levels, leading to cyclin-dependent kinase 5 activation by calpain proteolysis of p35 to p25 and glycogen synthase kinase 3beta activation by its phosphorylation at tyrosine 216. The D2 receptor agonism or lowering cAMP levels has no effect in our experimental settings. Moreover, we do not observe any association between phosphorylated tau and cellular damage. These data unravel novel mechanisms of tau hyperphosphorylation during G-protein-coupled receptor activation and are the first to show that stimulation of D1 receptors could have a profound influence on the neuronal cytoskeletal constituent tau.

Increased expression of the dopamine transporter leads to loss of dopamine neurons, oxidative stress and l-DOPA reversible motor deficits

The dopamine transporter is a key protein responsible for regulating dopamine homeostasis. Its function is to transport dopamine from the extracellular space into the presynaptic neuron. Studies have suggested that accumulation of dopamine in the cytosol can trigger oxidative stress and neurotoxicity. Previously, ectopic expression of the dopamine transporter was shown to cause damage in non-dopaminergic neurons due to their inability to handle cytosolic dopamine. However, it is unknown whether increasing dopamine transporter activity will be detrimental to dopamine neurons that are inherently capable of storing and degrading dopamine. To address this issue, we characterized transgenic mice that over-express the dopamine transporter selectively in dopamine neurons. We report that dopamine transporter over-expressing (DAT-tg) mice display spontaneous loss of midbrain dopamine neurons that is accompanied by increases in oxidative stress markers, 5-S-cysteinyl-dopamine and 5-S-cysteinyl-DOPAC. In addition, metabolite-to-dopamine ratios are increased and VMAT2 protein expression is decreased in the striatum of these animals. Furthermore, DAT-tg mice also show fine motor deficits on challenging beam traversal that are reversed with l-DOPA treatment. Collectively, our findings demonstrate that even in neurons that routinely handle dopamine, increased uptake of this neurotransmitter through the dopamine transporter results in oxidative damage, neuronal loss and l-DOPA reversible motor deficits. In addition, DAT over-expressing animals are highly sensitive to MPTP-induced neurotoxicity. The effects of increased dopamine uptake in these transgenic mice could shed light on the unique vulnerability of dopamine neurons in Parkinsons disease.

Intrastriatal inhibition of extracellular signal-regulated kinases impaired the consolidation phase of motor skill learning.

It is well known that motor skill learning is characterized by rapid improvement in performances within the first training session and a slower progression in the following sessions that is correlated to the consolidation phase. Our goal was to establish the regional mapping of neural activity in relation to the motor skill learning included in the accelerating rotarod task using Zif268, c-Fos and ERK 1/2. As ERK 1/2 activity is also a marker of adaptive response to synaptic activation for newly learned events, its role was also verified. Learning the rotarod task did not affect levels of Zif268, but induced a selective upregulation of c-Fos in the cerebellum, motor cortex M1 and M2, cingulate cortex CG1 and CG2 as well as dorsal striatum. Notably, levels of phosphorylated ERK 1/2 were selectively increased in this later region during consolidation phase. To further study this effect, we injected inhibitors of ERK activation, the SL327 intraperitoneally or the PD98059 directly into the dorsal striatum, and observed that motor performances were exclusively impaired in this phase. These findings indicate that ERK 1/2 activity of the dorsal striatum is critical for the consolidation of late but not early phase of motor skill memory.

Blockade of NMDA receptors 2A subunit in the dorsal striatum impairs the learning of a complex motor skill.

Accumulating evidence proposes that the striatum, known to control voluntary movement, may also play a role in learning and memory. Striatum learning is thought to require long-lasting reorganization of striatal circuits and changes in the strength of synaptic connections during the memorization of a complex motor task. Whether the ionotropic glutamate receptor N-methyl-D-aspartate (NMDAR) contributes to the molecular mechanisms of these memory processes is still unclear. The aim of the present study was to investigate the role of striatal NMDAR and its subunit composition during the learning of the accelerating rotarod task in mice. To this end, we injected directly into the dorsal striatum of mice, via chronically implanted cannula, the NMDAR channel blocker MK-801 as well as the NR2A and NR2B subunit-selective antagonists NVP-AAM077 and Ro 25-6981, respectively, before rotarod training. There was no effect in the motor performances of mice treated with 1.0 μg/side of MK-801, 0.1 μg/side of NVP-AAM077, or 5 and 10 μg/side of Ro 25-6981. In contrast, injections of 2.5 and 5 μg/side of MK-801 or 0.5 and 1 μg/side of NVP-AAM077 impaired motor learning at Day 3 and 8. Interestingly, treatments with MK-801 and NVP-AAM077 did not alter the general motor capacities of mice as revealed by the stepping, wire suspension, and pole tests. Our study demonstrates that the NMDAR of the dorsal striatum contributes to motor learning, especially during the slow acquisition phase, and that NR2A subunits play a critical role in this process.

Partial dopamine depletion in MPTP-treated mice differentially altered motor skill learning and action control.

Recent findings suggest that the neurotransmitter dopamine (DA) system plays a role in motor control and the acquisition of habits and skills. However, isolating DA-mediated motor learning from motor performance remains challenging as most studies include often severely DA-depleted mice. Using the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we investigated the effect of various degrees of DA-depletion in mice on three tests of motor behaviors: the accelerating rotarod, wire suspension and pole tests. Three protocols were performed to decrease DA synthesis to various extents: 4 injections (i.p.) of 9 mg/kg in 1 day; 4 injections (i.p.) of 15 mg/kg in 1 day; or 5 injections (s.c.) of 30 mg/kg in 5 days. Severity of DA-depletion was assessed by the evaluation of tyrosine hydroxylase (TH) and dopamine transporter levels in the striatum using the Western blot technique. Mice were gathered into four different groups according their TH levels: mild, moderate, marked and severe. In these mice, the general motor abilities such as coordination, motion speed and muscular strength were relatively intact whereas impaired acquisition of skilled behavior occurred in mice with marked and severe reduction in TH levels. Marked and severely DA-depleted mice exhibited lower scores within the first trials of the first training day as well as a much slower progression in the following days on the accelerating rotarod. Based on these results, we conclude that the learning of a skilled behavior is more vulnerable to DA depletion than the DA-mediated control of motor activity.

Dopamine D1 receptor–mediated aggregation of N-terminal fragments of mutant huntingtin and cell death in a neuroblastoma cell line

Huntingtons disease (HD) is an inherited neurodegenerative disorder caused by abnormal CAG repeat expansion in the IT15 gene encoding huntingtin protein (htt). Mutated htt is predicted to acquire toxic properties in specific brain regions. For instance, striatal neurons expressing dopamine receptors predominantly degenerate in HD patients. Although the basis of this specific vulnerability remains unclear, a great deal of evidence has documented the ability of the dopamine system to modulate the toxicity of expanded htt. To investigate the relationship between dopamine receptors and expanded htt, we transfected enhanced green fluorescent proteins (EGFP) tagged to normal (25 CAG) or mutant (103 CAG) htt in SK-N-MC neuroblastoma cells that endogenously express D1 receptors. Forming nuclear and cytoplasmic aggregates, mutant htt-EGFP was toxic to cells beyond 24 h post-transfection. Remarkably, low doses of a selective D1 receptors agonist or forskolin, an activator of adenylate cyclase, accelerated the formation of mutant htt nuclear aggregates, whereas the number of cytoplasmic aggregates was decreased. These effects were associated with a minor increase in cell death. Understanding the functional bases of these effects may further elucidate the role of dopamine receptors signaling in the complex pathophysiology of HD.

NMDA reduces Tau phosphorylation in rat hippocampal slices by targeting NR2A receptors, GSK3β, and PKC activities.

The molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. In the present study, pharmacological inhibitors were deployed to investigate potential processes by which the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors modulates Tau phosphorylation in rat hippocampal slices. Our results demonstrated that Tau phosphorylation at Ser199-202 residues was decreased in NMDA-treated hippocampal slices, an effect that was not reproduced at Ser262 and Ser404 epitopes. NMDA-induced reduction of Tau phosphorylation at Ser199-202 was further promoted when NR2A-containing receptors were pharmacologically isolated and were completely abrogated by the NR2A receptor antagonist NVP-AAM077. Compared with nontreated slices, we observed that NMDA receptor activation was reflected in high Ser9 and low Tyr216 phosphorylation of glycogen synthase kinase-3 beta (GSK3β), suggesting that NMDA receptor activation might diminish Tau phosphorylation via a pathway involving GSK3β inhibition. Accordingly, we found that GSK3β inactivation by a protein kinase C- (PKC-) dependent mechanism is involved in the NMDA-induced reduction of Tau phosphorylation at Ser199-202 epitopes. Taken together, these data indicate that NR2A receptor activation may be important in limiting Tau phosphorylation by a PKC/GSK3β pathway and strengthen the idea that these receptors might act as an important molecular device counteracting neuronal cell death mechanisms in various pathological conditions.

Canadian Association of Neurosciences Review: the role of dopamine receptor function in neurodegenerative diseases.

Dopamine (DA) receptors, which are heavily expressed in the caudate/putamen of the brain, represent the molecular target of several drugs used in the treatment of various neurological disorders, such as Parkinsons disease. Although most of the drugs are very effective in alleviating the symptoms associated with these conditions, their long-term utilization could lead to the development of severe side-effects. In addition to uncovering novel mediators of physiological DA receptor functions, recent research advances are suggesting a role of these receptors in toxic effects on neurons. For instance, accumulating evidence indicates that DA receptors, particularly D1 receptors, are central in the neuronal toxicity induced by elevated synaptic levels of DA. In this review, we will discuss recent findings on DA receptors as regulators of long term neuronal dysfunction and neurodegenerative processes.

Striatal inhibition of calpains prevents levodopa-induced neurochemical changes and abnormal involuntary movements in the hemiparkinsonian rat model

Pharmacological dopamine replacement with l-3,4-dihydroxyphenylalanine (L-DOPA) remains the most effective approach to treat the motor symptoms of Parkinsons disease (PD). However, as the disease progresses, the therapeutic response to L-DOPA gradually becomes erratic and is associated with the emergence of dyskinesia in the majority of patients. The pathogenesis of L-DOPA-induced dyskinesia (LID) is still unknown. In the current study, using the 6-hydroxydopamine (6-OHDA)-lesioned rat model of PD, we demonstrated that the calcium-dependent proteins calpains and cdk5 of the striatum play a critical role in the behavioral and molecular changes evoked by L-DOPA therapy. We first confirmed that L-DOPA reversed PD symptoms, assessed by the cylinder, stepping and vibrissae-elicited reaching tests in this animal model, and elicited robust abnormal involuntary movements (AIMs) reminiscent of LID. Interestingly, intrastriatal infusion of the calpains inhibitor MDL28170, and to a lower extent the cdk5 inhibitor roscovitine, reduced the severity and amplitude of AIMs without affecting L-DOPAs antiparkinsonian effects. Notably, the calpains and cdk5 inhibitors totally reversed the striatal molecular changes attributed to L-DOPA therapy, such as ERK1/2 and dynamin phosphorylation. Another fascinating observation was that L-DOPA therapy, in combination with intrastriatal infusion of MDL28170, augmented tyrosine hydroxylase levels in the striatum of lesioned rats without affecting the number of dopaminergic cells in the substantia nigra. These findings disclose a novel mechanism underlying the maladaptive alterations induced by L-DOPA therapy in the 6-OHDA rat model of PD.