Emanuela Santini

Critical Involvement of cAMP/DARPP-32 and Extracellular Signal-Regulated Protein Kinase Signaling in l-DOPA-Induced Dyskinesia

The molecular basis of l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID), one of the major hindrances in the current therapy for Parkinsons disease, is still unclear. We show that attenuation of cAMP signaling in the medium spiny neurons of the striatum, achieved by genetic inactivation of the dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), reduces LID. We also show that, in dyskinetic mice, sensitized cAMP/cAMP-dependent protein kinase/DARPP-32 signaling leads to phosphorylation/activation of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). The increase in ERK1/2 phosphorylation associated with dyskinesia results in activation of mitogen- and stress-activated kinase-1 (MSK-1) and phosphorylation of histone H3, two downstream targets of ERK involved in transcriptional regulation. In line with these observations, we found that c-Fos expression is abnormally elevated in the striata of mice affected by LID. Persistent enhancement of the ERK signaling cascade is implicated in the generation of LID. Thus, pharmacological inactivation of ERK1/2 achieved using SL327 (α-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile), an inhibitor of the mitogen-activated kinase/ERK kinase, MEK, during chronic l-DOPA treatment counteracts the induction dyskinesia. Together, these results indicate that a significant proportion of the abnormal involuntary movements developed in response to chronic l-DOPA are attributable to hyperactivation in striatal medium spiny neurons of a signaling pathway including sequential phosphorylation of DARPP-32, ERK1/2, MSK-1, and histone H3.

Distinct subclasses of medium spiny neurons differentially regulate striatal motor behaviors

The direct and indirect pathways of the basal ganglia have been proposed to oppositely regulate locomotion and differentially contribute to pathological behaviors. Analysis of the distinct contributions of each pathway to behavior has been a challenge, however, due to the difficulty of selectively investigating the neurons comprising the two pathways using conventional techniques. Here we present two mouse models in which the function of striatonigral or striatopallidal neurons is selectively disrupted due to cell type–specific deletion of the striatal signaling protein dopamine- and cAMP-regulated phosphoprotein Mr 32kDa (DARPP-32). Using these mice, we found that the loss of DARPP-32 in striatonigral neurons decreased basal and cocaine-induced locomotion and abolished dyskinetic behaviors in response to the Parkinsons disease drug L-DOPA. Conversely, the loss of DARPP-32 in striatopallidal neurons produced a robust increase in locomotor activity and a strongly reduced cataleptic response to the antipsychotic drug haloperidol. These findings provide insight into the selective contributions of the direct and indirect pathways to striatal motor behaviors.

Exaggerated translation causes synaptic and behavioural aberrations associated with autism

Autism spectrum disorders (ASDs) are an early onset, heterogeneous group of heritable neuropsychiatric disorders with symptoms that include deficits in social interaction skills, impaired communication abilities, and ritualistic-like repetitive behaviours. One of the hypotheses for a common molecular mechanism underlying ASDs is altered translational control resulting in exaggerated protein synthesis. Genetic variants in chromosome 4q, which contains the EIF4E locus, have been described in patients with autism. Importantly, a rare single nucleotide polymorphism has been identified in autism that is associated with increased promoter activity in the EIF4E gene. Here we show that genetically increasing the levels of eukaryotic translation initiation factor 4E (eIF4E) in mice results in exaggerated cap-dependent translation and aberrant behaviours reminiscent of autism, including repetitive and perseverative behaviours and social interaction deficits. Moreover, these autistic-like behaviours are accompanied by synaptic pathophysiology in the medial prefrontal cortex, striatum and hippocampus. The autistic-like behaviours displayed by the eIF4E-transgenic mice are corrected by intracerebroventricular infusions of the cap-dependent translation inhibitor 4EGI-1. Our findings demonstrate a causal relationship between exaggerated cap-dependent translation, synaptic dysfunction and aberrant behaviours associated with autism.

Inhibition of mTOR Signaling in Parkinson’s Disease Prevents l-DOPA–Induced Dyskinesia

Dyskinetic side effects of a Parkinson’s disease medication may involve dopamine D1 receptor–mediated activation of mTORC1. Dyskinesia Relief In its role as a regulator of cell growth, the mammalian complex of rapamycin (mTOR) phosphorylates several proteins involved in protein synthesis, such as 4E-BP (eukaryotic initiation factor 4E binding protein) and S6K (p70 S6 kinase), in response to growth factors and nutrient availability. Santini et al. show that l-DOPA, the most commonly used medication to alleviate the immobility and rigidity (akinesia) characteristic of Parkinson’s disease (PD), also stimulates the rapamycin-sensitive mTOR complex 1 (mTORC1). In a mouse model of PD, l-DOPA treatment increased phosphorylation of several direct and indirect mTOR targets, including S6K, its substrate ribosomal protein S6 (S6), 4E-BP, and eukaryotic initiation factor 4E (eIF4E). These phosphorylation increases required the activity of dopamine D1 receptors and extracellular signal–regulated kinase (ERK). Furthermore, increased phosphorylation of S6K, S6, 4E-BP, and eIF4E correlated with stronger abnormal involuntary movements (AIMs), a measure of dyskinesia (a side effect of l-DOPA that limits its clinical use). Administration of rapamycin, which predominantly inhibits mTORC1, decreased the severity of AIMs without affecting the ability of l-DOPA to reduce akinesia. Thus, the mTORC1 signaling pathway could be targeted in PD patients suffering from the dyskinesia associated with l-DOPA treatment. Parkinson’s disease (PD), a disorder caused by degeneration of the dopaminergic input to the basal ganglia, is commonly treated with l-DOPA. Use of this drug, however, is severely limited by motor side effects, or dyskinesia. We show that administration of l-DOPA in a mouse model of Parkinsonism led to dopamine D1 receptor–mediated activation of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is implicated in several forms of synaptic plasticity. This response occurred selectively in the GABAergic medium spiny neurons that project directly from the striatum to the output structures of the basal ganglia. The l-DOPA–mediated activation of mTORC1 persisted in mice that developed dyskinesia. Moreover, the mTORC1 inhibitor rapamycin prevented the development of dyskinesia without affecting the therapeutic efficacy of l-DOPA. Thus, the mTORC1 signaling cascade represents a promising target for the design of anti-Parkinsonian therapies.

L-DOPA activates ERK signaling and phosphorylates histone H3 in the striatonigral medium spiny neurons of hemiparkinsonian mice

In the dopamine‐depleted striatum, extracellular signal‐regulated kinase (ERK) signaling is implicated in the development of l‐DOPA‐induced dyskinesia. To gain insights on its role in this disorder, we examined the effects of l‐DOPA on the state of phosphorylation of ERK and downstream target proteins in striatopallidal and striatonigral medium spiny neurons (MSNs). For this purpose, we employed mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoters for the dopamine D2 receptor (Drd2‐EGFP mice) or the dopamine D1 receptor (Drd1a‐EGFP mice), which are expressed in striatopallidal and striatonigral MSNs, respectively. In 6‐hydroxydopamine‐lesioned Drd2‐EGFP mice, l‐DOPA increased the phosphorylation of ERK, mitogen‐ and stress‐activated kinase 1 and histone H3, selectively in EGFP‐negative MSNs. Conversely, a complete co‐localization between EGFP and these phosphoproteins was observed in Drd1a‐EGFP mice. The effect of l‐DOPA was prevented by blockade of dopamine D1 receptors. The same pattern of activation of ERK signaling was observed in dyskinetic mice, after repeated administration of l‐DOPA. Our results demonstrate that in the dopamine‐depleted striatum, l‐DOPA activates ERK signaling specifically in striatonigral MSNs. This regulation may result in ERK‐dependent changes in striatal plasticity leading to dyskinesia.

Parkinson's disease: levodopa-induced dyskinesia and signal transduction.

l‐3,4‐Dihydroxyphenylalanine (l‐dopa) remains the most effective pharmacological treatment for relief of the severe motor impairments of Parkinson’s disease. It is very effective in controlling parkinsonian symptoms in the initial phase of the disease, but its action wanes with time. Such ‘wearing‐off’ imposes an escalation in the dosage of the drug, which ultimately fails to provide stable control of motor symptoms and results in the appearance of abnormal involuntary movements or dyskinesia. ‘Peak‐dose’l‐dopa‐induced dyskinesia (LID) currently represents one of the major challenges in the treatment of Parkinson’s disease. Accumulating evidence suggests that LID derives from overstimulation of dopamine receptors located on the GABAergic medium spiny neurons (MSNs) of the dorsal striatum. These neurons form two distinct projection pathways, which exert opposite effects on motor activity: the direct, striatonigral pathway promotes locomotion, whereas the indirect, striatopallidal pathway depresses locomotion. In order to understand the mechanisms underlying LID, it is important to identify molecular adaptations produced by chronic administration of l‐dopa, at the level of one or the other of these two neuronal populations. This review summarizes the results of recent studies indicating that LID is associated with abnormal dopamine D1 receptor signaling affecting the MSNs of the direct pathway. The role of this pathological adaptation and of the consequent changes in signaling in the development and expression of LID are discussed.

Distinct Changes in cAMP and Extracellular Signal-Regulated Protein Kinase Signalling in L-DOPA-Induced Dyskinesia

Background In rodents, the development of dyskinesia produced by L-DOPA in the dopamine-depleted striatum occurs in response to increased dopamine D1 receptor-mediated activation of the cAMP - protein kinase A and of the Ras-extracellular signal-regulated kinase (ERK) signalling pathways. However, very little is known, in non-human primates, about the regulation of these signalling cascades and their association with the induction, manifestation and/or maintenance of dyskinesia. Methodology/Results We here studied, in the gold-standard non-human primate model of Parkinsons disease, the changes in PKA-dependent phosphorylation of DARPP-32 and GluR1 AMPA receptor, as well as in ERK and ribosomal protein S6 (S6) phosphorylation, associated to acute and chronic administration of L-DOPA. Increased phosphorylation of DARPP-32 and GluR1 was observed in both L-DOPA first-ever exposed and chronically-treated dyskinetic parkinsonian monkeys. In contrast, phosphorylation of ERK and S6 was enhanced preferentially after acute L-DOPA administration and decreased during the course of chronic treatment. Conclusion Dysregulation of cAMP signalling is maintained during the course of chronic L-DOPA administration, while abnormal ERK signalling peaks during the initial phase of L-DOPA treatment and decreases following prolonged exposure. While cAMP signalling enhancement is associated with dyskinesia, abnormal ERK signalling is associated with priming.

Dopamine D2 receptor dysfunction is rescued by adenosine A2A receptor antagonism in a model of DYT1 dystonia.

DYT1 dystonia is an inherited disease linked to mutation in the TOR1A gene encoding for the protein torsinA. Although the mechanism by which this genetic alteration leads to dystonia is unclear, multiple lines of clinical evidence suggest a link between dystonia and a reduced dopamine D2 receptor (D2R) availability. Based on this evidence, herein we carried out a comprehensive analysis of electrophysiological, behavioral and signaling correlates of D2R transmission in transgenic mice with the DYT1 dystonia mutation. Electrophysiological recordings from nigral dopaminergic neurons showed a normal responsiveness to D2-autoreceptor function. Conversely, postsynaptic D2R function in hMT mice was impaired, as suggested by the inability of a D2R agonist to re-establish normal corticostriatal synaptic plasticity and supported by the reduced sensitivity to haloperidol-induced catalepsy. Although an in situ hybridization analysis showed normal D1R and D2R mRNA expression levels in the striata of hMT mice, we found a significant decrease of D2R protein, coupled to a reduced ability of D2Rs to activate their cognate Go/i proteins. Of relevance, we found that pharmacological blockade of adenosine A2A receptors (A2ARs) fully restored the impairment of synaptic plasticity observed in hMT mice. Together, our findings demonstrate an important link between torsinA mutation and D2R dysfunction and suggest that A2AR antagonism is able to counteract the deficit in D2R-mediated transmission observed in mutant mice, opening new perspectives for the treatment of this movement disorder.

The GTP-binding protein Rhes modulates dopamine signalling in striatal medium spiny neurons

Rhes is a small GTP-binding protein prominently localized in the striatum. Previous findings obtained in cell culture systems demonstrated an involvement of Rhes in cAMP/PKA signalling pathway, at a level proximal to the activation of heterotrimeric G-protein complex. However, its role in the striatum has been, so far, only supposed. Here we studied the involvement of Rhes in dopaminergic signalling, by employing mice with a null mutation in the Rhes gene. We demonstrated that the absence of Rhes modulates cAMP/PKA signalling in both striatopallidal and striatonigral projection neurons by increasing Golf protein levels and, in turn, influencing motor responses challenged by dopaminergic agonist/antagonist. Interestingly, we also show that Rhes is required for a correct dopamine-mediated GTP binding, a function mainly associated to stimulation of dopamine D2 receptors. Altogether, our results indicate that Rhes is an important modulator of dopaminergic transmission in the striatum.

Metabotropic glutamate receptor-dependent long-term depression is impaired due to elevated ERK signaling in the ΔRG mouse model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) and fragile X syndrome (FXS) are caused by mutations in negative regulators of translation. FXS model mice exhibit enhanced metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD). Therefore, we hypothesized that a mouse model of TSC, ΔRG transgenic mice, also would exhibit enhanced mGluR-LTD. We measured the impact of TSC2-GAP mutations on the mTORC1 and ERK signaling pathways and protein synthesis-dependent hippocampal synaptic plasticity in ΔRG transgenic mice. These mice express a dominant/negative TSC2 that binds to TSC1, but has a deletion and substitution mutation in its GAP-domain, resulting in inactivation of the complex. Consistent with previous studies of several other lines of TSC model mice, we observed elevated S6 phosphorylation in the brains of ΔRG mice, suggesting upregulated translation. Surprisingly, mGluR-LTD was not enhanced, but rather was impaired in the ΔRG transgenic mice, indicating that TSC and FXS have divergent synaptic plasticity phenotypes. Similar to patients with TSC, the ΔRG transgenic mice exhibit elevated ERK signaling. Moreover, the mGluR-LTD impairment displayed by the ΔRG transgenic mice was rescued with the MEK-ERK inhibitor U0126. Our results suggest that the mGluR-LTD impairment observed in ΔRG mice involves aberrant TSC1/2-ERK signaling.