RGS9-2 rescues dopamine D2 receptor levels and signaling in DYT1 dystonia mouse models
Ontology highlight
ABSTRACT:
SUBMITTER: Dr. Paola Bonsi
PROVIDER: S-SCDT-EMM-2018-09283 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Dopamine D2 receptor signaling is central for striatal function and movement, while abnormal activity is associated with neurological disorders including the severe early-onset DYT1 dystonia. Nevertheless, the mechanisms that regulate D2 receptor signaling in health and disease remain poorly understood. Here, we identify a reduced D2 receptor binding, paralleled by an abrupt reduction in receptor protein level, in the striatum of juvenile Dyt1 mice. This occurs through increased lysosomal degradation, controlled by competition between β-arrestin 2 and D2 receptor binding proteins. Accordingly, we found lower levels of striatal RGS9-2 and spinophilin. Further, we show that genetic depletion of RGS9-2 mimics the D2 receptor loss of DYT1 dystonia striatum, whereas RGS9-2 overexpression rescues both receptor levels and electrophysiological responses in Dyt1 striatal neurons. This work uncovers the molecular mechanism underlying D2 receptor downregulation in Dyt1 mice and in turn explains why dopaminergic drugs lack efficacy in DYT1 patients despite significant evidence for striatal D2 receptor dysfunction. Our data also open up novel avenues for disease-modifying therapeutics to this incurable neurological disorder.
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Project description:The histamine H3 receptor (H3R) is highly enriched in the spiny projection neurons (SPNs) of the striatum, in both the D1 receptor (D1R)-expressing and D2 receptor (D2R)-expressing populations. A crossantagonistic interaction between H3R and D1R has been demonstrated in mice, both at the behavioral level and at the biochemical level. Although interactive behavioral effects have been described upon coactivation of H3R and D2R, the molecular mechanisms underlying this interaction are poorly understood. Here, we show that activation of H3R with the selective agonist R-(-)-α-methylhistamine dihydrobromide mitigates D2R agonist-induced locomotor activity and stereotypic behavior. Using biochemical approaches and the proximity ligation assay, we demonstrated the existence of an H3R-D2R complex in the mouse striatum. In addition, we examined consequences of simultaneous H3R-D2R agonism on the phosphorylation levels of several signaling molecules using immunohistochemistry. H3R agonist treatment modulated Akt (serine/threonine PKB)-glycogen synthase kinase 3 beta signaling in response to D2R activation via a β-arrestin 2-dependent mechanism in D2R-SPNs but not in D1R-SPNs. Phosphorylation of mitogen- and stress-activated protein kinase 1 and rpS6 (ribosomal protein S6) was largely unchanged under these conditions. As Akt-glycogen synthase kinase 3 beta signaling has been implicated in several neuropsychiatric disorders, this work may help clarify the role of H3R in modulating D2R function, leading to a better understanding of pathophysiology involving the interaction between histamine and dopamine systems.
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Project description:Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D1-like family of dopamine receptors and inputs of aversion coming from neurons containing the D2-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D1 reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D2 receptors function, we present evidence of ?1 receptor molecular and functional interaction with dopamine D2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D2 receptors (the long isoform of the D2 receptor) can complex with ?1 receptors, a result that is specific to D2 receptors, as D3 and D4 receptors did not form heteromers. We demonstrate that the ?1-D2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to ?1 -D2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from ?1 knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D1 and D2 receptor containing neurons in the brain.
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Project description:BackgroundDYT1 dystonia is a heritable, early-onset generalized movement disorder caused by a GAG deletion (?GAG) in the DYT1 gene. Neuroimaging studies and studies using mouse models suggest that DYT1 dystonia is associated with dopamine imbalance. However, whether dopamine imbalance is key to DYT1 or other forms of dystonia continues to be debated.Methodology/principal findingsWe used Dyt1 knock out (Dyt1 KO), Dyt1 ?GAG knock-in (Dyt1 KI), and transgenic mice carrying one copy of the human DYT1 wild type allele (DYT1 hWT) or human ?GAG mutant allele (DYT1 hMT). D1R, D2R, and G?(olf) protein expression was analyzed by western blot in the frontal cortex, caudate-putamen and ventral midbrain in young adult (postnatal day 60; P60) male mice from all four lines; and in the frontal cortex and caudate putamen in juvenile (postnatal day 14; P14) male mice from the Dyt1 KI and KO lines. Dopamine receptor and G?(olf) protein expression were significantly decreased in multiple brain regions of Dyt1 KI and Dyt1 KO mice and not significantly altered in the DYT1 hMT or DYT1 hWT mice at P60. The only significant change at P14 was a decrease in D1R expression in the caudate-putamen of the Dyt1 KO mice.Conclusion/significanceWe found significant decreases in key proteins in the dopaminergic system in multiple brain regions of Dyt1 KO and Dyt1 KI mouse lines at P60. Deletion of one copy of the Dyt1 gene (KO mice) produced the most pronounced effects. These data offer evidence that impaired dopamine receptor signaling may be an early and significant contributor to DYT1 dystonia pathophysiology.
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Project description:Trihexyphenidyl, a nonselective muscarinic receptor antagonist, is the small molecule drug of choice for the treatment of DYT1 dystonia, but it is poorly tolerated due to significant side effects. A better understanding of the mechanism of action of trihexyphenidyl is needed for the development of improved treatments. Because DTY1 dystonia is associated with both abnormal cholinergic neurotransmission and abnormal dopamine regulation, we tested the hypothesis that trihexyphenidyl normalizes striatal dopamine release in a mouse model of DYT1 dystonia using ex vivo fast scan cyclic voltammetry and in vivo microdialysis. Trihexyphenidyl increased striatal dopamine release and efflux as assessed by ex vivo voltammetry and in vivo microdialysis respectively. In contrast, ʟ-DOPA, which is not usually effective for the treatment of DYT1 dystonia, did not increase dopamine release in either Dyt1 or control mice. Trihexyphenidyl was less effective at enhancing dopamine release in Dyt1 mice relative to controls ex vivo (mean increase WT: 65% vs Dyt1: 35%). Trihexyphenidyl required nicotinic receptors but not glutamate receptors to increase dopamine release. Dyt1 mice were more sensitive to the dopamine release decreasing effects of nicotinic acetylcholine receptor antagonism (IC50: WT = 29.46 nM, Dyt1 = 12.26 nM) and less sensitive to acetylcholinesterase inhibitors suggesting that nicotinic acetylcholine receptor neurotransmission is altered in Dyt1 mice, that nicotinic receptors indirectly mediate the differential effects of trihexyphenidyl in Dyt1 mice, and that nicotinic receptors may be suitable therapeutic targets for DYT1 dystonia.
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Project description:RationaleAccumulating evidence indicates that the cognitive effects of dopamine depend on the subtype of dopamine receptor that is activated. In particular, recent work with animals as well as current theorizing has suggested that cognitive flexibility depends on dopamine D2 receptor signaling. However, there is no evidence for similar mechanisms in humans.ObjectivesWe aim to demonstrate that optimal dopamine D2 receptor signaling is critical for human cognitive flexibility.MethodsTo this end, a pharmacological pretreatment design was employed. This enabled us to investigate whether effects of the dopamine receptor agonist bromocriptine on task-set switching were abolished by pretreatment with the D2 receptor antagonist sulpiride. To account for individual (genetic) differences in baseline levels of dopamine, we made use of a common variable number of tandem repeat (VNTR) polymorphism in the 3'-untranslated region of the dopamine transporter gene, DAT1.ResultsBromocriptine improved cognitive flexibility relative to placebo, but only in subjects with genetically determined low levels of dopamine (n?=?27). This beneficial effect of bromocriptine on cognitive flexibility was blocked by pretreatment with the selective dopamine D2 receptor antagonist sulpiride (n?=?14).ConclusionsThese results provide strong evidence in favor of the hypothesis that human cognitive flexibility implicates dopamine D2 receptor signaling.
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Project description:A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. Compared to the wild type D2 receptor, the novel allelic variant D2-I212F activates a Gαi1β1γ2 heterotrimer with higher potency and modestly enhanced basal activity in human embryonic kidney (HEK) 293 cells and has decreased capacity to recruit arrestin3. We now report that omitting overexpressed G protein-coupled receptor kinase-2 (GRK2) decreased the potency and efficacy of quinpirole for arrestin recruitment. The relative efficacy of quinpirole for arrestin recruitment to D2-I212F compared to D2-WT was considerably lower without overexpressed GRK2 than with added GRK2. D2-I212F exhibited higher basal activation of GαoA than Gαi1 but little or no increase in the potency of quinpirole relative to D2-WT. Other signs of D2-I212F constitutive activity for G protein-mediated signaling, in addition to basal activation of Gαi/o, were enhanced basal inhibition of forskolin-stimulated cyclic AMP accumulation that was reversed by the inverse agonists sulpiride and spiperone and a ∼4-fold increase in the apparent affinity of D2-I212F for quinpirole, determined from competition binding assays. In mouse midbrain slices, inhibition of tonic current by the inverse agonist sulpiride in dopamine neurons expressing D2-I212F was consistent with our hypothesis of enhanced constitutive activity and sensitivity to dopamine relative to D2-WT. Molecular dynamics simulations with D2 receptor models suggested that an ionic lock between the cytoplasmic ends of the third and sixth α-helices that constrains many G protein-coupled receptors in an inactive conformation spontaneously breaks in D2-I212F. Overall, these results confirm that D2-I212F is a constitutively active and signaling-biased D2 receptor mutant and also suggest that the effect of the likely pathogenic variant in a given brain region will depend on the nature of G protein and GRK expression.
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Project description: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.
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Project description:BackgroundWe describe a 4-generation Dutch pedigree with a unique dominantly inherited clinical phenotype of a combined progressive chorea and cervical dystonia carrying a novel heterozygous dopamine D2 receptor (DRD2) variant.ObjectivesThe objective of this study was to identify the genetic cause of the disease and to further investigate the functional consequences of the genetic variant.MethodsAfter detailed clinical and neurological examination, whole-exome sequencing was performed. Because a novel variant in the DRD2 gene was found as the likely causative gene defect in our pedigree, we sequenced the DRD2 gene in a cohort of 121 Huntington-like cases with unknown genetic cause (Germany). Moreover, functional characterization of the DRD2 variant included arrestin recruitment, G protein activation, and G protein-mediated inhibition of adenylyl cyclase determined in a cell model, and G protein-regulated inward-rectifying potassium channels measured in midbrain slices of mice.ResultWe identified a novel heterozygous variant c.634A > T, p.Ile212Phe in exon 5 of DRD2 that cosegregated with the clinical phenotype. Screening of the German cohort did not reveal additional putative disease-causing variants. We demonstrated that the D2S/L -I212 F receptor exhibited increased agonist potency and constitutive activation of G proteins in human embryonic kidney 239 cells as well as significantly reduced arrestin3 recruitment. We further showed that the D2S -I212 F receptor exhibited aberrant receptor function in mouse midbrain slices.ConclusionsOur results support an association between the novel p.Ile212Phe variant in DRD2, its modified D2 receptor activity, and the hyperkinetic movement disorder reported in the 4-generation pedigree. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
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Project description:Substantial spine loss in striatal medium spiny neurons (MSNs) and abnormal behaviors are common features of Parkinson's disease (PD). The caudate putamen (CPu) mainly contains MSNs expressing dopamine D1 receptor (dMSNs) and dopamine D2 receptor (iMSNs) exerting critical effects on motor and cognition behavior. However, the molecular mechanisms contributing to spine loss and abnormal behaviors in dMSNs and iMSNs under parkinsonian state remain unknown. In the present study, we revealed that Cell division control protein 42 (Cdc42) signaling was significantly decreased in the caudate putamen (CPu) in parkinsonian mice. In addition, overexpression of constitutively active Cdc42 in the CPu reversed spine abnormalities and improved the behavior deficits in parkinsonian mice. Utilizing conditional dopamine D1 receptor (D1R) or D2 receptor (D2R) knockout mice, we found that such a decrease under parkinsonian state was further reduced by conditional knockout of the D2R but not D1R. Moreover, the thin spine loss in iMSNs and deficits in motor coordination and cognition induced by conditional knockout of D2R were reversed by overexpression of constitutively active Cdc42 in the CPu. Additionally, conditional knockout of Cdc42 from D2R-positive neurons in the CPu was sufficient to induce spine and behavior deficits similar to those observed in parkinsonian mice. Overall, our results indicate that impaired Cdc42 signaling regulated by D2R plays an important role in spine loss and behavioral deficits in PD.
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