Rhes regulates dopamine D2 receptor transmission in striatal cholinergic interneurons

Highlights

• Rhes mRNA is localized in cholinergic interneurons of both human and mouse striata.

• Lack of Rhes causes an aberrant excitatory response to D2R activation in cholinergic interneurons.

• PI3K inhibitors rescue the aberrant D2R response in Rhes knockout mice.

Abstract

Ras homolog enriched in striatum (Rhes) is highly expressed in striatal medium spiny neurons (MSNs) of rodents. In the present study, we characterized the expression of Rhes mRNA across species, as well as its functional role in other striatal neuron subtypes. Double in situ hybridization analysis showed that Rhes transcript is selectively localized in striatal cholinergic interneurons (ChIs), but not in GABAergic parvalbumin- or in neuropeptide Y-positive cell populations. Rhes is closely linked to dopamine-dependent signaling. Therefore, we recorded ChIs activity in basal condition and following dopamine receptor activation. Surprisingly, instead of an expected dopamine D2 receptor (D2R)-mediated inhibition, we observed an aberrant excitatory response in ChIs from Rhes knockout mice. Conversely, the effect of D1R agonist on ChIs was less robust in Rhes mutants than in controls. Although Rhes deletion in mutants occurs throughout the striatum, we demonstrate that the D2R response is altered specifically in ChIs, since it was recorded in pharmacological isolation, and prevented either by intrapipette BAPTA or by GDP-β-S. Moreover, we show that blockade of Cav2.2 calcium channels prevented the abnormal D2R response. Finally, we found that the abnormal D2R activation in ChIs was rescued by selective PI3K inhibition thus suggesting that Rhes functionally modulates PI3K/Akt signaling pathway in these neurons. Our findings reveal that, besides its expression in MSNs, Rhes is localized also in striatal ChIs and, most importantly, lack of this G-protein, significantly alters D2R modulation of striatal cholinergic excitability.

Introduction

Ras homolog enriched in striatum (Rhes) is a small monomeric GTP-binding protein, discovered by a subtractive hybridization procedure, predominantly localized throughout dopaminoceptive neurons of dorsal striatum and nucleus accumbens (Errico et al., 2008, Vargiu et al., 2001, Vargiu et al., 2004). Rhes expression is developmentally regulated by thyroid hormone in rodents (Falk et al., 1999, Vallortigara et al., 2008, Vallortigara et al., 2009), and by dopamine (DA) innervation in adult rats (Harrison and LaHoste, 2006, Harrison et al., 2008). Studies in cell lines (Agretti et al., 2007, Harrison and LaHoste, 2006, Harrison et al., 2008, Vargiu et al., 2004), have indicated that Rhes, most likely through its binding to G_αi, reduces G-protein coupled receptor (GPCR)-mediated cAMP accumulation (Harrison and He, 2011). Accordingly, we have shown that lack of Rhes increases striatal cAMP/PKA activity in mutant mice (Errico et al., 2008). In addition, earlier in vitro evidence has revealed that Rhes influences N-type/Cav2.2 calcium channels activity (Thapliyal et al., 2008). In particular, the authors found that Rhes, by modulating G_αi-dependent signaling, reduces basal calcium current density and triggers a voltage-dependent inhibition of Cav2.2 channels in HEK293-trasfected cell culture. Aside from its influence on cAMP accumulation and regulation of Cav2.2 channels activity, it has been found that Rhes acts as a selective striatal E3 ligase of mutant Huntingtin (mHtt) (Mealer et al., 2013, Mealer et al., 2014, Subramaniam et al., 2009, Subramaniam et al., 2010). On the other hand, it has been shown that Rhes also activates mammalian target of rapamycin complex1 (mTORC1), a critical signaling pathway associated, among other processes (Wullschleger et al., 2006), with l-DOPA-induced dyskinesia (LID) in hemiparkinsonian animal models (Santini et al., 2010, Subramaniam et al., 2012). Finally, different in vitro settings have shown that Rhes modulates PI3K/Akt signaling pathway (Bang et al., 2012, Vargiu et al., 2004). Moreover, recent in vivo findings have revealed that Rhes is also able to interact with β-arrestins (Harrison et al., 2013), a scaffolding protein involved in the striatal modulation of DA D2R-dependent Akt/GSK3-β signaling (Beaulieu et al., 2005).

Despite the growing interest for the involvement of Rhes in striatal medium spiny neurons (MSNs) dysfunction, many basic issues still need to be addressed, including specific localization and activity of Rhes in other distinct striatal neuronal subtypes. Therefore, in the present work we explored the striatal cell-type expression of Rhes and RASD2 mRNAs in mouse and human brain, respectively. Interestingly, we found a selective expression of Rhes in the striatal cholinergic interneurons (ChIs), but not in those expressing GABAergic parvalbumin (Parv) or neuropeptide Y (NPY). Considering the pivotal role of acetylcholine (ACh) in the modulation of striatal circuit function (Pisani et al., 2007), here we also investigated the functional role of Rhes in modulating dopaminergic transmission in ChIs, by using knockout animals with targeted deletion of Rhes gene. Notably, our data clearly show that lack of Rhes affects the dopamine-dependent ChIs activity, thus highlighting a regulatory role of this small G-protein on dopaminergic responses in such a striatal neuronal subpopulation.