Strength of cholinergic tone dictates the polarity of dopamine D2 receptor modulation of striatal cholinergic interneuron excitability in DYT1 dystonia

Abstract

Balance between cholinergic and dopaminergic signaling is central to striatal control of movement and cognition. In dystonia, a common disorder of movement, anticholinergic therapy is often beneficial. This observation suggests there is a pathological increase in cholinergic tone, yet direct confirmation is lacking. In DYT1, an early-onset genetic form of dystonia caused by a mutation in the protein torsinA (TorA), the suspected heightened cholinergic tone is commonly attributed to faulty dopamine D2 receptor (D2R) signaling where D2R agonists cause excitation of striatal cholinergic interneurons (ChIs), rather than the normal inhibition of firing observed in wild-type animals, an effect known as “paradoxical excitation”. Here, we provide for the first time direct measurement of elevated striatal extracellular acetylcholine (ACh) in a knock-in mouse model of human DYT1 dystonia (TorAΔE/+ mice), confirming a striatal hypercholinergic state. We hypothesized that this elevated extracellular ACh might cause chronic over-activation of muscarinic acetylcholine receptors (mAChRs) and disrupt normal D2R function due to their shared coupling to Gi/o-proteins. We tested this concept in vitro first using a broad-spectrum mAChR antagonist, and then using a M2/M4 mAChR selective antagonist to specifically target mAChRs expressed by ChIs. Remarkably, we found that mAChR inhibition reverses the D2R–mediated paradoxical excitation of ChIs recorded in slices from TorAΔE/+ mice to a typical inhibitory response. Furthermore, we recapitulated the paradoxical D2R excitation of ChIs in striatal slices from wild type mice within minutes by simply increasing cholinergic tone through pharmacological inhibition of acetylcholinesterase (AChE) or by prolonged agonist activation of mAChRs. Collectively, these results show that enhanced mAChR tone itself is sufficient to rapidly reverse the polarity of D2R regulation of ChI excitability, correcting the previous notion that the D2R mediated paradoxical ChI excitation causes the hypercholinergic state in dystonia. Further, using a combination of genetic and pharmacological approaches, we found evidence that this switch in D2R polarity results from a change in coupling from the preferred Gi/o pathway to non-canonical β-arrestin signaling. These results highlight the need to fully understand how the mutation in TorA leads to pathologically heightened extracellular ACh. Furthermore the discovery of this novel ACh-dopamine interaction and the participation of β-arrestin in regulation of cholinergic interneurons is likely important for other basal ganglia disorders characterized by perturbation of ACh-dopamine balance, including Parkinson and Huntington diseases, l-DOPA-induced dyskinesia and schizophrenia.

TorsinB overexpression prevents abnormal twisting in DYT1 dystonia mouse models

“…TorsinB overexpression did not change cortical thickness, striatal volume, striatal Nissl+ small and medium cell number, or striatal GFAP immunoreactivity in Dlx-CKO and Cre control mice ( Figure S6A-D). TorsinB overexpression completely prevented the loss of dorsal striatal ChIs that have been linked to motor and electrophysiologic abnormalities in dystonia mouse models [23,[29][30][31][32][33][34]. Analysis using unbiased stereology demonstrated that Dlx-CKO mice had 33.5% fewer ChIs compared to Cre controls, whereas the B-OE allele completely prevented this loss ( Figure 4C-D).…”

“…These mutants develop abnormal limb and trunk twisting movements as juveniles that are responsive to anti-muscarinic drugs, mimicking key features of the human phenotype. Coincident with the emergence of these movements, these animals exhibit a highly selective loss of dorsal striatal cholinergic interneurons (ChIs), cells that have been implicated in dystonia pathophysiology in electrophysiological studies [29][30][31]. The disease relevant time course of motor features, clear endpoints, and predictive validity of this model provides a critical system for evaluating the effects of torsinB overexpression in a torsinA LOF pathophysiological in vivo setting.…”

Biased Receptor Signaling in Drug Discovery

“…The natural signaling bias of the calcium-sensing receptor is also altered in disease states (for review, see Leach et al, 2015). Models of dystonia (a common movement disorder) involving mutation of the protein torsinA demonstrate a pathologic increase in cholinergic tone to affect dopamine interneurons, and there is a change in dopamine signaling polarity and a bias introduced into dopamine signaling from primarily G i/o to noncanonical b-arrestin signaling (Scarduzio et al, 2017). In general, biased signaling is an obvious mechanism to exploit for drug therapy and the existing data with characterized biased ligands certainly show different phenotypical signaling profiles in vivo.…”

Cholinergic modulation of striatal microcircuits

“…Impairment of striatal ChIs is central in the production of movement disorders (Pisani et al., ); altered cholinergic signaling is seen in a diverse class of syndromes that include Parkinson's disease (PD; Brichta et al., ; Kalia et al., ; Ztaou et al., ), dystonia (Peterson et al., ; Eskow Jaunarajs et al., ; Scarduzio et al., ), Tourette's syndrome (Xu et al., ; Albin et al., ), and Huntington's disease (Di Filippo et al., ).…”