M4 Muscarinic Acetylcholine Receptor Modulation of Dopamine Receptor Functions

M₄ muscarinic acetylcholine receptors (mAChRs) belong to the Rhodopsin family of G protein-couple receptors. These receptors are found most abundantly in the striatum and are implicated in a number of central nervous system disorders, including schizophrenia. Indeed, a M₁/M₄ mAChR subtype-preferring agonist, xanomeline, has been shown in clinical trials to alleviate psychotic symptoms and improve cognitive deficits associated with both Alzheimer’s disease and schizophrenia. The antipsychotic effects of xanomeline were found to be predominantly M₄ mAChR-mediated, which is in contrast with the multi-targeted mode of action of current antipsychotics, which display poly-pharmacology but have the D₂ dopamine receptor (DR) as a common therapeutic target. In the striatum, M₄ mAChRs are co-expressed with D₁ DRs in direct GABAergic output medium spiny neurons and with D₂ DRs in cholinergic interneurons. M₄ mAChRs have been shown to modulate striatal dopaminergic activity, and many M₄ mAChR positive allosteric modulators have been developed as potential antipsychotics.
 
   In Chapter 2, the ability of a label-free technology to detect and quantify the positive allosteric modulation of endogenous M₄ mAChR in a rodent neuronal cell line was established. The allosteric parameters estimated using this approach are comparable to those estimated from endpoint-based assays, demonstrating that label-free technologies can be used to screen for allosteric modulators, including those with no known G protein-coupling preferences.
 
   Chapters 3 and 4 explored the modulation of endogenous D₂ DRs and D₁ DRs by M₄ mAChRs in vitro and in vivo, respectively. In Chapter 3, it was first established that the NG108-15 cell line endogenously expresses both M₄ mAChRs and D₂ DRs, and that allosteric modulation of ACh by LY2033298, a M₄ mAChR-selective positive allosteric modulator in the presence of ACh, can be detected with end-point based signalling assays, as well as with label-free technology. The presence of functional cross-talk between M₄ mAChRs and D₂ DRs was determined by performing interaction studies with an M₄ mAChR orthosteric agonist, inverse agonist and positive allosteric modulator combined with D₂ DR ligands in two end-point based signalling assays. Though some small changes to efficacy were observed in some interactions, overall, there was no apparent functional cross-talk between these two receptors. This suggests that the cell line and the assays used for this study was unsuitable for detecting functional cross-talk between M₄ mAChRs and D₂ DRs.
 
   In Chapter 4, the cross-talk between M₄ mAChRs and D1 DRs in vivo was investigated, using mouse models of aspects of psychosis. R(+)-6-Br-APB, a selective D₁ DR agonist, was used to induce D₁ DR-mediated disruption of prepulse inhibition and increases in locomotor activity in C57Bl/6J mice. LY2033298 in combination with donepezil, an acetylcholinesterase inhibitor, showed a trend to reverse the R(+)-6-Br-APB-induced disruption of prepulse inhibition. In locomotor activity experiments, combined LY2033298 and donepezil treatment significantly reduced the R(+)-6-Br-APB-induced increase in locomotor activity.
 
   Chapter 5 describes the investigation of the role of M₄ mAChRs in the reversal effects of LY2033298 and donepezil using whole-body M₄ mAChR knockout mice. However, the results were inconclusive.
 
   Finally, Chapter 6 provides a summary of the findings and discusses the potential future directions of this study.