title: Characterisation of M2 muscarinic acetylcholine receptor signalling in dental pulp stem cells
Creators: name: Alqahtani, Saeed Saad H.
Publisher: name: University of Glasgow
Year: 2023

description: Cholinergic signalling is hypothesised to occur in stem cells, and there is evidence that mesenchymal stem cells (MSCs) express a functional cholinergic system. Expression of functional acetylcholine receptors (AChRs) have been reported in several types of MSC, which suggests that MSCs have non-neuronal cholinoceptive properties that may play a role in their regenerative potential. However, this remains relatively unexplored, particularly, in Dental pulp stem cells (DPSCs). This project commenced by reviewing AChRs in MSCs, highlighting DPSCs characteristics, and then investigated the presence of functional AChRs and their role in modulating DPSCs regenerative potential.

This study commenced by identifying gene expression of both classes of AChRs, the muscarinic (mAChRs) and the nicotinic (nAChRs), in DPSCs. Protein expression of detected AChRs was assessed via western blotting and immunofluorescence. Functionality of expressed AChRs was assessed using an array of AChRs agonists and antagonists and DPSCs viable count was measured via MTT assay. Subtype selective agonist was used to study the role of the targeted AChR and its influence on DPSCs regenerative potential. Proliferation of DPSCs in response to that stimulation was assessed via measuring viable cell count using MTT assay, Cell Counting Kit-8 (CCK-8), and cell cycle analysis. Survival of DPSCs was assessed via detecting proliferation recovery, measuring Lactate dehydrogenase (LDH) levels, and detecting Annexin V/Propidium iodide staining. Stemness potential of DPSCs was assessed via detecting gene expression of MSCs stemness markers and pluripotency markers. Migration of DPSCs was investigated using a wound healing assays. Osteogenic differentiation of DPSCs was assessed via phenotypic mineralisation stains. Gene expression of cell cycle markers, stemness markers, osteogenic markers were assessed via Real-time polymerase chain reaction (q-PCR). Whole RNA sequencing (RNA-seq) was undertaking to measure transcriptome changes and enriched signalling pathways. Follow-up analysis was undertaking via measuring the phosphorylation and transcripts levels of ERK1 and ERK2 of the Mitogen-activated protein kinase (MAPK) pathway.

The results showed transcripts expression for the M2, M3 and M5 mAChRs, and expression of subunits that support the formation of α7 and α4β2-nAChRs. Subtype selective agonists/antagonists results suggest DPSCs to express functional M2 mAChR, α7 nAChRs, and α4β2-nAChRs. This was based on the ability of the agonists to influence DPSCs viable count and the subtype selective antagonist to cancel that effect. The project then focussed on mAChRs and protein expression of M2, M3 and M5 mAChRs were detected. The subsequent work focused on investigating the role of the M2 mAChRs in modulating the function of DPSCs via activating this receptor through its selective agonist Arecaidine propargyl ester (APE). Activation of the M2 mAChR inhibited DPSCs proliferation, in a reversable manner, without affecting DPSCs viability or survival. Further evidence showed that the M2 mAChR inhibits DPSCs proliferation by arresting cell cycle progression. This was further corroborated via expression analysis of key genes involved in the regulating cell cycle. The results also showed that M2 mAChR activation inhibited DPSCs migration and differentiation potential but did not interfere with DPSCs stemness. This was further corroborated via expression analysis of key genes involved in stemness and osteogenesis. The data obtained suggests that M2 mAChR activation induce DPSCs to go into a quiescent state.

The RNA-seq results showed that DPSCs responded differently to M2 mAChR activation 4 and 24 hours post activation, with different sets of differentially expressed genes (DEGs). The analysis of the enriched pathways suggested that M2 mAChR activation regulates cellular processes involved in metabolism, growth, adhesion, and response to stimuli. These processes function in proliferation, migration, and cell cycle through several metabolic pathways associated with response to cellular and oxidative stress. Follow up analysis showed upregulation of ERK1 and ERK2 phosphorylation and transcripts, which are downstream effectors of the MAPK pathway. The data obtained suggests that the transcriptomic data support the observed inhibitory effect of the M2 mAChR on DPSCs functions and highlights the many downstream effectors involved in the M2 mAChR downstream signalling.

In conclusion, this thesis presents evidence for the expression of a functional M2 mAChR in DPSCs, indicating the involvement of ACh signalling in modulating DPSCs behaviour. It also provides a promising route ultimately to pharmacologically control the regenerative output of DPSCs.