Phosphorylation of the M1 muscarinic acetylcholine receptor provides neuroprotection in mouse prion disease

The M1 muscarinic acetylcholine receptor (mAChR) plays a crucial role in learning and memory and is a validated drug target for the treatment of Alzheimer’s disease (AD). Pharmacological activation of the M1 mAChR can not only improve cognitive symptoms in AD patients but has also been proven to slow down disease progression in preclinical mouse models of neurodegeneration. Thus, the M1 mAChR has a promising potential as drug target for diseasemodifying therapies of AD (Scarpa et al., 2020). However, development of clinically effective M1 mAChR-targeted ligands has been challenging due to associated adverse effects, highlighting the need to dissect clinically relevant M1 mAChR-mediated pathways from those leading to undesirable outcomes. By employing a novel transgenic mouse model expressing a phosphorylationdeficient mutant of the M1 mAChR (M1-PD) (Butcher et al., 2016), our group previously found that adverse effects can be minimised through pathways favouring receptor phosphorylation (Bradley et al., 2020). This thesis aimed to extend this study and explore the role of M1 mAChR phosphorylation/arrestindependent pathways in the disease modification potential of the M1 mAChR. I combined M1-PD transgenic mice with prion neurodegenerative disease, a model of terminal neurodegeneration, leading to the discovery that disease is accelerated in M1-PD mice, thereby revealing an inherent neuroprotective property of the M1 mAChR that is dependent on receptor phosphorylation. To provide insight into the potential signalling mechanisms of the M1-PD, in vitro functional assays were performed on cell lines expressing the M1-PD version or wild-type of the M1 mAChR. Lack of receptor phosphorylation significantly impaired agonist-induced receptor internalisation, which is an important process in the desensitisation of G protein-dependent signalling. However, removal of M1 mAChR receptor phosphorylation was shown to have little impact on phosphoinositide accumulation, which is indicative of Gαq protein activation. The mouse prion disease model was then investigated through behavioural observations and histological and biochemical studies to characterise neurodegenerative disease progression through the detection of markers of disease. Mouse prion disease is caused by neurotoxic aggregates of misfolded prion proteins, and shares key hallmarks with human neurodegenerative diseases such as AD. These include memory and hippocampal function decline, disease markers such as APO-E, clusterin and serpinA3N, and widespread neuroinflammation, as indicated by the upregulation of astrocytic and microglial markers GFAP, Vimentin, Iba1 and CD86. Importantly, the appearance of misfolded, neurotoxic prion was shown to occur prior to the start of dosing studies of M1 mAChR-selective ligands (Bradley et al., 2017, Dwomoh et al., 2021), establishing that the therapeutic effects exerted by the M1 mAChR are not due to prevention of disease, but disease-modification. In this thesis, mouse prion disease model was also demonstrated to feature the significant upregulation of pro-inflammatory cytokines TNF-α, IL-1β and IL-6, similar to other neurodegenerative disorders characterised by chronic neuroinflammation. Removal of M1 mAChR phosphorylation in mice caused a significant acceleration of prion neurodegenerative disease progression. This was evident from behavioural changes such as faster hippocampal decline and symptom onset and shorter lifespan compared to wild-type animals, but also significantly elevated accumulation of misfolded prion and upregulation of markers of disease and neuroinflammation. Particularly, the pro-inflammatory cytokine TNF-α was significantly upregulated in prion-infected M1-PD mice with compared to wildtype mice, suggesting the M1 mAChR might be involved in the regulation of TNF-α. These findings unravelled an important neuroprotective property that is inherent to the M1 mAChR and depends on the receptor’s phosphorylation/arrestin-dependent signalling. In addition, these data have important implications for development of new drug treatments for neurodegenerative diseases, especially, M1 mAChR ligands that maintain receptor phosphorylation will more likely deliver neuroprotection that could not only improve memory symptoms but slow disease progression. Given the parallels between mouse prion disease and human proteinopathies, the neuroprotective mechanism observed here mediated by the M1 mAChR, is likely to be relevant to other human neurodegenerative conditions such as AD