Investigating the potential role of the M1 muscarinic acetylcholine receptor in misfolded protein propagation

Alepuz Guillen, Jose Andres (2024) Investigating the potential role of the M1 muscarinic acetylcholine receptor in misfolded protein propagation. PhD thesis, University of Glasgow.

Abstract

Prion diseases are a variety of fatal neurodegenerative diseases characterised by the misfolding, aggregation and propagation of the prion protein. There are currently no disease modifying treatments available that can slow the progression of the disease (Scarpa et al., 2020). However, previous data from our group suggests that the activation of the M1 muscarinic acetylcholine receptor (mAChR) can both restore memory deficits as well as slow the accumulation of misfolded scrapie prion protein (PrPˢᶜ) in a in vivo murine prion disease model (Bradley et al., 2017; Dwomoh, Rossi, et al., 2022). These findings pose a question around the mechanism via which the M1 mAChR signalling acts in a neuroprotective mode against neurodegeneration by slowing down the propagation of misfolded prion.

In this study, PrPˢᶜ from diseased animals was purified and administered to primary neuronal cultures to evaluate prion infection and propagation. In prion protein overexpressing neurons, infection with PrPˢᶜ resulted in the progressive accumulation and propagation of PrPˢᶜ over time. In contrast, prion protein knockout cultures showed a gradual degradation of PrPSc after infection due to the lack of endogenous cellular prion (PrPᶜ) to act as a substrate for propagation. In this study, results confirmed the successful infection and propagation of PrPˢᶜ in vitro suggesting that PrPᶜ is required for PrPˢᶜ propagation but not for infection.

To understand the role of the M1 mAChR in prion infection and propagation, neurons from M1 wildtype, M1 knockout, M1-DREADD (designer receptor exclusively activated by designer drugs) and M1-PD (phosphorylation deficient) mice were used. These primary neuronal cultures were also chronically treated for 7 days, after infection, with several muscarinic ligands with different potencies, efficacies and affinities to test the impact of muscarinic downstream signalling on prion infection and misfolding. No significant differences in PrPˢᶜ accumulation were observed in all strains, with or without ligand treatment. This data suggests that the action of the receptor could be more important in the presence of glia, requiring the expression of these cell types for a reduction in PrPˢᶜ to be observed.

A proteomic study of this novel prion model in primary neuronal cultures showed several dysregulations in mitochondrial, ribosomal and neuronal-associated proteins as a consequence of PrPˢᶜ infection. Results revealed dysregulation of neuronal proteins which had not been previously observed in vivo prion disease mouse models, possibly hindered by the high upregulation of neuroinflammatory-associated proteins. It also highlighted several cytoskeletal-associated proteins that could potentially be involved in the mechanisms of PrPˢᶜ infection and propagation.

These findings validate this model to specifically study infection and propagation of PrPˢᶜ in neurons, with the advantage of no neuronal degeneration and disease. Further research into the role of muscarinics and other proteins in prion infection and propagation utilising this in vitro primary neuronal cell model may allow to develop novel drug candidates with disease modifying effects.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: R Medicine > RC Internal medicine > RC0321 Neuroscience. Biological psychiatry. Neuropsychiatry
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Tobin, Professor Andrew and Milligan, Professor Graeme
• Date of Award: 2024
• Depositing User: Theses Team
• Unique ID: glathesis:2024-84698
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 15 Nov 2024 11:31
• Last Modified: 15 Nov 2024 11:31
• Thesis DOI: 10.5525/gla.thesis.84698
• URI: https://theses.gla.ac.uk/id/eprint/84698