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Orji, Oliver Chukwuma (2025) Glutamate receptor-mediated m6A-RNA regulation of synaptic plasticity and neuronal function. PhD thesis, University of Nottingham.

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Abstract

Coordinated neuronal activity is fundamental for brain function and relies on tightly regulated molecular processes. Epitranscriptomic mechanisms, particularly N6-methyladenosine (m6A) RNA modification, have emerged as important regulators of RNA stability and translation. This modification is implicated in synaptic plasticity, the activity-dependent changes in synaptic strength that underly learning, memory, and cognition. Dysregulation of such processes contributes to neurological disorders. Increasing evidence links altered m6A dynamics to neurodegenerative diseases, including Parkinson’s disease and Dementia with Lewy bodies, both of which are characterised by α-synuclein pathology. However, the precise molecular mechanisms linking m6A to synaptic dysfunction and disease remain poorly understood.

In this thesis, I tested the hypothesis that transcripts central to synaptic plasticity, including those relevant to synucleinopathies, are differentially m6A-modified across the brain, and that activation of glutamate receptors alters the interactions between m6A-RNAs, their effector proteins, and α-synuclein. The thesis further explored whether m6A regulation of plasticity occurs within a broader framework of cross-talk with other RNA modifications and mitochondrial processes.

Analysis of high-throughput m6A-RNA sequence data of multiple human brain regions revealed region-specific modification patterns. The cerebellum displayed the highest enrichment of m6A-modified transcripts, while ionotropic glutamate receptor subunits GRIN2A and GRIA2 were among the most highly and multi-modified. These findings suggest that m6A modification may display regional diversity in brain function regulation.

In vitro studies using differentiated SH-SY5Y (dSH-SY5Y) neuronal cells demonstrated that m6A modifications are highly dynamic in response to receptor stimulation. Activation of NMDA, AMPA, and kainate receptors (NMDAR, AMPAR and KAR) revealed distinct temporal patterns of m6A-RNA colocalisation with m6A effector proteins. Notably, KAR activation for 5 minutes increased YTHDF2 colocalisation with m6A in both cytoplasmic and postsynaptic compartments, whereas NMDAR and AMPAR activation produced significant reductions. The m6A demethylase, ALKBH5 showed increased postsynaptic region colocalisation with m6A after activation of all receptor subtypes, and NMDAR stimulation enhanced demethylase activity in both cytoplasmic and synaptic fractions. These results suggest that m6A-RNA is dynamically regulated during early phases of synaptic plasticity.

Transcriptome-wide profiling of NMDA-stimulated dSH-SY5Y cells further identified differential m6A modification of synapse-relevant transcripts, including those encoding glutamate receptors, SNARE proteins, cytoskeletal elements, and mitochondrial respiratory chain components. This highlights the role of m6A in regulating transcripts critical for both synaptic signalling and cellular energetics.

The relationship between m6A and α-synuclein was also investigated. While the SNCA transcript was not extensively modified, SNCG, which encodes γ-synuclein, was highly multi-modified. Colocalisation studies revealed that phosphorylated α-synuclein associates with m6A-RNAs at presynaptic sites, and that this association increases following oxidative stress. These findings suggest a functional link between m6A and α-synuclein biology, with potential implications for synaptic vulnerability in neurodegenerative disease.

Finally, evidence was obtained for cross-system regulation between m6A and other RNA modifications. Reciprocal modification of transcripts encoding m6A and m5C effector proteins was observed, and proteomic analyses revealed shared functional pathways involving phosphorylation, SUMOylation, ubiquitination, and mitochondrial processes. Colocalisation of m6A with m5C and m3C after NMDAR activation further supported the view that multiple RNA modifications act cooperatively to regulate neuronal plasticity.

In conclusion, this thesis demonstrates that m6A-RNA modification is a rapid, dynamic regulator of glutamate receptor-mediated synaptic plasticity. It acts on key synaptic and mitochondrial transcripts, interacts with α-synuclein at presynaptic sites under stress conditions, and engages in crosstalk with other RNA modification systems. Together, these findings establish m6A-RNA regulatory dynamics as a central epitranscriptomic mechanism that integrates neurotransmission, mitochondrial function, and RNA modification networks, providing novel insight into synaptic regulation and disease mechanisms.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Knight, Helen Miranda Fray, Rupert
Keywords:	Synaptic plasticity, m6A, m5C, m3C, RNA methylation, Epitranscriptomics, Cross regulation, brain function.
Subjects:	QS-QZ Preclinical sciences (NLM Classification) > QT Physiology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	82748
Depositing User:	Orji, Oliver
Date Deposited:	31 Dec 2025 04:40
Last Modified:	31 Dec 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/82748

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