Dynamic nanoclustering of synaptic proteins in health and disease: an examination of pre- and post-synaptic protein mobility in cell-to-cell communication

Abstract

Cell to cell communication within the brain occurs at synaptic junctions that link adjacent neurons. These connections are formed from a pre and postsynapse. Neurotransmission is driven by the fusion of synaptic vesicles with the plasma membrane, which causes the release of specialised chemicals, termed neurotransmitters, from the presynapse. Neurotransmitters bind receptors at the postsynapse, which causes influx of ions leading to depolarisation of the intracellular environment and initiation of action potential. Within presynapses, a multitude of proteins come together to coordinate the release of neurotransmitter. This process is rapid and highly regulated, involving a number of proteins within the pre and postsynapse acting in concert with one another. In recent years, the development of super-resolution imaging has allowed us to examine the local recruitment and nanoclustering of synaptic machinery. Studies have highlighted the importance of nanoclustering in facilitating the activity and trafficking of key proteins that drive neurotransmission at the pre and postsynapse. Perturbations in nanoclustering events are also linked to disease pathology. In this study, we examine the impact of nanoclustering of the presynaptic molecules synaptotagmin-1 (Syt1) and synaptic vesicle protein 2A (SV2A) and how clustering and mobility impacts the recycling of both molecules into synaptic vesicles (SVs). We also look at how nanoclustering is linked to the activity of the kinase Fyn, which is involved in strengthening postsynaptic neurotransmission but also plays a role in the pathology of Alzheimer’s Disease (AD) and Frontotemporal Dementia (FTD).The research presented here is divided into seven parts. The first chapter provides a short summary of neurotransmission and the role of nanoclustering in neurotransmission. The second chapter describes the methodological approaches used to examine the nanoscale clustering and mobility of key synaptic molecules during neurotransmission. The third chapter reviews the literature surrounding Syt1 and SV2A, and their involvement in neurotransmission. The fourth chapter presents a manuscript in preparation in which examine the nanoscale organization of Syt1 and SV2A during neurotransmission. We conclude that interaction with SV2A primes the nanoclustering of Syt1 on the plasma membrane and in synaptic vesicles, with implications on the recycling of both molecules in nerve terminals. The fifth chapter describes the impact of the R383Q mutation, which confers epilepsy, on the trafficking and nanoscale organization and trafficking of SV2A and Syt1. The sixth chapter deals with nanoclustering during postsynaptic neurotransmission and provides a submitted manuscript in which the mechanisms responsible for controlling the nanoclustering of the molecule Fyn kinase at the postsynapse are characterised. The study presented in this chapter determines that the phospho-dependent entry into an open conformation drives nanoclustering of Fyn, and that this nanoclustering is exacerbated in FTD. The final chapter provides a conclusion and discussion to the work presented in this thesis.