PSD-95 is an abundant scaffolding protein found in the postsynaptic densities (PSDs) of excitatory synapses throughout the mammalian brain, and plays a critical role in innate and learned behaviours. PSD-95 assembles with numerous other proteins, including glutamate receptors, adhesion molecules and signalling proteins, into postsynaptic supercomplexes that are then organised into nanoclusters that comprise the postsynaptic density of excitatory synapses. While the subcellular localisation of PSD-95 has been widely studied, much less is known about its turnover. In this thesis, I present novel insights into PSD-95 synthesis and degradation at synapses of cultured primary neurons gained using the HaloTag technology. The HaloTag consists of an engineered bacterial protein domain that covalently binds synthetic ligands labelled with fluorescent and affinity moieties. Hence, cells expressing proteins fused to the HaloTag can be used to study protein levels, complexes and turnover using these different ligands. This project was based upon a knock-in mouse line expressing the HaloTag fused to endogenous PSD-95 using gene targeting. After demonstrating that these mice were phenotypically normal and that PSD95-HaloTag fusion proteins normally assembled into supercomplexes in the PSD, hippocampal primary cultures were grown from this mouse line. Fluorescent HaloTag ligands were then used to label live neurons, allowing for the visualisation of PSD-95 at synapses by confocal microscopy. Next, I established a pulse-chase labelling method, where one ligand is used to label all existing PSD-95 first, before a second ligand can then be used to label any newly synthesised PSD-95. This allows for the identification and characterisation of subpopulations of PSD-95, which can be separately analysed. I find that PSD-95 has a half-life of 36 hours at synapses, consistent with previous literature. I was also able to observe synaptic heterogeneity in PSD-95 turnover, and classify synapses into types according to their PSD-95 expression profile. Finally, a range of chemical compounds known to modulate protein turnover and neuronal activity was applied over a 24-hour period, and their effects on PSD-95 turnover analysed. It was found that inhibiting either the proteasome or protein synthesis led to significant reductions in PSD-95 degradation as well as inhibiting PSD-95 synthesis. Thus, this project established a method offering a unique way of investigating the turnover of a specific, tagged protein, as well as gaining novel insights into the turnover of PSD-95 at individual synapses.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:743826 |
| Date | January 2018 |
| Creators | Kratschke, Maximilian Moritz |
| Contributors | Grant, Seth ; Meijer, Dingenus |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/31131 |
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