Teo, Zhi Yao Samuel; (2024) Palmitoylation of the Frizzled-5 receptor and its role in Wnt signalling and synapse assembly. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Synapses are critical microstructures required for many brain functions. Thus, the proper formation of synapses is an integral developmental process. Wnts are secreted proteins that promote synapse assembly through Frizzled (Fz) receptors. In particular, Fz5 is required on the presynaptic side for Wnt7a-mediated synaptogenic activity. Neuronal activity is also a key modulator of synapse assembly. Indeed, the pattern of neuronal activity regulates the surface and synaptic localisation of Fz5 in hippocampal neurons. However, the precise mechanisms regulating the localisation of synaptogenic receptors such as Fz5 are not well understood. In this thesis, I focus on a novel post-translational modification on Frizzled receptors, palmitoylation. I show that Frizzled receptors with known roles in synapse assembly, including Fz5, are endogenously palmitoylated in the hippocampus. I also demonstrate the palmitoylation of Fz5 using different methods. This post-translational modification occurs at three consecutive cysteines of the Fz5 C-terminus. By examining the contribution of palmitoylation to Fz5’s role in Wnt signalling and synapse assembly, I show that palmitoylation-deficient Fz5 fails to activate the canonical Wnt signalling pathway, which is required for Fz5-mediated synaptogenic activity. Consistent with these results and other in vivo results from our laboratory, palmitoylation-deficient Fz5 does not promote excitatory presynaptic assembly in hippocampal neurons. In this thesis, I also demonstrate that the pattern of neuronal activity regulates the levels of Fz5 palmitoylation, possibly by modulating Wnt7a levels. I show that palmitoylation-deficient Fz5 cannot localise to the cell surface in response to neuronal activity. Consequently, palmitoylation-deficient Fz5 blocks activity-mediated synapse assembly. Finally, I identify zDHHC5 as the enzyme required for Fz5 palmitoylation and demonstrate that zDHHC5 knockout phenocopies the behaviour of palmitoylation-deficient Fz5 in hippocampal neurons. Together, these results reveal that palmitoylation is an important mechanism regulating the function of Frizzled receptors, opening new avenues to modulate Wnt receptors in health and disease.

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