Bossio, Alessandro; (2019) The role and regulation of Frizzled receptors in synapse formation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The formation of synapses is crucial for brain function. Secreted Wnt proteins signal through Frizzled and other receptors to regulate synaptogenesis. In particular, Wnt7a promotes synaptogenesis in the hippocampus. The receptor Fz5 mediates Wnt7a-induced presynaptic assembly, but the mechanisms underlying Fz5 regulation are not well understood. How Wnt7a signals at postsynaptic sites is also unknown. Fz7, another receptor binding Wnt7a, is hypothesised to have a role in this process. To address these questions, I used biochemical and cell biology techniques combining in vitro and in vivo approaches. My findings demonstrate that Fz5 and Fz7 have distinct synaptic localisation. Fz5 is absent from dendritic spines - excitatory postsynaptic structures - and is not required for spine development. In contrast, Fz7 localises in spines and is required for Wnt7a-induced spine formation. Our preliminary data suggested that Fz5 is palmitoylated, a post-translational lipid modification that affects protein distribution and function. I demonstrated that all Frizzled receptors can be palmitoylated. Using a palmitoylation-deficient Fz5 receptor, I showed that palmitoylation is required for Fz5 interaction with the scaffold protein Dishevelled, a key component of the Wnt signalosome, but has no impact on Fz5 degradation rate and lateral mobility at the plasma membrane. Palmitoylation-deficient Fz5 exhibits impaired axonal distribution, increased endocytosis and decreased surface levels. Expression of wild-type Fz5 in the hippocampus promotes presynaptic assembly, whereas palmitoylation-deficient Fz5 lacks synaptogenic activity. Palmitoylation is therefore a critical molecular mechanism that underpins Fz5 regulation and function in vivo. These findings demonstrate that two distinct Frizzled receptors act pre- and postsynaptically to promote synaptogenesis, and reveal a previously uncharacterised lipid modification of Frizzled receptors, which is of critical functional importance. This work opens up new avenues to study the role of Frizzled palmitoylation in different biological contexts, from cell fate decisions to neuronal circuit formation and plasticity.

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