Investigating the structural and functional role of the Shank3 N-terminal region in neurons

Shank3 is a scaffolding protein expressed within the postsynaptic density (PSD) of glutamatergic neurons. Mutations throughout Shank3 have been linked with neurodevelopmental and neuropsychiatric conditions notably, autism. However, the function of Shank3 and how these mutations lead to these pathological phenotypes are unclear. Recent research has revealed many key protein components of the PSD which undergo liquid- liquid phase separation (LLPS) form biomolecular condensates. The Shank3 N-terminal region consists of the SPN (Shank/ProSAP) N-terminal domain and ARR (Ankyrin Repeat Region). This region harbours dynamic and multivalent properties, key features of proteins capable of undergoing LLPS, as well as an actin-binding region. Due to the increasing connection between LLPS and PSD regulation, we investigated the ability of the N-terminal region to undergo LLPS under various in vitro conditions. These experiments revealed a propensity for the N-terminal region to form stable nanoclusters, often precursors to biomolecular condensates. We then examined mutations in the N-terminal region which disrupt the interaction between the SPN-ARR fragments, including the autism-associated P141A mutation and the artificial N52R mutation. We found the most intrinsically unstable mutant, N52R, had the highest propensity to form nanoclusters. Next, we sought to examine the effect of these mutations on the cytoskeletal protein actin. SPN-ARR WT and P141A displayed similar binding affinities while N52R significantly increased binding to actin according to a co-sedimentation assay. Further Cryo-EM analysis revealed SPN-ARR N52R induced cross-linking of polymerised actin, a property not observed for the WT fragment. Finally, we sought to understand if these observations occur in a neuronal context by expressing Shank3 WT and mutants in Drosophila melanogaster primary neuronal cell culture. Microscopy image analysis of neuronal cell culture revealed distinct differences in localisation of Shank3 WT, P141A, N52R and R12D mutations. Overall, this work highlights the functional significance of the Shank3 N-terminal region and implications of mutations disrupting the intra-domain interaction. Further research is required to characterise the impact of mutations disrupting this interaction on actin dynamics in neurons.