Argonaute-2 Serine-388 Phosphorylation is Essential for Metabotropic Glutamate Receptor-Dependent Long-Term Depression and Spatial Memory

Abstract

Local and rapid changes in neuronal protein synthesis are necessary for plasticity. The RNA induced silencing complex (RISC) contributes to the translational repression of mRNA. Argonaute 2 (Ago2) is a highly conserved RISC component that is phosphorylated at serine-387 (S387) following NMDAR stimulation in primary neurons. Ago2 S387 phosphorylation promotes RISC assembly, culminating in the targeted downregulation of Limk1 expression and dendritic spine shrinkage in vitro. Since these findings suggest a role for S387 phosphorylation in plasticity, there is a need to investigate how this could contribute to intact brain function. Using a novel Ago2^S388A/S388A mouse model, in which the murine homologue of S387 is globally mutated to non-phosphorylatable alanine, this thesis investigates how blocking Ago2 S388 phosphorylation affects the hippocampus and perirhinal cortex in vivo.

Behavioural paradigms performed here reveal that S388 phosphorylation is required for hippocampal-dependent short- and long-term forms of spatial memory, but not for perirhinal cortex-dependent novel object recognition memory. It is also shown using ex vivo extracellular field recordings that Ago2 phosphorylation is necessary for mGluR-dependent long-term depression (LTD), but not NMDAR-dependent LTD, in both brain regions. Blocking S388 phosphorylation unexpectedly results in the basal ‘thinning’ of dendritic spines on CA1 pyramidal neurons visualised using Golgi-Cox staining, whilst spines on cortical neurons are unaffected. Importantly, immunoblotting suggests that the downstream regulation of Limk1 expression by Ago2 S387 phosphorylation previously seen in vitro does not take place in vivo. Immunoblotting of synaptic proteins in hippocampal synaptosomes prepared from Ago2^S388A/S388A mice also reveals that blocking phosphorylation downregulates the synaptic expressions of the AMPAR subunits GluA1 and GluA2, and upregulates components of the AP2 complex, which drives the clathrin-mediated endocytosis of AMPARs and other transmembrane proteins.

This thesis defines a novel role for the regulation of RISC function by Ago2 phosphorylation in neuronal processes underlying learning and memory.

Date of Award	17 Mar 2026
Original language	English
Awarding Institution	University of Bristol
Supervisor	Jonathan G Hanley (Supervisor), Elizabeth Warburton (Supervisor) & Jonathan Brown (Supervisor)