Activity deprivation modulates the Shank3/Homer1/mGluR5 signaling pathway to enable synaptic upscaling

Shank3 is an autism spectrum disorder-associated postsynaptic scaffold protein that links glutamate receptors to trafficking and signaling networks within the postsynaptic density. Shank3 is required for synaptic scaling (Tatavarty et al., 2020), a form of homeostatic plasticity that bidirectionally modulates post-synaptic strength in the right direction to stabilize neuronal activity. Shank3 undergoes activity-dependent phosphorylation/dephosphorylation at S1586/S1615, and dephosphorylation at these sites is critical for enabling synaptic upscaling (Wu et al., 2022). Here, we probe the molecular machinery downstream of Shank3 dephosphorylation that allows for synaptic upscaling. We first show that a phosphomimetic mutant of Shank3 has reduced binding ability and interaction with long-form Homer1, a postsynaptic protein also crucial for scaling, and a known binding partner of Shank3. Since metabotropic glutamate receptor 5 (mGluR5) has been shown to associate with Shank3 through long-form Homer1, we manipulated mGluR5 signaling with either noncompetitive or competitive inhibitors and found that only competitive inhibition (which targets agonist-dependent signaling) impairs synaptic upscaling. Further, we found that mGluR5 activation rescues synaptic upscaling in the presence of phosphomimetic Shank3, thus is downstream of Shank3 phosphorylation. Finally, we identify necessary signaling pathways downstream of group I mGluR. Taken together, these data show that activity-dependent dephosphorylation of Shank3 remodels the Shank3/Homer1/mGluR signaling pathway to favor agonist-dependent mGluR signaling, which is necessary to enable synaptic upscaling. More broadly, because downscaling depends on agonist-independent mGluR5 signaling, these findings demonstrate that synaptic up and downscaling rely on distinct functional configurations of the same signaling elements. SIGNIFICANCE STATEMENTSynaptic scaling is a bidirectional, homeostatic form of synaptic plasticity that allows neural circuits to maintain stable function in the face of experience-dependent or developmental perturbations. Synaptic scaling up requires dephosphorylation of the Autism Spectrum Disorder (ASD)-associated synaptic scaffold protein Shank3, but how this dephosphorylation event enables scaling up was unknown. Here we show that dephosphorylation of Shank3 rearranges interactions between synaptic proteins to drive agonist-dependent signaling through metabotropic glutamate receptors (mGluRs), and that this signaling is necessary for scaling up. These findings show that altered mGluR signaling is downstream of Shank3 during homeostatic plasticity, and raise the possibility that some human Shankopathies impair signaling through this important signaling pathway.