The Shank3-S1615 Phosphosite is Necessary for Synaptic Downscaling

Neurons maintain stable firing rates through synaptic scaling, a form of homeostatic plasticity that multiplicatively adjusts the strength of a neuron's excitatory synapses in response to changes in activity. When activity deviates from a neuron-specific baseline for an extended period, AMPA receptors (AMPAR) are added to or removed from the postsynaptic density to restore baseline firing rates, scaling synaptic strength up or down, respectively. Our lab previously showed that the scaffolding protein Shank3 is required for this process, and that changes in the phosphorylation state of Shank3 at residues S1586 and S1615 control the direction of synaptic scaling. A chronic increase in activity results in increased phosphorylation at these sites, which permits scaling down; similarly, a decrease in activity results in decreased phosphorylation of these sites and subsequent scaling up. Additionally, replacing serine with aspartic acid to mimic phosphorylation at both sites (S1586D and S1615D) prevents scaling up, while replacing serine with alanine to block phosphorylation at both sites (S1586A and S1615A) prevents downscaling. However, the individual contribution of each phosphosite to synaptic scaling is unknown. Here, I began to investigate this by testing whether the S1586A or S1615A mutations independently prevent downscaling in cultured pyramidal neurons. I overexpressed wild-type Shank3, Shank3-S1586A, or Shank3-S1615A in cultured neurons and treated the cells with PTX, a GABAA receptor antagonist that increases neuronal firing rates to induce downscaling. I then used confocal microscopy to measure changes in surface GluA2 intensity at putative synapses as a proxy for AMPAR abundance. I found that the S1615A mutation fully blocked scaling down, but the S1586A mutation did not, suggesting that only the S1615 phosphosite is required for synaptic downscaling.