A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

Abstract

Homeostatic synaptic plasticity requires widespread remodeling of synaptic signaling and scaffolding networks, but the role of post-translational modifications in this process has not been systematically studied. Using deep-scale quantitative analysis of the phosphoproteome in mouse neocortical neurons, we found widespread and temporally complex changes during synaptic scaling up and down. We observed 424 bidirectionally modulated phosphosites that were strongly enriched for synapse-associated proteins, including S1539 in the autism spectrum disorder-associated synaptic scaffold protein Shank3. Using a parallel proteomic analysis performed on Shank3 isolated from rat neocortical neurons by immunoaffinity, we identified two sites that were persistently hypophosphorylated during scaling up and transiently hyperphosphorylated during scaling down: one (rat S1615) that corresponded to S1539 in mouse, and a second highly conserved site, rat S1586. The phosphorylation status of these sites modified the synaptic localization of Shank3 during scaling protocols, and dephosphorylation of these sites via PP2A activity was essential for the maintenance of synaptic scaling up. Finally, phosphomimetic mutations at these sites prevented scaling up but not down, while phosphodeficient mutations prevented scaling down but not up. These mutations did not impact baseline synaptic strength, indicating that they gate, rather than drive, the induction of synaptic scaling. Thus, an activity-dependent switch between hypo- and hyperphosphorylation at S1586 and S1615 of Shank3 enables scaling up or down, respectively. Collectively, our data show that activity-dependent phosphoproteome dynamics are important for the functional reconfiguration of synaptic scaffolds and can bias synapses toward upward or downward homeostatic plasticity.

Clustering of CaV1.3 L‐type calcium channels by Shank3

“…In non-transfected neurons (NT) or neurons expressing control nonsense shRNA (nssh), punctate staining for endogenous Shank3 was readily detected in the soma and dendrites (Figure S4a), consistent with previous studies (Lutz et al, 2022;Perfitt et al, 2020;Wu et al, 2022;Zhang et al, 2005). Moreover, the intensity of somatic Shank3 puncta was essentially identical in non-transfected neurons and neurons expressing the control RNA (nssh/NT ratios: 1.19 ± 0.14 and 1.14 ± 0.12 in neurons co-expressing β3 and β2a subunits, respectively) (Figure S4b).…”

Microglial TNFα orchestrates protein phosphorylation in the cortex during the sleep period and controls homeostatic sleep

“…The latter is a central kinase in postsynaptic plasticity whose activity is controlled by phosphorylation at Thr 286 (Bayer & Schulman, 2019), also shown to be modified by TNFα. Another target phosphosite is Ser 1539 on the postsynaptic scaffold Shank3, recently shown to control synaptic availability of the protein and thereby expression of homeostatic plasticity (preprint: Wu et al , 2021). Examples can also be drawn on the regulation of presynaptic proteins by specific phosphorylations, herein identified as microglial TNFα‐dependent during the light period (Fig 4 and Dataset EV2).…”

Synaptopodin regulates denervation-induced plasticity at hippocampal mossy fiber synapses

“…We therefore conclude that the function of the spine apparatus organelle and its related protein synaptopodin might not be restricted to the mediation of receptor accumulation at postsynaptic sites but rather comprises the implementation of plasticity directionality (Vlachos, 2012). However, the link between synaptopodin and recently described mechanisms that switch plasticity directionality from homeostatic to associative rules remain to be determined (Wu et al, 2022). On the other hand, the lack of synaptopodin might decrease synaptic resilience due to the lack of stabilizing features (Yap et al, 2020), which is required to maintain synaptic transmission upon lesion.…”