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A Bidirectional Switch in the Shank3 Phosphorylation State Is Necessary to Enable Synaptic Scaling Up and Down

ABSTRACT: Wu C, Tatavarty V, Jean-Beltran PM, Guerrero A, Keshishian H, Krug K, MacMullan M, de Arce KP, Carr SA, Cottrell J, Turrigiano GG. 2021 Homeostatic synaptic plasticity requires widespread remodeling of synaptic signaling and scaffolding networks, but the role of posttranslational modifications in this process has not been systematically studied. Here we analyzed changes in the phosphoproteome during synaptic scaling up and down and found wide-spread and temporally complex changes. These included 424 bidirectionally modulated phosphosites that were strongly enrichment for synapse-associated proteins, including the ASD-associated synaptic scaffold protein Shank3. Shank3 was dephosphorylated at two highly conserved sites (rat S1586 and S1615) during scaling up, and hyperphosphorylated during scaling down. These changes modified the synaptic localization of Shank3 during scaling, and phosphomimetic or deficient mutants of Shank3 prevented scaling up or down, respectively. Finally, we found that dephosphorylation of these sites via PP2A activity was essential for the maintenance of synaptic scaling up. Thus Shank3 undergoes an activity-dependent switch between hypo- and hyperphosphorylation at S1586/ S1615, that is necessary to enable scaling up or down, respectively. More broadly, our data suggest that widespread and bidirectional changes in the synaptic phosphoproteome are essential for the functional reconfiguration of synaptic scaffolds during homeostatic plasticity.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Steven A. Carr

PROVIDER: MSV000087926 | MassIVE | Fri Jul 30 13:48:00 BST 2021

REPOSITORIES: MassIVE

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Project description:Neuronal networks are subject to fluctuations in both the magnitude and frequency of inputs, requiring plasticity mechanisms to stabilize network activity. Homeostatic synaptic scaling is a form of synaptic plasticity that adjusts the strength of neuronal connections up or down in response to large changes in input. Although homeostatic plasticity requires changes in gene expression, there is only limited data describing the molecular changes associated with homeostatic scaling, focusing mostly on the expression mechanisms involving glutamate receptors. The fact that neuronal networks can be scaled up (in response to reduced activity) or down (in response to enhanced activity) provides a unique opportunity to examine the molecular and proteomic response to opposite ends of the phenotypic spectrum of synaptic plasticity. Here we first demonstrate that homeostatic scaling required protein synthesis. We then examined the plasticity-induced changes in the newly-synthesized neuronal proteome of neurons to identify the landscape of proteomic changes that contribute to opposing forms of homeostatic plasticity. Cultured rat hippocampal neurons (21 DIV) underwent homeostatic upscaling or downscaling (treatments with TTX and Bicucculine, respectively). We used BONCAT (BioOrthogonal Non-Canonical Amino acid Tagging) to metabolically label, capture and identify newly-synthesized proteins, detecting and analysing 5940 newly-synthesized proteins using liquid chromatography-coupled tandem mass spectrometry and label-free quantitation. Neither up- or down-scaling produced changes in the number of different proteins translated. Rather, our findings indicate that synaptic up- and down-scaling elicit opposing translational regulation of several molecular pathways, producing targeted adjustments in the neuronal proteome. We detected ~ 300 differentially regulated proteins involved in neurite outgrowth, reorganization of nerve terminals, axon guidance and targeting, neurotransmitter transport, filopodia assembly, excitatory synapses and glutamate receptor complexes. These proteins include well-characterized mediators of synaptic plasticity, e.g. the ionotropic glutamate receptor complex that is down-regulated during down-scaling and coordinately upregulated during upscaling. We also identified differentially regulated proteins that in addition to their regulation in homeostatic plasticity, are also associated with multiple diseases and disorders, including intellectual disability, schizophrenia, epilepsy, and Parkinson’s disease.

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A Bidirectional Switch in the Shank3 Phosphorylation State Is Necessary to Enable Synaptic Scaling Up and Down

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