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Dynamic bi-directional phosphorylation events associated with the reciprocal regulation of synapses during homeostatic up- and down-scaling.

ABSTRACT: Homeostatic synaptic scaling allows for bi-directional adjustment of the strength of synaptic connections in response to changes in their input. Protein phosphorylation modulates many neuronal processes, but it has not been studied on a global scale during synaptic scaling. Here, we use liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses to measure changes in the phosphoproteome in response to up- or down-scaling in cultured cortical neurons over minutes to 24 h. Of ~45,000 phosphorylation events, ~3,300 (associated with 1,285 phosphoproteins) are regulated by homeostatic scaling. Activity-sensitive phosphoproteins are predominantly located at synapses and involved in cytoskeletal reorganization. We identify many early phosphorylation events that could serve as sensors for the activity offset as well as late and/or persistent phosphoregulation that could represent effector mechanisms driving the homeostatic response. Much of the persistent phosphorylation is reciprocally regulated by up- or down-scaling, suggesting that mechanisms underlying these two poles of synaptic regulation make use of a common signaling axis.

SUBMITTER: Desch K

PROVIDER: S-EPMC8411114 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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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Dynamic bi-directional phosphorylation events associated with the reciprocal regulation of synapses during homeostatic up- and down-scaling.

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