Owing to their morphological complexity and dense network connections, neurons modify their proteomes locally, using mRNAs and ribosomes present in the neuropil (tissue enriched for dendrites and axons). Although ribosome biogenesis largely takes place in the nucleus and perinuclear region, neuronal ribosomal protein (RP) mRNAs have been frequently detected remotely, in dendrites and axons. Here, using imaging and ribosome profiling, we directly detected the RP mRNAs and their translation in the neuropil. Combining brief metabolic labeling with mass spectrometry, we found that a group of RPs rapidly associated with translating ribosomes in the cytoplasm and that this incorporation was independent of canonical ribosome biogenesis. Moreover, the incorporation probability of some RPs was regulated by location (neurites vs. cell bodies) and changes in the cellular environment (following oxidative stress). Our results suggest new mechanisms for the local activation, repair and/or specialization of the translational machinery within neuronal processes, potentially allowing neuronal synapses a rapid means to regulate local protein synthesis.
Highlights d Global proteome and phosphoproteome dynamics following homeostatic synaptic scaling d Approximately 3,300 activity-sensitive, synapse-associated phospho-events d Persistent signaling of~25% of initial phospho-events (min to 24 h) d Persistent and reciprocal phosphoregulation links synaptic up-and down-scaling
The proteasome, the major protein-degradation machine in cells, regulates neuronal synapses and long-term information storage. Here, using super-resolution microscopy, we found that the two essential subcomplexes of the proteasome, the regulatory (19
S
) and catalytic (20
S
) particles, are differentially distributed within individual rat cortical neurons. We discovered an unexpected abundance of free 19
S
particles near synapses. The free neuronal 19
S
particles bind and deubiquitylate lysine 63–ubiquitin (Lys
63
-ub), a non–proteasome-targeting ubiquitin linkage. Pull-down assays revealed a significant overrepresentation of synaptic molecules as Lys
63
-ub interactors. Inhibition of the 19
S
deubiquitylase activity significantly altered excitatory synaptic transmission and reduced the synaptic availability of AMPA receptors at multiple trafficking points in a proteasome-independent manner. Together, these results reveal a moonlighting function of the regulatory proteasomal subcomplex near synapses.
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 and synaptic processes, but it has not been studied on a global, proteome-wide scale during synaptic scaling. To examine this, we used 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 hours. Out of 45,000 phosphorylation events measured, ~3,300 (associated with 1,280 phospho-proteins) were regulated by homeostatic scaling. The activity-sensitive phosphoproteins were predominantly located at synapses and involved in cytoskeletal reorganization. We identified many early transient phosphorylation events which 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 was reciprocally regulated by up- or down-scaling, suggesting that the mechanisms underlying these two poles of synaptic regulation make use of a common signaling axis.
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