Activity-Induced Cortical Glutamatergic Neuron Nascent Proteins

Abstract

Neuronal activity initiates signaling cascades that culminate in diverse outcomes including structural and functional neuronal plasticity, and metabolic changes. While studies have revealed activity-dependent neuronal cell type-specific transcriptional changes, unbiased quantitative analysis of cell-specific activity-induced dynamics in newly synthesized proteins (NSPs) synthesis in vivo has been complicated by cellular heterogeneity and a relatively low abundance of NSPs within the proteome in the brain. Here we combined targeted expression of mutant MetRS (methionine tRNA synthetase) in genetically defined cortical glutamatergic neurons with tight temporal control of treatment with the noncanonical amino acid, azidonorleucine, to biotinylate NSPs within a short period after pharmacologically induced seizure in male and female mice. By purifying peptides tagged with heavy or light biotin-alkynes and using direct tandem mass spectrometry detection of biotinylated peptides, we quantified activity-induced changes in cortical glutamatergic neuron NSPs. Seizure triggered significant changes in ∼300 NSPs, 33% of which were decreased by seizure. Proteins mediating excitatory and inhibitory synaptic plasticity, including SynGAP1, Pak3, GEPH1, Copine-6, and collybistin, and DNA and chromatin remodeling proteins, including Rad21, Smarca2, and Ddb1, are differentially synthesized in response to activity. Proteins likely to play homeostatic roles in response to activity, such as regulators of proteastasis, intracellular ion control, and cytoskeleton remodeling proteins, are activity induced. Conversely, seizure decreased newly synthetized NCAM, among others, suggesting that seizure induced degradation. Overall, we identified quantitative changes in the activity-induced nascent proteome from genetically defined cortical glutamatergic neurons as a strategy to discover downstream mediators of neuronal plasticity and generate hypotheses regarding their function. SIGNIFICANCE STATEMENT Activity-induced neuronal and synaptic plasticity are mediated by changes in the protein landscape, including changes in the activity-induced newly synthesized proteins; however, identifying neuronal cell type-specific nascent proteome dynamics in the intact brain has been technically challenging. We conducted an unbiased proteomic screen from which we identified significant activity-induced changes in ∼300 newly synthesized proteins in genetically defined cortical glutamatergic neurons within 20 h after pharmacologically induced seizure. Bioinformatic analysis of the dynamic nascent proteome indicates that the newly synthesized proteins play diverse roles in excitatory and inhibitory synaptic plasticity, chromatin remodeling, homeostatic mechanisms, and proteasomal and metabolic functions, extending our understanding of the diversity of plasticity mechanisms.

Neuronal membrane proteasomes regulate neuronal circuit activity in vivo and are required for learning-induced behavioral plasticity

“…Increased neuronal activity significantly increases protein synthesis in neurons compared with constitutive levels of protein synthesis ( 45 , 48 ). Based on the observation that NMPs preferentially degrade nascent proteins ( 13 ), one might expect that the total amount of nascent proteins degraded by NMPs would be higher under stimulated conditions than that under the basal condition because of increased substrate availability.…”

“…Synthesis of the proteasome subunit PSMD2 is up-regulated in the tadpole brain following 4 h of enhanced visual stimulation ( 11 ). The synthesis and degradation of proteasome components are known to be regulated by neuronal activity in the brain ( 19 , 48 ). In the brief pharmacological stimulation paradigm we employed here, we observed no change in nascent PSMD2 in the stimulated samples compared with the basal condition with normal NMP function.…”

Cell-selective proteomics segregates pancreatic cancer subtypes by extracellular proteins in tumors and circulation

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Cell type–specific labeling of newly synthesized proteins by puromycin inactivation

“…A Cre-inducible mouse line expressing a mutant methionyl-tRNA synthetase is currently available, opening up the possibility to specifically label newly synthesized proteins in a variety of cell types, limited by the Cre driver lines available ( 25 ). However, labeling times in the range of days (or weeks) have limited its application for tracking short-term changes in the proteome ( 24 , 26 ). More recently, owing to excellent temporal resolution and absence of bias toward amino acid composition of target proteins, PMY and its derivatives have found extended use for the efficient labeling of nascent peptides.…”