Local protein synthesis is a ubiquitous feature of neuronal pre- and postsynaptic compartments

INTRODUCTION The regulation of synaptic proteins by posttranslational modifications and by ongoing protein synthesis and degradation drives homeostasis and plasticity at synapses. A key question is where synaptic proteins are made. Most of the neuron’s volume comprises axons and dendrites that can be micrometers or millimeters long. Thus, a substantial fraction of proteomic remodeling could potentially occur locally within both axons and dendrites. Whereas there is wealth of data indicating that protein synthesis occurs in mature dendrites, there has been much less evidence in support of local translation in mature axons. RATIONALE Efforts to localize molecules or cell biological events to neuronal pre- or postsynaptic compartments by using fluorescence microscopy are limited by the tight association of the axonal bouton and the dendritic spine; the synaptic cleft, the only clear region of separation, is only ~20 nm wide. In this study, to increase the resolving power to visualize RNA molecules in pre- and postsynaptic compartments, we optimized fluorescence in situ hybridization (FISH) and nascent protein detection methods for use with expansion microscopy. To characterize transcripts and translational machinery in excitatory presynaptic terminals, we used a recently developed platform that couples fluorescence sorting with biochemical fractionation to sort and purify fluorescently labeled synaptosomes. To obtain direct evidence for protein synthesis in synaptic compartments, particularly presynaptic boutons, we adapted the puromycin-based metabolic labeling strategy for detection with electron or expansion microscopy. We also examined the translational signature of these three different forms of local protein synthesis–dependent plasticity in three different synaptic compartments: dendritic spines, excitatory terminals, and inhibitory terminals. RESULTS In adult rodent brain slices and cultured neurons, we found that >75% of both excitatory and inhibitory presynaptic terminals contain the machinery for protein synthesis: rRNA, ribosomes, and polyadenylated [poly (A) + ] mRNA. Using mature mouse forebrain synaptosomes that are enriched for vGLUT1 + presynaptic terminals, we identified ~450 transcripts that were enriched (relative to the generic synaptosome transcriptome). These included many mRNAs that code for proteins that regulate neurotransmitter release. Both light microscopy (confocal and super-resolution) and electron microscopy revealed that in the absence of overt stimulation, there was a notably high level of ongoing protein synthesis in both pre- and postsynaptic compartments. After just 5 min of metabolic labeling, ~40% of both excitatory and inhibitory presynaptic terminals and ~60% of dendritic spines exhibited active translation. Three different forms of synaptic plasticity resulted in distinct patterns of protein synthesis stimulation in dendritic spines and in excitatory and inhibitory presynaptic axon terminals. CONCLUSION In this study, we investigated the localization and stimulation of protein synthesis in mature synapses. We unambiguously identified protein synthesis machinery and mRNA translation in individual synaptic compartments. Both excitatory and inhibitory presynaptic boutons (as well as postsynaptic spines) carry out protein synthesis regularly, in the absence of any exogenous stimulation. Synthesis within these three compartments is differentially recruited to modify local proteomes during synaptic plasticity. Local protein synthesis adds spatial and temporal precision for proteome remodeling that can be exploited to rapidly modify synapses in specific subcellular compartments.