Final Report Summary - NEURONAL DYNAMICS (Dynamics of local transcriptomes and proteomes in neurons)
Brains are dynamic organs - changing their properties to encode the behavioral experiences of animals and allowing them to adapt to future situations. The ability of the brain to respond adaptively relies on modifications to existing proteins as well as changes in gene transcription and protein translation. It is now clear that synapses, the points of communication between neurons, possess the capacity for local protein synthesis, owing to the localization of ribosomes and mRNAs within dendrites. Previous studies have identified a relative small number of localized mRNAs and an even smaller number of locally synthesized proteins. Here we discovered a near-complete list of mRNAs that are localized, “the local transcriptome”. Using the deep RNA sequencing technology, we identified over 2500 mRNAs that are present in the dendrites and/or axons of neurons. Using a new high-resolution platform that makes use of “fluorescent barcodes” we visualized individual mRNAs and quantified their abundance in the dendrites. We discovered that brain mRNAs have extended non-translated regions that harbor binding platforms for regulatory elements. In a related project, we discovered that the small non-coding family of microRNAs can be generated locally within dendrites upon synaptic stimulation. MicroRNAs inhibit the translation of mRNAs by binding to complementary sequences, usually present in the untranslated region of the mRNA. This spatial maturation of the miRNA allows for coordinated regulation of mRNA translation in space and time. We further developed a bioorthogonal labeling strategy that we invented earlier with Daniela Dieterich and Dave Tirrell. In this labeling technique, newly synthesized proteins are labeled with a non-canonical amino acid, charged onto tRNAs with a mutant methionyl tRNA synthetase, and then incorporated into protein. By expressing the mutant synthetase with cell type-specific promoters we are able to identify the newly synthesized proteins in a given cell type. We used this to examine the proteins that are synthesized. We can also discover the proteins that are synthesized in dendrites, “the local proteome”, using a novel chemical tagging strategy that we developed. Using this approach, we discovered how homeostatic scaling of synapses alters the neuronal proteome. We also developed a technique that allows one to visualize a newly synthesized protein of interest in intact tissue - something that was not possible before. These results close a critical gap in our understanding of how different pattern of activity are read out by rapid modifications of the local proteome. These data begin to give us a “systems” view of the synapse.