The main focus of the grant was to discover the diversity of ribosomes in neurons, neuronal compartments and synapses, which includes (i) comparing the ribosome population of neurons to other somatic cells, (ii) discovering the diversity of ribosomes in neuronal cell bodies vs. dendrites and of ribosomes associated with synapses, and (iii) examining the nature of ribosomes heterogeneity. In the project, we successfully isolated cell-type specific assembled ribosomes from different brain samples. For the subsequent analysis of the ribosomal and ribosomal-associated proteins, we have optimized the protocol for mass spectrometry (MS), and with this we obtained MS data comparing inhibitory (Gad2+) and excitatory (CamK2a+) neurons from the cortex of adult mice. In order to access the ribosomal diversity in neuronal compartments, we isolated ribosomes from cortical neurons cultured on membrane inserts in which the cell bodies and the processes can be separately harvested. The nature of ribosomes undergoing translation was addressed via polysome profiling. We have also profiled the ribosomes that are singly associated with a transcript (monosomes) and those that are multiply associated with transcripts (polysomes). We found that monosomes were a surprisingly dominant format for translation in the neuropil. These monosomes carry out active protein synthesis (Biever et al., Science, 2020).
In a second work package, we analyzed the nature and impact of dendritically localized ribosomal protein mRNAs. We found a dendritic enrichment of 16 different RP mRNAs in hippocampal slices and cultured neurons and quantified those via fluorescence in vitro hybridization (FISH). Using 3’UTR sequencing and bioinformatic approaches to describe and identify the ribosomal protein mRNAs, we discovered that 69 (out of 80) RP mRNAs were localized in the neuropil. Many of these exhibit novel and multiple 3’UTR isoforms, with some enriched in neurons and neuropil (Tushev et al., 2018). When asking the question whether ribosomal mRNAs can be translated into proteins in dendrites, we detected and quantified via FUNCAT-PLA/Puro-PLA method ~ 20 unique RPs that were synthesized in hippocampal cultured neurons (Fusco et al., Nature Communications, 2021).
We also examined the regulation of the local RP transcripts and whether they are incorporated into pre-existing ribosomes. We discovered, for the first time into and translation by synaptic activity and plasticity, and asked if synaptic plasticity changes the quality or quantity globally and locally. So far we have tested for the ribosomal protein RPL26 with a protocol for pharmacologically induced plasticity, but we did not observe any change in RPL26 synthesis. In order to determine how the global RP proteome is modified by synaptic activity and plasticity, we are measuring the turnover of ribosomal proteins after action-potential blockade, e.g. with tetrodoxin (TTX). In this context, we could also recently show that ribosomal proteins exhibit different half-lives, suggesting that there is remodeling in ribosomes (Doerrbaum et al., 2018).
