Periodic Reporting for period 4 - NeuroRibo (Specialized Ribosomes for Neuronal Protein Synthesis) Reporting period: 2022-02-01 to 2022-07-31 Summary of the context and overall objectives of the project The ability of the nervous system to respond adaptively relies on modifications to existing proteins as well as changes in gene transcription and mRNA translation. In this scenario, changes at the synapses play a key role in learning and memory. It is now clear that synapses possess the capacity for local protein synthesis, owing to the localization of ribosomes and mRNAs within dendrites. There is emerging interest in the possibility that ribosomes, as cellular machines, are not as static as typically assumed, and may be heterogeneous in composition and specialized for particular functions and cellular compartments. Intriguingly, several studies have noted the presence of ribosomal protein mRNAs in the dendrites and axons, raising the possibility that locally synthesized ribosomal proteins (RPs) might serve to modify the local ribosome population. Amongst the players in protein synthesis, many signaling proteins, RNA-binding proteins, and most translation factors have been considered as potential regulatory hubs. The ribosomes, thus far, have not been considered. The objective of this project was to discover the nature and diversity of ribosomes present in neurons, their dendrites and their synapses in the rodent brain. Using a combination of RNA-sequencing, mass spectrometry, fluorescence in situ hybridization, and new labelling approaches to visualize nascent proteins, we examined whether neuronal ribosomes are heterogeneous and specialized in different subcellular compartments. These experiments will allow us determine the relationship between the ribosome composition and the neuronal mRNAs that undergo translation. We have also examined how plasticity sculpts the ribosome population with an eye towards understanding the regulation of the proteome by ongoing synaptic events and plasticity. Our studies have provided further insights into the cellular and molecular mechanisms allowing synapses to continuously change and thus will contribute to our understanding of learning and memory formation. In addition, many diseases involve dysregulation of protein synthesis and in some cases, “ribosomopathies” may also be involved. As such, our studies shed light on both the functioning and dysfunctioning of ribosomes in neurons. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far 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). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) As part of our studies on the ribosomal diversity in neuronal compartments, we discovered the presence of ribosomes in axon terminals and have analyzed their localization with light microscopy and electron microscopy (Hafner et al., Science, 2019). In addition, we discovered that nascent ribosomal proteins can actively exchange with pre-existing ribosomes (Fusco et al., 2021)- over-turning the dogma that ribosomes are static structures. Another highlight was the discovery of monosomes as a dominant source of translation in the neuropil.