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Specialized Ribosomes for Neuronal Protein Synthesis

Periodic Reporting for period 3 - NeuroRibo (Specialized Ribosomes for Neuronal Protein Synthesis)

Reporting period: 2020-08-01 to 2022-01-31

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 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 will determine whether neuronal ribosomes are heterogeneous and specialized in different subcellular compartments. The proposed experiments will determine the relationship between the ribosome composition and the neuronal mRNAs that undergo translation. We will also examine how plasticity sculpts the ribosome population to regulate the changes in the proteome that are dynamically regulated by ongoing synaptic events and plasticity. Our studies will provide 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 will shed light on both the functioning and dysfunctioning of ribosomes in neurons.
Our main focus during this first reporting period 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 undergoing translation. So far, we have 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 preliminary 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 managed to isolate 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. To this point, we have preliminary mass spec data from different polysome profiled fractions of cultured cortical neurons (e.g. representing different levels of translation) which will be further analyzed.
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 the FUNCAT-PLA/Puro-PLA method 9 different RPs that were synthetized in hippocampal cultured neurons.
We have also started to examine the regulation of the local RP transcripts 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).
As part of our studies on the ribosomal diversity in neuronal compartments, we have recently discovered the presence of ribosomes in axon terminals and have analyzed their localization with light microscopy and electron microscopy (Hafner et al., Science, in press). Until the end of the project, we expect to further support and expand the data described above. In addition, we aim to also discover the diversity of ribosomes associated with synapses, to determine the spatial and functional fate of locally synthesized ribosomal proteins, to determine how the local RP proteome is modified by synaptic activity and plasticity, and to understand the interaction between the ribosome-proteome and translation targets (translatome) in cell bodies, dendrites and synapses under basal and plasticity conditions.