An abundance of free regulatory (19S) proteasome particles regulates neuronal synapses.

The proteasome, the major protein-degradation machine in cells, regulates neuronal synapses and long-term information storage. Here, using super-resolution microscopy, we found that the two essential subcomplexes of the proteasome, the regulatory (19S) and catalytic (20S) particles, are differentially distributed within individual rat cortical neurons. We discovered an unexpected abundance of free 19S particles near synapses. The free neuronal 19S particles bind and deubiquitylate lysine 63-ubiquitin (Lys63-ub), a non-proteasome-targeting ubiquitin linkage. Pull-down assays revealed a significant overrepresentation of synaptic molecules as Lys63-ub interactors. Inhibition of the 19S deubiquitylase activity significantly altered excitatory synaptic transmission and reduced the synaptic availability of AMPA receptors at multiple trafficking points in a proteasome-independent manner. Together, these results reveal a moonlighting function of the regulatory proteasomal subcomplex near synapses.

Cell-type Specific Protein Purification and Identification from Complex Tissues using a Mutant Methionine tRNA Synthetase Mouse Line.

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.

The translatome of neuronal cell bodies, dendrites, and axons.

To form synaptic connections and store information, neurons continuously remodel their proteomes. The impressive length of dendrites and axons imposes logistical challenges to maintain synaptic proteins at locations remote from the transcription source (the nucleus). The discovery of thousands of messenger RNAs (mRNAs) near synapses suggested that neurons overcome distance and gain autonomy by producing proteins locally. It is not generally known, however, if, how, and when localized mRNAs are translated into protein. To investigate the translational landscape in neuronal subregions, we performed simultaneous RNA sequencing (RNA-seq) and ribosome sequencing (Ribo-seq) from microdissected rodent brain slices to identify and quantify the transcriptome and translatome in cell bodies (somata) as well as dendrites and axons (neuropil). Thousands of transcripts were differentially translated between somatic and synaptic regions, with many scaffold and signaling molecules displaying increased translation levels in the neuropil. Most translational changes between compartments could be accounted for by differences in RNA abundance. Pervasive translational regulation was observed in both somata and neuropil influenced by specific mRNA features (e.g., untranslated region [UTR] length, RNA-binding protein [RBP] motifs, and upstream open reading frames [uORFs]). For over 800 mRNAs, the dominant source of translation was the neuropil. We constructed a searchable and interactive database for exploring mRNA transcripts and their translation levels in the somata and neuropil [MPI Brain Research, The mRNA translation landscape in the synaptic neuropil. https://public.brain.mpg.de/dashapps/localseq/ Accessed 5 October 2021]. Overall, our findings emphasize the substantial contribution of local translation to maintaining synaptic protein levels and indicate that on-site translational control is an important mechanism to control synaptic strength.

LIMK, Cofilin 1 and actin dynamics involvement in fear memory processing.

Long-term memory has been associated with morphological changes in the brain, which in turn tightly correlate with changes in synaptic efficacy. Such plasticity is proposed to rely on dendritic spines as a neuronal canvas on which these changes can occur. Given the key role of actin cytoskeleton dynamics in spine morphology, major regulating factors of this process such as Cofilin 1 (Cfl1) and LIM kinase (LIMK), an inhibitor of Cfl1 activity, are prime molecular targets that may regulate dendritic plasticity. Using a contextual fear conditioning paradigm in mice, we found that pharmacological induction of depolymerization of actin filaments through the inhibition of LIMK causes an impairment in memory reconsolidation, as well as in memory consolidation. On top of that, Cfl1 activity is inhibited and its mRNA is downregulated in CA1 neuropil after re-exposure to the training context. Moreover, by pharmacological disruption of actin cytoskeleton dynamics, the process of memory extinction can either be facilitated or impaired. Our results lead to a better understanding of the role of LIMK, Cfl1 and actin cytoskeleton dynamics in the morphological and functional changes underlying the synaptic plasticity of the memory trace.

Proteome dynamics during homeostatic scaling in cultured neurons.

Protein turnover, the net result of protein synthesis and degradation, enables cells to remodel their proteomes in response to internal and external cues. Previously, we analyzed protein turnover rates in cultured brain cells under basal neuronal activity and found that protein turnover is influenced by subcellular localization, protein function, complex association, cell type of origin, and by the cellular environment (Dörrbaum et al., 2018). Here, we advanced our experimental approach to quantify changes in protein synthesis and degradation, as well as the resulting changes in protein turnover or abundance in rat primary hippocampal cultures during homeostatic scaling. Our data demonstrate that a large fraction of the neuronal proteome shows changes in protein synthesis and/or degradation during homeostatic up- and down-scaling. More than half of the quantified synaptic proteins were regulated, including pre- as well as postsynaptic proteins with diverse molecular functions.

Cell-type-specific metabolic labeling of nascent proteomes in vivo.

Although advances in protein labeling methods have made it possible to measure the proteome of mixed cell populations, it has not been possible to isolate cell-type-specific proteomes in vivo. This is because the existing methods for metabolic protein labeling in vivo access all cell types. We report the development of a transgenic mouse line where Cre-recombinase-induced expression of a mutant methionyl-tRNA synthetase (L274G) enables the cell-type-specific labeling of nascent proteins with a non-canonical amino-acid and click chemistry. Using immunoblotting, imaging and mass spectrometry, we use our transgenic mouse to label and analyze proteins in excitatory principal neurons and Purkinje neurons in vitro (brain slices) and in vivo. We discover more than 200 proteins that are differentially regulated in hippocampal excitatory neurons by exposing mice to an environment with enriched sensory cues. Our approach can be used to isolate, analyze and quantitate cell-type-specific proteomes and their dynamics in healthy and diseased tissues.

Direct visualization of newly synthesized target proteins in situ.

Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the proximity ligation assay to visualize specific newly synthesized proteins and monitor their origin, redistribution and turnover in situ.

Deep sequencing and high-resolution imaging reveal compartment-specific localization of Bdnf mRNA in hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) is a small protein of the neurotrophin family that regulates various brain functions. Although much is known about how its transcription is regulated, the abundance of endogenous BDNF mRNA and its subcellular localization pattern are matters of debate. We used next-generation sequencing and high-resolution in situ hybridization in the rat hippocampus to reexamine this question. We performed 3' end sequencing on rat hippocampal slices and detected two isoforms of Bdnf containing either a short or a long 3' untranslated region (3'UTR). Most of the Bdnf transcripts contained the short 3'UTR isoform and were present in low amounts relative to other neuronal transcripts. Bdnf mRNA was present in the somatic compartment of rat hippocampal slices or the somata of cultured rat hippocampal neurons but was rarely detected in the dendritic processes. Pharmacological stimulation of hippocampal neurons induced Bdnf expression but did not change the ratio of Bdnf isoform abundance. The findings indicate that endogenous Bdnf mRNA, although weakly abundant, is primarily localized to the somatic compartment of hippocampal neurons. Both Bdnf mRNA isoforms have shorter half-lives compared with other neuronal mRNAs. Furthermore, the findings show that using complementary high-resolution techniques can provide sensitive measures of endogenous transcript abundance.