Dataset Information

Local translation of mitochondrial proteins at the synapse

ABSTRACT: To systematically explore the dynamics of the synaptic proteome upon neuronal stimulation we employed a simple model system which is based on isolated synaptoeurosomes (SN). Synaptoneurosomes are preparations freshly obtained from the brain and enriched in synapses, containing both pre and postsynaptic compartments. After the isolation they were in vitro stimulated. Protocol was set to induce NMDARs on the synaptoneurosomes which initiate calcium signaling in neurons and lead to long lasting responses such as long term potentiation (LTP). The control experiments in which we incubated SN with RNAse and proteinase K have shown that the translation is taking place inside the SN vesicles. Having established that translation in synaptoeurosomes prepared by us relay on endogenous mRNA we assumed that all proteins which abundance is increased after in vitro stimulation must be locally synthetized on base of mRNA already transported into synapses. In order to identify newly synthetized proteins we in vitro stimulated synaotoneurosomes and combined it with high-resolution quantitative MS techniques (Figure 1A). It is well established that quantitative mass spectrometry heavily depends on physico-chemical properties of peptides and dynamic range (~4 orders of magnitude) of sample which limits a depth of analysis. Some of tryptic peptides would be better detectable unmodified, other when modified for example with stable isobaric labeling techniques iTRAQ or TMT tag. We begin with label-free experiments, but it was mainly limited by dynamic range of protein in SN, we overcome that issue using iTRAQ 8-plex (4 x controls, 4 x stimulation) coupled with sample fractionation prior to LC-MS.. For this reason we used also TMT 10 plex, which allows us to measure 5 control and 5 stimuli samples at once. Using all three techniques we quantified (FDR 0.01) 2187 proteins. Number of identified proteins could be considered as relatively low for shotgun MS experiment. But it should be noticed that synaptical proteome is way different then “normal” cellular proteome since is depleted of nuclear, many of cytoplasmic and other organelles proteins. Finally some of proteins could be heavily modified. . Importantly, we identified 439 of which were significantly (Whitney-Mann p<0.05) upregulated in synaptoneurosomes in response to the stimulation which corresponds to 20% of identified proteins. The average fold change was relatively low which was however expected because it is not possible to quantitatively change a proteome of synapse through short pule of in vitro stimulation. But four replicates are often not enough for reliable statistic calculation of protein level changes. That is because of low level of proteins that could be synthetase at the synapse, due to its low ATP/syntethic potential. Which results in changes on average 3% of protein level.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Dominik Cysewski

LAB HEAD: Magdalena Dziembowska

PROVIDER: PXD012707 | Pride | 2020-08-12

REPOSITORIES: Pride

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Mitochondrial protein biogenesis in the synapse is supported by local translation.

Kuzniewska Bozena B Cysewski Dominik D Wasilewski Michal M Sakowska Paulina P Milek Jacek J Kulinski Tomasz M TM Winiarski Maciej M Kozielewicz Pawel P Knapska Ewelina E Dadlez Michal M Chacinska Agnieszka A Dziembowski Andrzej A Dziembowska Magdalena M

EMBO reports 20200618 8

Synapses are the regions of the neuron that enable the transmission and propagation of action potentials on the cost of high energy consumption and elevated demand for mitochondrial ATP production. The rapid changes in local energetic requirements at dendritic spines imply the role of mitochondria in the maintenance of their homeostasis. Using global proteomic analysis supported with complementary experimental approaches, we show that an essential pool of mitochondrial proteins is locally produc ...[more]

PMID: 32558077

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Project description:To systematically explore the dynamics of the synaptic proteome upon neuronal stimulation we employed a simple model system which is based on isolated synaptoeurosomes (SN). Synaptoneurosomes are preparations freshly obtained from the brain and enriched in synapses, containing both pre and postsynaptic compartments. After the isolation they were in vitro stimulated. Protocol was set to induce NMDARs on the synaptoneurosomes which initiate calcium signaling in neurons and lead to long lasting responses such as long term potentiation (LTP). The control experiments in which we incubated SN with RNAse and proteinase K have shown that the translation is taking place inside the SN vesicles. Having established that translation in synaptoeurosomes prepared by us relay on endogenous mRNA we assumed that all proteins which abundance is increased after in vitro stimulation must be locally synthetized on base of mRNA already transported into synapses. In order to identify newly synthetized proteins we in vitro stimulated synaotoneurosomes and combined it with high-resolution quantitative MS techniques (Figure 1A). It is well established that quantitative mass spectrometry heavily depends on physico-chemical properties of peptides and dynamic range (~4 orders of magnitude) of sample which limits a depth of analysis. Some of tryptic peptides would be better detectable unmodified, other when modified for example with stable isobaric labeling techniques iTRAQ or TMT tag. We begin with label-free experiments, but it was mainly limited by dynamic range of protein in SN, we overcome that issue using iTRAQ 8-plex (4 x controls, 4 x stimulation) coupled with sample fractionation prior to LC-MS.. For this reason we used also TMT 10 plex, which allows us to measure 5 control and 5 stimuli samples at once. Using all three techniques we quantified (FDR 0.01) 2187 proteins. Number of identified proteins could be considered as relatively low for shotgun MS experiment. But it should be noticed that synaptical proteome is way different then “normal” cellular proteome since is depleted of nuclear, many of cytoplasmic and other organelles proteins. Finally some of proteins could be heavily modified. . Importantly, we identified 439 of which were significantly (Whitney-Mann p<0.05) upregulated in synaptoneurosomes in response to the stimulation which corresponds to 20% of identified proteins. The average fold change was relatively low which was however expected because it is not possible to quantitatively change a proteome of synapse through short pule of in vitro stimulation. But four replicates are often not enough for reliable statistic calculation of protein level changes. That is because of low level of proteins that could be synthetase at the synapse, due to its low ATP/syntethic potential. Which results in changes on average 3% of protein level.

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