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.

Project description:BackgroundThe key role in the dynamic regulation of synaptic protein turnover belongs to the Fragile X Mental Retardation Protein, which regulates the efficiency of dendritic mRNA translation in response to stimulation of metabotropic glutamate receptors at excitatory synapses of the hippocampal pyramidal cells. Its activity is regulated via positive and negative regulatory loops that function in different time ranges, which is an absolute factor for the formation of chaotic regimes that lead to disrupted proteome stability. The indicated condition may cause a number of neuropsychiatric diseases, including autism and epilepsy. The present study is devoted to a theoretical analysis of the local translation system dynamic properties and identification of parameters affecting the chaotic potential of the system.ResultsA mathematical model that describes the maintenance of a specific pool of active receptors on the postsynaptic membrane via two mechanisms - de novo synthesis of receptor proteins and restoration of protein function during the recycling process - has been developed. Analysis of the model revealed that an increase in the values of the parameters describing the impact of protein recycling on the maintenance of a pool of active receptors in the membrane, duration of the signal transduction via the mammalian target of rapamycin pathway, influence of receptors on the translation activation, as well as reduction of the rate of synthesis and integration of de novo synthesized proteins into the postsynaptic membrane - contribute to the reduced complexity of the local translation system dynamic state. Formation of these patterns significantly depends on the complexity and non-linearity of the mechanisms of exposure of de novo synthesized receptors to the postsynaptic membrane, the correct evaluation of which is currently problematic.ConclusionsThe model predicts that an increase of "receptor recycling" and reduction of the rate of synthesis and integration of de novo synthesized proteins into the postsynaptic membrane contribute to the reduced complexity of the local translation system dynamic state. Herewith, stable stationary states occur much less frequently than cyclic states. It is possible that cyclical nature of functioning of the local translation system is its "normal" dynamic state.

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

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

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