Dataset Information

Local translation of mitochondrial proteins at the synapse, LFQ expriment

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: PXD012746 | Pride | 2020-08-12

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 10

Publications

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

Similar Datasets

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:Action of alcohol on synaptic mRNA in the amygdala of mice Chronic alcohol consumption induces changes in gene expression, causing persistent long-term neuro-adaptations and the remodeling of synaptic structures. These alcohol-induced synaptic changes may rely specifically on the local translation of mRNAs in the synaptic compartments of the cell. We profiled the transcriptome from synaptoneurosomes (SN) and paired total homogenates (TH) of amygdala to analyze the synaptic adaptations induced by chronic voluntary alcohol consumption in mice. In the SN both the number of alcohol-responsive mRNAs and the magnitude of fold-change were greater than in the TH. Accordingly, the SN detected many genes with coordinated patterns of expression producing a highly connected mRNA network in gene co-expression analysis. The greater sensitivity of the SN preparation allowed for improved cell-type specificity analysis, revealing an up-regulation of alcohol-responsive astrocytic and microglial modules that correlated with alcohol consumption. Alcohol was found to induce changes in the SN functionally important biological pathways, including long-term potentiation, long-term potentiation depression, glutamate pathway, neuro-immune, RNA-processing and translational machineries and provided overlap with changes seen in human alcoholic brain. Transposable elements were responsive to alcohol and found in the down-regulated neuronal mRNA module, which may underlie some of the coordinated gene expression changes associated with alcohol. We provide evidence that enrichment of synaptic components reveals a more intricate network of coordinated gene expression. Increased resolution captures the molecular effects of synaptic manipulations and provides an improved technique for identifying therapeutic targets for alcohol abuse. Local translation of mRNAs in the synapse plays a major role in synaptic structure and function. Chronic alcohol use causes persistent changes in synaptic mRNA expression, possibly mediated by microRNAs localized in the synapse. We profiled the transcriptome of synaptoneurosomes (SN) from the amygdala of mice chronically consuming 20% ethanol in a continuous two-bottle choice test to identify the microRNAs that target alcohol-induced mRNAs. SN are membrane vesicles containing pre- and post-synaptic compartments of neurons and astroglia and are a unique model for studying the synaptic transcriptome. We previously showed that chronic alcohol regulates mRNA expression in a coordinated manner. Here, we examine microRNAs and mRNAs from the same samples to define alcohol-responsive synaptic microRNAs and their predicted interactions with targeted mRNAs. We used a combination of unbiased bioinformatic methods (differential expression, correlation, co-expression, microRNA-mRNA target prediction, co-targeting and cell type specific analyses) to identify key alcohol-sensitive microRNAs. Bidirectional mRNA-microRNA prediction analysis showed that a subset of alcohol-responsive microRNAs were predicted to target many alcohol-responsive mRNAs. We found microRNAs-mRNAs with overlapping patterns of expression that correlated with the amount of alcohol consumed. Cell-type specific analysis revealed a significant number of the alcohol-responsive mRNAs and microRNAs that were unique to glutamate neurons and were highly predicted to target each other. Chronic alcohol appears to perturb the coordinated microRNA regulation of mRNAs in SN, a mechanism that may explain the aberrations in synaptic plasticity affecting the alcoholic brain.

Project description:The recent development within high-throughput technologies for expression profiling has allowed for parallel analysis of transcriptomes and proteomes in biological systems such as comparative analysis of transcript and protein levels of tissue regulated genes. Until now, such studies of have only included microarray or short length sequence tags for transcript profiling. Furthermore, most comparisons of transcript and protein levels have been based on absolute expression values from within the same tissue and not relative expression values based on tissue ratios. Presented here is a novel study of two porcine tissues based on integrative analysis of data from expression profiling of identical samples using cDNA microarray, 454-sequencing and iTRAQ-based proteomics. Sequence homology identified 2.541 unique transcripts that are detectable by both microarray hybridizations and 454-sequencing of 1.2 million cDNA tags. Both transcript-based technologies showed high reproducibility between sample replicates of the same tissue, but the correlation across these two technologies was modest. Thousands of genes being differentially expressed were identified with microarray. Out of the 306 differentially expressed genes, identified by 454-sequencing, 198 (65 %) were also found by microarray. The relationship between the regulation of transcript and protein levels was analyzed by integrating iTRAQ-based proteomics data. Protein expression ratios were determined for 354 genes, of which 148 could be mapped to both microarray and 454-sequencing data. A comparison of the expression ratios from the three technologies revealed that differences in transcript and protein levels across heart and muscle tissues are positively correlated. We show that the reproducibility within cDNA microarray and 454-sequencing is high, but that the agreement across these two technologies is modest. We demonstrate that the regulation of transcript and protein levels across identical tissue samples is positively correlated when the tissue expression ratios are used for comparison. The results presented are of interest in systems biology research in terms of integration and analysis of high-throughput expression data from mammalian tissues. Keywords: tissue comparison, platform comparison Tissue samples of heart (HEA) and Longissimus dorsi (LDO) were prepared by pooling from five healthy Hampshire gilts at age four to six months and division into six sub-samples, three for each tissue named HEA1, HEA2, HEA3, LDO1, LDO2 and LDO3. The exact same six tissue samples were used for subsequent expression profiling with cDNA microarray, 454-sequencing and iTRAQ-based proteomics. A reference sample for the cDNA microarray experiment and iTRAQ-based proteomics was constructed by combining the six samples. In the microarray experiment, three cDNA microarray slides were used per sample.

Project description:Implications for neuroprotection in Parkinson's disease Parkinson’s disease and its characteristic symptoms are thought to arise from the progressive degeneration of specific midbrain dopamine (DA) neurons. In humans, DA neurons of the substantia nigra (SN) and their projections to the striatum show selective vulnerability, while neighboring DA neurons of the ventral tegmental area (VTA) are relatively spared from degeneration. This pattern of cell loss is mimicked in humans, primates, and certain rodents by the neurotoxin MPTP. In this study, we aimed to test the hypothesis that there are factors in the VTA that are potentially neuroprotective against MPTP and that these factors change over time. We have found a differential transcriptional response within the cells of the SN and VTA to sustained exposure to a low dose of MPTP. Specifically, the VTA has increased expression of 148 genes as an early response to MPTP and 113 genes as a late response to MPTP toxicity. This response encompasses many areas of cellular function, including protein regulation (Phf6) and ion/metal regulation (PANK2, Car4). Notably, these responses were largely absent from the cells of the SN. Our data show a clear dynamic response in maintaining the homeostasis and viability of the neurons in the VTA that is lacking in the SN after neurotoxin challenge. We used microarrays to analyze the differential response of the substantia nigra (SN) and ventral tegmental area (VTA) to a chronic low dose of the neurotoxin MPTP. Transgenic hTH-GFP mice were treated with MPTP (4mg/kg) for either 2 or 10 days. Control mice were given an equal volume of saline for 10 days. Dopamine neurons from the substantia nigra and ventral tegmental areas of control and MPTP treated animals were laser captured. The RNA was isolated and processed for microarray hybridization. Each group had three biological replicates, for a total of 18 samples. Three each in the following: Control SN, Control VTA, 2 day MPTP SN, 2 day MPTP VTA, 10 day MPTP SN, 10 day MPTP VTA. Samples were log2 transformed and RMA normalized using Agilent Genespring 10.0 GX.

			Action	DRS
	50109cys_K1.raw	Raw
	50109cys_K2.raw	Raw
	50109cys_K3.raw	Raw
	50109cys_K4.raw	Raw
	50109cys_N1.raw	Raw