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. 2016 Oct 17:6:35456.
doi: 10.1038/srep35456.

Dynamics of the mouse brain cortical synaptic proteome during postnatal brain development

Miguel A Gonzalez-Lozano  1 , Patricia Klemmer  1 , Titia Gebuis  1 , Chopie Hassan  2 , Pim van Nierop  1 , Ronald E van Kesteren  1 , August B Smit  1 , Ka Wan Li  1
Affiliations

Dynamics of the mouse brain cortical synaptic proteome during postnatal brain development

Miguel A Gonzalez-Lozano et al. Sci Rep. .

Abstract

Development of the brain involves the formation and maturation of numerous synapses. This process requires prominent changes of the synaptic proteome and potentially involves thousands of different proteins at every synapse. To date the proteome analysis of synapse development has been studied sparsely. Here, we analyzed the cortical synaptic membrane proteome of juvenile postnatal days 9 (P9), P15, P21, P27, adolescent (P35) and different adult ages P70, P140 and P280 of C57Bl6/J mice. Using a quantitative proteomics workflow we quantified 1560 proteins of which 696 showed statistically significant differences over time. Synaptic proteins generally showed increased levels during maturation, whereas proteins involved in protein synthesis generally decreased in abundance. In several cases, proteins from a single functional molecular entity, e.g., subunits of the NMDA receptor, showed differences in their temporal regulation, which may reflect specific synaptic development features of connectivity, strength and plasticity. SNARE proteins, Snap 29/47 and Stx 7/8/12, showed higher expression in immature animals. Finally, we evaluated the function of Cxadr that showed high expression levels at P9 and a fast decline in expression during neuronal development. Knock down of the expression of Cxadr in cultured primary mouse neurons revealed a significant decrease in synapse density.

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Figures

Figure 1
Figure 1. Venn diagram showing the distribution of the number of quantified proteins over the experiments.
Represented are all 1978 proteins quantified with a high confidence protein score (ProteinPilot unused ProtScore ≥3, see main text) across all three experiments. In total 1560 proteins are common between the data sets.
Figure 2
Figure 2. Protein expression relationship between samples of different developmental stages.
(A) The average values of the protein levels of the different age groups were hierarchically clustered. The vertical distance connecting two samples within the dendrogram reflects similarity between the samples. (B) Number of proteins that were significantly different in abundance between P280 and other developmental stages (one-sample student’s t-test, p-≤0.05). P280 was taken as reference for the relative quantification of the other 7 age groups.
Figure 3
Figure 3. Relative iTRAQ levels and SDS-PAGE immunoblot analysis of selected synaptic proteins.
(A) The abundance profile of each protein depicted is presented as the ratio of signal intensity (fold difference on log2 scale) of the mean of three biological independent iTRAQ sets compared to the reference sample (P280). (B) SDS-PAGE immunoblot analysis of the selected proteins with an increasing (left) or decreasing (right) expression pattern across time points.
Figure 4
Figure 4. Unbiased construction of protein sub-networks by IPA from proteins showing significant developmental changes.
(A,B) show mainly the networks of post- and pre-synaptic proteins, respectively. Green color intensity indicates the level of down-regulation and red for up-regulation in P9/P280 comparison. Continuous and discontinuous connections indicate direct or indirect interaction, respectively.
Figure 5
Figure 5. SDS-PAGE immunoblot and stain-free gel images of different biochemical fractions of adult mouse cortex.
Samples of different enriched subfractions were resolved on SDS-PAGE, and then immunoblotted for specific synaptic markers. NMDA receptor 2b (Grin2b) and Dlg4 proteins are markers of postsynaptic density fraction; synaptophysin (Syp) is a marker of the presynaptic terminal. Hom: homogenate; P2: pellet 2; Syn: synaptosome; Sym, synaptic membrane; PSD: Triton X-100 insoluble postsynaptic density fraction. Normalization of the protein input was performed using the stain-free gel.
Figure 6
Figure 6. Relative levels of typical presynaptic proteins.
The abundance profile of each protein depicted is presented as the ratio of signal intensity (fold difference on log2 scale) of the mean of three biological independent iTRAQ sets compared to the reference sample (P280). Synaptic proteins were assigned to specific functional groups, such as synaptic vesicle proteins (A), proteins important for docking (D), proteins involved in priming and exocytosis (E), and proteins implicated in clathrin-mediated endocytosis and recycling (G). In addition, the expression profile of the different subunits of the vacuolar ATPase is shown (B), vesicular transporters (C) and calcium channels (F). The asterisks at the protein names indicate significant difference in level over at least two time points (BETR ≤ 0.001; Supplemental Table 1).
Figure 7
Figure 7. Relative levels of typical postsynaptic proteins.
The abundance profile of each protein depicted is presented as the ratio of signal intensity (fold difference on log2 scale) of the mean of three biological independent iTRAQ sets compared to the reference sample (P280). Synaptic proteins were assigned to specific functional groups, such as ionotropic glutamate receptors; NMDA- (A), AMPA- (B), kainate-type and metabotropic glutamate receptors (C), scaffolding proteins of the PSD (D,E), inhibitory GABAB and GABAA receptor subunits and gephyrin (F,G). The asterisks indicate proteins significantly regulated over at least two time points (BETR ≤ 0.001; Supplemental Table 1).
Figure 8
Figure 8. Relative levels of cell adhesion molecules.
The abundance profile of each protein depicted is presented as the ratio of signal intensity (fold change on log2 scale) of the mean of three biological independent iTRAQ sets compared to the reference sample (P280). Major classes of cell adhesion molecules were grouped based on their expression patterns into (A) overall decreasing levels (ephrin receptors, NCAM), (B) increase in levels (SynCAM, Contactin, ICAM), or (C) not changed (cadherins, neuroligins, neurexins and contactins). The asterisks indicate proteins significantly regulated over at least two time points (BETR ≤ 0.001; Supplemental Table 1).
Figure 9
Figure 9. Cxadr knockdown affects synapse formation in vitro.
(A) Images of primary mouse hippocampal neurons transduced with a negative control shRNA or with one of three shRNAs against Cxadr. Top panels show image selections with MAP2 staining (red) to identify dendrites and VAMP staining (green) to identify presynaptic spots. The MAP2 channel (row 2) was used to create a dendritic mask, and the VAMP channel (row 3) was used to identify synaptic spots within the dendritic mask region. Examples of the dendritic masks and identified synaptic spots are shown in row 4 on top of the VAMP staining. Scale bars: 10 μm. (B) Transduction with shRNAs did not affect neuron numbers. (C) All three shRNAs against Cxadr reduced synapse densities. Cxadr shRNAs, n = 5; control shRNA, n = 8; untreated cells, n = 15. *p < 0.05; **p < 0.01.
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References

    1. Chua J. J., Kindler S., Boyken J. & Jahn R. The architecture of an excitatory synapse. J Cell Sci 123, 819–823 (2010). - PubMed
    1. Bayes A. & Grant S. G. Neuroproteomics: understanding the molecular organization and complexity of the brain. Nat Rev Neurosci 10, 635–646 (2009). - PubMed
    1. Meredith R. M. Sensitive and critical periods during neurotypical and aberrant neurodevelopment: a framework for neurodevelopmental disorders. Neuroscience and biobehavioral reviews 50, 180–188 (2015). - PubMed
    1. Rice D. & Barone S. Jr. Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ Health Perspect 108 Suppl 3, 511–533 (2000). - PMC - PubMed
    1. Li M. et al.. Synaptogenesis in the developing mouse visual cortex. Brain Res Bull 81, 107–113 (2010). - PubMed

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