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.
Highlights
To establish insight into synaptic protein expression profiles that cover the diversity of the synaptic proteome during the entire span of development, a comprehensive proteomics approach is required
As the postsynaptic density is crucial for the biochemical enrichment of synaptic membranes, the main target of this study, the juvenile age postnatal days 9 (P9) was chosen as the earliest time point
Cortical synaptic membrane fractions were isolated, and peptides derived from trypsin protein digestion were fractionated by Strong Cation Exchange (SCX) chromatography, to remove the bulk of free iTRAQ label and other reagents that might interfere in subsequent analysis
Summary
To establish insight into synaptic protein expression profiles that cover the diversity of the synaptic proteome during the entire span of development, a comprehensive proteomics approach is required. An iTRAQ reagent-based proteomics workflow was employed for the relative quantitation of synaptic membrane proteins across 8 time points of juvenile, adolescent and adult mouse brain cortex development in a single 8-plex set of experiments. Using the 8-plex iTRAQ reagents, we performed a time-series relative quantification of the brain cortical synaptic membrane proteome of juvenile postnatal days 9 (P9), P15, P21 and P27, adolescent P35 and adult P70, P140 and P280 mice.
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