Protein expression dynamics during postnatal mouse brain development

Abstract

We explored differential protein expression profiles in the mouse forebrain at different stages of postnatal development, including 10-day (P10), 30-day (P30), and adult (Ad) mice, by large-scale screening of proteome maps using two-dimensional difference gel electrophoresis. Mass spectrometry analysis resulted in the identification of 251 differentially expressed proteins. Most molecular changes were observed between P10 compared to both P30 and Ad. Computational ingenuity pathway analysis (IPA) confirmed these proteins as crucial molecules in the biological function of nervous system development. Moreover, IPA revealed Semaphorin signaling in neurons and the protein ubiquitination pathway as essential canonical pathways in the mouse forebrain during postnatal development. For these main biological pathways, the transcriptional regulation of the age-dependent expression of selected proteins was validated by means of in situ hybridization. In conclusion, we suggest that proteolysis and neurite outgrowth guidance are key biological processes, particularly during early brain maturation.

Keywords: 2-D DIGE; biological process; brain; development; mouse; proteomics.

Figure 1

Overview of differentially expressed protein spots in mouse forebrain development A) 2-D DIGE overview image of the internal standard mouse brain tissue lysates separated on a pH 3–11 (non-linear) 2-D gel. Protein spots that showed statistically significant differences in fluorescence levels (P ≤ 0.01) after DeCyder analysis and that were identified, are numbered on this 2-D DIGE gel. Red: significant differential expression between P10 and P30 (P10/P30); yellow: P10/adult; blue: adult/P30; green: P10/P30 and P10/adult; magenta: P10/P30 & adult/P30; cyan: P10/adult and adult/P30; white: P10/P30, adult/P30, P10/adult. B) Venn diagram depicting similarities and differences in the differentially expressed spots between P10, P30, and Ad. Color codes correspond to panel A. The number of spots differentially expressed between all age conditions and corresponding to the white numbers in panel A are indicated in the center of the Venn diagram.

Figure 2

Gel view of the differentially expressed spots for the selected proteins implicated in ‘Semaphorin signaling in neurons’ and ‘Protein ubiquitination pathway’. A) 2-D gel of the 10-day (P10) mouse brain sample. B) 2-D DIGE overview image of the 30-day old (P30) sample. C) 2-D gel image of the adult mouse brain sample. The selected differential spots are marked by their spot number on the gels.

Figure 3

Western analysis as validation of 2-D DIGE results. A) Overview of 2-D DIGE ratios for 3 differentially expressed proteins FSCN1, DYN1, and ENOG over the course of postnatal forebrain development. B) For FSCN1, a single 55 kDa protein band was detected, for DYN1 a 100 kDa band, and for ENOG a 48 kDa band. The optical densities are plotted against postnatal age. In accordance with 2-D DIGE results, FSCN1 expression diminished with age, while DYN1 and ENOG expression gradually increased.

Figure 4

Differential mRNA distribution of 6 proteins implicated in Semaphorin signaling in neurons. In general, mRNA expression for cfl1, and crmp1-5 was higher in P10 forebrain compared to P30 and Ad brain. Corresponding oligonucleotide sequences and Swiss-Prot entries can be found in Supplemental Table 1. Major brain subdivisions are indicated (Cx, neocortex; DG, dentate gyrus; H, hippocampus; Hyp, hypothalamus; Th, thalamus). Scale bar = 2 mm.

Figure 5

Differential mRNA distribution of 7 proteins implicated in the protein ubiquitination pathway. Forebrain development is accompanied by a clear decrease in the mRNA expression of proteolysis-related proteins, ie, psma1, 4, 7, psmb1, 2, 4, and uba1, from P10 to P30 and Ad. Corresponding oligonucleotide sequences and Swiss-Prot entries can be found in Supplemental Table 1. Subcortical structures correspond to those indicated in Figure 4. S and I indicate supra- and infragranular layers of the neocortex, respectively, and highlight the switch in relative intensity with age. Scale bar = 2 mm.