Alterations in the Proteome of Developing Neocortical Synaptosomes in the Absence of MET Signaling Revealed by Comparative Proteomics

Abstract

Alterations in the expression of genes encoding proteins involved in synapse formation, maturation, and function are a hallmark of many neurodevelopmental and psychiatric disorders. For example, there is reduced neocortical expression of the MET receptor tyrosine kinase (MET) transcript and protein in Autism Spectrum Disorder (ASD) and Rett syndrome. Preclinical in vivo and in vitro models manipulating MET signaling reveal that the receptor modulates excitatory synapse development and maturation in select forebrain circuits. The molecular adaptations underlying the altered synaptic development remain unknown. We performed a comparative mass spectrometry analysis of synaptosomes generated from the neocortex of wild type and Met null mice during the peak of synaptogenesis (postnatal day 14; data are available from ProteomeXchange with identifier PXD033204). The analyses revealed broad disruption of the developing synaptic proteome in the absence of MET, consistent with the localization of MET protein in pre- and postsynaptic compartments, including proteins associated with the neocortical synaptic MET interactome and those encoded by syndromic and ASD risk genes. In addition to an overrepresentation of altered proteins associated with the SNARE complex, multiple proteins in the ubiquitin-proteasome system and associated with the synaptic vesicle, as well as proteins that regulate actin filament organization and synaptic vesicle exocytosis/endocytosis, were disrupted. Taken together, the proteomic changes are consistent with structural and functional changes observed following alterations in MET signaling. We hypothesize that the molecular adaptations following Met deletion may reflect a general mechanism that produces circuit-specific molecular changes due to loss or reduction of synaptic signaling proteins.

Keywords: Developing brain with SNARE complex; Isobaric tag for relative and absolute quantitation proteomics; MET; Neocortex; Synaptosome.

Fig. 1.

Characterization of synaptosomes generated from the P14 neocortex. (a) Western blot illustrating enrichment of synaptophysin and depletion of lamin B in the synaptosome fraction prepared from two independent P14 neocortical samples. Lanes were loaded with equal amounts of protein from homogenate (H), cytoplasm (C), and synaptosome (S) fractions. Uncropped images of the membrane are shown in Supplementary Figure S2. (b) Venn diagram displaying overlap in proteins detected in experiments 1 and 2 in the current study. (c) Venn diagram displaying overlap in proteins detected in both experiments in the current study and in (32). (d) Top 20 enriched Gene ontology (GO): cell component terms based on p-values using DAVID. Red bars: terms associated with the synapse; blue bars: terms associated with synapse-associated organelles and structural elements. Numbers in parentheses indicate the number of proteins for each term. The complete set of significantly enriched GO terms, and the proteins therein, are listed in Supplemental Table S3. (e) 530 proteins were categorized in SynGO cellular component. Sunburst plot with color-coded enrichment significance for top-level and second-level GO: cellular component terms. The number of counts compared to the total number of hits for each term is indicated. The proteins associated with the significant GO terms can be found in Supplemental Table S4.

Fig. 2.

(a) Distribution of protein ratios for each sample from neocortical Met^−/− synaptosomes normalized to wild type ratio. A single iTRAQ experiment included either samples 1 & 2 or samples 3 & 4. (b) Representation of distribution of mean fold change (FC) protein ratios across the four experiments.

Fig. 3.

(a) Gene ontology (cellular component, black bars) and (b) KEGG pathway (blue bars) analyses in DAVID of proteins expressed differentially in neocortical synaptosomes in the absence of Met. The proteins associated with each significant term are listed in Supplemental Table S7. (c) Sunburst plot with color-coded gene counts for top-level and second-level SynGO: cellular component terms that include proteins exhibiting differential expression in the absence of Met. No term was overrepresented. The number of counts compared to the total number of hits for each term is indicated. The proteins associated with each term can be found in Supplemental Table S8.

Fig. 4.

(a) Representation of the members of the MET interactome identified in (27). Undetected members are in grey, detected unchanged members in dark green and changed detected members in light green. (b) Identity and mean fold change (FC) in expression of MET-interacting proteins impacted by the absence of MET signaling. (c) Representation of the proteins encoded by ASD risk genes identified in SFARI gene. Undetected members are in grey, detected unchanged members in dark green and detected changed members in light green. (d) Identity and mean fold change (FC) in expression proteins encoded by ASD risk genes impacted by the absence of MET signaling.

Fig. 5.

Validation of iTRAQ proteomics. (a) Western blot analysis of neocortical synaptosomes generated from independent P14 wild type and Met-null neocortex using antibodies against 5 proteins exhibiting significantly changed expression by iTRAQ. Uncropped images of the cut membranes are shown in Supplementary Figure S1. Semi-quantitative analysis reveals that VAMP2 (b), syntaxin-1 (c), TRIM3 (e) and GNAZ (f) meet the mean fold change criteria in the same direction as for iTRAQ. CDC42BP4 does not meet this criterion (d). Data are expressed relative to mean wild type value. Blue circles, wild type; red circles, Met-null. *Hedge’s g > 1.0 (large effect size).