Comparative study of human and mouse postsynaptic proteomes finds high compositional conservation and abundance differences for key synaptic proteins

Abstract

Direct comparison of protein components from human and mouse excitatory synapses is important for determining the suitability of mice as models of human brain disease and to understand the evolution of the mammalian brain. The postsynaptic density is a highly complex set of proteins organized into molecular networks that play a central role in behavior and disease. We report the first direct comparison of the proteome of triplicate isolates of mouse and human cortical postsynaptic densities. The mouse postsynaptic density comprised 1556 proteins and the human one 1461. A large compositional overlap was observed; more than 70% of human postsynaptic density proteins were also observed in the mouse postsynaptic density. Quantitative analysis of postsynaptic density components in both species indicates a broadly similar profile of abundance but also shows that there is higher abundance variation between species than within species. Well known components of this synaptic structure are generally more abundant in the mouse postsynaptic density. Significant inter-species abundance differences exist in some families of key postsynaptic density proteins including glutamatergic neurotransmitter receptors and adaptor proteins. Furthermore, we have identified a closely interacting set of molecules enriched in the human postsynaptic density that could be involved in dendrite and spine structural plasticity. Understanding synapse proteome diversity within and between species will be important to further our understanding of brain complexity and disease.

Figure 1. Validation of fractionation method and quantitative comparison of mouse and human cortical PSDs.

a. Immunoblots of known postsynaptic density components. Mouse synaptosomes (S) and PSD (P) fractions are analyzed for each of the three replicates. All proteins show enrichment in the PSD compared to synaptosomes. The presynaptic marker synaptophysin (Syn) is only detected in the synaptosomal fraction. Only genes identified in triplicate in both species were considered for the quantitative analysis. Correlations are calculated based on linear models. Coefficients of correlations are represented by r. Examples of intra-species protein abundance correlation: b. human PSD first replica against second; c. mouse PSD first replica against second. d. Plot of average values of PSD proteins abundance from human and mouse cortex. e. Protein abundance correlation of PSD components found in the NMDA receptor complex ; the PSD-95 complex (f), and the mGluR5 complex (g). h. Clustering of PSD protein abundance values for human and mouse replicates.

Figure 2. Analysis of evolutionary conservation of PSD proteins in mouse and humans.

a. Median dN/dS of proteins identified in the human PSD, mouse PSD and mouse consensus set (cPSD) compared with the median genomic value. b. Comparison of dN/dS values in proteins exclusive or enriched in one species with all PSD proteins.

Figure 3. Inter-species differences on postsynaptic density proteins.

a. Venn diagrams showing the total amount of proteins in mPSD and hPSD as well as the overlap between them relative to the hPSD. b. Venn diagrams showing the proteins identified in triplicate for the mPSD and hPSD, the overlap relative to hPSD between the two sets is also shown. c. Immunoblot validation of proteins identified only in the hPSD by mass spectrometry. The following samples were analyzed: H-M, mouse brain homogenate; H-H, human brain homogenate; and SX, each of the three synaptosomal fractions, PX, each of the three postsynaptic density fractions for both species. d. The abundance fold difference (AFD) between mouse and human is shown for members of adaptors, channels and neurotransmitter receptors protein families. AFD is calculated as the mouse to human ratio of normalized and log2 transformed average abundance values. For each protein family the number of proteins displayed is given in brackets.

Figure 4. Functional analysis of proteins enriched in the human PSD.

a. Ontology hierarchical tree leading to the Biological Process ‘Cellular Component Morphogenesis’. In red are the terms significantly enriched in proteins abundant in the human PSD. b. Molecular interaction map of proteins from the human PSD that belong to the Biological Process ‘Cellular Component Morphogenesis’ (CCM) or are known to be involved in semaphorin signaling. Only CCM proteins enriched in the human PSD are considered. Some proteins related to semaphorin signaling are not enriched in either species, these are identified with a white name. Proteins highly interconnected are represented in clusters of different colors. Clustering was done using the Markov Clustering Algorithm (MCL) in BioLayout Express 3D . Node edges are coloured according to a confidence score, from red (100% confidence) to blue (40% confidence).

Figure 5. Functional protein groups with the mammalian cortical PSD.

The mammalian cortical PSD was defined by combining all proteins identified in mouse and human. This set of PSD proteins were then functionally classified using the Panther Classification descriptor ‘Protein Class’. Enrichment analysis was done comparing the number of PSD proteins per class with the genome. The Panther Protein Class ontology is hierarchically organized, only end-branch classes are displayed to remove redundancy from the graph (all data in Table S8). Protein classes that did not show difference with the genome after correction for multiple testing are shown in blue. Classes overrepresented are shown in red and classes underrepresented are shown in green.