Systems approach to explore components and interactions in the presynapse

Abstract

The application of proteomic techniques to neuroscientific research provides an opportunity for a greater understanding of nervous system structure and function. As increasing amounts of neuroproteomic data become available, it is necessary to formulate methods to integrate these data in a meaningful way to obtain a more comprehensive picture of neuronal subcompartments. Furthermore, computational methods can be used to make biologically relevant predictions from large proteomic data sets. Here, we applied an integrated proteomics and systems biology approach to characterize the presynaptic (PRE) nerve terminal. For this, we carried out proteomic analyses of presynaptically enriched fractions, and generated a PRE literature-based protein-protein interaction network. We combined these with other proteomic analyses to generate a core list of 117 PRE proteins, and used graph theory-inspired algorithms to predict 92 additional components and a PRE complex containing 17 proteins. Some of these predictions were validated experimentally, indicating that the computational analyses can identify novel proteins and complexes in a subcellular compartment. We conclude that the combination of techniques (proteomics, data integration, and computational analyses) used in this study are useful in obtaining a comprehensive understanding of functional components, especially low-abundance entities and/or interactions in the PRE nerve terminal.

Figure 1

Biochemical validation of the separation protocol used to identify proteins from the mouse hippocampal presynaptic (PRE) fraction. To demonstrate the purity of the fractions, equal amounts (10 to 30 μg) of protein from hippocampal synaptosomes, synaptic junctions, PSD, and PRE fractions were separated by SDS-PAGE and probed with antibodies to presynaptic markers (clathrin heavy chain (HC), syntaxin I, and SNAP25) and postsynaptic markers (GluR1, PSD95, and CAMKIIα). Protein bands were quantified using the Odyssey infrared imaging system. Bar graphs (right) show the integrated intensity of protein bands in each fraction relative to the synaptosomal fraction.

Figure 2

A) To characterize the proteins identified by proteomics, we compiled lists of proteins from our proteomic studies with other studies (Morciano et al., 2005; Phillips et al., 2005) and our literature-based network. The majority (67%) of proteins in the merged list (containing 306 proteins) were identified only once in proteomic studies. The subset of proteins detected in two or more independent lists was taken as the “core list” (101 proteins). B) Contributions of each individual list to the core list and to the merged list. “Not detected” indicates proteins that were not identified in the list. “Not in core list” indicates proteins that were identified in the list but did not contribute to the core list. “In core list” indicates proteins that were identified in the list and contributed to the core list. C) Schematic illustrating the data compilation process used to generate the final core presynaptic list of 117 proteins. Protein lists from our proteomic studies, two other published studies, and our literature-based presynaptic network were combined to form a merged list containing 306 proteins. We placed proteins that were identified two or more times in a core list containing 101 proteins. To enrich this list with additional proteins, we used network analysis to identify 16 intermediates from the merged list that interact directly with proteins from the core list. These proteins were added to the core list to generate the final core presynaptic list of 117 proteins (see Table 1).

Figure 3

Prediction and experimental validation of novel presynaptic components and complexes. A) To validate the presence of some of the predicted proteins in the PRE fraction, 50 μg of protein from homogenate, synaptosomes, and PRE fractions were separated by SDS-PAGE and probed with selective antibodies to IQGAP, GEF-H1, RIN1, and PCTK1 by Western Blotting. B) To further confirm the presence of these proteins at the presynapse, immunofluorescence studies were performed using cultured primary cortical neurons. The cells were fixed with PFA, permeabilized, and probed for the localization of RIN1 and PCTK1, and their co-localization with the presynaptic markers SV2 or synaptophysin (SYP) using immunocytochemistry. C) To predict a novel presynaptic complex, proteins from the merged list were analyzed for their ability to interact indirectly (via shared neighbors). A schematic of the predicted complex (containing 17 proteins) is shown. Proteins are indicated by their gene names and links represent indirect interactions. D) Validation of the predicted presynaptic protein complex by co-immunoprecipitation. Left panels: Mouse hippocampal synaptosomal fractions were immunoprecipitated using anti-synapsin I antibody. MAP2, dynamin, and CAMKIIα were detected in the immunoprecipitate by Western Blotting (lane 1). Lane 2 represents immunoprecipitation without synapsin antibody, and lane 3 represents immunoprecipitation without synaptosomal lysate. Right panels: Western Blotting was carried out on the synaptosomal lysate as a control. Bottom panels: Blots were reprobed with synapsin I antibody as a control.