We chose to perform these experiments at P28 because synaptogenesis is mostly complete at this time, and synapse loss in CSPα KOs is not yet pronounced (Chandra et al., 2005 and Fernández-Chacón et al., 2004). This time point, therefore, avoids nonspecific changes in synaptic proteins that occur once more synapses are lost in CSPα KO mice. Wild-type and VE822 CSPα KO brains were homogenized to prepare synaptosomes and further fractionated to obtain purified synaptic plasma membranes, cytosol, and synaptic

vesicles (Figure 1A). This fractionation procedure allowed us to increase our signal-to-noise ratio and delve deeper into the synaptic proteome. The synaptic plasma membrane, cytosol, and vesicle fractions of the two genotypes were subjected to DIGE and iTRAQ in a pairwise fashion. We carried out multiple, independent DIGE and iTRAQ experiments, and analyzed over 1,500 synaptic proteins in the three fractions (Table 1). By analyzing these ∼1,500 proteins, we sampled nearly the entire synaptic proteome. All protein changes CP-690550 ic50 over 40% were scored and identified by mass spectrometry. Figure 1B shows a DIGE experiment on the synaptic plasma membrane fraction of wild-type and CSPα KO brains. The gels revealed only a few protein changes between the two genotypes, supporting our hypothesis that deletion of CSPα leads initially only to the loss of its clients. Similar to the

DIGE runs, the iTRAQ experiments also showed select changes in protein levels (Figure 1C; see Figure S1 available online). The most prominent changes were, as expected,

for CSPα and for the t-SNARE SNAP-25 (Figures 1B, 1C, S1A, and S1C), the previously characterized CSPα client (Chandra et al., 2005 and Sharma et al., 2011), validating this approach to identify other CSPα clients. We considered a synaptic protein 3-mercaptopyruvate sulfurtransferase to be a potential CSPα client if its levels were changed significantly in CSPα KO samples in at least two independent proteomic experiments. Based on these stringent criteria, we identified a total of 37 proteins (Table 1). This set of candidate client proteins has striking features: (1) Most of the identified proteins are presynaptic, as opposed to postsynaptic, as would be expected for CSPα clients. The 27 proteins we identified in our proteomic screen, besides the 10 chaperones, are potential CSPα clients (Table 1). These include exocytic proteins that are components of the SNARE machinery (SNAP-25, complexin I, NSF) and endocytic proteins that regulate vesicle fission (dynamin 1, Necap 1). Cytoskeletal proteins include regulators of the actin and microtubule cytoskeleton (Crmp2, Crmp3, and BASP1) and GTP binding cytoskeletal proteins (Septin 3, 5, 6, and 7). Many of these proteins are represented in Gene Ontology shortest pathway networks emanating from CSPα that are linked by a maximum of three interactions (Figure 1D), adding further credence that they may indeed be direct CSPα clients.