Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins

Esperanza Fernández¹, Mark O Collins², Rachel T Uren¹, Maksym V Kopanitsa¹, Noboru H Komiyama¹, Mike D R Croning¹, Lysimachos Zografos³, J Douglas Armstrong³, Jyoti S Choudhary² & Seth G N Grant¹

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Synopsis

Systems biology has the potential to explain physiological processes as emergent properties of sets of genes and proteins. Beyond simple cellular systems, the challenge of delivering systems biology into the intact and freely behaving animal will require new methods. Currently, the most widely used approach is immunoprecipitation of the target protein and its associate binding proteins. This method suffers from the drawbacks of single step purification strategies that include a high level of non-specific background proteins amongst other limitations. To overcome these limitations we demonstrate that the tandem affinity purification (TAP) technology originally developed in yeast (Rigaut et al, 1999), when combined with gene targeting, can be used to efficiently isolate highly specific complexes from mouse. The 'targeted TAP tagging' strategy combines the two major advantages of each system. The first advantage is that the insertion of two tags into the protein of interest allows two consecutive purification steps that facilitate the recovery of protein complexes with high confidence and decreases the recovery of non-specific proteins or weak interactors. The second advantage, conferred by targeting the endogenous gene, is that the tagged protein is expressed under its natural regulatory mechanisms. We have designed an endogenous TAP targeting strategy to isolate complexes from mouse brain excitatory synapses. The brain is the most complex organ from a cellular and molecular perspective and thus an ideal model to explore the TAP method. Post Synaptic Density 95 (PSD-95/Dlg4) is an adaptor protein comprised of PDZ, SH3 and GK domains and is expressed in the postsynaptic terminal of excitatory synapses where it organizes signaling from neurotransmitter receptors to downstream pathways (Kornau et al, 1995; Hunt et al, 1996; Tu et al, 1999; Husi et al, 2000; Nehring et al, 2000; Dosemeci et al, 2007; Carlisle et al, 2008). Mice carrying a knockout mutation in PSD-95 show it is essential for synaptic plasticity and a range of important behaviours (Migaud et al, 1998; El-Husseini et al, 2000; Beique et al, 2006). Here, a new TAP tag was fused to the carboxyl terminus of PSD-95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD-95 expression, subcellular localization or synaptic electrophysiological function (Figure 2). As a result of four independent tandem purifications and mass spectrometry analysis, we were able to define PSD-95 core complexes with high sensitivity and reproducibility. The four purifications show an average of 12519 proteins, having 118 proteins (94%) common in at least three of four replicates. This reproducibility rate is among the highest rate reported for systematic protein complex isolation. To further validate this interaction data we compared it to information from public datasets. Of the 118 proteins, 22% were proteins that directly bind PSD-95 and 18% were proteins not previously found in other PSD-95 analysis. All together, these data show robust reproducibility and sensitivity of this method for purifying synaptic complexes. These PSD-95 core complexes comprise key functional components of synapses including the glutamate neurotransmitter receptors, K⁺ channels, scaffolding and signaling proteins. These complexes contain ionotropic glutamate receptors of the NMDA, AMPA and kainate subtypes as well as major K⁺ channels that together are the major postsynaptic constituents responsible for synaptic transmission and shaping the postsynaptic electrophysiological response to presynaptic input (Watanabe et al, 2002; Chen et al, 2006; Kim et al, 2007). We believe that this is the first method that has allowed the robust copurification of these proteins. To explore functional organization using network models, we manually curated interactions (Pocklington et al, 2006) and the UniHi database (http://www.mdc-berlin.de/unihi) to identify 119 interactions between 50 proteins (excluding self-interactions) of the PSD-95 core complexes. Network clustering of the interacting proteins showed 40 out of the 50 proteins formed a large connected component (major connected component, MCC) and a modular structure that was segregated into 5 clusters referred to as cluster a (Cla) to cluster e (Cle) (Figure 5A). In addition to the 5 MCC clusters, 2 further disconnected clusters ('Clf' and 'Clg') were found. Of great interest is the location and proximity of the receptors and channels responsible for the postsynaptic depolarization and subsequent action potential generation. All NMDA, AMPA and kainate glutamate receptors were restricted to Cla and Clb and the voltage-dependent K⁺ channels were found in Cla and Clc (entirely comprised of K⁺ channels). It therefore appears that Cla, Clb and Clc are enriched with membrane proteins responsible for electrical properties of the postsynaptic terminal. The central role of PSD-95 was supported by calculation of the shortest path from each protein to every other protein and PSD-95 showed the lowest. Annotation of clusters with human disease associations revealed that multiple disorders map onto the network with a highly significant correlation of schizophrenia within the glutamate receptor clusters (P<10-6). 20 genes involved in schizophrenia were significantly associated with the clusters Cla and Clb that contains all the glutamate receptors and MAGUK/Dlg proteins (Figure 5B). Mapping the primary interactors of these schizophrenia proteins recruited many other proteins found in the other modules of the network. This suggests that the overall network and its different modules are a substrate for schizophrenia, and not simply the glutamate receptors, as was generally considered in the 'glutamate hypothesis' of schizophrenia (Greene, 2001; Coyle, 2006; Lisman et al, 2008). This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease. TAP tagged mice are a valuable resource and useful for a wide range of physiological studies and whole animal studies.

Figure 2

Analysis of TAP-tagged PSD-95 localization and synaptic plasticity in PSD-95^TAP/TAP mice. (A) Immunohistochemical staining of PSD-95 in sagittal brain sections from PSD-95^TAP/TAP and wt mice. B, brainstem; C, cortex; CB, cerebellum; H, hippocampus; S, striatum. Scale bar=1 mm. (B) Immunohistochemical staining of PSD-95 in sagittal hippocampus sections from PSD-95^TAP/TAP and wt mice showing CA1, CA3 and dentate gyrus (DG). Scale bar=1 mm. (C) Synaptic localization of TAP-tagged PSD-95 in primary hippocampus neurons. DIV14 neurons from wt and PSD-95^TAP/TAP mice were stained with PSD-95 and MAP2B antibodies (top panels). Three lower panel show PSD-95 and FLAG antibody staining in a culture from PSD-95^TAP/TAP mice (bottom panels). Inset panels show higher magnification of synaptic puncta labeling with each antibody and merged image. Scale bar=10 m. (D) Long-term potentiation of fEPSPs induced by theta-burst stimulation in CA1 area of hippocampal slices is similar in PSD-95^TAP/TAP (13 slices from 4 animals) and wild-type mice (15 slices from 4 animals).

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Figure 5

Protein interaction network of PSD-95 interacting proteins. (A) 50 proteins of the PSD-95 core complex were connected, with 119 interactions segregated into 5 clusters (Cla–Cle) forming the MCC and two separate small clusters Clf and Clg. PSD-95/Dlg4 is showed in red, primary interactors of PSD-95/Dlg4 are shown in blue and secondary interactors are shown in yellow. The glutamate receptors (NMDA, AMPA and kainate receptors) and potassium channels are bracketed. (B) Schizophrenia susceptibility genes are shown in orange.

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