In vivo mapping of protein-protein interactions of schizophrenia risk factors generates an interconnected disease network

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   Aug 5, 2024

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   Manuscript number: RC-2023-02306

   Corresponding author(s): John, Yates

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   1. General Statements [optional]

   We greatly appreciate the reviewers taking time from their busy scientific careers to evaluate our manuscript. We were elated to read all the positive comments, such as …

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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   Reply to the reviewers

   Manuscript number: RC-2023-02306

   Corresponding author(s): John, Yates

   [Please use this template only if the submitted manuscript should be considered by the affiliate journal as a full revision in response to the points raised by the reviewers.

   If you wish to submit a preliminary revision with a revision plan, please use our "Revision Plan" template. It is important to use the appropriate template to clearly inform the editors of your intentions.]

   1. General Statements [optional]

   We greatly appreciate the reviewers taking time from their busy scientific careers to evaluate our manuscript. We were elated to read all the positive comments, such as “the conclusions are well-supported and convincing”, “should contribute to a more nuanced understanding of SCZ pathogenesis”; “The potential implications for drug development underscore the broader significance of the study in advancing our knowledge of neurobiology and its relevance to neurological disorders like schizophrenia”, and “The study is informative, and has great potential to enrich the specific literature of this field”. We also found the constructive criticism very helpful for improving our manuscript. We performed additional experiments and bioinformatic analyses, as requested. We modified the manuscript to answer the reviewers’ questions. Due to its complexity, it is difficult to describe the different and sometimes conflicting hypotheses of SCZ pathogenesis in a single manuscript. This complexity is reflected in the conflicting requests from the reviewers. One reviewer requested we investigate and highlight the role of non-neuronal cells in SCZ while another reviewer suggested we did not focus enough on synaptic proteins. We believe we have achieved a balance to represent the intricacy of SCZ biology and the different opinions of the reviewers.

   Thanks again.

   2. Point-by-point description of the revisions

   This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   *Summary: Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). *In this manuscript, McClatchy and colleagues used a conventional approach combining immunoprecipitation (IP) of endogenous target proteins (baits) followed by liquid chromatography mass spectrometry (MS) analysis of the co-immunoprecipitating proteins to map protein-protein interaction (PPI). This interaction network is centered around baits that had been annotated as susceptibility factors for schizophrenia (SCZ). A variety of previous studies have identified thousands of such SCZ susceptibility factors. Mostly based on the availability of antibodies, 8 bait proteins were selected in this study. The authors reasoned that immunoprecipitating endogenous proteins from tissues using specific antibodies was a more accurate view of physiological conditions than epitope tagging followed by affinity purification (AP) from cells in culture. The model system from which proteins were extracted was the hippocampus dissected from mice that had been treated or not by phencyclidine (PCP), a drug that has been shown to induce SCZ symptoms in humans and animals. By comparing the proteins identified and quantified from the PCP-treated samples against control IPs and/or saline-injected mouse controls, a large number of PPI were deemed statistically significant. Most of these potential interactors were not present in PPI databases (BioGRID), most likely because such databases are populated with large-scale APMS datasets from cell cultures, with very few studies using brain tissue. Strikingly, many of the co-immunoprecipitated proteins were also known as SCZ susceptibility factors, which lend weight to the hypothesis that these factors form a large protein interaction network, localized at the synapses.

   *Major comments:

   • Are the key conclusions convincing?* Overall, the conclusions drawn from the experimental design, data analysis, and corroboration with existing literature are well-supported and convincing. When selecting the SCZ susceptibility factors, the authors clearly state their goal, the databases used for gene selection, and the rationale for choosing proteins with synaptic localization. The inclusion of evidence from genetic studies and previous publications strengthens the credibility of the selected genes. The methodology used to establish the novel SCZ PPI network is mostly well-described (see minor comments below). The use of an 15N internal standard also adds rigor to the quantitation of PPI. The GO enrichment analysis provides valuable insights into the biological functions and cellular components associated with the SCZ PPI network. The annotation of identified proteins using the SynGo synaptic database and the distribution of annotated synaptic proteins among different baits further support the biological relevance of this PPI network. The cross-referencing of the PPI network with published genetic studies on SCZ susceptibility genes adds robustness to the findings. Specifically, the observation that 68% of protein interactors have evidence of being potential SCZ risk factors is a strong corroboration of the prevailing hypothesis in the field. Finally, the significant changes induced by PCP that were identified for all baits except Syt1, along with the comparison of altered proteins with SAINT-identified PPI, add depth to the understanding of PCP modulation.

   *- Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? *No, but note that APMS/IPMS has been around for more than a decade (Introduction page 3).

   We agree and did not mean to imply that IP-MS is new technology. We tried to convey that IP-MS is not new technology, but the number of IP-MS studies employed to study the PPI of endogenous proteins in brain tissue is a small percentage of all the published PPI MS studies.

   We added the following to the Conclusions to clarify this point:* “Although IP-LC-MS technology has been employed for more than a decade, quantitation of proteins using this strategy in mammalian tissue is scarce in the literature.””*

   *- Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. *One piece of data that is missing are Western blots using the 8 selected antibodies against the proteins extracted from their experimental samples to validate the antibodies recognize 1 protein of the expected size from these tissue extracts.

   We took your suggestion and performed immunoblots with our 8 IP antibodies using the starting material (i.e. rat brain hippocampus). All antibodies recognized a single band of the approximate molecular weight of the target except for the Gsk3b, which produced a doublet instead of a single band. This image is similar to what has been observed with the phosphorylation of Gsk3b(Krishnankutty, Kimura et al. 2017, Vainio, Taponen et al. 2021). To provide evidence that the additional band observed for Gsk3b is the phosphorylated target protein, we searched our Gsk3b IP dataset for a differential phosphorylation (i.e. 79.9663) on S,T, or Y. Even though we did not perform phosphorylation enrichment, we identified S389 as abundantly phosphorylated in all Sal and PCP samples consistent with our immunoblot. Images of these immunoblots are now Supplementary Figure 1.

   • *Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. *Running SDS-PAGE and Western blotting should be straightforward and cheap.

   *- Are the data and the methods presented in such a way that they can be reproduced? *Yes

   *- Are the experiments adequately replicated and statistical analysis adequate? *Yes

   *Minor comments:

   • Specific experimental issues that are easily addressable. *The rationale for the short duration between PCP injection and animal sacrifice is only explained in the discussion section (page 17). The fact that this short treatment of less than 30 min should prevent any change in transcription or translation should be introduced earlier (in the experimental procedures).

   We agree this is an important aspect of the study and that it suggests that the effect of PCP is independent of changes in transcription and translation as stated in the Discussion.

   We added the following to the Introduction:

   *“PCP was administered for less than 30min., which precluded any changes in transcription or translation and allowed us to focus on PPI.” *

   Note that the duration is written as 26 min on page 4 and 25 min on page 9. Please reconcile these numbers*. *

   We have corrected this typo. It was 26min.
   Is there any biological significance for this SCZ study that the mice were maintained on a reverse day-night cycle?

   Rats are nocturnal animals, i.e. active at night and sleep during the day. In this study, rats were housed on a reverse day-night cycle so that assessment of the response to PCP could be evaluated during their active phase. This is not specific SCZ research and is the routine protocol for behavioral testing in the Powell laboratory. It is not clear from reading Experimental Procedures/Bioinformatic Analysis section (page 6) if normalized N14/N15 protein ratios measured in the bait-IPs and control-IPs were used for the SAINT analysis? Or did the authors used label-free quantitation with spectral counts?

   We apologize for not making the methods clearer. In the results, it is stated that the N14 identifications are used in the SAINT analysis, and we state in the Discussion that SAINT uses spectral counts. We modified the Experimental Procedures/Bioinformatic Analysis section (page 6) to state: The input for SAINT was only the 14N identifications.

   *- Are prior studies referenced appropriately? Yes

   • Are the text and figures clear and accurate? *Fig1C: The workflow is a little too simple, the authors might want to add more details.

   We revised Fig1C with more details as suggested.

   FigS1C: Please add x-axis title (spectral counts) directly to the figure.

   “Spectral counts” was added to the x-axis. FigS1C is now FigS2C ,with the addition of the immunoblots you suggested. Fig2B-D: The color scale bar should have number values to denote lower and upper limits in % (as opposed to "lowest" and "highest"). Numerical values were added to replace the upper and lower limits. *- Do you have suggestions that would help the authors improve the presentation of their data and conclusions? *No *

   Reviewer #1 (Significance (Required)):

   • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. In this study, the authors have drastically expanded the protein interaction landscape around 8 known SCZ susceptibility factors by using a conventional IPMS approach. Performing the IPs on protein extracted from hippocampus dissected from mice treated with phencyclidine to model SCZ increases the biological significance of such lists of proteins. Furthermore, the co-immunoprecipitation of many other SCZ susceptibility factors along with the 8 selected baits supports the hypothesis that these proteins of varied functions are part of large interaction networks. Overall, the integration of experimental data with in silico networks, along with the quantification of PPI changes in response to PCP, should contribute to a more nuanced understanding of SCZ pathogenesis. The potential implications for drug development underscore the broader significance of the study in advancing our knowledge of neurobiology and its relevance to neurological disorders like schizophrenia.

   • Place the work in the context of the existing literature (provide references, where appropriate). Overall, this study contributes to the existing literature by providing experimental data on in vivo PPI networks related to SCZ risk factors. Not only do the authors validate 124 known interactions but also they identify many novel PPI, due to a gap in the existing literature regarding the comprehensive mapping of PPI directly from tissue extracts, especially brain tissue. The authors advocate for more IPMS studies in mammalian tissues to generate robust tissue-specific in silico networks, which agrees with the growing understanding of the importance of tissue-specific networks for identifying disease mechanisms and potential drug targets. Furthermore, the SCZ PPI network reported here is enriched in proteins previously associated with SCZ, which aligns with the existing literature emphasizing the involvement of certain proteins and pathways in the pathogenesis of SCZ [References: 78-85]. The authors also investigate the response of the SCZ network to PCP treatment, hence providing insights into the potential effects of post-translational modifications, protein trafficking, and PPI alterations in a model of schizophrenia, which adds to existing knowledge about the impact of PCP on the molecular processes associated with SCZ [References: 88, 89, 92].

   • State what audience might be interested in and influenced by the reported findings. Overall, the findings reported in this manuscript have implications for both basic research in molecular biology and potential translational applications in the development of targeted therapies for neurological disorders, particularly schizophrenia. The study delves into in vivo protein-protein interaction (PPI) networks related to genes implicated in schizophrenia (SCZ) risk factors. Researchers in neuroscience, molecular biology, and psychiatry would find the information valuable for understanding the molecular basis of SCZ. The study highlights the potential for identifying disease "hubs" that could be drug targets. Pharmacologists and drug developers interested in targeting protein complexes for drug development, especially in the context of neurological disorders, may find the study relevant.

   • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. Technical Expertise | biochemistry, liquid chromatography mass spectrometry, proteomics, computational biology, protein engineering, protein interaction networks, post-translational modifications, protein crosslinking, proximity labeling, limited proteolysis, thermal shift assay, label-free and isotope-labeled quantitation. Biological Applications | human transcriptional complexes, apicomplexan parasites, viruses, nuclear envelope, ubiquitin ligases, non-model organisms.

   Reviewer #2 (Evidence, reproducibility and clarity (Required)):

   Summary: McClatchy, Powell and Yates aimed at identifying a protein interactome associated to schizophrenia. For that, they treated rats (N14 and N15) with PCP, which disturbs gutamatergic transmission, as a model for the disease and co-immunoprecipitated hippocampi proteins, which were further analyzed by standard LC-MS.

   The study is new, considering not much has been done in this direction in the field of schizophrenia. This justifies its publication. On the other hand, a major flaw of the is the lack of information on the level of interaction of the so called protein interactome. Meaning, we cannot distinguish, as the study was performed, which proteins are directly interacting with the targets of interest from proteins which are interacting with targets´ interactors. The different shells of interaction are crucial information in protein interactomics.

   Major: most of I am pointing below must be at least discussed or better presented in the paper, as It may not be solvable considering how the study has been conducted.

   1. The study fails in defining the level of interaction of the protein interactome with the considered targets. This has been shortly mentioned in the discussion, but must be more explicit to readers, for instance, in the abstract, introduction and in the methods sections. We agree this is crucial information that is absent from our dataset. As we explained in the Discussion, we cannot distinguish between PPI that are direct interactors with the target protein and PPI that reside in a multi-protein complex that includes the protein (i.e. indirect). This is an inherent problem with any IP-MS study. We amended the Introduction to highlight the ambiguity of the interaction data produced by the IP-MS approach, as you suggested.

   Text added to the Introduction:

   “Regardless of whether Ab or tagged proteins are employed to identify PPI from a biological sample, it cannot be determined if the identified interactor binds directly to the target protein or reside in a complex of proteins that includes the target protein (i.e. indirect).”

   Since this important information is routinely missing from IP-MS studies, we decided to try to determine the level of interaction by using the artificial intelligence algorithm AlphaFold3(AF3). We believe it is not yet optimized for PPI, but AF3 is a big leap forward in the field of structural biology. For example, we observed AF3 did not predict high confident structures for our large membrane target proteins and was unable to validate known direct PPI of these targets. In addition, analyzing data with AF3 is currently not automated or streamlined so with ~1600 PPI identified in our dataset, we chose to look at one target protein, Ppp1ca. AF3 identified many known direct binding proteins in our Ppp1ca PPI dataset, which gives high confidence to the novel PPI predicted to be direct interactors. The AF3 data is encompassed in an additional Figure 6.

   The following was added to the Results Section:

   *“A disadvantage of IP-MS studies is that it cannot distinguish between a PPI that binds directly to the target protein, and a PPI in which the interactor and target protein reside the same multiprotein complex (i.e. indirect). We sought to predict which PPI may be directly interacting with its target protein by using the artificial intelligence algorithm AlphaFold3(AF3) (Abramson, Adler et al. 2024). First, we analyzed the predicted AF3 structure of the targets using the pTM score and the fraction of each structure calculated to be disordered (Figure 6A and Supplementary Table7). Our reasoning was that if targets have a poorly resolved structures, it will be difficult to screen them for direct PPI. A pTM score >0.5 suggests that the structure may be correct (the highest confidence score is 1). Undefined or disordered regions hinder the accuracy of the prediction. All targets possessed a pTM score > 0.5 except Syt1. The disordered fraction negatively correlated with the pTM score, as expected. Gsk3b, Ppp1ca, and Map2k1 had the highest pTM scores and were also the smallest of our target proteins (Figure 6B). Ppp1ca had the most confident structure (i.e. pTM 0.9) and the smallest disordered fraction (i.e. 0.07). Next, we determined the AF3 prediction of previously reported direct interactions of the targets. We used the iPTM score to determine interaction confidence. An iPTM score >0.8 is considered a highly confident direct interaction, whereas 0.8. These eight PPI have all previously been reported to form a direct interaction with Ppp1ca, except Phactr3 (Zhang, Zhang et al. 1998, Terrak, Kerff et al. 2004, Hurley, Yang et al. 2007, Marsh, Dancheck et al. 2010, Ragusa, Dancheck et al. 2010, Ferrar, Chamousset et al. 2012, Choy, Srivastava et al. 2024, Xu, Sadleir et al. 2024). Phactr3 is structurally similar to, but less studied than, the reported direct interactor Phactr1. These interactors are all inhibitors of PP1 except Ppp1r9b which targets Ppp1ca to specific subcellular compartments. Nine PPI were assigned a score The following has been added to the Discussion:

   “Our SCZ PPI network consists of two types of PPI: direct physical interactions and “co-complex” or indirect interactions. Typically, the nature of the interaction can be distinguished in IP-MS studies. We decided to employ the new AF3 algorithm to screen the PPI of Ppp1ca to provide evidence for direct interactors. We chose to examine the PPI assigned to Ppp1ca, because its structure was the most confident among our target proteins and AF3 correctly predicted a known direct interactor with high confidence. Ppp1ca is a catalytic subunit of the phosphatase PP1, which is required to associate with regulatory subunits to create holoenzymes (Li, Wilmanns et al. 2013). Eighteen PPI were predicted to be directly interacting with Ppp1ca using a 0.6 or higher iPTM filter. This filter may be too conservative and generate false negatives, because another study employed a 0.3 filter followed by additional interrogation to screen for direct PPI (Weeratunga, Gormal et al. 2024). Forty-four percent of these predictions were confirmed by previous publications. Most of the validated direct interactions are inhibitors of the phosphatase, but one, Ppp1r9b (aka spinophilin), is known to target Ppp1ca to dendrite spines to enhance its activity to specific substrates (Allen, Ouimet et al. 1997, Salek, Claeboe et al. 2023). This high correlation with the literature provides substantial confidence in the novel PPI predicted to be direct Ppp1ca interactors. The AF3 screen predicted that NDRG2 directly interacts with Ppp1ca. This protein is known to regulate many phosphorylation dependent signaling pathways by directly interacting with other phosphatases including Pp1ma and PP2A (Feng, Zhou et al. 2022, Lee, Lim et al. 2022). Actin binding protein Capza1 was also predicted to directly interact with Ppp1ca and Ppp1ca interacts with actin and its binding proteins to maintain optimal localization for efficient activity to specific substrates (Foley, Ward et al. 2023). Hsp1e is a heat shock protein predicted to directly interact with Ppp1ca. Although there is no direct connection to Ppp1ca, other heat shock proteins have been reported to regulate Ppp1ca (Mivechi, Trainor et al. 1993, Flores-Delgado, Liu et al. 2007, Qian, Vafiadaki et al. 2011). We also observed that many of these direct PPI were altered with PCP treatment. One direct interactor, Ppp1r1b (aka DARPP-32), is phosphorylated at Thr34 by PKA in the brain upon PCP treatment. This phosphorylation event converts Ppp1rb to a potent inhibitor of Ppp1ca(Svenningsson, Tzavara et al. 2003). Importantly, manipulation of Thr34 attenuated the behavioral effects of PCP. Consistent with this report, Ppp1r1b-Ppp1ca interaction was only observed with PCP in our study. Further investigation is needed to determine if our novel direct interactors regulate the PCP phenotype. We conclude that AF3 can provide important structural insights into the nature of PPI obtained from large scale IP-MS studies.”

   1. Considering the protein extraction protocol, it is fair to mention that only the most soluble proteins are being considered here. I am bringing this up since the importance of membrane receptors is clear in the studied context. This is an interesting point. It has been predicted that transmembrane proteins constitute 25-30% of the proteome(Dobson, Remenyi et al. 2015). Thus, we would predict our dataset will have more soluble proteins than membrane proteins. Half of our target proteins were transmembrane proteins, so in designing the protocol for this study we ensured that these membrane proteins could be significantly enriched compared to the control IPs (Supplementary Figure 2C). In addition, compared to soluble proteins, membrane proteins are notoriously difficult to identify by bottom-up proteomics (Savas, Stein et al. 2011). We decided to investigate how many of our protein interactors were transmembrane proteins. Using Uniprot, 199 (20%) of our protein interactors were determined to have a transmembrane domain. Therefore, this data does not support the statement that only the most soluble proteins are being considered in our study. We added this percentage of transmembrane proteins in our network to the text of the Results section.

   2. It is not clear from the methods description if antibodies from all 8 targets were all together in one Co-IP or have been incubated separately in 8 different hippocampi samples. It seems the first, given how results have been presented. If so, this maximizes the major issue raised above (in 1). We apologize for not clearly describing our experimental design. All the targets were immunoprecipitated separately and analyzed separately on the mass spectrometer. With all the biological replicates and two conditions (i.e. Saline and PCP), we performed 48 individual, separate IPs. There were an additional 48 individual, separate IPs run in parallel that were the control IPs.

   We modified the schematic of our experimental design in Figure 1C to clarify that the 8 targets IPs were analyzed separately. In addition, we modified the Results to read:

   “In total, 96 (48 bait and 48 control) IPs were performed, and each was analyzed separately by LC-MS analysis.”

   1. Definitely, results here are not representing a "SCZ PPI network". PCP-treated animals, as any other animal model, are rather limited models to schizophrenia. As a complex multifactorial disease, synaptic deficits, which is the focus of this study, can no longer be considered "the pivot" of the disease. Synaptic dysfunction is only one among many other factors associated to schizophrenia.

   We do agree that synaptic dysfunction is only one factor associated with SCZ and we will discuss this more in our response to your next comment.

   We understand the limitations of PCP as an animal model of SCZ. It is quite difficult to model a specific human complex multifactorial neurological disease in rodents and we would contend that there is no single universal SCZ model that everyone agrees with. We addressed this by adding the following to the Introduction:

   “Since many SCZ symptoms are uniquely human, this is no single animal model that truly replicates all the complex human SCZ phenotypes(Winship, Dursun et al. 2019). In this respect, all SCZ animal models can be considered limited.* “ *

   We respectfully disagree, however, with the term SCZ PPI network. This study is focused on SCZ by choosing proteins implicated in SCZ, quantitating how the PPI changes in a SCZ model, and discussing how our findings are relevant to SCZ pathogenesis. So, it seems logical to call our dataset a SCZ PPI network. We do concede that without further experimentation we do not know if these PPI play a causal role in SCZ. Furthermore, our novel PPI may involve biological pathways unrelated to SCZ and that have relevance to other biological conditions.

   We added the following statement to the Discussion to address this comment:

   “Even though our network was constructed in the context of SCZ, our dataset has relevance to other neurological diseases where our targets have been implicated in the pathogenesis.”

   1. Authors should look for protein interactions that might be happening also in glial cells. They are not the majority in hippocampus, but are present in the type of tissue analyzed here. Thus, some of the interactions observed might be more abundantly present in those cells. Maybe enriching using bioinformatics tools the PPI network to different cell types.

   As mentioned above, we agree that synaptic dysfunction is just one of the hypotheses of SCZ pathogenesis and emerging evidence suggests that dysfunction in astrocytes and microglia are factors. Since these non-neuronal cells can regulate synapses, these hypotheses are not mutually exclusively and suggests that at the cellular level SCZ etiology involves multiple cell types.

   We addressed your query by comparing our PPI network to an RNA-seq analysis of different cell types in the rodent brain(Zhang, Chen et al. 2014). First, we analyzed our target proteins, and found that they were expressed in all cell types to varying degrees except Syngap which was not in the RNA-seq database. This data is now represented in Figure 3E. We then determined the RNA abundance distribution of all the protein interactors, which is represented in Figure 3D as a heatmap. From a bird’s eye view, it suggests that some PPI exist in non-neuronal cells. Next, we determine how many of our protein interactors were enriched in one cell type, which is shown in Figure 3F. We defined an enriched protein as having >50% of the RNA signal in one cell type. We identified 175 proteins that were enriched in one cell type compared to the entire RNA-seq dataset which had 4008 enriched proteins. In the entire RNA-seq dataset, 24% of the enriched proteins were in neurons whereas 47% of our protein interactors were enriched in neurons. This is consistent with the enrichment of synaptic proteins in our network. There was also an increased percentage of astrocytes (19%) and oligodendrocytes (6%) in our network compared to the entire database (i.e. astrocytes-11% and oligodendrocytes-4%). In other cell types, such as microglia, there was less protein enrichment in our network compared to the database. We have amended this cell type analysis to our manuscript and concluded that a portion of our PPI network may occur in non-neuronal cells. We also created a supplementary table of our network with its associated RNA-seq data.

   Text added to the Results:

   “Non-synaptic proteins represented 59% of our network suggesting that some PPI may occur in non-neuronal cells. To investigate this possibility, we annotated our network with a transcriptome rodent brain database of eight cell types(Zhang, Chen et al. 2014). All the targets were detected in all cell types but there was obvious enrichment in specific cell types for some targets (Figure 3E). Syngap1 was not in the database. We also observed a large variation of cellular distributions for the interactors (Figure 3D). Next, we sought to determine how many interactors are enriched in a particular cell type by defining cell enrichment as a protein having >50% RNA signal in one cell type. We identified 175 protein interactors enriched in one cell type, whereas the entire database had 4008 proteins enriched (Figure 3F). Consistent with our synaptic enrichment, 47% of the enriched protein interactors were in neurons whereas only 24% of the enriched protein in the entire database were in neurons. We also observed an increase in protein interactors enriched in astrocytes compared to the database. Overall, this analysis provides evidence that our identified PPI may occur in non-neuronal cells.”

   Text added to the Discussion:

   “The exact etiology of SCZ, however, remains unclear and synaptic dysfunction is only one hypothesis (Misir and Akay 2023). There is evidence for the involvement of non-neuronal cell types, including endothelial cells, astrocytes, and microglia(Tarasov, Svistunov et al. 2019, Rodrigues-Neves, Ambrosio et al. 2022, Stanca, Rossetti et al. 2024). Although we observed an enrichment of synaptic proteins in our SCZ network, we provided evidence that a portion of our network may occur in non-neuronal cells. Since non-neuronal cells can regulate synapses(Vilalta and Brown 2018, Bauminger and Gaisler-Salomon 2022)*, synaptic dysfunction and perturbations in non-neuron cells in SCZ etiology are not mutually exclusive. Our data corresponds with emerging evidence that pathogenesis is multifaceted, involving dysfunction in multiple cell types. *“

   Minor:

   1. in the abstract, it is not clear if 90% of the PPI are novel to brain tissue in general or specifically schizophrenia. We apologize for the confusing sentence. 90% are novel meaning the PPI have not been reported in any study. We changed the abstract to read:

   “Over 90% of the PPI have not been previously reported.”

   1. authors refer to LC-MS-based proteomics as "MS" all across the text. Who am I to say this to Yates et al, but I think it is rather simplified use "Mass Spectrometry Analysis", when this is a typical LC-MS type of analysis We agree with you. We have replaced MS analysis with LC-MS analysis in the manuscript.

   1. Several references used to construct the hypothesis of the paper are rather outdated: several from 10-15 years ago. It would be interesting to provide to the reader up to date references, given the rapid pace science has been progressing. We agree many of the references are 10-15 years old. Many of the hypotheses and biological mechanisms we discussed can be supported by too many studies to cite them all, due to space. If we could, we would. We also agree that there are many more recent studies that have confirmed and added more details to the original discovery or hypothesis cited. We cite the first study to support our conclusions because it deserves the most credit.

   2. "UniProt rat database". Please, state the version and if reviewed or unreviewed.

   This information was added to the Methods section. UniProt reviewed rat database with isoforms 03-25-2014.

   Reviewer #2 (Significance (Required)):

   The study is informative, and has great potential to enrich the specific literature of this field. But should tone down some arguments, given the experimental limitations of the PPI network (as described above) and should state PCP-treated rats as a limited model to schizophrenia.

   Reviewer #3 (Evidence, reproducibility and clarity (Required)):

   Summary

   It is now widely accepted that schizophrenia is polygenic disorder in which a large fraction of the genetic risk is in variants affecting the expression of synaptic proteins. Moreover, it is known that these synaptic proteins are found in multiprotein complexes and that many proteins encoded by schizophrenia risk genes interact directly or indirectly in these complexes. It is also known that some drugs including phencyclidine, which binds to NMDA receptors and to Dopamine D2 receptors (not mentioned by the authors) can induce schizophreniform psychosis. The authors have set out to advance on this position by performing proteomic mass spectrometry studies on proteins identified as encoded by schizophrenia risk genes. They target 8 proteins for immunoprecipitation from rat brain and identify coisolated proteins and perform various network analyses. In the most interesting part of the paper they ask if PCP-treatment altered protein interactions and report various changes.

   Major comments:

   1. Choice of target proteins. It was not until the first paragraph of the results section that the authors first name the 8 synaptic proteins that have chosen to study. This information should be in the abstract.

   This information was added to the abstract as requested.

   The authors then use figure 1A and 1B as evidence that these 8 "baits" are schizophrenia-relevant proteins. Figure 1A does not provide any evidence at all and Figure 1B is about as weak a line of evidence imaginable - a histogram of the number of papers that have the search term "schizophrenia" and the protein name. I tried this search for Grin2B and almost immediately found papers that reported no association between Grin2B and schizophrenia (e.g. PMID: 33237434). Figure 1B should be scrapped.

   The purpose of Figure 1A was not to demonstrate that there is evidence that our proteins are involved in SCZ. The purpose of this figure is to show that these proteins are diverse in function and structure (blue = membrane proteins; yellow = soluble proteins), and that there are published studies reporting physical and functional interactions between these 8 proteins. This suggests that a more extensive network may exist.

   We agree that Figure 1B does not specifically describe how each protein is related to SCZ but demonstrates how many papers investigating their connection to SCZ have been published. We understand how by itself, this can be considered weak. We still think it is important to show that multiple laboratories have published papers connecting these proteins to SCZ. Instead of scrapping this figure, we have moved it to the Supplementary Figure 2A.

   We read PMID: 33237434 and interpret their findings quite differently than you. This report examined whether one single nucleotide mutation (SNV) in Grin2b is associated with the cognitive dysfunction in SCZ but did not examine if this mutation is associated with the other major SCZ phenotypes (i.e. psychotic and emotional). Specifically, the study selected 117 “patients in whom cognitive dysfunctions are present despite effective antipsychotic treatment of other schizophrenia symptoms.” The study concluded that Grin2B SNV was not associated with this subset of patients but concluded that they need to search for other NMDAR variants and study their association with SCZ. We would argue that the only reason this group performed these experiments was the well-known association between Grin2b and SCZ. Many studies have found SNVs in Grin2B that are associated with SCZ, but there are conflicting reports. It is unclear if the discrepancies are connected to different cohorts, complexity of SCZ phenotype, or small sample sizes. Regardless of Grin2B mutations significantly associated with SCZ, there are several lines of evidence that Grin2B is involved in SCZ. Most importantly, Grin2b is a component of the NMDAR, which is a key player to the SCZ hypo-glutamate hypothesis and the receptor that binds PCP. By immunoprecipitating Grin2b, we are analyzing the PPI network of NMDAR, which is arguably the most studied complex in SCZ research.

   The remaining part of paragraph 1 of the results does not provide an adequate, let alone systematic, justification for the use of the 8 baits. It would be appropriate to construct a table with the 8 proteins and cite relevant papers and identify the basis for why they are implicated in schizophrenia (is it a direct mutation or some other evidence?). What makes these 8 proteins better than many others that are cited as synaptic schizophrenia relevant proteins?

   We apologize for not clearly and thoroughly describing the reasons for choosing our baits. As stated in the first paragraph of the Results, we chose the proteins that had evidence of being a SCZ risk factor in SCZ databases that included a plethora of human genomic studies. This criterion by itself results in ~5000 genes. To further narrow our candidates, we chose targets that were synaptic and were observed to have phosphorylation changes in response to PCP in an SCZ animal model. Since protein-protein interactions (PPI) are often dependent on phosphorylation, we believe this is an important criterion for quantitation of PPI in response to PCP. These requirements still resulted in a list of hundreds of proteins. So, what makes these better than any other SCZ relevant protein? As stated in the manuscript, the major limiting criterion was identifying commercial antibodies that can efficiently immunoprecipitate their target in brain tissue. Since there are many reports associating our targets with SCZ, we directed the reader to SCZ databases that compile large genomic association studies. We understand, however, the request for more specific information regarding the biological connection between these proteins and SCZ. We took your suggestion and constructed a table with our 8 targets, and it is now Figure 1A. In this table, we selected references to indicate if the target has reported changes in expression and/or activity in SCZ samples (i.e. human and animal model) or genetic association with SCZ in human studies.

   The methods of protein extraction are particularly concerning. The postsynaptic density of excitatory synapses (which contains several of the target proteins in this study) has been notoriously difficult to solubilise unless one uses high pH (9) and harsh detergent extraction (1% deoxycholate). The authors use pH 7 and weak detergent conditions, which are likely to be inefficient for solubilising at least several of the target proteins. Nowhere do the authors report how much of the total of their target protein is being solubilised. Indeed, there are no figures showing biochemical conditions at all. What if only a small percentage of the target protein is being immunoprecipitated - what does this mean for the interaction data? How do we know if the fraction being immunoprecipitated is from the synapse? (why did they not use synaptosomes).

   How do we know if the fraction being immunoprecipitated is from the synapse? (why did they not use synaptosomes). The absence of this kind of data undermines the reader's confidence in the findings.

   We apologize for not clearly explaining our experimental design We were not interested in identifying the PPI of the PSD. All these proteins have been localized to the synapse, but they are also localized to other neuronal compartments and non-neuronal cell types. Synaptic dysfunction is one hypothesis of SCZ pathogenesis, but there is evidence of other cell types, including astrocytes, microglia, and oligodendrocytes(Kerns, Vong et al. 2010, Ma, Abazyan et al. 2013, Goudriaan, de Leeuw et al. 2014, Park, Noh et al. 2020). For these reasons, we chose an unbiased approach to identifying PPI.

   The Results have been amended to read: “All the targets are localized to the synapse, but also localized to non-synaptic compartments and expressed in non-neuronal cells. Thus, since there is also evidence for non-synaptic perturbations contributing to SCZ pathogenesis, we chose to perform an unbiased analysis in unfractionated brain tissue (Tarasov, Svistunov et al. 2019, Rodrigues-Neves, Ambrosio et al. 2022, Stanca, Rossetti et al. 2024). “

   Why do we choose a specific solubilization strategy? Harsh detergents can disrupt PPI and prevent efficient enrichment of the target by disrupting the target-antibody interaction(Pankow, Bamberger et al. 2015). To identify protein interactions, mild detergent conditions are typically employed in PPI studies. We used a combination of “weak” detergents (i.e. 0.5% NP-40, 0.5% Triton, and 0.01% Deoxycholate) to help prevent non-specific PPI, but still allowing efficient enrichment of the target proteins. We do agree that with our conditions the targets were not completely solubilized. It is a balancing act to find the correct conditions for IP-MS analysis. Since we are unable to immunoprecipitate all the target protein, we did not identify all the PPI for each target, and we did not make this claim. Importantly, we did identify known interactions for all our targets. Our mild detergent protocol is similar to other PPI studies and our results validates results reported in previous studies. It is more important to significantly enrich the target protein over control than to achieve complete solubilization (Supplementary Figure 2D). This allows us to use control IPs to successfully employ the SAINT algorithm to determine which proteins are confident PPI using a 5% FDR.

   How do we know protein are being immunoprecipitated from the synapse? As we show in Figures 2B and 3A, multiple proteins are annotated to the synapse with different databases, Gene ontology and SynGO. Well-known synaptic PPI were also observed, such as Grin2B-Dlg4(i.e. PSD-95), providing further evidence for proteins being immunoprecipitated for the synapses. Besides validating over a hundred published PPI interactions, we also identified many reciprocal interactions between the target datasets demonstrating the reproducibility of our protocol. Thus, we respectfully disagree with you and assert that our PPI network is very confident.

   The immunoprecipitation protocol is unusual in that the homogenates were incubated overnight (twice), which is a very long period compared to most published protocols. This is a concern because spurious protein interactions could form during this long incubation.

   There are many different immunoprecipitation protocols in the literature. The IP conditions depend upon the target protein and the antibody employed. Specifically, the abundance of the target and the affinity of the antibody to the target will dictate the IP conditions. We routinely perform overnight incubation for our IP-MS studies(Pankow, Bamberger et al. 2016, McClatchy, Yu et al. 2018). In our experience with brain tissue, this results in the highest enrichment of the target protein and the best reproducibility between biological replicates compared to IP protocols with shorter incubation times. Many other laboratories use overnight incubations(Lin and Lai 2017, Iqbal, Akins et al. 2018, Lagundzin, Krieger et al. 2022), so we do not consider our protocol unusual. We do find that IPs with tagged proteins in cell culture are more amenable to short incubation times. We have no evidence that overnight incubation causes spurious protein interactions nor could find any in the literature. Non-specific interactions are a concern with IP-MS experiments regardless of the incubation time. We took multiple steps to reduce the non-specific PPI from affecting our dataset. The first overnight incubation was incubating the brain lysate with agarose beads linked to IgGs to preclear the lysate from “sticky” non-specific interactors binding to IgGs and the beads. In addition, control IPs with IgG crosslinked to beads were incubated with brain lysate in parallel to each target IP. We computationally compared the non-specific control IPs with the target IPs using the SAINT algorithm to generate a confident list of PPI with a stringent 5% FDR. Therefore, our pipeline is specifically designed to prevent spurious PPI.

   In the section "Biological interpretation of scz PPI network". Surprisingly the authors found that synaptic proteins that are exclusively postsynaptic (Grin2B, SynGAP) or exclusively presynaptic (Syt1) show very high percentages of their interacting proteins are from the synaptic compartments where the target protein is not expressed. The authors offer no explanation for this paradox. One explanation for this could be that spurious PPIs have formed in the protein extraction/immunoprecipitation protocol. These findings need validation by biochemical fractionation of synapses into pre and post synaptic fractions and immunohistochemistry to demonstrate the subsynaptic localisation of the proteins. Grin2b is traditionally described as exclusively post-synaptic, but there is evidence for other localizations, including presynaptic(Berretta and Jones 1996, Sjostrom, Turrigiano et al. 2003, Bouvier, Larsen et al. 2018) and expression in astrocytes(Serrano, Robitaille et al. 2008, Lee, Ting et al. 2010, Lalo, Koh et al. 2021, Kim, Choi et al. 2024). Syngap has been localized to non-synaptic sites and glia expression in addition to its heavily studied role at the post synapse(Moon, Sakagami et al. 2008, Araki, Zeng et al. 2015, Birtele, Del Dosso et al. 2023). Syt1 is commonly used as a presynaptic marker, but along with other proteins previously reported to be exclusively presynaptic (such as SNAP-25), it has been localized to the postsynapse (Selak, Paternain et al. 2009, Tomasoni, Repetto et al. 2013, Hussain, Egbenya et al. 2017, Madrigal, Portales et al. 2019, Sumi and Harada 2023). Similarly, SynGo database assigns both post-synaptic and pre-synaptic localizations to Grin2b as stated in the manuscript. Thus, our data is not paradoxical, but supports the emerging evidence against the canonical exclusivity of the pre- and post-synaptic compartments. Determining subsynaptic localization of a protein is a huge undertaking and requires expertise we do not possess. This is why we relied on synaptic databases and the literature for our interpretation of our data, as other publications have done.

   We added the following to the Discussion to address this issue:

   “Using the SynGo database, 418 proteins (i.e. 41% of our network) were identified as synaptic proteins consistent with the targets having a synaptic localization. Defining the synaptic proteome is inherently difficult because the synapse is an “open organelle”, and many synaptic proteins also have non-synaptic localizations and are expressed in non-neuronal cells. We further attempted to define our synaptic PPI by differentiating between pre- and post- synaptic compartments via SynGo. Half of our targets were annotated to both compartments and all targets had PPI that were annotated to both. This data supports the emerging evidence against the canonical localization exclusivity of the pre and post synapse(Bouvier, Larsen et al. 2018, Madrigal, Portales et al. 2019).”

   My concerns about spurious interactions are raised again because the authors say that 92% of their interactions are novel (I note that they authors have not compared their interaction data of the NMDA receptor with published datasets from Dr Seth Grant's laboratory). BioGrid itself is good but not enough for comparison, maybe at this point it worth taking String, which accumulates several sources of PPIs, just select the direct PPIs.

   Since the MS-IP experiments in our study have never been performed before, we are not surprised by the extent of novel data we produced. As described above, we took many steps to prevent spurious PPI from entering our final dataset, including the use of detergents, preclearing and stringent bioinformatic filtering. Our entire dataset is very large, so the 8% of PPI that we replicated from other studies represents 124 interactions. We believe this to be an impressive number which correlates to the confidence of our data. Providing more confidence, we identified many reciprocal PPI where shared protein interactors between target proteins were identified in both target protein datasets.

       The PPI described for our targets in BioGrid encompassed 713 publications.  Two of the BioGrid datasets that were compared to our Grin2b PPI data were from the laboratory of Seth Grant.  Arbuckle et al (2010) is a low-throughout paper that describes a Grin2b and DLG4 PPI (that we also identified) and Husi et al (__2000__) is a seminal paper using high-throughput LC-MS to identify PPI in the PSD of mouse brain.  There were many differences between Husi et al and our pipeline.  Husi et al employed the C-terminal Grin2b peptide to pull down interactors from the PSD fraction whereas we employed Grin2b antibody to enrich Grin2b and its interactors from unfractionated brain tissue.  Despite these differences, our studies found 8 proteins in common. 
   

   We took your suggestion and compared our data to String which includes direct PPI and functional PPI. Our input was the high confidence PPI identified by SAINT with 5% FDR as with the BioGrid comparison. The PPI network for each target protein had a more significant enrichment (p We think the problem you suggest with SynGO is more of an inherent problem with characterizing the synaptic proteome. The synaptic proteome is difficult to define since it is an “open organelle” with proteins transporting in and out. In addition, most synaptic proteins, such as mitochondrial and translational proteins, also have non-synaptic localizations. It is not possible to isolate a contaminant-free “pure” synaptic preparation by biochemical fractionation. Recently, SynGO was used in a meta-analysis of previously published PSD datasets(Kaizuka, Hirouchi et al. 2024). Kaizuka et al. found 123 proteins identified in 20 PSD datasets. SynGo annotated proteins with post-synaptic localization from this list. To a lesser extent they also identified presynaptic localizations, but it is unclear if the presynaptic proteins are novel localizations. Kaizuka et al. continued the investigation and identified a novel PSD protein, thus demonstrating that our knowledge of pre- and post- synaptic proteomes is incomplete.

   Minor comments

   1. A number of papers have reported protein interactions of native NMDA receptor complexes and their associated proteins isolated from rodent brain and are neither referenced in this paper. It would be relevant to compare these published datasets with the Grin2B IP datasets.

   We employed BioGrid as a reference of reported PPI for each of our target proteins. For Grin2B, the PPI came from 142 different publications. For eight target proteins, we decided *BioGrid * was the best resource for determining the novelty of our PPI because it is routinely used for large-scale unbiased PPI analysis. To determine the novelty of our network, we compared our PPI network to 713 publications via BioGrid. We are unsure whether the papers you are referring to are included in the BioGrid database. To make it easier for readers with similar queries, we added an additional supplementary table (TableS4) including all the publications (i.e. PMID numbers) included in BioGrid comparison for each target protein.

   We amended the Results with the following sentence, so the readers realized the extensiveness of the Biogrid comparison analysis:

   “There were 713 publications in BioGrid that describe at least one interaction with one of our targets (Supplementary Table4).”

   The use of the term "bait" in purification experiments typically refers to a protein and not an antibody. I suggest removing the word bait to avoid ambiguity and simply use the word target. We took your suggestion and used “target” instead of “bait” to avoid ambiguity.

   26 mins of treatment gives completely different set of PPIs between PCP and saline which is very interesting, so both networks should be included in Supplementary. Also, it would be useful to have a list of modulated (phosphorylated in their case, but also ubiquitinated etc) proteins, which is not presented. Table S1 lists the PPI for each target, and we designated whether the interactors were for Sal, PCP, or both. Phosphorylated and ubiquitinated proteins are very hard to reproducibly identify without an additional enrichment step. Since we did not perform this enrichment step, we did not search for these modifications and do not have any modified proteins to report.

   As they say their final network is composed of "direct physical and "co-complex" interactors and they cannot distinguish between them. This is particularly bad for the postsynapse, where all the PSD components can be co-IP-ed in different combinations. It can explain the Figure 5C, where most of the proteins have FDR = 1, which means they do not reproduce. Figure 5C represents the intersection of 15N quantification and SAINT analysis. The x-axis is the FDR reported for SAINT analysis, and the y-axis is the significant proteins from the N15 analysis. This figure demonstrates that some proteins that were significantly different with PCP via N15 quantification also were annotated as PPI by SAINT (i.e. 5%. As stated in the Discussion, we concluded that the SAINT analysis and N15 quantitation are complementary in identifying PPI and that the quantification of a biological perturbation may aid the identification of PPI. Figure 5C is not related to whether our PPI are direct physical or "co-complex" interactors. Distinguishing between direct physical and co-complex interactors is an inherent problem for all IP studies. Since another reviewer also highlighted this deficit in our manuscript, we decided to analyze our PPI dataset with the artificial intelligence algorithm AlphaFold 3(AF3). The AF3 data is encompassed in Figure 6.

   The following AF3 data was added to the Results Section:

   *“A disadvantage of IP-MS studies is that it cannot distinguish between a PPI that binds directly to the target protein, and a PPI in which the interactor and target protein reside in the same multiprotein complex (i.e. indirect). We sought to predict which PPI may be directly interacting with its target protein by using the artificial intelligence algorithm AlphaFold3(AF3) (Abramson, Adler et al. 2024). First, we analyzed the predicted AF3 structure of the targets using the pTM score, and determined the fraction of each structure that was calculated to be disordered (Figure 6A and Supplementary Table7). Our reasoning was that if our targets have a poorly resolved structures then it will be difficult to screen for direct PPI. A pTM score >0.5 suggests that the structure may be correct, with the highest confidence equaling 1. Undefined or disordered regions hinder the accuracy of the prediction, and all our targets possessed a pTM score > 0.5 except Syt1. The fraction of disordered negatively correlated with the pTM score, as expected. Gsk3b, Ppp1ca, and Map2k1 were the target proteins with the highest pTM scores and were also the smallest of our targets (Figure 6B). Ppp1ca had the most confident structure (i.e. pTM 0.9) and the least fraction disordered (i.e. 0.07). Next, we determined the AF3 prediction of previously reported direct interactions of the targets. We used the iPTM score to determine an interaction confidence. An iPTM score >0.8 is a highly confident direct interaction, whereas 0.8. These eight PPI have all previously been reported to form a direct interaction with Ppp1ca, except Phactr3 (Zhang, Zhang et al. 1998, Terrak, Kerff et al. 2004, Hurley, Yang et al. 2007, Marsh, Dancheck et al. 2010, Ragusa, Dancheck et al. 2010, Ferrar, Chamousset et al. 2012, Choy, Srivastava et al. 2024, Xu, Sadleir et al. 2024). Phactr3 is structurally similar to, but less studied than, the reported direct interactor, Phactr1. These interactors are all inhibitors of PP1 except for Ppp1r9b which targets Ppp1ca to specific subcellular compartments. Nine PPI were assigned a score The following AF3 interpretation was added to the Discussion:

   “Our SCZ PPI network consists of two types of PPI: direct physical interactions and “co-complex” or indirect interactions. Typically, the nature of the interaction cannot be distinguished in IP-MS studies. We decided to employ the new AF3 algorithm to screen the PPI of Ppp1ca to provide evidence for direct interactors. We chose to examine the PPI assigned to Ppp1ca, because its structure was the most confident among our target proteins and AF3 correctly predicted a known direct interactor with high confidence. Ppp1ca is a catalytic subunit of the phosphatase PP1, which is required to associate with regulatory subunits to create holoenzymes (Li, Wilmanns et al. 2013). Eighteen PPI were predicted to be directly interacting with Ppp1ca using a 0.6 or higher iPTM filter. This filter may be too conservative and may generate false negatives, because another study employed a 0.3 filter followed by additional interrogation to screen for direct PPI (Weeratunga, Gormal et al. 2024). Forty-four percent of these predictions were confirmed by previous publications. Most of these validated direct interactions are inhibitors of the phosphatase, but one, Ppp1r9b (aka spinophilin), is known to target Ppp1ca to dendritic spines (Allen, Ouimet et al. 1997, Salek, Claeboe et al. 2023). This high correlation with the literature provides substantial confidence to the novel PPI predicted to be direct Ppp1ca interactors. The AF3 screen predicted that NDRG2 directly interacts with Ppp1ca. This protein is known to regulate many phosphorylation dependent signaling pathways by directly interacting with other phosphatases including Pp1ma and PP2A (Feng, Zhou et al. 2022, Lee, Lim et al. 2022). Actin binding protein Capza1 was also predicted to directly interact with Ppp1ca and Ppp1ca interacts with actin and its binding proteins to maintain optimal localization for efficient activity to specific substrates (Foley, Ward et al. 2023). Hsp1e is a heat shock protein predicted to directly interact with Ppp1ca. Although there is no direct connection to Ppp1ca, other heat shock proteins have been reported to regulate Ppp1ca (Mivechi, Trainor et al. 1993, Flores-Delgado, Liu et al. 2007, Qian, Vafiadaki et al. 2011). We also observed that many of the direct PPI were altered with PCP treatment. One direct interactor, Ppp1r1b (aka DARPP-32), is phosphorylated at Thr34 by PKA in the brain upon PCP treatment. This phosphorylation event converts Ppp1rb to a potent inhibitor of Ppp1ca(Svenningsson, Tzavara et al. 2003). Importantly, the manipulation of Thr34 attenuated the behavioral effects of PCP. Consistent with this report, Ppp1r1b-Ppp1ca interaction was only observed with PCP in our study. Further investigation is needed to determine if our novel direct interactors regulate the PCP phenotype. We conclude that AF3 can provide important structural insights into the nature of PPI obtained from large scale IP-MS studies.”

   The way PPI data is reported can be improved so that I does not have to be extracted from Table 1 and 2. It would be good if they provide just two columns PPI list, with names or IDs, plus PSP/saline/both conditions in third column, for ease of comparison with other sources and building the graph. They can add it as another spreadsheet to Table 2. We generated this table (TableS2) as you requested.

   Is Figure 2 built for Sal or PCP conditions? as they have only 23% interactions in common (Figure 4A) the Figure 2 should be pretty different for two conditions. Are the 1007 interactors combined from SAL and PCP?

   Figure 2 contains ALL the unique PPI for each target regardless of Sal or PCP conditions. The 1007 protein interactors shown in Figure 2Awhere Sal and PCP were combined to generate a non-redundant list of proteins for each target.

   We amended the Results to make this clearer:

   “When the PCP and SAL datasets were combined, there were 1007 unique proteins.”

   This sentence was added to Figure 2A:

   “For this comparison, Sal and PCP PPI were combined into a unique PPI list for each target.”

   Figure 1F is mentioned but no figure is shown. We apologize for this oversight, and we have corrected the manuscript.

   1. Overall the paper could be edited and made more concise, especially the introduction and discussion. We extensively edited the manuscript to be more concise.

   Reviewer #3 (Significance (Required)):

   General assessment

   Proteomic mass spectrometry of immunoprecipitated complexes from synapses has been extensively studied since Husi et al (2000) first study of NMDA receptor and AMPA receptor complexes. Since then, a wide variety of methods have been employed to purify synaptic protein complexes including peptide affinity, tandem-affinity purification of endogenous proteins tagged with FLAG and Histine-affinity tags amongst other methods. Purification of protein complexes and the postsynaptic density from the postsynaptic terminal of mammalian excitatory synapses have been crucial for establishing that schizophrenia is a polygenic disorder affecting synapses (e.g. Fernandez et al, 2009; Kirov et al, 2012; Purcell et al, 2014, Fromer et al, 2014 etc). Network analyses of the postsynaptic proteome have described networks of schizophrenia interacting proteins (e.g. Pocklington et al, 2006; Fernandez et al, 2009) and other neuropsychiatric disorders.

   Hundreds of synaptic protein complexes have been identified (Frank et al, 2016), but very few have been characterised using proteomic mass spectrometry. This paper has chosen 8 protein targets for such analysis and identified many proteins that a putative interactors of the target protein. At this level the current manuscript does not represent a conceptual advance and the value of the data lies in its utility as a resource that may be used in future studies.

   The findings from the 8 target proteins from normal adult rat brain were used for a secondary study that describes the effects that PCP has on the interaction networks. Interestingly, this work shows that 26 minutes of drug treatment leads to considerable changes in the interactomes of the target proteins. These descriptive data could be used in future studies to understand the cell biological mechanisms that mediate these rapid changes in the proteome. PCP and drugs that interact with NMDA receptors are known to induce changes in synaptic proteome phosphorylation including modifications in protein-protein interaction sites, which may explain the PCP effects.

   The study would benefit from validation of experimental protocols for solubilisation and immunoprecipitation and validation of described interactions using orthogonal biochemical or localisation experiments.

   Audience Specialists in synapse proteins and mechanisms of schizophrenia.

   Expertise

   The reviewers' expertise is in molecular biology of synapses including synapse proteomics, protein interaction and network analysis, and genetics of schizophrenia and other brain disorders.

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   Referee #3

   Evidence, reproducibility and clarity

   Summary

   It is now widely accepted that schizophrenia is polygenic disorder in which a large fraction of the genetic risk is in variants affecting the expression of synaptic proteins. Moreover, it is known that these synaptic proteins are found in multiprotein complexes and that many proteins encoded by schizophrenia risk genes interact directly or indirectly in these complexes. It is also known that some drugs including phencyclidine, which binds to NMDA receptors and to Dopamine D2 receptors (not mentioned by the authors) can induce schizophreniform psychosis. The authors have set out to advance on this position by performing …

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   Referee #3

   Evidence, reproducibility and clarity

   Summary

   It is now widely accepted that schizophrenia is polygenic disorder in which a large fraction of the genetic risk is in variants affecting the expression of synaptic proteins. Moreover, it is known that these synaptic proteins are found in multiprotein complexes and that many proteins encoded by schizophrenia risk genes interact directly or indirectly in these complexes. It is also known that some drugs including phencyclidine, which binds to NMDA receptors and to Dopamine D2 receptors (not mentioned by the authors) can induce schizophreniform psychosis. The authors have set out to advance on this position by performing proteomic mass spectrometry studies on proteins identified as encoded by schizophrenia risk genes. They target 8 proteins for immunoprecipitation from rat brain and identify coisolated proteins and perform various network analyses. In the most interesting part of the paper they ask if PCP-treatment altered protein interactions and report various changes.

   Major comments:

   1. Choice of target proteins. It was not until the first paragraph of the results section that the authors first name the 8 synaptic proteins that have chosen to study. This information should be in the abstract. The authors then use figure 1A and 1B as evidence that these 8 "baits" are schizophrenia-relevant proteins. Figure 1A does not provide any evidence at all and Figure 1B is about as weak a line of evidence imaginable - a histogram of the number of papers that have the search term "schizophrenia" and the protein name. I tried this search for Grin2B and almost immediately found papers that reported no association between Grin2B and schizophrenia (e.g. PMID: 33237434). Figure 1B should be scrapped. The remaining part of paragraph 1 of the results does not provide an adequate, let alone systematic, justification for the use of the 8 baits. It would be appropriate to construct a table with the 8 proteins and cite relevant papers and identify the basis for why they are implicated in schizophrenia (is it a direct mutation or some other evidence?). What makes these 8 proteins better than many others that are cited as synaptic schizophrenia relevant proteins?
   2. The methods of protein extraction are particularly concerning. The postsynaptic density of excitatory synapses (which contains several of the target proteins in this study) has been notoriously difficult to solubilise unless one uses high pH (9) and harsh detergent extraction (1% deoxycholate). The authors use pH 7 and weak detergent conditions, which are likely to be inefficient for solubilising at least several of the target proteins. Nowhere do the authors report how much of the total of their target protein is being solubilised. Indeed, there are no figures showing biochemical conditions at all. What if only a small percentage of the target protein is being immunoprecipitated - what does this mean for the interaction data? How do we know if the fraction being immunoprecipitated is from the synapse? (why did they not use synaptosomes). The absence of this kind of data undermines the reader's confidence in the findings.
   3. The immunoprecipitation protocol is unusual in that the homogenates were incubated overnight (twice), which is a very long period compared to most published protocols. This is a concern because spurious protein interactions could form during this long incubation.
   4. In the section "Biological interpretation of scz PPI network". Surprisingly the authors found that synaptic proteins that are exclusively postsynaptic (Grin2B, SynGAP) or exclusively presynaptic (Syt1) show very high percentages of their interacting proteins are from the synaptic compartments where the target protein is not expressed. The authors offer no explanation for this paradox. One explanation for this could be that spurious PPIs have formed in the protein extraction/immunoprecipitation protocol. These findings need validation by biochemical fractionation of synapses into pre and post synaptic fractions and immunohistochemistry to demonstrate the subsynaptic localisation of the proteins.
   5. My concerns about spurious interactions are raised again because the authors say that 92% of their interactions are novel (I note that they authors have not compared their interaction data of the NMDA receptor with published datasets from Dr Seth Grant's laboratory). BioGrid itself is good but not enough for comparison, maybe at this point it worth taking String, which accumulates several sources of PPIs, just select the direct PPIs.
   6. A major concern is that they use SynGO as a reference database, and even test the enrichment against it. SynGO is about ~ 2000 genes in size and was built around the presynaptic datasets, so it is biased and incomplete in terms of the whole synapse. This may be one of the reasons there is the strangely high percentage of presynaptic proteins interacting with postsynaptic proteins as noted above.

   Minor comments

   1. A number of papers have reported protein interactions of native NMDA receptor complexes and their associated proteins isolated from rodent brain and are neither referenced in this paper. It would be relevant to compare these published datasets with the Grin2B IP datasets.
   2. The use of the term "bait" in purification experiments typically refers to a protein and not an antibody. I suggest removing the word bait to avoid ambiguity and simply use the word target.
   3. 26 mins of treatment gives completely different set of PPIs between PCP and saline which is very interesting, so both networks should be included in Supplementary. Also, it would be useful to have a list of modulated (phosphorylated in their case, but also ubiquitinated etc) proteins, which is not presented.
   4. As they say their final network is composed of "direct physical and "co-complex" interactors and they cannot distinguish between them. This is particularly bad for the postsynapse, where all the PSD components can be co-IP-ed in different combinations. It can explain the Figure 5C, where most of the proteins have FDR = 1, which means they do not reproduce.
   5. The way PPI data is reported can be improved so that I does not have to be extracted from Table 1 and 2. It would be good if they provide just two columns PPI list, with names or IDs, plus PSP/saline/both conditions in third column, for ease of comparison with other sources and building the graph. They can add it as another spreadsheet to Table 2.
   6. Is Figure 2 built for Sal or PCP conditions? as they have only 23% interactions in common (Figure 4A) the Figure 2 should be pretty different for two conditions. Are the 1007 interactors combined from SAL and PCP?
   7. Figure 1F is mentioned but no figure is shown.
   8. Overall the paper could be edited and made more concise, especially the introduction and discussion.

   Significance

   General assessment

   Proteomic mass spectrometry of immunoprecipitated complexes from synapses has been extensively studied since Husi et al (2000) first study of NMDA receptor and AMPA receptor complexes. Since then, a wide variety of methods have been employed to purify synaptic protein complexes including peptide affinity, tandem-affinity purification of endogenous proteins tagged with FLAG and Histine-affinity tags amongst other methods. Purification of protein complexes and the postsynaptic density from the postsynaptic terminal of mammalian excitatory synapses have been crucial for establishing that schizophrenia is a polygenic disorder affecting synapses (e.g. Fernandez et al, 2009; Kirov et al, 2012; Purcell et al, 2014, Fromer et al, 2014 etc). Network analyses of the postsynaptic proteome have described networks of schizophrenia interacting proteins (e.g. Pocklington et al, 2006; Fernandez et al, 2009) and other neuropsychiatric disorders.

   Hundreds of synaptic protein complexes have been identified (Frank et al, 2016), but very few have been characterised using proteomic mass spectrometry. This paper has chosen 8 protein targets for such analysis and identified many proteins that a putative interactors of the target protein. At this level the current manuscript does not represent a conceptual advance and the value of the data lies in its utility as a resource that may be used in future studies.

   The findings from the 8 target proteins from normal adult rat brain were used for a secondary study that describes the effects that PCP has on the interaction networks. Interestingly, this work shows that 26 minutes of drug treatment leads to considerable changes in the interactomes of the target proteins. These descriptive data could be used in future studies to understand the cell biological mechanisms that mediate these rapid changes in the proteome. PCP and drugs that interact with NMDA receptors are known to induce changes in synaptic proteome phosphorylation including modifications in protein-protein interaction sites, which may explain the PCP effects.

   The study would benefit from validation of experimental protocols for solubilisation and immunoprecipitation and validation of described interactions using orthogonal biochemical or localisation experiments.

   Audience

   Specialists in synapse proteins and mechanisms of schizophrenia.

   Expertise

   The reviewers' expertise is in molecular biology of synapses including synapse proteomics, protein interaction and network analysis, and genetics of schizophrenia and other brain disorders.

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   Referee #2

   Evidence, reproducibility and clarity

   Summary: McClatchy, Powell and Yates aimed at identifying a protein interactome associated to schizophrenia. For that, they treated rats (N14 and N15) with PCP, which disturbs gutamatergic transmission, as a model for the disease and co-immunoprecipitated hippocampi proteins, which were further analyzed by standard LC-MS.

   The study is new, considering not much has been done in this direction in the field of schizophrenia. This justifies its publication. On the other hand, a major flaw of the is the lack of information on the level of interaction of the so called protein interactome. Meaning, we cannot distinguish, as the study was …

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   Referee #2

   Evidence, reproducibility and clarity

   Summary: McClatchy, Powell and Yates aimed at identifying a protein interactome associated to schizophrenia. For that, they treated rats (N14 and N15) with PCP, which disturbs gutamatergic transmission, as a model for the disease and co-immunoprecipitated hippocampi proteins, which were further analyzed by standard LC-MS.

   The study is new, considering not much has been done in this direction in the field of schizophrenia. This justifies its publication. On the other hand, a major flaw of the is the lack of information on the level of interaction of the so called protein interactome. Meaning, we cannot distinguish, as the study was performed, which proteins are directly interacting with the targets of interest from proteins which are interacting with targets´ interactors. The different shells of interaction are crucial information in protein interactomics.

   Major: most of I am pointing below must be at least discussed or better presented in the paper, as It may not be solvable considering how the study has been conducted.

   1. The study fails in defining the level of interaction of the protein interactome with the considered targets. This has been shortly mentioned in the discussion, but must be more explicit to readers, for instance, in the abstract, introduction and in the methods sections.
   2. Considering the protein extraction protocol, it is fair to mention that only the most soluble proteins are being considered here. I am bringing this up since the importance of membrane receptors is clear in the studied context.
   3. It is not clear from the methods description if antibodies from all 8 targets were all together in one Co-IP or have been incubated separately in 8 different hippocampi samples. It seems the first, given how results have been presented. If so, this maximizes the major issue raised above (in 1).
   4. Definitely, results here are not representing a "SCZ PPI network". PCP-treated animals, as any other animal model, are rather limited models to schizophrenia. As a complex multifactorial disease, synaptic deficits, which is the focus of this study, can no longer be considered "the pivot" of the disease. Synaptic dysfunction is only one among many other factors associated to schizophrenia.
   5. Authors should look for protein interactions that might be happening also in glial cells. They are not the majority in hippocampus, but are present in the type of tissue analyzed here. Thus, some of the interactions observed might be more abundantly present in those cells. Maybe enriching using bioinformatics tools the PPI network to different cell types.

   Minor:

   1. in the abstract, it is not clear if 90% of the PPI are novel to brain tissue in general or specifically schizophrenia.
   2. authors refer to LC-MS-based proteomics as "MS" all across the text. Who am I to say this to Yates et al, but I think it is rather simplified use "Mass Spectrometry Analysis", when this is a typical LC-MS type of analysis
   3. Several references used to construct the hypothesis of the paper are rather outdated: several from 10-15 years ago. It would be interesting to provide to the reader up to date references, given the rapid pace science has been progressing.
   4. "UniProt rat database". Please, state the version and if reviewed or unreviewed.

   Significance

   The study is informative, and has great potential to enrich the specific literature of this field. But should tone down some arguments, given the experimental limitations of the PPI network (as described above) and should state PCP-treated rats as a limited model to schizophrenia.

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   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

   In this manuscript, McClatchy and colleagues used a conventional approach combining immunoprecipitation (IP) of endogenous target proteins (baits) followed by liquid chromatography mass spectrometry (MS) analysis of the co-immunoprecipitating proteins to map protein-protein interaction (PPI). This interaction network is centered around baits that had been annotated as susceptibility factors for schizophrenia (SCZ). A variety of previous studies have identified thousands of such SCZ susceptibility …

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   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

   In this manuscript, McClatchy and colleagues used a conventional approach combining immunoprecipitation (IP) of endogenous target proteins (baits) followed by liquid chromatography mass spectrometry (MS) analysis of the co-immunoprecipitating proteins to map protein-protein interaction (PPI). This interaction network is centered around baits that had been annotated as susceptibility factors for schizophrenia (SCZ). A variety of previous studies have identified thousands of such SCZ susceptibility factors. Mostly based on the availability of antibodies, 8 bait proteins were selected in this study. The authors reasoned that immunoprecipitating endogenous proteins from tissues using specific antibodies was a more accurate view of physiological conditions than epitope tagging followed by affinity purification (AP) from cells in culture. The model system from which proteins were extracted was the hippocampus dissected from mice that had been treated or not by phencyclidine (PCP), a drug that has been shown to induce SCZ symptoms in humans and animals. By comparing the proteins identified and quantified from the PCP-treated samples against control IPs and/or saline-injected mouse controls, a large number of PPI were deemed statistically significant. Most of these potential interactors were not present in PPI databases (BioGRID), most likely because such databases are populated with large-scale APMS datasets from cell cultures, with very few studies using brain tissue. Strikingly, many of the co-immunoprecipitated proteins were also known as SCZ susceptibility factors, which lend weight to the hypothesis that these factors form a large protein interaction network, localized at the synapses.

   Major comments:

   • Are the key conclusions convincing?

   Overall, the conclusions drawn from the experimental design, data analysis, and corroboration with existing literature are well-supported and convincing. When selecting the SCZ susceptibility factors, the authors clearly state their goal, the databases used for gene selection, and the rationale for choosing proteins with synaptic localization. The inclusion of evidence from genetic studies and previous publications strengthens the credibility of the selected genes. The methodology used to establish the novel SCZ PPI network is mostly well-described (see minor comments below). The use of an 15N internal standard also adds rigor to the quantitation of PPI. The GO enrichment analysis provides valuable insights into the biological functions and cellular components associated with the SCZ PPI network. The annotation of identified proteins using the SynGo synaptic database and the distribution of annotated synaptic proteins among different baits further support the biological relevance of this PPI network. The cross-referencing of the PPI network with published genetic studies on SCZ susceptibility genes adds robustness to the findings. Specifically, the observation that 68% of protein interactors have evidence of being potential SCZ risk factors is a strong corroboration of the prevailing hypothesis in the field. Finally, the significant changes induced by PCP that were identified for all baits except Syt1, along with the comparison of altered proteins with SAINT-identified PPI, add depth to the understanding of PCP modulation.

   • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

   No, but note that APMS/IPMS has been around for more than a decade (Introduction page 3).

   • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

   One piece of data that is missing are Western blots using the 8 selected antibodies against the proteins extracted from their experimental samples to validate the antibodies recognize 1 protein of the expected size from these tissue extracts.

   • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

   Running SDS-PAGE and Western blotting should be straightforward and cheap.

   • Are the data and the methods presented in such a way that they can be reproduced?

   Yes

   • Are the experiments adequately replicated and statistical analysis adequate?

   Yes

   Minor comments:

   • Specific experimental issues that are easily addressable.

   The rationale for the short duration between PCP injection and animal sacrifice is only explained in the discussion section (page 17). The fact that this short treatment of less than 30 min should prevent any change in transcription or translation should be introduced earlier (in the experimental procedures). Note that the duration is written as 26 min on page 4 and 25 min on page 9. Please reconcile these numbers. Is there any biological significance for this SCZ study that the mice were maintained on a reverse day-night cycle? It is not clear from reading Experimental Procedures/Bioinformatic Analysis section (page 6) if normalized N14/N15 protein ratios measured in the bait-IPs and control-IPs were used for the SAINT analysis? Or did the authors used label-free quantitation with spectral counts?

   • Are prior studies referenced appropriately?

   Yes

   • Are the text and figures clear and accurate?

   Fig1C: The workflow is a little too simple, the authors might want to add more details. FigS1C: Please add x-axis title (spectral counts) directly to the figure. Fig2B-D: The color scale bar should have number values to denote lower and upper limits in % (as opposed to "lowest" and "highest").

   • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

   No

   Significance

   • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

   In this study, the authors have drastically expanded the protein interaction landscape around 8 known SCZ susceptibility factors by using a conventional IPMS approach. Performing the IPs on protein extracted from hippocampus dissected from mice treated with phencyclidine to model SCZ increases the biological significance of such lists of proteins. Furthermore, the co-immunoprecipitation of many other SCZ susceptibility factors along with the 8 selected baits supports the hypothesis that these proteins of varied functions are part of large interaction networks. Overall, the integration of experimental data with in silico networks, along with the quantification of PPI changes in response to PCP, should contribute to a more nuanced understanding of SCZ pathogenesis. The potential implications for drug development underscore the broader significance of the study in advancing our knowledge of neurobiology and its relevance to neurological disorders like schizophrenia.

   • Place the work in the context of the existing literature (provide references, where appropriate).

   Overall, this study contributes to the existing literature by providing experimental data on in vivo PPI networks related to SCZ risk factors. Not only do the authors validate 124 known interactions but also they identify many novel PPI, due to a gap in the existing literature regarding the comprehensive mapping of PPI directly from tissue extracts, especially brain tissue. The authors advocate for more IPMS studies in mammalian tissues to generate robust tissue-specific in silico networks, which agrees with the growing understanding of the importance of tissue-specific networks for identifying disease mechanisms and potential drug targets.

   Furthermore, the SCZ PPI network reported here is enriched in proteins previously associated with SCZ, which aligns with the existing literature emphasizing the involvement of certain proteins and pathways in the pathogenesis of SCZ [References: 78-85]. The authors also investigate the response of the SCZ network to PCP treatment, hence providing insights into the potential effects of post-translational modifications, protein trafficking, and PPI alterations in a model of schizophrenia, which adds to existing knowledge about the impact of PCP on the molecular processes associated with SCZ [References: 88, 89, 92].

   • State what audience might be interested in and influenced by the reported findings.

   Overall, the findings reported in this manuscript have implications for both basic research in molecular biology and potential translational applications in the development of targeted therapies for neurological disorders, particularly schizophrenia. The study delves into in vivo protein-protein interaction (PPI) networks related to genes implicated in schizophrenia (SCZ) risk factors. Researchers in neuroscience, molecular biology, and psychiatry would find the information valuable for understanding the molecular basis of SCZ. The study highlights the potential for identifying disease "hubs" that could be drug targets. Pharmacologists and drug developers interested in targeting protein complexes for drug development, especially in the context of neurological disorders, may find the study relevant.

   • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

   Technical Expertise | biochemistry, liquid chromatography mass spectrometry, proteomics, computational biology, protein engineering, protein interaction networks, post-translational modifications, protein crosslinking, proximity labeling, limited proteolysis, thermal shift assay, label-free and isotope-labeled quantitation. Biological Applications | human transcriptional complexes, apicomplexan parasites, viruses, nuclear envelope, ubiquitin ligases, non-model organisms.

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5. 

   Version published to 10.1101/2023.12.12.571320v1 on bioRxiv

   Dec 13, 2023