Synaptic protein interaction networks encode experience by assuming stimulus-specific and brain-region-specific states.

A core network of widely expressed proteins within the glutamatergic post-synapse mediates activity-dependent synaptic plasticity throughout the brain, but the specific proteomic composition of synapses differs between brain regions. Here, we address the question, how does proteomic composition affect activity-dependent protein-protein interaction networks (PINs) downstream of synaptic activity? Using quantitative multiplex co-immunoprecipitation, we compare the PIN response of in vivo or ex vivo neurons derived from different brain regions to activation by different agonists or different forms of eyeblink conditioning. We report that PINs discriminate between incoming stimuli using differential kinetics of overlapping and non-overlapping PIN parameters. Further, these "molecular logic rules" differ by brain region. We conclude that although the PIN of the glutamatergic post-synapse is expressed widely throughout the brain, its activity-dependent dynamics show remarkable stimulus-specific and brain-region-specific diversity. This diversity may help explain the challenges in developing molecule-specific drug therapies for neurological disorders.

A Repurposed Drug Screen Identifies Compounds That Inhibit the Binding of the COVID-19 Spike Protein to ACE2.

Repurposed drugs that block the interaction between the SARS-CoV-2 spike protein and its receptor ACE2 could offer a rapid route to novel COVID-19 treatments or prophylactics. Here, we screened 2,701 compounds from a commercial library of drugs approved by international regulatory agencies for their ability to inhibit the binding of recombinant, trimeric SARS-CoV-2 spike protein to recombinant human ACE2. We identified 56 compounds that inhibited binding in a concentration-dependent manner, measured the IC50 of binding inhibition, and computationally modeled the docking of the best inhibitors to the Spike-ACE2 binding interface. The best candidates were Thiostrepton, Oxytocin, Nilotinib, and Hydroxycamptothecin with IC50's in the 4-9 µM range. These results highlight an effective screening approach to identify compounds capable of disrupting the Spike-ACE2 interaction, as well as identify several potential inhibitors of the Spike-ACE2 interaction.

Remodeling of the Homer-Shank interactome mediates homeostatic plasticity.

Neurons maintain stable levels of excitability using homeostatic synaptic scaling, which adjusts the strength of a neuron's postsynaptic inputs to compensate for extended changes in overall activity. Here, we investigated whether prolonged changes in activity affect network-level protein interactions at the synapse. We assessed a glutamatergic synapse protein interaction network (PIN) composed of 380 binary associations among 21 protein members in mouse neurons. Manipulating the activation of cultured mouse cortical neurons induced widespread bidirectional PIN alterations that reflected rapid rearrangements of glutamate receptor associations involving synaptic scaffold remodeling. Sensory deprivation of the barrel cortex in live mice (by whisker trimming) caused specific PIN rearrangements, including changes in the association between the glutamate receptor mGluR5 and the kinase Fyn. These observations are consistent with emerging models of experience-dependent plasticity involving multiple types of homeostatic responses. However, mice lacking Homer1 or Shank3B did not undergo normal PIN rearrangements, suggesting that the proteins encoded by these autism spectrum disorder-linked genes serve as structural hubs for synaptic homeostasis. Our approach demonstrates how changes in the protein content of synapses during homeostatic plasticity translate into functional PIN alterations that mediate changes in neuron excitability.

A repurposed drug screen identifies compounds that inhibit the binding of the COVID-19 spike protein to ACE2.

Repurposed drugs that block the interaction between the SARS-CoV-2 spike protein and its receptor ACE2 could offer a rapid route to novel COVID-19 treatments or prophylactics. Here, we screened 2701 compounds from a commercial library of drugs approved by international regulatory agencies for their ability to inhibit the binding of recombinant, trimeric SARS-CoV-2 spike protein to recombinant human ACE2. We identified 56 compounds that inhibited binding by <90%, measured the EC 50 of binding inhibition, and computationally modeled the docking of the best inhibitors to both Spike and ACE2. These results highlight an effective screening approach to identify compounds capable of disrupting the Spike-ACE2 interaction as well as identifying several potential inhibitors that could serve as templates for future drug discovery efforts.

Plasma From Recovered COVID-19 Patients Inhibits Spike Protein Binding to ACE2 in a Microsphere-Based Inhibition Assay.

We present a microsphere-based flow cytometry assay that quantifies the ability of plasma to inhibit the binding of spike protein to angiotensin-converting enzyme 2. Plasma from 22 patients who had recovered from mild coronavirus disease 2019 (COVID-19) and expressed anti-spike protein trimer immunoglobulin G inhibited angiotensin-converting enzyme 2-spike protein binding to a greater degree than controls. The degree of inhibition was correlated with anti-spike protein immunoglobulin G levels, neutralizing titers in a pseudotyped lentiviral assay, and the presence of fever during illness. This inhibition assay may be broadly useful to quantify the functional antibody response of patients recovered from COVID-19 or vaccine recipients in a cell-free assay system.

Plasma from recovered COVID19 subjects inhibits spike protein binding to ACE2 in a microsphere-based inhibition assay.

High throughput serological tests that can establish the presence and functional activity of anti-SARS-COV2 antibodies are urgently needed. Here we present microsphere-based Flow Cytometry assays that quantify both anti-spike IgGs in plasma, and the ability of plasma to inhibit the binding of spike protein to angiotensin converting enzyme 2 (ACE2). First, we detected anti-trimer IgGs in 22/24 and anti-RBD IgGs in 21/24 COVID+ subjects at a median of 36 (range 14-73) days following documented SARS-CoV-2 RNA (+) secretions. Next, we find that plasma from all 22/24 subjects with anti-trimer IgGs inhibited ACE2-trimer binding to a greater degree than controls, and that the degree of inhibition correlated with anti-trimer IgG levels. Depletion of trimer-reactive Igs from plasma reduced ACE2-trimer inhibitory capacity to a greater degree than depletion of RBD-reactive Igs, suggesting that inhibitory antibodies act by binding both within and outside of the RBD. Amongst the 24 subjects, presence of fever was associated with higher levels of anti-trimer IgG and inhibition of binding to human ACE2. This inhibition assay may be broadly useful to quantify the functional antibody response of recovered COVID19 patients or vaccine recipients in a cell-free assay system.

Activity-dependent changes in synaptic protein complex composition are consistent in different detergents despite differential solubility.

At the post-synaptic density (PSD), large protein complexes dynamically form and dissociate in response to synaptic activity, comprising the biophysical basis for learning and memory. The use of detergents to isolate the PSD and release its membrane-associated proteins complicates studies of these activity-dependent protein interaction networks, because detergents can simultaneously disrupt the very interactions under study. Despite widespread recognition that different detergents yield different experimental results, the effect of detergent on activity-dependent synaptic protein complexes has not been rigorously examined. Here, we characterize the effect of three detergents commonly used to study synaptic proteins on activity-dependent protein interactions. We first demonstrate that SynGAP-containing interactions are more abundant in 1% Deoxycholate (DOC), while Shank-, Homer- and mGluR5-containing interactions are more abundant in 1% NP-40 or Triton. All interactions were detected preferentially in high molecular weight complexes generated by size exclusion chromatography, although the detergent-specific abundance of proteins in high molecular weight fractions did not correlate with the abundance of detected interactions. Activity-dependent changes in protein complexes were consistent across detergent types, suggesting that detergents do not isolate distinct protein pools with unique behaviors. However, detection of activity-dependent changes is more or less feasible in different detergents due to baseline solubility. Collectively, our results demonstrate that detergents affect the solubility of individual proteins, but activity-dependent changes in protein interactions, when detectable, are consistent across detergent types.

Clustering the autisms using glutamate synapse protein interaction networks from cortical and hippocampal tissue of seven mouse models.

Background: Autism spectrum disorders (ASDs) are a heterogeneous group of behaviorally defined disorders and are associated with hundreds of rare genetic mutations and several environmental risk factors. Mouse models of specific risk factors have been successful in identifying molecular mechanisms associated with a given factor. However, comparisons among different models to elucidate underlying common pathways or to define clusters of biologically relevant disease subtypes have been complicated by different methodological approaches or different brain regions examined by the labs that developed each model. Here, we use a novel proteomic technique, quantitative multiplex co-immunoprecipitation or QMI, to make a series of identical measurements of a synaptic protein interaction network in seven different animal models. We aim to identify molecular disruptions that are common to multiple models. Methods: QMI was performed on 92 hippocampal and cortical samples taken from seven mouse models of ASD: Shank3B, Shank3Δex4-9, Ube3a2xTG, TSC2, FMR1, and CNTNAP2 mutants, as well as E12.5 VPA (maternal valproic acid injection on day 12.5 post-conception). The QMI panel targeted a network of 16 interacting, ASD-linked, synaptic proteins, probing 240 potential co-associations. A custom non-parametric statistical test was used to call significant differences between ASD models and littermate controls, and Hierarchical Clustering by Principal Components was used to cluster the models using mean log2 fold change values. Results: Each model displayed a unique set of disrupted interactions, but some interactions were disrupted in multiple models. These tended to be interactions that are known to change with synaptic activity. Clustering revealed potential relationships among models and suggested deficits in AKT signaling in Ube3a2xTG mice, which were confirmed by phospho-western blots. Conclusions: These data highlight the great heterogeneity among models, but suggest that high-dimensional measures of a synaptic protein network may allow differentiation of subtypes of ASD with shared molecular pathology.