A Microbead-Based Multiplex Immunoassay to Measure Dynamic Protein Interaciton Networks at the Glutamate Synapse

Background: Protein-protein interaction (PPI) networks are thought to represent a system with emergent network properties, integrating signals from a variety of inputs into coordinated responses. At the glutamatergic synapse, interacting networks of proteins, consisting of structural proteins, membrane channels, kinases, ubiquitin ligases and regulatory proteins influence action potential generation and cell fate decisions, including synapse development, maintenance and elimination. Many of these proteins are associated with autism, leading to the current “synaptic hypothesis of autism”.  Current technology allows the detailed study of individual members of this synaptic interaction network, but network-scale activity of protein-protein interactions, both at rest and following signaling, is incompletely understood.

Objectives: To develop a technique to quantitatively measure protein-protein interactions at the network level, and to quantify how these networks change in response to biological signaling events and autism-linked gene mutations.

Methods: We have developed a technique called multiplex immunoprecipitation detected by flow cytometry (mIP-FCM).  In mIP-FCM, protein complexes are immunoprecipitated onto Luminex microspheres conjugated to antibodies specific for 20+ different proteins, then probed with fluorophore-conjugated antibodies to the same protein set and read on a flow cytometer. In total, 400+ protein-protein interactions are assessed, producing a network-scale analysis of protein interactome dynamics. Our group uses mIP-FCM to study the signaling networks engaged in response to agonist vs. antagonist stimuli of either the T cell immunologic synapse or the neuronal glutamatergic synapse.  Here, we present a new mIP-FCM array focused on autism-linked proteins from the glutamate synapse.

Results: Using the well-characterized T cell immunologic synapse that forms between a T cell and an antigen presenting cell as a model system, mIP-FCM is capable of assessing 400+ dynamic interactions during a T cell signaling event.  We have developed high-sensitivity statistical methods capable of identifying small changes in protein interactions, and visualization methods to make sense of the vast amount of data collected (~7000+ data points per timepoint).  Analysis of proteins from the T cell synapse has revealed new network protein interaction patterns that could be decisive in agonist vs. antagonist signal transduction. In our new mIP-FCM array focused on the glutamate synapse, we have targeted 25 autism-related proteins that form a highly interconnected protein interaction network.  Our preliminary data focuses on detecting changes in the interaction network specific to excitation or inhibition of synaptic activity.

Conclusions: Our goal is to better understand the network-scale alterations in protein-protein interactions during glutamatergic signaling, and how different autism-associated risk factors may play important, perhaps convergent, roles in these synaptic network processes.  The data presented here are an important first step towards this goal.