""""""""Evaluating the synaptic hypothesis of autism using protein-protein interaction network analysis"""""""" Protein-protein interaction (PPI) networks of a cell are thought to represent a system with emergent network properties, integrating signals for a variety of inputs into coordinated responses. A deeper understanding of protein complexes at the synapse will enhance our knowledge of normal brain function and may highlight new therapeutic targets for diseases, including autism. The applicant has recently developed a novel technology, multiplex immunoprecipitation measured by flow cytometry (mIP-FCM) that allows the rapid, simultaneous, quantitative measurement of hundreds of protein-protein interactions (PPIs) from small amounts of biological samples. This proposal involves translating mIP-FCM technology to target synaptic proteins relevant to autism, and investigating the quantitative changes in protein complexes that occur under physiological and disease-associated conditions. The observation of the dynamic activity of protein complexes at the mouse and human glutamate synapse will contribute to a greater understanding of normal brain function, and may highlight pathological molecular mechanisms of autism-associated genes. The applicant proposes three specific aims: 1) Generate a multiplex IP-FCM assay targeted to synaptic proteins, to be completed during year 1 of the K99 portion of the award;2) Map synaptic PPIs in mice carrying autism-associated mutations. This portion will be completed in years 2-5, during the K99/R00 transition. 3) Map synaptic PPIs in human iPS neurons derived from autistic and typically developing individuals, to be completed during years 3-5 of the R00 phase of the award. The overall goal of this proposal is to define specific changes in the PPI network of the glutamatergic synapse associated with different autism risk factors, and to investigate the extent to which there is convergence of autism risk genes into specific common molecular pathways at the synapse. During the K99 phase of the award, the applicant will 1) Develop the mIP-FCM assay targeted to the glutamatergic synapse, and learn appropriate statistical and visualization methods for the large datasets;2) Learn to manage a research team, including management of technical support staff and students, overseeing budgets, integrating data from multiple individual projects, and acting as a mentor of junior scientists. The applicant has assembled a group of experienced co-mentors to help with this career development training. Overall, this training will facilitate the achievement of the applicant's long-term goal to develop an internationally-renowned independent research program focused on the molecular mechanisms of autism risk factors.
At a very basic level, a cell is a collection of proteins that interact with each other to perform biological functions. This project involves developing a new technology, mIP-FCM, to quantitatively measure these protein interactions as they change during biological signaling processes. The proposed mIP-FCM assay will measure 484 protein-protein interactions among autism-linked proteins at the neuronal synapse, with the goal of understanding how these proteins interact with each other to contribute to both normal and pathological neuronal function.
|Lautz, Jonathan D; Brown, Emily A; Williams VanSchoiack, Alison A et al. (2018) Synaptic activity induces input-specific rearrangements in a targeted synaptic protein interaction network. J Neurochem 146:540-559|
|Smith, Stephen E P; Neier, Steven C; Reed, Brendan K et al. (2016) Multiplex matrix network analysis of protein complexes in the human TCR signalosome. Sci Signal 9:rs7|
|Southwell, Amber L; Smith, Stephen E P; Davis, Tessa R et al. (2015) Ultrasensitive measurement of huntingtin protein in cerebrospinal fluid demonstrates increase with Huntington disease stage and decrease following brain huntingtin suppression. Sci Rep 5:12166|