We will develop a new method to measure the time courses of activation of biochemical regulatory networks that control changes in synaptic strength which underlie processing and storage of information in neural networks. The proposed method will permit unprecedented time resolution and will enable measurement of the time courses of activation of at least 20, and eventually as many as 50 to 100 enzymes in brain tissue that has been rapidly frozen at intervals as small as one second following an electrical or pharmacological stimulus. The method will be immediately applicable to basic research on, and target development for, mental illnesses and Alzheimer's disease. Upon scale-up, it will be applicable to screening for drugs to treat these diseases. The method will involve substantial adaptation of two existing technologies: """"""""plunge-freezing"""""""" and """"""""Selected/ Multiple Reaction Monitoring"""""""" (S/MRM) by mass spectrometry. Once developed, both technologies can be scaled up for medium or high throughput screening. The project has three aims. First, we will develop a plunge freeze apparatus to rapidly freeze slices of hippocampal tissue at accurate time intervals following application of a stimulus to the perfused slice. We will accomplish this by making modifications and additions to a plunge-freeze apparatus now commercially available from Leica (Leica EM GP). We will devise an optimal design for a sample chamber to maintain the health of slices during perfusion, and to deliver electrical stimuli to the Schaffer collateral pathway, a major hippocampal axon tract, prior to rapidly freezing the slice by plunging it into a -1900 C liquid propane/ethane bath. We estimate that freezing time to the center of the slice upon plunge will be ~ 200 msecs or less. This freezing time is compatible with a resolution of one second for time intervals following application of a discrete stimulus. Second, we will develop methods to measure the activation of a panel of 20-25 protein kinases or their key substrate proteins located at positions in the regulatory networks that are believed to control synaptic plasticity in excitatory synapses in the hippocampus. Each enzyme or substrate that we will measure is regulated by addition of a phosphate group to key residues in the protein structure. Mass spectrometry will be used to measure changes in the levels of these phosphorylated sites in the frozen slice tissue. Third, once the assays are developed, we will carry out """"""""proof of principle"""""""" experiments by combining the technologies developed in Aims 1 and 2 to acquire time courses of activation of each the enzymes in hippocampal slices after delivery of stimuli that alter synaptic plasticity.

Public Health Relevance

This project will develop a new method for measuring activity in biochemical pathways that do not function properly in individuals with mental illnesses or Alzheimer's disease. The method will be used to support research on the causes of mental illnesses and Alzheimer's disease and for efficient screening for new drugs to treat these diseases.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
Research Project (R01)
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Special Emphasis Panel (ZMH1-ERB-C (05))
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Asanuma, Chiiko
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California Institute of Technology
Schools of Arts and Sciences
United States
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Kennedy, Mary B (2017) Biochemistry and neuroscience: the twain need to meet. Curr Opin Neurobiol 43:79-86
Kennedy, Mary B; Mastro, Tara L (2017) Liquid Phase Transition in the Postsynaptic Density? Trends Biochem Sci 42:2-4
Walkup, Ward G; Mastro, Tara L; Schenker, Leslie T et al. (2016) A model for regulation by SynGAP-?1 of binding of synaptic proteins to PDZ-domain 'Slots' in the postsynaptic density. Elife 5:
Walkup 4th, Ward G; Kennedy, Mary B (2015) Protein purification using PDZ affinity chromatography. Curr Protoc Protein Sci 80:9.10.1-37
Walkup 4th, Ward G; Washburn, Lorraine; Sweredoski, Michael J et al. (2015) Phosphorylation of synaptic GTPase-activating protein (synGAP) by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5) alters the ratio of its GAP activity toward Ras and Rap GTPases. J Biol Chem 290:4908-27
Walkup 4th, Ward G; Kennedy, Mary B (2014) PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands. Protein Expr Purif 98:46-62
Kennedy, Mary B (2013) Synaptic Signaling in Learning and Memory. Cold Spring Harb Perspect Biol 8:a016824