Synapses are dynamic, with respect to their magnitude of synaptic transmission, their morphologic structure and their protein components. Dynamic changes in the synaptic proteome, mediated by changes in protein synthesis, degradation, distribution and post- translational modifications affect synaptic function and the capacity for synaptic plasticity. Despite the appreciation of the biological importance of synaptic protein dynamics, methods to enrich for and identify the dynamic synaptic proteome are not yet an integral part of neuroscience research. Studies conducted largely in prokaryotes and non-neuronal cells have demonstrated the promise of using the incorporation of non- canonical amino acids into newly synthesized proteins followed by bio-orthogonal click chemistry to tag the non-canonical amino acid labeled protein with probes, such as biotin, that can be used for purification and identification of newly synthesized proteins. We propose experiments to optimize the use of non-canonical amino acid incorporation and bio-orthogonal click chemistry in neuronal cell cultures, and in the visual system of intact rats and Xenopus tadpoles to demonstrate the feasibility of metabolic labeling to identify and quantify dynamic components of the synaptic proteome. These experiments are meant to represent proof of principal for comparing synthesis of synaptic proteins between different conditions and in different experimental systems.

Public Health Relevance

Newly synthesized synaptic proteins regulate synaptic function and plasticity. We propose to improve feasibility of using metabolic labeling with non-canonical amino acids to identify and quantify changes in the synaptic proteome in neuronal cultures and intact animals.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21MH099799-02
Application #
8544504
Study Section
Special Emphasis Panel (ZMH1-ERB-S (05))
Program Officer
Asanuma, Chiiko
Project Start
2012-09-12
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
2
Fiscal Year
2013
Total Cost
$227,400
Indirect Cost
$107,400
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Shen, Wanhua; Liu, Han-Hsuan; Schiapparelli, Lucio et al. (2014) Acute synthesis of CPEB is required for plasticity of visual avoidance behavior in Xenopus. Cell Rep 6:737-47
Schiapparelli, Lucio Matias; McClatchy, Daniel B; Liu, Han-Hsuan et al. (2014) Direct detection of biotinylated proteins by mass spectrometry. J Proteome Res 13:3966-78
Sharma, Pranav; Schiapparelli, Lucio; Cline, Hollis T (2013) Exosomes function in cell-cell communication during brain circuit development. Curr Opin Neurobiol 23:997-1004