Elucidating the Hippocampal Dopaminergic Subproteome with Novel Bioorthogonal Techniques

Citation

Hodas, Jennifer Jin Lee (2010) Elucidating the Hippocampal Dopaminergic Subproteome with Novel Bioorthogonal Techniques. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/DBGH-J143. https://resolver.caltech.edu/CaltechTHESIS:10012009-223603699

Abstract

Both synaptic and behavioral plasticity require de novo protein synthesis. Dopamine is a critical neuromodulator, and abnormalities in dopaminergic regulation underlie disorders like Parkinson’s disease, Alzheimer’s disease, and schizophrenia— diseases that impair the ability of the brain to perform complex processes, including the formation and retrieval of memories. The stimulation of D1/D5 dopaminergic receptors in the hippocampus is critical for protein synthesis-dependent long-term potentiation (LTP), a process important for long-term synaptic plasticity and memory. The proteins synthesized upon activation of dopaminergic pathways, the dopaminergic subproteome, however, remain unknown.

Here, we describe the development of two sister technologies that employ bioorthogonal chemistry to effectively and specifically identify and visualize a proteome in an unbiased, nontoxic manner. In both bioorthogonal noncanonical amino acid tagging (BONCAT) and fluorescent noncanonical amino acid tagging (FUNCAT), we utilize methionine surrogates, either azidohomoalanine (AHA) or homopropargylglycine (HPG), which are conjugated via [3+2] copper (I)-catalyzed cycloaddition to either a biotin or fluorescent molecule-bearing probe. We demonstrate the utility of these methods by showing that both AHA and HPG can be used to examine two temporally distinct protein populations. Furthermore, we visualize the dendrite-specific contribution to the neuronal proteome by taking advantage of the spatial control achievable by FUNCAT. We then combine these techniques to address the question of the identity of the proteins in the specifically dendritic subproteome of the hippocampus. We confirm that upon stimulation with a D1/D5 dopamine receptor-specific agonist, there are significantly increased levels of protein synthesis in dendrites when compared to unstimulated dendrites. By utilizing a combination of these novel methods and more traditional techniques, we are able to provide the first comprehensive list of the dopaminergic dendritic subproteome of the hippocampus. These data suggest that the initial stages of D1/D5 receptor activation lead to the translation of proteins that may play a role in synaptic strengthening.

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