James A. Bibb, Department of Psychiatry, The University of Texas Southwestern Medical Center
It is now clear that addiction results from usurpation of biochemical and molecular mechanisms that underlie normal learning processes in the reward circuitry of the brain. These involve both glutamatergic and dopaminergic neurotransmission mediated by the second messengers Ca2+ and cAMP, respectively. Our research has revealed that the neuronal protein kinase Cdk5 plays a critical synaptic role in learning, and regulates behavioral responses to cocaine. Cdk5 associates with NMDA receptor complexes. It regulates the constituency of these postsynaptic complexes and is itself regulated by NMDA receptors in a Ca2+-dependent manner. The downstream effectors of this regulation appear to be multiple components of the dopamine/cAMP/PKA signaling cascade. The key to understanding Cdk5’s role in addition is to better understand the regulatory relationship that exists between it and NMDA receptors, and how chronic exposure to cocaine affects this relationship. Therefore the goal of this research is to combine the powerful approach of neuroproteomics with transgenic technology to explore this important interaction. We propose to (1) characterize the effects of Cdk5 conditional knockout and transgenic overexpression of the Cdk5 activating cofactor p25 on the NMDA receptor complex in adult mouse brain. We will then (2) assess the effect that chronic cocaine exposure upon this profile and the contribution of Cdk5 to these effects. NMDA receptor complexes will be isolated from control, Cdk5 knockout, and p25 overexpressing mice and analyzed with regard to the spectrum of NMDA receptor constituents and phosphoproteins using an array of proteomic technologies including IMAC, DIGE, nLC-MS/MS, and LTQ-Orbitrap MS. Bioinformatic analysis will be conducted to define phosphorylation sites. The effects of chronic cocaine exposure will be assessed using similar samples from drug versus saline treated control, Cdk5 conditional knockout, and p25 overexpressing mice. From these studies we delineate how Cdk5 regulates NMDA receptor complexes and gain basic knowledge of the precise biochemical mechanisms of addiction. These findings of these important analyses will be pursued as part of an ongoing and aggressive addiction research program.