Elizabeth A. Eipper, Department of Neuroscience, University of Connecticut Health Center
Chronic cocaine exposure results in structural changes in the medium spiny neurons of the brain reward centers. Significant progress has been made in understanding the signaling pathways involved in short-term responses to cocaine. Our long-term goal is to build on this knowledge to understand how chronic cocaine exposure leads to the changes in spine number that presumably underlie the associated behavioral changes. Spine formation, which requires carefully coordinated changes in the actin cytoskeleton, is a complex process known to involve the activation of Rac, a small GTPase of the Rho subfamily. Recent proteomic studies identified Kalirin7 as the major guanine nucleotide exchange factor for Rac in the PSD. The fact that levels of Kalirin7 increase in response to chronic cocaine led to the hypothesis that Kalirin7 plays an essential role in the structural changes observed. Consistent with this, mice lacking Kalirin7 (Kal7KO) have fewer dendritic spines and exhibit increased sensitivity to chronic cocaine.
The technologies available to us through the Yale/NIDA Neuroproteomics Center will allow us to determine the phosphorylation state of Kalirin7 in the mouse striatum/nucleus accumbens under basal conditions and after acute and chronic exposure to cocaine. A targeted method will be developed so that phosphorylation at all key sites can be determined simultaneously. Cdk5, whose role in addiction is studied by Center Investigator James Bibb, alters Kalirin7 function by phosphorylating a single site near its C-terminus. Methods for assessing the phosphorylation state of activated Kalirin7 and Kalirin7 that is engaged in interactions with the NR2B subunit of the NMDA receptor will be developed. Lfc, a RhoGEF found in dendritic spines but not concentrated at the PSD, is studied by Center Co-Director Angus Nairn; comparisons of Kalirin7 and Lfc will be especially informative. The 8-plex iTraq capabilities of the Center will allow us to compare the composition of PSDs isolated from wildtype and Kal7KO mice under control conditions and after chronic exposure to cocaine. Kalirin7, with its nine spectrin-like repeats and PDZ-binding motif, is known to interact with multiple proteins; its absence may result in the loss of specific components of the PSD. The Kal7KO mice, with their heightened sensitivity to cocaine, should provide a valuable resource to Center Investigators studying synaptic maintenance, protein complexes that mediate synapse formation and NMDA Receptor complexes.