Cellular and Molecular Neurobiology
Author: Victoria Vaccaro | Email: victoria.vaccaro@unc.edu.ar
Victoria Vaccaro1°, Julieta Boezio1°, Daiana Rigoni1°, Liliana Cancela1°, Mariano Bisbal2°, Flavia Bollati1°
1° Instituto de Farmacología Experimental de Córdoba (IFEC-CONICET), Departamento de Farmacología Otto Orsingher, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina
2° Instituto de Investigación Médica Mercedes y Martin Ferreyra, INIMEC-CONICET, Córdoba, Argentina
Previous studies from our laboratory demonstrated that chronic stress enhances behavioral sensitization to cocaine and that the Rho GTPase Rac1 in the Nucleus Accumbens core (NAc) plays a crucial role in this phenomenon. Rho GTPases are guanine nucleotide-binding proteins cycling between an active, GTP-bound state and an inactive, GDP-bound state. In its active form, Rac1 activates p21-activated kinase 1 (PAK1), which regulates downstream signaling by phosphorylation. Rac1 controls actin cytoskeleton dynamics, essential for dendritic spine morphogenesis and synaptic plasticity, contributing to the long-lasting effects of drug sensitization.
The aim of this project is to characterize and quantify Rac1 activity in the NAc during stress-induced cocaine sensitization. To achieve this goal, we developed a laboratory kit for a Rac1 activity assay based on the downstream signaling pathway of this protein. The assay uses the p21 Binding Domain, PAK-PBD of the Rac effector protein, that binds specifically to the GTP-bound form of Rac. Our experiments involved the production of the recombinant protein PAK-PBD and its subsequent conjugation with agarose beads to conduct a pulldown assay that exclusively recognizes the active form of Rac1, GTP-bound Rac1. We will also evaluate structural plasticity in the NAc to provide further insight into the molecular mechanisms underlying the structural changes associated with cocaine sensitization induced by chronic stress.