Parkinson's disease (PD) results from the degeneration of nigrostriatal dopaminergic neurons. This process ultimately leads to a progressive decrease in dopamine mediated striatal signaling which manifests as a clinical syndrome characterized by bradykinesia, rigidity, tremor, and gait abnormalities. Traditional therapy for PD has aimed at restoring dopamine levels in the striatum through administration of the dopamine precursor, L-dopa. With advanced disease, L-dopa leads to dyskinesias and periods of marked fluctuation in motor activity ('on-off effect'). Alleviation of these side effects has been a major challenge and has prompted a search for alternative strategies which can provide a more stable level of dopaminergic signaling. A previously unexplored option to restore striatal dopaminergic activity and at the same time to potentially avoid the consequences of long term L-dopa administration, is through the introduction of constitutively active dopamine receptors. The laboratory of the PI has extensive experience in generating receptors with ligand independent (or constitutive) activity through the introduction of activating point mutations. These receptors have the potential to maintain dopaminergic signaling even in the absence of dopamine and/or dopaminergic agonist drugs. The premise of this application is that constitutively active dopamine receptors can be identified using in vitro assays and expressed in the striatum of rats to enhance dopaminergic signaling over an extended time interval. The objective of Specific Aim 1 is to generate and pharmacologically characterize in vitro a series of constitutively active dopamine 1 and dopamine 2 receptors. Using recombinant adeno-associated virus, the functional consequences of striatal overexpression of constitutively active dopamine receptors will be explored in rats (Specific Aim 2). Circling behavior after unilateral viral administration will be used as an index of construct activity. The methodologies utilized will include molecular (generation of constitutively active mutant receptors, expression of recombinant proteins), pharmacologic (radioligand binding, second messenger signaling assays), and behavioral approaches (assessment of circling behavior). These experiments will provide additional insight into the role of dopaminergic receptors in the striatum as well as potentially take the first steps toward the development of a new therapeutic option for Parkinson's disease. ? ?
