Agents that enhance cholinergic transmission or activate muscarinic acetylcholine receptors have been developed to ameliorate the loss of cognitive function in patients with Alzheimer's disease (AD) and have been suggested to have modest success in slowing disease progression. The development of selective orthosteric agonists for the M1 receptor has been relatively unsuccessful due to the high conservation of the orthosteric binding site across all five muscarinic receptor subtypes (M1-M5). We have discovered several different classes of allosteric modulators of G protein-coupled receptors: allosteric agonists and positive allosteric modulators (PAMs). Our preliminary data suggest that targeting allosteric sites on the receptor is a viable approach for developing highly selective activators of the M1 muscarinic acetylcholine receptor, since these allosteric sites are thought to be less conserved between receptor subtypes. Furthermore, evidence suggests that there are multiple allosteric binding sites on the M1 receptors. However, the locations of these secondary binding sites and the residues that are critical for binding have not been fully characterized. We propose to determine and characterize the location of the allosteric binding sites for BQCA (PAM), and TBPB and VU0357017 (allosteric agonists) using a combination of chimeric receptors and site-directed mutagenesis. We will then determine if these compounds display agonist-directed signaling, a phenomenon in which there is selective activation of particular signaling cascades, downstream of the M1 muscarinic acetylcholine receptor in cortical tissue slice preparations. Furthermore, we will determine the ability of these compounds to alter the electrophysiological properties of pyramidal neurons in the medial prefrontal cortex using whole-cell patch clamp recordings. In addition, we will utilize a mouse model of AD to try to understand how muscarinic receptor activation might be altered in pathological states. These studies will provide greater insight into the mechanism of action of allosteric ligands at the M1 receptor and potentially the possibility of selectively targeting activation of particular signaling cascades that are beneficial for disease treatment, while avoiding the activation of other cascades that may result in dose-limiting or adverse side effects.
Studies of the function of muscarinic receptors in the medial prefrontal cortex may lead to an increased understanding of the changes in muscarinic receptor signaling that contribute to the cognitive symptoms of Alzheimer's disease. The resuts of these studies will have important implications for the development of novel approaches to treat Alzheimer's disease and other neurodegenerative diseases.
