Neil M. Nathanson

Molecular Studies of Muscarinic Acetylcholine Receptors and Neuronal Differentiation Factors:

Our laboratory is interested in the regulation of and mechanisms responsible for signal transduction in excitable cells. We have had a long-standing interest in the muscarinic acetylcholine receptors (mAChR), which comprise a family of related receptor proteins which are the products of distinct genes. Muscarinic receptors can regulate the activity of enzymes involved in intracellular second messenger pathways, such as adenylyl and guanylyl cyclases, phospholipase C, phosphodiesterases, and protein kinases, and can also regulate the function of ion channels. The mAChR gene family produces these effects by interacting with the members of a second gene family, the GTP-binding coupling proteins (G-proteins), which are required for receptor function. We are using a combination of molecular genetic, proteomic, immunological, biochemical, physiological, and behavioral studies to study the regulation of expression and mechanisms of action of the mAChR in the nervous and cardiovascular systems.

There are several model systems currently in use. Neuronal cell lines are being used because they provide a ready source of large numbers of homogeneous populations of neural cells which can be maintained under controlled conditions in culture to determine what regulates the expression and function of the receptors in nerve cells. We are also using both primary neuronal cultures derived from mouse brain and intact cultured brain slices to study these processes in more 'native' fully differentiated neurons. The developing chick embryo is also under investigation because it is amenable to experimental manipulation in ovo and because neurons can be grown under defined conditions in cell culture. We are using antibody and nucleic acid probes to determine what regulates the expression of the genes encoding mAChR, G-proteins, and their effectors both in vivo and in vitro, and are combining these with physiological studies to detect functional changes in receptor-effector coupling. We have isolated the genes encoding both mouse and chick muscarinic receptors, and are using these to study the mechanisms for regulation of receptor gene expression by innervation, developmental factors, synaptic activity, etc. We are also introducing wildtype and mutant cloned genes for the mAChR into cells lacking the receptors to compare the function and regulation of the different subtypes of receptor. We are particularly interested in the ways that receptor expression and function are altered by various physiological stimuli, and have identified several discrete pathways for both transcriptional and posttranslational regulation of the muscarinic receptors. We have shown that different subtypes of mAChR are differentially localized in different regions on the cell surface and utilize different pathways to undergo internalization from the cell surface; we have identified specific amino acid sequences of the receptors which are responsible either for this subcellular targeting or which mediate specific internalization pathways. We have also demonstrated the presence of a novel factor which is responsible for the developmentally regulated cell-type specific induction of muscarinic receptor gene transcription in neurons in the retina. We are identifying this novel neurotrophic factor and studying its mechanism of action in order to both identify the mechanisms which regulate mAChR gene expression and to understand the molecular and cellular pathways which control the establishment of specific synaptic pathways in the retina.

We have also used the technique of gene disruption by homologous recombination to generate strains of mice that are deficient in individual receptor subtypes, to determine the role that the various mAChR play in development, learning and memory, seizures, nociception, control of movement, etc. Our results demonstrate that the M1 receptor plays a crucial role in the regulation of certain neuronal ion channels, in the initiation of seizures in a widely used animal model of epilepsy, and in cognition. We are currently using these and other strains of knockout mice to determine the role of the various muscarinic receptor in seizure initiation, striatal function, memory and learning, and regulation of cardiovascular function. Finally, we are creating strains of mice with mutant mAChR genes containing subtle mutations in sites which we have shown regulate receptor expression and function in cells in culture; these mice will allow us to determine both the physiological and behavioral roles that these regulatory pathways play in vivo.

We are also interested in mechanisms for the regulation of neuronal function by trophic and differentiation factors. We are concentrating on the receptors for the neurokines LIF (leukemia inhibitory factor) and CNTF (ciliary neurotrophic factor), which are members of the cytokine receptor superfamily. In the nervous system, the receptors for LIF and CNTF have been implicated both in neuronal survival and in the determination of neuronal phenotype: they are required for the survival of certain types of neurons and induce other classes of neurons to alter the expression of neurotransmitter-synthesizing and neuropeptide genes. We are using cell and molecular biological techniques to study the regulation and action of the receptors for LIF and CNTF. These receptors activate multiple intracellular signal transduction cascades, and we are determining the roles of these various signalling pathways in the regulation of neuronal function. We have also identified novel feedback regulatory pathways involving the phosphorylation-dependent regulation of LIF receptor signaling following activation of both theneurokine receptors as well as heterologous receptor systems. We are also analyzing the mechanisms for retrograde signaling by these receptors to determine how stimulation of neurokine receptors on the nerve terminal result sin altered neuronal gene expression. These studies should provide new insights into the molecular mechanisms regulating long-term plasticity and synaptic function in the nervous system.