Michael Francis, Ph.D. - US grants

The main goal of the proposal is to determine the mechanism of action and structural basis for cocaine inhibition of neuronal nicotinic receptors (nAChRs). Although evidence from a number of studies makes clear the importance of cocaine inhibition of the dopamine transporter in mediating many of the addictive and locomotor effects of cocaine, our preliminary data indicate that cocaine can inhibit nicotinic receptors in a subtype-dependent manner at concentrations which are potentially relevant in mediating some of the toxicity associated with cocaine use. It is becoming increasingly apparent that the primary function of brain nicotinic receptors is to modulate the release of other neurotransmitters via presynaptic effects. Thus, it is possible that some of cocaine's effects could be related to inhibition of transmitter release via direct interactions with neuronal nAChRs. The proposal makes use of the Xenopus oocyte expression system in conjunction with single channel recording, laser pulse photolysis and molecular biology to characterize cocaine inhibition of nicotinic receptors. The applicant will gain training in the analysis of single channel and macroscopic receptor kinetics as well as additional experience in molecular biology. In order to understand the wide spectrum of cocaine effects on brain function, it will be necessary to consider the potential for cocaine effects at sites in addition to those associated with inhibition of the dopamine transporter, including binding to neuronal nicotinic receptors.

DESCRIPTION (provided by applicant): A number of drugs of abuse and therapeutics act by altering the efficacy of synaptic transmission. Likewise, the pathophysiology of many neurological disorders is rooted in synaptic dysfunction. However, the processes that regulate the organization and maintenance of the synaptic architecture remain largely unknown. Receptor tyrosine kinases (RTKs) have been shown to regulate neurite outgrowth and the clustering of post-synaptic neurotransmitter receptors. The C. elegans RTK CAM-1 is related to both Muscle-specific kinase (MUSK), an RTK required for clustering of post-synaptic acetylcholine receptors (AChRs) at the mammalian neuromuscular junction (NMJ), and the mammalian Ror family of RTKs. Ror RTKs are highly conserved across species and are widely expressed in the mammalian nervous system. However, the functional roles of Ror RTKs in the nervous system are unknown. Our preliminary results show that CAM-1 is localized to the C. elegans NMJ and that targeted mutation of the cam-1 gene results in altered synaptic transmission at C. elegans neuromuscular synapses. The experiments in this proposal are designed to test the functional role of CAM-1 at the C. elegans NMJ. We will determine the subcellular distribution of CAM-1 and assess whether it is colocalized with specific neurotransmitter receptors. Furthermore, we will determine if CAM-1 physically interacts with specific synaptic proteins involved in neurotransmission. Finally, to identify potential ligands and downstream effectors for Ror RTKS, we will screen for suppressors of the paralysis caused by overexpression of CAM-1 in C. elegans muscles. This research program will provide novel insights into potential synaptic functions for CAM-1 and Ror kinases in general. The proposed experiments will also provide valuable training for the applicant in C. elegans research techniques. Through a diverse training program including formal lectures, informal presentations and research, the applicant will gain new experience in the design and implementation of C. elegans molecular genetic approaches. These approaches will complement the applicant's previous training in ion channel physiology, ultimately enabling the applicant to direct an independent research program focused upon the biological regulation of synaptic function in C. elegans. The application of genetic techniques in concert with electrophysiological approaches in the Maricq laboratory makes this an ideal location for the proposed training.

DESCRIPTION: Nicotinic cholinergic signaling plays key roles in the mammalian nervous system. Nicotinic acetylcholine receptors mediate excitatory signaling between neurons as post-synaptic receptors and, from extrasynaptic sites, modulate neurotransmitter release at diverse synapse types across virtually every area of the brain and spinal cord. Alterations in nicotinic cholinergic signaling are associated with a number of debilitating neurological disorders including Alzheimer's disease, schizophrenia and certain forms of epilepsy. Moreover, nicotine binding to nicotinic receptors in the nervous system initiates the cellular and molecular cascade that results in nicotine addiction. Despite the clear importance of nicotinic signaling in normal brain physiology and neuronal dysfunction, there are major gaps in our understanding of the molecular mechanisms by which nicotinic signaling is achieved, and the regulatory pathways that impact cholinergic signaling in the nervous system remain poorly defined. This proposal employs a highly tractable model system, the nematode C. elegans, to investigate the molecular details of cholinergic signaling in a defined nervous system. Our preliminary data show that nicotinic receptors play key roles in regulating the excitability of motor neurons in a well-characterized C. elegans motor circuit. In Aim 1, we will test the hypothesis that the expression and localization of specific receptor types are restricted to subsets of motor neurons, determine the molecular nature of pathways important for proper localization of nicotinic receptors on neurons, and test the roles of specific receptor types in the control of C. elegans behavior. In Aim 2, we will use patch clamp electrophysiology to directly measure cholinergic currents from motor neurons and assess the roles of these receptors in motor neuron physiology. In Aim 3, we will use a powerful genetic approach to uncover components of novel molecular pathways that regulate cholinergic signaling onto neurons. We expect that our studies will provide fundamental insights into the mechanisms of nicotinic receptor function in the central nervous system. Additionally, the identification and functional characterization of genetic pathways that regulate synapse formation and function in our experiments will ultimately yield novel drug targets for therapeutic strategies designed to treat neurological disorders involving cholinergic signaling.

? DESCRIPTION (provided by applicant): Cholinergic signaling mediated by ionotropic (nicotinic) acetylcholine receptors (iAChRs) plays key roles in the mammalian nervous system. iAChRs modulate neurotransmitter release at diverse synapse types across virtually every area of the brain and spinal cord, and in addition, regulate the activity of inhibitory interneurons in critical brain areas. Alterations in iAChR signaling are associated with a number of debilitating neurological disorders including Alzheimer's disease, schizophrenia and certain forms of epilepsy. Moreover, nicotine binding to nicotinic receptors in the nervous system initiates the cellular and molecular cascade that results in nicotine addiction. Despite the clear importance of iAChR signaling in normal brain physiology and health, there are major gaps in our understanding of the cellular mechanisms that regulate cholinergic signaling in the brain. We have made the exciting observation that iAChRs also regulate inhibitory neuron activity in the genetically tractable model organism Caenorhabditis elegans. We have developed a powerful system to study the trafficking, localization and function of these receptors in the dendrites of inhibitory neurons located in a simple 3-layer circuit that controls C. elegans movement. This proposal aims to use the genetic tools we have generated for manipulation of activity levels in this circuit, along with the powerful array of molecular genetic approaches available in C. elegans, to explore fundamental questions in iAChR biology. In this project we will (Aim 1) characterize iAChR subcellular localization, subunit composition, in vivo dynamics, and molecular pathways responsible for delivery of iAChRs to specific subcellular domains on inhibitory neurons; (Aim 2) investigate how altered cholinergic innervation of GABA neurons leads to defects in the development or maintenance of GABA synapses; (Aim 3) determine the role of a novel neurexin signaling pathway in shaping GABA synapse development. We expect that our studies of this experimentally tractable circuit in the worm will provide exciting new insights into mechanisms for biological regulation of iAChRs in the brain, and their important roles in regulating inhibitory signaling. Additionally, the identification and functional characterization of conserved genetic pathways that regulate synapse formation and function in our experiments will ultimately yield novel drug targets for therapeutic strategies designed to treat neurological disorders involving cholinergic signaling.

? DESCRIPTION (provided by applicant): Altered signaling by neuromodulators, such as neuropeptides, are linked with debilitating human brain diseases including anxiety and panic disorders, schizophrenia, and epilepsy, yet we have a surprisingly limited understanding of how neuromodulators shape proper neural circuit function. How do neuromodulators produce specific patterns of neural circuit activity and how do changes in circuit activity produce specifi behavioral states? Our aim is to dissect molecular and neuronal mechanisms by which a conserved neuromodulatory system functions to shape neural circuit activity and behavior in response to changing sensory information (context). We have recently demonstrated that the neuropeptide NLP-12, a C. elegans ortholog of mammalian cholecystokinin (CCK), regulates context-dependent transitions between behavioral states, and we have begun to define the neural circuit basis for these effects. CCK is among the most abundant neuropeptides expressed in the mammalian brain, and CCK knockout mice display heightened anxiety, yet we do not have a clear understanding of how CCK functions in the context of the circuits that it modulates, or how CCK-mediated changes in activity alter anxiety levels. We are now well-positioned to gain a completely new level of understanding of how CCK shapes circuit activity and behavior, using the circuit we have defined in C. elegans as a model. We have developed tools for cell-specific manipulation of NLP-12/CCK levels, and for optogenetic stimulation and inhibition of NLP-12/CCK-expressing neurons. Further, we have defined 2 receptors, CKR-1 and CKR-2 that are required for NLP-12/CCK activity, and share significant homology with mammalian brain CCK1 and CCK2 receptors. In the first aim, we will determine how NLP-12 release from the interneuron DVA cooperates with descending sensory information (e.g., from olfactory neurons) to initiate context-dependent changes in behavioral output. In the second aim, we will define the circuitry involved and investigate how CKR- 1 and CKR-2 modulate the activity of the neurons in which they are expressed. By making a detailed functional investigation of a conserved neuromodulatory system in the context of this powerful model circuit, and linking functional changes to transitions between behavioral states, we expect to achieve a completely new level of understanding of how neuromodulators, in particular CCK, regulate neural circuit activity and modify behavior. We anticipate our findings will accelerate a path toward the development of effective therapeutic approaches for brain disorders associated with altered neuromodulatory signaling.

Dendritic spines are essential structures in brain synaptic connectivity and plasticity, and spine defects are associated with a host of neurodevelopmental and neuropsychiatric disorders. However, we know relatively little about the basic biology of how spines form, are maintained over time, and are altered during plasticity. The discovery and characterization of new molecules regulating fundamental aspects of spine biology may open entirely new avenues of research into how spine defects arise in neurological disorders. We recently made the key discovery that excitatory synaptic contacts onto a subset of C. elegans GABAergic neurons occur at spine-like protrusions from the dendrite, enabling for the first time unbiased genetic analysis of spine development and plasticity in this genetically tractable system. In this proposal we aim to discover new regulators of spines using a novel and high throughput unbiased forward genetic screening approach recently developed in an exciting collaborative effort between the Francis and Bénard labs. This approach allows us to assay spine morphology, number, and plasticity with unprecedented single dendrite and even single spine resolution in vivo. Newly identified genes in the regulation of spines will then be characterized using an array of new tools we have developed and optimized, and we will determine precise cell biological mechanisms underlying spine plasticity in vivo. Given that spine defects are strongly associated with neurodevelopmental and neuropsychiatric disorders, it is likely that a number of these genes will have causal and/or accessory roles in these diseases. This effort represents (to the best of our knowledge) the first high-throughput forward genetic screen for molecules required for spine development and maintenance in vivo. Thus, a wealth of novel regulators of spine biology, which have potential roles in neurological disease, are likely to be revealed.