Liqun Luo - US grants

DESCRIPTION: The long-term goal of the research is to understand the molecular mechanism by which neurons acquire their characteristic pattern of connectivity during development. Recent evidence suggests that Rac and Cdc42, members of small GTPase of the Rho subfamily, mediate signaling from extracellular factors to the actin cytoskeleton in the regulation of neuronal arborization. The PI identified a Cdc42-binding protein, C17, in Drosophila. C17 binds to Cdc42 in a GTP-dependent manner and is highly enriched in the nervous system at the time of axonal and dendritic outgrowth. The amino third of C17 shares homology with human myotonic dystrophy protein kinase. In addition, C17 also contains domains suggestive of cytoskeletal association and regulation by other signaling molecules. The c17 mutants display defects in actin cytoskeleton and neuronal function. In this proposal, the role of C17 in the morphogenesis of neurons will be assessed by genetic interaction with other genes required in axon guidance, by biochemical studies of the regulation of its kinase activity, and by subcellular localization studies. In addition, the function of mouse C17 homolog in the morphogenesis of cerebellar Purkinje cells will be analyzed by expressing dominant negative mutants of mouse C17. Knowledge of how neurons generate axons and dendrites in development may help us understand regeneration following nerve injury. The high degree of sequence similarity between C17 and myotonic dystrophy protein kinase suggests functional similarities of the two proteins. Thus understanding the biological function C17 may shed light on the molecular pathogenesis of myotonic dystrophy, the most common adult form of muscular dystrophy.

DESCRIPTION (provided by applicant): Pruning of exuberant neuronal connections is a fundamental and widespread mechanism to develop the diversity and specificity of neuronal connections evident in both vertebrate and invertebrate nervous systems. Mechanisms used for developmental pruning of axons and dendrites could also be used for structural plasticity of adult neurons in response to experience, learning, and injuries of the nervous system. Misregulation of such mechanisms may contribute to pathogenesis of neurodegenerative diseases. We are using axon pruning of Drosophila mushroom body (MB) y neurons during metamorphosis as a model to investigate the mechanisms of developmental axon pruning. During the past grant period, we found that MB 7 axon pruning utilizes a degenerative mechanism to eliminate specific axon branches, and that degenerating axons are engulfed for endosome-lysosome degradation by nearby glial cells. We showed that axon pruning requires cell autonomous transcriptional regulation by steroid hormone ecdysone receptor (EcR), and cell-autonomous action of the ubiquitin-proteasome system (UPS). We have also initiated several screens to identify other genes involved in MB y axon pruning. These include a forward genetic screen using chemical mutagen EMS that identified two new pruning mutants so far, a gain-of-function screen for genes that when over-expressed would block axon pruning, and a microarray-based screen that identified hundreds of genes whose transcription is up- or down-regulated by EcR in MB neurons during metamorphosis. The last two screens converged on the identification of a protein whose expression in MB neurons is down-regulated 17 fold by EcR during metamorphosis, and when over-expressed blocks axon pruning. Here we propose a series of experiments to follow the existing evidence, and to continue our investigation of molecular mechanisms of developmental axon pruning. We have provided evidence that MB y axon pruning shares striking similarities to axon degeneration in injury and neurological diseases. Thus, we believe these studies will contribute to our understanding of axon degeneration in diseases

DESCRIPTION (provided by applicant): From insects to mammals, olfactory receptor neurons (ORNs) expressing the same odorant receptors converge their axonal projections to specific glomerular targets in the antennal lobe/olfactory bulb, creating an odor map in these first olfactory structures of the central nervous system. In the fruit fly Drosophila, antennal lobe projection neurons (PNs, equivalent to vertebrate mitral/tufted cells) send dendrites to glomeruli and synapse with ORN axons;PN axons then relay the olfactory information to higher brain centers the mushroom body and the lateral horn. Over the past five years and thanks to the support of the previous grant, we have made significant progress towards understanding how wiring specificity in the Drosophila olfactory circuit is established, both in terms of cellular and developmental events and their molecular mechanisms. In this renewal, we propose a series of molecular, genetic and biochemical experiments to continue our effort in understanding the mechanisms by which each PN or ORN chooses one of 50 alternative areas to target its dendrites or axons, ultimately resulting in the formation of a stereotyped and highly precise circuit for flies to sense and discriminate odors and pheromones. We utilize both candidate gene approaches and forward genetic screen to identify molecules that are required for these targeting processes, and then investigate their detailed mechanisms of action. Emphasis will be placed on studying ligands and receptors that guide PN dendrites and ORN axons to appropriate areas in the antennal lobe and faciliate their synaptic matching. We expect that completion of the proposed experiments in this grant will significantly enrich and expand our understanding of the logic and mechanisms of olfactory circuit assembly. These studies will contribute to our understanding of a number of important neurobiological questions, including neuronal fate specification, dendritic guidance and targeting, and the logic of the assembly of the olfactory circuits and neural circuits in general. Our studies will also provide insight into how olfactory information is transferred and transformed along the central pathways. PUBLIC HEALTH RELEVANCE Understanding how neural circuits are wired during normal development is a prerequisite for understanding the nature of pathological wiring, which underlie many human neurological and psychiatric disorders. In addition, knowledge of insect olfactory system organization and development can be used to help design strategies to combat malaria, transmitted by mosquitos that utilize primarily olfaction to find their mates and human host.

[unreadable] DESCRIPTION (provided by applicant): The Gordon Research Conference (GRC) on Molecular and Cellular Neurobiology, although held only three times, has already emerged as a key meeting in the field. Two of the main features of the meetings are the breadth it covers in neurobiology and the international interactions it promotes (especially for American and Asian scientists). The conference is intended to bring together mixture of groups working on molecular and cellular mechanisms of a wide range of neurobiological problems, from neural development, neuronal communication, sensory systems, neural plasticity and behavior, to diseases of the nervous system. At the same time, the Chair and Vice-Chair will strive to ensure that these diverse topics form nice connections among each other and each topic has sufficient depth. Thus the conference will be an exceptional opportunity for scientists working in different areas of neurobiology to learn the latest advance from each other, and to bridge basic and clinical neuroscience research. It also provides an overview to graduate students and postdocs who wish to or just enter the field. Feedbacks from participants (both speakers and students/postdocs) in the past three conferences were overwhelmingly positive. The meeting will be held at Hong Kong University of Science and Technology, which has been chosen by the GRC Secretariat as a permanent Gordon operating site in Asia. As with the tradition of GRC, the meeting is small (approximately 150 participants) and will focus on the discussion of cutting-edge, unpublished research. Forty minutes will be allowed for each speaker's topic, a third of which will be devoted for discussion. Much of the afternoons are usually open for more discussions and for poster sections, which we expect the majority of participants will present. Thus the meeting will serve as an excellent opportunity for education of young scientists. Financial support is requested from NIH to cover a small fraction of travel expense for US speakers. Majority of which be used to support conference fees and/or travel of students and postdocs from US, especially minority and women participants to increase their number. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We propose to develop a genetic mosaic system in mice that allows simultaneous labeling and genetic manipulation of defined neuronal populations, down to the level of single isolated neurons in vivo. This system, which we have named "MADM" (for Mosaic Analysis with Double Markers), utilizes two hybrid marker genes knocked-in at identical locations on homologous chromosomes. Each marker gene is interrupted by a loxP-containing intron and neither expresses a functional protein. Only upon Cre-mediated recombination between the two loxP sites on the homologous chromosomes are functional marker genes restored. Depending on the cell-cycle stage at which recombination takes place and the segregation pattern of the chromosomes after recombination, daughter cells are labeled with one or both markers. We have preliminary results indicating that MADM can be used to generate, with high efficiency, inter-chromosomal exchanges in both postmitotic neurons and in dividing neural precursors. By further developing this method and its variations we will be able to label defined neuronal populations and single neurons with genetically encoded markers, in live or fixed brains. It will also be possible to create genetic mosaics such that cells expressing the first functional marker are homozygous mutant for a gene of interest, whereas cells expressing the second functional marker are homozygous wild type, while the rest of the animal is heterozygous. MADM will allow investigation of the relationship between cell lineage and neural circuits during development, tracing of neural circuits in the adult nervous system, and conditional knock-out of candidate genes of interest as well as overexpression of transgenes in single isolated neurons. This method can also be used to create mouse models for human diseases such as loss of heterozygosity in cancer and neurological diseases.

The mammalian brain consists of hundreds of millions to billions of nerve cells (neurons) that form complex networks. How perception, cognition, and action are reflected by the activities of neurons is a central question in modern neuroscience. The investigators have previously developed a genetic method to mark neurons that are activated by a specific sensory experience or behavioral episode, so they can visualize their connections and measure their activities. In this research, the investigators will develop new methods to improve the signal-to-noise ratio to more effectively identify the active neurons, and to mark two separate experiences differentially in the same animal so they can directly compare physiological properties of two populations of neurons, such as those before and after learning. The success of these approaches will enable scientists to compare brain representations of different stimuli and behavior, and what changes occur after learning.

The method used to mark active neurons (TRAP, for targeted recombination in active populations) utilizes the property of immediate early genes, whose transcription is activated by neuronal activity. This was achieved using mouse genetics to place a drug-inducible Cre recombinase under the control of immediate early gene promoters, such that experience in the drug-active period turns on Cre reporter transgenes permanently. This research will utilize a combination of viral transduction and mouse genetics to differentially label neurons that are activated by two separate experiences. In addition, a strategy of using light to locally silence inhibitory neurons will be used to enhance excitation-to-inhibition ratio, and thereby enhance TRAP efficiency, in a much narrower time window during the drug-active period. The new transgenic mice and viral vectors will be deposited in public repositories after validation.

PROJECT SUMMARY A key question in neurobiology is how individual neurons precisely connect with each other to form functional circuits during development. Understanding the mechanisms of neural circuit assembly in the mammalian brain may provide insights into the etiology of human brain disorders. In the mammalian brain, each neuron on average forms connection with thousands of other neurons. The assembly of these complex circuits depends on cell-cell communication during many steps of neural development. In the previous two cycles of this grant, we have focused on developing MADM (Mosaic Analysis with Double Markers) in mice. MADM labels with two distinct colors isolated individual neurons or groups of neurons that share a common lineage. At the same time, MADM can render neurons labeled with one of the colors homozygous mutant for a gene of interest and neurons labeled with the second color wild type as internal controls. MADM has allowed researchers to examine gene function in mammalian neural development (as well as other processes) with single-cell resolution, and enabled many new discoveries. In this renewal, we will utilize MADM and other tools we have developed in the previous grant cycles to study cell-cell communications in neural circuit assembly in the mouse brain. Specifically, we focus on two classes of proteins: neurotrophin receptors and teneurins. Using MADM analysis of the neurotrophin receptor TrkC, we have previously shown that sparse but not global knockout of TrkC in Purkinje cells reduces dendritic growth and branching, suggesting a competitive mechanism for dendrite morphogenesis. We will investigate the cellular mechanisms by which TrkC-mediated competitive dendrite morphogenesis using in vivo imaging, test whether postsynaptic activity required for Purkinje cell dendrite development, and the relationship between activity and TrkC signaling. We will also explore the function of neurotrophin receptor TrkB in neuronal morphogenesis. From our studies in Drosophila olfactory circuit assembly, we identified two teneurins, which are evolutionally conserved type II transmembrane proteins, that instruct synaptic partner matching via homophilic attraction. We will test whether teneurins also mediate cell-cell interaction in neural circuit assembly in the mouse brain using a combination of MADM analysis, conditional knockout, virus-mediated misexpresssion, and in vitro assays.  

PROJECT SUMMARY The monoamines, which include dopamine, norepinephrine, and serotonin, are evolutionarily conserved neurotransmitters that modulate the activity of excitatory and inhibitory neurons throughout the entire brain, and are thus essential for diverse aspects of physiology and behavior. Abnormalities of monoamine systems contribute to numerous brain disorders including schizophrenia, depression, and Parkinson's disease. We recently developed viral-genetic tools to determine the input, output, and input?output relationships of a given neuronal population at the scale of the entire mouse brain, and discovered contrasting input?output architectures between locus coeruleus norepinephrine neurons and midbrain dopamine neurons. Here, we apply these tools to study the organization and function of the dorsal raphe (DR) serotonin system, which provides major serotoninergic input to the forebrain to regulate diverse . functions and brain states including mood, impulsivity, anxiety, as well as hunger and thirst. Using rabies-mediated trans-synaptic tracing, we previously defined the input architecture to the entire populations of DR-serotonin and DR-GABA neurons However, our unpublished work revealed considerable heterogeneity within the DR serotonin system and suggests that it consists of parallel sub- systems that differ in input, output, and neurotransmitter phenotypes. We propose that each DR serotonin sub-system may carry out a specific subset of the diverse functions ascribed to the DR-serotonin neurons. We plan to complete our characterization of the anatomical organization of the DR serotonin sub- systems, addressing the questions of how axons of each sub-system divide up the projections of the entire DR serotonin system, and what is the input?output relationship for each DR serotonin sub-system. These will lay a foundation for all future studies of DR-serotonin neurons. We also propose to identify behavioral functions of a subset of these sub-systems by manipulating and recording serotonin neuron subtypes in anxiety- and depression-like states known to involve serotonin, as well as new behavioral paradigms. Finally, because previous studies and our own unpublished data suggest a strong link between serotonin and thirst, we will explore the circuit and cellular mechanisms by which serotonin regulates thirst-motivated behavior using quantitative and sensitive assays we have established based on a technique we developed to gain genetic access of thirst-activated neurons. The integration of anatomical, physiological, and behavioral studies on genetic-, projection-, and activity-defined neuronal populations proposed here will help dissect the complex serotonin system into specific sub-systems and advance our understanding of how serotonin modulates diverse physiological functions and behaviors.