Marc Hammarlund - US grants

DESCRIPTION (provided by applicant): Living organisms are highly efficient and often reuse the same genes multiple times for different purposes. If one function of a gene is essential, death or arrest of the mutant masks other, later functions. This blind spot to later functions of essential genes is particularly troublesome in the nervous system. Most neurons can't be renewed from stem cell populations and therefore must survive and preserve their information- processing capabilities throughout the life of the organism. To accomplish this feat of survival, neurons must maintain their structure and repair it when damaged. But because maintenance and regeneration occur after development, the contribution of essential genes to maintaining and regenerating aging neurons is poorly understood. This project develops a novel strategy in C. elegans for achieving spatial and temporal control of gene inactivation. By using this strategy, it is possible to circumvent the initial requirement for essential genes. Further, the strategy has a critical advantage over existing techniques for making conditional or inducible knockouts: it is compatible with genetic screens. Thus, this strategy breaks down the barrier that prevents genetic screens from finding the later functions of essential genes. This important advance will enable multiple functions of essential genes to be teased apart wherever they occur. This project deploys this strategy to discover the function of essential genes specifically in neurons, focusing on cell survival, development, maintenance of axons and synapses, and regeneration. These experiments should identify a set of common factors that help preserve and restore neural function in all nervous systems, long after development has ended. A hallmark of many neurological diseases is delayed onset. Further, age-related decline in nervous system function occurs even in the absence of known disease. These observations suggest that post-developmental changes in the mature nervous system result in disease susceptibility and loss of function. By inactivating genes in neurons after their development is complete, this project will identify critical functions for conserved genes in aging neurons, with broad relevance for aging, disease susceptibility, and drug target discovery. PUBLIC HEALTH RELEVANCE: As neurons age, they need to maintain their function and structure, and repair themselves when damaged. This project will develop a novel genetic technology for the spatial and temporal regulation of gene inactivation. By inactivating genes in neurons after their development is complete, this project will identify critical functions for conserved genes in aging neurons, with broad relevance for aging, disease susceptibility, and drug target discovery.

DESCRIPTION (provided by applicant): There is currently no effective treatment to improve axon regeneration in humans. Thus, basic research in model organisms such as nematodes, flies, and mice is needed to provide a better understanding of the biological mechanisms that regulate regeneration. Recent work has demonstrated that the conserved DLK-1 signaling pathway is a critical regulator of regeneration. In nematodes, flies, and mice, the DLK-1 pathway regulates regeneration by modulating gene expression in injured neurons. This proposal seeks to identify the genes that are regulated by DLK-1 signaling (Aim 1), and to determine which of these genes are important for determining the regenerative potential of the injured neuron (Aim 2). These findings will expand understanding of the DLK-1 pathway, and have the potential to identify novel mechanisms for axon regeneration. This proposal uses an innovative approach in the model organism C. elegans to identify transcriptional targets of DLK-1 signaling that function in regeneration. Studies of gene transcription typically identify large numbers of targets, but it s often difficult to analyze the function of more than a few selected candidates. This proposal uses four strategies to address this difficulty. First, by using novel genetic backgrounds, analysis is focused on the transcriptional effects of a single signaling pathway-the DLK-1 pathway. Second, a novel approach is used to purify cells for transcriptional profiling, enabling analysis to be directed to a single neuronal type, the GABA motor neurons. Third, by using a novel RNAi technique, genes are knocked down only in GABA neurons for functional analysis, avoiding confounding effects and enabling the study of even essential genes. Fourth, functional analysis is performed using single-neuron laser axotomy in GABA neurons. These experiments will provide a detailed analysis of how modulation of gene expression by the DLK-1 pathway affects axon regeneration. In addition, this study will serve as a blueprint for future investigations into the mechanisms that link nerve injury, cellular signaling, gene transcription, and axon regeneration.

DESCRIPTION (provided by applicant): Axon regeneration is a fundamental and conserved property of neurons. A key question in the field is to discover what determines the regenerative capacity of injured neurons. The overarching goal of this proposal is to discover how three novel factors-Notch, Mint/LIN-10, and Rab6-function to inhibit regeneration. The three central hypotheses are: 1) that Mint/LIN- 10 and Rab6 work together in injured neurons to inhibit regeneration; 2) that they do so by promoting the intracellular trafficking of one or more inhibitory factors; and 3) that Notch signaling regulates one or more components of this inhibitory system. These hypotheses are supported by substantial preliminary data, and will be tested in the three Aims. 1) Define the regeneration function of Mint and Rab6 in motor and sensory neurons. This aim will establish how Mint/LIN-10 and Rab6 work together to inhibit axon regeneration, and how they function in different neurons. 2) Analyze Mint/LIN-10 binding partners in regeneration. This aim will identify the effector molecules that mediate Mint/LIN-10's regeneration function. 3) Investigate the cell biology of Mint, Rab6, and effectors in regulating axon regeneration. This aim will use in vivo real time imaging to determine the link between intracellular trafficking of these inhibitory factors and axon regeneration. These aims use a number of innovative techniques, and the proposal as a whole provides a major conceptual advance in our understanding of protein function and intracellular trafficking in relation to axon regeneration. By describing a novel neuronal mechanism that inhibits the ability of neurons to regenerate after injury, this proposal has the potential to significantly affect and improve curren efforts to find new treatments for damaged axons.

PROJECT SUMMARY Axon regeneration is a fundamental and conserved property of nervous systems. But axon regeneration often fails to restore function after nerve injury. Thus, a key question in the field is to discover what determines the capacity of injured neurons to rebuild functional circuits. This proposal investigates new mechanisms that function in the injured neuron and that help determine whether or not effective regeneration occurs. The long-term goal is to gain a comprehensive understanding of the cellular functions that link neuronal injury to successful functional regeneration. The specific goal of this project is to analyze the process of synapse regeneration. The project uses a combination of in vivo approaches aimed at understanding how synapse regeneration works, why it fails to restore normal function, and how it can be improved. Completion of these Aims will describe fundamental cellular mechanisms that mediate functional axon regeneration after nerve injury.

SUMMARY The CNS of adult mammals, as compared to the peripheral nervous system of mammals or the nervous system of other organisms, has extremely limited capacity for axonal regeneration. Specific factors limiting adult mammalian regeneration of axons have been identified, but they provide an incomplete explanation for poor adult mammalian CNS regeneration. We have completed a genome-wide shRNA-based screen for endogenous genes limiting the repair of axons in the mammalian CNS. We have also conducted experiments to identify conserved genes that affect axon regeneration in the model organism C. elegans. Factors common to both experimental systems are expected to identify fundamental mechanisms in regeneration that are likely to affect the equivalent process in human patients. We aim to study and develop the translational potential of those evolutionarily conserved mechanisms here. From our studies we have selected one evolutionarily conserved pathway identified both in mouse cell culture and in C. elegans axon regeneration. It is bioinformatically the most enriched gene set in the primary mammalian screen data, with multiple family members identified, and also regulates regeneration in C. elegans. The relevance of the pathway will be tested in preclinical models of traumatic spinal cord injury. Multiple steps in the pathway will be assessed in rodent spinal cord injury models. Both gene deletion strains and pharmacological inhibition will be studied to provide a validated pathway for future therapeutic development. While we will focus on one particular pathway regulating membrane traffic in the axon, we will utilize both laser axotomy and mouse spinal cord traumatic injury to explore additional pathways identified in the primary screen. This project builds on genetic screens in the mature mammalian central nervous system and C. elegans to analyze novel mechanisms that promote axon regeneration after mammalian spinal cord injury. The findings will have high relevance for the development of novel therapeutics for neurological disorders.

? DESCRIPTION (provided by applicant): The nervous system is highly susceptible to the effects of age. Aging affects the nervous system in specific and deleterious ways. Aging neurons are less able to repair injury, less able to transmit information, less able to maintain their normal morphology, and are more prone to degeneration and neurodegenerative disease. However, despite the clear evidence for functional loss, the molecular mechanisms that mediate neuronal aging are obscure. This proposal addresses aging within neurons-that is, how do individual neurons respond to age, and how do they regulate this response? At the same time, the ways in which neuronal aging may affect overall lifespan are investigated. The project focuses in particular on the dramatic loss of neuronal ability to repair damaged axons that occurs with age, as well as other aspects of neuronal aging. Preliminary data identify novel and specific cell-biological processes that affect neuronal aging. The project will elucidate the detailed mechanisms that contribute to neuronal aging, test their interdependence, and examine whether different functional aspects of neuronal aging are regulated by different or by common mechanisms. Each Aim is supported by extensive preliminary data, as well as by novel biological concepts and experimental approaches. Completion of these Aims will describe fundamental cellular mechanisms that mediate neuronal aging, and contribute to a more complete understanding of aging in complex organisms with multiple tissue types.

PROJECT SUMMARY There is a current lack of understanding of differential gene expression within the nervous system. Ideally one would like to know, across all neuron types, exactly how the genome is transcribed and processed into functional RNAs. This information is fundamentally important because differential gene expression defines the form and function of individual neurons, determines how individual neurons contribute to circuit physiology and behavior, and influences how individual neurons are affected by injury and disease. Further, detailed and complete knowledge of differential gene expression within the nervous system would help elucidate the logic and cellular mechanisms that generate neuronal diversity, including regulation of gene expression, alternative splicing, and miRNA function. Yet progress in this area has been limited: For most nervous systems, the exact number of distinct types of neurons is unknown and therefore a global map of neuron-specific gene expression is not achievable. Here we propose to address this problem in a project to discover and analyze the C. elegans Neuronal Gene Expression Map & Network (CeNGEN). The C. elegans nervous system contains precisely 302 total neurons comprising 118 classes of distinct neuronal types. We propose to exploit this unique attribute to analyze gene expression with high accuracy in every individual neuronal type. CeNGEN proceeds in four specific aims. Aim 1) Establish 118 transgenic strains, each one expressing fluorescent markers that uniquely label a single type of neuron. Aim 2) Use innovative cell dissociation and FACS methods to isolate each type of neuron from age-matched adults, and use RNA-seq approaches to assess global coding transcript and miRNA expression, as well as splicing diversity. Aim 3) Utilize single cell sequencing technology to precisely map gene expression over multiple parameter spaces. Aim 4) Build cell-centered and gene-centered expression maps, and seek connections with other uniquely known features of the C. elegans nervous system including the wiring diagram, the cell lineage, neurotransmitter identity, and function. CeNGEN represents a paradigmatic advance in neurogenetics, and provides a unique opportunity to elucidate the global control of neuron-specific gene expression and to relate gene expression to neuronal wiring and function. Expected significant outcomes include: Identification of conserved regulatory mechanisms that generate neuronal specificity and diversity; Detailed understanding of alternative splicing and miRNA function across the nervous system; Relationship of differential gene expression to neuronal lineage, anatomy, function and connectivity. CeNGEN will also serve as a resource for future studies in C. elegans neuroscience, and will provide a framework for addressing global differential gene expression in more complex nervous systems that are currently not amenable to this comprehensive approach.