Marc Tessier-Lavigne - US grants

A major challenge in the study of mammalian brain development is to identify the extracellular ligands and receptors that direct the assembly of the complex wiring pattern of the brain. A recent modification of the gene trap technique in embryonic stem cells, the "secretory trap", allows the systematic trapping of genes encoding ligands and receptors, and the generation of mice harboring mutations in these genes, providing an important tool to elucidate the functions of these genes. However, the problem with studying brain wiring is that it can be extremely difficult to identify changes in wiring resulting from experimental perturbation because of the difficulty of tracing the axonal projections of neurons. In particular, it is usually extremely difficult to identify wiring defects in mutant mice, whether the mutation arose spontaneously or was generated by gene targeting or insertional mutagenesis. We have conceived a further modification of the gene trap technique that should now dramatically facilitate the identification and characterization of receptors involved in wiring the mammalian nervous system. In our method, a histochemical axonal marker is targeted to neurons that normally express the trapped gene. This enables direct visualization of the connections made by these neurons, and direct visualization of wiring defects in homozygous mutant animals, through a simple histochemical stain. In this way the role of the trapped gene in brain wiring can be rapidly assessed. Of equal importance, and of great benefit to the Neuroscience community at large, the method will also simultaneously result in the generation of a bank of mice expressing the histochemical marker in different populations of axons, which will provide an important tool for researchers interested in elucidating the normal pattern of neuronal connections in the mammalian brain. A request is made here to support the isolation of a large number of secretory trap ES cell lines using this modified vector. It is proposed that 80 of these lines per year will be put through the germ line for expression and phenotypic analysis to identify those involved in wiring the nervous system, and to provide a resource of mice expressing the axonal marker in different populations of neurons for use by the Neuroscience community. We expect that this method will help greatly accelerate the pace of discovery of mechanisms that direct the wiring of the mammalian nervous system.

The long-term aim of this proposal is to understand the mechanisms that direct axons to their target fields in the trigeminal sensory system. In the last cycle of this grant, we studied and identified molecularly a chemotropic activity, Maxillary Factor, that was implicated in directing the gross pattern of trigeminal sensory axon projections to the periphery. Here, we propose to use single-cell cDNA differential screening technology and cDNA microarray technology to study the mechanisms responsible for the more discrete pathway choices made by subsets of trigeminal sensory neurons to form (1) the three trigeminal nerve branches, (2) the topographic projections to individual tactile hairs of the whisker pad, and (3) the divergent central terminations of large and small diameter sensory neurons. The approach will be to identify genes that are differentially expressed in subpopulations of trigeminal neurons projecting among distinct pathways, and to test the involvement in axon guidance of any of these genes that encode transmembrane proteins. The long-term aim is to define transmembrane receptors involved in guiding axons along different pathways, though the experiments should also reveal other molecular differences among the neurons, including in expression of transcription factors that regulated axon guidance receptor expression. This work will help define the molecular differences that regulate the distinct pathway choices of trigeminal sensory axons. In addition, it should be of more general importance in providing insight into the mechanisms through which discrete axonal pathway choices and topographic axonal projections are formed, two fundamental issues in axon guidance that are still poorly understood.

DESCRIPTION (provided by applicant): Neuronal cell death and axonal degeneration are major pathological features of most neurodegenerative diseases16. Because neuronal cell loss is most commonly associated with symptom onset, considerable research efforts have been focused on understanding the mechanisms that regulate neuronal cell death in order to prevent neuronal loss in these diseases. However, it is now appreciated that axon degeneration can often precede both neuronal cell death and symptom onset, suggesting that it may be an underlying etiology of neurodegenerative diseases thus prompting researchers to pursue it as a novel therapeutic point of intervention 16. One commonly observed form of axon degeneration, termed Wallerian degeneration, is best known as the program responsible for destroying and clearing axon segments distal to a physical injury site. However, the Wallerian degeneration program operates not only in injury induced axon degeneration but also during the pathological axon degeneration associated with several neurodegenerative diseases1,2. Our collaborators and we have recently identified the first endogenous gene, SARM1, responsible for executing the Wallerian degeneration program in both invertebrates and vertebrates7. Here, we propose to further our understanding of axon degeneration in the mammalian nervous system by using SARM1 as a novel entry point for revealing new molecular mechanisms of Wallerian degeneration of axons. We will first focus on examining SARM1 localization and function in injured neurons and their axons, and determine how SARM1 promotes Wallerian degeneration of axons following injury. We next aim to identify and characterize novel components of the Wallerian degeneration pathway by isolating SARM1 interacting proteins and using a candidate approach to screen through genes that may be part of an evolutionary conserved SARM1 signaling pathway. Finally, we will assess the role of the SARM1 signaling pathway in vivo in injury-induced and chemically-induced neuropathies. We expect that the experiments outlined in this proposal will lead to a better understanding of mechanisms that regulate axon degeneration and will likely be helpful in identifying novel targets for treating a number of neurodegenerative diseases.

DESCRIPTION (provided by applicant): Axon pruning or degeneration occurs widely during development, as part of axonal rearrangements following injury and in adult plasticity, and is a major pathological feature of most neurodegenerative diseases. However, the molecular mechanisms that initiate and execute axon degeneration in these diverse settings remain incompletely understood. Recently, we identified a caspase-dependent pathway that regulates axon degeneration both in vitro and in vivo. These findings have provided a new entry point for determining the molecular components that regulate axon degeneration. This proposal is focused on identifying key components of the biochemical pathway of caspase-dependent axon degeneration that lead to degeneration in the developing and diseased peripheral nervous system. We will use complementary approaches to (i) rapidly screen mice mutant for various pathway components to determine which ones regulate degeneration of developing sensory and motor axons in response to trophic deprivation in vitro, (ii) identify additional components of the degeneration pathway through biochemical analysis and siRNA screening, (iii) elucidate mechanisms of caspase regulation (iv) determine the contribution of the apoptotic pathway to developmental axon pruning in vivo, and (v) test for disease relevance by crossing relevant mutants to a mouse model of sensory axon degeneration. These studies will identify novel mechanisms regulating developmental and pathological axon degeneration, including potential therapeutic targets.