Maureen L. Condic - US grants

The adult central nervous system (CNS) does not readily support axonal regeneration following injury. In contrast, there is extensive regeneration and functional recovery in embryos following CNS damage. The inability of the adult CNS to regenerate appears to be due both to changes in the extracellular environment of the nervous system that did make it less permissive to neuronal growth and to changes in the intrinsic ability of adult neurons to regenerate damage axons. By the end of embryonic development, the expressions of growth-promoting or stimulatory extracellular matrix molecules in the CNS is quite low. Myelin-associated factors found in mature brain tissue actively inhibit axonal growth and following injury, several additional inhibitory proteins are up-regulated in regions of scarring. Compounds these less than ideal extracellular conditions, the regeneration of adult neurons is somehow compromised by changes associated with neuronal maturation. Recent work has shown that embryonic neurons readily adapt both to low availability of growth promoting molecules and to the presence of inhibitory compounds by regulating the expression of integrins--the receptors extracellular matrix proteins that mediate axon extension. The compensatory regulation of integrin by components of the matrix has not been previous described for any cell type, may contribute to the superior regenerative performance of embryonic neurons under less than ideal extracellular conditions. Integrins are known to interact with both extracellular and cytoskeletal proteins. In Aim 1, the contributions of ligand and cytoskeletal binding to the regulation of integrin expression will be examined using antibodies, pharmacological reagents and integrin mutant constructs. The contribution of integrin regulation to the adaptation of embryonic neurons to inhibitory matrix components will be characterized in Aim 2. Neuronal response to members of two major inhibitory families that are expressed in the CNS following injury will be determined. The role of one inhibitory proteoglycan (aggrecan) in neuronal development will be examined using mutant animals that do not express aggrecan. Experiments proposed in Aims 3 and 4 will determine whether increased integrin expression is sufficient to mediate neuronal adaptation to both low availability of ligand and to inhibitory molecules (conditions similar to those in the adult CNS following injury). Integrin expression will be increased in both embryonic and adult neurons using replication- deficient adenoviral constructs. The performance of neurons with increased integrin expression will be examined on substrata that would otherwise not support neuronal growth. In summary, these experiments will greatly strengthen our understanding of two novel forms of integrin regulation, and potentially provide a basis for improving the regenerative performance of adult neurons.

DESCRIPTION (provided by applicant): In contrast to our growing understanding of spinal cord development, the factors controlling specification and patterning of peripheral sensory neurons are poorly understood. Recent work from our laboratory provides evidence that sensory neurons have unique phenotypes prior to their extension into the periphery. Using a new method to enrich for populations of early sensory neurons with different developmental fates, we have shown that sensory neuron subtypes (e.g. cutaneous versus proprioceptive) have unique patterns of gene expression and differing abilities to interact with matrix proteins shortly after they are born. This work provides evidence for the early specification of sensory neurons, independent of target interactions, and thereby limits the developmental window over which sensory neuron specification can occur. In addition, our results have, for the first time, provided valuable prospective markers for sensory neuron subtypes that will greatly enhance future studies of sensory neuron development. We will build on our recent findings in three ways. In Aim 1, we will exploit the new method we have developed to screen gene expression profiles of different sensory subtypes. We will focus on axon guidance molecules known to direct the pathfinding of central neurons. This work will both expand our available markers for further study of sensory development and provide useful information regarding sensory pathfinding, a question about which very little is currently known. In Aim 2, we will pursue our observation that sensory specification must occur early in the development. Preliminary evidence presented in this proposal suggests that molecules involved in the patterning and specification of central neurons also influence the developing dorsal root ganglia. We will examine gain and loss of function experimental conditions to determine whether specific patterning molecules are necessary and sufficient to alter sensory neuron proliferation, death and patterning. In Aim 3, we will pursue our initial findings that members of the netrin-1 signaling pathway are differentially expressed between different prospective classes of sensory neurons to ask what role netrin-1 signaling may play in the development of central and peripheral sensory projections. We will experimentally test three potential roles for netrin-1 in sensory development; the regulation of initial proprioceptive outgrowth, control of the timing for proprioceptive central projections and the establishment of dorsal projections of motor and sensory neurons in the dorsal ramus.