This is a competing continuation grant seeking five years of support. The objectives of the present grant period were to investigate the effects of soluble trophic factors and glial cell surface molecules in regulating the survival and development of hypothalamic neurons. In the next project period we will extend and amplify this work to the role of neural/glial interactions in shaping the development and regeneration of the nervous system. We have identified a population of type 1 astrocytes (named """"""""rocky"""""""") which are strongly inhibitory to neuronal adhesion and growth, in contrast to the vast majority of """"""""flat"""""""" astrocytes which are permissive to both neuronal adhesion and growth. The rocky astrocyte extracellular matrix (ECM) contains high levels of tenascin and chondroitin sulfate-6- proteoglycan (CS-6-PG), molecules that have been associated with boundary formation in the brain. In response to certain cytokines, flat astrocytes synthesize tenascin and CS-6-PG and become unfavorable for neuronal growth, resembling rocky astrocytes. We propose that, during development and following injury, cytokines have significant actions on astrocytes to alter their expression of cell surface molecules that then alter neuronal migration and neuronal growth. We have three related Specific Aims.
Aim 1 will test the hypothesis that cytokines alter permissive flat astrocytes in ways that make them inhibitory to neuronal growth and regeneration. Cultured rat cerebral cortical astrocytes will be exposed to gamma-IFN, TNF-alpha, and TGF-beta1. We will evaluate changes in the composition of the ECM of these astrocyte monolayers using Western blotting and immunocytochemical techniques and also evaluate the adhesion and outgrowth of dissociated embryonic neurons to these cells as well as to ECM preparations of these cells. The dynamic movement of neuronal growth cones on these monolayers will be evaluated using time-lapse video enhanced microscopy and laser scanning confocal microscopy.
Specific Aim 2 will test whether the expression of human tenascin in rat astrocytes (from a transgene) leads directly to inhibition of neuronal growth in vitro. Neuronal adhesion and growth will be evaluated on cells with different levels of tenascin expression. We will also test the hypothesis that the balance of expression of permissive vs. non-permissive ECM molecules on cells regulates neuronal adhesion and growth. Finally, we will evaluate the effects of the expression of the alternately spliced variants of human tenascin.
Specific Aim 3 will test the effects of blocking tenascin and CS-6-PG with antibodies in order to evaluate the hypothesis that these ECM components have a synergistic, inhibitory effect on neuronal adhesion and neurite outgrowth. We will also evaluate whether the sugar or the protein moieties of the proteoglycans are involved in modulation of neuronal adhesion/growth. We will also test the hypothesis that blocking tenascin and CS-6-PG will reduce or eliminate the ability of growth cones to sense boundaries between permissive and non-permissive astrocytes. The experiments proposed provide a unified approach to a major problem of neural/glial interactions during development and may also apply to the lack of regeneration following injury.
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