Proteoglycans (PGs) are a structurally diverse class of molecules that interact with many extracellular matrix (ECM) and cell surface components. PGs play a role in many cellular processes, including inhibition of retinal ganglion and dorsal root ganglion cell outgrowth. The mechanisms by which PGs inhibit neurite outgrowth are unknown. Therefore, the goal of this proposal is to investigate the hypothesis that PGs may inhibit growth cone migration by two fundamental mechanisms: 1) limiting access to growth promoting adhesive molecules, and 2) triggering the reorganization of the growth cone cytoskeleton through transient rises in [Ca2+]. First, we will use 35S-CSPG and 3H-laminin to determine the amounts of these molecules bound to the substrata in DRG cultures. We will then do behavioral assays to determine whether PGs limit cell surface access to the growth-promoting adhesive molecule laminin, and to determine whether such masking can lead to growth cone turning at a PG border. Second, we will determine if there are changes in the cytoskeletal proteins tubulin and actin, or in related proteins, as growth cones contact and are inhibited by PGs. We will use injection of fluorescent actin and tubulin, or immunocytochemistry of fixed cultures, and treatment with cytochalasin to inactivate growth cone filopodia. Third, contact with PGs induces a large rise in [Ca2+]i within growth cones. Therefore, we will determine the role of the transient elevation of [Ca2+]i in growth cones that contact CSPG, and test whether changes in [Ca2+]i can induce inhibition of growth cone migration. Fluorescence imaging of the calcium indicator fura-2/AM will be used in combination with pharmacological reagents to block the influx or release of Ca2+. Elevation of [Ca2+]i may affect the morphology and trajectory of the elongating growth cone, 1) through direct action on cytoskeletal components of the growth cone, or 2) through second messenger systems that regulate the dynamic motile apparatus of growth cones. These experiments are important because: 1) they will lead to an understanding of the mechanisms by which PGs regulate growth cone guidance, and 2) they may provide molecular methods by which to manipulate PGs in adult tissue to enable CNS regeneration, since PGs are expressed and are inhibitory to regenerating nerve cells, following CNS injury.
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