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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29EY010545-04
Application #
2545870
Study Section
Visual Sciences C Study Section (VISC)
Project Start
1994-09-30
Project End
1999-09-29
Budget Start
1997-09-30
Budget End
1998-09-29
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Kentucky
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
832127323
City
Lexington
State
KY
Country
United States
Zip Code
40506
Heron, P M; Sutton, B M; Curinga, G M et al. (2007) Localized gene expression of axon guidance molecules in neuronal co-cultures. J Neurosci Methods 159:203-14
Hynds, Dianna L; Rangappa, Nagarathnamma; Ter Beest, Julia et al. (2004) Microglia enhance dorsal root ganglion outgrowth in Schwann cell cultures. Glia 46:218-23
Snow, Diane M; Smith, Jeffrey D; Cunningham, Andrew T et al. (2003) Neurite elongation on chondroitin sulfate proteoglycans is characterized by axonal fasciculation. Exp Neurol 182:310-21
Hynds, DiAnna L; Spencer, Mike L; Andres, Douglas A et al. (2003) Rit promotes MEK-independent neurite branching in human neuroblastoma cells. J Cell Sci 116:1925-35
Hynds, DiAnna L; Snow, Diane M (2002) A semi-automated image analysis method to quantify neurite preference/axon guidance on a patterned substratum. J Neurosci Methods 121:53-64
Johnson, William E B; Caterson, Bruce; Eisenstein, Stephen M et al. (2002) Human intervertebral disc aggrecan inhibits nerve growth in vitro. Arthritis Rheum 46:2658-64
Snow, Diane M; Smith, Jeffrey D; Gurwell, Julie A (2002) Binding characteristics of chondroitin sulfate proteoglycans and laminin-1, and correlative neurite outgrowth behaviors in a standard tissue culture choice assay. J Neurobiol 51:285-301
Hynds, D L; Takehana, A; Inokuchi, J et al. (2002) L- and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) inhibit neurite outgrowth from SH-SY5Y cells. Neuroscience 114:731-44
Snow, D M; Smith, J D; Booze, R M et al. (2001) Cocaine decreases cell survival and inhibits neurite extension of rat locus coeruleus neurons. Neurotoxicol Teratol 23:225-34
Snow, D M; Mullins, N; Hynds, D L (2001) Nervous system-derived chondroitin sulfate proteoglycans regulate growth cone morphology and inhibit neurite outgrowth: a light, epifluorescence, and electron microscopy study. Microsc Res Tech 54:273-86

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