Astrocytes respond to injury of the central nervous system with a dramatic change in morphology, resulting in the formation of a dense glial scar that can inhibit axonal regeneration. The molecular basis for the """"""""reactive astrocyte"""""""" phenotype is not understood. We recently identified a novel cytoskeleton-associated protein named palladin, which plays an essential role in maintaining the actin cytoskeleton in many cell types, This proposal explores the role of palladin in the response of astrocytes to mechanical injury, both in vitro and in vivo. Our hypothesis is that palladin functions as a molecular scaffold to organize the actin cytoskeleton and promote a change in cell shape of astrocytes in response to a specific stimulus. We obtained preliminary evidence that palladin is rapidly upregulated in cultured astrocytes in response to mechanical wounding of the cell monolayer. In addition, we show that palladin upregulation occurs along a similar and rapid time-course following injury to the cerebral cortex in adult rats. The goals of the proposed research are to answer the following questions: (1) Palladin upregulation correlates closely with a change in cell shape from stellate to flattened. Is palladin expression directly responsible for this change in shape? This question will be answered using transient transfection techniques to increase and decreasepalladin expression in cultured astrocytes. (2) What are palladin's binding partners in astrocytes, and are they coordinately upregulated in astrocytes following mechanical injury? Based on sequence homologies, we have compiled a list of five proteins that are likely to bind directly to palladin. We will explore these interactions in cultured astrocytes and also search more broadly using a yeast two-hybrid screen. (3) Is palladin upregulated in astrocytes following injury to the cortex in vivo? Double-label immunofluorescence will be used to definitively identify the cell types that upregulate palladin and to quantify the expression of palladin, in these cells, at the margins of the wound. Finally, viral vectors will be used to ask if glial scar formation is attenuated or inhibited when palladin expression is reduced in astrocytes in the site of injury. These experiments are expected to produce new insights into the basic cellular processes that underlie the response of astrocytes to an injury stimulus. A long-term goal of this research is to provide new therapeutic approaches for controlling the astrocytic response and glial scar formation in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS043253-05
Application #
7086788
Study Section
Molecular, Cellular and Developmental Neurosciences 2 (MDCN)
Program Officer
Kleitman, Naomi
Project Start
2002-09-30
Project End
2009-07-31
Budget Start
2006-08-01
Budget End
2009-07-31
Support Year
5
Fiscal Year
2006
Total Cost
$303,698
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Physiology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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Dixon, Richard D S; Campbell, Sharon L (2008) 1H, 15N, and 13C NMR chemical shift assignments for the Ig3 domain of palladin. Biomol NMR Assign 2:51-3
Dixon, Richard D S; Arneman, Daniel K; Rachlin, Andrew S et al. (2008) Palladin is an actin cross-linking protein that uses immunoglobulin-like domains to bind filamentous actin. J Biol Chem 283:6222-31
Ronty, Mikko; Taivainen, Anu; Heiska, Leena et al. (2007) Palladin interacts with SH3 domains of SPIN90 and Src and is required for Src-induced cytoskeletal remodeling. Exp Cell Res 313:2575-85
Rachlin, Andrew S; Otey, Carol A (2006) Identification of palladin isoforms and characterization of an isoform-specific interaction between Lasp-1 and palladin. J Cell Sci 119:995-1004
Pogue-Geile, Kay L; Chen, Ru; Bronner, Mary P et al. (2006) Palladin mutation causes familial pancreatic cancer and suggests a new cancer mechanism. PLoS Med 3:e516
Goicoechea, Silvia; Arneman, Daniel; Disanza, Andrea et al. (2006) Palladin binds to Eps8 and enhances the formation of dorsal ruffles and podosomes in vascular smooth muscle cells. J Cell Sci 119:3316-24
Otey, Carol A; Carpen, Olli (2004) Alpha-actinin revisited: a fresh look at an old player. Cell Motil Cytoskeleton 58:104-11