Experiments will be carried out to characterize the molecular mechanisms that regulate and coordinate the expression of the neurofilament (NF) triplet proteins. Cis-acting elements in the 5' flanking (and other) regions of the three mouse NF genes will be mapped by cell transfection and by in vitro transcription. Binding sites of trans-acting factors that are located in the same regions of the NF genes will be identified by gelshift, methylation interference and DNase I footprinting. The effects of the respective binding factors on NG transcription will be examined by comparing transcriptional activities of NF constructs bearing wild vs mutant (non-binding) binding sites. The presence of binding factors will be compared in cells and in tissues that express varying levels of endogenous NF genes, including neuronal (PC12, and Neuro 2a) vs non- neuronal (HeLa and L cells) cell lines and in high- (DRG), low- (brain) and non- (liver) expressing tissues of the rat (or mouse). Transcriptional factors that are unique to high-expressing tissues and bind to regulatory elements in one or more NF genes will be cloned and further characterized. The extent of transcriptional vs post-transcriptional control of NF expression will be analyzed in primary cultures of rat DRG neurons. The ability of axonal factors (or signals) to regulate NF expression will be studied by further examining the downregulation of NF expression in rat DRG neurons following sciatic nerve transection under conditions in which the microenvironment of the severed axons are controlled and manipulated. The overall intent of the research project is to elucidate the factors that regulate NF expression and to begin to identify and characterize these factors. NF proteins represent the most abundant neuron-specific gene product. In addition, NF expression is highly instrumental in several basic neuronal events, such as neuronal differentiation and development, the maintenance of steady-state neuronal gene expression and the regenerative response following neuronal injury. Insight into the factors that regulate NF gene expression is likely to reflect on the nature of fundamental phenomena that underlie and determine the structure and function of the neuron.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS015722-16
Application #
3396451
Study Section
Neurology B Subcommittee 2 (NEUB)
Project Start
1979-02-01
Project End
1996-05-31
Budget Start
1993-06-01
Budget End
1994-05-31
Support Year
16
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Wu, Junhua; Zhai, Jinbin; Lin, Hong et al. (2003) Cytoplasmic retention sites in p190RhoGEF confer anti-apoptotic activity to an EGFP-tagged protein. Brain Res Mol Brain Res 117:27-38
Zhai, Jinbin; Lin, Hong; Nie, Zhenying et al. (2003) Direct interaction of focal adhesion kinase with p190RhoGEF. J Biol Chem 278:24865-73
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Canete-Soler, R; Silberg, D G; Gershon, M D et al. (1999) Mutation in neurofilament transgene implicates RNA processing in the pathogenesis of neurodegenerative disease. J Neurosci 19:1273-83
Schwartz, M L; Hua, Y; Canete-Soler, R et al. (1998) Characterization of the mouse neurofilament light (NF-L) gene promoter by in vitro transcription. Brain Res Mol Brain Res 57:21-30
Schwartz, M L; Hua, Y; Schlaepfer, W W (1997) In vitro activation of the mouse mid-sized neurofilament gene by an NF-1-like transcription factor. Brain Res Mol Brain Res 48:305-14
Schwartz, M L; Bruce, J; Shneidman, P S et al. (1995) Deletion of 3'-untranslated region alters the level of mRNA expression of a neurofilament light subunit transgene. J Biol Chem 270:26364-9
Schwartz, M L; Shneidman, P S; Bruce, J et al. (1994) Stabilization of neurofilament transcripts during postnatal development. Brain Res Mol Brain Res 27:215-20

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