Abstract

Epithelial fatty acid-binding protein (E-FABP) is expressed in the dorsal root ganglia following sciatic nerve injury and in migrating and differentiating neurons during neuronal development. It was hypothesized that E-FABP expression is required for the robust outgrowth of axons from developing and regenerating neurons. To test this hypothesis, E-FABP expression in both PC12 cells and primary retinal neurons was examined. In PC12 cells, NGF induces E-FABP mRNA and protein during the period of neurite outgrowth, and E-FABP localizes to the perinuclear cytoplasm, nucleus, and growth cone. Furthermore, E-FABP-deficient cell lines exposed to NGF were less differentiated and had shorter neurites relative to control cells. In developing retina, EFABP localized to retinal ganglion cells (RGCs) from E14 through P10, being confined to RGC somas on E14 but localized to axons by E15. Western immunoblotting revealed that on E18, P1, and P10, E-FABP levels were 3-4 times greater than adult levels, but decreased by 50% on P15. Furthermore, dissociated P15 retinal cells cultured in the presence of neurotrophins contained 6-fold more E-FABP-positive RGCs relative to controls. Taken together, these results indicate that: (1) E-FABP is expressed in differentiating PC12 cells and RGCs, (2) E-FABP plays a functional role in the elaboration of neurites/axons in PC12 cells and RGCs.

A vaccinia virus (VV) gene transduction system was developed to facilitate future gene transfer experiments in primary neurons. VV was able to infect retinal and PC12 cells in a virus dose-dependent manner. 50-100% of retinal cells were positive for virus infection at multiplicities of infection (MOI) between 10 and 100 while over 50% of PC12 cells were infected at a MOI of 10. The production of foreign mRNA and protein by VV was verified, as was the simultaneous production of two transgenes from a dual promoter viral construct. Retinal cultures maintained for 0.5 days in vitro (DIV) showed greater than 90% survival at 24 h post-infection, while 14 DIV cultures were equally viable for 48 h. These data suggest that VV may be a useful vector for delivering foreign genes to neuronal cells with a high degree of efficiency over a short time course.

LLU Discipline

Physiology

Department

Physiology

School

Graduate School

First Advisor

Marino De León

Second Advisor

Istvan Fodor

Third Advisor

Michael Kirby

Fourth Advisor

Lawrence D. Longo

Fifth Advisor

Barry Taylor

Sixth Advisor

Andrew Welcher

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

2000

Date (Title Page)

6-2000

Language

English

Library of Congress/MESH Subject Headings

Protein Binding; Fatty Acids -- metabolism; Gene Expression Regulation, Developmental; Neurons -- growth and development; Fatty Acids -- metabolism.

Type

Dissertation

Page Count

x; 169

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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