NGF rescues substance P expression but not neurofilament or tubulin gene expression in axotomized sensory neurons

Abstract

A reduction in the supply of retrogradely transported NGF has been proposed as a possible signal for the axotomy response in dorsal root ganglion (DRG) neurons. Components of the axotomy response that have previously been well characterized in axotomized DRG cells include changes in cytoskeletal gene expression and changes in the expression of neurotransmitters/neuromodulators such as substance P. In this study, we examined the role of NGF in the axotomy response by examining protein synthesis and mRNA levels of the low-MW neurofilament protein (NF-L) and beta-tubulin in DRG cells at 1, 7, and 12 d after axotomy with and without continuous administration of exogenous NGF. We also examined substance P levels in the DRG by immunocytochemistry under the same experimental conditions. Sciatic nerves of adult male rats were unilaterally transected at the midthigh level, and the proximal nerve stumps were placed into Silastic tubes connected to osmotic minipumps that were filled with biologically active NGF. NGF (0.5 mg/ml in saline) was continuously infused (0.5 microliter/hr) onto the proximal stumps of transected sciatic nerves for 1-12 d. Control animals were prepared in an identical fashion except that the nerves were treated with saline alone. At death, DRGs were removed from the animals; the L4 experimental DRGs (axotomized) and contralateral L4 DRGs (uninjured) were used immediately for protein synthesis experiments, while the experimental and contralateral L5 DRGs were fixed in 4% paraformaldehyde and subsequently used for in situ hybridization and immunocytochemistry. From another set of experimental animals, the L4 and L5 DRGs were harvested and used for total RNA isolation and RNA blotting experiments. Immunocytochemical studies using a polyclonal antibody to substance P showed that the immunodetectable levels of this peptide decreased to undetectable levels in DRG neurons after axotomy and saline administration. However, in axotomized neurons treated with NGF, the level of immunodetectable substance P did not decrease, but instead, increased over even that present in normal DRG neurons. Pulse labeling of DRGs with 35S-methionine:cysteine followed by 2-dimensional (2D) gel electrophoresis and fluorography revealed that the synthesis of neurofilament (NF) proteins was decreased, while that of tubulin was increased, 12 d after sciatic nerve transection. NGF administration to axotomized neurons did not alter this pattern. Quantitative analysis of in situ hybridizations of DRG neurons and RNA blot analysis with cDNA probes specific for NF-L and beta-tubulin mRNAs showed that NGF treatment of axotomized DRGs did not significantly affect cytoskeletal gene expression at the mRNA level.(ABSTRACT TRUNCATED AT 400 WORDS)