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)

A Transcription-Dependent Switch Controls Competence of Adult Neurons for Distinct Modes of Axon Growth

“…3A), and subpopulations of DRG neurons differ in their propensity to extend arborizing processes under various conditions. DRG cells are a heterogeneous population of neurons that innervate distinct targets and, in the absence of injury, express different neurotrophin receptors, synaptic components, and growth-associated proteins (Schreyer and Skene, 1991;Wong and Oblinger, 1991;Weise et al, 1992;Zhang et al, 1995;Snider and Wright, 1996). Thus, although genes expressed in adult DRG neurons are sufficient for axonal arborization, differential expression of additional growthrelated genes may account for significant variation among subpopulations of DRG neurons in the type and extent of arborizing growth they can support and in the types of neurotrophins or Figure 8.…”

Differential expression of estrogen receptor alpha and beta in rat dorsal root ganglion neurons

“…Neuropeptide expression in the DRG system is known to be modulated by nerve growth factor (NGF; Fitzgerald et al, 1985;Verge et al, 1995;Wong and Oblinger, 1991) and several reports have shown that estrogen treatment alters the mRNA levels of trkA, the high-affinity NGF receptor, in the DRG (Liuzzi et al, 1999b;Sohrabji et al, 1994). Interestingly, acute vs. chronic estrogen treatment has very different effects on trkA mRNA levels (decreases vs. increases, respectively) in the small neurons of the DRG which express this neurotrophin receptor (Liuzzi et al, 1999a,b).…”

Neuropeptides and neurotrophin receptor mRNAs primary sensory neurons of aged rats

“…Neurotrophins can also influence the cellular expression of neuropeptides such as CGRP, SP, and NPY (e.g., Goedert et al, 1981;Schwartz et al, 1982;Fitzgerald et al, 1985;Lindsay and Harmar, 1989;Wong and Oblinger, 1991 Jones et al, 1994;Mulderry, 1994;Verge et al, 1995;Watson, 1995). It can therefore not be excluded that the changed expression of neuropeptides during aging, or as a result of axotomy, is mediated by changes in the availability of neurotrophins and/or the expression of the neurotrophin receptors.…”

“…With regard to sensory neurons, it has been demonstrated that axonal injury also influences expression of neurotrophin receptors (Verge et al, 1989(Verge et al, , 1992. Moreover, several studies have shown that the expression of a number of neuropeptides such as CGRP, SP, and NPY in sensory neurons can be influenced by neurotrophic factors (e.g., Goedert et al, 1981;Schwartz et al, 1982;Fitzgerald et al, 1985;Lindsay and Harmar, 1989;Wong and Oblinger, 1991;Jones et al, 1994;Mulderry, 1994;Verge et al, 1995;Watson, 1995). Based on these evidences, it has been suggested that the changes in neuropeptide expression seen in sensory neurons following axonal lesions may at least in part be the result of decreased neurotrophic signalling.…”