THE NON-IMMUNOSUPPRESSIVE IMMUNOPHILIN LIGAND GPI-1046 POTENTLY STIMULATES REGENERATING AXON GROWTH FROM ADULT MOUSE DORSAL ROOT GANGLIA CULTURED IN MATRIGEL Z. KHAN, a G. FERRARI, a M. KASPER, a D. A. TONGE, b J. P. STEINER, c G. S. HAMILTON a and P. R. GORDON-WEEKS aà a MRC Centre for Developmental Neurobiology, King’s College London, Room 4.26B, New Hunts House, Guy’s Campus, London SE1 1UL, UK b Physiology Group, Division of Biomedical Sciences, King’s College London, Hodgkin Building, Guy’s Campus, London SE1 1UL, UK c Guilford Pharmaceuticals Inc., 6611 Tributary Street, Baltimore, MD 21224, USA AbstractöWe used explant cultures of adult mouse dorsal root ganglia with spinal nerve attached growing in Matrigel to assess the e¡ects of the non-immunosuppressive immunophilin ligand GPI-1046 [Snyder et al. (1998) TIPS 19, 21^26] on the growth rate of regenerating sensory axons and found a potent stimulation of axon growth. In these explant cultures, naked, unfasciculated axons emerge from the cut end of the spinal nerve and continue to grow in the Matrigel for up to eight days [Tonge et al. (1996) Neuroscience 73, 541^551]. Some axons are entirely smooth whilst others show prominent varicosities. Some of the former express the phosphorylated neuro¢lament epitope recognised by monoclonal antibody RT97, a marker for large calibre, myelinated axons, whilst the latter express calcitonin gene-related peptide, predom- inantly a marker for unmyelinated, and small diameter myelinated sensory axons. Many of the axons in these cultures also express the low-a⁄nity neurotrophin receptor p75. GPI-1046 has been shown to have striking stimulatory e¡ects on embryonic primary sensory axons growing in vitro and it was therefore of interest to see whether it could also enhance regenerating sensory axon growth from the adult ganglia in our cultures. GPI-1046 potently stimulated axon growth in our cultures in a dose-dependent manner. The stimulatory e¡ect was not dependent on the class of sensory axon. These observations show that GPI-1046 is a potent stimulator of regenerating axons from adult, primary sensory neurones. The cellular site of action of GPI-1046 is unknown. To distinguish between a direct e¡ect of the drug on neurones and an indirect e¡ect we compared the e¡ects of GPI-1046 on explant and dissociated cultures. In con¢rmation of previous results, we found that GPI-1046 potently stimulated axon outgrowth from explants of embryonic chick dorsal root ganglia. However, the drug was without e¡ect on dissociated embryonic dorsal root ganglion neurones, suggesting that non-neuronal cells are important for axon growth stimulation. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: axonogenesis, growth cone, regeneration. The immunosuppressant drugs, cyclosporin A and FK506 exert their immunosuppressive e¡ects through high a⁄nity binding to the cytoplasmic proteins called immunophilins: cyclophilin and FK506 binding protein (FKBP), respectively (reviewed by Marks, 1996). The drug^immunophilin complex binds to the calcium and calmodulin-dependent protein phosphatase 2B (calci- neurin), inhibiting its activity and causing the accumula- tion of phosphorylated calcineurin substrates (Liu et al., 1991). In T lymphocytes, one of the calcineurin sub- strates is nuclear factor of activated T cells (NFAT), which enters the nucleus to stimulate interleukin 2 for- mation only when dephosphorylated. Thus, inhibition of calcineurin prevents NFAT from entering the nucleus and thereby stimulating interleukin 2 formation and the mounting of an immune response. The immunophilins are far more abundant in the adult nervous system than the immune system (Maki et al., 1990; Steiner et al., 1992; Lyons et al., 1995). In the brain, there is a striking regional variation in the expres- sion of mRNA for FKBP and binding sites for [ 3 H]FK506, which is similar to that for calcineurin mRNA. Although the function of the immunophilins in the nervous system is not known, several lines of evi- dence suggest that they play a role in axonogenesis (reviewed by Snyder and Sabatini, 1995; Snyder et al., 1998). The expression of mRNA for FKBP-12 is up- regulated in the neuronal cell bodies of the facial nucleus and the lumbar motor neurone pool and dorsal root 601 *Corresponding author. Tel.: +44-20-78486467; fax: +44-20- 78486798. E-mail address: phillip.gordon-weeks@kcl.ac.uk (P. R. Gordon-Weeks). Abbreviations: BDNF, brain-derived neurotrophic factor; CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglion; FKBP-12, FK506 binding protein 12; GDNF, glial-derived neu- rotrophic factor; HEPES, N-(2-hydroxyethyl)piperazine-NP-(2- ethanesulfonic acid); mAb, monoclonal antibody; NFAT, nuclear factor of activated T cells; NGF, nerve growth factor; PBS, phosphate-bu¡ered saline; SDS^PAGE, sodium dodecyl sulphate^polyacrylamide gel electrophoresis. NSC 5747 22-8-02 Cyaan Magenta Geel Zwart www.neuroscience-ibro.com Neuroscience Vol. 114, No. 3, pp. 601^609, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain PII:S0306-4522(02)00314-7 0306-4522/02 $22.00+0.00