Effect of ciliary neurotrophic factor (CNTF) on motoneuron survival MICHAEL SENDTNER, YOSHIHIRO ARAKAWA, KURT A. STÖCKLI, GEORG W. KREUTZBERG and HANSTHOENEN Max-Planck Institute for Psychiatry, Am Klopferspitz i8a, W-8033 Planegg-Martinsried, Germany Summary We have demonstrated that the extensive degener- ation of motoneurons in the rat facial nucleus after transection of the facial nerve in newborn rats can be prevented by local ciliary neurotrophic factor (CNTF) administration. CNTF differs distinctly from known neurotrophic molecules such as NGF, BDNF and NT-3 in both its molecular characteristics (CNTF is a cytosolic rather than a secretory molecule) and its broad spectrum of biological activities. CNTF is expressed selectively by Schwann cells and astro- cytes of the peripheral and central nervous system, respectively, but not by target tissues of the great variety of CNTF -responsive neurons. CNTF mRNA is not detectable by Northern blot or PCR analysis during embryonic development and immediately after birth. However, during the second post-natal week, a more than 30-fold increase in CNTF mRNA and pro tein occurs in the sciatic nerve. Since the period of low CNTF levels in peripheral nerves coincides with that of high vulnerability of moto- neurons (i.e. axonallesion results in degeneration of motoneuron cell bodies), insufficient availability of CNTF may be the reason for the rate of lesion- induced cell death of early post-natal motoneurons. Highly enriched embryonic chick motoneurons in culture are supported at survival rates higher than 60% by CNTF, even in single cell cultures, indicating that CNTF acts directly on motoneurons. In contrast to CNTF, the members of the neurotrophin gene family (NGF, BDNF and NT-3) do not support the survival of motoneurons in culture. However, aFGF and bFGF show distinct survival activities which are additive to those of CNTF, resulting in the survival of virtually all motoneurons cultured in the presence of CNTF and bFGF. Key words: motoneurons, ciliary neurotrophic factor, CNTF, nerve lesion, rat, chick, neurotrophic factor. Introduction Survival of spinal motoneurons during the embryonic period of naturally occurring cell death has been shown to depend on the presence of the targets of these neurons, i.e. the skeletal muscle cells (Holliday and Hamburger, 1975; Oppenheim, 1.989). Similarly, ciliary neurons, which innervate smooth and striated muscle within the eye ( Pilar et al. 1980) die after removal ofthe eye during early development (Landmesser and Pilar , 1974). Ciliary neuro- trophic factor (CNTF) was first identified and partially Journal of Cell Science, Supplement 15 , 103-109 (1991) Printed in Great Britain © The Compa ll Y of Biologists Limited 1991 purified from late embryonic chick eye tissue ( Barbin et al. 1984). The adult rat sciatic nerve was subsequently identified as the most abundant source of CNTF (Man- thorpe et al. 1986) wherefrom it was purified to homogen- eity as a basis for microsequencing and finally molecular cloning (Stöckli et al. 1989, 1991). In contrast to nerve growth factor (NGF) and other members ofthe NGF-gene family, i.e. brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), CNTF supports the survival of both cultured ciliary neurons from eight day old chick embryos (Barbin et al. 1984) and purified spinal mo tone ur- ons from six day old chick embryos (Arakawa et al. 1990) . Moreover, it has recently been shown that CNTF supports the survival of spinal motoneurons in vivo when applied daily onto the chorioallantoic membrane of chick embryos from E6 to E9 (Oppenheim et al. 1991), a time-period during which natural cell death of motoneurons occurs. The analysis of CNTF expression during embryonic development (Stöckli et al. 1991) and the perinatal period (Stöckli et al. 1989) suggests that it is highly unlikely that CNTF regulates the survival ofneurons during the per iod of regulated physiological motoneuron death: CNTF mRNA is not expressed by embryonic or adult skeletal muscle cells or any other target tissues of CNTF . responsive neurons (Stöckli et al. 1991). Moreover, the expression of CNTF by rat peripheral nerve Schwann cells does not start before postnatal day four, and the high levels found in adult animals are not reached before postnatal week three. During the early postnatal period, lesion of peripheral nerves leads to degeneration of the corresponding motoneurons. We have therefore investi- gated whether the low levels of CNTF expressed during that time period might be responsible for these high rates of lesion-mediated motoneuron death. Indeed, CNTF application to the proximal stump oflesioned facial nerves in newborn rats rescued virtually all of the axotomized motoneurons (Sendtner et al. 1990). These data indicate that CNTF might act as a 'lesion factor' in postnatal animals rather than a target-derived neurotrophic factor regulating the survival of ciliary and/ or motoneuron's during embryonic development. CNTF supports the survival of a great variety of embryonie chick neurons in culture CNTF differs significantly from target-derived neuro- trophic factors such as NGF, BDNF and NT-3, by the great variety of different neurons whose survival CNTF can support in culture. These neurons include NGF-sensitive chick EI0 sympathetic neurons and sensory neurons, but also NGF-insensitive E8 ciliary neurons or neurons from 103