BIOLOGICAL STUDIES OF A PUTATIVE AVIAN MUSCLE-DERIVED NEUROTROPHIC FACTOR THAT PREVENTS NATURALLY-OCCURRING MOTONEURON DEATH IN-VIVO

A series of in vivo studies have been carried out using the chick embr yo to address several critical questions concerning the biological, an d to a lesser extent, the biochemical characteristics of a putative av ian muscle-derived trophic agent that promotes motoneuron survival in vivo. A partially purified fraction of muscle extract was shown to be heat and trypsin sensitive and rescued motoneurons from naturally occu rring cell death in a dose-dependent fashion. Muscle extract had no ef fect on mitotic activity in the spinal cord and did not alter cell num ber when administered either before or after the normal cell death per iod. The survival promoting activity in the muscle extract appears to be developmentally regulated. Treatment with muscle extract during the cell death period did not permanently rescue motoneurons. The motoneu ron survival-promoting activity found in skeletal muscle was not prese nt in extracts from a variety of other tissues, including liver, kidne y, lung, heart, and smooth muscle. Survival activity was also found in extracts from fetal mouse, rat, and human skeletal muscle. Conditione d medium derived from avian myotube cultures also prevented motoneuron death when administered in vivo to chick embryos. Treatment of embryo s in ovo with muscle extract had no effect on several properties of de veloping muscles. With the exception of cranial motoneurons, treatment with muscle extract did not promote the survival of several other pop ulations of neurons in the central and peripheral nervous system that also exhibit naturally occurring cell death. Initial biochemical chara cterization suggests that the activity in skeletal muscle is an acidic protein between 10 and 30 kD. Examination of a number of previously c haracterized growth and trophic agents in our in vivo assay have ident ified several molecules that promote motoneuron survival to one degree or another. These include S100beta, brain-derived neurotrophic factor (BDNF), neurotrophin 4/5 (NT-4/5), ciliary neurotrophic factor (CNTF) , transforming growth factor beta (TGFbeta), platelet-derived growth f actor-AB (PDGF-AB), leukemia inhibitory factor (CDF/LIF), and insulin- like growth factors I and II (IGF). By contrast, the following agents were ineffective: nerve growth factor (NGF), neurotrophin-3 (NT3), epi dermal growth factor (EGF), acidic and basic fibroblast growth factors (aFGF, bFGF), and the heparin-binding growth-associated molecule (HB- GAM). Of those agents that were effective, CDF/LIF, IGF-1 and -2, BDNF , and TGF are reported to be expressed in developing or adult muscle. Studies are underway to determine whether the survival activity found in avian muscle extract can be accounted for by one or more of these g rowth factors. Of all the tissue extracts and purified proteins tested here, only the neurotrophins-NGF, NT-3, and BDNF (but not NT-4/5)-res cued sensory neurons from naturally occurring cell death. (C) 1993 Joh n Wiley & Sons, Inc.