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Motoneurons played an essential role in establishing the concept of target-mediated support of innervating neurons. However, it took several decades until molecules were identined which trophically support motoneurons in vitro and in vivo. The most potent molecule identined so far is ciliary neurotrophic factor (CNTF). It is expressed as a cytosolic molecule in myelinating Schwann cells rather than in skeletal muscle in the postnatal period and therefore does not qualify as a target-derived neurotrophic factor regulating motoneuron survival during embryonic development. However, the inactivation of CNTF by gene targeting experiments results in progressive atrophy and degeneration of motoneurons, demonstrating that CNTF plays an essential role as a maintenance factor for motoneurons postnatally. Secretory molecules which are expressed in skeletal muscle during embryonic development and which support motoneurons in culture and partially also in vivo include members of the NGF gene family (BDNF, NT-3, NT-4/S) , FGF-S, IGF-I, and UF. The evaluation of the physiological importance of these molecules is under investigation.
Ciliary neurotrophic factor (CNTF) is expressed in high quantities in Schwann cells of peripheral nerves during postnatal development of the rat. The absence of a hydrophobic leader sequence and the immunohistochemical localization of CNTF within the cytoplasm of these cells indicate that the factor might not be available to responsive neurons under physiological conditions. However, CNTF supports the survival of a variety of embryonic neurons, including spinal motoneurons in culture. Moreover we have recently demonstrated that the exogenous application of CNTF protein to the lesioned facial nerve of the newborn rat rescued these motoneurons from cell death. These results indicate that CNTF might indeed play a major role in assisting the survival of lesioned neurons in the adult peripheral nervous system. Here we demonstrate that the CNTF mRNA and protein levels and the manner in which they are regulated are compatible with such a function in lesioned peripheral neurons. In particular, immunohistochemical analysis showed significant quantities of CNTF at extracellular sites after sciatic nerve lesion. Western blots and determination of CNTF biological activity of the same nerve segments indicate that extracellular CNTF seems to be biologically active. After nerve lesion CNTF mRNA levels were reduced to <5 % in distal regions of the sciatic nerve whereas CNTF bioactivity decreased to only one third of the original before-lesion levels. A gradual reincrease in Schwann cells occurred concomitant with regeneration.
Ciliary neurotrophic factor (CNTF) is a potent survival molecule for a variety of embryonic neurons in culture. The developmental expression of CNTF occurs clearly after the time period of the physiological cell death of CNTF-responsive neurons. This, together with the sites of expression, excludes CNTF as a target-derived neuronal survival factor, at least in rodents. However, CNTF also participates in the induction of type 2 astrocyte differentiation in vitro. Here we demonstrate that the time course of the expression of CNTF-mRNA and protein in the rat optic nerve (as evaluated by quantitative Northern blot analysis and biological activity, respectively) is compatible with such a glial differentiation function of CNTF in vivo. We also show that the type 2 astrocyte-inducing- activity previously demonstrated in optic nerve extract can be precipitated by an antiserum against CNTF. Immunohistochemical analysis of astrocytes in vitro and in vivo demonstrates that the expression of CNTF is confined to a subpopulation of type 1 astrocytes. The olfactory bulb of adult rats has comparably high levels of CNTF to the optic nerve, and here again, CNTF-immunoreactivity is localized in a subpopulation of astrocytes. However, the postnatal expression of CNTF in the olfactory bulb occurs later than in the optic nerve. In other brain regions both CNTF-mRNA and protein levels are much lower.
The structure of the rat ciliary neurotrophic factor (CNTF) gene and the regulation ofCNTF mRNA levels in cultured glial cells were investigated. The rat mRNA is encoded by a simple two-exon transcription unit. Sequence analysis of the region upstream of the transcription start-site did not reveal a typical TATA-box consensus sequence. Low levels of CNTF mRNA were detected in cultured Schwann cells, and CNTF mRNA was not increased by a variety of treatments. Three-week-old astrocyteenriched cell cultures from new-born rat brain contained easily detectable CNTF mRNA. In astrocyte-enriched cultures, upregulation of CNTF mRNA levels was observed after treatment with IFN-gamma. CNTF mRNA levels were down-regulated in these cells by treatments that elevate intracellular cyclic AMP and by members of the fibroblast growth factor (FGF) family. The implications of these results for potential in vivo functions of CNTF are discussed.
DURING vertebrale development, many neurons depend for survival and differentiation on their target cells\(^{1-3}\). The best documented mediator of such a retrograde trophic action is the neurotrophin nerve growth factor (NGF)\(^1\). NGF and the other known members of tbe neurotrophin family, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT -3) and neurotrophin-4/5 (NT -4/5) are conserved as distinct genes over large evolutionary distances\(^{4 -6}\). Here we report the cloning of neurotrophin-6 (NT -6), a new member of this family from the teleost fish Xiphophorus. NT -6 distinguishes itself from the other known neurotrophins in that it is not found as a soluble protein in the medium of producing cells. The addition of heparin (but not chondroitin) effects the release of NT -6 from cell surface and extracellular matrix molecules. Recombinant purified NT -6 has a spectrum of actions similar to NGF on chick sympathetic and sensory neurons, albeit with a lower potency. NT -6 is expressed in tbe embryonie valvulla cerebelli; expression persists in some adult tissues. The interaction of NT-6 with heparin-binding molecuJes may modulate its action in the nervous system .
The purpose of the experiments reported is to provide an unambiguous demonstration that embryonie skeletal muscle contains factors that act directly on embryonie spinal motor neurons both to support their survival and to stimulate the outgrowth of neurites. Cells of lumbar and brachial ventral spinal cords from 6-day-old chick embryos were separated by centrifugation in a two-step metrizamide gradient, and a motor neuron enriched fraction was obtained. Motor neurons were identified by retrogradely labeling with rhodamine isothiocyanate, and were enriched fourfold in the motor neuron fraction relative to unfractionated cells. In culture, the isolated motor neurons died within 3-4 days unless they were supplemented with embryonie chick skeletal muscle extract. Two functionally distinct entities separable by ammonium sulfate precipitation were responsible for the effects of muscle extracts on motor neurons. The 0-25% ammonium sulfate precipitate contained molecules that alone bad no effect on neuronal survival but when bound to polyornithine-coated culture substrata, stimulated neurite outgrowth and potentiated the survival activity present in muscle. Most of this activity was due to a laminin-like molecule being immunoprecipitated with antisera against laminin, and immunoblotting demonstrated the presence of both the A and B chains of laminin. A long-term survival activity resided in the 25-70% ammonium sulfate fraction, and its apparent total and specific activities were strongly dependent on the culture substrate. In contrast to the motor neurons, the cells from the other metrizamide fraction (including neuronal cells) could be kept in culture for a prolonged time without addition of exogenous factor(s).
More on motor neurons
(1992)