Leukemia inhibitory factor and ciliary neurotrophic factor cause dendritic retraction in cultured rat sympathetic neurons.
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Dendritic retraction occurs in many regions of the developing brain and also after neural injury. However, the molecules that regulate this important regressive process remain largely unknown. Our data indicate that leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) cause sympathetic neurons to retract their dendrites in vitro, ultimately leading to an approximately 80% reduction in the size of the arbor. The dendritic retraction induced by LIF exhibited substantial specificity because it was not accompanied by changes in cell number, in the rate of axonal growth, or in the expression of axonal cytoskeletal elements. An antibody to gp130 blocked the effects of LIF and CNTF, and both cytokines induced phosphorylation and nuclear translocation of stat3. Moreover, addition of soluble interleukin-6 (IL-6) receptor to the medium endowed IL-6 with the ability to cause dendritic regression. These data indicate that ligands activating the gp130 pathway have the ability to profoundly alter neuronal cell shape and polarity by selectively causing the retraction of dendrites. (+info)
CNTF, not other trophic factors, promotes axonal regeneration of axotomized retinal ganglion cells in adult hamsters.
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PURPOSE: To investigate the in vivo effects of trophic factors on the axonal regeneration of axotomized retinal ganglion cells in adult hamsters. METHODS: The left optic nerve was transected intracranially or intraorbitally, and a peripheral nerve graft was apposed or sutured to the axotomized optic nerve to enhance regeneration. Trophic factors were applied intravitreally every 5 days. Animals were allowed to survive for 3 or 4 weeks. Regenerating retinal ganglion cells (RGCs) were labeled by applying the dye Fluoro-Gold to the distal end of the peripheral nerve graft 3 days before the animals were killed. RESULTS: Intravitreal application of ciliary neurotrophic factor substantially enhanced the regeneration of damaged axons into a sciatic nerve graft in both experimental conditions (intracranial and intraorbital optic nerve transections) but did not increase the survival of distally axotomized RGCs. Basic fibroblast growth factor and neurotrophins such as nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 failed to enhance axonal regeneration of distally axotomized RGCs. CONCLUSIONS: Neurons of the adult central nervous system can regenerate in response to trophic supply after injury, and ciliary neurotrophic factor is at least one of the trophic factors that can promote axonal regeneration of axotomized RGCs. (+info)
Receptor recognition sites of cytokines are organized as exchangeable modules. Transfer of the leukemia inhibitory factor receptor-binding site from ciliary neurotrophic factor to interleukin-6.
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Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4-helical bundle" cytokines of the IL-6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology. (+info)
Repeated injections of a ciliary neurotrophic factor analogue leading to long-term photoreceptor survival in hereditary retinal degeneration.
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PURPOSE: To determine whether ciliary neurotrophic factor (CNTF) or brain-derived neurotrophic factor (BDNF) treatment leads to long-term photoreceptor survival in hereditary retinal degeneration. METHODS: An autosomal dominant feline model of rod-cone dystrophy was used throughout the study with two normal animals. In the first experiment, intravitreal injections of a human CNTF analogue (Axokine; Regeneron Pharmaceuticals, Tarrytown, NY) were administered to one eye of each animal (n = 10) beginning on postnatal day 10 and were repeated every 4 weeks. Clinical and histopathologic examinations were performed at 5.5, 9.5, and 13.5 weeks. In the second experiment, animals (n = 17) were randomly assigned to receive intravitreal injections of either Axokine (at half the initial dose), human BDNF, or the vehicle for Axokine to one eye at 5.5 weeks. The same therapy was repeated every 4 weeks in each group. Clinical and histopathologic examinations were performed at 9.5, 13.5, and 17.5 weeks. Photoreceptor survival was assessed by cell counting. Apoptotic cells were identified by morphology and a modified TdT-dUTP terminal nick-end labeling (TUNEL) technique. In the third experiment, two normal animals were treated with Axokine as in the first experiment. Glial fibrillary acidic protein ((GFAP) immunohistochemistry was performed to assess glial cell reaction. RESULTS: In the first two experiments, Axokine significantly prolonged photoreceptor survival (P < 0.01) and reduced the presence of apoptotic cells (P < 0.05) and TUNEL-positive cells (P < 0.05). In the second experiment, results in the the BDNF- and sham-injected eyes were not significantly different from those in the untreated eyes. Minimal posterior subcapsular cataract and mild retinal folds were found in all Axokine-treated eyes in both dystrophic and normal animals. These complications were milder in the second experiment when injections were started later and at a reduced dose. GFAP immunolabeling was also increased in all Axokine-treated eyes. CONCLUSIONS: Axokine, but not BDNF, delays photoreceptor loss in this hereditary retinal degeneration. Repeated injections maintain the protective effect. (+info)
Signalling by the RET receptor tyrosine kinase and its role in the development of the mammalian enteric nervous system.
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RET is a member of the receptor tyrosine kinase (RTK) superfamily, which can transduce signalling by glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) in cultured cells. In order to determine whether in addition to being sufficient, RET is also necessary for signalling by these growth factors, we studied the response to GDNF and NTN of primary neuronal cultures (peripheral sensory and central dopaminergic neurons) derived from wild-type and RET-deficient mice. Our experiments show that absence of a functional RET receptor abrogates the biological responses of neuronal cells to both GDNF and NTN. Despite the established role of the RET signal transduction pathway in the development of the mammalian enteric nervous system (ENS), very little is known regarding its cellular mechanism(s) of action. Here, we have studied the effects of GDNF and NTN on cultures of neural crest (NC)-derived cells isolated from the gut of rat embryos. Our findings suggest that GDNF and NTN promote the survival of enteric neurons as well as the survival, proliferation and differentiation of multipotential ENS progenitors present in the gut of E12.5-13.5 rat embryos. However, the effects of these growth factors are stage-specific, since similar ENS cultures established from later stage embryos (E14. 5-15.5), show markedly diminished response to GDNF and NTN. To examine whether the in vitro effects of RET activation reflect the in vivo function(s) of this receptor, the extent of programmed cell death was examined in the gut of wild-type and RET-deficient mouse embryos by TUNEL histochemistry. Our experiments show that a subpopulation of enteric NC undergoes apoptotic cell death specifically in the foregut of embryos lacking the RET receptor. We suggest that normal function of the RET RTK is required in vivo during early stages of ENS histogenesis for the survival of undifferentiated enteric NC and their derivatives. (+info)
Target-dependent regulation of acetylcholine secretion at developing motoneurons in Xenopus cell cultures.
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1. Myocyte-dependent regulation of acetylcholine (ACh) quantal secretion from developing motoneurons was studied in day-3 Xenopus nerve-muscle co-cultures. Spontaneous synaptic currents (SSCs) were measured in manipulated synapses by using whole-cell voltage-clamped myocytes. Changes in SSC amplitude were assumed to reflect changes in the ACh content of secreted quantal packets. Compared with natural synapses, motoneurons without any contact with a myocyte (naive neurons) released ACh in smaller quantal packets. 2. Bipolar cultured motoneurons, which were in contact with a myocyte with one axon branch (contact-end) but remained free at another axon branch (free-end), were further used to examine quantal ACh secretion. The ACh quantal size recorded at free-end terminals was similar to that of naive neurons and was smaller than that at the contact-end, indicating that myocyte contact exerts differential regulation on quantal secretion in the same neuron. 3. Some of the neurons that formed a natural synapse with a myocyte continued to grow forward and ACh quantal secretion from the free growth cone was examined. The ACh quantal size recorded at free growth cones was inversely proportional to the distance to the natural synapse, implying localized regulation of quantal secretion by the myocyte. 4. Chronic treatment of day-1 cultures with veratridine and d-tubocurarine, respectively, increased and decreased the neurotrophic action of myocytes when assayed on day 3. 5. Taken together, these findings suggest that the myocyte is an important postsynaptic target in the regulation of quantal secretion and that the trophic action is spatially restricted to the neighbourhood of the neuromuscular junction. (+info)
Dynamic regulation of expression and phosphorylation of tau by fibroblast growth factor-2 in neural progenitor cells from adult rat hippocampus.
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The nature of the extracellular signals that regulate the expression and the phosphorylation of the microtubule-associated protein tau, which is aberrantly hyperphosphorylated in Alzheimer disease and other adult-onset neurodegenerative diseases, is not known. We have found that neural progenitor cells from adult rat hippocampus express adult isoforms of tau and that the expression and the phosphorylation of tau are regulated by fibroblast growth factor-2 (FGF-2). Astrocytes that are differentiated from these cells by stimulation with ciliary neurotrophic factor express phosphorylated tau similarly when cultured in the presence of FGF-2. In fetal progenitor cells that express only the fetal tau isoform, expression, but not the phosphorylation, of this protein is regulated by FGF-2 in cultures of higher passages. The FGF-2-mediated tau hyperphosphorylation is inhibited by lithium, an inhibitor of glycogen synthase kinase-3 (GSK-3), but not by inhibitors of mitogen-activated protein kinase or the cyclin-dependent kinases. Furthermore, both GSK-3 activity and the phosphorylation of tau increase when the concentration of FGF-2 is increased up to 40 ng/ml. These results demonstrate that proliferating adult rat hippocampal progenitor cells express adult isoforms of tau stably and that FGF-2 upregulates the expression and, by upregulating GSK-3 activity, the phosphorylation of tau. (+info)
Activation of TrkA by nerve growth factor upregulates expression of the cholinergic gene locus but attenuates the response to ciliary neurotrophic growth factor.
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Nerve growth factor (NGF) stimulates the expression of the cholinergic gene locus, which encodes choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), the proteins necessary for the synthesis and storage of the neurotransmitter acetylcholine (ACh). To determine whether this action of NGF is mediated by the p140TrkA NGF receptor (a member of the Trk tyrosine kinase family) we used a murine basal forebrain cholinergic cell line, SN56, stably transfected with rat trkA cDNA. Treatment of these transfectants with NGF activated mitogen-activated protein kinase and increased cytosolic free calcium concentrations, confirming the reconstitution of TrkA-mediated signalling pathways. The expression of ChAT and VAChT mRNA, as well as ACh content, were coordinately up-regulated by NGF in SN56-trkA transfectants. None of these responses occurred in the parental SN56 cells, which do not express endogenous TrkA, indicating that these actions of NGF required TrkA. We previously reported that ciliary neurotrophic factor (CNTF) upregulates the expression of ChAT and VAChT, as well as ACh production, in SN56 cells. The combined treatment of SN56-trkA cells with CNTF and NGF revealed a complex interaction of these factors in the regulation of cholinergic gene locus expression. At low concentrations of CNTF (<1 ng/ml), the upregulation of ACh synthesis evoked by these factors was additive. However, at higher concentrations of CNTF (>1 ng/ml), NGF attenuated the stimulatory effect of CNTF on ChAT and VAChT mRNA and ACh content. This attenuation was not due to interference with early steps of CNTF receptor signalling, as pre-treatment of SN56-trkA cells with NGF did not affect the nuclear translocation of the transcription factor, Stat3, evoked by CNTF. (+info)