Evidence for Multiple, Local Functions of Ciliary Neurotrophic Factor (CNTF) in Retinal Development: Expression of CNTF and Its Receptor and In Vitro Effects on Target Cells

Abstract: There is increasing, although largely indirect, evidence that neurotrophic factors not only function as target-derived survival factors for projection neurons, but also act locally to regulate developmental processes. We studied the expression of ciliary neurotrophic factor (CNTF) and the CNTF-specific ligand-binding α-subunit of the CNTF receptor complex (CNTFRα) in the rat retina, a well-defined CNS model system, and CNTF effects on cultured retinal neurons. Both CNTF and CNTFRα (mRNA and protein) are expressed during phases of retinal neurogenesis and differentiation. Retina-specific Müller glia are immunocytochemically identified as the site of CNTF production and CNTFRα-expressing, distinct neuronal cell types as potential CNTF targets. Biological effects on corresponding neurons in culture further support the conclusion that locally supplied CNTF plays a regulatory role in the development of various retinal cell types including ganglion cells and interneurons.     

Galectin-1 expression in innervated and denervated skeletal muscle

Galectin-1 is a soluble carbohydrate-binding protein with a particularly high expression in skeletal muscle. Galectin-1 has been implicated in skeletal muscle development and in adult muscle regeneration, but also in the degeneration of neuronal processes and/or in peripheral nerve regeneration. Exogenously supplied oxidized galectin-1, which lacks carbohydrate-binding properties, has been shown to promote neurite outgrowth after sciatic nerve sectioning. In this study, we compared the expression of galectin-1 mRNA and immunoreactivity in innervated and denervated mouse and rat hind-limb and hemidiaphragm muscles. The results show that galectin-1 mRNA expression and immunoreactivity are up-regulated following denervation. The galectin-1 mRNA is expressed in the extrasynaptic and perisynaptic regions of the muscle, and its immunoreactivity can be detected in both regions by Western blot analysis. The results are compatible with a role for galectin-1 in facilitating reinnervation of denervated skeletal muscle.     

睫状神经营养因子对体外培养骨骼肌细胞的促增殖效应

目的 :探讨睫状神经营养因子 (CNTF)对骨骼肌细胞的直接营养作用 ,从而为神经肌肉系统损伤和退行性病变的治疗提供新的思路。结果 :CNTF可以促进体外培养的L6 TG肌母细胞和新生SD大鼠原代骨骼肌细胞增殖。结论 :CNTF对体外骨骼肌细胞具有营养作用。CNTF的神经和肌肉双重营养性能使其可能在神经肌肉损伤和退行性病变的治疗上发挥重要作用。     

Improved Neurological Outcome by Intramuscular Injection of Human Amniotic Fluid Derived Stem Cells in a Muscle Denervation Model

PurposeThe skeletal muscle develops various degrees of atrophy and metabolic dysfunction following nerve injury. Neurotrophic factors are essential for muscle regeneration. Human amniotic fluid derived stem cells (AFS) have the potential to secrete various neurotrophic factors necessary for nerve regeneration. In the present study, we assess the outcome of neurological function by intramuscular injection of AFS in a muscle denervation and nerve anastomosis model.ResultsNT-3 (Neurotrophin 3), BDNF (Brain derived neurotrophic factor), CNTF (Ciliary neurotrophic factor), and GDNF (Glia cell line derived neurotrophic factor) were highly expressed in AFS cells and supernatant of culture medium. Intra-muscular injection of AFS exerted significant expression of several neurotrophic factors over the distal end of nerve and denervated muscle. AFS caused high expression of Bcl-2 in denervated muscle with a reciprocal decrease of Bad and Bax. AFS preserved the muscle morphology with high expression of desmin and acetylcholine receptors. Up to two months, AFS produced significant improvement in electrophysiological study and neurological functions such as SFI (sciatic nerve function index) and Catwalk gait analysis. There was also significant preservation of the number of anterior horn cells and increased nerve myelination as well as muscle morphology.ConclusionIntramuscular injection of AFS can protect muscle apoptosis and likely does so through the secretion of various neurotrophic factors. This protection furthermore improves the nerve regeneration in a long term nerve anastomosis model.     

Cholinergic neuronal differentiation factors: evidence for the presence of both CNTF-like and non-CNTF-like factors in developing rat footpad.

Catecholaminergic sympathetic neurons are able to change their transmitter phenotype during development and to acquire cholinergic properties. Cholinergic sympathetic differentiation is only observed in fibers innervating specific targets like the sweat glands in the rat footpad. A function for ciliary neurotrophic factor (CNTF) in this process has been implied as it is able to induce cholinergic properties (ChAT, VIP) in cultured chick and rat neurons. We show here that a CNTF-like, VIP-inducing activity is present in rat footpads and that its increases 6-fold during the period of cholinergic sympathetic differentiation. Immunohistochemical analysis of P21 rat footpads demonstrated CNTF-like immunoreactivity in Schwann cells but not in sweat glands, the target tissue of cholinergic sympathetic neurons. The expression of this factor in footpads seems to be dependent on the presence of intact nerve axons, as nerve transection results in a loss of CNTF-like cholinergic activity and immunoreactivity. Immunoprecipitation experiments with rat footpad extracts provided evidence for the presence of ChAT-inducing factors other than CNTF, which may independently or together with CNTF be involved in the determination of sympathetic neuron phenotype.     

Nerve sprouting in innervated adult skeletal muscle induced by exposure to elevated levels of insulin-like growth factors

Partial denervation or paralysis of adult skeletal muscle is followed by nerve sprouting, probably due to release of diffusible sprout-inducing activity by inactive muscle. Insulin-like growth factors (IGF1 and IFG2) are candidates for muscle-derived sprouting activity, because (a) they induce neurite growth from peripheral neurons in vitro; and (b) their mRNA levels in adult skeletal muscle increase severalfold after denervation or paralysis. We sought to determine whether the presence of elevated levels of IGFs in innervated adult skeletal muscle was sufficient to produce intramuscular nerve growth. Low concentrations of IGFs induced massive neurite growth from enriched embryonic chick motoneurons in vitro. Half-maximal responses required 0.2 nM IGF2 or IGF1, or 20 nM insulin. Similar hormone binding properties of motoneuron processes in vitro were observed. Exposure of adult rat or mouse gluteus muscle in vivo to low quantities of exogenous IGF2 or IGF1 led to intramuscular nerve sprouting. Numbers of sprouts in IGF-exposed muscles were 10-fold higher than in vehicle-exposed or untreated muscles, and 12.2% of the end plates in IGF-exposed muscle (control: 2.7%) had sprouts growing from them. The nerve growth reaction was accompanied by elevated levels of intramuscular nerve-specific growth-associated protein GAP43. Additional properties of IGF-exposed muscle included modest proliferation of interstitial cells and elevated interstitial J1 immunoreactivity. These results suggest that elevated levels of IGFs in denervated or paralyzed muscle might trigger coordinate regenerative reactions, including nerve sprouting and expression of nerve growth-supporting substrate molecules by activated interstitial cells.     

Adult Rat Mesenchymal Stem Cells Delay Denervated Muscle Atrophy

To evaluate the function of rat mesenchymal stem cells (rMSCs) on denervated gastrocnemius muscles and to address the role of ciliary neurotrophic factor (CNTF) in rMSCs, denervated Wistar rats were separately injected with culture media (sham control), CNTF protein, 2.5?×?10⁵ siCNTF-treated rMSCs, 2.5?×?10⁵ GFP-transfected rMSCs, or 2.5?×?10⁵ untreated rMSCs. Muscle function was assessed at different time points post-surgery. Tibial nerve and gastrocnemius muscle samples were taken at 4, 8, and 12?weeks for histochemistry, and neuromuscular junction repair was also examined by electron microscopy. Fluorescence immunocytochemistry on tissue sections confirmed neurotrophin expression in rMSCs but with little evidence of neuronal differentiation. The engraftment of rMSCs significantly preserved the function of denervated gastrocnemius muscle based both on evaluation of muscle function and direct examination of muscle tissue. Further, the density and depth of the junctional folds were visibly reduced 12?weeks after surgery and transplantation, especially in control group. Knockdown of CNTF expression in rMSCs failed to block muscle preservation, although administration of CNTF protein alone inhibited muscle atrophy, which indicating that delivery of rMSCs could preserve gastrocnemius muscle function following denervation and post-junctional mechanisms involved in the repairing capability of rMSCs.     

Dedifferentiation of adult human myoblasts induced by ciliary neurotrophic factor in vitro

Ciliary neurotrophic factor (CNTF) is primarily known for its important cellular effects within the nervous system. However, recent studies indicate that its receptor can be highly expressed in denervated skeletal muscle. Here, we investigated the direct effect of CNTF on skeletal myoblasts of adult human. Surprisingly, we found that CNTF induced the myogenic lineage-committed myoblasts at a clonal level to dedifferentiate into multipotent progenitor cells--they not only could proliferate for over 20 passages with the expression absence of myogenic specific factors Myf5 and MyoD, but they were also capable of differentiating into new phenotypes, mainly neurons, glial cells, smooth muscle cells, and adipocytes. These "progenitor cells" retained their myogenic memory and were capable of redifferentiating into myotubes. Furthermore, CNTF could activate the p44/p42 MAPK and down-regulate the expression of myogenic regulatory factors (MRFs). Finally, PD98059, a specific inhibitor of p44/p42 MAPK pathway, was able to abolish the effects of CNTF on both myoblast fate and MRF expression. Our results demonstrate the myogenic lineage-committed human myoblasts can dedifferentiate at a clonal level and CNTF is a novel regulator of skeletal myoblast dedifferentiation via p44/p42 MAPK pathway.     

Spinal cord-muscle relations: their role in neuro-muscular development in birds

In the present study we focused our attention on the role of spinal cord-muscle interactions in the development of muscle and spinal cord cells. Four experimental approaches were used: 1) muscle fiber-spinal cord co-culture; 2) chronic spinal cord stimulation in chick embryos; 3) direct electrical stimulation of the denervated chick muscle; 4) skeletal muscle transplantation in close apposition to the spinal cord in chick embryos. The characteristics of mATPase and energetic metabolism enzyme activities and of myosin isoform expression were used as markers for fiber types in two peculiar muscles, the fast-twitch PLD and the slow-tonic ALD. In vitro, in the absence of neurons, myoblasts can express some characteristics of either slow or fast muscle types according to their origin, while in the presence of neurons, muscle fiber differentiation seems to be related to the spontaneous rhythm delivered by the neurons. The in ovo experiments of chronic spinal cord stimulation demonstrate that the differentiation of the fast and slow muscle features appears to be rhythm dependent. In the chick, direct stimulation of denervated muscles shows that the rhythm of the muscle activity is also involved in the control of muscle properties. In chick embryos developing ALD, the changes induced by modifications of muscle tension demonstrate that this factor also influences muscle development. Other experiments show that muscle back-transplantation can alter the early spinal cord development.     

Temporal Expression of mRNAs for Neuropoietic Cytokines, Interleukin-11 (IL-11), Oncostatin M (OSM), Cardiotrophin-1 (CT-1) and Their Receptors (IL-11Rα and OSMRβ) in Peripheral Nerve Injury

The mRNA expression pattern of the neuropoietic cytokines, interleukin-11 (IL-11), oncostatin M (OSM) and cardiotrophin-1 (CT-1), and their receptor components (IL-11R and OSMR) was examined in peripheral nerves on two different types of injury, crush and transection. The IL-11 mRNA increased after nerve damage and immediately returned to control levels. The OSM mRNA expression increased rapidly after nerve injury and relatively high expressions were maintained for at least 14 days. The CT-1 mRNA was not expressed in any time before and after the injury. Interestingly, IL-11R was expressed in the intact nerve and decreased after injury. The expression of OSMR increased slightly after the injury. Moreover, temporal mRNA expression pattern of these neuropoietic cytokines and receptors was similar between the crushed and transected models. Each neuropoietic cytokine of IL-11, OSM and CT-1 has its own specific temporal mRNA expression pattern, which is also different from those of ciliary neuro-trophic factor (CNTF), leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). These results suggest that all neuropoietic cytokines have distinctive functions in nerve degeneration and repair process in response to peripheral nerve injury.     

Expression of SERCA2a is independent of innervation in regenerating soleus muscle

The speed of contraction of a skeletal muscle largely depends on the myosin heavy chain isoforms (MyHC), whereas the relaxation is initiated and maintained by the sarcoplasmic reticulum Ca²⁺-ATPases (SERCA). The expression of the slow muscle-type myosin heavy chain I (MyHCI) is entirely dependent on innervation, but, as we show here, innervation is not required for the expression of the slow-type sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) in regenerating soleus muscles of the rat, although it can play a modulator role. Remarkably, the SERCA2a level is even higher in denervated than in innervated regenerating soleus muscles on day 7 when innervation is expected to resume. Later, the level of SERCA2a protein declines in denervated regenerated muscles but it remains expressed, whereas the corresponding mRNA level is still increasing. SERCA1 (i.e., the fast muscle-type isoform) expression shows only minor changes in denervated regenerating soleus muscles compared with innervated regenerating controls. When the soleus nerve was transected instead of the sciatic nerve, SERCA2a and MyHCI expressions were found to be even more uncoupled because the MyHCI nearly completely disappeared, whereas the SERCA2a mRNA and protein levels decreased much less. The transfection of regenerating muscles with constitutively active mutants of the Ras oncogene, known to mimic the effect of innervation on the expression of MyHCI, did not affect SERCA2a expression. These results demonstrate that the regulation of SERCA2a expression is clearly distinct from that of the slow myosin in the regenerating soleus muscle and that SERCA2a expression is modulated by neuronal activity but is not entirely dependent on it. slow type sarcoplasmic reticulum Ca²⁺ pump; MyHCI; nerve influence     

Primary structure and functional expression of the alpha-, beta-, gamma-, delta- and epsilon-subunits of the acetylcholine receptor from rat muscle.

The isolation and characterization of five clones carrying sequences of the alpha-, beta-, gamma-, delta- and epsilon-subunit precursors of the rat muscle acetylcholine receptor (AChR) are described. The deduced amino acid sequences indicate that these polypeptides contain 457-519 amino acids and reveal the structural characteristics common to subunits of ligand-gated ion channels. The pattern of subunit-specific mRNA levels in rat muscle shows characteristic changes during development and following denervation, suggesting that innervation of muscle reduces the expression of the alpha-, beta- and delta-subunit mRNAs, suppresses the expression of the gamma-subunit mRNA, and induces expression of epsilon-subunit mRNA. Subunit-specific cRNAs generated in vitro were injected into Xenopus laevis oocytes, resulting in the assembly of two functionally different AChR channel subtypes. The AChR gamma, composed of the alpha-, beta-, gamma- and delta-subunits, has functional properties similar to those of the native AChRs in fetal muscle. The AChR epsilon, composed of alpha-, beta-, delta- and epsilon-subunits, corresponds to the end-plate channel of the adult muscle. Thus in rat skeletal muscle the motor nerve regulates the expression of two functionally different AChR subtypes with different molecular composition by the differential expression of subunit-specific mRNAs.     

Expression of CNTF/LIF-receptor components and activation of STAT3 signaling in axotomized facial motoneurons: evidence for a sequential postlesional function of the cytokines

Several lines of evidence suggest that ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are important for the survival and regeneration of axotomized motoneurons. To investigate the role of CNTF/LIF signaling in regenerative responses of motoneurons, we studied the expression of the three receptor components, CNTF receptor alpha (CNTFRalpha), LIF receptor beta (LIFRbeta), and gp130, and the activation of the STAT3 signal transduction pathway in the rat facial nucleus following peripheral nerve transection. As shown by in situ hybridization and immunoblotting, axotomy resulted in a rapid down-regulation of CNTFRalpha mRNA expression within 24 h and a concomitant massive up-regulation of LIFRbeta mRNA and protein in the lesioned motoneurons. The altered mRNA levels were maintained for 3 weeks but had returned back to control levels by 6 weeks postlesion after successful regeneration. In contrast, mRNA levels remained in the lesioned state during the 6-week period studied, when regeneration was prevented by nerve resection. Significant lesion-induced changes in gp130 mRNA levels were not detectable. Rapid (within 24 h) and sustained (for at least 5 days) activation of STAT3 in axotomized facial motoneurons was revealed by demonstrating the phosphorylation and nuclear translocation of the protein using immunocytochemistry and immunoblotting. In agreement with previous studies showing a complementary regulation of CNTF and LIF in the lesioned facial nerve, our observations on the postlesional regulation of CNTF/LIF receptor components in the facial nucleus indicate a direct and sequential action of the two neurotrophic proteins on axotomized facial motoneurons.     

Ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) was first identified and partially purified from embryonic chick eye tissues. Subsequently, it was shown that CNTF is also present in large amounts in sciatic nerves of adult rats and rabbits, which led to its final purification and cloning. CNTF is not secreted by the classical secretory pathway involving the endoplasmatic reticulum and Golgi complex, but can be detected in high quantities within the cytoplasm of myelinating Schwann cells and astrocytes using immunohistochemistry. CNTF supports survival and / or differentiation of a variety of neuronal cell types including sensory, sympathetic and motoneurons. Also, nonneuroanl cells, such as oligodendrocytes, microglial cells, liver cells, and skeletal muscle cells, respond to exogenously administered CNTF, both in vitro and in vivo. During development, expression of CNTF is very low, if indeed it is expressed at all, and the phenotype of mice lacking endogenous CNTF, suggesting that CNTF after inactivation of the CNTF gene by homologous recombination suggests that CNTF does not play a crucial role for responsive cells during embryonic development. However, motoneurons are lost postnatally in mice lacking endogenous CNTF, suggesting that CNTF acts physiologically on the maintenance of these cells. The ability of exogenous CNTF to protect against motoneuron loss following lesion or in other animal models indicates that CNTF might be useful in the treatment of human motoneuron disorders, provided appropriate means of administration can be found. 1994 John Wiley & Sons, Inc.     

Ciliary neurotrophic factor (CNTF) affects the excitable and contractile properties of innervated skeletal muscles

The well-established trophic role of CNTF upon neurons led to performing clinical trials in patients of neurodegenerative diseases. However, trials were suspended due to side effects such as severe weight loss, hyperalgesia, coughing, muscle cramps and pain. So far it is not known how CNTF triggers the problems related to skeletal muscle cramps and pain. CNTF has also been described as a myotrophic factor for denervated skeletal muscles, but the possibility that it affects innervated muscles has also been considered. Since a myotrophic factor could be a valuable tool for treatment of several muscle diseases, we studied the effects of low doses of CNTF delivered systemically by an osmotic pump, over the electrical and mechanical properties of innervated and denervated fast and slow muscles. CNTF induced spontaneous electrical discharges and slowed twitches in innervated muscles, but did not prevent the changes induced by denervation. We postulate that the spontaneous discharges induced by CNTF in innervated muscles may be the cause of the cramps, coughing, and muscle ache reported by patients. At low doses, CNTF does not exert its myotrophic role over denervated muscles but clearly affects the excitable and contractile properties of innervated muscles.     

Regulation of skeletal muscle dihydropyridine receptor gene expression by biomechanical unloading.

Biomechanical unloading of the rat soleus by hindlimb unweighting is known to induce atrophy and a slow- to fast-twitch transition of skeletal muscle contractile properties, particularly in slow-twitch muscles such as the soleus. The purpose of this study was to determine whether the expression of the dihydropyridine (DHP) receptor gene is upregulated in unloaded slow-twitch soleus muscles. A rat DHP receptor cDNA was isolated by screening a random-primed cDNA lambda gt10 library from denervated rat skeletal muscle with oligonucleotide probes complementary to the coding region of the rabbit DHP receptor cDNA. Muscle mass and DHP receptor mRNA expression were assessed 1, 4, 7, 14, and 28 days after hindlimb unweighting in rats by tail suspension. Isometric twitch contraction times of soleus muscles were measured at 28 days of unweighting. Northern blot analysis showed that tissue distribution of DHP receptor mRNA was specific for skeletal muscle and expression was 200% greater in control fast-twitch extensor digitorum longus (EDL) than in control soleus muscles. A significant stimulation (80%) in receptor message of the soleus was induced as early as 24 h of unloading without changes in muscle mass. Unloading for 28 days induced marked atrophy (control = 133 +/- 3 vs. unweighted = 62.4 +/- 1.8 mg), and expression of the DHP receptor mRNA in the soleus was indistinguishable from levels normally expressed in EDL muscles. These changes in mRNA expression are in the same direction as the 37% reduction in time to peak tension and 28% decrease in half-relaxation time 28 days after unweighting. Our results suggest that muscle loading necessary for weight support modulates the expression of the DHP receptor gene in the soleus muscle.     

Expression of ARPP-16/19 in rat denervated skeletal muscle

It is known that denervation of rat skeletal muscle causes atrophy and this is often adopted as a model for human muscle atrophy. To understand the molecular changes that occur, it is important to identify the profiles of differential gene expression. In the present study, we investigated differentially expressed genes in denervated muscle using DNA microarrays with printed genes preferentially expressed in skeletal muscle. We found that several genes are differentially expressed. Of these genes, ARPP-16/19 (cAMP-regulated phosphoprotein 16/19) is selectively enhanced after denervation. The expression of ARPP-16/19 in denervated muscles starts to increase from two days after denervation surgery. On the other hand, the expression of ARPP-16/19 does not change in hind-limb suspended muscles, such as EDL and soleus muscles. These results suggest that the increase in ARPP-16/19 mRNA expression is regulated by unknown factor(s) secreted from nerves, and not by electrical muscle activity.