Expression Of Ciliary Neurotrophic Factor Research Articles

Systemic administration of nilvadipine delays photoreceptor degeneration of heterozygous retinal degeneration slow (rds) mouse

To investigate the effect of nilvadipine, a calcium channel blocker, upon the retina of retinal degeneration slow (rds) mouse, nilvadipine was intraperitoneally injected into heterozygous rds mice for up to 200 days. The effect of nilvadipine was evaluated by electroretinography (ERG), light and electron microscopies, DNA microarray, quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and western-blot analysis. After nilvadipine treatment, both a- and b-waves of ERG were significantly higher than in the control group ( p < 0.01). Although there was no difference in histological findings by light microscopy between the nilvadipine treated group and control group, apparent preservation of photoreceptor disc was demonstrated by electron microscopy in the treated group. Rhodopsin level was also increased in the treated group comparing to the control group. The DNA microarray analysis detected increased expression of genes encoding proteins which function in protein synthesis, growth factors and neurotrophic factor like ciliary neurotrophic factor (CNTF) and fibroblast growth factors (FGFs22 and 13). Decreased expression of genes coding for proteins related to proteolysis, apoptosis and growth factor (FGF18) was also demonstrated. Increased expression of CNTF, FGF22 and FGF13 and decreased expression of FGF18 were confirmed by both quantitative RT-PCR and western-blot analysis. In addition, FGF2 was constitutively expressed in both treated and control groups. Since CNTF has been known to retard retinal degeneration by rds mouse or other models of inherited retinal degeneration, it is possible that nilvadipine has a photoreceptor survival effect on rds retinal degeneration partly by enhancing expression of endogenous CNTF in the retina.

Effect of ammonia, glutamine, and serum on calcineurin, p38MAPK-diP, GADD153/CHOP10, and CNTF in primary rat astrocyte cultures

Primary astrocyte cultures were subjected to different experimental schedules using several concentrations of ammonia (1, 3, and 5 mM ammonium chloride), serum (2.5%, 5%, and 12%), and glutamine (0.5, 1, and 3 mM) to analyze the involvement of calcineurin (CaN) in hyperammonemia and its relation with p38MAPK-diP and ciliary neurotrophic factor (CNTF). We demonstrated that exposure to ammonia affects CaN content, and confirmed the ammonia-induced reduction of CNTF expression; however, the involvement of CaN and p38MAPK-diP in CNTF reduction could not be confirmed. On the contrary, an inverse relationship between CaN and p38MAPK-diP contents was clearly demonstrated. GADD153/CHOP10 content was always higher under hyperammonemic conditions as well as under glutamine exposure, probably due to the osmotic stress provoked by glutamine accumulation, which was induced after exposure to ammonia. Statistical analysis demonstrated significant interactions of ammonia and serum for CaN, GADD153/CHOP10 and CNTF contents. The exposure to glutamine also induced changes in GADD153/CHOP10 and CaN; however, CNTF content was not affected. In conclusion, CaN content was affected by exposure to ammonia and glutamine; the serum content of the culture medium had a strong influence on the astroglial response to ammonium chloride, and glutamine exposure only reproduced some of the ammonia effects.

Leukemia inhibitory factor participates in the expansion of neural stem/progenitors after perinatal hypoxia/ischemia

Subsequent to perinatal hypoxia/ischemia there is an increase in the number of neural stem/progenitor cells (NSP) within the subventricular zone (SVZ). Gene expression analyses have implicated Notch signaling in the expansion of these tripotential cells but there are limited data as to which signals are stimulating Notch activation. There is evidence that the leukemia inhibitory factor receptor (LIFR)/gp130 receptor heterodimer induces Notch1 to maintain NSP populations during normal development. LIF and ciliary neurotrophic factor (CNTF) bind to these receptor components and they coordinate injury responses in the CNS. Therefore, the aim of these studies was to investigate whether CNTF and/or leukemia inhibitory factor (LIF) participate in NSP expansion in the rat SVZ after hypoxia/ischemia (H/I) as well as to characterize the downstream events that regulate NSP numbers. We report that LIF mRNA is induced 48 h post-insult by 13-fold but that it returns almost to baseline by 72 h. Commensurate with increased LIF expression there is a corresponding increase in phosphorylated Stat-3 within the SVZ. Modeling the changes that occur in vivo, we show that LIF induces Stat-3 phosphorylation in neurospheres to enhance Delta-like-1 and Notch1 expression as well as to increase Notch1 activation. LIF also expands neurosphere number and size in vitro. Whereas CNTF can mimic the effects of LIF in vitro, CNTF expression in the SVZ was unchanged during recovery from H/I. Cumulatively, these data implicate LIF and not CNTF in the expansion of NSPs in the rat SVZ after perinatal brain injury. As both LIF expression and the endogenous regenerative response after brain injury are time-delimited, these findings provide insights into strategies to expand the endogenous pool of NSPs to repopulate the damaged brain.

Expression and histochemical localization of ciliary neurotrophic factor in cultured adult rat dorsal root ganglion neurons

Ciliary neurotrophic factor (CNTF) is abundantly expressed in Schwann cells in adult mammalian peripheral nerves, but not in neurons. After peripheral nerve injury, CNTF released from disrupted Schwann cells is likely to promote neuronal survival and axonal regeneration. In the present study, we examined the expression and histochemical localization of CNTF in adult rat DRG in vivo and in vitro. In contrast to the restricted expression in Schwann cells in vivo, we observed abundant CNTF mRNA and protein expression in DRG neurons after 3 h, 2, 7, and 15 days in dissociated cell culture. At later stages (7 and 15 days) of culture, CNTF immunoreactivity was detected in both neuronal cell bodies and regenerating neurites. These results suggest that CNTF is synthesized and transported to neurites in cultured DRG neurons. Since we failed to observe CNTF immunoreactivity in DRG neurons in explant culture, disruption of cell-cell interactions, rather than the culture itself, may be an inducible factor for localization of CNTF in the neurons.

Neuroprotection in the Juvenile Rat Model of Light-Induced Retinopathy: Evidence Suggesting a Role for FGF-2 and CNTF

In a former study, it was demonstrated that the retina of juvenile Sprague-Dawley (SD) rat has a remarkable intrinsic resistance to light-induced retinopathy compared with the adult retina. The purpose of the present study was to investigate the cellular and molecular mechanisms underlying this endogenous resistance to light-induced damage. Juvenile SD rats were exposed for 6 (from P14 to P20) or 14 (from P14 to P28) days to a bright, cyclic, luminous environment of 10,000 lux. Retinal histology was examined immediately after exposure to light or at 2 months of age, and photoreceptor cell death was quantified by measuring the thickness of the outer nuclear layer (ONL) and by TUNEL assays. Changes in protein levels and cellular localization of fibroblast growth factor (FGF)-2, ciliary neurotrophic factor (CNTF), and brain-derived neurotrophic factor (BDNF) were determined by Western blot analysis and retinal immunohistochemistry, respectively. The data demonstrate that although the rate of photoreceptor loss was different after 6 and 14 days of exposure to light, similar ONL thickness was reached at 2 months of age--that is, 4 to 5 weeks after exposure to light. A large number of TUNEL-positive photoreceptors was visualized immediately after 6 and 14 days of exposure to light, reflecting the intense cell death that was occurring in the ONL. Western blot analysis showed that exposure to light induced a strong upregulation of the neurotrophic factors FGF-2 and CNTF in juvenile retinas, whereas no change in BDNF protein expression was noted. Of interest, after exposure to light, endogenous FGF-2 and CNTF were selectively upregulated in Müller cells. The results show that endogenous expression of FGF-2 and CNTF by Müller glia may play a role in protecting the juvenile retina from light-induced damage.

Molecular and Cellular Alterations Induced by Sustained Expression of Ciliary Neurotrophic Factor in a Mouse Model of Retinitis Pigmentosa

To characterize molecular and cellular changes induced by sustained expression of ciliary neurotrophic factor (CNTF) in the rds mutant mouse retina. Recombinant adeno-associated virus (rAAV) expressing CNTF was injected subretinally, for transduction of peripherin/rds(+/)(-) transgenic mice that carry the P216L mutation found in human retinitis pigmentosa. Characterization of retinal neurons and glia was performed by immunocytochemistry with cell-type-specific markers. Activation of signaling molecules was examined by Western blot and immunostaining. Alterations of gene transcription profiles were studied by microarray analyses. CNTF viral transduction maintained rhodopsin expression in surviving rod photoreceptors, but greatly reduced both S- and M-opsin normally expressed in cones. In addition, CNTF treatment resulted in increased numbers and dispersion of Müller glia and Chx10-positive bipolar cells within the inner nuclear layer. Persistent CNTF signaling also caused enhanced phosphorylation of STAT1, STAT3, and p42/44 ERK, as well as their levels of expression. Moreover, altered transcription profiles were detected for a large number of genes. Among these, Crx and Nrl involved in photoreceptor differentiation and several genes involved in phototransduction were suppressed. Despite the rescue from cell death, continuous exposure to CNTF changed photoreceptor cell profiles, especially resulting in the loss of cone immunoreactivity. In addition, the Müller glia and bipolar cells became disorganized, and the number of cells expressing Müller and bipolar cell markers increased. Constitutive CNTF production resulted in sustained activation of cytokine signal transduction and altered the expression of a large number of genes. Therefore, stringent regulation of CNTF may be necessary for its therapeutic application in preventing retinal degeneration.

Neuroma-in-Continuity Model in Rabbits

The present study presents a model for creation of neuroma-in-continuity in the rabbit, confirmed by histologic study, electrophysiology, and examination of molecular markers. Twelve New Zealand rabbits were used. The lateral fascicule of the peroneal nerve with 15-mm in length was resected. In the intervals from 4 to 6 weeks postoperatively, the histology showed the typical pathologic changes of neuroma by hematoxylin-eosin (HE), Luxol fast blue, and Van-Gieson staining. As compared with the healthy nerve, the motor nerve conduction velocity (MCV) and compound motor action potential (CMAP) were found significantly slowed and reduced. The expression of ciliary neurotrophic factor (CNTF) in nerve and calcitonin gene-related peptide (CGRP) mRNA in L7, S1 dorsal root ganglia were downgraded and upgraded, respectively, with formation of neuroma-in-continuity. These electrophysiologic and molecular markers' expression data can be used as reliable parameters for evaluation of management of the neuroma-in-continuity.

Continued Administration of Ciliary Neurotrophic Factor Protects Mice from Inflammatory Pathology in Experimental Autoimmune Encephalomyelitis

Multiple sclerosis is an inflammatory disease of the central nervous system that leads to loss of myelin and oligodendrocytes and damage to axons. We show that daily administration (days 8 to 24) of murine ciliary neurotrophic factor (CNTF), a neurotrophic factor that has been described as a survival and differentiation factor for neurons and oligodendrocytes, significantly ameliorates the clinical course of a mouse model of multiple sclerosis. In the acute phase of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein peptide 35-55, treatment with CNTF did not change the peripheral immune response but did reduce the number of perivascular infiltrates and T cells and the level of diffuse microglial activation in spinal cord. Blood brain barrier permeability was significantly reduced in CNTF-treated animals. Beneficial effects of CNTF did not persist after it was withdrawn. After cessation of CNTF treatment, inflammation and symptoms returned to control levels. However, slight but significantly higher numbers of oligodendrocytes, NG2-positive cells, axons, and neurons were observed in mice that had been treated with high concentrations of CNTF. Our results show that CNTF inhibits inflammation in the spinal cord, resulting in amelioration of the clinical course of experimental autoimmune encephalomyelitis during time of treatment.

AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells

We compared the effects of intravitreal injection of bi-cistronic adeno-associated viral (AAV-2) vectors encoding enhanced green fluorescent protein (GFP) and either ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43) on adult retinal ganglion cell (RGC) survival and regeneration following (i) optic nerve (ON) crush or (ii) after ON cut and attachment of a peripheral nerve (PN). At 7 weeks after ON crush, quantification of betaIII-tubulin immunostaining revealed that, compared to AAV-GFP controls, RGC survival was not enhanced by AAV-GAP43-GFP but was increased in AAV-CNTF-GFP (mean RGCs/retina: 17 450+/-358 s.e.m.) and AAV-BDNF-GFP injected eyes (10 200+/-4064 RGCs/retina). Consistent with increased RGC viability in AAV-CNTF-GFP and AAV-BDNF-GFP injected eyes, these animals possessed many betaIII-tubulin- and GFP-positive fibres proximal to the ON crush. However, only in the AAV-CNTF-GFP group were regenerating RGC axons seen in distal ON (1135+/-367 axons/nerve, 0.5 mm post-crush), some reaching the optic chiasm. RGCs were immunoreactive for CNTF and quantitative RT-PCR revealed a substantial increase in CNTF mRNA expression in retinas transduced with AAV-CNTF-GFP. The combination of AAV-CNTF-GFP transduction of RGCs with autologous PN-ON transplantation resulted in even greater RGC survival and regeneration. At 7 weeks after PN transplantation there were 27 954 (+/-2833) surviving RGCs/retina, about 25% of the adult RGC population. Of these, 13 352 (+/-1868) RGCs/retina were retrogradely labelled after fluorogold injections into PN grafts. In summary, AAV-mediated expression of CNTF promotes long-term survival and regeneration of injured adult RGCs, effects that are substantially enhanced by combining gene and cell-based therapies/interventions.

Ciliary neurotrophic factor is expressed in the magnocellular neurosecretory system of the rat in vivo: Evidence for injury- and activity-induced upregulation

Although ciliary neurotrophic factor (CNTF) has been shown to promote the survival of magnocellular neurons when applied exogenously to explants of the paraventricular and supraoptic nuclei (SON) in vitro, little is known regarding its expression or regulation in the adult magnocellular neurosecretory system (MNS) following injury in vivo. Therefore, we utilized in situ hybridization and immunocytochemical analysis in conjunction with quantitative optical densitometric analysis to identify the cellular source of CNTF and examine the temporal pattern of its expression, following unilateral transection of the hypothalamo-neurohypophysial tract in the adult rat. In intact rats, CNTF immunoreactivity (CNTF-ir) was predominantly localized within identified astrocytes within the ventral glial limitans subjacent to the SON. Quantitative optical densitometric analysis of CNTF-ir levels in the axotomized SON demonstrated that the proportional area of CNTF-ir was significantly elevated between 3 and 30 days following injury. A significant but more limited increase was also observed in the non-injured contralateral SON. In situ hybridization confirmed the expression and upregulation of CNTF in the axotomized SON. These results demonstrate the expression of CNTF in the adult rodent MNS in vivo and provide evidence that levels of CNTF are upregulated in response to both direct injury, and heightened metabolic activity, within the lesioned and sprouting SON, respectively.

Time Course of Neurotrophic Factor Upregulation and Retinal Protection against Light-Induced Damage after Optic Nerve Section

To assess neurotrophic factor upregulation in the retina after damage to the optic nerve and relate that regulation to changes in photoreceptor stability and function. Retinas of adult pigmented (Long-Evans) rats were examined at successive times (1-60 days) after unilateral optic nerve section. The distribution and expression of ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (FGF-2) and their receptor elements FGFR1 and CNTFRalpha were studied with immunohistochemistry and Western blot analysis. FGF-2 and CNTF mRNA levels were also assessed, with semiquantitative reverse transcription-PCR. Levels and localization of the intracellular signaling molecule ERK and its activated, phosphorylated form pERK, were examined by immunohistochemistry. To assess the correlation between neurotrophic factor levels and their protective effect against light damage, albino (Sprague-Dawley) rats were exposed to bright continuous light (1000 lux) for 24 or 48 hours at successive times after nerve section. The TUNEL technique was used to visualize neuronal cell death in the retina. CNTF upregulation was detected 1 week after optic nerve section, peaked at 2 weeks, and fell to control levels at 4 weeks. CNTF appeared first in the inner retina in the ganglion cells, then in the Muller cells in which it became prominent at the outer limiting membrane (OLM) and in the outer segment (OS) region of photoreceptors. FGF-2 upregulation became prominent, particularly in photoreceptors, 21 to 28 days after surgery, continued to 2 months, and slowly declined thereafter. Double labeling with antibodies to ligand and the receptor showed colocalization of CNTF to its receptor at the OS region, whereas FGF-2-to-FGFR1 binding was found in the outer nuclear (ONL) and outer plexiform (OPL) layers. Optic nerve section provided a significant protective effect against light-induced damage in the first 2 weeks. There was no protection when animals were exposed to damaging light 1 month after nerve section. The upregulation of CNTF 7 to 14 days after nerve section correlates with a reduction in the a-wave described previously. Colocalization of CNTF and CNTFRalpha on the outer segments suggests that CNTF acts at the photoreceptor membrane. The slower upregulation of FGF-2 correlates with a reduction of the b-wave. FGF-2/FGFR1 colocalization in the OPL suggests that this factor acts at the synaptic terminals of photoreceptors, modulating the release of neurotransmitters. The time course of pERK upregulation suggests that the successive upregulation of CNTF and FGF-2 activates the ERK pathway. Based on the time course of protection against bright continuous light, it seems that CNTF plays a major role in this effect, and FGF-2 has a less important role in the protection against light-induced damage.

Expression of ciliary neurotrophic factor (CNTF), CNTF receptor alpha (CNTFR-α) following experimental intracerebral hemorrhage in rats

Ciliary neurotrophic factor (CNTF) is known as a neuro-survival factor in the developing and developed CNS, as well as in the CNS following injury. However, little is known about the expression of CNTF or that of its receptor (CNTFR-α) in cases of intracerebral hemorrhage (ICH). We investigated the temporal and spatial profiles of CNTF and CNTFR-α expression using a collagenase-induced ICH rat model. CNTF expression was up-regulated from the day following ICH induction and reached a peak level at 5 to 14 days, with increased expression observed in brain tissue surrounding the hematoma lesion and white matter structures in association with astroglial proliferation. Further, CNTFR-α was transiently expressed in the cerebral cortex surrounding the hematoma, with a peak at 5 days. Administration of exogenous CNTF into the lesion following initiation of ICH resulted in a prolonged expression of CNTFR-α on cortical neurons neighboring the hematoma. Our findings suggest differential regulation of CNTF and CNTFR-α, and the possibility of a therapeutic strategy using CNTF administration for ICH.

Ciliary neurotrophic factor and its receptors are differentially expressed in the optic nerve transected adult rat retina

Ciliary neurotrophic factor (CNTF) has been implicated in the survival and regeneration of adult rat retinal ganglion cells (RGCs). Expression of CNTF and its receptors, ciliary neurotrophic factor receptor alpha (CNTFRα) and leukemia inhibitory factor receptor (LIFR) was studied in normal, 6-h, 1-, and 3-day optic nerve transected retinas. Optic nerve transection was performed on the right eye in one group. In the other group, it was performed 5 days after intratectal Fluorogold injection. Retinas were studied using immunohistochemical labeling and Western blot analysis. Astrocytes were labeled with GFAP and Müller cells with glutamine synthetase. CNTF was observed in identifiable astrocytes and Müller cells, and its intensity increased in the experimental retinas. CNTFRα was localized on RGCs in experimental retinas. LIFR was localized on RGCs and Müller cells in normal and experimental retinas. CNTF and its receptors are differentially expressed after optic nerve transection and could help delay RGC death in such a stressful environment.

Expression of ciliary neurotrophic factor (CNTF) in charcot‐marie‐tooth type 1A (CMT1A) disease

cDNA microarray experiments on sciatic nerves from 30‐day‐old PMP22 transgenic (PMP22tg) rats were performed to detect genes modulated in CMT1A. Among the downregulated genes, CNTF mRNA was significantly reduced, whereas other neurotrophic factors (BDNF, NT3, NGF) and cognate receptors (TRKA, TRKB, TRKC) were unchanged. We further studied CNTF expression in transgenic nerves using an ELISA technique. We observed significantly (p &lt; 0.001) lower concentrations of the protein in sciatic nerves from homozygous (59 ± 7.8 pg/mg of proteins) and heterozygous (140.8 ± 17.56 pg/mg of proteins) PMP22tg nerves compared to normal controls (713.8 ± 168.7 pg/mg of proteins). Moreover, using real time PCR, we studied CNTF expression in human sural nerves from 2 CMT1A patients and in 3 control nerves. According to the animal results, CNTF mRNA expression was absent in CMT1A patients whereas it was detectable in control nerves (1.99 ± 0.9). Finally, we studied CNTF mRNA expression in aging PMP22tg rats; preliminary results show a further decrease of CNTF mRNA in heterozygous transgenic rats compared to normal age‐matched littermates. Our results suggest that reduced CNTF expression may account for the development of axonal atrophy in CMT1A.

Differential modulation of energy balance by leptin, ciliary neurotrophic factor, and leukemia inhibitory factor gene delivery: microarray deoxyribonucleic acid-chip analysis of gene expression.

Most obese animal models, whether associated with genetic, diet-induced, or age-related obesity, display pronounced leptin resistance, rendering leptin supplement therapy ineffective in treating obesity. Ciliary neurotrophic factor (CNTF) has been recently used to invoke leptin-like signaling pathways, thereby circumventing leptin resistance. In the current study, we characterize immediate and long-term molecular events in the hypothalamus of rats exposed to the sustained ectopic expression of leptin, CNTF, or leukemia inhibitory factor, another neurocytokine of IL-6 family, all delivered centrally via a viral vector. The respective transgene-encoded ligands induced similar but not identical metabolic responses as assessed by the reduction in body weight gain and changes in food intake. To define molecular mechanisms of weight-reducing and anorexigenic action of cytokines, we have analyzed the gene expression profiles of 1300 brain-specific genes in the hypothalami of normal rats subjected to the prolonged cytokine action for 10 wk. We present evidence that constitutive expression of cytokines in the brain induces changes in gene expression characteristic of chronic inflammation leading to either temporal weight reduction (CNTF) or severe cachexia (leukemia inhibitory factor). Our results convey a cautionary note regarding potential use of the tested cytokines in therapeutic applications.

Long-term lentiviral-mediated expression of ciliary neurotrophic factor in the striatum of Huntington's disease transgenic mice

Ciliary neurotrophic factor (CNTF) has been shown to prevent behavioral deficits and striatal degeneration in neurotoxic models of Huntington's disease (HD), but its effect in a genetic model has not been evaluated. Lentiviral vectors expressing the human CNTF or LacZ reporter gene were therefore injected in the striatum of wild-type (WT) and transgenic mice expressing full-length huntingtin with 72 CAG repeats (YAC72). Behavioral analysis showed increased locomotor activity in 5- to 6-month-old YAC72-LacZ mice compared to WT-LacZ animals. Interestingly, CNTF expression reduced the activity levels of YAC72 mice compared to control animals. In both WT and YAC72 mice, CNTF expression was demonstrated in striatal punches, up to a year after lentiviral injection. Stereological analysis revealed that the number of LacZ and DARPP-32-positive neurons were decreased in YAC72-LacZ mice compared to WT-LacZ animals. Assessment of the benefit of CNTF expression in the YAC72 mice was, however, complicated by a down-regulation of DARPP-32 and to a lesser extent of NeuN in all mice treated with CNTF. The expression of the neuronal marker NADPH-d was unaffected by CNTF, but expression of the astrocytic marker glial fibrillary acidic protein (GFAP) was increased. Finally, a reduction of the number of striatal dark cells was observed in YAC mice treated with CNTF compared to LacZ. These data indicate that sustained striatal expression of CNTF can be achieved with lentiviruses. Further studies are, however, needed to investigate the intracellular signaling pathways mediating the long-term effects of CNTF expression on dopamine signaling, glial cell activation and how these changes may affect HD pathology.

Endogenous and exogenous ciliary neurotrophic factor enhances forebrain neurogenesis in adult mice

Neurogenesis in the adult mammalian CNS occurs in the subventricular zone (SVZ) and dentate gyrus. The receptor for ciliary neurotrophic factor (CNTF), CNTFRα, is expressed in the adult subventricular zone. Because the in vitro effects of CNTF on neural precursors have been varied, including proliferation and differentiation into neurons or glia, we investigated its role in vivo. Injection of CNTF in the adult C57BL/6 mice forebrain increased the number of cells labeled with ip BrdU in both neurogenic regions. In the dentate gyrus, CNTF also appeared to enhance differentiation of precursors into neurons, i.e., increased the proportion of NeuN+/BrdU+ cells from ∼14 to ∼29%, but did not affect differentiation into astrocytes (GFAP+) or oligodendrocytes (CNPase+). In the SVZ, CNTF increased the proportion of GFAP+/BrdU+ cells from ∼1 to ∼2%. CNTF enhanced the distance of migration of new neurons into the granule cell layer. Intraventricular injection of neutralizing anti-CNTF antibodies reduced the number of BrdU-labeled cells in the SVZ. These results suggest that endogenous CNTF regulates adult neurogenesis by increasing proliferation of neural stem cells and/or precursors. Alternatively, CNTF could maintain cells longer in the S-phase, resulting in increased BrdU labeling. In the neurogenic region of the SVZ, CNTFRα was exclusively present in GFAP-positive process-bearing cells, suggesting that CNTF affects neurogenesis indirectly via neighboring astroglia. Alternatively, these cells may be part of the neural precursor lineage. The restricted expression of CNTF within the nervous system makes it a potential selective drug target for cell replacement strategies.

Ciliary neurotrophic factor inhibits differentiation of photoreceptor-like cells in rat pineal glands in vitro

Ciliary neurotrophic factor (CNTF) is a unique member of the interleukin-6 (IL-6) family, whose receptor subunit for ligand binding is exclusively expressed in the nervous system and muscle. The role of CNTF in mammalian development remains unknown. We recently reported the specific expression of CNTF in the pineal gland and eyes. To further examine the expression pattern and role of CNTF in development, we prepared a polyclonal antibody against rat CNTF, performed western blotting with this antibody, and confirmed a strong and specific expression of the CNTF protein in pineal glands and a moderate expression in the eyes among the various tissues examined in newborn rats. In pineal organ cultures of newborn rats, exogenously added recombinant rat CNTF potently inhibited the differentiation of photoreceptor-like cells in a dose-dependent manner, while CNTF did not influence the survival of pineal cells. Among several cell growth factors known to have a similar effect in retinal cultures examined, strong inhibitory effects were seen only with CNTF and the leukemia inhibitory factor (LIF), both of which belong to the IL-6 cytokine family. This inhibitory effect was the strongest during three to 6 days of culture when CNTF was added to these cultures. These results suggest that CNTF plays an inhibitory role in the development of photoreceptor-like cells in early postnatal rat pineal glands.

GFAP knockout mice have increased levels of GDNF that protect striatal neurons from metabolic and excitotoxic insults.

In response to injury and degeneration, astrocytes hypertrophy, extend processes, and increase production of glial fibrillary acidic protein (GFAP), an intermediate filament protein located within their cytoplasm. The present study tested the hypothesis that GFAP expression alters the vulnerability of neurons to excitotoxic and metabolic insult induced by 3-nitroproprionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II activity or the excitotoxin quinolinic acid (QA). In this respect, adult GFAP knockout mice (KO) and wild-type control mice (WT) received unilateral intrastriatal injections of 3-NP (200 nmol/microl) or QA (100 nmol/microl) and were killed 1, 2, or 4 weeks later. Lesion volume and neuronal counts were quantified using unbiased stereologic principles. For both QA and 3-NP lesions, a significant decrease in lesion volume and an increase in striatal projection neurons were seen in GFAP KO mice compared with WT mice. Enzyme-linked immunoassay analysis revealed increased basal levels of glial cell derived neurotrophic factor (GDNF) relative to WT mice. In contrast, no differences were observed in the expression of ciliary neurotrophic factor or nerve growth factor. These data strongly suggest that the expression of GFAP is implicated with the production of GDNF to a degree that confers neuroprotection after an excitotoxic or metabolic insult.

Retinoic acid downregulates the expression of ciliary neurotrophic factor in rat Schwann cells

Neuropoietic cytokines, which serve as mediators in neuroglial interactions, are differentially regulated after peripheral nerve injury. In Schwann cells, the expression of ciliary neurotrophic factor (CNTF) decreases. Pursuing the hypothesis that retinoic acid (RA) serves as a regulator of lesion-induced cytokine signaling we found that all RA receptors and retinoid X receptors are expressed in Schwann cell primary cultures. Using quantitative reverse transcription-polymerase chain reaction, we have investigated the effect of RA on the expression of CNTF in these cells. After treatment with 10 nM all- trans RA for 22 h the concentration of CNTF mRNA was reduced to 63% of the control level, reminiscent of the regulation after nerve injury in vivo. In addition to CNTF, the mRNAs of leukemia inhibitory factor, interleukin-6, ciliary neurotrophic factor receptor component α and gp130 were detected in the Schwann cells.