Abdominal Aortic Transplantation of Bone Marrow Mesenchymal Stem Cells Regulates the Expression of Ciliary Neurotrophic Factor and Inflammatory Cytokines in a Rat Model of Spinal Cord Ischemia-Reperfusion Injury.

BACKGROUND This study aimed to investigate the effects of abdominal aortic transplantation of bone marrow mesenchymal stem cells (BMMSCs) on the expression of inflammatory cytokines in a rat model of spinal cord ischemia-reperfusion injury. MATERIAL AND METHODS Adult female Sprague-Dawley rats (N=160) were divided into five groups: the sham operation group (N-32); the control group (N=32); the BMMSC transplanted group (N=32); the anti-ciliary neurotrophic factor (CNTF)-treated BMMSC transplanted group (N=32); and the CNTF small interfering RNA (siRNA)-treated BMMSC transplanted group (N=32). Motor behavior was assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. Motor evoked potentials (MEPs) and cortical somatosensory evoked potentials (CSEPs) were measured. Immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot analysis evaluated the expression of spinal inflammatory cytokines. RESULTS Following surgery, compared with the control group the findings in the BMMSC transplant groups included significantly increased BBB scores; the latency and the amplitude of MEP and CSEP were reduced and increased, respectively; spinal neuronal necrosis was reduced; the number of normal neurons increased; CNTF mRNA and protein expression levels increased; expression levels of interleukin-6 (IL-6) were reduced and IL-10 levels were significantly increased (P<0.05). The effects of abdominal aortic BMMSC transplantation were at least partially reversed by both anti-CNTF and CNTF siRNA treatment. CONCLUSIONS In a rat model of spinal cord ischemia-reperfusion injury, abdominal aortic transplantation of BMMSCs increased the expression of CNTF, which improved hindlimb locomotor recovery by regulating the expression of IL-6 and IL-10 to reduce inflammation of the spinal cord.

Ciliary neurotrophic factor is a key sex-specific regulator of depressive-like behavior in mice.

Ciliary neurotrophic factor (CNTF) is produced by astrocytes and promotes neurogenesis and neuroprotection. Little is known about the role of CNTF in affective behavior. We investigated whether CNTF affects depressive- and anxiety-like behavior in adult mice as tested in the forced swim, sucrose preference and elevated-T maze tests. Female wild type CNTF+/+ mice more readily developed behavioral despair with increased immobility time and decreased latency to immobility in the forced swim test than male CNTF+/+ littermates. The lack of CNTF in CNTF-/- mice had an opposite effect on depressive-like behavior in female mice (reduced immobility time and increased sucrose preference) vs. male mice (increased immobility time). Female wildtype mice expressed more CNTF in the amygdala than male mice. Ovariectomy increased CNTF expression, as well as immobility time, which was significantly reduced in CNTF-/- mice, suggesting that CNTF mediates overiectomy-induced immobility time, possibly in the amygdala. Progesterone but not 17-ß estradiol inhibited CNTF expression in cultured C6 astroglioma cells. Progesterone treatment also reduced CNTF expression in the amygdala and decreased immobility time in female CNTF+/+ but not in CNTF-/- mice. Castration did not alter CNTF expression in males nor their behavior. Lastly, there were no effects of CNTF on the elevated T-maze, a behavioral test of anxiety, suggesting that a different mechanism may underlie anxiety-like behavior. This study reveals a novel CNTF-mediated mechanism in stress-induced depressive-like behavior and points to opportunities for sex-specific treatments for depression, e.g. progesterone in females and CNTF-stimulating drugs in males.

mTORC1 pathway disruption abrogates the effects of the ciliary neurotrophic factor on energy balance and hypothalamic neuroinflammation.

Ciliary neurotrophic factor (CNTF) potently decreases food intake and body weight in diet-induced obese mice by acting through neuronal circuits and pathways located in the arcuate nucleus (ARC) of the hypothalamus. CNTF also exerts pro-inflammatory actions within the brain. Here we tested whether CNTF modifies energy balance by inducing inflammatory responses in the ARC and whether these effects depend upon the mechanistic target of rapamycin complex 1 (mTORC1) pathway, which regulates both energy metabolism and inflammation. To this purpose, chow- and high fat diet (HFD)- fed mice lacking the S6 kinase 1 (S6K1-/-), a downstream target of mTORC1, and their wild-type (WT) littermates received 12 days continuous intracerebroventricular (icv) infusion of the CNTF analogue axokine (CNTFAx15). Behavioral, metabolic and molecular effects were evaluated. Central chronic administration of CNTFAx15 decreased body weight and feed efficiency in WT mice only, when fed HFD, but not chow. These metabolic effects correlated with increased number of iba-1 positive microglia specifically in the ARC and were accompanied by significant increases of IL-1ß and TNF-α mRNA expression in the hypothalamus. Hypothalamic iNOS and SOCS3 mRNA, molecular markers of pro-inflammatory response, were also increased by CNTFAx15. All these changes were absent in S6K1-/- mice. This study reveals that CNTFAx15 requires a functional S6K1 to modulate energy balance and hypothalamic inflammation in a diet-dependent fashion. Further investigations should determine whether S6K1 is a suitable target for the treatment of pathologies characterized by a high neuroinflammatory state.

Maintenance of membrane organization in the aging mouse brain as the determining factor for preventing receptor dysfunction and for improving response to anti-Alzheimer treatments.

Although a major risk factor for Alzheimer's disease (AD), the "aging" parameter is not systematically considered in preclinical validation of anti-AD drugs. To explore how aging affects neuronal reactivity to anti-AD agents, the ciliary neurotrophic factor (CNTF)-associated pathway was chosen as a model. Comparison of the neuroprotective properties of CNTF in 6- and 18-month old mice revealed that CNTF resistance in the older animals is associated with the exclusion of the CNTF-receptor subunits from rafts and their subsequent dispersion to non-raft cortical membrane domains. This age-dependent membrane remodeling prevented both the formation of active CNTF-receptor complexes and the activation of prosurvival STAT3 and ERK1/2 pathways, demonstrating that age-altered membranes impaired the reactivity of potential therapeutic targets. CNTF-receptor distribution and CNTF signaling responses were improved in older mice receiving dietary docosahexaenoic acid, with CNTF-receptor functionality being similar to those of younger mice, pointing toward dietary intervention as a promising adjuvant strategy to maintain functional neuronal membranes, thus allowing the associated receptors to respond appropriately to anti-AD agents.

Neurotrophin signaling in a genitofemoral nerve target organ during testicular descent in mice.

BACKGROUND/AIM: It has been proposed that androgens control inguinoscrotal testicular descent via release of calcitonin gene-related peptide (CGRP) from a masculinised genitofemoral nerve (GFN). As there are androgen receptors in the inguinoscrotal fat pad (IFP) during the window of androgen sensitivity (E14-17 in mouse embryos), we tested the hypothesis that neurotrophins in the IFP may masculinise the sensory fibers of the GFN supplying the gubernaculum and IFP prior to gubernacular migration. METHODS: Androgen-receptor knockout (ARKO) and wild-type (WT) mouse embryos were collected at E17, with ethical approval (AEC 734). Sagittal sections of IFP, mammary area and bulbocavernosus (BC) muscle were processed for standard histology and fluorescent immunohistochemistry for ciliary neurotrophic factor (CNTF), ciliary neurotrophic factor receptor (CNTFR) and cell nuclei (DAPI). RESULTS: In the ARKO mouse CNTFR immunoreactivity (CNTFR-IR) was increased in the IFP but decreased in BC. Perinuclear staining of CNTF-IR was seen in mouse sciatic nerve but only weakly in IFP. In the mammary area, also supplied by GFN, there were no differences in IR staining. CONCLUSION: This study found CNTFR-IR in the IFP was negatively regulated by androgen, suggesting that CNTF signaling may be suppressed in GFN sensory nerves to enable CGRP expression for regulating gubernacular migration in the male, but not the female. The indirect action of androgen via the GFN required for testicular descent may be one of the sites of anomalies in the putative multifactorial cause of cryptorchidism.

Myokines (muscle-derived cytokines and chemokines) including ciliary neurotrophic factor (CNTF) inhibit osteoblast differentiation.

Muscle and bone are intimately linked by bi-directional signals regulating both muscle and bone cell gene expression and proliferation. It is generally accepted that muscle cells secrete factors (myokines) that influence adjacent bone cells, but these myokines are yet to be identified. We have previously shown that osteocyte-specific deletion of the co-receptor subunit utilized by IL-6 family cytokines, glycoprotein 130 (gp130), resulted in impaired bone formation in the trabecular bone, but enhanced periosteal expansion, suggesting a gp130-dependent periosteum-specific inhibition of osteoblast function, potentially induced by the local muscle fibres. We report here that differentiated primary calvarial osteoblasts cultured in myotube-conditioned media (CM) from myogenic C2C12 cells show reduced mRNA levels of genes associated with osteoblast differentiation. Alkaline phosphatase protein activity and all mRNA markers of osteoblast differentiation in the tested panel (runx2, osterix, alkaline phosphatase, parathyroid hormone (PTH) receptor, osteoprotegerin, osteocalcin, sclerostin) were reduced following culture with myotube CM. The exception was RANKL, which was significantly elevated in differentiated primary osteoblast cultures expressing osteocytic genes. A cytokine array of the C2C12 myotube-conditioned media identified TIMP-1 and MCP-1 as the most abundant myokines, but treatment with recombinant TIMP-1 or MCP-1 did not inhibit osteoblast gene expression. Rather, the IL-6 family cytokine ciliary neurotrophic factor (CNTF), which we found abundantly expressed by mouse muscle at the transcript and protein level, reduced osteoblast gene expression, although not to the same extent as the myotube-conditioned media. These data indicate that muscle cells secrete abundant TIMP-1, MCP-1, and CNTF, and that of these, only CNTF has the ability to suppress osteoblast function and gene expression in a similar manner to myotube-conditioned medium. This suggests that CNTF is an inhibitory myokine for osteoblasts.

Neurotrophins and the regulation of energy balance and body weight.

Complex interactions between the brain and peripheral tissues mediate the effective control of energy balance and body weight. Hypothalamic and hindbrain neural circuits integrate peripheral signals informing the nutritional status of the animal and in response regulate nutrient intake and energy utilization. Obesity and its many medical complications emerge from the dysregulation of energy homeostasis. Excessive weight gain might also arise from alterations in reward systems of the brain that drive consumption of calorie dense, palatable foods in the absence of an energy requirement. Several neurotrophins, most notably brain-derived neurotrophic factor, have been implicated in the molecular and cellular processes underlying body weight regulation. Here, we review investigations interrogating their roles in energy balance and reward centers of the brain impacting feeding behavior and energy expenditure.

Motoneuron disease.

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) represent the two major forms of motoneuron disease. In both forms of disease, spinal and bulbar motoneurons become dysfunctional and degenerate. In ALS, cortical motoneurons are also affected, which contributes to the clinical phenotype. The gene defects for most familial forms of ALS and SMA have been discovered and they point to a broad spectrum of disease mechanisms, including defects in RNA processing, pathological protein aggregation, altered apoptotic signaling, and disturbed energy metabolism. Despite the fact that lack of neurotrophic factors or their corresponding receptors are not found as genetic cause of motoneuron disease, signaling pathways initiated by neurotrophic factors for motoneuron survival, axon growth, presynaptic development, and synaptic function are disturbed in ALS and SMA. Better understanding of how neurotrophic factors and downstream signaling pathways interfere with these disease mechanisms could help to develop new therapies for motoneuron disease and other neurodegenerative disorders.

Mechanisms for axon maintenance and plasticity in motoneurons: alterations in motoneuron disease.

In motoneuron disease and other neurodegenerative disorders, the loss of synapses and axon branches occurs early but is compensated by sprouting of neighboring axon terminals. Defective local axonal signaling for maintenance and dynamics of the axonal microtubule and actin cytoskeleton plays a central role in this context. The molecular mechanisms that lead to defective cytoskeleton architecture in two mouse models of motoneuron disease are summarized and discussed in this manuscript. In the progressive motor neuropathy (pmn) mouse model of motoneuron disease that is caused by a mutation in the tubulin-specific chaperone E gene, death of motoneuron cell bodies appears as a consequence of axonal degeneration. Treatment with bcl-2 overexpression or with glial-derived neurotrophic factor prevents loss of motoneuron cell bodies but does not influence the course of disease. In contrast, treatment with ciliary neurotrophic factor (CNTF) significantly delays disease onset and prolongs survival of pmn mice. This difference is due to the activation of Stat-3 via the CNTF receptor complex in axons of pmn mutant motoneurons. Most of the activated Stat-3 protein is not transported to the nucleus to activate transcription, but interacts locally in axons with stathmin, a protein that destabilizes microtubules. This interaction plays a major role in CNTF signaling for microtubule dynamics in axons. In Smn-deficient mice, a model of spinal muscular atrophy, defects in axonal translocation of ß-actin mRNA and possibly other mRNA species have been observed. Moreover, the regulation of local protein synthesis in response to signals from neurotrophic factors and extracellular matrix proteins is altered in motoneurons from this model of motoneuron disease. These findings indicate that local signals are important for maintenance and plasticity of axonal branches and neuromuscular endplates, and that disturbances in these signaling mechanisms could contribute to the pathophysiology of motoneuron diseases.

Ciliary neurotrophic factor (CNTF) promotes skeletal muscle progenitor cell (MPC) viability via the phosphatidylinositol 3-kinase-Akt pathway.

Muscle progenitor cells (MPCs) are currently being investigated as cellular vectors to deliver neurotrophic factor (NF) for the promotion of re-innervation after axonal injury. Ideally NF delivery in such a model would enhance axonal regeneration while simultaneously promoting MPC viability. To date, insulin-like growth factor 1 (IGF-1) is one of the few NFs known to promote both re-innervation and MPC viability. We herein identify ciliary neurotrophic factor (CNTF) as a factor that promotes MPC viability in culture, and demonstrate CNTF to impart greater viability effects on MPCs than IGF-1. We demonstrate that pharmacological inhibition via LY294002 results in abrogation of CNTF-mediated viability, suggesting that the CNTF-mediated MPC viability benefit occurs via the PI3-Akt pathway. Finally, we employ a genetic model, establishing MPC cultures from mice deficient in class IA PI-3 K (p85α(-/-) ) mice, and demonstrate that the viability benefit imparted by CNTF is completely abrogated in PI-3 K-deficient MPCs compared to wild-type controls. In summary, our investigations define CNTF as a promoter of MPC viability beyond IGF-1, and reveal that the CNTF-mediated MPC viability effects occur via the PI3-Akt pathway.

Role of ciliary neurotrophic factor in the proliferation and differentiation of neural stem cells.

Ciliary neurotrophic factor (CNTF) is a pleiotropic cytokine that has been fully studied for its structure, receptor, and signaling pathways and its multiplex effects on neural system, skeletal muscle, and weight control. Recent research demonstrates that CNTF also plays an important role in neurogenesis and the differentiation of neural stem cells. In this article, we summarize the general characteristics of CNTF and its function on neural stem cells, which could be a valuable therapeutic strategy in treating neurological disorders.

Cerebellar neural stem cells differentiate into two distinct types of astrocytes in response to CNTF and BMP2.

Neural stem cells (NSCs) are present in postnatal murine cerebellum. The detailed characteristics of these NSCs have never been reported. This study isolated NSC-like cells from postnatal mouse cerebellum. These cells proliferated in response to epidermal growth factor, expressed various NSC markers, and had the ability to self-renew. Neurosphere assays revealed that these cells could differentiate into neurons, astrocytes, and oligodendrocytes, indicating multipotency as NSCs. Although possessing multipotency, most of these cells differentiated into astrocytes spontaneously in vitro. Both ciliary neurotrophic factor (CNTF) and bone morphogenetic protein 2 (BMP2) facilitated expression of glial fibrillary acidic protein (GFAP) and some other characteristics of mature astrocytes by these cells. However, the shape and expression of glutamine transporter GLT-1 of GFAP(+) cells generated in the presence of CNTF or BMP2 differed significantly, suggesting that CNTF and BMP2 induced differentiation of these NSCs into two distinct types of astrocytes.

Misguidance and modulation of axonal regeneration by Stat3 and Rho/ROCK signaling in the transparent optic nerve.

The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological blockade of ROCK, a key signaling component for myelin-associated growth inhibitors, reduced axonal U-turns and potentiated AAV2.Stat3-ca-induced regeneration. Similar results were obtained after the sustained delivery of CNTF in the axotomized retina. These results show the important role of Stat3 in the activation of the neuronal growth program for regeneration, and they reveal that axonal misguidance is a key limiting factor that can affect long-distance regeneration and target interaction after trauma in the CNS. The correction of axonal misguidance was associated with improved long-distance axon regeneration in the injured adult CNS.

Hypothalamic neurogenesis in the adult brain.

Adult-born new neurons are continuously added to the hippocampus and the olfactory bulb to serve aspects of learning and perceptual functions. Recent evidence establishes a third neurogenic niche in the ventral hypothalamic parenchyma surrounding the third ventricle that ensures the plasticity of specific brain circuits to stabilize physiological functions such as the energy-balance regulatory system. Hypothalamic lesion studies have demonstrated that regions associated with reproduction-related functions are also capable of recruiting newborn neurons to restore physiological functions and courtship behavior. Induced by lesion or other stimulation, elevated neurotrophic factors trigger neurogenic cascades that contribute to remodeling of certain neural circuits to meet specific transient functions. This insight raises the possibility that event-specific changes, such as increased GnRH, may be mediated by courtship-sensitive neurotrophic factors. We will discuss the potentially integral and ubiquitous roles of neurogenesis in physiological and biological phenomena, roles that await future experimental exploration.

Neuroprotection in glaucoma: present and future.

OBJECTIVE: To review the updated research on neuroprotection in glaucoma, and summarize the potential agents investigated so far. DATA SOURCES: The data in this review were collected from PubMed and Google Scholar databases published in English up to September 2012, with keywords including glaucoma, neuroprotection, and retinal ganglion cells, both alone and in combination. Publications from the past ten years were selected, but important older articles were not excluded. STUDY SELECTION: Articles about neuroprotection in glaucoma were selected and reviewed, and those that are cited in articles identified by this search strategy and judged relevant to this review were also included. RESULTS: Although lowering the intraocular pressure is the only therapy approved as being effective in the treatment of glaucoma, increasing numbers of studies have discovered various mechanisms of retinal ganglion cells death in the glaucoma and relevant neuroprotective strategies. These strategies target neurotrophic factor deprivation, excitotoxic damage, oxidative stress, mitochondrial dysfunction, inflammation, activation of intrinsic and extrinsic apoptotic signals, ischemia, and protein misfolding. Exploring the mechanism of axonal transport failure, synaptic dysfunction, the glial system in glaucoma, and stem cell used in glaucoma constitute promising research areas of the future. CONCLUSIONS: Neuroprotective strategies continue to be refined, and future deep investment in researching the pathogenesis of glaucoma may provide novel and practical neuroprotection tactics. Establishing a system to assess the effects of neuroprotection treatments may further facilitate this research.

Ciliary neurotrophic factor controls progenitor migration during remyelination in the adult rodent brain.

Ciliary neurotrophic factor (CNTF) has been shown to be expressed after brain lesions and in particular after demyelination. Here, we addressed the role of this cytokine in the regulation of neural progenitor migration in the adult rodent brain. Using an acute model of demyelination, we show that CNTF is strongly re-expressed after lesion and is involved in the postlesional mobilization of endogenous progenitors that participate in the myelin regenerative process. We show that CNTF controls the migration of subventricular zone (SVZ)-derived neural progenitors toward the demyelinated corpus callosum. Furthermore, an ectopic source of CNTF in adult healthy brains changes SVZ-derived neural progenitors' migratory behavior that migrate toward the source by activation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway. Using various in vitro assays (Boyden chambers, explants, and video time-lapse imaging), we demonstrate that CNTF controls the directed migration of SVZ-derived progenitors and oligodendrocyte precursors. Altogether, these results demonstrate that in addition to its neuroprotective activity and its role in progenitor survival and maturation, CNTF acts as a chemoattractant and participates in the recruitment of endogenous progenitors during myelin repair.

CNTF-mediated preactivation of astrocytes attenuates neuronal damage and epileptiform activity in experimental epilepsy.

Activated astrocytes display a broad spectrum of properties, ranging from neuroprotection to active contribution to demise of neural tissue. To investigate if activation of astrocytes by a single, defined stimulus enhances neuroprotective properties, we tested whether injection of ciliary neurotrophic factor (CNTF) can ameliorate epilepsy-related brain damage. Intrahippocampal CNTF injection in mice induced a rapid (within 2 days) and persistent (3 weeks) activation of astrocytes reflected by strong upregulation of glial fibrillary acidic protein (GFAP) mRNA synthesis and GFAP immunoreactivity. Moreover, CNTF signaling via phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3) was specifically activated in GFAP-positive astrocytes. CNTF-mediated activation of astrocytes 2 days prior to an epileptogenic intrahippocampal injection of kainate (KA) resulted in strongly reduced cell death in the hilus and CA3 region of the hippocampus, revealed by Fluoro-Jade B staining. Granule cell dispersion, the pathological widening of the granule cell layer, was also significantly reduced 16 days after KA injection. Importantly, intrahippocampal in vivo recordings 3 weeks after KA injection showed that the occurrence of high frequency oscillations (fast ripples, FR), a surrogate marker for epileptic activity, was significantly reduced in CNTF+KA-injected mice as compared to KA-injected animals. However, when CNTF was applied in the chronic epileptic phase at 3 weeks after KA injection, no reduction of FR activity was observed. In summary, our results indicate that the activation of astrocytes prior to an excitotoxic injury effectively reduces neuronal damage and the severity of epileptiform activity, whereas activation in the chronic phase is no longer protective.

Endogenous ciliary neurotrophic factor modulates anxiety and depressive-like behavior.

On a molecular level, depression is characterized by an altered monoaminergic neurotransmission as well as a modulation of cytokines and other mediators in the central nervous system. In particular, neurotrophic factors may influence affective behavior including depression and anxiety. Ciliary neurotrophic factor (CNTF) plays an important role in the regulation of neuronal development, neuroprotection and may also influence cognitive processes. Here we investigate the affective behavior in mice deficient for CNTF (CNTF -/- mice) at young age of 10-20 weeks. CNTF -/- mice displayed an increased anxiety-like behavior with a 30% reduction of the time spent in the bright compartment of the light/dark box as well as a significantly increased startle response. In the learned helplessness paradigm, CNTF -/- mice are more prone to depressive-like behavior. In the hippocampus of 20 weeks old, but not 10 weeks old, CNTF -/- mice, these changes correlated with a loss of parvalbumin immunoreactive GABAergic interneurons and a reduction of serotonin levels as well as 5-HT receptor 1A expression. Modulation of monoaminergic neurotransmitter levels via chronic application of the antidepressants amitriptyline and citalopram did not exert beneficial effects. These data imply that endogenous CNTF plays a pivotal role for the structural maintenance of hippocampal functions and thus has an important impact on the modulation of affective behavior in rodent models of anxiety and depression.

CNTF and retina.

Ciliary neurotrophic factor (CNTF) is one of the most studied neurotrophic factors for neuroprotection of the retina. A large body of evidence demonstrates that CNTF promotes rod photoreceptor survival in almost all animal models. Recent studies indicate that CNTF also promotes cone photoreceptor survival and cone outer segment regeneration in the degenerating retina and improves cone function in dogs with congenital achromotopsia. In addition, CNTF is a neuroprotective factor and an axogenesis factor for retinal ganglion cells (RGCs). This review focuses on the effects of exogenous CNTF on photoreceptors and RGCs in the mammalian retina and the potential clinical application of CNTF for retinal degenerative diseases.

Functional duality of astrocytes in myelination.

Astrocytes undergo major phenotypic changes in response to injury and disease that directly influence repair in the CNS, but the mechanisms involved are poorly understood. Previously, we have shown that neurosphere-derived rat astrocytes plated on poly-L-lysine (PLL-astrocytes) support myelination in dissociated rat spinal cord cultures (myelinating cultures). It is hypothesized that astrocyte reactivity can affect myelination, so we have exploited this culture system to ascertain how two distinct astrocyte phenotypes influence myelination. Astrocytes plated on tenascin C (TnC-astrocytes), a method to induce quiescence, resulted in less myelinated fibers in the myelinating cultures when compared with PLL-astrocytes. In contrast, treatment of myelinating cultures plated on PLL-astrocytes with ciliary neurotrophic factor (CNTF), a cytokine known to induce an activated astrocyte phenotype, promoted myelination. CNTF could also reverse the effect of quiescent astrocytes on myelination. A combination of microarray gene expression analysis and quantitative real-time PCR identified CXCL10 as a potential candidate for the reduction in myelination in cultures on TnC-astrocytes. The effect of TnC-astrocytes on myelination was eliminated by neutralizing CXCL10 antibodies. Conversely, CXCL10 protein inhibited myelination on PLL-astrocytes. Furthermore, CXCL10 treatment of purified oligodendrocyte precursor cells did not affect proliferation, differentiation, or process extension compared with untreated controls, suggesting a role in glial/axonal ensheathment. These data demonstrate a direct correlation of astrocyte phenotypes with their ability to support myelination. This observation has important implications with respect to the development of therapeutic strategies to promote CNS remyelination in demyelinating diseases.