Novel analogs of allopregnanolone show improved efficiency and specificity in neuroprotection and stimulation of proliferation.

The natural neurosteroid allopregnanolone exerts beneficial effects in animal models of neurodegenerative diseases, nervous system injury and peripheral neuropathies. It not only has anti-apoptotic activity, but also promotes proliferation of progenitor cells. With respect to using it as a therapeutic tool, such pleiotropic actions might create unwanted side effects. Therefore, we have synthesized allopregnanolone analogs and analyzed their neuroprotective and proliferative effects to identify compounds with higher efficiency and less ambiguous biological actions. Proliferation-promoting effects of 3α and 3ß isomers of 3-O-allyl-allopregnanolone and 12 oxo-allopregnanolone were studied in adult subventricular zone stem cell cultures and in primary hippocampal cultures by measuring 5-ethynyl-2'-deoxyuridine incorporation. Neuroprotective activity against amyloid beta 42-induced cell death was determined by quantifying caspase 3/7 activity. The 3α isomers significantly stimulated proliferation in all culture systems, whereas the 3ß isomers were ineffective. The stimulatory effect of 3α-O-allyl-allopregnanolone was significantly higher than that of allopregnanolone. In neural stem cell cultures, 3α-O-allyl-allopregnanolone specifically enhanced proliferation of Nestin-positive progenitors. In addition, it promoted the differentiation of doublecortin-positive neurons. In neural stem cell cultures treated with amyloid beta 42, both the α and ß isomers of O-allyl- allopregnanolone showed increased neuroprotective activity as compared to allopregnanolone, completely preventing amyloid-induced caspase 3/7 activation. The 12 oxo-allopregnanolone analogs were ineffective. These results identify structural allopregnanolone analogs with higher anti-apoptotic and proliferation-promoting activity than the natural neurosteroid. Interestingly, stereoisomers of the analogs were found to have distinct profiles of activity raising the possibility of exploiting the neuroprotective properties of neurosteroids with or without simultaneously stimulating neurogenesis. Cover Image for this issue: doi: 10.1111/jnc.13344.

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.

JAK2-STAT3 signaling: A novel function and a novel mechanism.

The function of JAK-STAT signaling in the central nervous system has been widely studied in the context of neural cell development and differentiation and in neuronal and glial responses to CNS injury. A study published recently in Neuron by Nicolas et al. now demonstrates that the JAK2-STAT3 pathway also plays an important role in the regulation of synaptic transmission. By using a combination of biochemical, pharmacological and genetic approaches they show that induction of long-term depression (LTD), an activity-dependent rapid and long-lasting decrease in synaptic strength, via NMDA receptors depends on STAT3 activation by JAK2 that can be localized specifically to postsynaptic structures. Most interestingly, they find that induction of LTD requires STAT3 phosphorylation and dimerization but is independent of nuclear translocation and transcriptional activity of STAT3. Although it remains to be clarified how NMDA receptor-mediated postsynaptic processes lead to JAK2-STAT3 activation and how this in turn translates into persistent changes in synaptic strength, these results provide evidence for a novel mechanism of signal transduction.

Involvement of gp130-associated cytokine signaling in Müller cell activation following optic nerve lesion.

Ciliary neurotrophic factor (CNTF) and the related cytokine leukemia inhibitory factor (LIF) have been implicated in regulating astrogliosis following CNS lesions. Application of the factors activates astrocytes in vivo and in vitro, and their expression as well as their receptors is upregulated after brain injury. Here, we investigated their function by studying Müller cell activation induced by optic nerve crush in CNTF- and LIF-deficient mice, and in animals with deficiencies in cytokine signaling pathways. In the retina of CNTF(-/-) mice, basal GFAP expression was reduced, but unexpectedly, injury-induced upregulation in activated Müller cells was increased during the first 3 days after lesion as compared to wild-type animals and this corresponded with higher phosphorylation level of STAT3, an indicator of cytokine signaling. The observation that LIF expression was strongly upregulated in CNTF(-/-) mice but not in wild-type animals following optic nerve lesion provided a possible explanation. In fact, additional ablation of the LIF gene in CNTF/LIF double knockout mice almost completely abolished early lesion-induced GFAP upregulation in Müller cells and STAT3 phosphorylation. Early Müller cell activation was also eliminated in LIF(-/-) mice, despite normal CNTF levels, as well as in mutants deficient in gp130/JAK/STAT signaling and in conditional STAT3 knockout mice. Our results demonstrate that LIF signaling via the gp130/JAK/STAT3 pathway is required for the initiation of the astrogliosis-like reaction of retinal Müller cells after optic nerve injury. A potential role of CNTF was possibly masked by a compensatory increase in LIF signaling in the absence of CNTF.

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice.

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1(-/-) mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75(NTR)) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.

Neurogenesis in the dentate gyrus depends on ciliary neurotrophic factor and signal transducer and activator of transcription 3 signaling.

In the neurogenic areas of the adult rodent brain, neural stem cells (NSCs) proliferate and produce new neurons throughout the lifetime. This requires a permanent pool of NSCs, the size of which needs to be tightly controlled. The gp130-associated cytokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) have been implicated in regulating NSC self-renewal and differentiation during embryonic development and in the adult brain. To study the relevance of the two cytokines in vivo, we analyzed precursor cell proliferation and neurogenesis in the dentate gyrus of CNTF- and LIF-deficient mouse mutants. The number of radial glia-like NSCs, proliferative activity, and generation of new neurons were all reduced in CNTF(-/-) mutants but unaltered in LIF(-/-) animals. Conditional ablation of the signal transducer and activator of transcription 3 (STAT3) gene under the control of the human glial fibrillary acidic protein promoter resulted in a reduction of neurogenesis similar to that in CNTF(-/-) mice. The size of the granule cell layer was decreased in both mutants. Treatment of neurosphere cultures prepared from adult forebrain with CNTF inhibited overall proliferative activity but increased the number of NSCs as indicated by enhanced secondary neurosphere formation and upregulated expression of stem cell markers. Knockdown of STAT3 with short interfering RNA inhibited CNTF effects on neurospheres, and knockdown of suppressor of cytokine signaling 3 (SOCS3) enhanced them. Our results provide evidence that CNTF-induced STAT3 signaling is essential for the formation and/or maintenance of the neurogenic subgranular zone in the adult dentate gyrus and suggest that CNTF is required to keep the balance between NSC self-renewal and the generation of neuronal progenitors.

Regulation of cytokine signaling components in developing rat retina correlates with transient inhibition of rod differentiation by CNTF.

Ciliary neurotrophic factor (CNTF) is known to inhibit the differentiation of rod photoreceptors from postmitotic precursor cells. During early postnatal development, photoreceptor precursors lose their responsiveness to CNTF. The underlying events causing this change in responsiveness are unknown. Moreover, whether rods express CNTF receptor alpha, a prerequisite for a direct response to the factor, is controversial. Since morphological studies have previously produced conflicting results, we have analyzed the expression of cytokine receptor components and potential ligands in the rat photoreceptor layer by real-time reverse transcription with the polymerase chain reaction after laser microdissection and by immunoblotting. Cytokine effects on rods were studied in explant cultures from newborn rat retina. CNTF receptor alpha (CNTFR alpha) and leukemia inhibitory factor receptor ss (LIFRss) were expressed in immature photoreceptors. Expression of the CNTF-specific alpha-subunit (but not of LIFRss) was downregulated specifically in the photoreceptor layer in parallel with the appearance of opsin-positive rods. The decrease of CNTFR alpha levels in explant cultures was closely correlated with the loss of precursor cell responsiveness to CNTF. Increasing the CNTF concentration in the culture medium led to prolonged CNTFR alpha expression and, concomitantly, to persistent inhibition of rod differentiation. Application of CNTF and LIF in vitro induced phosphorylation of STAT3. Inducibility of STAT3 activation by CNTF decreased with photoreceptor maturation, whereas the LIF effect persisted. Our results thus indicate that CNTF acts directly on photoreceptor precursors inhibiting their differentiation via activation of the JAK/STAT3 signal transduction pathway, and that this effect is temporally limited because of the downregulation of CNTFR alpha.

Altered neuronal responses and regulation of neurotrophic proteins in the medial septum following fimbria-fornix transection in CNTF- and leukaemia inhibitory factor-deficient mice.

Degeneration of axotomized GABAergic septohippocampal neurones has been shown to be enhanced in ciliary neurotrophic factor (CNTF)-deficient mice following fimbria-fornix transection (FFT), indicating a neuroprotective function of endogenous CNTF. Paradoxically, however, the cholinergic population of septohippocampal neurones was more resistant to axotomy in these mutants. As leukaemia inhibitory factor (LIF) has been identified as a potential neuroprotective factor for the cholinergic medial septum (MS) neurones, FFT-induced responses were compared in CNTF(-/-), LIF(-/-) and CNTF/LIF double knockout mice. In CNTF(-/-) mice, FFT-induced cholinergic degeneration was confirmed to be attenuated as compared with wildtype mice. The expression of both LIF and LIF receptor beta was increased in the MS providing a possible explanation for the enhanced neuronal resistance to FFT in these animals. However, ablation of the LIF gene also produced paradoxical effects; following FFT in LIF(-/-) mice no loss of GABAergic or cholinergic MS neurones was detectable during the first postlesional week, suggesting that other efficient neuroprotective mechanisms are activated in these animals. In fact, enhanced activation of astrocytes, a source of neurotrophic proteins, was indicated by increased up-regulation of glial fibrillary acidic protein and vimentin expression. In addition, mRNA levels for neurotrophin signalling components (e.g. nerve growth factor, p75(NTR)) were differentially regulated. The positive effect on axotomized cholinergic neurones seen in CNTF(-/-) and LIF(-/-) mice as well as the increased up-regulation of astrogliose markers was abolished in CNTF/LIF double knockout animals. Our results indicate that endogenous CNTF and LIF are involved in the regulation of neuronal survival following central nervous system lesion and are integrated into a network of neurotrophic signals that mutually influence their expression and function.

Activation of STAT3 signaling in axotomized neurons and reactive astrocytes after fimbria-fornix transection.

It is an open question to what extent neuroprotective mechanisms involving neurotrophic proteins are activated after central nervous system (CNS) lesions. Results from previous studies have indicated that ciliary neurotrophic factor (CNTF) and other members of the family of gp130-associated cytokines have neuroprotective effects on septohippocampal projection neurons axotomized by fimbria-fornix transection (FFT). Here we demonstrate that the transcription factor STAT3, a component of the primary cytokine signal transduction pathway, is transiently activated after FFT in the medial septum and in the lateral septum deafferented by the lesion. Immunocytochemical double-labeling showed nuclear signals for phosphorylated STAT3 in both types (GABAergic and cholinergic) of axotomized medial septal neurons around day 4 postlesion. This response temporally coincided with the cell-type-specific upregulation of the cytokine receptor components CNTF receptor alpha and LIF receptor beta in the same neurons. However, neuronal STAT3-activation was not abolished in CNTF- or LIF-deficient mouse mutants. Furthermore, lesion-induced STAT3 signaling was also found in reactive GFAP-positive astrocytes of the medial and lateral septum. Interestingly, basal GFAP expression was reduced but postlesional upregulation was markedly enhanced in CNTF(-/-) animals. These results demonstrate transient activation of cytokine signaling after CNS lesion and suggest that gp130-associated cytokines have multiple functions in the lesioned CNS by directly acting on axotomized neurons and on reactive astrocytes.

The peptides ADNF-9 and NAP increase survival and neurite outgrowth of rat retinal ganglion cells in vitro.

PURPOSE: Recent studies demonstrated that short peptides derived from activity-dependent neurotrophic factor (ADNF) and activity-dependent neuroprotective protein (ADNP) are neuroprotective at femtomolar concentrations. We evaluated these findings in cultures of purified rat retinal ganglion cells (RGCs) using two such peptides: ADNF-9 and NAP. In a second step, the influence of these peptides on neurite outgrowth in retinal explants was investigated. METHODS: Retinal ganglion cells (RGCs) were purified from newborn (postnatal day [P]0-P2) rat retina by immunopanning with antibodies against Thy1.1 and were cultured in serum-free N2 medium for 2 days. RGCs were treated with ADNF-9 and NAP at concentrations ranging from 10(-18) to 10(-10) M. Survival was quantified by counting viable cells by phase-contrast microscopy. Retinal explants from postnatal (P9-P11) rats were cultured in three-dimensional fibrin clots in serum-free medium for 3 days. Explants were treated with 1 microM NAP or 1 microM ADNF-9. Neurite outgrowth was visualized by staining with Sudan black and quantified by measuring axonal length. RESULTS: Both peptides enhanced survival of RGCs in a dose-dependent manner. ADNF-9 showed a maximum effect at 0.1 pM with an increase in survival to 177% (95% confidence interval: 149-204) of the control level. The EC(50) was 10.9 fM. NAP showed a maximum effect at 5 pM with an increase in survival to 167% (146-189) and an EC(50) of 6.1 fM. In the explants, 1 microM ADNF-9 enhanced axonal outgrowth to 126% (118-133) and 1 microM NAP to 117% (98-137) compared with the control. CONCLUSIONS: Both peptides, ADNF-9 and NAP, not only increase RGC survival in vitro but also support neurite outgrowth in retinal explants. These peptides deserve further attention as potential neuroprotective compounds in retinal and optic nerve diseases.

Retinal ganglion cell survival is enhanced by gabapentin-lactam in vitro: evidence for involvement of mitochondrial KATP channels.

BACKGROUND: Recently, gabapentin-lactam (GBP-L) was shown to be neuroprotective in vivo. It has been suggested that GBP-L may act by opening mitochondrial ATP-sensitive potassium (K(ATP)) channels. We tested this hypothesis by quantifying the effect of GBP-L on the survival of purified retinal ganglion cells (RGCs). METHODS: RGCs were purified from early postnatal rat retinae by immunopanning with antibodies against Thy1.1 and cultured in serum-free medium for 2 days. Cell survival was quantified by counting vital cells under phase-contrast optics. Results were normalized to controls. RGCs were treated with various concentrations (3.2-320 microM) of GBP-L with and without 1 microM glibenclamide, blocking both plasmalemmal and mitochondrial K(ATP) channels, or 100 microM 5-hydroxydecanoate (5-HD), antagonizing selectively mitochondrial K(ATP) channels. For comparison, additional cultures were treated with 32 microM gabapentin, the parent drug of GBP-L. A combination of the neurotrophic factors BDNF and CNTF (50 ng/ml each) served as a positive control. RESULTS. GBP-L increased RGC survival to a maximum of 145+/-5% (mean +/- SEM) in a concentration-dependent manner. The pEC(50) was 5.0, CI95 [4.7, 5.3]. Preincubation with glibenclamide changed the dose-response of GBP-L, indicating that it acted as a competitive antagonist with a pA2 value of 6.8, CI95 [5.9, 7.5]. 5-HD completely blocked the survival-promoting effect of GBP-L. Gabapentin had no effect, whereas the combination of CNTF and BDNF enhanced survival to 177+/-9%. CONCLUSIONS: GBP-L, but not gabapentin, can promote the survival of cultured central nervous system neurons, possibly by opening mitochondrial K(ATP) channels. These results suggest further testing of GBP-L as a potentially neuroprotective drug.

Expression of CNTF receptor-alpha in chick violet-sensitive cones with unique morphologic properties.

PURPOSE: Application of ciliary neurotrophic factor (CNTF) can rescue mature photoreceptors from lesion-induced and hereditary degeneration. In the chick retina, expression of the CNTF receptor is present in a subpopulation of photoreceptor cells. The present study was undertaken to identify the CNTF receptor-expressing photoreceptors and to describe the subcellular localization of the receptor protein. METHODS: The localization of the CNTF receptor was analyzed by light and electron microscopic immunocytochemistry in chick retinal wholemount preparations, with an antibody for CNTF receptor alpha (CNTFRalpha). Immunoreactive cells were identified by double labeling with immunocytochemical markers for photoreceptor subpopulations. RESULTS: The CNTFRalpha antibody labeled evenly distributed outer segments (OS) of a photoreceptor subpopulation. CNTFRalpha-positive OS were associated with oil droplets of uniform size. Receptor immunoreactivity did not colocalize with markers for rods and red-green cones. Complete overlap was found after double labeling with the antibody CERN 933, which recognizes violet-sensitive cones in the chick retina. Ultrastructurally, the CNTFRalpha-immunoreactive OS showed rodlike properties: an elongated shape and stacks of membrane discs separated from the plasma membrane. Immunoreactivity was completely restricted to the plasma membrane of the OS and the inner membrane sheet of the photoreceptor calices present in avian retinas. CONCLUSIONS: CNTFRalpha expression identifies a unique type of photoreceptors in the avian retina which does not fit into the classic morphologic definition of rods and cones. The specific expression in violet-sensitive photoreceptors suggests that CNTF may have a neuroprotective role related to the specific function of these cells.

Ciliary neurotrophic factor is an early lesion-induced retrograde signal for axotomized facial motoneurons.

To investigate the involvement of ciliary neurotropic factor (CNTF) in the postlesional response of motoneurons, we studied the activation of STAT3 signaling, the main signal transduction pathway of CNTF-like cytokines, in the facial nucleus of wildtype and CNTF-deficient mice following peripheral nerve transection. As shown by immunocytochemistry and immunoblot analysis, phosphorylation and nuclear translocation of STAT3 was maximally induced within 12 h postlesion in motoneurons of the ipsilateral facial nucleus of wildtype mice and is maintained for at least 3 days. In CNTF(-/-) mouse mutants, activation of STAT3 signaling was delayed by 10-12 h. Application of CNTF to the transected nerve restored rapid STAT3 activation in CNTF-deficient animals, whereas application of colchicine suppressed STAT3 signaling in wildtype mice for at least 24 h. These results identify CNTF as an early retrograde signal in axotomized facial motoneurons by showing that CNTF released at the lesion site is responsible for the initial induction of STAT3 signaling. Other cytokines like leukemia inhibitory factor obviously become active at later time points.

Endogenous ciliary neurotrophic factor protects GABAergic, but not cholinergic, septohippocampal neurons following fimbria-fornix transection.

Application of neurotrophic proteins including ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), members of the family of gp130-associated cytokines, can rescue CNS neurons from injury-induced degeneration. However, it is not clear so far if these effects reflect a physiological function of the endogenous cytokines. Using fimbria-fornix transection as a model, we examined whether responses of GABAergic and cholinergic septohippocampal neurons to axotomy are altered in mice lacking CNTF. In addition, we studied the cellular expression of CNTF, LIF and related cytokine receptor components in the septal complex following lesion. Degeneration of septohippocampal GABAergic neurons in the medial septum as indicated by the loss of parvalbumin-immunoreactive neurons was accelerated and permanently enhanced in CNTF(-/-) mice as compared to wild-type animals. Unexpectedly, the number of axotomized cholinergic MS neurons was significantly higher in CNTF-deficient mice during the first 2 weeks postlesion. Both in wild-type and in CNTF(-/-) mutants, expression of mRNA for the CNTF-specific alpha-subunit of the cytokine receptor complex was specifically upregulated in axotomized GABAergic septal neurons, whereas enhanced expression of the LIF-binding beta-subunit was specifically observed in axotomized cholinergic neurons. Following lesion, CNTF expression in wild-type mice was induced in activated astrocytes surrounding the axotomized neurons and at the lesion site. Expression of LIF mRNA was localized in the GABAergic and cholinergic septohippocampal neurons. These results strongly indicate that endogenous CNTF, supplied by reactive glia cells, acts as a neuroprotective factor for axotomized CNS neurons. In the septum, endogenous CNTF specifically supports lesioned GABAergic projection neurons, whereas LIF may play a similar role for the cholinergic counterparts.

Glial reactivity in ciliary neurotrophic factor-deficient mice after optic nerve lesion.

There is evidence that ciliary neurotrophic factor (CNTF), in addition to its neurotrophic activity, positively regulates astrogliosis after CNS injury. CNTF and its receptor, CNTFRalpha, are strongly upregulated in activated astrocytes. Application of CNTF upregulates GFAP expression in cultured astrocytes and induces various aspects of gliosis in the intact brain. Here we examined whether inactivation of the CNTF gene results in the expected changes in glial reactivity by analyzing gliosis in the superior colliculus (SC) after optic nerve crush. Basal expression levels of GFAP and vimentin in unlesioned CNTF-deficient mice were reduced by 66 and 37%, respectively. Absolute numbers of astrocytes were found not to be different. Surprisingly, however, lesion induced robust activation of astrocytes in CNTF-deficient mice; the time course of activation was even accelerated as compared with wild-type animals. At later time points, activation reached the same level. With respect to microglial cells, basal expression of microglial markers was unaltered in CNTF-knock-out animals. Lesion-induced upregulation of Iba-1, ICAM-1, and F4/80 in microglial cells was unaffected in CNTF-deficient animals. Differences were observed with respect to the time course of microglial activation, different markers being affected differentially. We further demonstrate that lesion induces upregulation of CNTF-related cytokines (LIF, NNT-1) and, interestingly, a more pronounced upregulation of cytokine receptor components (LIF receptor beta, gp130) and TGFbeta in CNTF-deficient animals. Our results thus indicate that CNTF is required for the development and maintenance of the mature astrocyte phenotype and provide evidence that CNTF is part of the complex regulatory network modulating lesional glial reactivity after lesion.

Distribution of CNTF receptor alpha protein in the central nervous system of the chick embryo.

Ciliary neurotrophic factor (CNTF) promotes the survival and differentiation of various neuronal and glial cell populations in the nervous system of vertebrates. In mammals, the ligand-binding alpha-subunit of the CNTF receptor (CNTFRalpha) is expressed in a variety of neuronal populations, including all CNTF-responsive cells. Previous studies suggested that functional differences in the CNTF/CNTF receptor system between chicks and mammals exist. The purpose of the present study was to examine the temporal and spatial expression pattern of the chick CNTFRalpha protein during CNS development. Receptor expression was detectable by immunoblotting in all CNS areas tested but showed area-specific developmental regulation. Interestingly, two variants of CNTFRalpha, 69 and 65 kD, were identified by immunoblotting with a shift from the higher to the lower molecular mass species occurring during development. Immunoreactivity for CNTFRalpha protein was preferentially observed in neuropil and white matter structures of the developing CNS while neuronal somata generally appeared unlabeled. For example, expression was observed in the olfactory system, in the telencephalon, in parts of the somatosensory system, in components of the tectofugal pathway, in the cerebellum, and in auditory brainstem nuclei. Fiber tracts that exhibit CNTFRalpha immunoreactivity were the lateral forebrain bundle, occipitomesencephalic tract, quintofrontal tract, and vestibular nerve. Our study identifies potential new targets of a chick CNTF-related molecule and reveals significant regional differences of CNTFRalpha protein expression between chick and mammals. These results suggest that the CNTF receptor performs distinct developmental functions in different animals.

Induction of STAT3 signaling in activated astrocytes and sprouting septal neurons following entorhinal cortex lesion in adult rats.

Entorhinal cortex lesion (ECL) leads to the activation of astrocytes and reactive axonal sprouting in the denervated fascia dentata. Previous studies indicated that CNTF or related cytokines are involved in the regulation of these processes. Therefore, we studied (1). whether the cytokine-associated STAT3 signaling pathway is activated in response to ECL and (2). which CNTF/cytokine receptor components are available for signal transduction. Lesion-induced STAT3 phosphorylation was found in reactive astrocytes of the fascia dentata. Intriguingly, rapid and transient activation of STAT3 signaling was also observed in sprouting neurons of the medial septum. Messenger RNAs for the three components of the CNTF/cytokine receptor complex were expressed and differentially regulated following ECL both in septal neurons and in reactive astrocytes of the fascia dentata. These data indicate that CNTF or related gp130-associated cytokines play a dual role after brain lesion: (1). regulation of astrocytic responses to deafferentation and (2). regulation or modulation of axonal sprouting.

Ciliary neurotrophic factor as a transient negative regulator of rod development in rat retina.

PURPOSE: Ciliary neurotrophic factor (CNTF) has been shown to inhibit the developmental expression of rod differentiation markers in rat retinal cultures. The present study was undertaken to investigate whether CNTF transiently inhibits rod differentiation or induces irreversible changes in the developmental fate of rod precursors. METHODS: The effects of CNTF on rod differentiation were monitored in organotypic slice cultures from early postnatal rat retinas by quantification of opsin-immunoreactive cells. The in vitro formation of the photoreceptor layer was analyzed by light and electron microscopy. The developmental expression of the CNTF receptor in photoreceptors was determined by immunoblot analysis. RESULTS: CNTF did not interfere with the generation of rod precursors, their morphologic differentiation, or the formation of the outer nuclear layer. Inhibition of rod differentiation was reversible. In the continuous presence of CNTF the number of opsin-positive cells increased at a normal rate but with a delay of 3 to 4 days. Developing rods became resistant to CNTF, both in vivo and in vitro, and this correlated temporally with the downregulation of CNTF receptor expression. Receptor downregulation was inhibited by CNTF in a dose-dependent manner. At higher CNTF concentrations with sustained receptor expression, the CNTF-induced decrease in opsin expression was accompanied by an increase in the expression of a bipolar cell marker in rod precursors located in the photoreceptor layer. CONCLUSIONS: These data indicate that, although rod precursors exhibit some phenotypic plasticity in the presence of CNTF, the factor does not induce a switch in the developmental fate of rod precursors but plays a role as a transient and reversible negative regulator of rod differentiation.

CNTF and CNTF receptor alpha are constitutively expressed by astrocytes in the mouse brain.

Ciliary neurotrophic factor (CNTF) is regarded as one of the signals that lead to astrocyte activation following central nervous system lesion. However, it is not clear if CNTF can directly initiate astrocytic responses to injury because CNTF levels are very low in most areas of the unlesioned brain and CNTF receptor (CNTFRalpha) expression by astrocytes has not yet been demonstrated in the intact brain. In the present study, the expression patterns of CNTF protein and CNTFRalpha mRNA were studied in the intact mouse brain using immunocytochemistry and in situ hybridization, respectively. These procedures were combined with immunocytochemistry for glial fibrillary acidic protein in order to identify CNTF- and CNTFRalpha-expressing astrocytes. CNTF-immunoreactive astrocytes were exclusively found in white matter structures such as the optical tract, the corticospinal tract, and the fimbria-fornix. Gray matter astrocytes did not exhibit CNTF immunoreactivity. In contrast, CNTFRalpha mRNA-expressing astrocytes were found in gray matter areas, preferentially in the molecular layers of the cortex and hippocampus. White matter astrocytes did not show a detectable CNTFRalpha mRNA signal. These data demonstrate that both CNTF and its receptor are constitutively expressed by astrocytes in mouse brain. The nonoverlapping locations of astrocytes expressing detectable levels of CNTF and CNTFRalpha, respectively, may be related to distinct postlesional functions of these two glial cell populations.