Hans-Dieter Hofmann

Evidence for multiple, local functions of ciliary neurotrophic factor (CNTF) in retinal development: Expression of CNTF and its receptor and in vitro effects on target cells

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

Differential Regulation of Ciliary Neurotrophic Factor (CNTF) and CNTF Receptor α Expression in Astrocytes and Neurons of the Fascia Dentata after Entorhinal Cortex Lesion

Neurotrophic factors have been implicated in reactive processes occurring in response to CNS lesions. Ciliary neurotrophic factor (CNTF), in particular, has been shown to ameliorate axotomy-induced degeneration of CNS neurons and to be upregulated at wound sites in the brain. To investigate a potential role of CNTF in lesion-induced degeneration and reorganization, we have analyzed the expression of CNTF protein and CNTF receptor α (CNTFRα) mRNA in the rat dentate gyrus after unilateral entorhinal cortex lesions (ECLs), using immunocytochemistry and nonradioactive in situhybridization, respectively. In sham-operated as in normal animals, CNTF protein was not detectable by immunocytochemistry. Starting at 3 d after ECL, upregulation of CNTF expression was observed in the ipsilateral outer molecular layer (OML). Expression was maximal at around day 7, and at this stage immunoreactivity could be specifically localized to astrocytes in the ipsilateral OML. By day 14 postlesion, CNTF immunoreactivity had returned to control levels. CNTFRα mRNA was restricted to neurons of the granule cell layer in controls. Three days postlesion, prominent CNTFRα expression was observed in the deafferented OML. A similar but less prominent response was noticed in the contralateral OML. After 10 d, CNTFRα expression had returned to control levels. Double labeling for CNTFRα mRNA and glial fibrillary acidic protein (GFAP) showed that upregulation of CNTFRα occurred in reactive, GFAP-immunopositive astrocytes of the OML. A substantial reduction of CNTFRα expression in the deafferented granule cells was transiently observed at 7 and 10 d postlesion. Our results suggest a paracrine or autocrine function of CNTF in the regulation of astrocytic and neuronal responses after brain injury.

Upregulation of ciliary neurotrophic factor in reactive Müller cells in the rat retina following optic nerve transection.

We have investigated the expression and cellular localization of ciliary neurotrophic factor (CNTF) in the rat retina following optic nerve transection. In the normal retina, CNTF immunoreactivity was restricted to profiles in the ganglion cell layer. Following optic nerve transection, immunoreactivity appeared in Müller cell somata and processes and its intensity increased between three and seven days post-lesion. Quantitative evaluation by immunoblotting confirmed that CNTF expression increased continuously up to 7 days after optic nerve transection (to 430% of control levels), but decreased again to 250% of controls at 4 weeks post-lesion. Our findings suggest that CNTF supplied by Müller cells may play a protective role for axotomized ganglion cells in the rat retina.

Expression of CNTF in Müller cells of the rat retina after pressure-induced ischemia.

We have investigated the expression and cellular localization of ciliary neurotrophic factor (CNTF) in the rat retina following ischemia induced by transiently increasing the intraocular pressure. In the normal retina, CNTF immunoreactivity was restricted to profiles in the ganglion cell layer. Following ischemia and reperfusion, immunoreactivity appeared in Müller cell somata and processes and its intensity increased between 1 day and 2 weeks post-lesion. Quantitative evaluation by immunoblotting confirmed that CNTF expression continuously increased up to 2 weeks after ischemic injury (to 600% of control levels), but had declined again to 250% of controls at 4 weeks post-lesion. Our findings suggest that CNTF supplied by Müller cells has a protective function for lesioned neurons following transient ischemia.

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. STEM CELLS 2008;27:431–441

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

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

Involvement of P2 purinoceptors in the regulation of DNA synthesis in the neural retina of chick embryo.

The activation of P2 purinoceptors induces Ca2 mobilization in the earlyembryonic chick neural retina. This purinergic Ca2 response declines parallel with thedecrease in mitotic activity during retinal development. To investigate the role of P2purinoceptors in the regulation of retinal cell proliferation, we studied the effects of the P2purinoceptor antagonists suramin and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), and of the agonist ATP on DNA synthesis in retinal organcultures from embryonic day 3 (E3) chick. Suramin inhibited [3H]‐thymidineincorporation in a dose‐dependent manner (IC50 : ∼70 μM). PPADS alsoreduced [3H]‐thymidine incorporation with maximum inhibition of 46% at 100 μM. Exogenous ATP enhanced [3H]‐thymidine incorporation in adose‐dependent manner to maximally 200% of control (EC50 : ∼70 μM). Indissociated retinal cultures from E7 chick, both antagonists showed similar inhibitory effects on [3H]‐thymidine incorporation without affecting cell viability. In line with theseobservations, the presence of extracellular ATP was demonstrated both in vitro and in vivo. In the medium of E3 retinal organ cultures, the concentration of ATP increased25‐fold within 1 h of incubation and this concentration was kept for at least 24 h. In the chickamniotic fluid, the ATP concentration was nearly 3 μM at E3 and declined to 0.15 μM at E7. The results indicate that P2 purinoceptors activated by autocrine or paracrinerelease of ATP are involved in the regulation of DNA synthesis in the neural retina at earlyembryonic stages.

Expression of ciliary neurotrophic factor receptor-α messenger RNA in neonatal and adult rat brain : An in situ hybridization study

Ciliary neurotrophic factor is a pleiotropic molecule thought to have multiple functions in the developing and adult nervous system. To investigate the role of ciliary neurotrophic factor in the developing and mature brain by defining putative target cells the expression of the ligand-binding alpha-subunit of the ciliary neurotrophic factor receptor was studied in neonatal and adult rat brains using a digoxygenin-labelled probe for in situ hybridization. Neuronal populations expressing ciliary neurotrophic factor receptor-alpha messenger RNA were found in many functionally diverse brain areas including the olfactory bulb (mitral cells and other neurons) neocortex (layer V) and other cortical areas (pyramidal cell layers in the piriform cortex and hippocampus, granule cell layer of the dentate gyrus) and distinct nuclei in the thalamus, hypothalamus and brainstem. In the latter, reticular nuclei and both cranial motor and sensory nerve nuclei showed intense hybridization signals in the neonatal brain. The nucleus ruber, substantia nigra pars reticularis, deep cerebellar nuclei and a subpopulation of cells in the internal granular layer of the cerebellum were also labelled. In many areas (e.g. in thalamic, midbrain and pontine nuclei) ciliary neurotrophic factor receptor-alpha expression became undetectable with maturation; however, there were other areas (e.g., olfactory bulb, cerebral cortex and hypothalamus) where expression was higher in the adult. The neuroepithelium of the neonatal rat displayed a highly selective expression of ciliary neurotrophic factor receptor-alpha in areas which are known to exhibit high rates of postnatal cell proliferation in the germinal zones. Generally, neurons which have been reported to respond to exogenous ciliary neurotrophic factor were labelled by the ciliary neurotrophic factor receptor-alpha probe. This was not the case, however, for striatal and septal neurons. The results of this study suggest that ciliary neurotrophic factor receptor-alpha ligands have even broader functions than previously thought, acting on different neuronal populations in the developing and mature brain, respectively.

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 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.