Muscle ciliary neurotrophic factor receptor α contributes to motor neuron STAT3 activation following peripheral nerve lesion.

Expression of the ciliary neurotrophic factor (CNTF) receptor essential ligand binding subunit, CNTF receptor α (CNTFRα), is induced in motor neurons and skeletal muscle following peripheral nerve lesion. We previously found muscle CNTFRα promotes motor neuron axon regeneration post-lesion. Both nerve lesion and CNTF administration activate motor neuron signal transducer and activator of transcription 3 (STAT3), a transcription factor implicated in axon growth, suggesting CNTF receptors may contribute to the lesion-induced STAT3 activation. However, many receptor types signal through STAT3, and if CNTF receptors contribute, motor neuron receptors seemed most likely to regulate motor neuron STAT3. To determine the role played by muscle CNTFRα, we used in vivo, muscle-specific CNTFRα depletion in mice and report here that this selectively impairs the second phase, sustained motor neuron STAT3 activation post-lesion. Thus, muscle CNTFRα makes an essential contribution to motor neuron STAT3 activation during axon regeneration and may thereby promote axon regeneration through such signaling. We also report CNTFRα quantitative PCR suggesting involvement of many denervated muscle types, as well as muscle damaged at the lesion site. The present data add to the evidence suggesting that enhancing muscle CNTFRα expression may promote motor neuron regeneration in trauma and disease.

Muscle and motor neuron ciliary neurotrophic factor receptor α together maintain adult motor neuron axons in vivo.

The molecular mechanisms maintaining adult motor innervation are comparatively unexplored relative to those involved during development. In addition to the fundamental neuroscience question, this area has important clinical ramifications given that loss of neuromuscular contact is thought to underlie several adult onset human neuromuscular diseases including amyotrophic lateral sclerosis. Indirect evidence suggests that ciliary neurotrophic factor (CNTF) receptors may contribute to adult motor neuron axon maintenance. To directly address this in vivo, we used adult onset mouse genetic disruption techniques to deplete motor neuron and muscle CNTF receptor α (CNTFRα), the essential ligand binding subunit of the receptor, and incorporated reporters labelling affected motor neuron axons and terminals. The combined depletion of motor neuron and muscle CNTFRα produced a large loss of motor neuron terminals and retrograde labelling of motor neurons with FluoroGold indicated axon die-back well beyond muscle, together revealing an essential role for CNTFRα in adult motor axon maintenance. In contrast, selective depletion of motor neuron CNTFRα did not affect motor innervation. These data, along with our previous work indicating no effect of muscle specific CNTFRα depletion on motor innervation, suggest that motor neuron and muscle CNTFRα function in concert to maintain motor neuron axons. The data also raise the possibility of motor neuron and/or muscle CNTFRα as therapeutic targets for adult neuromuscular denervating diseases.

Ciliary neurotrophic factor receptor regulation of adult forebrain neurogenesis.

Appropriately targeted manipulation of endogenous neural stem progenitor (NSP) cells may contribute to therapies for trauma, stroke, and neurodegenerative disease. A prerequisite to such therapies is a better understanding of the mechanisms regulating adult NSP cells in vivo. Indirect data suggest that endogenous ciliary neurotrophic factor (CNTF) receptor signaling may inhibit neuronal differentiation of NSP cells. We challenged subventricular zone (SVZ) cells in vivo with low concentrations of CNTF to anatomically characterize cells containing functional CNTF receptors. We found that type B "stem" cells are highly responsive, whereas type C "transit-amplifying" cells and type A neuroblasts are remarkably unresponsive, as are GFAP(+) astrocytes found outside the SVZ. CNTF was identified in a subset of type B cells that label with acute BrdU administration. Disruption of in vivo CNTF receptor signaling in SVZ NSP cells, with a "floxed" CNTF receptor α (CNTFRα) mouse line and a gene construct driving Cre recombinase (Cre) expression in NSP cells, led to increases in SVZ-associated neuroblasts and new olfactory bulb neurons, as well as a neuron subtype-specific, adult-onset increase in olfactory bulb neuron populations. Adult-onset receptor disruption in SVZ NSP cells with a recombinant adeno-associated virus (AAV-Cre) also led to increased neurogenesis. However, the maintenance of type B cell populations was apparently unaffected by the receptor disruption. Together, the data suggest that endogenous CNTF receptor signaling in type B stem cells inhibits adult neurogenesis, and further suggest that the regulation may occur in a neuron subtype-specific manner.

Muscle ciliary neurotrophic factor receptor α helps maintain choline acetyltransferase levels in denervated motor neurons following peripheral nerve lesion.

Systemic ciliary neurotrophic factor (CNTF) administration protects motor neurons from denervating diseases and lesions but produces non-neuromuscular side effects. Therefore, CNTF related therapeutics will need to specifically target motor neuron protective receptor mechanisms. Expression of the essential ligand binding subunit of the CNTF receptor, CNTF receptor α (CNTFRα), is induced in skeletal muscle by denervating lesion and in human denervating diseases. We show here, with muscle-specific in vivo genetic disruption, that muscle CNTFRα makes an essential/non-redundant contribution to maintaining choline acetyltransferase levels in denervated motor neurons following nerve crush, suggesting the muscle CNTFRα induction is an endogenous denervation-induced neuroprotective response that could be enhanced to treat nerve lesion and denervating diseases. Notably, unlike motor neuron gene expression, skeletal muscle gene expression can be specifically targeted with human gene therapy vectors already approved for market.

Conditional, genetic disruption of ciliary neurotrophic factor receptors reveals a role in adult motor neuron survival.

Indirect evidence suggests that endogenous ciliary neurotrophic factor (CNTF) receptor signaling can promote motor neuron (MN) survival in the adult. If so, proper targeting of this signaling may selectively counteract the effects of adult MN diseases. However, direct evidence for CNTF receptor involvement in adult MN survival is lacking, presumably because the unconditional blockade of the mouse CNTF receptor in vivo [through genetic disruption of the essential CNTF receptor alpha (CNTFRalpha) gene] leads to uniform perinatal death of the mice. To overcome this limitation, we have developed a method to selectively disrupt CNTF receptor function in a targeted subset of adult MNs that are not required for survival. A 'floxed CNTFRalpha' mouse line was generated and characterized. In addition, an adeno-associated virus (AAV) vector that drives Cre recombinase (Cre) expression was constructed and shown, with reporter mouse lines, to selectively excise floxed genes in facial MNs following its stereotaxic injection into the facial motor nucleus. Adult floxed CNTFRalpha mice were then injected with the AAV-Cre vector to excise the CNTFRalpha gene in the targeted MNs. The resulting data indicate that adult CNTF receptor signaling, likely by the MNs themselves, can play an essential role in MN survival. The data further indicate that this role is independent of any developmental contributions CNTF receptor signaling makes to MN survival or function.

Immunohistochemical localization of CNTFRalpha in adult mouse retina and optic nerve following intraorbital nerve crush: evidence for the axonal loss of a trophic factor receptor after injury.

Ciliary neurotrophic factor (CNTF) is important for the survival and outgrowth of retinal ganglion cells (RGCs) in vitro. However, in vivo adult RGCs fail to regenerate and subsequently die following axotomy, even though there are high levels of CNTF in the optic nerve. To address this discrepancy, we used immunohistochemistry to analyze the expression of CNTF receptor alpha (CNTFRalpha) in mouse retina and optic nerve following intraorbital nerve crush. In normal mice, RGC perikarya and axons were intensely labeled for CNTFRalpha. At 24 hours after crush, the immunoreactivity normally seen on axons in the nerve was lost near the lesion. This loss radiated from the crush site with time. At 2 days postlesion, labeled axons were not detected in the proximal nerve, and at 2 weeks were barely detectable in the retina. In the distal nerve, loss of axonal staining progressed to the optic chiasm by 7 days and remained undetectable at 2 weeks. Interfascicular glia in the normal optic nerve were faintly labeled, but by 24 hours after crush they became intensely labeled near the lesion. Double labeling showed these to be both astrocytes and oligodendrocytes. At 7 days postlesion, darkly labeled glia were seen throughout the optic nerve, but at 14 days labeling returned to normal. It is suggested that the loss of CNTFRalpha from axons renders RGCs unresponsive to CNTF, thereby contributing to regenerative failure and death, while its appearance on glia may promote glial scarring.

Adult ciliary neurotrophic factor receptors help maintain facial motor neuron choline acetyltransferase expression in vivo following nerve crush.

Exogenous ciliary neurotrophic factor (CNTF) administration promotes the survival of motor neurons in a wide range of models. It also increases the expression of the critical neurotransmitter enzyme choline acetyltransferase (ChAT) by in vitro motor neurons, likely independent of its effects on their survival. We have used the adult mouse facial nerve crush model and adult-onset conditional disruption of the CNTF receptor α (CNTFRα) gene to directly examine the in vivo roles played by endogenous CNTF receptors in adult motor neuron survival and ChAT maintenance, independent of developmental functions. We have previously shown that adult activation of the CreER gene construct in floxed CNTFRα mice depletes this essential receptor subunit in a large subset of motor neurons (and all skeletal muscle, as shown in this study) but has no effect on the survival of intact or lesioned motor neurons, indicating that these adult CNTF receptors play no essential survival role in this model, in contrast to their essential role during embryonic development. Here we show that this same CNTFRα depletion does not affect ChAT labeling in nonlesioned motor neurons, but it significantly increases the loss of ChAT following nerve crush. The data suggest that, although neither motor neuron nor muscle CNTF receptors play a significant, nonredundant role in the maintenance of ChAT in intact adult motor neurons, the receptors become essential for ChAT maintenance when the motor neurons are challenged by nerve crush. Therefore, the data suggest that the receptors act as a critical component of an endogenous neuroprotective mechanism. J. Comp. Neurol. 525:1206-1215, 2017. © 2016 Wiley Periodicals, Inc.

The S1P2 sphingosine 1-phosphate receptor is essential for auditory and vestibular function.

Sphingosine 1-phosphate (S1P) is an endogenous growth factor with potent effects on many different cell types. Most of these effects are produced by activation of one or more of a family of G-protein coupled receptors. The S1P2 receptor can mediate S1P-induced proliferation, differentiation and survival in a wide variety of cells in culture. However, identifying essential in vivo functions for S1P2 has been hampered by its ubiquitous expression and the failure to detect any anatomical abnormalities in initial analyses of S1P2 knockout mice. We report here that all S1P2 knockout mice are profoundly deaf from postnatal day 22 and approximately half display a progressive loss of vestibular function with aging. Anatomically, both the auditory and vestibular systems appear to develop normally but then degrade. Morphological defects associated with hearing are first detected at 3 weeks postnatal as deformations of the organ of Corti/Nuel's space. By one year of age structures within the scala media are dramatically altered. The S1P2 knockout mice also display a loss of otoconia consistent with the vestibular impairment. The present data are the first to indicate that S1P signaling plays critical roles, in vivo, in auditory and vestibular functions. The data further establish that the S1P signaling occurs through the S1P2 receptor and makes an essential contribution to the structural maintenance of these systems, raising the possibility that properly targeted enhancement of this signaling may prove to be clinically beneficial.

Penetrating brain injury leads to activation of ciliary neurotrophic factor receptors.

Endogenous injury response mechanisms likely reduce secondary neuronal loss following CNS trauma by activating growth factor receptors. Therefore, it is important to determine which growth factor receptors are activated in vivo by CNS trauma and which signal transduction pathways are affected in which cell types. We present a model of penetrating brain injury utilizing stereotaxic insertion of a fine needle. This procedure can be used to anatomically characterize injury response mechanisms through immediate, local application of pharmacological agents. We find, through immunohistochemistry, that injury of the rat facial motor nucleus leads to activation of STAT3, a neuronal survival factor, in the dendrites, nuclei and cytoplasm of the motor neurons. A similar response was observed with the trigeminal motor nucleus. Use of the ciliary neurotrophic factor (CNTF) receptor antagonist, AADH-CNTF, indicated that the STAT3 activation resulted largely, and perhaps entirely, from injury-induced activation of CNTF receptors.

STAT3 phosphorylation in injured axons before sensory and motor neuron nuclei: potential role for STAT3 as a retrograde signaling transcription factor.

STAT3 is a latent transcription factor that is activated by plasma membrane growth factor receptor complexes. Conditional gene disruption data indicate that it contributes to the survival of cranial motor neurons after peripheral nerve lesion. In agreement, levels of activated STAT3 (Tyr705-phosphorylated STAT3) have been shown to increase in the nuclei of adult cranial motor neurons during their regeneration after the same injury. The data presented here demonstrate that STAT3 is similarly but not identically affected in sciatic motor neurons after sciatic nerve injury. In addition, we find that sensory neuron nuclei also display an analogous increase in activated STAT3, thereby supporting a role for STAT3 in the survival and regeneration of these cells. Most interesting, the present data indicate that peripheral nerve lesion leads to a very rapid activation of STAT3 in axons at the lesion site. This response increases during the first 24 hours after injury and extends back to the motor and sensory neurons such that phospho-STAT3-immunoreactive axons are first detected in the dorsal root ganglia and ventral spinal cord at the same postlesion time intervals at which the activated STAT3 is first detected in the neuronal nuclei. Together these data raise the possibility that axonal STAT3, activated at the injury site, acts as a retrograde signaling transcription factor, which promotes the survival and regeneration of both sensory and motor neurons.

The contribution of ciliary neurotrophic factor receptors to adult motor neuron survival in vivo is specific to insult type and distinct from that for embryonic motor neurons.

Exogenous ciliary neurotrophic factor (CNTF) promotes motor neuron (MN) survival following trauma and in genetic models of MN disease. Unconditional disruption of the mouse CNTF receptor α (CNTFRα) gene leads to MN loss, demonstrating a developmental role for endogenous CNTF receptor signaling. These data also suggest that CNTF receptors may promote adult MN survival and that appropriately manipulating the receptors could effectively treat adult MN disorders. This effort would greatly benefit from a better understanding of the roles played by CNTF receptors in adult MNs. We have previously found that adult onset disruption of CNTFRα in facial MNs of "floxed CNTFRα" mice by AAV-Cre vector injection leads to significantly more MN loss than in identically treated controls. While indicating that CNTF receptors can promote adult MN survival, the data did not distinguish between potential roles in MN maintenance versus roles in protecting MNs from the injection associated trauma or the toxicity of the chronic Cre recombinase (Cre) produced by the AAV-Cre. Here we used an inducible Cre gene construct to produce adult-onset CNTFRα disruption in facial MNs without the traumatic and toxic effects of the AAV-Cre procedure. The MNs survive without CNTFRα, even when challenged by facial nerve crush or the injection-associated trauma, thereby suggesting, in conjunction with our previous study, that endogenous CNTF receptor signaling can protect MNs against toxic insult, such as that produced by chronic Cre. The data also indicate that in vivo CNTF receptors play very different roles in adult and embryonic MNs.

Muscle ciliary neurotrophic factor receptor α promotes axonal regeneration and functional recovery following peripheral nerve lesion.

Ciliary neurotrophic factor (CNTF) administration maintains, protects, and promotes the regeneration of both motor neurons (MNs) and skeletal muscle in a wide variety of models. Expression of CNTF receptor α (CNTFRα), an essential CNTF receptor component, is greatly increased in skeletal muscle following neuromuscular insult. Together the data suggest that muscle CNTFRα may contribute to neuromuscular maintenance, protection, and/or regeneration in vivo. To directly address the role of muscle CNTFRα, we selectively-depleted it in vivo by using a "floxed" CNTFRα mouse line and a gene construct (mlc1f-Cre) that drives the expression of Cre specifically in skeletal muscle. The resulting mice were challenged with sciatic nerve crush. Counting of nerve axons and retrograde tracing of MNs indicated that muscle CNTFRα contributes to MN axonal regeneration across the lesion site. Walking track analysis indicated that muscle CNTFRα is also required for normal recovery of motor function. However, the same muscle CNTFRα depletion unexpectedly had no detected effect on the maintenance or regeneration of the muscle itself, even though exogenous CNTF has been shown to affect these functions. Similarly, MN survival and lesion-induced terminal sprouting were unaffected. Therefore, muscle CNTFRα is an interesting new example of a muscle growth factor receptor that, in vivo under physiological conditions, contributes much more to neuronal regeneration than to the maintenance or regeneration of the muscle itself. This novel form of muscle-neuron interaction also has implications in the therapeutic targeting of the neuromuscular system in MN disorders and following nerve injury. J. Comp. Neurol. 521: 2947-2965, 2013. © 2013 Wiley Periodicals, Inc.

Targeted disruption of the S1P2 sphingosine 1-phosphate receptor gene leads to diffuse large B-cell lymphoma formation.

S1P(2) sphingosine 1-phosphate receptor signaling can regulate proliferation, survival, morphology, and migration in many cell types in vitro. Here, we report that S1P(2)(-/-) mice develop clonal B-cell lymphomas with age, such that approximately half of the animals display this neoplasm by 1.5 to 2 years of age. Histologic, immunophenotypic, and molecular analyses revealed a uniform tumor phenotype with features of germinal center (GC)-derived diffuse large B-cell lymphoma (DLBCL). Tumor formation was preceded by increases in GC B cells and CD69(+) T cells, as well as an increased formation of spontaneous GCs, suggesting that S1P(2) loss may promote lymphomagenesis in part by disrupting GC B-cells homeostasis. With the sole exception of rare lung tumors, the effect of S1P(2) gene disruption is remarkably restricted to DLBCL. In humans, 28 of 106 (26%) DLBCL samples were found to harbor multiple somatic mutations in the 5' sequences of the S1P(2) gene. Mutations displayed features resembling those generated by the IgV-associated somatic hypermutation mechanism, but were not detected at significant levels in normal GC B cells, indicating a tumor-associated aberrant function. Collectively, our data suggest that S1P(2) signaling may play a critical role in suppressing DLBCL formation in vivo. The high incidence of DLBCL in S1P(2)(-/-) mice, its onset at old age, and the relative lack of other neoplasms identify these mice as a novel, and potentially valuable, model for this highly prevalent and aggressive human malignancy.

Vascular dysfunction in S1P2 sphingosine 1-phosphate receptor knockout mice.

There is growing evidence that sphingosine 1-phosphate (S1P) plays an important role in regulating the development, morphology, and function of the cardiovascular system. There is little data, however, regarding the relative contribution of endogenous S1P and its cognate receptors (referred to as S1P(1-5)) to cardiovascular homeostasis. We used S1P(2) receptor knockout mice (S1P(2)(-/-)) to evaluate the role of S1P(2) in heart and vascular function. There were no significant differences in blood pressure between wild-type and S1P(2)(-/-) mice, measured in awake mice. Cardiac function, evaluated in situ by using a Millar catheter, was also not different in S1P(2)(-/-) mice under baseline or stimulated conditions. In vivo analysis of vascular function by flowmetry revealed decreases in mesenteric and renal resistance in S1P(2)(-/-) mice, especially during vasoconstriction with phenylephrine. In intact aortic rings, the concentration-force relations for both KCl and phenylephrine were right shifted in S1P(2)(-/-) mice, whereas the maximal isometric forces were not different. By contrast, in deendothelialized rings the concentration-force relations were not different but the maximal force was significantly greater in S1P(2)(-/-) aorta. Histologically, there were no apparent differences in vascular morphology. These data suggest that the S1P(2) receptor plays an important role in the function of the vasculature and is an important mediator of normal hemodynamics. This is mediated, at least in part, through an effect on the endothelium, but direct effects on vascular smooth muscle cannot be ruled out and require further investigation.