A motor neuron is nothing without its axons. Several growth factors can return degenerating neurons from the brink of death, but they don’t necessarily rescue shriveled axons. Ciliary neurotrophic factor (CNTF) differs from other trophins in that it not only boosts cell survival, but also protects axons, and thus neuronal function. Now, researchers know why. According to a paper in today’s Journal of Cell Biology online, CNTF’s magic lies in stabilizing mouse neuron cytoskeletons. The study authors, led by Michael Sendtner at the University of Würzburg, Germany, hope to find a CNTF-based treatment that would also shore up the cytoskeleton in people. Since microtubule damage has been linked to many neurodegenerative conditions, such a drug might have broad applications, Sendtner suggested.

Sendtner first discovered two decades ago that CNTF protects motor neurons (Sendtner et al., 1992). In a mouse model of motor neuron disease, the growth factor not only extended survival, but also improved motor function; he showed that the treatment improved the ability to grab onto and balance atop a ruler. The dying back of axons, a key initiator of many neurodegenerative conditions, slowed in the treated mice as well. In the current study, first authors Bhuvaneish Selvaraj and Nicholas Frank finally explain how CNTF works.

The Sendtner lab studies a model of progressive motor neuronopathy (PMN). This mouse strain spontaneously developed a mutation in tubulin-specific chaperone E (Tbce), resulting in unstable, irregular microtubules (see ARF related news story on Martin et al., 2002; Bömmel et al., 2002). “This causes a disorder which is predominantly a motor neuron disease,” Sendtner said. The mice develop normally for the first two and a half weeks of life, and then rapidly deteriorate, losing weight and weakening, until they die at five to seven weeks. No one has found Tbce mutations in people, but Sendtner likes the model because it represents a pure microtubule defect. CNTF also protects motor neurons in mice carrying a mutant human superoxide dismutase 1 transgene that induces a type of amyotrophic lateral sclerosis (Pun et al., 2006).

The researchers studied the CNTF pathway in primary motor neuron cultures from embryonic PMN mice. These cells exhibit stunted axon growth compared to wild-type neurons, but CNTF treatment boosted axon length. Engagement of the CNTF receptor triggers kinases that phosphorylate signal transducer and activator of transcription 3 (STAT3; see Rajan et al., 1996). As its name suggests, STAT3 shuttles to the nucleus to activate genetic targets. Knocking out STAT3 in PMN mice abolished the effects of CNTF on axon growth, confirming that STAT3 mediates the growth factor’s signals.

However, the researchers were surprised to see that STAT3 did not promote axon growth as a transcription factor. They observed that it stayed in the cytosol in neurons treated with CNTF. And when the team used a mutation to disable STAT3’s DNA-binding ability, it still responded to the trophin and protected PMN neurons. These data contradict the standard “dogma” of STAT activation and translocation to the nucleus, noted Stanley Halvorsen of the State University of New York at Buffalo, who was not involved in the study.

If it is not acting as a transcription factor, how does STAT3 protect axons? Sendtner’s team found a cytoplasmic partner for STAT3 in another protein called stathmin. It binds to tubulin monomers, preventing them from polymerizing, and thus destabilizes the cytoskeleton. STAT3 grabs hold of stathmin, prompting it to drop the microtubule building blocks (Verma et al., 2009; Ng et al., 2006). The researchers showed the two co-immunoprecipitated from the PMN neurons, and that CNTF treatment doubled the amount of STAT3-stathmin pairings.

Finally, Selvaraj developed an assay to show CNTF’s effects on microtubules themselves. He treated cells with nocodazole to disassemble the cytoskeleton, and then washed out the drug and observed how quickly the microtubules re-formed. Neurons from PMN mice were slow to put their bones back together, but CNTF treatment sped up the reassembly. “CNTF has a very robust and rapid effect in restabilizing destabilized microtubules,” Sendtner concluded. “This is something which we think could be of therapeutic interest.”

In fact, Sendtner noted, researchers tried subcutaneous CNTF as a therapy for amyotrophic lateral sclerosis back in the 1990s (ALS CNTF Treatment Study Phase 1-2 Study Group, 1995). Unfortunately, more of the growth factor wound up in the liver than the brain, and recipients suffered fevers and other infection-like side effects, he said. Today, scientists are testing CNTF in several trials for eye conditions using direct delivery methods, and Sendtner thinks this approach might work better. His current paper suggests inhibitors of stathmin might be beneficial in motor neuron disease by mimicking STAT3 and keeping microtubules—and thus axons—strong.

The fact that CNTF preserves not just cell bodies but also axons makes it particularly appealing, commented Wilfried Rossoll of Emory University in Atlanta, who worked as a postdoc in the Sendtner lab but was not involved in the current work. “There is a lot of focus on factors and drugs that can increase the survival of motor neurons,” he noted. However, motor neuron death is preceded by dying back of axons from neuromuscular junctions (Fischer et al., 2004). “Once contact is lost at the muscle, it is questionable how helpful it is to preserve the cell body,” Rossoll said.

CNTF, or a stathmin inhibitor, might do more than just preserve existing axons. Sendtner has shown that CNTF also promotes axon sprouting (Simon et al., 2010), so it might help neurons rebuild damaged projections. Other research suggests that STAT3 activation and microtubule support promotes regeneration after nerve injury (Lee et al., 2004; Hellal et al., 2011). Stathmin inhibitors might then be a broad-reaching treatment for both motor neuron degeneration and injury, suggested John MacLennan of the University of Cincinnati, Ohio.

What about other neurodegenerative conditions such as Alzheimer’s or Parkinson’s? “It is a bit more of a stretch to go there because you are working with a different class of neurons,” MacLennan said. Sendtner said he is investigating microtubule dynamics in mouse models of motor neuron disease as well as Alzheimer’s to determine if cytoskeleton instability is an early event that would be amenable to treatment.—Amber Dance

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  1. This study by Selvaraj et al. has identified an unexpected signaling pathway by which the neurotrophic factor CNTF promotes axon regeneration of motor neurons in a mouse model of motor neuron disease. In this model (progressive motor neuronopathy, or PMN mouse), a point mutation on the TBCE gene results in destabilization of tubulin-specific chaperone E, leading to defects in microtubule assembly and axon degeneration of motor neurons. Despite the fact that different neurotrophic factors, such as glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF) can all promote survival of the cell bodies of motor neurons, this study shows that only CNTF can rescue the axon degeneration of motor neurons prepared from PMN mice. By generating conditional knockout mice that lack the transcription factor STAT3 and crossbreed with the PMN mutant mice, the authors further show that STAT3 is required for axon regeneration in response to CNTF. Surprisingly, the effect of STAT3 does not depend on its transcriptional activity. Rather, STAT3 interacts with the microtubule-destabilizing protein Stathmin in the cytoplasm after CNTF treatment, facilitating microtubule stabilization and polymerization of the PMN motor neurons. These observations strongly suggest that the transcription factor STAT3 can function outside the nucleus and acts locally to regulate microtubule assembly in the axon of motor neurons, which can have important implications in motor neuron diseases.

    The study is intriguing in a number of aspects. First, it demonstrates that STAT3 is crucial in mediating the survival effect of CNTF in motor neurons, which increases our understanding of the pathology of motor neuron diseases and identifies new molecular targets for therapeutic agents. Second, it reveals that STAT3 can function in a manner independent of its transcriptional activity during axon regeneration. This contrasts with a recent study demonstrating that the retrograde transport of STAT3 into the nucleus is required for axon regeneration of dorsal root ganglion neurons after sciatic nerve injury (Ben-Yaakov et al., 2012), and suggests that STAT3 is a multifaceted signaling molecule that performs distinct functions in axon regeneration under different conditions. Third, together with recent findings from our laboratory on a critical role of STAT3 in mediating β amyloid-induced neuronal death implicated in Alzheimer’s disease (Wan et al., 2010), this study reveals that STAT3 activation can lead to survival or death of neurons in different types of neurodegenerative diseases. Finally, we previously reported that STAT3 can also be activated by ephrin (Lai et al., 2004), which negatively regulates neurotransmission in the adult brain by reducing synaptic connectivity of neurons (Fu et al., 2007; 2011). Given the localization of STAT3 at the postsynaptic density (Nicolas et al., 2012), and the demonstration in this study that STAT3 can function outside the nucleus, it would be of interest to explore whether STAT3 also acts locally at the synapse to trigger synaptic loss in neurodegenerative diseases.

    References:

    . Axonal transcription factors signal retrogradely in lesioned peripheral nerve. EMBO J. 2012 Mar 21;31(6):1350-63. PubMed.

    . APC(Cdh1) mediates EphA4-dependent downregulation of AMPA receptors in homeostatic plasticity. Nat Neurosci. 2011 Feb;14(2):181-9. Epub 2010 Dec 26 PubMed.

    . Cdk5 regulates EphA4-mediated dendritic spine retraction through an ephexin1-dependent mechanism. Nat Neurosci. 2007 Jan;10(1):67-76. PubMed.

    . Identification of the Jak/Stat proteins as novel downstream targets of EphA4 signaling in muscle: implications in the regulation of acetylcholinesterase expression. J Biol Chem. 2004 Apr 2;279(14):13383-92. PubMed.

    . The Jak/STAT pathway is involved in synaptic plasticity. Neuron. 2012 Jan 26;73(2):374-90. PubMed.

    . Tyk2/STAT3 signaling mediates beta-amyloid-induced neuronal cell death: implications in Alzheimer's disease. J Neurosci. 2010 May 19;30(20):6873-81. PubMed.

    View all comments by Nancy Ip

References

News Citations

  1. Tubulin Chaperone Found to Cause Rare Motor Neuron Degeneration, and More

Paper Citations

  1. . Ciliary neurotrophic factor prevents degeneration of motor neurons in mouse mutant progressive motor neuronopathy. Nature. 1992 Aug 6;358(6386):502-4. PubMed.
  2. . A missense mutation in Tbce causes progressive motor neuronopathy in mice. Nat Genet. 2002 Nov;32(3):443-7. PubMed.
  3. . Missense mutation in the tubulin-specific chaperone E (Tbce) gene in the mouse mutant progressive motor neuronopathy, a model of human motoneuron disease. J Cell Biol. 2002 Nov 25;159(4):563-9. PubMed.
  4. . Selective vulnerability and pruning of phasic motoneuron axons in motoneuron disease alleviated by CNTF. Nat Neurosci. 2006 Mar;9(3):408-19. PubMed.
  5. . STAT proteins are activated by ciliary neurotrophic factor in cells of central nervous system origin. J Neurosci Res. 1996 Feb 15;43(4):403-11. PubMed.
  6. . STAT3-stathmin interactions control microtubule dynamics in migrating T-cells. J Biol Chem. 2009 May 1;284(18):12349-62. PubMed.
  7. . Stat3 regulates microtubules by antagonizing the depolymerization activity of stathmin. J Cell Biol. 2006 Jan 16;172(2):245-57. PubMed.
  8. A phase I study of recombinant human ciliary neurotrophic factor (rHCNTF) in patients with amyotrophic lateral sclerosis. The ALS CNTF Treatment Study (ACTS) Phase I-II Study Group. No To Shinkei. 1996 Apr;48(4):333-44. PubMed.
  9. . Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man. Exp Neurol. 2004 Feb;185(2):232-40. PubMed.
  10. . Ciliary neurotrophic factor-induced sprouting preserves motor function in a mouse model of mild spinal muscular atrophy. Hum Mol Genet. 2010 Mar 15;19(6):973-86. PubMed.
  11. . STAT3 phosphorylation in injured axons before sensory and motor neuron nuclei: potential role for STAT3 as a retrograde signaling transcription factor. J Comp Neurol. 2004 Jul 5;474(4):535-45. PubMed.
  12. . Microtubule stabilization reduces scarring and causes axon regeneration after spinal cord injury. Science. 2011 Feb 18;331(6019):928-31. PubMed.

External Citations

  1. several trials

Further Reading

Papers

  1. . Stathmin is required for stability of the Drosophila neuromuscular junction. J Neurosci. 2011 Oct 19;31(42):15026-34. PubMed.
  2. . Axonopathy and cytoskeletal disruption in degenerative diseases of the central nervous system. Brain Res Bull. 2009 Oct 28;80(4-5):217-23. PubMed.
  3. . Serum level of CNTF is elevated in patients with amyotrophic lateral sclerosis and correlates with site of disease onset. Eur J Neurol. 2008 Apr;15(4):355-9. PubMed.
  4. . Ciliary neurotrophic factor cell-based delivery prevents synaptic impairment and improves memory in mouse models of Alzheimer's disease. J Neurosci. 2010 Jun 2;30(22):7516-27. PubMed.
  5. . The Jak/STAT pathway is involved in synaptic plasticity. Neuron. 2012 Jan 26;73(2):374-90. PubMed.

Primary Papers

  1. . Local axonal function of STAT3 rescues axon degeneration in the pmn model of motoneuron disease. J Cell Biol. 2012 Oct 29;199(3):437-51. PubMed.