29 October 2012. 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.
Selvaraj BT, Frank N, Bender FL, Asan E, Sendtner M. 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. Abstract