What’s Another Year?—Testing the Limits of Axon Regeneration

The window of opportunity for regenerating nerves severed in spinal cord injuries may be more expansive than previously thought, says a new study. By modifying not just the environment around spinal cord injuries, but also jumpstarting the regenerative capacities of the neurons themselves, Mark Tuszynski and colleagues at the University of California, San Diego, have achieved axon regeneration in a model of spinal cord injury in adult rats, even when treatment was delayed up to 15 months after the original injury. The results, which appeared in the October 29 issue of Neuron, lengthen the time frame for effective regenerative healing considerably, and offer hope for future treatments based on similar combinatorial strategies.

The approach taken by Tuszynski and colleagues tries to simultaneously surmount multiple barriers to axon regeneration in chronic spinal cord injury. These hurdles include environmental factors such as lack of growth factors, non-permissive or inhibitory extracellular matrix proteins, ongoing inflammation, secondary damage, and scarring. Cell-intrinsic factors are also at play, including atrophy of injured neurons and retrograde degeneration. Their idea was to treat established spinal cord injury with a combination of growth factors and cell grafting to overcome the environmental issues, and conditioning lesions to awaken the cells’ regenerative capacities.

To test their idea, first author Ken Kadoya looked at regeneration after cutting ascending sensory neurons in the spinal cord of rats. Six weeks after the injury, the animals received a conditioning lesion. This procedure, a bilateral crush of the sciatic nerves has been shown to increase the capacity of neurons to regenerate. One week later, the researchers grafted bone marrow stromal cells mixed with the neurotrophic factor NT-3 at the injury site, and added more NT-3 above the lesion via a lentivirus expression vector injected into the white matter of the spinal cord. After waiting another six weeks, the investigators traced axons by injecting a subunit of cholera toxin into the sciatic nerve.

The results showed that among the animals that received all three treatments, 10 of 16 regenerated axons that bridged the injury site; some axons continued for two or more millimeters beyond the lesion. The regeneration required all of the treatments. If animals got one or two of the three interventions, there was little or no axon growth beyond the injury site.

Even if the treatments were given much later, at 15 months after injury, five of 11 rats who got the full treatment showed bridging regeneration, though the number of axons that made it across was reduced and axon length was shorter compared to earlier intervention. The fullest regeneration required the combination therapy.

The results go against the idea that an old injury constitutes an insurmountable obstruction to regeneration, Tuszynski told ARF. “There may well be a persistent, significant barrier, but that can be changed and then overcome by this combination of modifying the chronically injured site and then providing a positive growth stimulus. It also requires modification of the intrinsic growth state of the injured cells by the conditioning lesion. One has to address multiple mechanisms to achieve this kind of thing in this very refractory chronically injured state,” he said. Importantly, the treatment did not require recutting scar tissue at the injury site, a procedure that risks further spinal damage.

The researchers also looked at the timing of the conditioning lesion. Early studies suggested it had to happen before injury, but the current study shows that lesioning enhances neurite outgrowth from cultured motor neurons even when it is done six weeks after injury. Such conditioning results in an increase in number of neurons expressing the regeneration-linked genes GAP43 and c-Jun and long-lasting changes in the expression of associated genes in dorsal root ganglia, similar to changes seen with pre-lesioning.

Kadoya and colleagues did not report measures of functional regeneration, although the same group has previously published that chemotropic guidance from NT-3 can promote functional reconnections (see ARF related news story on Alto et al. 2009). Tuszynski said they are looking at the effect of the combination therapy on injured motor neurons in the rat, and the studies will include functional measures.

Although it is a long way from rodents to humans, and much remains to be done, Tuszynski says, “The demonstration that you can influence any type of chronically injured axon by treating these multiple mechanisms and overcome a hurdle as big as regeneration beyond the lesion site is an important proof of principle.” In place of the conditioning lesion, it may be possible to treat cells with cAMP-elevating compounds, which mimic the growth stimulatory effects of conditioning lesions (e.g., see Pearse et al., 2004, or a review by Hannila and Filbin, 2008).

The work may also have some lessons for treating Alzheimer disease or other neurodegenerative conditions. Tuszynski has been a leader in the study of growth factors including NGF (see ARF related news story on Tuszynski et al., 2005) and BDNF (see ARF related news story on Nagahara et al., 2009) as possible agents to promote regeneration in AD. “In the Alzheimer’s brain there is chronic cell degeneration, there is a chronic inflammatory response, there are likely molecules around that inhibit the reorganization of the degenerating brain,” Tuszynski said. “I think some of the principles shown in this chronic paradigm—that you can remodel existing connections with growth factors, for example, and that you can stimulate the endogenous repair response of the cell by these cAMP-dependent conditioning mechanisms—could well have relevance to Alzheimer’s disease.”

Clearly, regenerating axons need all the help they can get and two other papers out this week provide additional molecular targets to push along the process. A paper from Larry Benowitz and Nina Irwin at Children’s Hospital Boston, reports that the Mst3b kinase, which regulates axonal outgrowth during embryonic development, is also essential for axon regeneration from adult PNS and CNS neurons. That work was published online October 25 in Nature Neuroscience. In this week’s PNAS, Brett Langley and coworkers at Weill Medical College of Cornell University in New York identify histone deacetylase 6 (HDAC6) as a potential target for neuroprotection and regeneration. Langley had previously shown that HDAC inhibitors can protect cortical neurons against oxidative stress, but at a cost, since the pan inhibitors are very toxic. Their new work shows that HDAC6 in particular becomes upregulated in cultured neurons during oxidative insult or under conditions that inhibit neurite outgrowth. Moreover, they find that a specific HDAC6 inhibitor blocks oxidative stress-induced cell death, and promotes neurite outgrowth in vitro. Importantly, the selective inhibitor does all this without the toxicity associated with inhibiting HDACs generally.—Pat McCaffrey

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References

News Citations

  1. Bridges and Blues—Striking a Chord for Axon Protection
  2. Special Delivery: NGF Trial Puts Growth Factor Where It’s Needed
  3. BDNF the Next AD Gene Therapy?

Paper Citations

  1. Alto LT, Havton LA, Conner JM, Hollis Ii ER, Blesch A, Tuszynski MH. Chemotropic guidance facilitates axonal regeneration and synapse formation after spinal cord injury. Nat Neurosci. 2009 Sep;12(9):1106-13. PubMed.
  2. Pearse DD, Pereira FC, Marcillo AE, Bates ML, Berrocal YA, Filbin MT, Bunge MB. cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury. Nat Med. 2004 Jun;10(6):610-6. PubMed.
  3. Hannila SS, Filbin MT. The role of cyclic AMP signaling in promoting axonal regeneration after spinal cord injury. Exp Neurol. 2008 Feb;209(2):321-32. PubMed.
  4. Tuszynski MH, Thal L, Pay M, Salmon DP, U HS, Bakay R, Patel P, Blesch A, Vahlsing HL, Ho G, Tong G, Potkin SG, Fallon J, Hansen L, Mufson EJ, Kordower JH, Gall C, Conner J. A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nat Med. 2005 May;11(5):551-5. PubMed.
  5. Nagahara AH, Merrill DA, Coppola G, Tsukada S, Schroeder BE, Shaked GM, Wang L, Blesch A, Kim A, Conner JM, Rockenstein E, Chao MV, Koo EH, Geschwind D, Masliah E, Chiba AA, Tuszynski MH. Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer's disease. Nat Med. 2009 Mar;15(3):331-7. PubMed.

Further Reading

Primary Papers

  1. Kadoya K, Tsukada S, Lu P, Coppola G, Geschwind D, Filbin MT, Blesch A, Tuszynski MH. Combined intrinsic and extrinsic neuronal mechanisms facilitate bridging axonal regeneration one year after spinal cord injury. Neuron. 2009 Oct 29;64(2):165-72. PubMed.
  2. Lorber B, Howe ML, Benowitz LI, Irwin N. Mst3b, an Ste20-like kinase, regulates axon regeneration in mature CNS and PNS pathways. Nat Neurosci. 2009 Nov;12(11):1407-14. PubMed.
  3. Rivieccio MA, Brochier C, Willis DE, Walker BA, D'Annibale MA, McLaughlin K, Siddiq A, Kozikowski AP, Jaffrey SR, Twiss JL, Ratan RR, Langley B. HDAC6 is a target for protection and regeneration following injury in the nervous system. Proc Natl Acad Sci U S A. 2009 Nov 17;106(46):19599-604. PubMed.

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