Dying to Regrow: Dissecting the Role of Programmed Cell Death in Spinal Cord Regeneration

Q&A with Aravinthan D.T. Samuel, Ph.D.
Brenda Patoine
March 25, 2013

Aravinthan D.T. Samuel, Ph.D.
Professor, Department of Physics & Center for Brain Science
Harvard University
Dana Grantee: 2007-2010

In studying the molecular mechanisms of adult neural regeneration, your laboratory has found–paradoxically–that programmed cell death (apoptosis) may play an important role. What does cell death have to do with nerve regeneration?

portraitAravinthan Samuel: Before a neuron can repair itself, it probably has to expunge the damaged tissue–to get rid of damaged fragments in order to start over again. With that hypothesis as a starting point, we analyzed genes used for programmed cell death to see if they had any effect on regenerative properties.

In the nematode C. elegans, programmed cell death has been extremely well studied. The caspase gene, which plays a central role in programmed cell death, was discovered in C. elegans by H. Robert Horvitz decades ago, work that earned him the Nobel Prize in Physiology or Medicine. C. elegans is the best understood model for programmed cell death that we have, so we piggybacked our studies on that research. We discovered that genes for cell death have powerful roles in regeneration, and we were able to dissect those roles.

Were you surprised to find that programmed cell death played such a critical role in axon regeneration?

It was an educated guess. The proteins involved in cell death had been mostly studied in early development. As the animal develops from egg to adult through successive cell divisions, programmed cell death mechanisms get rid of cells that are not needed by the adult. Interestingly, these genes continue to be expressed at the adult stage even after all the developmental programs are complete. If they are there in the adult, they must have some role. What they are good at is removing tissue.

We guessed that caspase genes might support nerve replacement after damage at the adult stage, and that turned out to be the case.

Programmed cell death has traditionally been seen as a “bad guy” in spinal cord injury–a program that needs to be turned off. How does this research change that thinking?
One possible therapeutic response to spinal cord injury is to immediately apply inhibitors of programmed cell death. This would seem to make sense, because you don’t want any more cells dying after the initial injury. On the other hand, if programmed cell death pathways actually have a positive role in regenerating the fibers, it may not make such great sense to inhibit them.

During development, growing axons must find the right targets on nerve cells. When axons reach the wrong targets, they get pruned. Previous research had shown that some cell-death programs play a role in that pruning, so our hypothesis wasn’t completely a shot in the dark. We showed that these programs also play a positive role in adult-stage axon regeneration in C. elegans. That was novel.

There’s been quite a lot of talk about apoptosis inhibitors to stop cell death after injury, and even some clinical trials underway. Is that the wrong way to go in your view?

It’s hard to say it is the wrong approach. There may be some trade-off, and our work identified one potential cost of those kinds of therapies. These are exceedingly complicated pathways and the more we understand them, the better our ability to regulate them to achieve optimal regenerative therapies.

You’re also looking at the extent to which adult nerve regeneration may be a recapitulation of development. Where is the divergence from development to adult?

A lot is known in C. elegans and other model systems about the guidance cues and receptors that detect those cues to guide axons toward their targets during development. It is really puzzling that these cues continue to be expressed in the adult even though the axons have already reached their targets. Why would you need these cues? One possibility is to support regrowth if axons are damaged.

We found very interesting asymmetries between the developmental and adult systems for axon guidance. Even though the same genes were being expressed in the adult, and the same kind of guidance cues were being used (proteins like slit and netrin), different sets of receptors were being used in adult-stage regeneration. This was an interesting observation because it suggests there really had to be genetic programs for adult regeneration that are both overlapping and distinct from the pathways for early neural development. Adult-stage regeneration has its own machinery.

C. elegans is recognized as an important model system, but what can we really learn from a tiny worm about how spinal cord repair occurs in humans?

The CED3 programmed cell death pathway discovered in C. elegans was later found to be ubiquitous in the animal kingdom. The reason that Bob Horvitz won the Nobel Prize was not just because it was the cell-death pathway in C. elegans, but because it represented a highly conserved cell-death pathway in all animals. The conservation of these molecular signaling pathways is fundamental to biology, and they do have homologues in humans. That’s what it makes it important.

Scientists have been talking about harnessing growth-inhibiting molecules to induce spinal cord repair for 20 years, yet there is still no drug on the market that capitalizes on these discoveries. Why has progress been so difficult?

It’s a very complicated problem. The difficulty of molecular genetics in most established model systems for this kind of work would be one factor in the slow progress. C. elegans was the first animal in which the complete genome was sequenced and the first for which the complete wiring diagram of the nervous system was mapped out. Dissecting molecular pathways is vastly easier in something like C. elegans, where one knows the entire toolkit of genetics.

Vertebrates are of course very relevant to humans, but they are not the fastest vehicles for genetic analysis. In C. elegans, we can rapidly dissect molecular pathways. Once you have a sense of the molecules that are involved, you have candidates to test in larger animals.

For this research, you have employed a technique termed femtosecond laser ablation, or “optical scalpel.” Why is this important?

When I started this work in 2003, we teamed up with Eric Mazur, a Harvard scientist who had developed ultra-fast laser technology for applications in biology and other areas of science. One of the things he discovered is that these ultra-fast lasers can create holes in materials with nanometer scale resolution. At the time, the state of the art in laser ablation in C. elegans was micrometer scale resolution–you could blow out whole cell bodies but you could never snip specific nerve fibers. The first thing we did in our collaboration was to start snipping individual nerve fibers.

This research was initiated by a “seed grant” from the Dana Foundation. Why are these types of grants important in today’s research-funding environment?

Most of my lab works on neural circuitry and behavior–observing behavior in animals and correlating behavior to neural circuits. A post-doc in my lab, Chris Gabel, who is the first author on the papers we have published, got excited about studying axon regeneration. We teamed up with Chieh Chang, a post-doc at Rockefeller who studied axon development, and Monica Driscoll at Rutgers, an expert in cell death. We’re very good at the optical methods and Chang and Driscoll are good at molecular genetics, so the collaboration worked very well.

Gabel wrote two really nice papers from this research, and went on to take a job as a professor at Boston University School of Medicine, where this is his major research thrust. He already has a lot of new work of his own in the axon regeneration area, has obtained his first NIH grants, and now has his own post-docs and graduate students working with him.

Science isn’t just about projects, it’s about supporting people and pushing forward the careers of talented people. The Dana grant helped me to help a talented post-doc to successfully launch his career. All we needed was a little time and flexibility and that’s what the Dana Foundation grant allowed.