Dispatch from the Working 2 Walk Science & Advocacy Symposium
held in Orange County, CA. A cool meeting (sponsored in part by the Reeve Foundation), two days of talks and small group breakouts with the speakers, and one for touring the labs at the University of California, Irvine. It’s got a nice international flavor (nine other countries) and is large enough to attract a good range of scientists and yet cozy enough for lots of personal interaction between the researchers and 150 or so members of the community. This edition, the 7th, featured fresh, unpublished data coming out of several labs. The meeting was videotaped; the feeds should be online soon.
PTEN & Push for Clinical Relevance
In the last three or four years, the PTEN story has caught a wave in neuroscience. You’ve read about it here
and if you want to mine the basic science, I recommend this paper from Nature Neuroscience
, available in its entirety: “PTEN Deletion Enhances the Regenerative Ability of Adult Corticospinal Neurons
." Here’s another key paper on PTEN, also in full from the top journal Nature
: “Sustained Axon Regeneration Induced by Co-deletion of PTEN and SOCS3
.” Remove both PTEN and another growth regulator, SOCS3: much more robust axon regeneration.
The principal scientist of the Nature
papers was Zhigang He, of Harvard. One of his co-authors was Oswald Steward, who heads the Reeve-Irvine Research center at the University of California, Irvine (UCI). Steward was at W2W – indeed his center was a primary sponsor of the event – to present new PTEN data. His talk titled, “Regenerating Connections that Mediate Motor Function after Spinal Cord Injury,” was mainly based on work in his lab by staff scientist Gail Lewandowski.
Background: PTEN (stands for phosphatase and tensin homolog) is a tumor suppressor gene that was discovered by cancer researchers 15 years ago. This gene regulates cell proliferation and it turns out to be a molecular switch for axon growth. When scientists deleted PTEN in a complete spinal cord injury model, cortical spinal axons – the ones needed for major movement function – regenerated at unprecedented rates. PTEN is complicated; you can’t just get rid of it because it is the brake needed to stop certain kinds of cellular overgrowth (cancer). But there are ways to release it.
PTEN encourages regeneration. But there are several big challenges before translation of the research can consider larger animals, or humans.
• First, the He experiments used specially bred mice that had the PTEN gene knocked out before their injury. Is there a way to turn off or delete PTEN after SCI in a clinically relevant time frame? If so, what is the delivery system?
• The model was limited because mice don’t produce cavities after SCI, as people do. Will PTEN deletion work as well in rats?
• Post treatment there was a lot of growth crossing the lesion site but no evidence of functional recovery. Can the axons make functional connections? And if so, does that mean recovery?
Here’s what Lewandowski did: she and colleagues at the University of Pennsylvania came up with an adeno-associated virus (AAV) vector that, when injected, silences PTEN (using something called a short hairpin to sort of highjack the RNA codes that makes the PTEN protein). Adult rats received injections of the vector into their motor cortex. It worked. She found almost 100 percent inhibition of PTEN expression.
Another set of rats were trained in a skilled forepaw reaching task (using what is called a Montoya Staircase; this measures the rats' skills as they reach for food rewards). A week after administration of the PTEN inhibitor, the rats received a C6 dorsal hemisection spinal cord injury. Half of those animals got a salmon fibrin treatment at the lesion site (shown elsewhere to promote functional recovery). Lewandowski then tested forepaw motor function again in the same Montoya staircase for 10 weeks post-injury.
Did it work?
“Tentative yes….At least the answer is not ‘no,’” said Steward. The rat model seemed to work fine. The clinical relevance question remains; Lewandowski deleted PTEN before injury (Steward said other unpublished animal work at his lab has shown that an AAV injection at the time of injury is effective too. Meanwhile, Lewandowski is currently working on a post-injury AAV application).
Here is news that had not been reported before: data from the staircase test indicated that treated animals improved forepaw function. The animals treated with both PTEN deletion and the fish fibrin did better than those with PTEN deletion alone.
Issues remain: how and when to administer a PTEN deletion? How can the PTEN story scale up to larger animals and humans? Most crucial to the W2W audience: Can this work in chronic injury? The work is ongoing.
(Under the umbrella of Unite 2 Fight Paralysis, W2W began fundraising last year to boost PTEN work, including direct support to Reeve-Irvine and toward collaborations between Jerry Silver and Steward, and including researchers in Hong Kong and at Harvard.)
Novel Regeneration Gene Discovered
Murray Blackmore, formerly of the Miami Project, is now a researcher at Marquette University in Milwaukee – not exactly a hotbed of neuroscience but this is very interesting stuff he’s doing. Blackmore is young and modest, science-geeky in the right way; his hobby is collecting gene databases. When it comes to SCI research, “I get it,” said Blackmore. His mother has been a C5 tetraplegic for 25 years.
His work, involving genetics, is about “rebooting the growth machinery” of the injured nervous system. Blackmore’s hypothesis is that developmental changes in gene expression control nerve cell outgrowth, and that it is possible to identify potential new gene targets to promote such outgrowth in an adult animal. Using sophisticated micro-array screening techniques, Blackmore identified genes (the body’s codes for cell activity and behavior) that are related to axon regeneration. One of these, called KLF7 (Krüppel-like Factor 7), is present in the developing system but not so much in an adult animal. If introduced in an animal injury model KLF7 does indeed reboot the intrinsic ability of nerve cells to send out axons. He previously reported
that KLF7 promotes axon regeneration in the adult corticospinal tract.
Over-expression of this gene promoted both sprouting and regenerative axon growth in the CST of adult mice. In his newest studies, another unpublished W2W scoop, the technique “promoted a functional benefit.” Treated animals did better on a motor score called the BBB (stands for Beatty, Bresnahan, and Basso the scientists who developed the scale).
Said Blackmore, “We are thrilled.” He said he didn’t want to “oversell” the results; only a fraction of the total axons regrew, and for only a few millimeters, in a mouse. Still, this opens many new doors: Blackmore is on the case to screen for more regeneration-related genes. There appears to be a precisely coordinated transcriptional program in the transition between a high growth state (development) and a low growth state (injury response in the adult). As Blackmore better understands this programming, he plans to combine KLF7 treatment with other options, including PTEN deletion.
Silver: Getting Axons Unstuck
Case Western scientist Jerry Silver
gave two talks at W2W. He is an energetic and engaging speaker – opinionated but quite optimistic. He had several progress reports to offer, including some fascinating news about long distance regeneration and bladder improvement.
First talk: “Functional Recovery after SCI with the use of Injectable Peptides that Counteract Inhibitory Proteoglycans.” Silver begins his science talks with a nod to the godfather of neuroscience, Spaniard Santiago Ramón y Cajal. Cajal, working over a century ago, described nerves after injury; the ends of lesioned axons become swollen into what he called “dystrophic endballs” and were no longer capable of regeneration. However, recent studies from Silver’s lab revealed that these dystrophic growth cones are not so quiescent; they might actually be highly active but stalled in the hostile injured environment.
OK, so imagine these axons, stuck in the area of injury. “They don’t die back,” said Silver. “They sit there in the white matter.” Why don’t they grow? Might be the scar that seals off the injury area. Plus, there’s something about the axons, too. They seem to be “happy” in their stuck position. Silver says the axon endballs are surrounded by a type of stem cell native to the spinal cord. It’s like these axons are sitting on a beach chair – the stem cells, oligodendrocite precursors, are the chair. The question is, said Silver, is how do we get them off?
There may be several ways to get the axons off the beach chair. One might be using chrondoitinase, or chase. This is a molecule that breaks up the sugar chains forming the scar. There has been much work published about the potential for chase; Silver has shown it to be “rocket fuel” in his experiments, including one major 2011 study
(sponsored in part by the Reeve Foundation) that restored breathing ability in animals with a high cervical injury. Another way to lose the beach chair involves a peptide inhibitor Silver’s team came up with; it appears to loosen up the stuck axons and get them moving.
In an animal contusion model, Silver reported “unprecedented levels of functional recovery” and “unbelievable improvement” in some animals after the peptide was applied. What’s more, the delivery of the peptide is simple: it is injected under skin near the lesion area. Chase, alas, must be injected into the spinal cord, often stated as a major drawback for human use.
So, peptide alone, or maybe with chase? What about chronic injury? Not known. Lots more to work out. Silver said his university has a patent on the peptide and has been in discussions with GlaxoSmithKline to develop it. He said the company appears more interested in stroke, a much larger market than SCI.
Silver part II: “Functional Regeneration Into and Well Beyond the Glial Scar.” Here Silver showed a way to restore bladder function in animals with acute injuries, using a short piece of peripheral nerve to bypass the lesion area, along with acidic fibroblast growth factor, fibrin and chase on the ends stitched to the cord. Axons from above the injury grew all the way down the cord; bladder functioned returned. In a chronic model, there was no recovery after six months. But Silver found that proper “wound preparation” woke up the beach chair axons described previously. Bingo. Silver said clinically relevant recovery occurred; despite what the great Cajal said, "long disatance regeneration is possible."
Part two to come
, including presentations from Aileen Anderson and Stem Cells, Inc, Hans Keirstead, Mark Tuszynski, California Institute on Regenerative Medicine.