A paper came out a few days ago from the lab of Zhigang He at Harvard. This is the scientist who more or less broke the story on PTEN
and spinal cord injury a few years ago, and as you recall from previous reporting here, PTEN is a tumor suppressor gene discovered by cancer researchers almost 20 ago; it acts as a brake on axons that attempt to regenerate after injury and appears to play an especially critical role on corticospinal axons, the ones that are important for major functional activities such as walking. PTEN (short for phosphatase and tensin homolog), when knocked down, allows robust axon growth at unprecedented distances.
The new paper is titled “Short Hairpin RNA against PTEN Enhances Regenerative Growth of Corticospinal Tract Axons after Spinal Cord Injury
There are a couple of ways scientists have been able to knock down PTEN – Zhigang previously used genetic manipulation to delete it. In this case, he uses what’s called short hairpin RNA, a non-genetic (and therefore more clinically relevant) way to inactivate PTEN. It seemed to work just fine -- he reports that corticospinal axons grow, they seem to form synapses in the right areas. There was no measurable functional recovery, though that wasn’t set forth as a goal.
From the abstract:
Developing approaches to promote the regeneration of descending supraspinal axons represents an ideal strategy for rebuilding neuronal circuits to improve functional recovery after spinal cord injury (SCI). Our previous studies demonstrated that genetic deletion of phosphatase and tensin homolog (PTEN) in mouse corticospinal neurons reactivates their regenerative capacity, resulting in significant regeneration of corticospinal tract (CST) axons after SCI. However, it is unknown whether nongenetic methods of suppressing PTEN have similar effects and how regenerating axons interact with the extrinsic environment. Herein, we show that suppressing PTEN expression with short-hairpin RNA (shRNA) promotes the regeneration of injured CST axons, and these axons form anatomical synapses in appropriate areas of the cord caudal to the lesion.
To be sure, Zhigang isn’t promoting the current work as potential therapy, although that no doubt underpins his ambition. This short hairpin method is characterized more as a tool to understand why axons do and don’t grow.
There are two main reasons axons don’t regenerate: intrinsic and extrinsic factors. If we were to use a motorway analogy, the roads are destroyed and the axons can’t leave the area. That’s clearly an extrinsic factor and we know that after spinal cord injury, myelin debris and astrocytic scar are indeed blocking the way of axons. Many attempts have been made to unblock the blockers, including anti-NOGO, chrondroitnincase, etc.
But there’s more to axon failure than a toxic environment. Clearing the roads isn’t enough. The axons have an intrinsic weakness too. That’s where PTEN comes in, to give the axons a supercharge that might overcome the extrinsic toxicity; indeed, that has been the appeal of PTEN. Maybe this is the missing ingredient to jumpstart regeneration, and until a few years ago, nobody knew about it. But here’s the way Zhigang sees it now: PTEN itself isn’t enough.
More from the paper:
....we show that short-hairpin RNA (shRNA) against PTEN, similar to gene deletion, can be used to sufficiently knock down PTEN and enhance the intrinsic growth of CST axons after a crush spinal cord injury (SCI). Indeed, many CST axons are able to regenerate across the lesion and make synapses caudal to the injury. Taking advantage of this model of enhanced intrinsic growth, we analyzed what extrinsic factors appear to support or inhibit regeneration. We show that axons grow across the lesion in close association with astrocyte bridges but avoid dense clusters of fibroblasts and macrophages. Moreover, axons that do not cross the lesion stop at the sharp border formed by fibroblasts and astrocytes at the lesion’s edge. With this shRNA tool, we now have more flexibility to test hypotheses regarding the true role of these cell types on axon regeneration in not only the mouse, but also other mammalian models.
Interestingly, Zhigang notes that regenerating axons seem to prefer certain astrocyte bridges, a notion that is quite counter-intuitive to the repellant role usually assigned to astrocytes. He suggests these are a special type of astrocyte that may have an evolutionary link to animals that are able to repair their damaged spinal cords.
Again, from the paper:
We find that regenerating axons avoid dense clusters of fibroblasts and macrophages in the lesion, suggesting that these cell types might be key inhibitors of axon regeneration. Furthermore, most regenerating axons cross the lesion in association with astrocytes, indicating that these cells might be important for providing a permissive bridge for axon regeneration.
... the GFAP astrocytes in the lesion appear to have a different phenotype than those forming the glial scar. Instead of having thick, interwoven processes, they appear to have longer processes that run more parallel to each other and the direction axons must travel. This phenotype is reminiscent of the phenotype adopted by astrocytes in species that are able to regenerate their spinal cords, such as the newt and turtle, and thus suggests that the mammalian spinal cord has some competence to respond to an SCI in a way that supports regenerative healing. This phenotype is also similar to that of immature, bipolar astrocytes, which are known to be permissive for axon regeneration. This led us to speculate that the bridge-forming astrocytes might, therefore, be derived from local stem cell progeny, whereas the scar-forming astrocytes might be derived from mature astrocytes that have become reactive.
Zhigang notes that although his group did not observe significant gross behavioral improvements in animals with a T8 crush injury, "it is possible that more sensitive behavioral assays are needed to monitor functional recovery in these animals."
He notes that while PTEN deletion activates growth, many axons do not grow into the lesion after a “thin” T8 crush, and none of them grow through a “wide” crush. The therapeutic answer is going to include a combination to boost intrinsic cell behavior and reduce the extrinsic factors that limit growth.
From the paper:
Understanding the identity of these bridge-forming astrocytes and what drives these divergent responses will give insights into how to create more astrocyte bridges for axon regeneration.
... combining the PTEN suppression/deletion method of activating intrinsic growth with those aimed at modifying the lesion environment has great promise in improving axon regeneration. Our studies suggest that strategies that increase the formation of astrocyte bridges and/or break up the dense cluster of fibroblasts and macrophages in the lesion core are strong candidates for such combinatorial approaches. Our new shRNA tool will now allow us to use cell-specific Cre drivers to alter cellular responses and test the ability of the manipulation to improve axon regeneration. It will also allow us to translate findings into other mammalian models for which targeted mutations are not possible.
Two more notes on the latest PTEN work: Last year the Os Steward lab at UC Irvine presented data
on a rat model of PTEN deletion using the short hairpin RNA method. The rats gained functional forepaw skill compared to animals that did not have the PTEN knocked down. That work was not cited in the new Zhigang paper. I asked Steward why; it’s because his lab has not published the data yet. Coming soon.
Second, also from Steward’s group: they are presenting unpublished data at the Society for Neuroscience meeting next month that it is possible to delete PTEN after an animal gets a spinal cord injury. That bit of clinical relevance is obvious, but it should be noted, all the animal work to date, including the latest Zhigang paper and the Steward rat data, used models wherein PTEN was removed ahead of the animals being injured.
From Steward’s SFN abstract:
These results support the conclusion that PTEN deletion in a clinically relevant timeframe after a spinal cord injury can enhance motor recovery and CST axon growth