Glial Growth Factor 2, a Soluble Neuregulin, Directly Increases Schwann Cell Motility and Indirectly Promotes Neurite Outgrowth

by Nagesh K. Mahanthappa, Cambridge Neuroscience, Inc.
Eva S. Anton and William D. Matthew, Department of Neurobiology, Duke University Medical Center

Definitions

Growth Factor - A chemical secreted by neurons that promotes the migration and proliferation of Schwann cells if provided in low and high doses, respectively.

Neurite - A neural outgrowth, specifically an axon or dendrite. Most often, this is an axon.

Neuregulin - A single gene protein secreted by neurons that affects nearby support cells.

Schwann Cell - Support cell that provides a myelin sheath for axons.

Myelin sheath - A "ring" of fatty lipids surrounding the axons that speed up signal transmission.

Cryosection - A frozen section of a nerve, complete with all its support cells.

Introduction

Glial Growth Factor (GGF) is a secreted neuregulin that influences Schwann cells. At high concentrations, they cause proliferation, and at lower concentrations, migration. Neuregulins are required to keep Schwann cells alive. In contrast, neuregulins suppress overproduction of neurons, while supporting Schwann cells.

This report shows that Schwann cells respond to recombinant human Glial Growth Factor 2 (rhGGF2) in two ways: by time and dosage. Application of rhGGF2 shows that migration precedes proliferation, and a low application causes migration without proliferation. This was done on dorsal root ganglia (DRG) implanted on peripheral nerve cryosections. Overall, neuregulins recruit Schwann cells via chemotaxis, cause proliferation of these cells, and began local trophic support. This means that neuregulins play an integral part in the regeneration and support of damaged peripheral nerves.

Materials and Methods

Schwann cell migration and proliferation on sciatic nerve cryosections
DRGs were taken from the upper portion of the spine of one-day-old rats. They were incubated on various mediums, cleaned and then explanted onto cryostat sections of adult rat sciatic nerve. The rhGGF2 was added to the culture in various concentrations. After 72 hours the explants were incubated in 5(6)-carboxyfluorescein diacetate succinimidyl ester. This showed the extent of the Schwann cell migration. BrdU, a DNA precursor, was added to the medium to show the mitotic status of the migrating cells.

Purified Schwann cell culture
Schwann cells were taken from the sciatic nerves of 3-day-old rats, plated, given supplements and were placed in a tissue culture incubator. Fibroblast were eliminated by this process and the batches were screened for lack of cell migration. They were either frozen for later use or held in suspension.

SCG neuron culture
Superior cervical ganglion were taken from 0-1day old rats, cleaned under a dissecting microscope and then with enzymes to produce pure neuron cultures. The suspension was pelleted and suspended and was allowed to preplate like the cells in the above procedure to remove fibroblasts and macrophages. The cells were finally plated in media with and without recombinant human nerve growth factor, in 24 well plates that had been precoated with collagen.

Coated bead preparation
The Heparin Sepharose beads used in the experiments are between 24-44 um in size. In preparation of the coating proceedure, the beads are washed three times in PBS. Then the beads are made into a 50% volume to volume slurry of PBS with 1 mg/ml of BSA. The slurry is then incubated for 1 hour at 37 degrees celcius while being agittated. Now the slurry is separated into equal parts and incubated at the same temperature with various rhGGF2 solution concentrations for one hour in centrifuge tubes. After incubation the beads are washed three times by centrifugation and then susspended in Schwann cell medium. Final suspensions are stored at 4 degrees celcius.

Tube culture preparation
The tubes used in the experiment have an inner diameter of 1.19mm and are cut from a length of intramedic polythene tubing. The tubing was sterilized by rinsing in a 70% EtOH solution and being air dried. The tubing was then cut into 10mm segments and filled with a collagen supplimented low-glucose DME using a 1ml syringe with a 28 gauge hypodermic needle while being kept at a temperature of 4 degrees celcius. A Schwann cell graft is then placed at on end of each tube as to barely touch the surface of the medium. the surface tension of the medium pulled the tissue into the tube leaving it at the extreme end. The tubes were then placed in a 24-well plate and ioncubated at 37 degrees celcius until the medium gelled. BrdU test tubes were then immersed overnight in 1 ml of low glucose DME supplimented with 10 uM BrdU and a matching concentration of rhGGF2 as in the tube culture. The tubes were then placed in a BrdU free medium for 48 hours before complete removal from the immersion culture medium.

Results

Multiple migration assays
Three different methods were used to determine Schwann cell motility. Each of these can be used together to support the hypothesis.

rhGGF2 promotes Schwann cell migration on sciatic nerve cryosections
The first experiment used DRG explants on cryosections of adult sciatic nerve substrates in different concentrations of rhGGF2. It was found that the distance traveled by the Schwann cells increased by about 63% when grown in concentrations of 2.5 ng/ml or higher. It was also found that mitosis is not a factor in this movement by using BrdU treatments.

Focal application of rhGGF2 sequentially causes chemotaxis and then proliferation of cultured Schwann cells
The second assay involved pure Schwann cell cutures with rhGGF2 applied in localized areas using heparin beads. Robust motility was observed for beads coated with 6pg or more. The cells formed clumps around these beads. It is possible that some of th cells are making contact with the beads by extremely thin processes. BrdU was again used to test for miotic activity which was found to not be a contributor to the chemotactic responses.

A novel explant culture system
It is possible to regenerate nerves in vivo by attaching the nerve stumps to opposite ends of a biocompatible tube. To test this regeneration in vitro, an experiment was set up in which the geometric shape of the nerve was simulated. This regenerated simulation of nerves can be stained to tag Schwann cells and axons. Both the Schwann cells and the axons were grown in a directional manner - parallel to the tube. This may be due to the possibility that the collagen gel formed an oriented matrix.

Schwann cell outgrowth in the tube cultures increases in the presence of rhGGF2
Explants were exposed to 0, 5, 50, and 500 ng/ml rhGGF2. They were stained to tag Schwann cells at 2, 5, and 10 days. At 2 days, there was no difference between the groups in the total number of Schwann cells observed. At 5 days, it was noted that rhGGF2 at least doubles the number of Schwann cell growth. The distance travelled by the Schwann cells was 50% greater for those cultures exposed to rhGGF2. This is important because it reveals that mitotic effects were not solely responsible for cell growth. Schwann cell outgrowth, however, is not dependent on the presence of rhGGF2, as there were Schwann cells observed in the cultures with 0 dosage.

Initial schwann cell outgrowth takes place in the absence of cell division
A test was performed to determine whether the Schwann cell outgrowth could be attributed to mitotic division or to the presence of rhGGF2. The cultures were labelled with BrdU, a DNA precursor whose presence indicates division has occured. On days 2 and 5, the outgrowth contained no labelled cells. On day 10, there were a number of labelled cells. The appearance of the labelled cells occured at the same time that neurite outgrowth was detected. The Schwann cell outgrowth observed through day 5 is attributed to the presence of rhGGF2 and no to mitotic division.

Neurite outgrowth in the tube cultures increases in the presence of rhGGF2
Here the axons of superior cervical ganglia were stained with tubulin bIII in order to observe their growth in vitro. The axons were placed in different doses of rhGGF2. At 5 days there was no observable significant outgrowth, but at 10 days, significant neurite outgrowth was observed ditinctly different from the Schwann cell outgrowth. These dose-dependency cultures also showed differences the length of outgrowth. For example, cultures in 0-5 ng/ml rhGGF2 showed an average number of intersections falling from 10-15 to just one and terminated at approximately 850 microns while in the 50-500 ng/ml rhGGF2 culture demonstrated 30-40 intersections falling to 0 at 1.1-1.2 mm from the explant. This exercise illustrates that although at the lowest dose of rhGGF2, Schwann cells responded, a higher dose is required to promote the neuite outgrowth from the SCG.

Effects of rhGGF2 on neurite outgrowth are mediated by Schwann cells
In this exercise, two mechanisms accounted for different rhGGF2 dose-responses in Schwann cells and neurite outgrowth. The first mechanism was that each responds differently because each uses different receptors or receptor complexes. The second is that at low doses, Schwann cells migrate and proliferate while higher doses are required for neurite-promoting response. In testing the first mechanism with disassociated (depleted of non-neuronal cells) SCG, the experimentor exposed the culture to varying doses of both rhGGF2 and NGF. They observed that rhGGF2 neither promotes SCG neuron survival or creates a shift in NGF dose responsiveness. Thus it was concluded that because there was no obvious effect of rhGGF2 on neurite outgrowth in the 48 hour assay, it was unliklely that the effects of rhGGF2 directly effected neurite outgrowth. In the second mechanism they tested an indirect effect by plating Schwann cells at two densities on a conditioned media in varying doses of rhGGF2 and harvested them after 5 days. The result was that Schwann cell number increased with an increase in the dosage of rhGGF2. This differed from the control cultures where such activity was not present. This assured that the increased activity was due to the amount of individual cell activity rather than the total increase of Schwann cell number. The final conclusion was that the results demostrated in the absense of NGF, the ability of Schwann cells to promote neuron outgrowth and survival is greatly increased by the secreted activity of rhGGF2, therefore rhGGF2 indirectly promotes neurite outgrowth.

Discussion

Neuregulin-induced Schwann cell migration
Essentially, through the use of three distinct assays, it was demonstrated that rhGGF2 increases Schwann cell motility when applied at doses submaximal to that necessary to induce mitosis. In two of the experiments, the growth factor was applied globally, promoting migration of the Schwann cells on frozen pieces of peripheral nerves or in a collagen matrix. The third experiment demonstated chemeotaxis, as Schwann cells migrated towards beads coated with rhGGF2 in various concentrations. While proliferation did occur in these experiments,it was only after a considerable delay, thus proving that the Schwann cells' motility occurs in the absence of DNA synthesis.

Neuregulin-induced Schwann cell proliferation
In all three assays, the delay which occured before cell proliferation began can be directly atrributed to the presence (or absence) of neurons in the culture. In the absence of neurons, beads are capable of inducing proliferation, given that they are coated with a sufficient amount of rhGGF2. In the other two assays, however, the presence of neurons modified the response of the Schwann cells to the growth factor, such that their proliferation correlated with the appearance of outgrowing neurites.

Neuregulin induction of neurotrophic support
Although originally thought to directly stimulate neurite outgrowth, rhGGF2 actually promotes neuron survival or outgrowth in terms of Schwann cells. Also, the neurite outgrowth could be attributed to the tropic effect , especially because of the 5 to 10 day lag period for any observable neurites. The growth could be attributed to the accumulation of Schwann cell secretions. Schwann cells are suited to produce factors supporting neuron outgrowth and survival. In further research, the secretions of Schwann cells should be studied in addition to studying Schwann cells treated with supramitotic doses of neuregulins.

Neuregulins and peripheral nerve regeneration
Studies of in body samples have led to some interesting conclusions about Schwann cells. First, Schwann cell migration and proliferation occur in the fluid around the regenerating nerve. Also, Schwann cells reside in degenerated nerves and regenerate damaged axons. The presence or neuregulins is essential for these events to happen. Neuregulins result from neuronal secretions and attribute to the proliferation of Schwann cells. An example is the neuregulin receptor subunit erbB2. If this subunit is secreted in high numbers, a concentration gradient is formed, and Schwann cells are "recruited" by the low concentration. Overall, it appears that neurons and Schwann cells are essential to peripheral nerve regeneration, and a key component in the equation could be neuregulin.

Our Interpretation

So, what does the article really mean? It shows that with the proper neuregulin, the growth and sustainment of Schwann cells can be achieved. When this happens, the neuron they support can be regenerated. This definately has effects in such areas as replacing damaged peripheral nerves. In a recent case in Tucson, a young girl was mauled by a bear, and a peripheral nerve torn. Adding these growth factors can help her after a nerve transplant to sustain the new nerve. In addition, with further research, we may be on our way to "curing" a much larger problem - paralysis by a broken spinal cord. If this research continues, people like Christopher Reeve may walk again.

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