|
Peripheral nerve axon regeneration is regulated by the balance between growth promoting and inhibiting factors. The heparan sulfate proteoglycan (HSPG)
syndecan-3, normally a co-receptor for numerous growth factors, becomes growth inhibitory once its extracellular domain is shed from the plasma membrane.
Matrix metalloproteinases (MMPs), a class of zinc-dependent proteolytic enzymes, regulate syndecan shedding, and inhibition of MMPs (especially MMP-7) reduces
that rate. Degrading the glycosaminoglycan (GAG) side chains of all HSPGs promotes axon regeneration in peripheral nerves, but whether this effect is specific
to syndecan-3 is unknown. To analyze the role of syndecan-3 in axon regeneration, the fluorescent subset of axons in transgenic thy-1-YFP-H mice were studied.
The common fibular (CF) nerves were cut and repaired with grafts from syndecan-3 knockout mice. After one week, nerves were harvested and examined using
confocal microscopy. Lengths of YFP+ axon profiles, from the distal tip to the surgical repair site, were measured. Axon profile lengths measured in grafts from
syn-3-/- mice are significantly longer than those measured in grafts from wild type (WT) mice (controls), and similar to those measured in Heparinase III- treated
WT grafts. If cut CF nerves in thy-1-YFP-H/syn-3-/- mice were repaired with grafts from WT mice, lengths of regenerating axon profiles also were longer,
resembling those found in Heparinase I-treated WT grafts. If cut CF nerves in thy-1-YFP-H/syn-3-/- mice were repaired with grafts from syn-3-/- donors, axon
regeneration is enhanced relative to controls. More axons longer than 1000 mm were observed in this graft-host combination than with any other studied. If
cut CF nerves in thy-1-YFP-H mice are repaired with grafts from MMP-7-/- mice, axon regeneration is also enhanced relative to controls. Thus, syndecan-3 inhibits
axon growth both on the regenerating axons and Schwann cells in their environment, but does so in different manners.
• Functional recovery after peripheral nerve injury is poor largely because regenerating axons in peripheral nerves grow slowly.
• Growth of regenerating axons is the net effect of growth promoting and growth inhibiting molecules in the environment of the regenerating axons.
• Syndecan-3 is a cell surface heparan sulfate proteoglycan (HSPG) found in the environment of regenerating axons that can act as a growth promoting or growth
inhibiting factor.
• Binding of growth factors to glycosaminoglycan (GAG) side chains promotes binding of the growth factor to its cognate receptor.
• The same growth factor signalling produces a cleavage of the ectodomain of syndecan-3. Shed syndecan-3 becomes a growth factor sink and inhibits further
growth factor-receptor binding.
• Syndecan shedding is produced by the actions of matrix metalloproteinases (MMPs), especially MMP-7, which are themselves activated by growth factor binding.
• The goal of this project was to evaluate peripheral nerve axon regeneration in the absence or reduction of syndecan-3 shedding.
EXPERIMENT 1
- Cut CF nerve in Thy-1-YFP-H host was repaired with a nerve graft from a syn-3 knockout mouse.
- Distribution of axon profile lengths is shifted to the right of control.
- Distribution of axon profile lengths resembles that found in Heparinase III-treated WT grafts.
- The axons are longest in this graft/host combination than in any other.
- Median axon profile length resembles that found in Heparinase III-treated WT grafts, and not Heparinase I-treated WT grafts.
EXPERIMENT 2
- Cut CF nerve in Thy-1-YFP-H/syn-3-/- host was repaired with a nerve graft from a WT mouse.
- Distribution of axon profile lengths is shifted to the right of control.
- Median axon profile length resembles that found in Heparinase I-treated WT grafts.
- Distribution of axon profile lengths is shifted to the right of control, and most down of control.
- There is more of the long axon population in this graft/host combination than in any other.
EXPERIMENT 3
- Cut CF nerve in Thy-1-YFP-H host was repaired with a nerve graft from a MMP-7 knockout mouse.
- Distribution of axon profile lengths is shifted to the right of control.
- Distribution of axon profile lengths resembles that found in Syndecan-3-/- grafts.
Axon Branching
• By knocking out syndecan-3 directly, or by indirectly reducing the rate of MMP-7-dependent syndecan-3 shedding, there is increased regeneration.
• Data is consistent with the hypothesis that syndecan-3 shedding significantly reduces the rate of peripheral nerve axon regeneration.
• Future experiments include repairing syn-3-/- CF nerves with MMP-7-/- CF nerves (and the reverse), and further trials repairing syn-3-/- CF nerves with syn-3-/- CF nerves.
All Heparinase data on this poster were taken from Groves et al., Exp. Neurol. The syndecan-3 cartoon was modified from Carey, J. Biochem.
We would like to thank Gail Schwartz for her technical assistance. This work was supported by the Howard Hughes Medical Institute under Grant
No.52003727 and by the USPHS under HD 43356.
It's very common to injure the peripheral nervous system (PNS), but, unfortunately, gaining back PNS function is slow, poor, and painful. There are a variety
of different mechanisms involved in axon regeneration, so we decided to look at molecules called Heparan Sulfate Proteoglycans (HSPGs). Proteoglycans, in
general, are proteins that have sugar chains added to them. One type of HSPG is called syndecan, and when it binds other molecules called growth factors, part
of the syndecan (including its sugar chains) is cleaved into the space outside of the cell. The cleaved part then acts in an INHIBITORY manner, preventing axons
from growing back to their targets. This process is called, overall, syndecan shedding. If you inhibit, or reduce, syndecan shedding, increased regeneration
seems a likely outcome. To test this, we took mice that lack the syndecan-3 gene, aka syndecan knockouts. (Since they lack syndecan to begin with, cells can't
exactly shed it!) We grafted syndecan knockout nerves into normal mice with injured nerves, and measured how far the axons grew back. Indeed, axons grew
much longer when syndecan was absent from the graft (we literally measured the length of individual axons using a confocal microscope)! We then tested the
reverse, and put normal grafts in syndecan knockout mice, and, again, there was increased regeneration! Then, we took syndecan knockout nerves, and grafted
them into syndecan knockout host mice, and, presto, even more regeneration. The last thing we did was knockout a protein called MMP-7 that mediates shedding.
We took MMP-7 knockout nerves and grafted them into normal mice and, found, once again, increased axon regeneration. So, even though we didn't show that
syndecan shedding is actually taking place (since previous work already has), we showed data that are consistent with the hypothesis that syndecan shedding
reduces axon regeneration.
Mouse survival surgery, nerve grafting, nerve harvesting, mounting slides, confocal microscopy
Syndecan shedding, peripheral nervous system injury, Wallerian degeneration, matrix metalloproteinases, heparan sulfate proteoglycans
|
