Dynamic regulation of SCG10 in regenerating axons after injury☆
Introduction
The peripheral nervous system can often recover from mechanical, chemical, and pathological insults that injure axonal projections. Impaired sensory and motor function as a result of the destruction of damaged axons can be restored if the axons re-grow across the injured area and successfully reinnervate their target cells (Allodi et al., 2012). However, efficient recovery of neural function depends on the speed of reinnervation — if the target cell is denervated for a prolonged period, it may lose function even if the neural connection is eventually restored (Gordon et al., 2011). Therefore, there is much interest in identifying molecular mechanisms regulating axon regeneration and developing methods that facilitate the regenerative responses (Blackmore, 2012, Bradke et al., 2012, Liu et al., 2011, Patodia and Raivich, 2012). The need for such studies has encouraged the development of in vivo assays for axon regeneration that can be used in genetic and pharmacological models. Such anatomical assays require methods to selectively label regenerating axons, distinguishing them from the distal axons undergoing Wallerian degeneration. Since it takes ~ 40 h for distal axons to fragment following axotomy (Beirowski et al., 2005), the need for selective regeneration markers is particularly acute in the early phase of the injury response.
Transgenic expression of neuronal YFP is a method to visualize axon regeneration and degeneration as well as normal axon morphology. However, YFP remains in distal axon fragments even during axonal degeneration, so regenerating axons are obscured by the YFP-positive degenerative particles (Bareyre et al., 2005, Pan et al., 2003) unless a YFP-negative nerve graft is transplanted to avoid the background signal (Witzel et al., 2005). Neuronal tracers such as the lipophilic DiI (Honig and Hume, 1989) and BDA (biotinylated dextran amine) can be injected proximal to a lesion and will selectively label regenerating axons (Liu et al., 2010). However, these methods are much more technically difficult and time consuming than immunocytochemistry. Indeed, antibody staining for proteins that are selectively localized to regenerating rather than degenerating axons would be a powerful and simple method for labeling re-growing axons. One popular target is growth-associated protein 43 (GAP43), whose transcription is upregulated days after axon injury (Bisby and Tetzlaff, 1992, Skene and Willard, 1981a) leading eventually to intense GAP43 immunolabeling in regenerating axons (Abe et al., 2010, Ackermann et al., 2002).
Superior cervical ganglion 10 (SCG10), which is also known as stathmin 2 (STMN2), is a neuronally expressed stathmin family protein that regulates microtubule dynamics and protein trafficking (Ozon et al., 1997, Riederer et al., 1997, Wang et al., 2013). SCG10 is highly expressed during development and plays an important role in axonal outgrowth by modulating microtubule stability (Morii et al., 2006, Sugiura and Mori, 1995, Tararuk et al., 2006). Interestingly, axonal injury leads to an increase in SCG10 expression in adult sensory neurons (Mason et al., 2002, Voria et al., 2006). In contrast, we recently demonstrated that SCG10 is rapidly lost from distal axons within hours of an axonal injury (Shin et al., 2012b). The differential regulation of SCG10 in regenerating cell bodies and the distal axon segments led us to test the hypothesis that SCG10 may be an efficient and selective marker for re-growing axons in the early stage of axon regeneration. In the current study, we show that SCG10 levels are increased in the axon segments proximal to a lesion in vitro and in vivo within an hour after the injury. After nerve crush or nerve repair, the rise in the proximal SCG10 expression persists while the axons re-grow through the distal nerve segment, which is nearly devoid of SCG10. We demonstrate that the SCG10 immunolabeling is more selective for regenerating axons than either GAP43 or YFP, especially in the early stage of regeneration and in conditions where Wallerian degeneration is delayed. We show that SCG10 is preferentially expressed in sensory fibers, and demonstrate that axonal regeneration can be quantified using SCG10 labeling in a genetic model with slowed axon regeneration. Hence, axonal SCG10 is dynamically regulated upon nerve injury and is a selective marker for regenerating sensory axons, thereby providing a useful new method to assess regeneration after nerve injury and repair.
Section snippets
Mice
Adult C57BL6 mice were purchased from Jackson Laboratory or Harlan Laboratories and used for in vivo analysis of protein levels and regeneration assays. YFP 16 mice (Feng et al., 2000) were kindly provided by Dr. Joshua Sanes (Harvard University, Cambridge). Advillin-Cre mice (Zhou et al., 2010) or Chat-Cre mice (MMRRC, #017259) were crossed to Thy1-STOP-YFP mice (Bareyre et al., 2005) to label sensory or motor neurons, respectively. Cyt-NMNAT1 mice (gifted by Dr. Jeffrey Milbrandt, Washington
SCG10 levels rapidly increase in the proximal stump of injured axons
We previously demonstrated that SCG10 levels are dynamically regulated in response to axonal injury, with a rapid loss of SCG10 from axon segments distal to an axotomy (Shin et al., 2012b). Here we study the temporal regulation of SCG10 protein in proximal axons following injury. We transected axons of cultured murine embryonic DRG neurons and performed immunolabeling for SCG10. In uncut axons, SCG10 is localized throughout the processes in a punctate pattern (Fig. 1A, boxed area magnified in
Discussion
Axonal regeneration is essential for the recovery of function after neuronal injury. As such, there is much interest in dissecting the molecular mechanisms controlling the regenerative response, as well as identifying novel therapeutic agents to improve regeneration. Such analysis requires efficient assays of in vivo regeneration, however current methods are particularly poor for assessing the earliest stages of regeneration. Here we demonstrate that immunolabeling for SCG10 is a powerful
Acknowledgments
We thank Dr. Jeffrey Milbrandt and Dr. Joshua Sanes for sharing transgenic mouse lines. We appreciate Ying Yan and Matt Wood in the Susan Mackinnon lab at Washington University for teaching us the nerve repair technique. We are grateful to the members of the DiAntonio and Milbrandt laboratories for helpful discussions. We thank Sylvia Johnson for her technical assistance. This work was supported by NIH grant NS065053 (to A.D.) and Wings for Life fellowship (to J.E.S.). A.D., J.E.S., and
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Conflict of interest: A.D., J.E.S., and Washington University may receive income based on a license by the University to Novus Biologicals.