The complex nature of spinal cord injuries makes it difficult to identify potential therapeutic agents, but the conditioning lesion model gives us the opportunity to do exactly that by providing a scenario in which axons can regenerate under adverse conditions. Using this model, we have now identified SLPI as a new and promising means of promoting axonal regeneration in the adult mammalian CNS. Not only is SLPI an essential component of the conditioning lesion effect, but it can also promote CNS axonal regeneration in vivo when administered exogenously. In addition, we have shown that the effects of SLPI on regeneration can be abolished by overexpression of Smad2, and provided the first evidence that myelin-associated inhibitors induce phosphorylation of Smad2. The phosphorylation of Smad2 by myelin-associated inhibitors represents not only a new signaling pathway for these inhibitors but also a novel target for therapeutic intervention to promote axonal regeneration after injury.
There has been enormous interest in elucidating the underlying mechanism of the conditioning lesion effect, but to date, the genes responsible have remained elusive. Our data indicate that SLPI is necessary for the conditioning lesion effect, but it is not yet known if it is sufficient. Like SLPI, both activating transcription factor 3 (ATF3) and the cytokine IL-6 are significantly increased after a sciatic nerve lesion (Tsujino et al., 2000
; Cao et al., 2006
). Neurite outgrowth was significantly increased when DRG neurons from ATF3 transgenic mice were cultured on permissive substrates, but these neurons were not able to overcome inhibition by myelin in vitro
(Seijffers et al., 2007
). Furthermore, regeneration of dorsal column axons in these mice was not improved (Seijffers et al., 2007
). These findings indicate that increasing the intrinsic growth capacity of neurons is not sufficient to overcome inhibition by myelin and replicate the conditioning lesion effect. Cafferty and colleagues (2004)
performed conditioning lesions in IL-6 null mutant mice and reported that both neurite outgrowth and regeneration of dorsal column axons were impaired. In contrast, a similar study performed by our laboratory reported that the extent of regeneration was equivalent in wild type mice and IL-6 null mutants, which led us to conclude that IL-6 is sufficient but not necessary for the conditioning lesion effect (Cao et al., 2006
). Recently, we reported that up-regulation of arginase I-mediated polyamine synthesis contributes to the conditioning lesion effect and that polyamines can promote axonal regeneration in vivo
(Deng et al., 2009
). However, we have not yet established whether activation of this pathway is sufficient to mimic the conditioning lesion effect.
Spinal cord injury is most commonly associated with axonal damage, but there is also widespread inflammation, cell death, and demyelination. Recently it was reported that wild type mice treated with exogenous SLPI after spinal cord contusion displayed a significant improvement in locomotor function as early as 3 days after injury, an improvement that was maintained for the 28-day duration of the experiment (Ghasemlou et al., 2010
). Although axonal regeneration was not specifically examined in that study, an increase in serotonergic innervation of ventral motor neurons was reported (Ghasemlou et al., 2010
). The authors attribute the functional recovery and the increased innervation to the significant increases in tissue sparing, improved motor neuron survival, and decreased expression of TNFα that were observed (Ghasemlou et al., 2010
). Now we show that SLPI also has a direct effect on neurons and promotes axonal growth in an inhibitory environment. Together, these studies demonstrate that SLPI mediates a unique combination of pro-regenerative and neuroprotective effects that enhance the growth capacity of axons and create a more favorable environment within the CNS.
Chondroitin sulfate proteoglycans (CSPGs) expressed by reactive astrocytes also contribute to the inhibitory environment of the injured CNS (Asher et al., 2000
; Schachtrup et al., 2010
; Hellal et al., 2011
), and a recent spinal cord injury study has shown that injury-induced expression of CSPGs was reduced following administration of taxol (Hellal et al., 2011
). This effect was attributed to taxol’s ability to inhibit nuclear translocation of Smad2 (Hellal et al., 2011
). This finding adds to a growing body of evidence that TGFβ signaling contributes to astroglial scarring (Asher et al., 2000
; Schachtrup et al., 2010
), and our observation that CNS myelin induces phosphorylation of Smad2 now raises intriguing questions about the role of TGFβ signaling in myelin-mediated inhibition. Our experiments show that Smad2 is phosphorylated at serines 465 and 467, which are directly phosphorylated by the active type I TGFβ receptor (Abdollah et al, 1997
). This indicates that myelin proteins activate the TGFβ receptor complex, but it is not known how this would occur (). Given the tremendous structural differences between MAG, Nogo, and TGFβ, and the fact that the type II TGFβ receptor binds TGFβ very specifically (Massagué, 2000
), it is unlikely that myelin-associated inhibitors bind directly to the type II TGFβ receptor. Expression of active TGFβ is strongly upregulated in neurons and glia within 2 days of spinal cord injury (Buss et al., 2008
) and this could lead to activation of the receptor in vivo
; however, it is more likely that this up-regulation of TGFβ occurs as part of the acute inflammatory response (Buss et al., 2008
) and does not involve myelin-associated inhibitors. The He laboratory has shown that MAG, Nogo, and OMgp can transactivate the epidermal growth factor receptor (EGFR) via an unknown mechanism (Koprivica et al., 2005
), and so, it is possible that the binding of MAG or Nogo to the Nogo receptors could also lead to transactivation of the type II TGFβ receptor.
Schematic of myelin-mediated activation of the TGFβ signaling pathway and downregulation of Smad2 by SLPI
There is now substantial evidence that Smad2 protein is required to mediate inhibition by myelin, but what is the role of pSmad2 in this process? The Bonni laboratory has provided insight into this by describing a connection between pSmad2, the transcriptional co-repressor SnoN, and the E3 ubiquitin ligase Cdh1-anaphase-promoting complex (Cdh1-APC). In earlier studies they had shown that Cdh1-APC negatively regulates axonal growth in the cerebellum by ubiquitinating SnoN (Konishi et al., 2004
; Stegmüller et al., 2006
). They subsequently demonstrated that pSmad2 interacts with SnoN within the nucleus and presents it to Cdh1-APC for ubiquitination, which leads to impaired neuritogenesis (Stegmüller et al., 2008
). Knockdown of either Cdh1 or Smad2 enhanced neurite outgrowth on myelin substrates (Konishi et al., 2004
; Stegmüller et al., 2008
), and they have therefore proposed that the Cdh1-APC-SnoN pathway plays a role in myelin-mediated inhibition of axonal growth (Konishi et al., 2004
; Stegmüller et al., 2008
). Our data support and expand this hypothesis, as myelin-induced phosphorylation of Smad2 would increase SnoN degradation and thereby inhibit neurite outgrowth. It should be noted that the Cdh1-APC-SnoN pathway has been studied primarily in the context of embryonic and early post-natal development (Konishi et al., 2004
; Stegmüller et al., 2006
), and so, it would be interesting to explore the function of this pathway in the mature CNS, particularly after injury.
The primary function of pSmad2, together with pSmad3 and Smad4, is to regulate transcription by associating with DNA binding co-factors and recruiting transcriptional co-activators or co-repressors (Massagué et al., 2005
). This is a highly complex process affecting hundreds of genes (Massagué et al., 2005
), and the role of transcription in myelin-mediated inhibition is unknown; however, we should consider the possibility that myelin-induced activation of the Smad complex modulates transcription in a way that leads to inhibition of axonal growth. Deletion of phosphatase and tensin homolog (PTEN) activates the mammalian target of rapamycin (mTOR) pathway and promotes axonal regeneration in the optic nerve (Park et al., 2008
). Since the mTOR pathway facilitates protein translation, it was proposed that protein synthesis is essential for axonal regeneration (Park et al., 2008
). It is therefore possible that myelin-associated inhibitors suppress protein synthesis by activating Smad2 and inhibiting the transcription of genes that are required to promote growth.
Interestingly, Smad signaling through the bone morphogenic protein (BMP) pathway appears to have the opposite effect. The binding of BMPs to their cognate receptors leads to phosphorylation of Smad1, 5, and 8 (Massagué et al., 2005
). Like Smad2 and 3, these Smads form complexes with Smad4 and translocate to the nucleus where they act as transcriptional regulators (Massagué et al., 2005
). A recent study by Parikh and colleagues (2011)
has reported that pSmad1 plays an essential role in axonogenesis during embryonic development, and enhances the growth capacity of adult DRG neurons. In addition, they also show that intrathecal injection of a BMP4 adeno-associated virus leads to activation of Smad1 and increased axonal regeneration, even when administered after a dorsal hemisection (Parikh et al., 2011
). This indicates that Smad1 signaling enhances axonal regeneration, which suggests that pSmad1 may modulate the expression of pro-regenerative genes. It therefore appears that the TGFβ-Smad signaling pathway restricts axonal regeneration, while the BMP-Smad signaling pathway enhances it, and it is possible that a regenerating axon’s fate may ultimately be decided by the competing transcriptional events mediated by these two pathways.
Transcriptional regulation may also be central to SLPI’s ability to overcome inhibition by myelin. In 293 cells, it has been shown that once pSmad2 performs its function in the nucleus, it is ubiquitinated and degraded by the proteasome (Lo and Massagué, 1999
; Seo et al., 2004
), and so, it is likely that new Smad2 protein must be synthesized to sustain myelin-mediated inhibition in neurons. By binding to the Smad2 promoter, SLPI would prevent de novo
transcription of the Smad2 gene, and this would ultimately lead to a decrease in the amount of total Smad2 protein within the neuron (). Levels of pSmad2 would also be reduced because there is less protein available for phosphorylation, and this loss of Smad2 function would allow the neuron to overcome inhibition by myelin. In addition to Smad2, SLPI can also downregulate expression of the pro-inflammatory cytokine TNFα (Taggart et al., 2005
; Ghasemlou et al., 2010
), which is strongly upregulated after spinal cord injury and has been implicated in both neuronal and glial apoptosis (Lee et al., 2000
; Pearse et al., 2004
; Ghasemlou et al., 2010
). Thus, it appears that SLPI may be capable of downregulating a variety of genes that contribute to the pathophysiology of spinal cord injury. If these genes could be identified, it would greatly advance our understanding of the mechanisms underlying regenerative failure and potentially provide new targets for pharmacological intervention.