https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5090983/
Exp Neurol. Author manuscript; available in PMC 2017 Sep 1.
Published in final edited form as:
Published online 2016 Jun 2. doi: 10.1016/j.expneurol.2016.05.021
The publisher's final edited version of this article is available at Exp Neurol
See other articles in PMC that cite the published article.
Abstract
Although
previous studies have identified several strategies to stimulate
regeneration of CNS axons, extensive regeneration and functional
recovery have remained a major challenge, particularly for large
diameter myelinated axons. Within the CNS, myelin is thought to inhibit
axon regeneration, while modulating activity of the mTOR pathway
promotes regeneration of injured axons. In this study, we examined NT-3
mediated regeneration of sensory axons through the dorsal root entry
zone in a triple knockout of myelin inhibitory proteins or after
activation of mTOR using a constitutively active (ca) Rheb in DRG
neurons to determine the influence of environmental inhibitory or
activation of intrinsic growth pathways could enhance NT-3-mediate
regeneration. Loss of myelin inhibitory proteins showed modest
enhancement of sensory axon regeneration. In mTOR studies, we found a
dramatic age related decrease in the mTOR activation as determined by
phosphorylation of the downstream marker S6 ribosomal subunit.
Expression of caRheb within adult DRG neurons in vitro
increased S6 phosphorylation and doubled the overall length of neurite
outgrowth, which was reversed in the presence of rapamycin. In adult
female rats, combined expression of caRheb in DRG neurons and NT-3
within the spinal cord increased regeneration of sensory axons almost 3
fold when compared to NT-3 alone. Proprioceptive assessment using a grid
runway indicates functionally significant regeneration of
large-diameter myelinated sensory afferents. Our results indicate that
caRheb-induced increase in mTOR activation enhances neurotrophin-3
induced regeneration of large-diameter myelinated axons.
Keywords: Rheb, mTOR signaling pathway, neurotrophin-3 (NT-3), axonal regeneration, dorsal root ganglion (DRG)
Introduction
Of
the sensory axon populations examined large diameter myelinated axons
show the poorest regeneration through the dorsal root entry zone (DREZ)
and into the spinal cord. Proprioceptive axons are some of the largest
diameter sensory axons in the body and express the NT3 receptor trkC.
Numerous studies indicate that regenerative failure is most likely due
to both reduced intrinsic growth ability by adult neurons and the
inhibitory environment within the spinal cord. Spinal cord application
of neurotrophin-3 (NT-3) is thought to enhance the intrinsic growth
state of adult DRG neurons, as well as modify the extrinsic environment
(Hanna-Mitchell et al., 2008) and promote some but limited amount of
sensory afferent regeneration across the dorsal root entry zone after
dorsal root rhizotomies (Ramer et al., 2001; 2002).
Previous studies indicated that NT-3-induced regeneration is sufficient
to bypass the gliotic region at the DREZ, but regeneration fails when
axons encounter degenerative domain containing myelin debris (Ramer et al., 2001). Other studies have also shown that soluble Nogo receptor can promote regeneration of sensory afferents across the DREZ (Harvey et al., 2009); however, this regeneration was limited primarily within the dorsal columns.
On
the other hand, intrinsic activation of mTOR by deletion of an upstream
negative regulator PTEN increased axonal regeneration of retinal
ganglion neurons and cortical neurons. Likewise, inhibition of mTOR with
rapamycin blocked regeneration, suggesting that modulation of mTOR
activity affects intrinsic axonal growth capacity within the CNS (Park et al., 2008).
mTOR is a critical regulator of protein synthesis and plays an
important role in cell growth, proliferation, and survival during
development (Martin and Hall, 2005; Sarbassov et al., 2005). Previous studies have suggested that mTOR expression level is correlated with increased growth capacity (Abe et al., 2010; Park et al., 2008). mTOR activity is upregulated in DRG neurons after sciatic nerve lesion (Belin et al., 2015) and downregulated in retinal and cortical neurons after lesion (Park et al., 2008, Liu et al., 2010).
In cultured DRG neurons, following PTEN inhibition and neurite growth
induction, rapamycin did not completely block increased neurite
outgrowth, suggesting other mechanisms by which PTEN knockdown might
affect regenerative growth beyond mTOR activation alone (Christie et al., 2010).
Ras homolog enriched in brain (Rheb) is a direct activator of mTOR and
was shown to activate the mTOR pathway in many studies (Bai et al., 2007; Inoki et al., 2003; Long et al., 2005).
To date, it is not reported whether direct and specific activation of
mTOR pathway can affect axonal growth capacity after central branch
injury in DRG neurons.
In this study we
attempted to augment NT-3 mediated regeneration of large diameter
myelinated axons using two approaches. The first was to overexpress NT-3
in the spinal cord of Nogo/MAG/OMgp triple knockout mice to determine
if the elimination of extrinsic, myelin-associated inhibitors would
augment NT-3 mediated primary afferent regeneration. In the second part
we expressed a constitutively active form of Rheb to activate mTOR to
determine if further enhancement of intrinsic growth would enhance
NT-3-mediated proprioceptive sensory axon regeneration through the DREZ
and into the spinal cord. Here, we observed the loss of
myelin-associated inhibitors to have only a modest effect on enhancing
regeneration in the presence of NT-3. However, expression of ca-Rheb in
DRG neurons induced greater than a 2 fold increase in NT-3 mediated
regeneration.
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