Rho Signaling and Axon Regeneration - International Review of Neurobiology - page 121

( Park et al., 2008 ). PTEN was identified as a potent target to promote RGC
regeneration in the optic nerve where deletion experiments showed that
inactivation of PTEN is sufficient to promote long-distance axon regener-
ation of RGC axons ( Park et al., 2008 ). In CNS tissue, activated ROCK
binds to PTEN and can be detected by coimmunoprecipitation ( Kilic
et al., 2010 ). In further experiments, it was demonstrated that ROCK
phosphorylates PTEN directly, which activates it ( Li et al., 2005 ). By extrap-
olation to neurons, we can surmise that activation of PTEN by ROCK is a
key step in arresting axon regeneration, but this remains to be shown
directly. Therefore, inactivation of either Rho or ROCK should attenuate
PTEN activity, but it is not known if other intracellular signals may be
needed to keep PTEN inactive to promote robust regeneration.
While both ROCK inhibitors and Rho inhibitors appear to have similar
effects on neurite outgrowth tested in tissue culture, there may be significant
differences for axon regeneration in vivo . First, we know that although Rho
activates ROCK, Rho activation may differentially affect signaling proteins
that directly bind Rho, such as CRMP4 or PTEN. Experiments to examine
regeneration in vivo in the transected optic nerve suggest that there are indeed
differences between Rho and ROCK signaling. While both Rho and
ROCK inhibitors promote regeneration, regeneration appears more robust
with Rho inactivation: RGC axon regeneration after optic nerve injury is
very robust when cell-permeable C3 is used to promote growth ( Bertrand,
Di Polo, &McKerracher, 2007 ) compared to the ROCK inhibitor Y27632
( Ichikawa et al., 2008 ). Similarly, genetic manipulation of PTEN, which acts
in the Rho pathway ( Park et al., 2008 ), appears to be more powerful than
inhibiting ROCK. Direct, side-by-side comparisons of PTEN, Rho, and
ROCK are needed to sort out which targets are more powerful for eliciting
axon regeneration and if combinations may be synergistic, such as observed
when PTEN and suppressor of cytokine signaling 3 are genetically deleted
simultaneously ( Sun et al., 2011 ).
1.4. ROCK and Rho inhibitors
C3 transferase is an enzyme from Clostridium botulinum that blocks Rho by
locking Rho in an inactive state and keeping it bound to Rho dissociation
inhibitor ( Wilde & Aktories, 2001 ). C3 transferase reverses the effect of
extracellular inhibitory substrates on neurons in culture and enables the out-
growth of neurites ( Lehmann et al., 1999 ). However, cell penetration by the
wild-type C3 transferase is very low, limiting its clinical development as a
therapeutic agent. Cell-permeable C3 transferase was created to effectively