Discussion
Poor functional recovery from injuries to peripheral nerves is a
significant public health issue. Such injuries are relatively common and
despite the well-documented ability of axons to regenerate following
PNI, they do so poorly. The successes of experimental treatments that
promote axon regeneration after peripheral nerve injury in preclinical
studies, such as exercise (Udina et al. , 2011a; English et
al. , 2014) or electrical stimulation (Al-Majed et al. , 2000b;
Gordon & English, 2016) are dependent on signaling between BDNF and its
TrkB receptor (Al-Majed et al. , 2000a; Wilhelm et al. ,
2012). Treatments of mice with small molecule TrkB agonists or prodrugs
that generate them also result in enhanced regeneration (Englishet al. , 2013; English et al. , 2022). We hypothesized that
at least one target of all of these treatments is AEP. A prediction of
this hypothesis is that downstream effectors of BDNF-TrkB signaling
inhibit AEP, which then reduce or eliminate its cleavage of Tau and APP,
and in doing so promote axon regeneration.
In the present study, we evaluated our hypothesis using a specific AEP
inhibitor, compound 11 (CP11) (Zhang et al. , 2017). The main
findings reported here are: 1) oral treatments with CP11 inhibit AEP
activity at the site of nerve injury; and 2) systemic treatments with
CP11 enhance motor and sensory axon regeneration after PNI. When
administered either orally or by i.p. injection, CP11 treatments
resulted in successful axon regeneration and muscle reinnervation by
significantly more motor and sensory neurons than vehicle treated
controls. Restoration of compound muscle action potentials (M wave
amplitudes) was greater in CP11-treated mice than in controls. These
findings are consistent with our hypothesis that inhibition of AEP is a
prime target of experimental therapies for treating PNI, whether by
activity-dependent or pharmacological approaches.
To begin to investigate the cellular mechanisms that might be involved,
we compared the effects of treatment with 7,8-DHF and CP11 on neurite
outgrowth from cultured DRG neurons. Both the small molecule TrkB
ligand, 7,8-DHF, and the specific AEP inhibitor, CP11, significantly
increased the growth of neurites when added individually to cultures but
when added together they produced no significant increase over that
observed when either was used alone. Similarly, addition of 7,8-DHF or
CP11 to cultures from AEP-KO mice produced no significant effect on
neurite outgrowth relative to untreated cultures of DRG neurons from the
same animals. The extent of enhancement of neurite outgrowth in these
cultures also was not significantly different from the effects of
treatments with 7,8-DHF, CP11, or both on the lengths of neurites from
neurons derived from WT mice. These outcomes all are consistent with our
hypothesis that the effectiveness of treatments that enhance axon
regeneration by stimulating TrkB activation, such as treatments with
7,8-DHF (English et al. , 2013) or prodrugs (English et
al. , 2022), as well as activity-dependent experimental therapies that
increase BDNF and/or TrkB expression in rats (Al-Majed et al. ,
2000a) and mice (Wilhelm et al. , 2012), all do so primarily by
inhibiting AEP. Activation of the TrkB receptor in these scenarios
results in its phosphorylation and a downstream inhibition of AEP,
leading to a decrease in cleavage of the microtubule domain of Tau at
asparagine 368 and a promotion of axon regeneration (English et
al. , 2021). A similar effect is achieved by direct AEP inhibition via
CP11 treatments.
Of particular interest were the results of experiments in which the
effects of the treatments were evaluated in cells that expressed the
TrkB receptor. Treatments with CP11 stimulated neurite outgrowth in all
cells, regardless of phenotype, but, as might be predicted, treatments
with 7,8-DHF were effective only in neurons that expressed the TrkB
receptor. The mRNA for TrkB is widely expressed in alpha, but not gamma
motoneurons in rats (Buck et al. , 2000; Copray & Kernell, 2000).
The robust effects of treatments with 7,8-DHF or its prodrug, R13, and
the results presented here using CP11, on the regeneration of motor
axons or/and restoration of M wave amplitude in reinnervated muscles are
likely due to similar TrkB expression in mouse motoneurons. The
regeneration of axons of a subset of sensory neurons that express the
TrkB receptor, even as modified following PNI in rats (Karchewskiet al. , 2002) or mice (English et al. , 2007), also would
be expected to respond. However, sensory neurons not expressing TrkB
would not benefit from such a TrkB-dependent therapy. We believe that
these results suggest that direct AEP inhibition, such as that produced
by CP11, might be overall the most effective therapy for PNI to date.
This assertion does not discount the possibility that pathways
alternative to the BDNF-TrkB pathway that result in inhibition of AEP
may exist. Whether they are in play when activity-dependent treatments
are employed remains for future study.
Conclusion: Inhibition of AEP activity is the main focus of
activity-dependent and BDNF-TrkB dependent experimental therapies for
peripheral nerve injury. Direct inhibition of AEP by CP11 is a potential
treatment worthy of further consideration.