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.