PTEN inhibition to facilitate intrinsic regenerative outgrowth of adult peripheral axons

Abstract

In vivo regeneration of peripheral neurons is constrained and rarely complete, and unfortunately patients with major nerve trunk transections experience only limited recovery. Intracellular inhibition of neuronal growth signals may be among these constraints. In this work, we investigated the role of PTEN (phosphatase and tensin homolog deleted on chromosome 10) during regeneration of peripheral neurons in adult Sprague Dawley rats. PTEN inhibits phosphoinositide 3-kinase (PI3-K)/Akt signaling, a common and central outgrowth and survival pathway downstream of neuronal growth factors. While PI3-K and Akt outgrowth signals were expressed and activated within adult peripheral neurons during regeneration, PTEN was similarly expressed and poised to inhibit their support. PTEN was expressed in neuron perikaryal cytoplasm, nuclei, regenerating axons, and Schwann cells. Adult sensory neurons in vitro responded to both graded pharmacological inhibition of PTEN and its mRNA knockdown using siRNA. Both approaches were associated with robust rises in the plasticity of neurite outgrowth that were independent of the mTOR (mammalian target of rapamycin) pathway. Importantly, this accelerated outgrowth was in addition to the increased outgrowth generated in neurons that had undergone a preconditioning lesion. Moreover, following severe nerve transection injuries, local pharmacological inhibition of PTEN or siRNA knockdown of PTEN at the injury site accelerated axon outgrowth in vivo. The findings indicated a remarkable impact on peripheral neuron plasticity through PTEN inhibition, even within a complex regenerative milieu. Overall, these findings identify a novel route to propagate intrinsic regeneration pathways within axons to benefit nerve repair.

Figure 1.

PTEN and Akt mRNA and protein are present in the adult DRG and sciatic nerve. A, qRT-PCR for PTEN in normal and 3 d injured DRG and sciatic nerve. B, qRT-PCR for Akt in normal and 3 d injured DRG and sciatic nerve. C, Western blot analysis of pPTEN/total PTEN in normal and 3 d injured DRG and sciatic nerve. D, Western blot analysis of pAkt/total Akt in normal and 3 d injured DRG and sciatic nerve. Asterisks indicate significant differences (t test, p < 0.05, n = 3–6). Error bars represent SEM.

Figure 2.

PTEN is intensely expressed in small IB4-positive DRG neurons and in regenerating axon profiles. A–C, Transverse section of 3 d injured DRG showing NF200 (A) and PTEN (B) staining. The merge image (C) shows intense PTEN expression in the small neurons. D–F, Transverse section of 3 d injured DRG showing CGRP (D) and PTEN (E) staining. The merge image (F) shows that intense PTEN expression is not colocalized with CGRP neurons (arrowhead). G–I, Transverse section of 3 d injured DRG showing IB4 (G) and PTEN (H) staining. The merge image (I) shows that intense PTEN labeling colocalizes with IB4 neurons (arrow). J–L, Longitudinal section of normal sciatic nerve showing NF200 (J) and PTEN (K) staining and merge (L). M–O, NF200 (M), PTEN (N), and merge (O) in the regenerating sciatic nerve tip. Long arrows represent regenerating axons, arrowheads identify Schwann cells. Scale bars are 50 μm.

Figure 3.

pPTEN expression in DRG nuclei decreases after sciatic nerve injury. A–C, Transverse section of normal DRG showing NF200 (A) and pPTEN (B) staining and merge (C). D–F, Transverse section of 3 d injured DRG showing NF200 (D) and pPTEN (E) staining and merge (F). G, H, pPTEN staining merged with DAPI in sham (G) and injured (H) DRG. Panels to the right show magnified nuclear pPTEN expression (arrows). Scale bars are 50 μm.

Figure 4.

Akt is present in the DRG and sciatic nerve and pGSK3β is decreased at the injury site. A–C, Transverse section of normal DRG showing NF200 (A) and Akt (B) staining and merge (C). D–F, Transverse section of 3 d injured DRG showing NF200 (D) and Akt (E) staining and merge (F). G–L, Longitudinal sections of NF200 (G, J) and Akt (H, K) staining and merge (I, L) in normal (G–I) and 3 d injured (J–L) sciatic nerve. M, Western blot analysis of pGSK3β (Ser 9, 46 kDa)/GSK3β (52 kDa) in 3 d injured sciatic nerve. P, Proximal; D, distal; white asterisk indicates transection site.

Figure 5.

Inhibition of PTEN phosphatase activity increases neurite outgrowth in vitro in both sham-injured and preconditionally injured neurons. A–C, PTEN is expressed in cultured DRG neurons. NF200 (A) and PTEN (B) staining in sham-injured DRG neurons. D–F, NF200 (D) and PTEN (E) staining in injured DRG neurons. Merged images (C, F) show DAPI staining (blue). Arrow shows neuron expression, arrowhead shows glial expression. G–J, NF200 staining of sham DRG neurons with 0 nm (G), 10 nm (H), 50 nm (I), and 200 nm (J) of the PTEN phosphatase inhibitor, bpV(pic). K, Neurite outgrowth summary from sham neurons. Outgrowth was normalized to control cultures for each sample. L–O, NF200 staining of injured cultured DRG neurons with 0 nm (L), 10 nm (M), 50 nm (N), and 200 nm (O) of bpV(pic). P, Neurite outgrowth summary from injured neurons. Outgrowth was normalized to control cultures for each sample. Asterisks indicate significant differences (one-way ANOVA with Tukey post hoc analysis, p < 0.05, n = 3 separate cultures). Scale bars are 50 μm. Error bars represent SEM.

Figure 6.

Increased doses of bpV(pic) results in a graded rise in expression of pAkt in vitro. Western blot of pAkt (Ser 473, 60 kDa) and Akt in cultured 3 d injured and sham-injured neurons with increasing doses (10, 50, 200 nm) of PTEN inhibitor.

Figure 7.

Application of rapamycin (50 nm) alone or in combination with bpV(pic) does not alter neurite outgrowth in sham-injured or preconditionally injured cultured neurons. mTOR activity is assessed with pS6K staining. A–C, pS6K (A) and NF200 (B) staining and merge (C) in injured neurons exposed to carrier alone. D–F, pS6K (D) and NF200 (E) staining and merge (F) in injured neurons exposed to rapamycin alone. G–I, pS6K (G) and NF200 (H) staining and merge (I) in injured neurons with bpV(pic) alone. J–L, pS6K (J) and NF200 (K) staining and merge (L) in injured neurons with bpV(pic) and rapamycin. M, N, Summary plots of neurite outgrowth in sham (M) and injured (N) cultures. Outgrowth was normalized to control cultures for each sample. Asterisks indicate significant differences (one-way ANOVA with Tukey post hoc analysis, *p < 0.05, **p < 0.01, n = 3). Scale bars are 50 μm. Error bars represent SEM.

Figure 8.

PTEN siRNA knocks down PTEN mRNA and increases neurite outgrowth in vitro. A, PTEN siRNA localizes to DRG sensory neuron nucleus, soma, and neurites. B, qRT-PCR from cultured neurons with PTEN siRNA (p < 0.001, n = 8). C–E, NF200 staining of sham-injured neurons without siRNA (C) and with scrambled siRNA (D) and PTEN siRNA (E). F–H, NF200 staining of injured neurons without siRNA (F) and with scrambled siRNA (G) and PTEN siRNA (H). I, J, Summary plots of neurite outgrowth for sham-injured (I) and injured (J) cultured neurons. Asterisks indicate significant differences (one-way ANOVA with Tukey post hoc analysis, p < 0.0001, n = 4 separate cultures). Scale bar is 50 μm. Error bars represent SEM.

Figure 9.

In vivo inhibition of PTEN at both the phosphatase and mRNA levels increases the number and length of regenerating profiles from transected peripheral nerves. A, NF200 staining of a regenerative bridge treated with saline. B, NF200 staining of a regenerative bridge treated with bpV(pic) (200 nm). C, Summary of the regenerating axon profile count with bpV(pic). D, Summary of the regenerating axon profile count with PTEN siRNA. E, NF200 staining of a regenerative bridge treated with scrambled siRNA. F, NF200 staining of a regenerative bridge treated with PTEN siRNA. Asterisks indicate significant differences (t test, p < 0.05, n = 3). Error bars represent SEM.