Targeting adenosine monophosphate-activated protein kinase (AMPK) in preclinical models reveals a potential mechanism for the treatment of neuropathic pain

Abstract

Neuropathic pain is a debilitating clinical condition with few efficacious treatments, warranting development of novel therapeutics. We hypothesized that dysregulated translation regulation pathways may underlie neuropathic pain. Peripheral nerve injury induced reorganization of translation machinery in the peripheral nervous system of rats and mice, including enhanced mTOR and ERK activity, increased phosphorylation of mTOR and ERK downstream targets, augmented eIF4F complex formation and enhanced nascent protein synthesis. The AMP activated protein kinase (AMPK) activators, metformin and A769662, inhibited translation regulation signaling pathways, eIF4F complex formation, nascent protein synthesis in injured nerves and sodium channel-dependent excitability of sensory neurons resulting in a resolution of neuropathic allodynia. Therefore, injury-induced dysregulation of translation control underlies pathology leading to neuropathic pain and reveals AMPK as a novel therapeutic target for the potential treatment of neuropathic pain.

Figure 1

PNI induces a fundamental reorganization of translation signaling and machinery in the injured PNS. A) PNI in rats causes upregulation of translation machinery and signaling pathways associated with protein translation. These changes are observed in the injured sciatic nerve and in both injured (L5 and L6) and uninjured (L4) DRGs. B) PNI in mice increases proteins that enhance translation and associated signaling pathways. In both panels, up arrows indicate an increase while down arrows indicate a decrease; numbers indicate fold change in ipilateral vs. contralateral (right) and phospho-protein vs. total protein (left). C) Immunohistochemical colocalization of p-mTOR with sensory neuronal markers (peripherin + N52) show that activated mTOR localizes to axons of injured sciatic nerve from rats with SNL.

Figure 2

PNI enhances cap-dependent protein translation in the injured sciatic nerve. A) Western blot of eIF4A, 4EBP and eIF4E from sciatic nerve co-precipitated using 7-methyl-GTP conjugated beads. B) PNI induces only 50% increase in 4EBP (negative regulator of translation) association with cap-binding protein eIF4E, C) while injury induces a 1200% increase in the association of eIF4A (a component of eIF4F complex) with eIF4E. D) PNI induces a 750% increase in the ratio of eIF4A to 4EBP associated with eIF4E bound to 7-methyl-GTP conjugated beads. PNI increases nascent protein synthesis in injured sciatic nerve. E) Western blot of AHA incorporated into nascently synthesized proteins. F) PNI induces a 50% increase in the incorporation of AHA into nascently synthesized proteins. All samples taken 17 days post SNL from rats with n = 6 per condition. *p < 0.05.

Figure 3

AMPK activators reduce PNI-induced neuropathic allodynia. Daily intraperitoneal injections of metformin in mice starting 2 (A) or 7 (B) weeks post PNI leads to a reversal of mechanical allodynia. C) A769662 daily treatment also reversed PNI-induced allodynia when started 2 weeks post PNI. D) Treatment of SNL rats (2 weeks post surgery) with metformin also significantly reduces neuropathic allodynia. N = 6 per group. **p < 0.01 and ***p < 0.001.

Figure 4

Treatment with AMPK activators suppresses translation regulation signaling. A) Treatment of mouse sensory neurons cultured in the presence of NGF (50 ng/ml) with metformin (2 and 20 mM) for 1 hour induces a dose-dependent increase in the phosphorylation of AMPK. Metformin treatment abrogates the phosphorylation of mTOR, 4EBP and rS6 in a dose-dependent manner. Metformin does not suppress the ERK-eIF4E pathway. B) AMPK allosteric activator A769662 suppresses translation regulation signaling. Treatment of mouse sensory neurons with A769662 (50 and 500 μM) results in a dose dependent suppression of phosphorylation of ERK, eIF4E, mTOR, 4EBP, AKT and rS6. C) Treatment of mouse sensory neurons with AICAR (0.5 and 2 mM) for 1 hour results in a dose dependent activation of AMPK. Moreover, AICAR dose-dependently suppresses the phosphorylation of ERK, eIF4E, AKT, TSC2, 4EBP and rS6. N = 6 per group. *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 5

AMPK activators suppress translation in sensory neurons and the injured PNS. Treatment of mouse sensory neurons cultured in the presence of NGF (50 ng/ml) with A) Metformin (2 and 20 mM) or B) A769662 (50 and 500 μM) results in decreased binding of eIF4G and increased 4EBP binding to the m7GTP-cojugated sepharose beads in a dose-dependent manner consistent with a decrease in eIF4F complex formation. C) Metformin treatment of rats with SNL restores nascent protein synthesis in injured nerves to levels observed in uninjured nerves. N = 6 per group. *p < 0.05, ** p < 0.01.

Figure 6

AMPK activators suppress hyperexcitability of sensory neurons. A) Patch clamp analysis of mouse primary sensory neurons cultured in the presence of NGF (50 ng/ml, n = 13) demonstrate an increase in the B) number of ramp currentevoked action potentials vs. vehicle (n = 14) and C) reduced latency to first action potential in response to ramp currents. Metformin (1 hr, n = 14) and A769662 (1 hr, n =12) reverse these parameters. A769662 effect persists after washout (n = 8). Colored stars denote significant effects compared to the NGF + Vehicle group. *p < 0.05, **p < 0.01 and ***p < 0.001.