Muscarinic acetylcholine type 1 receptor antagonism activates TRPM3 to augment mitochondrial function and drive axonal repair in adult sensory neurons.

OBJECTIVE

Antagonism of the muscarinic acetylcholine type 1 receptor (MR) promotes sensory axon repair and is protective in peripheral neuropathy, however, the mechanism remains elusive. We investigated the role of the heat-sensing transient receptor potential melastatin-3 (TRPM3) cation channel in MR antagonism-mediated nerve regeneration and explored the potential of TRPM3 activation to facilitate axonal plasticity.

METHODS

Dorsal root ganglion (DRG) neurons from adult control or diabetic rats were cultured and treated with TRPM3 agonists (CIM0216, pregnenolone sulfate) and MR antagonists pirenzepine (PZ) or muscarinic toxin 7 (MT7). Ca transients, mitochondrial respiration, AMP-activated protein kinase (AMPK) expression, and mitochondrial inner membrane potential were analyzed. The effect of MR activation or blockade on TRPM3 activity mediated by phosphatidylinositol 4,5-bisphosphate (PIP) was studied. Metabolic profiling of DRG neurons and human neuroblastoma SH-SY5Y cells was conducted.

RESULTS

MR antagonism induced by PZ or MT7 increased Ca influx in DRG neurons and was inhibited by TRPM3 antagonists or in the absence of extracellular Ca. TRPM3 agonists elevated Ca levels, augmented mitochondrial respiration, AMPK activation and neurite outgrowth. MR antagonism stimulated TRPM3 channel activity through inhibition of PIP hydrolysis to activate Ca/calmodulin-dependent protein kinase kinase β (CaMKKβ)/AMPK, leading to augmented mitochondrial function and neuronal metabolism. DRG neurons with AAV-mediated shRNA knockdown of TRPM3 exhibited suppressed antimuscarinic drug-induced neurite outgrowth. TRPM3 agonists increased glycolysis and TCA cycle metabolites, indicating enhanced metabolism in DRG neurons and SH-SY5Y cells.

CONCLUSIONS

Activation of the TRPM3/CaMKKβ/AMPK pathway promoted collateral sprouting of sensory axons, positioning TRPM3 as a promising therapeutic target for peripheral neuropathy.