Abstract
Objective:
Antagonism of the muscarinic acetylcholine type 1 receptor (M1R) 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 M1R 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 M1R antagonists pirenzepine (PZ) or muscarinic toxin 7 (MT7). Ca2+ transients, mitochondrial respiration, AMP-activated protein kinase (AMPK) expression, and mitochondrial inner membrane potential were analyzed. The effect of M1R activation or blockade on TRPM3 activity mediated by phosphatidylinositol 4,5-bisphosphate (PIP2) was studied. Metabolic profiling of DRG neurons and human neuroblastoma SH-SY5Y cells was conducted.
Results:
M1R antagonism induced by PZ or MT7 increased Ca2+ influx in DRG neurons and was inhibited by TRPM3 antagonists or in the absence of extracellular Ca2+. TRPM3 agonists elevated Ca2+ levels, augmented mitochondrial respiration, AMPK activation and neurite outgrowth. M1R antagonism stimulated TRPM3 channel activity through inhibition of PIP2 hydrolysis to activate Ca2+/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.
Keywords:
Axonal regeneration; Bioenergetics; Diabetic neuropathy; GPCR; Metabolomics; Mitochondria; Neuronal metabolism.