Muscarinic Acetylcholine Type 1 Receptor Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons: A Phenotype Rescued by Antagonist Treatment
Authors: Sabbir Mohammad Golam , Calcutt Nigel A. , Fernyhough Paul 
Published: June 2021

ObjectiveThe muscarinic acetylcholine type 1 receptor (M1R) is a metabotropic G protein coupled receptor. Knockout of M1R or exposure to selective or specific receptor antagonists elevates neurite outgrowth in adult sensory neurons and blockade of associated cholinergic pathways is therapeutic in diverse models of peripheral neuropathy. We tested the hypothesis that endogenous M1R activation constrained neurite outgrowth via a negative impact on the cytoskeleton and subsequent mitochondrial trafficking and that this could be rescued by antagonist treatment.MethodologyWe overexpressed M1R and M1R DREADD (designer receptors exclusively activated by designer drugs) mutant in primary cultures of adult rat dorsal root ganglion (DRG) neurons and in tumor cell lines and studied the physiological and molecular consequences related to regulation of cytoskeletal/mitochondrial dynamics and neurite outgrowth. The M1R‐DREADD mutant contained two mutations of conserved orthosteric site residues that cause minimal responsiveness to ACh. In addition, we used CRISPR/Cas9 based Gα12/13 knockout HEK293 cells and siRNA mediated knockdown of Gα13 in cultured DRG neurons to study M1R Gα13 protein mediated tubulin cytoskeletal dynamics. No M1R ligand was added to the culture media.ResultsOverexpression of M1R by adult sensory neurons caused dissolution of the polymerized tubulin cytoskeleton, but not the actin network and significantly reduced neurite outgrowth. Conversely, over‐expression of a M1R‐DREADD mutant increased neurite outgrowth. These data suggest that endogenously released M1R ligand interacts with the M1R to suppress neurite outgrowth. M1R‐dependent disruption of the cytoskeleton significantly diminished mitochondrial abundance and trafficking in the distal neurites. Treatment with the specific M1R antagonist – muscarinic toxin 7 (MT7, 100 nM) or the selective M1R antagonist pirenzepine (1 μM) rescued cytoskeletal abnormalities caused by M1R overexpression by sequestering G‐proteins (Gα12/13) to the receptor. In addition, Gα12/13 null HEK293 cells exhibited increased polymerized tubulin and knockdown of Gα13 protected from tubulin cytoskeletal destabilization and restored growth in neurons overexpressing the M1R.ConclusionsOur study indicates that constitutively high expression of M1R destabilizes cytoskeletal dynamics through activation of Gα13 protein that increased tubulin de‐polymerization, impeded mitochondrial transport and reduced neurite outgrowth. This suggests a novel mechanism of M1R control of G protein‐dependent (Gα13) modulation of the tubulin cytoskeleton, mitochondrial trafficking and neurite outgrowth in axons of adult sensory neurons. Manipulation of this novel pathway could be used to treat dying‐back neuropathies since anti‐muscarinic drugs are currently utilized for other clinical conditions.Support or Funding InformationSupported by CIHR grant # MOP‐130282 (PF) and NIH grant NS081082 (NAC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.