Li Li

Project title: Carbachol Prevents Oxidative Stress-mediated Apoptosis Induced by Domoic Acid in Cerebellar Granule Cells

Degree: MS (Thesis) | Program: Environmental Toxicology (Tox) | Project type: Thesis/Dissertation
Completed in: 2007 | Faculty advisor: Lucio G. Costa


In cerebellar granule neurons (CGNs) domoic acid (DomA) induced neuronal cell death, either by apoptosis or by necrosis, depending on its concentration. The apoptotic cell death was mediated by the p38 and JNK MAP kinase pathways which were activated by oxidative stress following exposure to low concentrations of DomA (≤ 0.1 µM). It is generally accepted that the cholinergic system plays an important role in cognitive functions, especially memory and learning which are compromised by DomA. It was interesting for us to examine whether the cholinergic agonist carbachol could protect from DomA-induced apoptosis. Simultaneous exposure to carbachol and DomA revealed a concentration-dependent inhibition of apoptosis, which was abolished by atropine (a muscarinic receptor antagonist) but not by mecamylamine (a nicotinic receptor antagonist). Antagonists of M1 and M2 muscarinic receptors (mAchR) (pirenzepine and methoctramine, respectively) failed to reverse the effects of carbachol, while the M3 antagonist 4-diphenylacetoxyl-N- methylpiperidine methiodide completely blocked the survival-promoting effects of carbachol. The protective effect of carbachol was reversed by specific inhibitors of pro-survival intracellular signaling proteins, including an inhibitor of phosphatidylinositol 3-kinase (LY294002), of Akt (Akt inhibitor III) and of MAPK (UO126). Western blot analysis also revealed a rapid activation of p42/44 MAPK and p-Akt following exposure to carbachol. These results suggest that the mAchR survival effect is mediated by a pathway that leads to inhibition of DomA-induced apoptosis by activating the PI3K/Akt and Erk1/2 signaling cascades. Additionally, our results also showed that carbachol augmented GCL activity and GSH level, which represents strong defense against DomA-induced oxidative stress.