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Protective role of carbachol in domoic acid-induced apoptosis in cerebellar granule neurons
Toxicology, MS
Preceptor: Lucio Costa, PhD
In cerebellar granule neurons (CGN) domoic acid (DomA) induces 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 mM).
It is generally accepted that the cholinergic system plays an important role in cognitive functions, especially memory and learning which are compromised by DomA. Therefore is interesting for us to examine whether the cholinergic agonist carbachol could protect from DomA-induced apoptosis. Simultaneous exposure to different doses of carbachol with DomA revealed a dose-dependent prevention, which was abolished by atropine (a muscarinic receptor antagonist) but not by mecamylamine (a nicotinic receptor antagonist). The m1 and m2 mAchR antagonists (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 phosphatidylinositol 3-kinase (LY294002), Akt (Akt inhibitor III) and a MAPK inhibitor (UO126).
To determine whether the protection induced by the carbachol may involve extracellular signal-regulated kinases (ERK), we have investigated changes in the activity of p42/44 MAPK (ERK1, ERK2). Western blot analysis revealed a rapid activation of p42/44 MAPK and p-Akt following exposure to carbachol.
Members of the Bcl-2 family proteins (such as BimEL), which mediate apoptosis generated through the mitochondria-initiated, intrinsic cell death pathway, are phosphorylated by JNK and p38 MAP kinases. Exposure to 0.1 mM of DomA caused phosphorylation at Ser-65 of BimEL. Survival factors inhibit the apoptotic activity of Bad (another member of Bcl-2 family) by activating intracellular signaling pathways resulting in the phosphorylation of Bad at Ser112 and Ser136. Carbachol treatment induced phosphorylation of Bad at Ser136.
These results therefore suggest that the muscarinic survival effect is mediated by a pathway that leads to inhibition of DomA-induced apoptosis by activating the MAPK/ERK- and PI 3-kinase signaling cascades.
(Supported by ES012762/NSF-OCE-0434087, ES 07033, and ES 04696).
