Acetylcholinesterase inhibitors enhance its folding and assembly in the endoplasmic reticulum

Abstract

Acetylcholinesterase (AChE) is responsible for terminating synaptic transmission by the hydrolysis of acetylcholine (ACh) in the synaptic cleft. AChE is synthesized at a constant rate in the rough endoplasmic reticulum with only an estimated 20% of all synthesized enzyme assembling into catalytically active oligomeric forms. The overwhelming majority of the enzyme undergoes degradation through the ERAD. It is this large inactive pool we seek to understand through in‐vitro analysis using human embryonic kidney cells expressing mouse acetylcholinesterase (HEKmAChE). In particular we wished to determine whether the inhibitors could rescue inactive AChE protein into catalytically active enzyme. Galantamine and Donepezil, two commonly used therapeutics for Alzheimer's disease (AD), act as active site AChE inhibitors with the purpose of allowing ACh to persist for prolonged synaptic transmission. To understand the effects of Galantamine on inactive AChE, HEKmAChE cultures were first treated with diisopropylfluorophosphate (DFP), an irreversible and membrane permeable AChE inhibitor, and then allowed to synthesize new AChE in the presence of Galantamine during a 60 minute recovery period. Preliminary results indicated a significant increase in active AChE when exposed to Galantamine in comparison to control (defined medium alone). To focus on the mechanism by which Galantamine enhances AChE folding and assembly, HEKmAChE cultures were incubated with DFP and subsequently treated with Puromycin alone or in combination with either Galantamine or Donepezil. Using Ellman's assay for AChE we observed a significant increase in catalytically active AChE in the presence of Donepezil or Galantamine. These results suggest current therapeutic treatments may be acting as molecular chaperones during the protein folding process, enabling otherwise inactive AChE bound for degradation to become catalytically active. This research was supported by the Florida Department of Health, Biomedical research program, grant 7AZ04.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.