Acetylcholinesterase and Butyrylcholinesterase anchoring in murine ocular compartments, role of Colq and PRiMA

Abstract

Purpose : Acetylcholinesterase (AChE) is the key hydrolytic enzyme of acetylcholine (Ach) in cholinergic synapses. Butyrylcholinesterase (BChE) is a non-specific cholinesterase prominently synthesized in the liver and mainly found in the blood plasma. BChE is also expressed in the nervous system. Cholinesterases must be matured and anchored to plasma membranes in neural and non-neural cells. The purpose of our experiments was to determine what anchoring molecules PRiMA and/or ColQ are involved in the process of maturation and membrane anchoring of each cholinesterase in each ocular compartment.

Methods : We used ColQ KO, PRiMA KO, AChEdelE5+6 5, AChE KO, BChE KO and control mice. We visualized the enzymatic activity of AChE or BChE in adult mouse ocular tissue sections by the colorimetric method of Tsuji. AChE and BChE proteins were localized using specific antibodies on ocular tissue sections. We dissected the different ocular compartments and solubilized their respective proteins. Western Blot analysis on the protein extracts were used to quantify AChE and BChE proteins. Sucrose gradients were used to separate the molecular forms i.e. AChE or BChE tetramers associated with ColQ or PRiMA in each ocular compartment.

Results : AChE enzymatic activity was localized in all mouse ocular compartments. BChE activity was barely seen in the retina, whereas it was significantly detected in the other ocular compartments. AChE activity was absent in the retina of PRiMA KO and AChEdelE5+6 5 mice, whereas it was normally detected in in the retina of ColQ KO mice. The results obtained by the Tsuji method were supported by immunohistochemistry and western blots analysis. AChE and BChE were anchored by PRiMA and ColQ in all ocular compartments, except in the retina. Retinal AChE is only found as a tetramer organized by PRiMA. Despite the absence of AChE in the retina of PRiMA KO mice, Its morphology appears normal.

Conclusions : We report that AChE is matured and anchored solely by PRiMA in the retina, whereas in the other ocular structures AChE is anchored by PRiMA and ColQ. BChE is quite abundant in most ocular compartments, except in the retina. BChE molecules form complexes with PRiMA or are monomers and possible precursors of the enzyme. If the function of BChE is also the degradation of ACh, this may suggest that ACh may participate to unsuspected signaling in ocular structures.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.