Quantitative Measurement of AChE Activity

Quantitative measurements of AChE enzymatic activity were made using a modified Ellman method (Ellman et al., 1961 (link); Rosenfeld et al., 2001 (link)). Stock solutions were acetylthiocholine iodide, used as the enzymatic substrate (ATH; 1.7 mg/ml in PBS, Sigma-Aldrich), 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB; 0.8 mg/ml in PBS, Sigma-Aldrich). Briefly, brains were rapidly dissected from either WT or J20 mice. Neocortex was isolated, weighed, and then homogenized using a Pellet Pestle (Sigma, Z 359971) in nine volumes of 0.1 M sodium phosphate buffer (pH 7.4; Patel et al., 2014 (link)). Five microliters of brain homogenate was aliquoted into each well of a 96-well plate, volume made up to 200 µl with PBS. DTNB (50 µl from stock) was added, followed by 50 µl of ATH substrate from stock. Measurement of absorption at 450 nm began immediately (<2 h from dissection) and was measured every 5 min for up to 30 min using a MRX microplate reader (Dynex Technologies). Thiocholine production in the test wells was expressed in units of nmol/min, calibrated with reference to the absorbance change over a range of concentrations giving a linear response using glutathione as the DTNB reactant (Eyer et al., 2003 (link)). Neostigmine (10 µM, Sigma-Aldrich) was used to completely inhibit AChE activity and establish that there was no baseline drift during the measurements.