Alzheimer’s disease (AD) is characterized by a decrease in the enzymatic activity of the enzyme acetylcholinesterase (AChE). variants in AD patients compared to AG-490 controls. Similar increases were detected by Western blot using an antibody raised against the specific N-terminal domain, exclusive of alternative N-extended variants of AChE (N-AChE). In accordance with a subset of AChE-R monomers that display amphiphilic properties which are upregulated in the AD brain, we demonstrate that the increase of N-AChE species is due, at least in part, to N-AChE-R variants. In conclusion, we demonstrate selective alterations in specific AChE variants in AD cortex, with no correlation in enzymatic activity. Therefore, differential expression of AChE variants in AD may reflect changes in the pathophysiological role of AChE, independent of cholinergic impairment or its role in degrading acetylcholine. at 4 C for 1 h, and then the supernatants were collected and frozen AG-490 at -80 C until assayed. Pfkp Cell Culture SH-SY5Y neuroblastoma cells were grown in AG-490 D-MEM/F12+GlutaMAX?-I (Dulbecco’s Modified Eagle medium; GIBCO Invitrogen Corporation) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin solution (P/S; 100 U/mL) (Gibco). Cells were transfected using Lipofectamine? 2000 (Invitrogen?, Life technologies Paisley, UK) with 4 g AG-490 of AChE-T or AChE-R cDNAs under the cytomegalovirus (CMV) promoter-enhancer (a generous gift from Prof Hermona Soreq, Institute of Life Science, Hebrew University, Jerusalem, Israel). The PCI empty vector (Promega, Madison, USA) served as negative control. The cells were collected for analysis 48 hours after the transfection. AChE enzymatic activity and total protein determination AChE activity was determined by a modified microassay version of the colorimetric Ellman’s method [32]. AChE was assayed with 1 mM acetylthiocholine and 50 M tetraisopropyl pyrophosphoramide (Iso OMPA), a specific inhibitor of butyrylcholinesterase, a second cholinesterase that co-exists with AChE in brain. One milliunit (mU) of AChE activity was defined as the number of nmoles of acetylthiocholine hydrolyzed per min at 22 C. Protein concentrations were determined using the bicinchoninic acid method, with bovine serum albumin as standard (Pierce, Rockford, IL). Sedimentation analysis Molecular forms of AChE were separated according to their sedimentation coefficients by ultracentrifugation on 5-20% (w/v) sucrose gradients containing 0.5% (w/v) Triton X-100 [32]. Ultracentrifugation was performed at 150,000 g in a SW 41Ti Beckman rotor for 18 hr, at 4 C. Approximately 40 fractions were collected from the bottom of each tube and assayed for AChE activity to identify individual AChE forms (G4 = tetramers; G1 = monomers) by comparison with the position of molecular weight markers, catalase (11.4S) and alkaline phosphatase (6.1S). We defined the ratio of AChE forms G4/G1 as the proportion of G4 molecules versus the light form, G1. The sucrose fractions containing the light G1 peaks were separately pooled, dialyzed against Tris buffer, and concentrated by ultrafiltration (Amicon Ultra 10,000 MWCO, Millipore Corporation, Bedford, MA, USA). AG-490 Monomers of AChE were then characterized by a phenyl-agarose interaction and Western blot assays. Western blotting assays AChE subunits and PRiMA-1 levels were detected by immunoblotting. 50 micrograms of protein from brain extracts (equal amount of protein in each lane) were resolved by electrophoresis on 10% SDS-polyacrylamide slab gels. Samples were denatured at 98 C for 7 min. Following electrophoresis, proteins were blotted onto nitrocellulose membranes (Schleicher & Schuell Bioscience, GmbH), blocked with 5% bovine serum albumin and probed with the following primary antibodies: anti-PRiMA-1 antibody (C16, Santa Cruz Biotech, Santa Cruz, CA, USA), anti AChE-T variants antibody (Ab31276, Abcam, Cambridge, UK), anti AChE-R antibody raised to the unique C-terminus of human AChE-R, an anti N-AChE raised to the extended N-terminus of N-AChE variants (both were a generous gift from Prof Hermona Soreq). A rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Abcam).