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This article belongs to the Special Issue Molecular Advances in Alzheimer's Disease., In Alzheimer’s disease (AD), the reduction in acetylcholinesterase (AChE) enzymatic activity is not paralleled with changes in its protein levels, suggesting the presence of a considerable enzymatically inactive pool in the brain. In the present study, we validated previous findings, and, since inactive forms could result from post-translational modifications, we analyzed the glycosylation of AChE by lectin binding in brain samples from sporadic and familial AD (sAD and fAD). Most of the enzymatically active AChE was bound to lectins Canavalia ensiformis (Con A) and Lens culinaris agglutinin (LCA) that recognize terminal mannoses, whereas Western blot assays showed a very low percentage of AChE protein being recognized by the lectin. This indicates that active and inactive forms of AChE vary in their glycosylation pattern, particularly in the presence of terminal mannoses in active ones. Moreover, sAD subjects showed reduced binding to terminal mannoses compared to non-demented controls, while, for fAD patients that carry mutations in the PSEN1 gene, the binding was higher. The role of presenilin-1 (PS1) in modulating AChE glycosylation was then studied in a cellular model that overexpresses PS1 (CHO-PS1). In CHO-PS1 cells, binding to LCA indicates that AChE displays more terminal mannoses in oligosaccharides with a fucosylated core. Immunocytochemical assays also demonstrated increased presence of AChE in the trans-Golgi. Moreover, AChE enzymatic activity was higher in plasmatic membrane of CHO-PS1 cells. Thus, our results indicate that PS1 modulates trafficking and maturation of AChE in Golgi regions favoring the presence of active forms in the membrane., This study was funded by Instituto de Salud Carlos III (ISCIII), Fondo de Investigaciones Sanitaria (grants PI17/00261 to M.S.G.-A.), co-financed by Fondo Europeo de Desarrollo Regional and through CIBERNED, ISCIII, Spain. M.-Á.C.-G. was supported by a GVA-Predoctoral fellowship (grant ACIF2019/242) from Generalitat Valenciana, Spain.

Early View: Online Version of Record before inclusion in an issue., In Alzheimer's disease (AD), the enzyme acetylcholinesterase (AChE) co‐localizes with hyperphosphorylated tau (P‐tau) within neurofibrillary tangles. Having demonstrated that AChE expression is increased in the transgenic mouse model of tau Tg‐VLW, here we examined whether modulating phosphorylated tau levels by over‐expressing wild‐type human tau and glycogen synthase kinase‐3β (GSK3β) influences AChE expression. In SH‐SY5Y neuroblastoma cells expressing higher levels of P‐tau, AChE activity and protein increased by (20% ± 2%) and (440% ± 150%), respectively. Western blots and qPCR assays showed that this increment mostly corresponded to the cholinergic ACHE‐T variant, for which the protein and transcript levels increased ~60% and ~23%, respectively. Moreover, in SH‐SY5Y cells differentiated into neurons by exposure to retinoic acid (10 µM), over‐expression of GSK3β and tau provokes an imbalance in cholinergic activity with a decrease in the neurotransmitter acetylcholine in the cell (45 ± 10%). Finally, we obtained cerebrospinal fluid (CSF) from AD patients enrolled on a clinical trial of tideglusib, an irreversible GSK3β inhibitor. In CSF of patients that received a placebo, there was an increase in AChE activity (35 ± 16%) respect to basal levels, probably because of their treatment with AChE inhibitors. However, this increase was not observed in tideglusib‐treated patients. Moreover, CSF levels of P‐tau at the beginning measured by commercially ELISA kits correlated with AChE activity. In conclusion, this study shows that P‐tau can modulate AChE expression and it suggests that AChE may possibly increase in the initial phases of AD., MACG is supported by a GVA-Predoctoral fellowship from Generalitat Valenciana, Spain. This study was funded by Instituto de Salud Carlos III (ISCIII), Fondo de Investigaciones Sanitaria (grants CP11/00067 and PI17/00261 to MSGA); co-financed by Fondo Europeo de Desarrollo Regional and through CIBERNED, ISCIII, Spain.

Stability of the cerebrospinal fluid (CSF) composition under different pre-analytical conditions is relevant for the diagnostic potential of biomarkers. Our aim was to examine the pre-analytical stability of promising CSF biomarkers that are currently evaluated for their discriminative use in various neurological diseases. Pooled CSF was aliquoted and experimentally exposed to delayed storage: 0, 1, 2, 4, 24, 72, or 168 h at 4 °C or room temperature (RT), or 1–4 months at −20 °C; or up to 7 freeze/thaw (f/t) cycles, before final storage at −80 °C. Eleven CSF biomarkers were screened using immunoassays, liquid chromatography, or enzymatic methods. Levels of neurogranin (truncP75), chitinase-3-like protein (YKL-40), beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), acetylcholinesterase (AChE) enzymatic activity, theobromine, secreted protein acidic and rich in cysteine-like 1 (SPARCL-1) and homovanillic acid (HVA) levels were not affected by the applied storage conditions. 3-Methoxy-4-hydroxyphenylglycol (MHPG) levels linearly and strongly decreased after 4 h at RT (−10%) or 24 h at 4 °C (−27%), and with 6% after every f/t cycle. 5-Methyltetrahydrofolate (5-MTHF) (−29% after 1 week at RT) and 5-hydroxyindoleacetic acid levels (5-HIAA) (−16% after 1 week at RT) were reduced and 3,4-dihydroxyphenylacetic acid (DOPAC) levels (+22% after 1 week at RT) increased, but only after >24 h at RT. Ten out of eleven potential CSF novel biomarkers showed very limited change under common storage and f/t conditions, suggesting that these CSF biomarkers can be trustfully tested under the pre-analytical conditions present across different cohorts., This research was financially supported by Biobanking and BioMolecular resources Research Infrastructure the Netherlands (BBMRI-NL), a research infrastructure financed by the Dutch Government (NWO 184.021.007) under project CP2013-68. This study commenced within the BIOMARKAPD program of the EU Joint Programme – Neurodegenerative Disease Research (JPND). Project supported by the European Regional Development Fund (ERDF) through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia, I.P., under the projects POCI-01-0145-FEDER-007440, POCI-01-0145-FEDER-016428, and POCI-01-0145-FEDER-016795, and by The National Mass Spectrometry Network (RNEM) under the contract POCI-01-0145-FEDER-402-022125.

Acetylcholinesterase (AChE) is a key enzyme in the cholinergic nervous system and is one of the most studied proteins in the field of Alzheimer's disease (AD). Moreover, alternative functions of AChE unrelated with the hydrolysis of acetylcholine are suspected. Until now, the majority of investigations on AChE in AD pathology have been focused on the determination of its enzymatic activity level, which is depleted in the AD brain. Despite this overall decrease, AChE activity increases at the vicinity of the two hallmarks of AD, the amyloid plaques and the neurofibrillary tangles (NFT). In fact, AChE may directly interact with Aβ in a manner that increases the deposition of Aβ to form plaques. In the context of protein-protein interactions, we have recently reported that AChE can interact with presenilin-1, the catalytic component of γ-secretase, influencing its expression level and also its activity. However, the alteration of AChE protein in the AD brain has not been determined. Here, we demonstrated by Western blotting and immunohistochemistry that a prominent pool of enzymatically inactive AChE protein existed in the AD brain. The potential significance of these unexpected levels of inactive AChE protein in the AD brain was discussed, especially in the context of protein-protein interactions with β-amyloid and presenilin-1. © 2013 Springer Science+Business Media., MLC is supported by a Consolider-Predoctoral fellowship from the CSIC, Spain. This work was supported by grants from Fundación CIEN-Reina Sofía, Fondo de Investigaciones Sanitarias (FIS; Grant PS09/00684) from Spain to JSV and Fondo de Investigaciones Sanitarias (FIS; Grant CP11/00312) to MSGA

PMCID: PMC5013723, Alzheimer's disease (AD) is characterized by a decrease in the enzymatic activity of the enzyme acetylcholinesterase (AChE). AChE is expressed as multiple splice variants, which may serve both cholinergic degradative functions and non-cholinergic functions unrelated with their capacity to hydrolyze acetylcholine. We have recently demonstrated that a prominent pool of enzymatically inactive AChE protein exists in the AD brain. In this study, we analyzed protein and transcript levels of individual AChE variants in human frontal cortex from AD patients by western blot analysis using specific anti-AChE antibodies and by quantitative real-time PCR (qRT-PCR). We found similar protein and mRNA levels of the major cholinergic "tailed"-variant (AChE-T) and the anchoring subunit, proline-rich membrane anchor (PRiMA-1) in frontal cortex obtained from AD patients and non-demented controls. Interestingly, we found an increase in the protein and transcript levels of the non-cholinergic "readthrough" AChE (AChE-R) variants in AD patients compared to 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 that 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., MLC was supported by a Consolider-Predoctoral fellowship from the CSIC, Spain. This study was funded in part by Instituto de Salud Carlos III (ISCIII), Fondo de Investigaciones Sanitaria (grants CP11/00067 and PI14/00566 to MSGA and PS09/00684 and PI11/03026 for JSV); co-financed by Fondo Europeo de Desarrollo Regional) and Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (Fisabio) and through CIBERNED, ISCIII, Spain. Support also comes from NIH grant AG043375 (S. Ginsberg).

Classical studies of cholinesterase activity during liver dysfunction have focused on butyrylcholinesterase (BuChE), whereas acetylcholinesterase (AChE) has not received much attention. In the current study, liver and plasma AChE levels were investigated in rats with cirrhosis induced after 3 weeks of bile duct ligation (BDL). BDL rats showed a pronounced decrease in liver AChE levels (∼50%) compared with sham-operated (non-ligated, NL) controls; whereas liver BuChE appeared unaffected. A selective loss of tetrameric (G4) AChE was detected in BDL rats, an effect also observed in rats with carbon tetrachloride-induced cirrhosis. In accordance, SDS-PAGE analysis showed that the major 55-kd immunoreactive AChE band was decreased in BDL as compared with NL. A 65-kd band, attributed in part to inactive AChE, was increased as became the most abundant AChE subunit in BDL liver. The overall decrease in AChE activity in BDL liver was not accompanied by a reduction of AChE transcripts. The loss of G4 was also reflected by changes observed in AChE glycosylation pattern attributable to different liver AChE forms being differentially glycosylated. BDL affects AChE levels in both hepatocytes and Kupffer cells; however, altered AChE expression was mainly reflected in an alteration in hepatocyte AChE pattern. Plasma from BDL rats had approximately 45% lower AChE activity than controls, displaying decreased G4 levels and altered lectin-binding patterns. In conclusion, the liver is an important source of serum AChE; altered AChE levels may be a useful biomarker for liver cirrhosis., Supported by grants from la Caixa Foundation, Instituto de Salud Carlos III (Grants 03/0038, G03/155, C03/02) and Generalitat Valenciana (CTIDIB/ 2002/42, OPVI-21-2002 & GV04B-664) from Spain.

In this study we examined changes in acetylcholinesterase (AChE) pattern in the brain of adult Reelin Orleans (RelnOrl) homozygous mutant mice. The AChE histochemistry firstly revealed an abnormal distribution of AChE-positive cells in several areas of the reeler brain, including cortices; the strongest labelling was observed in cerebellum and hippocampus when compared with controls. Biochemical determinations demonstrated an increase of 80-90% in AChE specific activity from cerebellar and hippocampal extracts. We also report that the AChE tetrameric form (G4) was selectively increased in the RelnOrl brain. The relationship between AChE and Reelin and suggested morphogenetic functions are also discussed.

Biochemical changes in rat brain acetylcholinesterase (AChE) activity and molecular isoform distribution were studied using the middle cerebral artery occlusion model. After 24 h of permanent focal ischemia, AChE specific activity from infarcted cortex was found to decrease compared with contralateral cortex. Also a decrease in the minor monomeric (G1) form of AChE is response to ischemia was observed. Ischemia-induced AChE decrease was partially diminished by pre-treatment with dizocilpine (MK-801), a neuroprotective NMDA receptor blocker., This work has been supported, in part, from grants SAF99-0060 from CICYT, BFI2001-1565 from MCyT; G03/167 from MSCO; GC-02-019 and PAI-02-031 from JCCM and from Fundación Campollano-BSCH to V.C., BFI2001-1058 from MCyT and GC02-029 JCCM to C.G.-G. J.S.-V. was supported by a grant from the MCyT of Spain (Ramón y Cajal Program).

The identification of biochemical markers of Alzheimer's disease (AD) may help in the diagnosis of the disease. Previous studies have shown that Abeta(1-42) is decreased, and tau and phospho-tau are increased in AD cerebrospinal fluid (CSF). Our own studies have identified glycosylated isoforms of acetylcholinesterase (Glyc-AChE) and butyrylcholinesterase (Glyc-BuChE) that are increased in AD CSF. Glyc-AChE is increased in APP (SW) Tg2576 transgenic mice prior to amyloid plaque deposition, which suggests that Glyc-AChE may be an early marker of AD. The aim of this study was to determine whether Glyc-AChE or Glyc-BuChE is increased in CSF at early stages of AD and to compare the levels of these markers with those of Abeta(1-42), tau and phospho-tau. Lumbar CSF was obtained ante mortem from 106 non-AD patients, including 15 patients with mild cognitive impairment (MCI), and 102 patients with probable AD. Glyc-AChE, tau and phospho-tau were significantly increased in the CSF of AD patients compared to non-neurological disease (NND) controls. Abeta(1-42) was lower in the AD patients than in NND controls. A positive correlation was found between the levels of Glyc-AChE or Glyc-BuChE and disease duration. However, there was no clear correlation between the levels of tau, phospho-tau or Abeta(1-42) and disease duration. The results suggest that Glyc-AChE and Glyc-BuChE are unlikely to be early markers of AD, although they may have value as markers of disease progression.

Previous studies have shown that an abnormal salt-soluble form of G1 acetylcholinesterase (AChE) is increased in the Alzheimer's disease (AD) brain. The aim of the present study was to examine changes in AChE activity in an in vivo model of β-amyloid peptide (Aβ) administration. Rats received intracerebroventricular injections of Aβ25-35 (20 μg/day for seven days). Levels of AChE were measured in cerebral cortex and cerebrospinal fluid (CSF) after two months. Aβ25-35 administration did not alter total AChE activity in the cerebral cortex or CSF. However, analysis of salt-extractable AChE isoforms revealed an increase in the proportion of G1 in both cortex and CSF, similar to that previously observed in AD patients. The results support the view that changes in AChE isoform pattern in the AD brain are a direct consequence of Aβ accumulation. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Previous studies have shown that a minor glycoform of acetylcholinesterase (AChE) is increased in Alzheimer’s disease brain and cerebrospinal fluid. This glycoform can be distinguished from other AChE species by its lack of binding to concanavalin A (Con A). In this study, the temporal relationship between AChE glycosylation and Ab deposition was examined in Tg2576 mice. There was a significant (p < 0.05) difference in AChE glycosylation in Tg2576 mice compared with age-matched background strain control mice at 4 months of age. This difference in glycosylation was also observed in 8- and 12-month-old Tg2576 mice. In contrast, Ab plaques were only seen in the Tg2576 mice at 12 months of age, and were not detected at 4 and 8 months of age. Soluble humansequence Ab was detected as early as 4 months of age in the transgenic mice. The altered AChE glycosylation was due to an increase in a minor AChE isoform, which did not bind Con A, similar to that previously observed to be increased in Alzheimer’s disease brain and cerebrospinal fluid. The results demonstrate that in transgenic mice altered AChE glycosylation is associated with very early events in the development of AD-like pathology. The study supports the possibility that

The cholinergic enzyme acetylcholinesterase (AChE) and the catalytic component of the ¿-secretase complex, presenilin-1 (PS1), are known to interact. In this study, we investigate the consequences of AChE-PS1 interactions, particularly the influence of AChE in PS1 levels and ¿-secretase activity. PS1 is able to co-immunoprecipitate all AChE variants (AChE-R and AChE-T) and molecular forms (tetramers and light subunits) present in the human brain. Overexpression of AChE-R or AChE-T, or their respective inactive mutants, all trigger an increase in PS1 protein levels. The AChE species capable of triggering the biggest increase in PS1 levels is a complex of AChE with the membrane anchoring subunit proline-rich membrane anchor (PRiMA), which restricts the localization of the resulting AChE tetramer to the outer plasma membrane. Incubation of cultured cells with soluble AChE demonstrates that AChE is able to increase PS1 at both the protein and transcript levels. However, the increase of PS1 caused by soluble AChE is accompanied by a decrease in ¿-secretase activity as shown by the reduction of the processing of the amyloid-ß protein precursor. This inhibitory effect of AChE on ¿-secretase activity was also demonstrated by directly assessing accumulation of CTF-AßPP in cell-free membrane preparations incubated with AChE. Our data suggest that AChE may function as an inhibitor of ¿-secretase activity.

Presenilin-1 (PS1) is the catalytic component of the γ-secretase complex. In this study, we explore if PS1 participates in the processing of the cholinergic acetylcholinesterase (AChE). The major AChE variant expressed in the brain is a tetramer (G4) bound to a proline-rich membrane anchor (PRiMA). Overexpression of the transmembrane PRiMA protein in Chinese hamster ovary cells expressing AChE and treated with the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester have enabled us to study whether, through its γ-secretase activity, PS1 participates in the processing of PRiMA-linked AChE. γ-Secretase inhibition led to a notable increase in the level of PRiMA-linked AChE, suggesting that γ-secretase is involved in the cleavage of PRiMA. We demonstrate that cleavage of PRiMA by γ-secretase results in a C-terminal PRiMA fragment. Immunofluorescence labeling allowed us to identify this PRiMA fragment in the nucleus. Moreover, we have determined changes in the proportion of the raft-residing AChE-PRiMA in a PS1 conditional knockout mouse. Our results are of interest as both enzymes have therapeutic relevance for Alzheimer's disease. © 2014 Elsevier Inc., MLC is supported by a Consolider-Predoctoral fellowship from the CSIC, Spain. This work was supported by grants from Fundación CIEN—Reina Sofía, Fondo de Investigaciones Sanitaria (FIS), cofinanced by Fondo Europeo de Desarrollo Regional (FEDER) (Grant PS09/00684) and CIBERNED, ISC-III from Spain to JSV; FIS cofinanced by FEDER (CP11/00067) to MSGA; and PI08/0018 to AL.

Acetylcholinesterase (AChE) is encoded by a single gene, and the alternative splicing at the 3′ end produces different isoforms, including tailed (AChET), read-through (AChER), and hydrophobic (AChEH). Different forms of this enzyme exist in different cell types. Each AChE form has been proposed to have unique function, and all of them could be found in same cell type. Thus, the splicing process of different AChE forms remains unclear. Here, we aimed to establish a quantification method in measuring the absolute amount of each AChE splicing variants within a cell type. By using real-time PCR coupled with standard curves of defined copy of AChE variants, the copies of AChET transcript per 100 ng of total RNA were 5.7×104 in PC12 (rat neuronal cell), 1.3×104 in Caco-2 (human intestinal cell), 0.67×104 in TF-1 (human erythropoietic precursor), 133.3 in SH-SY5Y (human neuronal cell), and 56.7 in human umbilical vein endothelial cells (human endothelial cells). The copies of AChEH in these cell types were 0.3×104, 3.3×104, 2.7×104, 133.3, and 46.7, respectively, and AChER were 0.07×104, 0.13×104, 890, 3.3, and 2.7, respectively. Furthermore, PC12 and TF-1 cells were chosen for the analysis of AChE splicing pattern during differentiation. The results demonstrated a selective increase in AChE T mRNA but not AChER or AChEH mRNAs in PC12 upon nerve growth factor-induced neuronal differentiation. PC12 cells could therefore act as a good cell model for the study on alternative splicing mechanism and regulation of AChET. © 2014 Springer Science+Business Media., This research was supported by Hong Kong Research Grants Council Theme-based Research Scheme (T13-607/12R), GRF (S-HK015/09, 662911, 663012, 662713, N_HKUST629/13), The Hong Kong Jockey Club Charities Trust and Foundation of The Awareness of Nature (TAON12SC01) to Karl Tsim

The glycosylation of acetylcholinesterase (AChE) in CSF was analyzed by lectin binding. AChE from Alzheimer's disease (AD) patients was found to bind differently to two lectins, concanavalin A and wheat germ agglutinin, than AChE from controls. As multiple isoforms of AChE are present in both CSF and brain, we examined whether the abnormal glycosylation of AD AChE was due to changes in a specific molecular isoform. Globular amphiphilic dimeric (G2a) and monomeric (G1a) isoforms of AChE were found to be differentially glycosylated in AD CSF. Glycosylation of AChE was also altered in AD frontal cortex but not in cerebellum and was also associated with an increase in the proportion of light (G2 and G1) isoforms. This study demonstrates that the glycosylation of AChE is altered in the AD brain and that changes in AChE glycosylation in AD CSF may reflect changes in the distribution of brain isoforms. The study also suggests that glycosylation of AChE may be a useful diagnostic marker for AD.

Our previous studies have shown that a minor isoform of acetylcholinesterase (AChE) is increased in the cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients. In the present study, the glycosylation of butyrycholinesterase (BuChE) was found to be altered in AD CSF. By combining an analysis of CSF AChE and BuChE glycosylation, it is possible to identify cases of AD with more than 90% sensitivity and specificity.

The identification of a biochemical marker of Alzheimer's disease (AD) is a major research aim of many groups. Abnormal levels of tau and Aβ have been identified in the cerebrospinal fluid (CSF) of AD patients, although the sensitivity and specificity of the changes in these two biomarkers alone is not sufficient to be of diagnostic value. Recently, our group has identified an abnormality in the glycosylation of acetylcholinesterase (AChE). The increase in this glycoform of AChE is very specific for Alzheimer's disease and is not seen in many other neurological diseases including other dementias.

Acoustic neurinomas were sequentially extracted with saline and saline-Triton X-100 buffers. Detergent was required to detach the bulk of acetylcholinesterase (AChE), but butyrylcholinesterase (BuChE) was mostly released with saline buffer. Sedimentation analysis and hydrophobic chromatography revealed that neurinomas contain principally amphiphilic AChE tetramers, dimers and monomers, and hydrophilic BuChE tetramers. The AChE dimers and monomers remained amphiphilic after incubation with phosphatidylinositol-specific phospholipase C (PIPLC), after or without prior treatment with alkaline hydroxylamine, which shows that, in contrast to the meningioma AChE dimers and monomers, the neurinoma isoforms are devoid of glycolipid. Neurinoma AChE reacted with the antibodies HR2 and AE1 raised against AChE from human brain or erythrocyte, whereas BuChE bound to a sheep antiserum.

The structural properties of acetyl- (AChE) and butyrylcholinesterase (BuChE) in meningioma and the possible relationship with brain and plasma were investigated. Meningioma ChEs were extracted with saline and saline-Triton X-100 buffers. The tumor ChE forms were identified by sedimentation analysis, and their amphiphilic/hydrophilic behaviour was assessed by Triton X-114 phase-partitioning and hydrophobic chromatography. Meningioma contained amphiphilic globular AChE dimers (G2A) and monomers (G1A), and hydrophilic BuChE tetramers (G4A). The conversion of G2A into G1A AChE by reduction confirmed their structures. In contrast to the meningioma species, brain G1A AChE forms remained amphiphilic after incubation with alkaline hydroxylamine and phosphatidylinositol-specific phospholipase C (PIPLC). Meningioma G1A and PIPLC-converted G1H, and brain G1A AChE showed similar rate constants for thermal inactivation, and this suggested that the thermal stability of AChE subunits was unaffected by the presence or not of phosphatidylinositol residues. AChE in meningioma and brain did not differ in the interaction with the lectins Con A, LCA, WGA and RCA. BuChE in meningioma and brain bound to a similar extent to Con A, LCA and WGA-Agarose, whereas one-half of BuChE in the tumor, all in plasma and little in brain was fixed by RCA. Therefore, meningioma possesses RCA+- and RCA−-BuChE, the former predominating in brain and the latter in plasma. It remains to be clarified whether the tumor RCA+-BuChE is intrinsic or derived from plasma.

Background & Aims: There have been many studies on plasma butyrylcholinesterase in liver dysfunction. However, no data is available about acetylcholinesterase in human cirrhosis, although profound changes have been described in cirrhotic rat models. Methods: Human serum and liver acetylcholinesterase and its molecular forms were determined enzymatically, after butyrylcholinesterase immunodepletion. The distinct species of acetylcholinesterase, with a distinct C-terminus, were determined by western blotting, and the level of liver transcripts by real-time PCR. Liver acetylcholinesterase was also evaluated by immunocytochemistry. Results: In patients with liver cirrhosis, the activity of plasma acetylcholinesterase (rich in light species), appeared to be apparently unaffected. However, the levels of the soluble readthrough (R) acetylcholinesterase form, an acetylcholinesterase species usually associated with stress and pathology, was increased compared to controls. Human liver acetylcholinesterase activity levels were also unchanged, but protein levels of the acetylcholinesterase-R and other acetylcholinesterase subunit species were increased in the cirrhotic liver. This increase in acetylcholinesterase protein expression in the cirrhotic liver was confirmed by PCR analysis. Immunohistological examination confirmed that acetylcholinesterase immunoreactivity is increased in parenchymal cells of the cirrhotic liver. Conclusions: We demonstrate significant changes in acetylcholinesterase at the protein and mRNA levels in liver cirrhosis, with no difference in enzymatic activity. The altered expression of acetylcholinesterase protein may reflect changes in its pathophysiological role. © 2012 García-Ayllón et al., This work was supported by grants from Fundación CIEN-Reina Sofía, Fondo de Investigaciones Sanitarias (PS09/00684), and CIBERNED, Instituto de Salud Carlos III from Spain to JSV; and Fondo de Investigaciones Sanitarias (CP11/00312) to MSGA.

We have previously identified presenilin-1 (PS1), the active component of the γ-secretase complex, as an interacting protein of the amyloid-associated enzyme acetylcholinesterase (AChE). In this study, we have explored the consequences of AChE-PS1 interactions. Treatment of SH-SY5Y cells with the AChE-inhibitor tacrine decreased PS1 levels, in parallel with increase in the secretion of amyloid precursor protein APPα, whereas the cholinergic agonist carbachol had no effect on PS1. AChE knockdown with siRNA also decreased PS1 levels, while AChE overexpression exerted opposing effect. AChE-deficient also had decreased PS1. Mice administered with tacrine or donepezil displayed lower levels of brain PS1. However, sustained AChE inhibition failed to exert long-term effect on PS1. This limited duration of response may be due to AChE upregulation caused by chronic inhibition. Finally, we exposed SH-SY5Y cells to β-amyloid (Aβ)42 which triggered elevation of both AChE and PS1 levels. The Aβ42-induced PS1 increase was abolished by siRNA AChE pretreatment, suggesting that AChE may participate in the pathological feedback loop between PS1 and Aβ. Our results provide insight into AChE-amyloid interrelationships. © 2012 Elsevier Inc., This work was supported by grants from Fundación CIEN-Reina Sofía, FIS (Grant 06/0181 and PS09/00684) and CIBERNED, ISC-III from Spain to J.S.V.

A common feature in the Alzheimer's disease (AD) brain is the presence of acetylcholinesterase (AChE) which is commonly associated with β-amyloid plaques and neurofibrillary tangles (NFT). Although our understanding of the relationship between AChE and the pathological features of AD is incomplete, increasing evidence suggests that both β-amyloid protein (Aβ) and abnormally hyperphosphorylated tau (P-tau) can influence AChE expression. We also review recent findings which suggest the possible role of AChE in the development of a vicious cycle of Aβ and P-tau dysregulation and discuss the limited and temporary effect of therapeutic intervention with AChE inhibitors., This work was supported by grants from Foundatión CIEN-Reina Sofía, FIS (GrantPS09/00684), and CIBERNED, ISC-III from Spain to Javier Sáez-Valero.

Human brain acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were sequentially extracted, first with a Tris-saline buffer (S1) and then with 1% (w/v) Triton X-100 (S2). About 20 and 30% of the AChE and BuChE activities were recovered in S1 and most of the remaining enzymes in S2. Main molecular forms of about 10.5 S and 12.0 S, G4 forms of AChE and BuChE, and smaller amounts of 4.5 S and 5.5 S forms, G1 species of AChE and BuChE, were measured in S1. Application of Triton X-114 phase partitioning and affinity chromatography on phenyl-agarose to S1 revealed that 25% of the AChE and none of the BuChE molecules displayed amphiphilic properties. Analysis of the enzyme activity retained by the phenyl-agarose showed that G1 AChE constituted the bulk of the amphiphilic molecules released without detergent. Main G4 forms of AChE and BuChE were found in the S2 extract. Eighty and 45% of the AChE and BuChE activities in S2 were measured in the detergent-rich phase by Triton X-114 phase partitioning. Thus, most of the AChE and about half of the BuChE molecules in S2 displayed amphiphilic properties. The main peak of BuChE, a 12.0 S form in gradients made with Triton X-100, splits into two peaks of 9.5 S and 12.5 S in Brij 96-containing gradients. This suggests that hydrophilic G4 BuChE forms are predominant in S1 and that hydrophilic and amphiphilic isoforms coexist in S2. © 1993 Wiley-Liss, Inc.

Background Many studies have been conducted in an extensive effort to identify alterations in blood cholinesterase levels as a consequence of disease, including the analysis of acetylcholinesterase (AChE) in plasma. Conventional assays using selective cholinesterase inhibitors have not been particularly successful as excess amounts of butyrylcholinesterase (BuChE) pose a major problem. Principal Findings Here we have estimated the levels of AChE activity in human plasma by first immunoprecipitating BuChE and measuring AChE activity in the immunodepleted plasma. Human plasma AChE activity levels were ~20 nmol/min/mL, about 160 times lower than BuChE. The majority of AChE species are the light G1+G2 forms and not G4 tetramers. The levels and pattern of the molecular forms are similar to that observed in individuals with silent BuChE. We have also compared plasma AChE with the enzyme pattern obtained from human liver, red blood cells, cerebrospinal fluid (CSF) and brain, by sedimentation analysis, Western blotting and lectin-binding analysis. Finally, a selective increase of AChE activity was detected in plasma from Alzheimer's disease (AD) patients compared to age and gender-matched controls. This increase correlates with an increase in the G1+G2 forms, the subset of AChE species which are increased in Alzheimer's brain. Western blot analysis demonstrated that a 78 kDa immunoreactive AChE protein band was also increased in Alzheimer's plasma, attributed in part to AChE-T subunits common in brain and CSF. Conclusion Plasma AChE might have potential as an indicator of disease progress and prognosis in AD and warrants further investigation., This work was supported by grants from CIEN Foundation (http://www.fundacioncien.es/), Fondo de Investigaciónes Sanitarias (PI06/0181), and CIBERNED, Instituto de Salud Carlos III from Spain (http://www.isciii.es/).

Differences in glycosylation of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in human brain, plasma and cerebrospinal fluid (CSF) have been investigated by means of their interaction with agarose-immobilized lectins. Most of the AChE in brain and CSF was associated to concanavalin A (Con A), Lens culinaris (LCA) and Triticum vulgaris (WGA) agglutinins, but little activity was adsorbed to Ricinus communis agglutinin I (RCA I ). Brain, plasma and CSF BuChE was almost fully bound to Con A, LCA and WGA-agarose. Brain BuChE was unable to react with RCA (RCA − BuChE), the plasma enzyme was completely bound to the lectin (RCA + BuChE) and BuChE from CSF of normal children was partially fixed to RCA (RCA ± BuChE). BuChE in CSF of children with meningitis fully reacts with the lectin. The data suggest that the of RCA + BuChE in CSF of children with meningitis is increased, this enzyme probably coming from plasma.

Presenilin 1 (PS1) plays a critical role in the γ-secretase processing of the amyloid precursor protein to generate the β-amyloid peptide, which accumulates in plaques in the pathogenesis of Alzheimer's disease (AD). Mutations in PS1 cause early onset AD, and proteins that interact with PS1 are of major functional importance. We report here the coimmunoprecipitation of PS1 and acetylcholinesterase (AChE), an enzyme associated with amyloid plaques. Binding occurs through PS1 N-terminal fragment independent of the peripheral binding site of AChE. Subcellular colocalization of PS1 and AChE in cultured cells and coexpression patterns of PS1 and AChE in brain sections from controls and subjects with sporadic or familial AD indicated that PS1 and AChE are located in the same intracellular compartments, including the perinuclear compartments. A PS1-A246E pathogenic mutation expressed in transgenic mice leads to decreased AChE activity and alteration of AChE glycosylation and the peripheral binding site, which may reflect a shift in protein conformation and disturbed AChE maturation. In both the transgenic mice and humans, mutant PS1 impairs coimmunoprecipitation with AChE. The results indicate that PS1 can interact with AChE and influence its expression, supporting the notion of cholinergic-amyloid interrelationships. Copyright © 2008, American Society for Microbiology. All Rights Reserved., This study was supported by grants from the la Caixa Foundation, FIS (grants 03/0038 and 06/0181), and CIBERNED, ISC-III (Spain), to J.S.-V.

The cholinergic system is involved in specific behavioural responses and cognitive processes. Here, we examined potential alterations in the brain levels of key cholinergic enzymes in cirrhotic patients and animal models with liver failure. An increase (∼30%) in the activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE) is observed in the brain cortex from patients deceased from hepatic coma, while the activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase, remains unaffected. In agreement with the human data, AChE activity in brain cortical extracts of bile duct ligated (BDL) rats was increased (∼20%) compared to controls. A hyperammonemic diet did not result in any further increase of AChE levels in the BDL model, and no change was observed in hyperammonemic diet rats without liver disease. Portacaval shunted rats which display increased levels of cerebral ammonia did not show any brain cholinergic abnormalities, confirming that high ammonia levels do not play a role in brain AChE changes. A selective increase of tetrameric AChE, the major AChE species involved in hydrolysis of acetylcholine in the brain, was detected in both cirrhotic humans and BDL rats. Histological examination of BDL and non-ligated rat brains shows that the subcellular localization of both AChE and choline acetyltransferase, and thus the accessibility to their substrates, appears unaltered by the pathological condition. The BDL-induced increase in AChE activity was not parallelled by an increase in mRNA levels. Increased AChE in BDL cirrhotic rats leads to a pronounced decrease (∼50-60%) in the levels of acetylcholine. Finally, we demonstrate that the AChE inhibitor rivastigmine is able to improve memory deficits in BDL rats. One week treatment with rivastigmine (0.6 mg/kg; once a day, orally, for a week) resulted in a 25% of inhibition in the enzymatic activity of AChE with no change in protein composition, as assessed by sucrose density gradient fractionation and western blotting analysis. In conclusion, this study is the first direct evidence of a cholinergic imbalance in the brain as a consequence of liver failure and points to the possible role of the cholinergic system in the pathogenesis of hepatic encephalopathy. © 2008 The Author(s)., Generalitat Valenciana (GV04B-664); Instituto de Salud Carlos III (G03/155, PI05/1269, PI06/0181 and CIBERNED)

The altered expression of acetylcholinesterase (AChE) in the brains of patients with Alzheimer's disease (AD) has raised much interest of late. Despite an overall decrease in the AD brain, the activity of AChE increases around β-amyloid plaques and indeed, the β-amyloid peptide (Aβ) can influence AChE levels. Such evidence stimulated our interest in the possibility that the levels of AChE and amyloid might vary together in AD. We previously found that the different AChE forms present in both the brain and in the cerebrospinal fluid (CSF) of AD patients varied in conjunction with abnormal glycosylation. Thus, the alterations in glycosylation are correlated with the accumulation of a minor subspecies of AChE monomers. We also recently analysed whether long-term exposure to the cholinesterase inhibitor (ChE-I) donepezil influences the AChE species found in AD CSF. The marked increase in CSF-AChE activity in AD patients following long-term treatment with donepezil was not paralleled by a rise in this subset of light variants. Hence, the correlation with the levels of CSF-Aβ is unique to these AChE species in patients receiving such treatment. The aim of this report is to review the links between AChE and β-amyloid, and to discuss the significance of the responses of the distinct AChE species to ChE-I during the treatment of AD. © 2008 Elsevier Ireland Ltd. All rights reserved., This work was supported by grants from la Caixa Foundation and Instituto de Salud Carlos III (Grant 03/0038, PI06/0181 and CIBERNED) from Spain to JSV.

We analyzed whether donepezil differently influences acetylcholinesterase (AChE) variants from cerebrospinal fluid (CSF) in patients with Alzheimer's disease (AD) after long-term treatment. Overall CSF-AChE activity in AD patients before treatment was not different from controls, but the ratio between the major tetrameric form, G(4), and the smaller G(1) and G(2) species was significantly lower. AChE levels at study outset were found to correlate positively with beta-amyloid (1-42) (Abeta42). When patients were re-examined after 12 months treatment with donepezil, there was a remarkable increase in both the G(4) and the lighter species of CSF AChE. As compared with placebo, donepezil caused decreases in the percentage of AChE that failed to bind to the lectin concanavalin A and the antibody AE1. These non-binding species comprised primarily a small subset of G(1) and G(2) forms. In treated patients, these light variants were the only subset of CSF AChE that correlated with CSF-Abeta42 levels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that a 77-kDa band, attributed in part to inactive AChE, was lower in AD patients than in controls. Unlike enzyme activity, the intensity of this band did not increase after donepezil treatment. The varying responses of different AChE species to ChE-I treatment suggest different modes of regulation, which may have therapeutic implications., MSGA is supported by a post-doctoral Fellowships from the Juan de la Cierva Program of Spain. MXS is a fellow of the Consejo Superior de Investigaciones Científicas (Ministerio de Educación y Ciencia) from Spain. This work was supported by grants from la Caixa Foundation, Fondo de Investigaciones Sanitarias (Grant 03/0038) and Generalitat Valenciana (ACOMP06/074) from Spain to JSV.

Changes in the glycosylation pattern of brain proteins have been associated with Creutzfeldt–Jakob disease (CJD). We have investigated the glycosylation status of acetylcholinesterase (AChE) by lectin binding assay. Our data show that in lumbar CSF from definite and probable sporadic CJD cases AChE activity is lower compared with that in age-matched controls. We also show, for the first time, that AChE glycosylation is altered in CJD CSF and brain. Unlike Alzheimer's disease, in which an alteration in both the glycosylation and levels of AChE molecular forms is observed, the abnormal glycosylation of AChE in CJD appears to be unrelated to changes in molecular forms of this enzyme. These findings suggest that altered AChE glycosylation in CJD may be a consequence of the general perturbation of the glycosylation machinery that affects prion protein, as well as other proteins. The diagnostic potential of these changes remains to be explored., M-XS is a fellow of Conselo Superior de Investigaciones Científicas (Ministero de Educación y Cienica) from Spain. JS-V is supported by a Postdoctoral Fellowship from the Ramón y Cajal Program of Spain. This work was supported by grants from la Caixa Foundation, Fondo de Investigación Sanitaria (FIS) (grant 03/0038) and Instituto de Mayores y Servicios Sociales (IMERSO) (grant E-160) from Spain to JS-V; and FIS (grant C03/06. CIEN network) and MEC (EET 2002/05155) from Spain to MC.

In this study we examined changes in acetylcholinesterase (AChE) pattern in the brain of adult Reelin Orleans (RelnOrl) homozygous mutant mice. The AChE histochemistry firstly revealed an abnormal distribution of AChE-positive cells in several areas of the reeler brain, including cortices; the strongest labelling was observed in cerebellum and hippocampus when compared with controls. Biochemical determinations demonstrated an increase of 80-90% in AChE specific activity from cerebellar and hippocampal extracts. We also report that the AChE tetrameric form (G4) was selectively increased in the RelnOrl brain. The relationship between AChE and Reelin and suggested morphogenetic functions are also discussed.

Acetylcholinesterase (AChE) was analyzed in organotypic hippocampal slice cultures (OHSCs) during recovery from a brief period (20 min) of combined hypoxia and hypoglycemia. Simulated ischemia transiently increased AChE activity in OHSCs in a time-dependent manner reaching a 1.5 fold increase at 6 h post-ischemia. The ischemia-induced AChE increase was totally abolished by incubation with 10 μM dizocilpine (MK-801), a neuroprotective NMDA receptor blocker., This work has been supported, in part, by grants SAF99-0060 from CICYT, BFI2001-1565 from Ministerio de Ciencia y Tecnologia, G03/167 from Ministerio de Sanidad, GC-02-019 and PAI-02-031 from Consejeria de Ciencia y Tecnologia, JCCM and from Fundación Campollano-Banco Santander Central Hispano to V.C., and by grants BFI2001-1058 from Ministerio de Ciencia y Tecnologia and GC02-029 from Consejeria de Sanidad JCCM to C.G.-G. J.S.-V. was supported by a fellowship from MCyT of Spain (Ramón y Cajal Program).

The cellular origin of the acetylcholinesterase (AChE) associated with amyloid plaques in the Alzheimer’s disease (AD) brain is unknown. In this study we report that amyloid β-peptides (Aβ) increased AChE levels in both neuronal and astrocytic primary cultures, supporting the possibility that both neurons and glia may make a direct contribution to the pool of AChE seen around amyloid deposits in the AD brain., JS-V was supported by a grant from the MCyT of Spain (Ramón y Cajal Program). DHS was supported by grants from the NH&MRC of Australia, Axonyx Inc. (New York), and the Rebecca L. Cooper Medical Research Foundation Ltd. (Australia).

The identification of a biochemical marker in cerebrospinal fluid (CSF) that can discriminate between Alzheimer's disease (AD) and other dementia-causing diseases would be a major advance. Our previous studies have shown that the glycosylation of acetylcholinesterase (AChE) is altered in the post mortem brain and cerebrospinal fluid of AD patients. We have also found that the glycosylation of AChE is altered in lumbar CSF collected ante mortem. The change in glycosylation of AChE is very specific for AD and is not seen in many other neurological diseases including other dementias. The sensitivity of detection of AD using AChE glycosylation (60–80%) satisfactory diagnostic marker. However, more recently we have found that the glycosylation of the related enzyme butyrylcholinesterase (BuChE) is also altered in AD CSF. By combining the analysis of AChE glycosylation with that of BuChE glycosylation, improved sensitivity of detection is obtained. We propose that AChE and BuChE glycosylation may be of diagnostic value, especially when used in combination with other CSF markers such as Aβ or tau.

Previous studies have shown that a minor glycoform of acetylcholinesterase (AChE) is increased in Alzheimer's disease brain and cerebrospinal fluid. This glycoform can be distinguished from other AChE species by its lack of binding to concanavalin A (Con A). In this study, the temporal relationship between AChE glycosylation and Abeta deposition was examined in Tg2576 mice. There was a significant (p0.05) difference in AChE glycosylation in Tg2576 mice compared with age-matched background strain control mice at 4 months of age. This difference in glycosylation was also observed in 8- and 12-month-old Tg2576 mice. In contrast, Abeta plaques were only seen in the Tg2576 mice at 12 months of age, and were not detected at 4 and 8 months of age. Soluble human-sequence Abeta was detected as early as 4 months of age in the transgenic mice. The altered AChE glycosylation was due to an increase in a minor AChE isoform, which did not bind Con A, similar to that previously observed to be increased in Alzheimer's disease brain and cerebrospinal fluid. The results demonstrate that in transgenic mice altered AChE glycosylation is associated with very early events in the development of AD-like pathology. The study supports the possibility that glycosylation may also be a useful biomarker of AD.

Brain and non-brain tumors contain acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) transcripts and enzyme activity. AChE and BuChE occur in tissues as a set of molecular components, whose distribution in a cyst fluid from a human astrocytoma we investigated. The fluid displayed high BuChE and low AChE activities. Three types of cholinesterase (ChE) tetramers were identified in the fluid by means of sedimentation analyses and assays with specific inhibitors, and their sedimentation coefficients were 11.7S (ChE-I), 11.1S (ChE-II), and 10.5S (ChE-III). ChE-I was unretained, ChE-II was weakly retained and ChE-III was adsorbed to edrophonium-agarose, confirming the AChE nature of the latter. ChE-I and ChE-II tetramers contained BuChE subunits as shown by their binding with an antiserum against BuChE. The ChE activity of the immunocomplexes made with ChE-II and anti-BuChE antibodies decreased with the AChE inhibitor BW284c51, revealing that ChE-II was made of AChE and BuChE subunits, in contrast to ChE-I, which only contained BuChE subunits. The binding of an anti-AChE antibody (AE1) to ChE-II and ChE-III, but not to ChE-I, demonstrated the hybrid composition of ChE-II. A substantial fraction of the AChE tetramers and dimers of astrocytomas and oligodendrogliomas bound both to anti-AChE and anti-BuChE antibodies, which revealed a mixed composition of AChE and BuChE subunits in them. The AChE components of brain, meningiomas and neurinomas were only recognized by AE1. In conclusion, our results demonstrate that aberrant ChE oligomers consisting of AChE and BuChE subunits are generated in astrocytomatous cyst and gliomas but not in brain, meningiomas or neurinomas.

As clinical diagnosis of Alzheimer's disease is only 80%-90% accurate, there is a need to identify biochemical markers of Alzheimer's disease. Previous studies have shown an abnormality in the glycosylation of acetylcholinesterase (AChE) in the CSF collected postmortem from patients with Alzheimer's disease. This abnormality was very specific for Alzheimer's disease, as it was not detected in other illnesses causing dementia. We report here that the glycosylation of AChE is also altered in lumbar CSF collected antemortem. The altered glycosylation was due to increased concentrations of a minor AChE isoform that does not bind to concanavalin A (Con A). Glycosylation of AChE may eventually be of diagnostic value, especially when used in combination with other CSF markers.

Several neurodegenerative disorders present deficiencies in the cholinergic system. Scarce research on prion encephalopathies has examined the levels of cholinergic pathway-related enzymes. Acetylcholinesterase (AChE) is expressed as several molecular forms. The potential importance of these variants is increased by the possibility that AChE has roles other than acetylcholine hydrolysis. We investigated the levels of AChE, its molecular forms, and glycosylation in the cerebrospinal fluid (CSF) from Creutzfeldt-Jakob disease (CJD) patients.

Several studies have suggested a role for cholinesterases in regulating neurite outgrowth. Some acetylcholinesterase (AChE) inhibitors can inhibit neurite outgrowth, but it is unclear if this is due to inhibition of AChE. In this study, the effect of cholinesterase inhibitors on neurite outgrowth from chick sympathetic neurons was examined. Very high (micromolar) concentrations of tacrine and BW284c51 were needed to inhibit neurite outgrowth. In contrast, nanomolar concentrations were required to block cholinesterase activity. No correlation was found between the type of inhibitor or potency of cholinesterase inhibition and inhibition of neurite outgrowth. Both tacrine and BW 284c51 were neurotoxic at concentrations that inhibited outgrowth. Therefore, the action of cholinesterase inhibitors on neurite outgrowth may be due to non-specific toxicity rather than to cholinesterase binding.

Alzheimer’s disease (AD) is a progressive dementia that commonly occurs in the aged population. It has been estimated that 5–10% of the population over the age of 65 have some cognitive signs of AD (1). Diagnosis of AD remains problematic as clinical examination is only about 80% accurate. To date, no ante mortem biochemical tests have been found which unequivocally indicate a diagnosis of AD.

A diagnostic test for Alzheimer’s disease (AD) based on biochemical analysis of blood or cerebrospinal fluid (CSF) is needed because clinical examination is only 80% accurate. Analysis of CSF from AD patients has shown decreased amyloid protein (A) and increased tau 1 but neither marker is more specific or sensitive than clinical examination. The level and distribution of acetylcholinesterase (AChE; EC 3.1.1.7) isoforms are profoundly altered in the Alzheimer brain, 2 but no consistent change in CSF isoform patterns or activity has been identified. 2 As glycosylation patterns of proteins are altered in severe disease states, 3 we examined the glycosylation of AChE in CSF AD patients and controls by lectin binding. Lumbar or ventricular CSF was obtained after death from 18 controls with no clinical or pathological dementia and no evidence of brain pathology, 27 cases of AD, seven cases of dementia non-AD type (DNAT: five frontal lobe dementia, one Lewy body dementia/Parkinson’s disease, one multi-infarct dementia/congophilic amyloid angiopathy), and six cases of other neurological disorders (ND: four Huntington’s disease, one schizophrenia, one corticobasal degeneration). CSF was stored at –70oC and centrifuged at 1000 g for 15 min before analysis. AChE activity was assayed at 22oC by a modified Ellman method. 4

One of the characteristic changes that occurs in Alzheimer's disease is the loss of acetylcholinesterase (AChE) from both cholinergic and noncholinergic neurons of the brain. However, AChE activity is increased around amyloid plaques. This increase in AChE may be of significance for therapeutic strategies using AChE inhibitors. The aim of this study was to examine the effect of amyloid beta-protein (A beta), the major component of amyloid plaques, on AChE expression. A beta peptides spanning residues 1-40 or 25-35 increased AChE activity in P19 embryonal carcinoma cells. A peptide containing a scrambled A beta(25-35) sequence did not stimulate AChE expression. To examine the possibility that the increase in AChE expression was mediated by an influx of calcium through voltage-dependent calcium channels (VDCCs), drugs acting on VDCCs were tested for their effects. Inhibitors of L-type VDCCs (diltiazem, nifedipine, and verapamil), but not N- or P- or Q-type VDCCs, resulted in a decrease in AChE expression. Agonists of L-type VDCCs (maitotoxin and S(-)-Bay K 8644) increased AChE expression. As L-type VDCCs are known to be modulated by cyclic AMP-dependent protein kinase, the effect of the adenylate cyclase activator forskolin was also examined. Forskolin stimulated AChE expression, an action that was blocked by the L-type VDCC antagonist nifedipine. The A beta(25-35)induced increase in AChE expression was mediated by an L-type VDCC, as the effect was also blocked by nifedipine. The results suggest that the increase in AChE expression around amyloid plaques could be due to a disturbance in calcium homeostasis involving the opening of L-type VDCCs.

Specimens of astrocytoma, oligodendroglioma and medulloblastoma were sequentially extracted with saline and saline-Triton X-100 buffers. Acetyl- (AChE) and butyrylcholinesterase (BuChE) activities were assayed in the soluble fractions, these being further analyzed to establish the distribution of molecular forms. All the tumors tested showed AChE and BuChE activities, the measured AChE/BuChE ratios being unrelated to the malignant grading. Hydrophilic and amphiphilic AChE and BuChE tetramers, amphiphilic AChE dimers and monomers, and hydrophilic BuChE monomers were identified in all the tumors analyzed. The amphiphilic behavior of the enzyme forms was assessed by sedimentation analysis and hydrophobic chromatography on phenyl-Agarose. A small fraction of glioma AChE monomers was released as, or transformed into, hydrophilic forms by incubation with phosphatidylinositol-specific phospholipase C (PIPLC). These data suggest that AChE monomers bearing distinct hydrophobic domains coexist in human glioma.

Amphiphilic monomers and dimers of acetylcholinesterase (AChE) and hydrophilic tetramers of butyrylcholinesterase (BuChE) were released by extracting human meningioma with Tris-saline and Tris-saline-Triton X-100 buffers. The amphiphilic or hydrophilic behavior of the AChE and BuChE forms was assessed by sedimentation analysis, hydrophobic chromatography and Triton X-114 phase-partitioning. A significant fraction of the amphiphilic AChE species was converted into hydrophilic components by incubation of the soluble enzyme with phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus thuringiensis, this fraction being increased by a double treatment with PIPLC and alkaline hydroxylamine. A significant amount of the membrane-bound AChE was released by incubation with PIPLC. These results demonstrate that AChE forms in meningioma are attached to the membrane via glycosylphosphatidylinositol, although part of the enzyme forms are resistant to PIPLC.

Pathological hyperphosphorylation and aggregation of the tau protein is associated with dementia and can be the central cause of neurodegeneration. Here, we examined potential alterations in the level of the cholinergic enzyme acetylcholinesterase (AChE) in the brain of transgenic mice (Tg-VLW) expressing human tau mutations. Overexpression of mutant hyperphosphorylated tau (P-tau) led to an increase in the activity of AChE in the brain of Tg-VLW mice, paralleled by an increase in AChE protein and transcripts; whereas the levels of the enzyme choline acetyltransferase remained unaffected. VLW tau overexpression in SH-SY5Y cells also increased AChE activity levels. All major molecular forms of AChE were increased in the Tg-VLW mice, including tetrameric AChE, which is the major species involved in hydrolysis of acetylcholine in the brain. Colocalization of human P-tau and AChE supports the conclusion that P-tau can act to increase AChE. This study is the first direct evidence of a modulatory effect of P-tau on brain AChE expression. © 2012 Elsevier Inc., Fundación CIEN-Reina Sofía; FIS Grant PS09/00684; CIBERNED; ISCIII