Toxic Effects of Acetylcholinesterase on Neuronal and Glial-Like Cells in Vitro

Home , BW284C51

ORIGINAL RESEARCH ARTICLE Toxic effects of acetylcholinesterase on neuronal and glial-like cells in vitro ´ FH Calderon, R von Bernhardi, G De Ferrari, S Luza, R Aldunate and NC Inestrosa ´ ´ ´ Departamento de Biologıa Celular y Molecular, Facultad de Ciencias Biologicas, Pontiﬁcia Universidad Catolica de Chile, Santiago, Chile

Acetylcholinesterase (AChE), the enzyme involved in the hydrolysis of the neurotransmitter acetylcholine, has been implicated in non-cholinergic actions which may play a role in neuro- degenerative diseases such as Alzheimer’s disease. To study the potential cytotoxicity of brain AChE, the effects of affinity purified AChE were analyzed on neuronal (Neuro 2a) and glial-like (B12) cells. LDH release and MTT reduction assays showed that AChE was toxic; the toxicity was dependent on the enzyme concentration, time of incubation and cellular density. The toxic effect of AChE was not related to its catalytic activity, since the anti-cholinesterase drug BW284C51 and heat inactivation were unable to block the effects of the enzyme. When cells were incubated at 4؇C, toxicity was completely blocked, in contrast to cells incubated at -37؇C. The presence of serum in the culture medium inhibited the toxic effects of AChE. Cyto plasmic shrinkage, condensation and fragmentation of nucleus as well as DNA strand breaks detected with the TUNEL technique indicated that apoptotic cell death is involved in the effect of AChE. Considering that we have previously shown that AChE promotes the assembly of ␤-amyloid peptide into neurotoxic amyloid fibrils, it is conceivable that the neurotoxicity of AChE shown here may play a role in the neuronal degeneration observed in Alzheimer’s disease. Keywords: acetylcholinesterase; senile plaques; neurotoxicity; non-cholinergic action; Alzheimer’s disease

Introduction ronal degeneration arises in AD are still unclear.8 The demonstration that the amyloid-␤-peptide (A␤), a small Alzheimer’s disease (AD) is a progressive neurodegen- polypeptide of 40–42 amino acids, the principal con- erative disease that involves loss of memory and higher stituent of senile plaques and vascular deposits,3,9 is cognitive functions.1 Neurodegeneration involves spe- neurotoxic in cell cultures gave rise to the hypothesis cific systems such as the nucleus basalis of Meynert that A␤ may be the primary cause of neuronal degener- and the hippocampus.2–5 Neuropathologically, the dis- ation in AD.6,8,10 However, the selective nature of the ease is characterized by the presence of amyloid neuronal populations damaged in AD suggests that deposits in the brain, which are associated with senile other factors may also be involved.11–14 plaques and neurofibrillary tangles.3,5 AD is a complex Several studies on brains displaying AD lesions have hereditary and sporadic disorder, linked to mutations shown changes in the expression and distribution of in many genes, which give rise to similar clinical and acetylcholinesterase (AChE), the enzyme responsible pathological phenotypes. However, because known for the hydrolysis of the neurotransmitter acetylcho- mutations on three identified loci on chromosomes 14, line.15–17 Firstly, AChE activity is lost in specific 1, and 21 (presenilin I, presenilin II and amyloid pre- regions of the AD brain, such as the cortex, hippocam- cursor protein) only account for hereditary cases (10– pus, amygdala and nucleus basalis of Meynert.2,18,19 15% of all AD cases), there is a need to search for the Secondly, it was shown that transgenic mice overex- mechanisms responsible for the progressive cognitive pressing human AChE in brain neurons underwent decline observed in sporadic cases, which correspond progressive cognitive deterioration.20 Thirdly, the rela- to the great majority of AD patients.5–7 Although there tive proportions of the different forms of AChE change, is increasing interest in studying the molecular compo- with a decrease in the tetrameric globular G4 AChE nents of the lesions, the mechanisms by which neu- 21–23 24 form and a parallel increase in the monomeric G1 and the asymmetric AChE forms,23 although the latter normally corresponds to a small fraction of the total Correspondence: NC Inestrosa, PhD, Molecular Neurobiology enzyme present in human brain. Finally, AChE has Unit, Catholic University of Chile, PO Box 114-D, Santiago, Chile. ␤ E-mail: ninestrȰgenes.bio.puc.cl also been found to co-localize with A deposits, such Received 21 August 1997; revised 30 December 1997; accepted 5 as those present in pre-amyloid diffuse deposits, January 1998 mature senile plaques and cerebral blood vessels.18,25–27 AChE toxicity in CNS cells ´ FH Calderon et al 248 All these facts suggest that AChE may play a role in saturated humidity. Neuro 2a and B12 cells were dif- the pathogenesis of AD.11,28 ferentiated by incubation with 5 mM and 1 mM dibu- A␤ deposition is an important step in the neurodeg- tyryl-cAMP respectively for 48 h prior to the experi- enerative processes associated with AD.3,6,8 However, ment. there is as yet no explanation for the selective death of specific populations of neurons in AD brains. It is Purification of bovine AChE possible that components of senile plaques other than The enzyme was purified from bovine caudate nucleus A␤ are involved in this selective neuronal cell death using an N-methylacridinium-Sepharose affinity col- and AChE is one such candidate. In fact, among the umn as described previously.35 This procedure allowed

neurons that degenerate in AD, there are populations the purification of the G4 form of AChE, with a specific that do not contain choline acetyltransferase yet still activity of 6000 U mg−1 of protein and almost 100% express AChE,29,30 suggesting that a major character- purity according to SDS-polyacrylamide gel electro- istic of degenerative neurons could be the expression phoresis (SDS-PAGE). of AChE.11,30 Furthermore, most of the cortical AChE activity present in AD brain has been found associated AChE assay with senile plaques, where it co-localizes with A␤ The enzymatic activity of AChE was measured using deposits probably promoting the aggregation of A␤ into the Ellman assay,36 as described previously.37 amyloid fibrils, thus increasing its toxicity.13,14 In order to determine whether AChE plays an active role in cell Cytotoxic assays damage, the effect of exogenously applied AChE was Neuro 2a and B12 cells were dissociated by trypsiniz- examined on a neuronal and a glial-like cell line. A ation and seeded at a density of 3–4 × 103 cells per well preliminary account of this work has been presented on 96-well microtiter plates in 100 ␮l of growth elsewhere.31 medium and maintained at 37°C with an atmosphere

of 5% CO2. After 24 h culture media were changed for fresh media without Phenol red and fetal bovine serum Materials and methods (FBS). AChE was added with the final concentrations Cell lines and culture indicated in the text or figure legends. To assess cyto- Neuro 2a is a neuronal cell line32 derived from mouse toxicity, two assays were utilized: the modified 3-[4,5- neuroblastoma cells.33 B12 is a glial-like cell line, dimethylthiazol-2-yl]-2,5-dipheniltetrazolium bromide derived from a collection of cell lines obtained from (MTT) assay, which measures metabolic activity and induced brain tumors in rats.34 Neuro 2a and B12 cell the lactic dehydrogenase (LDH) release assay, which lines were grown in Dulbecco’s modified Eagle’s indicates cell viability.38 The MTT assay described by medium (DMEM) supplemented with 5% fetal bovine Mossman39 was carried out by adding 11 ␮lofa5mg serum (FBS) and containing 100 ␮gml−1 streptomycin, ml−1 MTT solution to each well (110 ␮l) and incubated 100 U ml−1 penicillin and 25 ␮gml−1 fungizone and for 2–4 h, after which the reaction was stopped with ° ␮ maintained at 37 C, with an atmosphere of 5% CO2 and 100 l of a solution containing 50% dimethyl-formam-

Figure 1 Toxicity of puriﬁed AChE on B12 cells. B12 cells were incubated with increasing concentrations of the G4 bovine form of AChE for 4 h. Toxicity was evaluated using the MTT reduction assay and LDH release into the medium. The graph corresponds to one representative experiment performed in triplicate. A total of eight experiments were carried out and the relative error was consistently less than 10%. AChE induced a concentration-dependent toxic effect as can be appreciated by the cytotoxicity parameters, with a gradual decrease in MTT reduction and an increase in LDH release. The control vehicle

gave no decrease in MTT reduction or rise in LDH release. On the right, an SDS-PAGE 12% shows the purified G4 enzyme (3 ␮g) from bovine caudate nucleus. AChE concentrations were calculated taking into account the total number of catalytic sub- units present in the tetrameric enzyme. AChE toxicity in CNS cells ´ FH Calderon et al 249 Morphological changes: phase contrast and fluorescence microscopy Neuro 2a and B12 cells in culture medium supplemented as described earlier were plated at a density of 3 × 104 cells per ml on glass coverslips. Cultures ° were left at 37 C with 5% CO2 for 7 h. The culture medium was then changed for fresh medium supplemented with 1% FBS and antibiotics containing AChE at the desired concentrations for 13–16 h. Con- trast phase micrographs of living cells were obtained using a Nikon phase inverted microscope. For phalloidin labeling experiments cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 1% Tween 20 in PBS and then labeled with 50 ␮M fluorescein iso- thiocyanate (FITC) conjugated phalloidin for 2 h at room temperature. After being washed with PBS, cells were mounted in 90% glycerol and labeling was observed under an Axioplan Zeiss fluorescent microscope. In situ detection of DNA cleavage Neuro 2a and B12 cells were plated at a density of 1.5 × 104 per well on 24-well plates in 0.5 ml of growth medium 24 h before the experiment. After incubation with different concentrations of AChE for 2 h, cells were fixed with 4% paraformaldehyde in PBS solution, pH 7.4 for 30 min at room temperature. Cells were then

rinsed with PBS and incubated with 0.3% H2O2 in methanol for 30 min at room temperature to block endogenous peroxidase. After rinsing with PBS, cells were incubated in permeabilization solution 0.1% Tri- ton X-100 in 0.1% sodium citrate for 2 min on ice (4°C). Then cell cultures were processed with the In Situ Cell Death Detection kit POD (Boehringer Mannheim, Mannheim, Germany) based on terminal deoxynucleotidil transferase (TdT) mediated fluor- 40,41 Figure 2 Toxicity of AChE is time- and cell density-depen- escein-dUTP nick end labeling (TUNEL) following dent. (a) B12 cells were incubated with 60 nM AChE for dif- the manufacturer’s instructions. The specificity of the ferent periods of time. For the MTT reduction assay, MTT method was determined by processing of duplicate was added to the culture dish 2 h before the end of each incu- samples in the absence of deoxynucleotide transferase. bation period. AChE induced a gradual decrease in MTT reduction and an increase in LDH release as time passed. Results (b) B12 cells were seeded at different densities and the next day were exposed to 60 nM AChE for 8 h. A greater decrease Toxicity of AChE: effect of enzyme concentration, in MTT reduction and increase in LDH release can be seen time of incubation and cell density at lower cell densities. In both cases, the figure corresponds To evaluate whether brain AChE plays a role in cell to one representative experiment performed in triplicate of a damage, the effect of exogenously-applied enzyme on series of 3–5 experiments carried out in each case and the neuronal and glial-like cell lines was examined. Cells relative error was consistently less than 10%. were cultured in the presence of the bovine affinity- purified brain AChE. Toxicity was evaluated using the MTT reduction assay, an indicator of cell redox activity which serves as a marker for cellular metabolic state, and the LDH release assay which measures the release ide and 20% SDS (pH 4.8). Absorption values at 540– of this cytoplasmic soluble enzyme into the media after 650 nm were determined the following day, using an cell lysis. Figure 1 shows the concentration-dependent automatic microtiter plate reader (Uniskan, Helsinki, toxic response of B12 cells incubated with increasing Finland) and MTT reduction results were expressed as concentrations of brain AChE. The increasing concen- a percentage of control (untreated) cells. LDH release trations caused cell death, as reflected by the increasing was measured according to the manufacturer’s instruc- LDH released. As a control, bovine serum albumin tions (Promega, WI, USA) and the results were does not show cell death even at high protein concen- expressed as a percentage of the total cellular LDH con- trations (500 ␮M) up to 5000 times the amount used tent of untreated cells. for AChE (data not shown). AChE toxicity in CNS cells ´ FH Calderon et al 250

Figure 3 Effect of AChE on B12 cells. Living cells were viewed by Contrast Phase microscopy with different concentrations of AChE. (a) Control cells are polyhedral and have conspicuous processes. Cells incubated with 15 nM AChE (b) appeared swollen, having lost their polyhedral shape and long processes. Cells often became round in shape. Cells incubated with 46 nM AChE (c) lost most of their processes and appeared much more rounded. Cells incubated with 80 nM AChE (d) developed blebs on plasma membrane or cell lysis. Bar = 60 ␮m. Fluorescein conjugated to phalloidin to reveal actin filaments. Phalloidin showed actin bundles forming clusters of adhesion foci, stress fibers and diffuse labeling of the cell cortex in control cells (e). Cells lost most of their processes, the periphery is devoid of actin filaments and stress fibers and actin fiber clusters are rare and smaller than control cells. Bar = 30 ␮m. AChE toxicity in CNS cells ´ FH Calderon et al 251

Figure 4 Effect of AChE on the morphology of neuronal Neuro 2a cells. Contrast Phase microscopy of differentiated Neuro 2a cells (a,b) and fluorescence microscopy of fluorescein-phalloidin labeled undifferentiated Neuro 2a cells (c,d). Differentiated control cells have an extended soma and long branched neurites (a). Differentiated Neuro 2a cells incubated with 46 nM AChE (2 h) are turned, rounded, lost a great number of neurites and seem unbranched (b). Undifferentiated control cells (c) have extended lamellipodia with terminal microspikes and some short filopodia. Cells incubated with AChE (d) lost most of their processes and appear more rounded. Phalloidin labeling reveals fewer and shorter microspikes. (a and b), bar = 60 ␮m. (c and d), bar = 30 ␮m.

The toxic effect of AChE was also time-dependent. Morphological changes after addition of AChE to Greater toxicity was observed when B12 cells treated cultured cells with 60 nM AChE were incubated for longer periods of The morphology of cells exposed to AChE was clearly time (Figure 2a). AChE toxicity was also dependent on affected in a concentration-dependent manner, the cellular density of the cultures. Exposure to AChE untreated B12 cells plated on plastic possessed good had a greater deleterious effect on cells cultured at low adhesive properties and normally showed several well cell densities (Figure 2b). The toxic effects of AChE developed extensions (Figure 3a). However at low observed on B12 cells were also seen on Neuro 2a cells, enzyme concentration, cells appeared swollen and a neuronal cell line as well as on differentiated Neuro gradually lost their extensions (Figure 3b,c). They 2a and B12 cells (Table 1). became rounded (Figure 3c) and some developed blebs on the plasma membrane or underwent cell lysis (Figure 3d, see arrows). More detailed studies using FITC conjugated phalloidin, showed changes in actin Table 1 Effect of AChE on the survival of neuronal and glial- filament arrangement after exposure to AChE. Normal like cells B12 cells showed actin filaments arranged in conspicuous bundles and forming clusters at focal contacts Cell type % MTT % LDH release reduction (Figure 3e, see arrows), whereas after treatment with AChE, phalloidin labeling became lighter and clusters Neuro 2a 4.40 ± 5.00 83.90 ± 2.13 were smaller than in control cells (Figure 3f). One of Neuro 2a-differentiated 14.41 ± 3.14 62.5 ± 2.29 the first events following AChE treatment was the dis- B12-differentiated 0.45 ± 9.07 89.00 ± 3.57 ruption of focal adhesion contacts. AChE was also toxic to differentiated Neuro 2a Values represent mean ± s.d. of one trial done by triplicate. (Figure 4a) and B12 cells. When these cells were Cells were differentiated during 48 h with 5 mM dibutyryl exposed to AChE, they showed loss of neurite exten- cAMP for Neuro 2a and 1 mM dibutyryl cAMP for B12 cells. sions and became rounded, often showing blebs on the Cells were then incubated with 92 nM AChE for 4 h, under surface or cell lysis (Figure 4b). Undifferentiated Neuro the conditions described under Materials and methods sec- 2a cells showed short microspikes projecting from the tion. edge of their lamellipodia which presented a brush-like AChE toxicity in CNS cells ´ FH Calderon et al 252 distribution under normal conditions (Figure 4c). apoptosis.42 No positive staining for apoptosis was Exposure to AChE changed the labeling pattern, cells observed in control cultures of Neuro 2a (Figure 5a) became rounded, displayed fewer processes and lighter and B12 (Figure 5b) cells which conserved the globular staining (Figure 4d). Even cells with elongated pro- shape of nuclei and the extended morphology of each cesses showed only few or no actin bundles. cell line after the histochemical procedure.

AChE induced DNA fragmentation The toxic effect of AChE is not related to its catalytic In order to approach the potential mechanism involved activity in the cell death triggered by AChE, both Neuro 2a and In order to elucidate whether or not the toxic effect of B12 glial-like cells were treated with AChE and then AChE depends on its catalytic activity, various treat- ﬁxed and processed with the TUNEL assay to detect ments inhibiting its enzymatic activity were tested. The DNA fragmentation. This technique allows the speciﬁc enzyme was inhibited with BW284C51, a potent labeling of 3′-OH ends of DNA generated in apoptotic reversible inhibitor of the active site.43 Pre-inhibited cells.40 The incubation of AChE with Neuro 2a and B12 AChE was applied to the culture media in the presence cells induced the appearance of round cells with of the inhibitor and the catalytic activity of AChE was intense nuclear staining, cytoplasmic shrinkage, controlled both before and during the experiment. Con- nuclear condensation and fragmentation (Figure 5b,d), trols with the inhibitors alone were also done. Under all these morphological features characteristic of such conditions, AChE was still toxic to B12 cells

a b

c d

Figure 5 In situ detection of DNA degradation (TUNEL) induced by AChE. Neuro 2a control cells (a) and B12 control cells (c) have a conserved morphology characteristic of each cell line, with an extended soma, polyhedral in shape and with globular and lightly stained nuclei. Neuro 2a cells exposed to 15 nM AChE (b) became rounded, they lost many of their neurites and the nuclei are smaller, intensely dark-stained and fragmented (arrows). Treatment of B12 cells with 15 nM AChE (d) shows a decrease in cell number due to the AChE treatment. Cells became rounded, the cytoplasm is shrunken and the nuclei are condensed and intensely dark-stained, arrows show apoptotic cells. The dark label in neurites in Neuro 2a control cells (a) is unspeciﬁc labeling due to the staining procedure. Bar = 60 ␮M. AChE toxicity in CNS cells ´ FH Calderon et al 253 (Figure 6). Decamethonium, an inhibitor of both the 4°C, the same concentrations of AChE did not induce active and peripheral sites of AChE,44 was also unable changes in MTT reduction or LDH release (Figure 8). to block the toxicity of AChE (data not shown). The enzymatic activity of AChE was also eliminated by Discussion boiling the enzyme prior to its addition to the cell cultures. The heated enzyme was equally toxic to B12 cell AChE accumulates in senile plaques, which are cultures (Figure 6), showing the same concentration- strongly associated with the neuronal degeneration and time-dependent effect as the native enzyme. that occurs in AD. In this context, AChE could have a toxic effect on brain cells that could be associated with AChE toxicity is inhibited by FBS and by the incubation of cells at low temperatures Fetal bovine serum (FBS) was found to decrease AChE toxicity. When cells were incubated with AChE in complete growth media, higher concentrations of AChE were required to show its toxic effect and this effect was almost completely blocked by 5 or 10% FBS (Figure 7). Preliminary chromatographic fractionation studies of FBS indicated that high molecular weight molecules are involved in the ability of FBS to protect against cell death. AChE toxicity was also dependent on the temperature at which cell cultures were incubated. After 1 h incubation at 37°C with increasing AChE concentrations, B12 cells showed a clear decrease in MTT reduction and an increase in LDH release. In contrast, when these cells were incubated at

Figure 7 Toxicity of AChE is prevented by FBS. B12 cells were incubated with media containing the indicated concentrations of FBS and 76 nM AChE for 12 h, after which the cytotoxicity parameters were evaluated. As can be seen, MTT reduction was almost totally eliminated and LDH release greatly inhibited, by concentrations of FBS over 5%.

Figure 6 Toxicity of AChE is independent of its catalytic activity. The catalytic activity of AChE was inhibited or inactivated using different treatments. Firstly AChE was either pre-inhibited using BW284C51 or inactivated by heating before being added to the cell media and incubated for 4 h. Cytotoxicity parameters were then evaluated. B12 cells alone (control), incubated with 46 nM AChE previously inhibited with BW284C51 (AChE + BW), with BW184C51 alone as a Figure 8 Toxicity of AChE is sensitive to temperature. B12 control (BW) or with AChE inactivated by heating (denatured cells were incubated with AChE at the indicated concen- AChE). The toxicity of AChE was not prevented either by the trations for 1 h at 37°Corat4°C and then LDH released was inhibition or inactivation of the catalytic site of the enzyme. measured and MTT reduction was determined for a period The catalytic activity of AChE was controlled using the Ell- of 2 h at 37°C. The figure corresponds to one representative man assay,36 at the beginning and at the end of the experi- experiment done in triplicate from a series of four experiment. ments. AChE toxicity in CNS cells ´ FH Calderon et al 254 neuronal degeneration.14 We analyzed the effects of related to neuronal degeneration.8 Third, it has been exogenously applied affinity purified brain AChE on reported that AChE is able to promote the aggregation neuronal and glial cells in culture. Our results show of the A␤ peptide,12 a condition that increases the A␤ that low concentrations of AChE (in the nM range) toxicity.8 All these facts suggest that AChE may indeed induced morphological and metabolic changes in be involved in the pathogenesis of AD. The fact that Neuro 2a and B12 cells. Both cell types degenerated AChE is toxic to cells in vitro, supports the conclusion and eventually underwent cell death when exposed to that AChE may indeed contribute to neurotoxicity in higher levels of AChE. These responses were not only vivo. dependent on enzyme concentration, but also on the time of exposure and cell density. Acknowledgements The toxic effects of AChE were independent of its catalytic active site. Neither an inhibitor blocking its We thank Dr David Schubert from the Salk Institute, active site nor heat inactivation of the enzyme modified La Jolla, CA for sharing with us the B12 cells. This its cytotoxic effect. Effects independent of the catalytic work was supported by Doctoral Thesis Grants from activity of AChE have described as non-cholinergic FONDECYT No. 29600-53 (to FHC), No. 29600-48 (to actions of the enzyme, which include cellular effects SL) and No. 29700-73 (G De F). This research was orig- such as the induction of neurite outgrowth,45 respir- inally proposed as part of a Presidential Chair in atory bursts in macrophages and behavioral changes in Science awarded to Dr Inestrosa by the Chilean rats11,46–48 Government. 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