Neuroscience Vol. 111, No. 3, pp. 649^656, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain PII: S0306-4522(02)00031-3 0306-4522 / 02 $22.00+0.00 www.neuroscience-ibro.com A NON-CHOLINERGIC, TROPHIC ACTION OF ACETYLCHOLINESTERASE ON HIPPOCAMPAL NEURONES IN VITRO: MOLECULAR MECHANISMS T. DAYÃ and S. A. GREENFIELD Department of Pharmacology, University of Oxford, Mans¢eld Road, Oxford OX1 3QT, UK AbstractöIn this study neurite outgrowth from cultured hippocampal neurones was increased by addition of acetylcho- linesterase acting in a non-cholinergic manner. Only monomeric acetylcholinesterase, a form of acetylcholinesterase dominant in development, increased neurite outgrowth (3^10 U/ml); moreover this e¡ect was not blocked by active site blockers (echothiophate and galanthamine) but was sensitive to the addition ofperipheral site blockers (fasciculinand BW284c51). It appears therefore that acetylcholinesterase has alternative, non-cholinergic functions, one of which could be in development, via a peripheral site. The possibility ofa causal relationship between neurite outgrowth and calcium in£ux was explored using a spectrum ofacetylcholinesterase variants, inhibitors and calcium channel blockers. Acetyl- cholinesterase regulation ofoutgrowth was shown to depend on an in£ux ofextracellular calcium speci¢cally via the L-type voltage-gated calcium channel. In summary, we propose that, independent ofits catalytic activity, a selective formofacetylcholinesterase has a role in the development ofhippocampal neurones via a selective voltage-gated calcium channel. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: neurite, outgrowth, neurotrophic, tissue culture, nimodipine. Acetylcholinesterase (AChE, EC 3.1.1.7) is well known used to demonstrate that AChE promotes neurite out- for its role in the hydrolysis of acetylcholine (ACh). growth at least in part through an adhesive function However, it has been shown in several brain regions (Grifman et al., 1998; Sharma et al., 2001). that there is a disparity between levels ofAChE and These studies have demonstrated that AChE is actively choline acetyltransferase, a more reliable marker of cho- involved in neurite outgrowth since selective action of linergic neurones (Silver, 1974). Moreover, a soluble AChE inhibitors retards neuritic outgrowth and neuro- form of AChE has been identi¢ed that is secreted, an nal migration. Whilst a tetrameric form (G4) is dominant action which is beyond the requirements ofcholinergic within the adult CNS, lower weight forms (monomeric transmission (Green¢eld, 1991b). Hence AChE may have [G1] and dimeric [G2]) are most abundant in the devel- a further role distinct from its enzymatic function (Silver, oping CNS. Thus, in addition to the proposal that AChE 1974; Green¢eld, 1984, 1991a,b, 1992; Balasubramanian has a non-cholinergic developmental role, it has also and Bhanumathy, 1993; Appleyard, 1994; Darboux et been postulated that speci¢c developmental forms of al., 1996; Grifman et al., 1998; Tsigelny et al., 2000; AChE exist (Drews, 1975). In an attempt to identify Soreq and Seidman, 2001). More direct evidence for a selective forms of AChE responsible for these non-clas- non-cholinergic, developmental role for AChE has sical actions, this study compared for the ¢rst time the emerged from studies of cultured neurones (Gupta and actions ofmonomeric and tetrameric AChE on the Bigbee, 1992; Layer et al., 1993; Dupree and Bigbee, development ofthe hippocampal neurones in vitro. 1994; Jones et al., 1995; Small et al., 1995; Holmes et The early survival and development ofneurones is al., 1997). More recently, antisense technology has been dependent on levels ofcalcium (Ca 2þ) in£ux through voltage-gated calcium channels (VGCC)(Meir et al., 1999). Neurotransmitter-controlled Ca2þ in£ux can also *Corresponding author. Tel.: +44-1865-271627; direct remodelling in the mature CNS, whilst Ca2þ £ux is fax: +44-1865-271853. a necessary step in several signal transduction pathways E-mail address: [email protected] (T. Day). controlling neurite outgrowth (Mattson, 1992; Sa¡ell et Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase; al., 1992). Based on biophysical and pharmacological BChE, butyrylcholinesterase; BW284c51, 1:5-bis(4-allydimethyl- data these channels can be divided into three distinct ammoniumphenyl)-pentan-3-one-dibromide; DIV, days in vitro; groups (Hagiwara et al., 1975; Usowicz et al., 1992; Fas, fasciculin; G1, monomeric AChE; G2, dimeric AChE; G4, tetrameric AChE; LDH, lactate dehydrogenase; MAP, microtu- Cherksey et al., 1991): (1) low-voltage-activated channels bule-associated protein; NGF, nerve growth factor; P, postnatal; blocked by nickel, (2) a set ofmoderate-conductance, PAS, peripheral anionic site; rAChE, recombinant AChE; high-voltage-activated channels (N, R, and P/Q) blocked VGCC, voltage-gated calcium channel; UV, ultraviolet. by funnel web spider peptide toxin, g-agatoxin VIA and 649 NSC 5485 6-5-02 650 T. Day and S. A. Green¢eld Fig. 1. Hippocampal cell culture maintained in serum-free medium 14 DIV and processed for MAP-2 immunohistochemistry. This immunochemistry formed the basis of this investigation. Scale bar = 50 Wm. (3) a large-conductance L-type channel blocked by the Drugs and solutions dihydropyridines, e.g. nimodipine. The present study investigated the e¡ect ofAChE and Cultures were maintained and all treatments were given in g prewarmed, fresh ¢ltered serum-free medium (Dulbecco’s mini- VGCC blockers, nimodipine, -agatoxin VIA, conotoxin mal essential medium plus B-12 supplement) containing antimi- GVIA and soluble spider toxin (sFTX), in the develop- totic agents (Sigma-Aldrich) to prevent the over-proliferation of ment ofhippocampal neurones using the following glia cells and Fungizone0 (Life Technologies, Paisley, UK) to parameters: neurite outgrowth, lactate dehydrogenase minimise the risk ofinfection.At this point it is relevant to state (LDH) release, cell body area and primary neurite num- that B-12 is su⁄cient to replace serum in the medium. All inhib- itors were solubilised in water at 10 mM and stored in 50-Wl ber (Holmes et al., 1995, 1997; Heng et al., 1999). aliquots at 320‡C. Fasciculin (Fas; Latoxan, France) was solu- bilised in water to give a 10 WM stock. Nimodipine, sFTX 3.3 and g-agatoxin (CN Biosciences, Nottingham, UK) were made EXPERIMENTAL PROCEDURES up as 1 M stock solutions in 0.1% dimethyl sulphoxide and stored as aliquoted fractions at 320‡C. g-Conotoxin GVIA (Tocris-Cookson, Bristol, UK) was made as 1 mM stock and Puri¢cation of AChE maintained at 320‡C. The puri¢cation oftetrameric AChE (G 4) was carried out by Dr Jane Loughlin (Department ofPharmacology, University of Hippocampal slice cultures Oxford), from a commercial stock of electric eel AChE (Sigma- Aldrich, Poole, UK; type V-S, 1000 U/ml) using procainamide The organotypic slice culture system was chosen to investigate ECH Sepharose a⁄nity chromatography (Novales-Li, 1994). the e¡ects ofcholinesterase due to the complexity ofthe prep- Mouse recombinant AChE (rAChE) was kindly provided by aration. These cultures resemble as closely as possible the phys- Professor Palmer Taylor (Department of Pharmacology, Uni- iological environment in the developing rat brain. versity ofCalifornia,San Diego, CA, USA). The enzymes Hippocampal slices were prepared from postnatal day 7 (P7) were produced from stable transfections of constructs from Wistar rats (Harlan UK, Oxford, UK) (Ga«hwiler, 1981, 1984). the mouse pcDNA-3 clone. All preparations were expressed in In brief, rats were decapitated by a quick scissor cut at the level a HEK 293 cell line and shipped to Oxford in foetal calf serum ofthe foramenmagnum. Excess cortex and adhering meninges with 20% glycerol on dry ice. were removed before the hippocampi were placed ventral sur- AChE puri¢cation was assessed using gel ¢ltration with ultra- face down on a McIlwain tissue chopper (The Mickle Lab., violet (UV) detection (SMART1, Fig. 1) and AChE activity for Engineering Company, Surrey, UK). Coronal sections (400 each molecular form was determined using the spectrophoto- Wm) were cut and attached to cleaned, sterile poly-D-lysine- metric Ellman assay (Ellman et al., 1961). coated coverslips by means ofa plasma clot, formedby mixing In brief, 25-Wl aliquots ofsample were added to 96-well a solution ofchicken plasma with bovine thrombin. microtitre plates, followed by the addition of 175 Wl Ellman Cultures were treated for 14 days in vitro (DIV) with serum- reagent. Absorbance was determined at 405 nm over a 10-min free medium supplemented with cholinesterase in the presence or time span using a Molecular Devices UV plate reader (Alpha absence ofcholinesterase inhibitors. Ca 2þ channel blockers were Laboratories, Hampshire, UK). All cholinesterases were added for 24 h prior to ¢xation. Cultures were changed biweekly expressed in U/ml. using supplemented serum-free medium, used in order to mini- The Ellman assay was used to determine cholinesterase levels mise the amount ofcholinesterase in the system. 14 DIV was in the culture system (culture medium and chicken plasma), to chosen as a time point prior to the cultures becoming less con¢rm the ¢nal concentration ofcholinesterase in the supple- healthy and cell necrosis occurring. mented culture medium and to con¢rm that the concentration of All experiments were conducted in accordance with the Ani- inhibitors added was su⁄cient to inhibit levels ofcholinesterase mals Scienti¢c Procedures Act (1986), approved by Her Majes- within the culture system. The Bradford method was used to ty’s Government Home O⁄ce. All e¡orts were made to determine protein content (Dang et al., 1997). minimise the number ofanimals used and their su¡ering. NSC 5485 6-5-02 AChE increases neurite outgrowth via voltage gated calcium channels 651 Fig. 2. Serum-free medium supplemented with 3 U/ml G1 rAChE signi¢cantly increased neurite outgrowth ofhippocampal cultures, maintained for 14 DIV when compared to a medium control group. Addition of Fas to the AChE peripheral site blocker alone had no e¡ect on any parameter studied.