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ARTICLE IN PRESSBA-6658; No. of Pages 17

Neurobiology of Aging xxx (2007) xxx–xxx

Inhibition of acetylcholinesterase in CSF versus brain assessed by11C-PMP PET in AD patients treated with galantamine

T. Darreh-Shori a, A. Kadir a, O. Almkvist b, M. Grut c, A. Wall d,G. Blomquist e, B. Eriksson f, B. Langstrom d, A. Nordberg a,∗

a Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Karolinska University Hospital Huddinge,Division of Molecular Neuropharmacology, NOVUM, 5th Floor, 141 86 Stockholm, Sweden

b Department of Geriatric Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Swedenc Geriatrics Clinic, Danderyd Hospital, Stockhom, Sweden

d Uppsala PET Centre/Uppsala Imanet, Uppsala University, Swedene Department of Oncology, Radiology and Clinical Immunology, Uppsala University, Sweden

f Janssen-Cilag, Sollentuna, Sweden

Received 11 April 2006; received in revised form 13 September 2006; accepted 28 September 2006

bstract

The relationship between acetylcholinesterase (AChE) activity in the CSF and brain of patients with Alzheimer’s disease (AD) wasnvestigated in 18 mild AD patients following galantamine treatment. The first 3 months of the study had a randomized double-blind placebo-ontrolled design, during which 12 patients received galantamine (16–24 mg/day) and six patients placebo. This was followed by 9 monthsalantamine treatment in all patients. Activities and protein levels of both the “read-through” AChE (AChE-R) and the synaptic (AChE-S)ariants in CSF were assessed in parallel together with the regional brain AChE activity by 11C-PMP and PET. The AChE-S inhibitionas 30–36% in CSF, which correlated well with the in vivo AChE inhibition in the brain. No significant AChE inhibition was observed

n the placebo group. The increased level of the AChE-R protein was 16% higher than that of AChE-S. Both the AChE inhibition and thencreased level of AChE-R protein positively correlated with the patient’s performance in cognitive tests associated with visuospatial ability
nd attention. In conclusion, AChE levels in CSF closely mirror in vivo brain AChE levels prior to and after treatment with the cholinesterasenhibitors. A positive cognitive response seems to dependent on the AChE inhibition level, which is balanced by an increased protein level ofhe AChE-R variant in the patients.
2007 Elsevier Inc. All rights reserved.

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eywords: Alzheimer’s disease; Acetylcholinesterase; Positron emission to

. Introduction

The prominent role of central cholinergic pathways inearning and memory and the correlation of severe cholin-rgic deficits with cognitive impairment of patients withlzheimer’s disease (AD) have contributed to the develop-

Please cite this article in press as: Darreh-Shori, T. et al., Inhibition of acin AD patients treated with galantamine, Neurobiol Aging (2007), doi:1

ent of symptomatic cholinergic therapies. Cholinesterasenhibitors (ChEIs) act by inhibiting acetylcholinesterase

∗ Corresponding author. Tel.: +46 8 585 854 67; fax: +46 8 585 854 70.E-mail address: [email protected] (A. Nordberg).

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197-4580/$ – see front matter © 2007 Elsevier Inc. All rights reserved.oi:10.1016/j.neurobiolaging.2006.09.020

hy (PET); Cerebrospinal fluid (CSF); Red blood cells (RBC); Galantamine

AChE), the principal enzyme that hydrolyzes the cholin-rgic neurotransmitter, acetylcholine (ACh). The ChEIs haveifferent pharmacological properties. Tacrine, donepezil andalantamine are reversible ChEIs, while rivastigmine isegarded as a pseudo-irreversible inhibitor, being slowlyeversible in the aspect of the enzyme reactivation (Darreh-hori, 2006). Long-term clinical studies of all ChEIs suggestlinical efficacy characterized by short-term mild functional

etylcholinesterase in CSF versus brain assessed by 11C-PMP PET0.1016/j.neurobiolaging.2006.09.020

nd global cognitive improvements in AD patients (Birks,006) and there is evidence that they may delay the progres-ion of dementia (Giacobini, 2003; Hashimoto et al., 2005;

ori et al., 2006). Consequently, it is important to evaluate

dx.doi.org/10.1016/j.neurobiolaging.2006.09.020

mailto:[email protected]


INNBA-6658; No. of Pages 17

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ARTICLET. Darreh-Shori et al. / Neuro

he long-term pharmacodynamic outcomes of the treatmentsing different approaches.

An increased CSF AChE activity has been observed in sev-ral studies on AD patients treated with the reversible ChEIsuch as tacrine (about 50% increase) (Nordberg et al., 1999),onepezil (a four-fold increase) (Davidsson et al., 2001) andalantamine (a two-fold increase) (Davidsson et al., 2001).ositron emission tomography (PET) studies in contrast havehown an in vivo AChE inhibition in cortical brain regions foratients treated with donepezil (24–27%) by measuring theydrolysis rate of the radiolabeled acetylcholine analogue, N-11C]methyl-piperidin-4-yl propionate (11C PMP) (Bohnent al., 2005; Kuhl et al., 2000).

A clinically relevant question is therefore whether thencreased CSF AChE activity in response to the reversiblehEIs reflects development of tolerance to the treatment or isconsequence of, at least partially, stimulation of the cholin-rgic and related neuronal networks. In a preceding study, weave shown that an increased CSF AChE activity in responseo donepezil may reflect the inhibition level of CSF AChEDarreh-Shori et al., 2006a).

In addition, evaluation of changes or differential expres-ion of AChE splice variants in CSF is also shown to bemportant in addressing the above question (Darreh-Shori etl., 2004).

Various globular (G1) AChE splice variants possess theame N-termini and catalytic domain, which is about 500esidues, and corresponds to a core domain common to allChE variants. Alternative mRNA splicing of exons 2, 3nd 4 yield the common core domain transcript, which isell conserved and is sufficient to generate an active AChE

Massoulie et al., 2005; Meshorer and Soreq, 2006). Thisommon core domain may then be associated with onef three types of short C-terminal peptides, of about 40esidues, which confer characteristic hydrodynamic prop-rties, cellular distribution patterns and capacities to formuaternary associations with membrane anchoring proteins,hereby conditioning generation of a series of homomeric andeteromeric AChE molecular isoforms and their functionalocalization (Massoulie et al., 2005). The major AChE vari-nt, present in brain and muscle is the synaptic variant (theChE-S, S for synaptic, also known as the AChE-T, T fortailed”) (Massoulie et al., 2005; Meshorer and Soreq, 2006).

20-kDa hydrophobic protein, known as PRiMA (prolineich membrane anchor), is responsible for anchoring theolecular isoforms of AChE-S at synaptic cleft in the CNS

Perrier et al., 2002). The AChE-S is encoded by mRNA car-ying the common core exons plus exon 6, whereas exon 5 andseudointron 4 encode the C-termini of hematopoietic vari-nt (AChE-H) on the red blood cells and the stress-associatedread-through” AChE variants (AChE-R), respectively. The-terminal of AChE-R variant lacks cysteine residues, nec-


ssary for binding to the PRiMA and hence is assumed toender the AChE-R variant a monomeric soluble AChE iso-orms, which is expressed and secreted by neurons undercute stress or exposure to ChEIs (Kaufer et al., 1998). How-

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ver, recent findings indicate that AChE-R subunits may bencorporated into heavier AChE complexes found in humanSF (Darreh-Shori, 2006) or may interact with an intracellar

caffold protein involved in signal transduction through therotein kinase PKC �II (Birikh et al., 2003).

In general, the various AChE variants seem to beatalytically equivalent, although their oligomeric stateDarreh-Shori et al., 2004) and/or distinct levels of gly-osylation may convey kinetic differences, which in turnay explain the reported differential sensitivity of G1 and4 forms of AChE to various inhibitors (Giacobini, 1997;assoulie et al., 1999).Numerous reports indicate that a peptide sequence in the

-terminus of human synaptic AChE-S variant shows novelioactivity with both neurotrophic and neurotoxic infer-nces in certain neuronal populations in the brain throughn interaction with the �7 nicotinic acetylcholine recep-ors (nAChRs) (Day and Greenfield, 2004; Greenfield et al.,004). In comparison, other reports attribute morphogenicroperties to the AChE-R and its C-terminal peptide (ARP)n hematopoietic homeostasis following stress responsesDeutsch et al., 2002; Grisaru et al., 2001). Ex vivo, ARP-eptide is found to promote expansion and differentiationf early hematopoietic progenitor cells (Grisaru et al., 2001,006; Pick et al., 2006).

In developing brain, both catalytic and non-catalytic prop-rties of the S and R variants of AChE seem to affect cellroliferation and differentiation in the subventricular zone,nd neuronal migration to cortex (Dori et al., 2005), indicat-ng that these AChE variants and their C-peptides may playistinctive roles in neuronal migration and plasticity. Therere also evidence that cholinergic neurotransmission andence ACh levels may directly be involved in regulation ofmmune system and suppression of inflammatory processesMetz and Tracey, 2005; Pavlov et al., 2006), most likelyhrough activation of �7 nAChRs present on both periph-ral immune cells and glial cells in CNS (Saeed et al., 2005;ang et al., 2003). Indeed, changes in memory functioning

fter endotoxin exposure are associated with induction ofroinflammatory cytokines and AChE-R cleavage in humanolunteers (Cohen et al., 2003). Interestingly, diminishedeurodeterioration correlates such as dendritic deformitiesnd reactive astrocytes is observed in the brain of transgenicice over-expressing the human AChE-R compared to con-

rol or S variant transgenics, suggesting that AChE-R mayxert a neuroprotective effect (Sternfeld et al., 2000). Theselues link AChE-R variant and/or AChE activity with thenflammatory processes, such as astrocytosis and gliosis inhe AD brain (Fukuyama et al., 2001; Sternfeld et al., 2000).

The potential interplay between the AChE-S and AChE-Rariants and their molecular isoforms has also been shown toe important for assessment of the clinical response in treated


D patients (Darreh-Shori et al., 2004). In untreated ADatients, a selective decline in the expression of the AChE-

variant was observed after 1-year follow-up, while the2 AChE-S isoform was up-regulated (Darreh-Shori et al.,


IN PRESSNBA-6658; No. of Pages 17

biology of Aging xxx (2007) xxx–xxx 3

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Table 1Subject characteristics (means ± S.E.M.)

Measures Groups

Placebo Galantamine Total subjects

Demographic variablesNumber of subjects 6 12 18Male/female 3/3 7/5 10/8Age (years) 65.8 ± 3.7 70.9 ± 2.7 69.2 ± 2.2Education (years) 12.8 ± 0.9 10.9 ± 1.1 11.4 ± 0.9Duration of disease (years) 2.2 ± 0.7 5.1 ± 1.1 4.1 ± 0.8ApoE �4 carriers (+/−) 3/2 7/3 10/5MMSE at baseline 27.3 ± 0.8 25.6 ± 1.0 26.2 ± 0.7

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004). The expression pattern of these two AChE isoformsas completely opposite in CSF of AD patients receiving

ivastigmine for 1 year (Darreh-Shori et al., 2002, 2004),hereas tacrine induced more general increases in the expres-

ion of the CSF AChE isoforms (Darreh-Shori et al., 2004).high ratio of AChE-R to AChE-S is found to confer a sus-

ained cognition in AD patients after 1-year ChEI therapyDarreh-Shori et al., 2004).

In the current study, we evaluated subchronic and chronichanges of AChE activity in CSF of AD patients treated withalantamine or placebo for up to 1 year, by determining thectivity and protein levels of AChE variants in CSF. Further-ore, we investigated the inter-relationships of the changes in

he RBC or CSF AChE activities and the in vivo AChE activityn the AD patients by parallel measurement of AChE activ-ty in both cortical and non-cortical brain regions using PETreported in detail elsewhere by Kadir et al., 2007). Then, wevaluated our findings in relation to cognitive performancesf the AD patients in different neuropsychological tests toddress the above objectives.

. Methods

.1. Study design and patients

The main aim of this study was to investigate the inter-eliance of AChE activity in CSF and that seen in vivo in therain assessed in parallel by positron emission tomographyPET) in patients with AD prior to and after galantaminereatment. The patients were admitted to geriatric clinicst the Karolinska University Hospital Huddinge and theanderyd Hospital, Stockholm, Sweden for memory impair-ents. They all underwent a thorough clinical investigation

ncluding medical history, global cognitive function (MMSEnd ADAS-cog), physical- and neurological examination,creening laboratory blood tests and lumbar puncture. Theiagnosis of AD was made by exclusion of other demen-ia, in accordance with the National Institute of Neurologicalnd Communication Disorders and Stroke-Alzheimer’s dis-ase and Related Disorders Association (NINCDS-ADRDA)riteria (McKhann et al., 1984).

Eighteen mildly demented AD patients were recruited tohe study (Table 1). The first 3 months of the study wasouble-blinded placebo-controlled, when six AD patientsere randomly assigned to receive placebo (the placeboroup) and 12 patients received galantamine in flexible doses16–24 mg daily, the galantamine group). The subjects wereandomly assigned in a ratio 2:1 in favor of galantamineo 1 of 2 treatment groups according to the randomizationode generated by the sponsor. The placebo tablets weredentical in appearance, taste, and smell. After 3 months,


he placebo group started taking galantamine for 9 monthsthe Pla/Gal group) while the galantamine group continuedn galantamine. All patients and their responsible caregiversrovided written informed consent to participate in the study

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ADAS-cog/13 at baseline 19.0 ± 1.1 28.0 ± 3.4 25 ± 2.5

poE: apolipoprotein E; MMSE: Mini-Mental State Examination; ADAS-og/13: Alzheimer’s Disease Assessment Scale–cognitive subscale.

nd the study was conducted according to the Declaration ofelsinki and subsequent revisions. The Ethics Committee ofuddinge University Hospital, Sweden approved the study.

.2. Neuropsychological assessments

.2.1. Global cognitionNeuropsychological tests were performed throughout the

tudy, at baseline, after 3 weeks, and three and 12 monthsf treatment. Global cognition was assessed using the Mini-ental State Examination (MMSE) (Folstein et al., 1975),

nd the cognitive subscale of the Alzheimer’s Disease Assess-ent Scale (ADAS-cog) (Rosen et al., 1984).

.2.2. Episodic memoryEpisodic memory was evaluated using two measures from

he Stockholm Gerontology Research Center (SGRC) testBackman and Forsell, 1994) of memory for words: (1) theumber of correct responses in free recall of words (FRW-est) and (2) the d-prime value (an integration of correctesponses and false alarms following decision theory) inecognition of words (RWd-prime). In particular, these mea-ures assess abilities related to medial temporal brain activityCabeza et al., 2000).

.2.3. AttentionAttention was assessed using three measures: (1) the

umber of correct responses in the Digit Symbol responseAttention-DS) test from the revised Wechsler Adult Intel-igence Scale (Wechsler, 1981); (2) the time needed toomplete the Trailmaking A (TMA-) (Lezak, 1995); (3) theumber of correct responses in the TMB-test (Lezak, 1995).hese measures are known to assess abilities associated with

rontal-subcortical brain activity (Cummings, 1993).


Visuospatial ability was evaluated by recording the num-er of correct responses in reading and setting a clock (Luria,966). This test reflects parietal lobe function (Cahn-Weinert al., 1999).



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.3. Determination of AChE level in blood and CSF

.3.1. Collection of samplesTo evaluate cholinesterase activities in RBC, plasma and

SF, whole blood and CSF samples were collected from allatients at baseline (pre-drug samples). Then, blood was alsoollected after 3 weeks, and 3 and 12 months during the study.SF samples were taken after three and 12 months of treat-ent, which were collected by lumbar puncture in the L3/L4

r L4/L5 interspace at mornings between 9 and 12 a.m. Thelood and CSF samples were centrifuged at 2000 × g for0 min at 4 ◦C, and the plasma was separated from red bloodells. All samples were kept frozen at −70 ◦C until the assay.lasma and CSF samples were thawed and centrifuged at5,000 × g for 3 min at 4 ◦C immediately before the assay.

.3.2. RBC preparation and DFP protection assayTo evaluate the inhibition of RBC AChE activity, the

FP (diisopropylfluorophosphate, Sigma, St. Louis, MO)rotection assay described by Giacobini et al. (1996) waserformed at 1.25 dilution of RBC samples. Briefly, theBC samples were thawed and 100 �L aliquots were pre-ared in flat bottom tubes. Then, either 25 �L of a freshFP working solution (9 �M; triplicates) or vehicle working

olution (Peanut oil, Sigma; duplicates, for measuring basalChE activity). After an individually timed incubation, eachBC aliquot was immediately diluted by addition of 5.0 mL

odium–potassium phosphate buffer (50 mM, pH 7.4), whichhen was immediately frozen on dry ice and kept at −70 ◦Cntil the colorimetric enzyme assay.

.3.3. Colorimetric enzyme assays

.3.3.1. AChE activity. Specific AChE activities in CSFnd blood/plasma were measured by the modified Ellman’solorimetric assay in presence of the selective butyryl-holinesterase inhibitor, ethopropazine (Sigma, with a finaloncentration of 0.1 mM) as described previously (Darreh-hori et al., 2002; Ellman et al., 1961).

For measurement of the RBC AChE activity, the originalethod (Giacobini et al., 1996) was modified to a non-

adioactive version by using the colorimetric Ellman assayEllman et al., 1961). Briefly, prior to the colorimetric assay,he DFP and the vehicle pretreated RBC aliquots were thawedn a 20 ◦C water-bath and diluted further (7 times) in theodium-potassium phosphate buffer. All samples were runn triplicate. The AChE activity measured in the DFP or theehicle pretreated aliquots was regarded as the DFP antago-ized and the basal RBC AChE activity, respectively. Inhibi-ion of RBC AChE activity at the follow-ups was calculateds percentage of the DFP antagonism (at 3 weeks, and 3 and2 months) to the basal RBC AChE activity at baseline. Atach time interval, values were calculated separately for each


atient in comparison with the patient’s own baseline value.

.3.3.2. BuChE activity. Specific BuChE activities inSF and plasma were measured by the Ellman’s colori-

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etric assay in presence of the selective AChE inhibitor,W284C51 [1,5-bis(4-allyldimethylammoniumphenyl)entan-3-one dibromide); Sigma] with a final concentrationf 1.0 �M and the butyrylthiocholine (BTC; Sigma) as theubstrate as described previously (Darreh-Shori et al., 2002;llman et al., 1961).

.4. CSF BuChE protein level

The CSF BuChE protein levels were quantified by usingwo selective mouse monoclonal antibodies against humanuChE in a sandwich ELISA as described before (Darreh-hori et al., 2006b). Briefly, all CSF samples were dilutedour-fold with TBS-T-BSA (10 mM Tris–HCl, pH 7.4; 0.9%aCl; 0.05% Tween 20; 0.1% Bovine serum albumin, BSA;

ll from Sigma) and were then frozen at −70 ◦C until thessay. Partially purified human serum BuChE with a statedpecific activity of 12.7 U/mg (c-9971, Sigma) was used ashe standard protein. This standard was diluted in the TBS-T-SA buffer to concentrations of 2.0–0.016 ng/�L (by serial

wo-fold steps). Because the standard was not a single homo-eneous protein, its own BuChE content was computed usinghe established specific activity of 740 U/mg (Lockridge anda Du, 1978) before calculating the amounts of BuChE pro-

ein in the CSF samples.

.5. Protein levels of CSF AChE variants

Two mouse monoclonal antibodies were used to measurerotein level of the AChE-R and AChE-S splice variants: (i)he MA3-042 Ab (Affinity BioReagents, CO), which is raisedgainst cerebellar AChE (Brimijoin and Hammond, 1988;esulam et al., 1991; Rakonczay and Brimijoin, 1988). (ii)

he MAB337 Ab (Chemicon International, CA), raised againstuman RBC AChE (Zahler et al., 1996). In the current studyowever, the selectivity of these antibodies were determinedn comparison with two other well characterized antibod-es, namely (i) anti-Core Ab, a goat polyclonal Ab whichetects a peptide sequence in the N-terminal of AChE com-on to all variants (Santa Cruz Biotech., Santa Cruz, CA;19) (Darreh-Shori et al., 2004; Dori et al., 2005) and (ii)nti-AChE-R Ab, a rabbit polyclonal Ab selective towards aeptide sequence at the C-terminal of AChE, which is uniqueo the AChE-R variant (Darreh-Shori et al., 2004; Dori et al.,005).

.5.1. Immunoprecipitation analysis for selectivity of thentibodies

Conjugates of antibodies and Protein G plus-Agaroseeads (sc-2002, Santa Cruz biotechnology, CA) were pre-ared for primary antibodies (the MAB337, the MA3-042,he anti-Core and the anti-AChE-R) as well as control IgGs


ccording to standard procedure. Then, pooled CSF sam-les (n > 80 patients, 500 �L aliquots) were pre-cleared byncubation with the control IgG–Protein G plus conjugatest 4 ◦C on a rotating device for 1 h. After centrifugation, the



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re-cleared CSF supernatants were transferred to the tubesontaining the primary antibody–Protein G plus conjugatesnd incubated overnight at 4 ◦C. The pellets were collected,ashed three times, and finally resuspended in 6× reducingr non-reducing sample loading buffer (Darreh-Shori et al.,004). The samples were heated and used for immunoblotnalysis essentially as described previously (Darreh-Shori etl., 2004). However, to remove interference of the proteinands (corresponding to the light and heavy chains of therimary antibodies) with the AChE bands on blots, a biotin-abeled antibody was used as detecting antibody [MAB303b (directed against human AChE (Fambrough et al., 1982),hemicon Int)], which was labeled using a biotin labelingit [Cat #1418165, Roche, Mannheim, Germany]. Then, thelots were blocked with M-PBS-T (PBS, pH 7.4; 5% (w/v)on-fat dried milk, and 0.3% Tween 20; for 1 h at RT), incu-ated with the Biotin-MAB303 Ab (1 �g/mL in M-PBS-T;or 2 h at RT), washed with PBS-T (3 × 10 min), incubatedith streptavidin-conjugated HRP (Santa Cruz Biotechnol-gy, diluted 1/3000 in M-PBS-T) for 1 h at RT, washed withBS-T (4 × 10 min) and finally the detected protein bands on

he blots were documented as described previously (Darreh-hori et al., 2004).

.5.2. ELISA-like assay of CSF AChE variants proteinxpression

We termed the current method as ELISA-like assayecause AChE protein are immuno-captured to the wells ofn ELISA plate like the conventional ELISA, but then thenherent AChE activity of the captured protein are used ashe detecting system as is described below.

All CSF samples were diluted five times in advancen dilution buffer (10 mM Tris–HCl, pH 7.4; 0.9% NaCl;.0 mM EDTA; 0.05% Triton X-100 and 1% BSA) andere kept frozen at −20 ◦C until the assay. Partially puri-ed human AChE with a specific activity of 2.0 U/mg totalrotein content (#C1682, Sigma) was used as the standardrotein. This standard was diluted in the dilution buffer (toconcentration ranging between 2.0 and 0.15 ng/�L, by

erial two-fold steps). Nunc maxisorb ELISA plates wereoated overnight at 4 ◦C with 150 �L/well of the MA3-42 Ab (1:1000) or the MAB337-Ab (1:3000) diluted inoating buffer (0.1 M Carbonate buffer, pH 9.6, contain-ng 0.2% (w/v) sodium azide). The plates were blocked at7 ◦C for 2 h with 250 �L/well of the coating buffer con-aining 5% (w/v) BSA (Fraction V, Sigma), washed 3×ith TBS-T (10 mM Tris–HCl, pH 7.4, containing 0.9%aCl and 0.05% Tween 20), then incubated overnight at◦C with 150 �L/well of the standards (in triplicates) or

he diluted CSF samples (in duplicates), washed five timesith TBS-T, and finally incubated with acetylthiocholine-

eagent mix (200 �L/well of Na/K-phosphate buffer, pH 7.4,


ontaining 1.3 mM acetylthiocholine, 0.5 mM DTNB) for–2 h at RT (22.5 ◦C). The absorbance was read at a wave-ength of 412 nm as is described before (Darreh-Shori etl., 2002). The possible affinity of the antibodies to BuChE

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as controlled by washing the plates once with TBS andncubation with the BTC reagent mix (Darreh-Shori et al.,002).

.6. Estimation of CSF AChE inhibition

To estimate inhibition of the AChE variants’ activities inhe CSF of the patients after treatment with the reversiblehE inhibitor, galantamine, we combined the data derived

rom the direct colorimetric assay and the ELISA-like methods follows. The CSF AChE activity was first normalized tohe total protein level of the enzymes in CSF, using the fol-owing two definitions. Firstly, a baseline-normalized (BN)ctivity level (expressed as nmol/min/�g of CSF AChE),.e. the CSF AChE activity (nmol/min/mL CSF) at theaseline was divided by the protein level of the enzyme�g AChE/mL CSF) at the baseline and at the follow-upntervals, respectively. Since, the BN-activity level may over-stimate the actual inhibition values, it was used to calculatehe upper limit of CSF AChE inhibition (the Imax). Secondly,e also calculated an interval-normalized (IN) estimation,y dividing the enzyme activity at each follow-up intervaly the protein level of the enzyme at the correspondingnterval. This may underestimate the actual level, and wassed to define the lower limit of the inhibition in CSF (themin). The Imax or the Imin inhibition boundaries for theSF AChE variants were then calculated using the follow-

ng formula [the Imax or Imin = ((1 − Af (BN or IN)/Ab) × 100),here Af is the BN or IN normalized activity at the

ollow-ups, and Ab is the BN normalized activity ataseline].

.7. In vivo brain AChE level by PET

The in vivo brain AChE activity was measured as theate constant K3 for hydrolysis of the AChE substrate ana-ogue, N-[11C]methyl-piperidin-4-yl propionate (11C PMP)sing positron emission tomography (PET) and the kineticodel developed by Koeppe et al. (1999) for 11C PMP. Theethodological details are described in Kadir et al. (2007).he changes in the in vivo brain AChE activity (K3, min−1)ere calculated as the percentages of K3 value (in differ-

nt brain regions) at the follow-up intervals compared tohe baseline. Mean cortical AChE activity was calculatedy averaging the K3 values from the “region of interest”ROI) in the anterior cingulate, frontal, frontal association,arietal, parietotemporal, temporal, primary visual, sensoryotor and temporal medial lobe cortices of the left and right

rain hemispheres. For simplicity in the current study, theercent changes in the K3 value in brain regions will be


ng sections, which was defined using the following formula100 − %K3 = 100 − (K3 (f)/K3 (b) × 100), where f and b indi-ate the K3 values at the follow-ups and the baseline,espectively].



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.8. Statistical analyses

Data are expressed as mean values and standard error ofhe mean (S.E.M.). The effects of treatment at different timentervals compared to baseline were assessed using two-wayepeated measures (RM) ANOVA performed on the raw data.he within-group factor was each follow-up interval ver-us baseline and the between-group factor was the treatmentroups. A significant ANOVA result (p < 0.05) was followedy the Bonferroni–Dunn post hoc analysis that tested theignificance of results at each time point compared with base-ine (p < 0.0125 for RBC AChE inhibition and p < 0.0167or the changes in the CSF AChE levels) or between groupsp < 0.0167). Two-tailed correlation Z-test together with theon-parametric Spearman Rank test were used in correla-ion analysis, which then visualized graphically using simpleegression plot.

. Results

.1. Selectivity of the antibodies for different AChEariants

The selectivity of the antibodies were assessed by com-ining the immunoprecipitation and the subsequent reducingnd non-reducing immunoblot detection on AChE proteinn pooled CSF. Under the non-reducing condition, sev-ral different heavy AChE complexes were observed in themmunoprecipitates of the anti-Core Ab (lane 1) and the

A3-042 Ab (lane 2), but only one heavy AChE complexn the immunoprecipitates of the MAB337-Ab (lane 3) orhe anti-AChE-R Ab (lane 4, Supplementary Fig. 1a). Theeducing immunoblot analysis indicated that the MA3-042b had a greater affinity for an AChE subunit with a Mwf 70–75 kDa (lane 2 in the Supplementary Fig. 1b). This isompatible with the Mw of the full-length synaptic AChE-subunit, which has been the antigen for the production of

his antibody (Brimijoin and Hammond, 1988; Mesulam etl., 1991; Rakonczay and Brimijoin, 1988). The MAB337b or the anti-AChE-R Ab did not show detectable affin-

ty for this AChE subunit under the condition used in thistudy (lane 3 and 4 in the Supplementary Fig. 1a). Thus,n the following section the proportion of CSF AChE pro-ein determined by the MA3-042 Ab will be simply referredo as the protein level of the synaptic AChE-S isoform.owever, the same reducing immunoblot analysis on the

mmunoprecipitates of the MAB337-Ab and the anti-AChE-Ab showed that both antibodies had strong affinity only

or the 50-kDa AChE-R subunit (lanes 3 and 4 in theupplementary Fig. 1b, respectively), in agreement with ourrevious studies (Darreh-Shori et al., 2002, 2004). In addi-


ion, the ELISA-like assay (using the MAB337-Ab as theapturing Ab) detected about three-fold higher CSF AChErotein (∼1.10 �g AChE/mL CSF), compared to the MA3-42 Ab as capturing Ab (∼0.37 �g/mL CSF), which is

a4an


onsistent with the secretory nature of AChE-R variant asell as our previous report showing that AChE-R is the majorChE variant in CSF (Darreh-Shori et al., 2004). Altogether,

hese analysis suggested that the MAB337-Ab should be moreelective toward the AChE-R variant and the heavier com-lexes containing the ARP polypeptide. Hence, the AChErotein level, determined by the MAB337-Ab, will be referredo as the overall protein level of the AChE-R variant in theollowing section. However, it should be noted that someverlap in the affinity of the antibodies could not be excludedue to possible heteromeric nature of the heavier AChE iso-orms (Supplementary Fig. 1) (Darreh-Shori et al., 2002,004).

Furthermore, the MA3-042, the MAB337 antibodies didot show any significant affinity to the CSF BuChE pro-ein, since no higher hydrolytic rate than the spontaneousydrolysis of BTC (the BuChE substrate) in control wellsas observed after re-incubation of the assay plates with theTC reagent mix.

.2. Demographic data

The demographic characteristics of patients are presentedn Table 1. Although the patients in the randomized placeboroup were younger, showed shorter duration of the dis-ase, higher MMSE and lower ADAS-cog scores at baselineompared to the galantamine group, the demographic charac-eristics (age, gender, level of education, duration of disease,

MSE score and ADAS-cog score at baseline) did not showny significant difference between placebo, galantaminer total subjects groups (all p > 0.10 except ADAS-cog,= 0.09).

.3. The patients dropout

Of the 18 subjects who entered the study, 13 completedhe 12-month study. During the double blind phase (day 23),ne subject (from the galantamine group) was withdrawn dueo second-degree antrioventricular block. The event was con-idered possibly related to the study drug. Of the 17 subjectsho entered the open label phase, four patients were with-rawn: one patient from the Pla/Gal group due to chronicymphocytic leukemia (this event was considered unrelated toalantamine), one patient due to cardiac arrhythmia and syn-ope (which was considered possibly related to galantamine),ne patient due to severe myocardial infarction (the relation-hip of this event to galantamine was considered doubtful),nd one patient due to non-compliance.

During the dose escalation periods, weeks 1–6 in the dou-le blind phase for the galantamine group and weeks 14–19comparable to weeks 1–6 in the galantamine group) in thepen label phase for the placebo group who converted to


ctive treatment (the Pla/Gal group), the patients receivedmg b.i.d. for the first week, 8 mg b.i.d. for the next 4 weeksnd 12 mg b.i.d. in the 6th week. For the patients who didot tolerate the higher dose of 24 mg daily due to side effects


IN PRESSNBA-6658; No. of Pages 17

biology of Aging xxx (2007) xxx–xxx 7

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Fig. 1. Inhibition level of RBC AChE activity in response to galantaminetreatment. Open circles: the placebo group. Filled diamonds: the galan-tamine group. Filled circles: the Pla/Gal group. The values are expressed asm **

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uch as nausea, vomiting or diarrhoea, the dose was reducedt the end of 6 weeks to 16 mg daily.

At 3 months, one patient from the placebo group and threeatients from the galantamine group did not comply with CSFampling. At 9–12 months, two patients from the Pla/Galroup and four patients from the galantamine group did notomply with CSF sampling.

.4. Changes in BuChE activity and protein

No significant increase in plasma BuChE activities werebserved in the galantamine group throughout the study,hile a mild significant decline was observed in thelacebo group at the 3-week follow-up (p < 0.03, n = 6,upplementary Fig. 2A).

At baseline, the levels of CSF BuChE activity or proteinid not differ between the placebo group and the galantamineroup (p > 0.1). After 3 months of treatment, both activity androtein levels of BuChE in CSF remained unchanged in thealantamine group, which is compatible with the selectivityf galantamine for AChE (Supplementary Fig. 3). Yet again aignificant decline in the CSF BuChE protein was observed inhe placebo group after 3 months compared to baseline (7%)r the galantamine group (12%, all p < 0.05, Supplementaryig. 3B).

Following 9–12 months treatment with galantamine, noignificant changes in the levels of CSF BuChE activity orrotein were observed compared to baseline (Supplementaryig. 3).

.5. Changes in AChE levels in blood and CSF

.5.1. Plasma AChE activityAt the 3-month follow-up during the double-blinded

hase, a mild rise (5%) in the plasma AChE activity wasbserved in the galantamine group compared to baselinep < 0.04, n = 11), whereas a significant regression in thectivity of this enzyme in plasma was appeared in thelacebo group compared to baseline (6%, p < 0.04, n = 6)r the galantamine group (11%, p < 0.006, Supplementaryig. 2B).

.5.2. Inhibition of RBC AChEDuring the double-blinded phase, a weak to moder-

te but significant inhibition of the RBC AChE activityas observed in the galantamine group at the 3-week

10 ± 0.9%, n = 12) and 3-month follow-ups (11 ± 1.4%,= 11). No significant changes in the RBC AChE activityas observed in the placebo group (n = 6) at these follow-ups

Fig. 1).During the following open-labeled phase, the RBC AChE

nhibition was similar in the Pla/Gal group (Fig. 1, 10 ± 4%,


= 6) and the galantamine group (10 ± 1.3%, n = 8) after 9nd 12 months galantamine treatment, respectively.

The RBC AChE inhibition highly correlated with thelasma galantamine concentration at 3 weeks (r = 0.81,

psmg

ean ± S.E.M. p < 0.001 indicates the significant difference compared toaseline at the specified treatment intervals. The Bonferroni–Dunn post hocitation was p < 0.0125. w: weeks and m: months. The number of availableamples at each follow-up is shown in the figure.

< 0.0001) and at 3-month follow-up (r = 0.88, p < 0.0001),hile no correlation between the RBC AChE inhibition and

he plasma galantamine concentration was observed after–12 months treatment.

.5.3. Increased AChE activity in CSFThe CSF AChE activity was measured at baseline and at

- and 12-month treatment.The baseline CSF AChE activity did not differ between

he placebo and the galantamine groups (p > 0.8). After 3-onth treatment, the CSF AChE activity was increased by

5 ± 9% in the galantamine group (p < 0.02, Fig. 2A) com-ared to the baseline level and with 32% compared to thelacebo group (p < 0.03). A positive correlation was observedetween the CSF galantamine concentration and changes inhe CSF AChE activity at the 3-month follow-up (r = 0.72,< 0.004).

At the 12 months, when all patients had received galan-amine for 9–12 months, the increased CSF AChE activityas less pronounced, but still significantly higher than

he baseline level (in the overall patients group 16 ± 12%,< 0.03, Fig. 2A).

.5.4. Increased protein level of AChE variants in CSFThe baseline protein level of the AChE-S

0.60 ± 0.03 �g/mL CSF) was one third of protein level ofhe AChE-R variant (1.73 ± 0.09 �g/mL CSF, p < 0.0001).his result is in agreement with our previous findings,uggesting that the AChE-R was the most abundant AChEariant in CSF and corresponded to 65% of the total AChErotein in CSF (Darreh-Shori et al., 2006a).

The protein levels of the AChE-R or AChE-S spliceariants did not differ significantly at baseline between the


lacebo and the galantamine group. The protein levels of bothplice variants however were significantly increased after 3onths in the galantamine group, but not in the placebo

roup. In the galantamine group, the CSF protein level was


ARTICLE IN PRESSNBA-6658; No. of Pages 17

8 T. Darreh-Shori et al. / Neurobiology of Aging xxx (2007) xxx–xxx

Fig. 2. Comparison between the increased protein levels of AChE variants and the total AChE activity in CSF in response to galantamine treatment. Note thatthe moderate increase of the CSF AChE activity (A) is highly disproportional to the stronger increases observed in the protein levels of the synaptic (B) andthe “read-through” AChE variants (C) in the galantamine-treated AD patients, while both protein and activity levels of the AChE show a similar decrease inthe placebo group. Note also that the changes in the AChE activity and protein level become similar between the groups when the placebo group receivedgalantamine (the Pla/Gal group). The values are given as mean ± S.E.M. The Bonferroni–Dunn post hoc citation was p < 0.0167. ***p < 0.001, **p < 0.01 and* a , bp < 0. c

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p < 0.05 compared to baseline at the specified treatment intervals. p < 0.05months. The number of available samples at each follow-up is shown in th

.91 ± 0.07 �g/mL for the AChE-S and 2.84 ± 0.22 �g/mLor the AChE-R variant, corresponding to a 48 ± 13%ncrease in AChE-S (p < 0.006, n = 8, Fig. 2B) and 65 ± 18%ncrease in AChE-R protein compared to baseline (p < 0.007,= 8, Fig. 2C). We also found that the increased protein levelf AChE-R was 16% higher than the AChE-S variant at the 3-onth follow-up in the galantamine group (p < 0.05, Fig. 2B

nd C).In the placebo group, no significant changes were

bserved at the protein level of AChE-S or the AChE-Rariant (−7 ± 8%, p < 0.4, n = 5, Fig. 2C) at the 3-monthollow-up. The protein level of the AChE-R variant howeveras reduced in three out of five AD patients in the placeboroup (13–27% reduction).

After 9–12 months galantamine treatment, the protein lev-ls of AChE-S and AChE-R variants in the overall patientsroup were 0.90 ± 0.09 and 2.73 ± 0.25 �g/mL CSF, respec-ively. The corresponding increase in the protein levels ofhe AChE-R and AChE-S variants in CSF was comparableo the increase in protein levels observed in the galantamineroup at the 3-month interval, namely 47 ± 7% for the AChE-(p < 0.002, n = 8, Fig. 2B) and 54 ± 7% for the AChE-R

ariant (p < 0.0006, n = 8, Fig. 2C).The CSF AChE activities positively correlated with the

rotein levels of both AChE-S (r = 0.82, n = 15, p < 0.0001t baseline; r = 0.91, n = 13, p < 0.0001 at 3 months and= 0.97, n = 8, p < 0.0001 at 12 months) and the AChE-Rariants (r = 0.85, p < 0.0001 at baseline; r = 0.85, p < 0.0001t 3 months and r = 0.94, p < 0.0001 at 12 months). Further-


ore, both the CSF and plasma galantamine concentrationsositively correlated with the changes in protein levels ofChE splice variants at the 3-month follow-up (all p-values0.0001). After 9–12 months of treatment with galantamine,

onn

01 and p < 0.001 indicate differences compared to the placebo group at the-hand columns.

he plasma (but not the CSF) galantamine concentration pos-tively correlated with the increased protein level of theChE-S variant (r = 0.74, p < 0.05, n = 8). These findings

pecifically relate the changes in the protein levels of theChE variants to the galantamine concentration.

.5.5. Inhibition of AChE variants in CSFThe elevation of the protein levels of the CSF AChE vari-

nts after treatment with galantamine was disproportionallyabout two-fold) higher than the increased CSF AChE activ-ty (Fig. 2). This fit with the fact that the direct colorimetricssay measures the enzyme activity at one-forth originalalantamine concentration in the samples whereas duringhe ELISA-like assay this reversible inhibitor is completelyashed away, as may be appreciated from the normalized

ctivity of the AChE variants at the baseline and after 3nd 12 months of treatment shown in the Table 2. Bothaseline- and interval-normalized activities of the AChEplice variants showed significant reduction at the follow-ps compared to the baseline levels (Table 2). Since bothhe direct colorimetric and the ELISA-like assays are depen-ent on the activity of AChE, a reduction in the normalizedctivities indicates the level of CSF AChE inhibition byalantamine.

The averaged percentages of inhibition for the synapticnd the read-through AChE variants are also shown in theable 2. In response to the galantamine treatment, the activi-

ies of CSF AChE-S and the AChE-R variants were inhibitedy 30–36% and 24–32%, respectively (Table 2).


The RBC AChE inhibition correlated with the inhibitionf the CSF AChE at the 3-month (Fig. 3, r = 0.67, p < 0.01,= 13) and the 12-month follow-ups (r = 0.89, p < 0.01,= 8), which supports the above notion.



T. Darreh-Shori et al. / Neurobiology of Aging xxx (2007) xxx–xxx 9

Table 2Normalized activity and the averaged percentages of inhibition of AChE in CSF (means ± S.E.M.)

AChE splicevariants in CSF

Follow-up The galantamine group The placebo group (and Pla/Gal group)a

BN (nmol/min/�g) IN (nmol/min/�g) Inhibitionb (%) BN (nmol/min/�g) IN (nmol/min/�g) Inhibitionb (%)

AChE-SBaseline 2.07 ± 0.11 2.07 ± 0.11 0 2.01 ± 0.10 2.01 ± 0.10 03 months 1.24 ± 0.13** 1.48 ± 0.09** 30 ± 7 2.25 ± 0.15 2.06 ± 0.04 −8 ± 812 months 1.19 ± 0.07*** 1.32 ± 0.10** 36 ± 3 1.34 ± 0.12## 1.53 ± 0.07## 26 ± 8

AChE-RBaseline 5.86 ± 0.26 5.86 ± 0.26 0 5.71 ± 0.10 5.71 ± 0.10 03 months 3.87 ± 0.38** 5.02 ± 0.20** 24 ± 6 6.20 ± 0.43 5.68 ± 0.15 −4 ± 612 months 3.73 ± 0.16** 4.13 ± 0.24** 32 ± 3 3.96 ± 0.28*,## 4.52 ± 0.14*,## 25 ± 5

BN: baseline normalized activity, IN: interval normalized activity. At the 3-month follow-up, n = 8 in the galantamine group and n = 5 in the placebo group.At the 12-month interval, n = 4 both in the galantamine group and the Pla/Gal group. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate significant differencescompared to the baseline level. ##p < 0.01 indicates a significant difference compared to the 3-month follow-up in the placebo group. The Bonferroni–Dunnpost hoc citation was p < 0.0167.

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After 9–12 months of galantamine treatment (similar tobaseline) the activity or protein levels of AChE in CSF posi-tively correlated with the mean cortical AChE activity (K3) ofboth brain hemispheres (r = 0.97, p < 0.005, n = 5) or with the

The AD patients received placebo treatment up to the 3-month follow-uproup).b The inhibition values are the average of the Imax and Imin as defined in t

.6. AChE by 11C PMP-PET

.6.1. Regional brain AChE inhibitionA detailed presentation of the changes in the in vivo AChE

ctivity in different brain regions, as measured by PET iseported separately in Kadir et al. (2007). Briefly, 30–37%n vivo AChE inhibition was observed in the cortical brainegion of the patients treated with galantamine throughouthe study, which is consistent with the 30–36% inhibition ofhe synaptic AChE variant in the CSF of the patients (seerevious section).

.6.2. RBC or CSF AChE in relation to the brain AChEctivity

Positive correlations were observed between the in vivorain AChE activity (K3) and the AChE activity or proteinevel in CSF.


.6.2.1. At the baseline. The activity of AChE in CSF posi-ively correlated with the mean in vivo AChE activity in theortical (Fig. 4, r = 0.7, p < 0.004, n = 15) or the overall brainegions (r = 0.6, p < 0.02, n = 15) in the left, but not the right,

ig. 3. Positive correlation between the changes in the AChE activities inSF and red blood cells at 3-month follow-up. Open squares: the placeboroup. Filled diamonds: the galantamine group.

Fcw

cebo group), then they started taking galantamine for 9 months (the Pla/Gal

on 2.

rain hemisphere. These findings suggest that an AD patientith a high in vivo AChE activity in the brain (i.e. a higher3 value) also had a high AChE activity in CSF.

.6.2.2. After 3–12 months. After 3 months galantaminereatment, the in vivo AChE inhibition in all brain regionsositively correlated with the changes in AChEs in CSF andBC (Table 3). A representative plot is shown in Fig. 5, which

llustrates the positive correlation between the mean in vivoChE inhibition in overall brain regions and the inhibitionf the synaptic variant of AChE in CSF (r = 0.85, p < 0.0002,= 12).


ig. 4. Positive correlation between the in vivo AChE activity in overallortical brain regions and the activity of the synaptic AChE in CSF of patientsith mild AD prior to commencing treatment with galantamine.


Please cite this article in press as: Darreh-Shori, T. et al., Inhibition of acetylcholinesterase in CSF versus brain assessed by 11C-PMP PETin AD patients treated with galantamine, Neurobiol Aging (2007), doi:10.1016/j.neurobiolaging.2006.09.020


10 T. Darreh-Shori et al. / Neurobiology of Aging xxx (2007) xxx–xxx

Table 3Correlations between the CSF or RBC and the brain AChE activities after 3 months

In vivo inhibition in thebrain regions (100 − %K3)

CSF AChE activity(% increased)

AChE-S variant(% inhibition)

AChE-R variant(% inhibition)

RBC AChE(% inhibition)

r (n = 12) p r (n = 12) p r (n = 12) p r (n = 16) p

Frontal association cortexRt 0.82 <0.0006 0.79 <0.002 0.77 <0.003 0.53 <0.04

Frontal cortexRt 0.83 <0.0003 0.81 <0.0007 0.77 <0.003 0.42 <0.11

Temporal medial lobeRt 0.64 <0.03 0.60 <0.04 0.52 <0.08 0.53 <0.04

Parietal cortexRt 0.76 <0.003 0.81 <0.0007 0.72 <0.007 0.42 <0.11

Parietotemporal cortexRt 0.69 <0.01 0.72 <0.006 0.63 <0.03 0.37 <0.17

Temporal cortexRt 0.70 <0.009 0.72 <0.007 0.67 <0.02 0.59 <0.02

Cingulate anteriorRt 0.83 <0.0003 0.80 <0.002 0.77 <0.003 0.65 <0.01

Primary visual cortexRt 0.80 <0.002 0.83 <0.0004 0.81 <0.0007 0.59 <0.02

Sensory motor cortexRt 0.83 <0.0004 0.89 <0.0001 0.83 <0.0004 0.50 <0.05

Mean cortical regionsLt 0.82 <0.0006 0.81 <0.0007 0.78 <0.002 0.50 <0.05Rt 0.82 <0.0005 0.80 <0.001 0.77 <0.002 0.51 <0.05

Mean overall cortical regions 0.83 <0.0004 0.81 <0.0007 0.78 <0.002 0.51 <0.05

Note: Rt and Lt—the right and left brain hemispheres. The in vivo AChE inhibition was defined using the following formula [100 − %K3 =100 − (K3(f)/K3(b) × 100); f and b indicate the K3 values at the follow-up and the baseline, respectively]; r = correlation coefficient.

Fig. 5. The changes in the CSF AChE activity reliably reflect the in vivo brainAChE inhibition. The graph illustrates the positive correlation between theaveraged in vivo AChE inhibition in overall brain regions and the estimatedinhibition of the synaptic AChE variant in CSF at the 3-month follow-up (seealso Table 3). The CSF AChE-S inhibition is the average of the estimatedImax and Imin values (as defined in Section 3). The in vivo AChE inhibitionis defined as “100 − %K3”. The values on both axes are percentages of theindividual baseline. Open squares: the placebo group. Filled diamonds: thegalantamine group.

Fig. 6. Positive correlation between the in vivo AChE activity in overall cor-tical brain regions and the protein level of the synaptic AChE variant in CSFof patients with mild AD after 9–12 months of treatment with galantamine.



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ean AChE activity in overall brain regions (Fig. 6, r = 0.996,< 0.0001, n = 5).

.7. AChE inhibition and the patients’ cognitiveerformance

We would like to emphasize that the observations in thisection should be interpreted with caution and only as a pat-ern of findings. This is due to: (i) a large number of correlativenalysis were possible between the neuropsychological datand the changes in the AChE levels in the RBC and CSF atifferent follow-up intervals, (ii) the relatively small numberf subjects and (iii) the fewer CSF samples were availableompared to the blood samples.

.7.1. After 3 weeksThe RBC AChE inhibition at the 3-week follow-up

ositively correlated with the patient’s performance inhe Attention-DS test in the galantamine group (r = 0.68,< 0.02, n = 12). At this follow-up, the RBC AChE inhibi-

ion also positively correlated with cognitive improvementf the AD patients in the galantamine group, as assessed byhe ADAS-cog test (r = −0.64, p < 0.03, n = 12).


.7.2. After 3 monthsSimilarly at the 3-month follow-up, the RBC AChE inhi-

ition positively correlated with the patient’s performance in

ig. 7. Positive correlations between the relative augmentations of the expressionest, associated with visuospatial ability (A) and attention (B and C) after up to 12 m

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he Attention-DS test (in the overall group: r = 0.68, p < 0.005,= 15 or in the galantamine group alone: r = 0.71, p < 0.03,= 9) and the clock drawing test (r = 0.51, p < 0.05, n = 15).

At the 3-month follow-up, the ratio of AChE-R variant tohe total AChE protein in CSF positively correlated with theercent changes in the clock drawing test (r = 0.85, p < 0.001,= 11, Fig. 7A) and the Attention-DS test (r = 0.68, p < 0.02,= 12, Fig. 7B). No significant correlations were observedetween the CSF AChE inhibition and the neuropsychologi-al tests associated with attention or visuospatial ability.

.7.3. After 9–12 monthsAfter 9–12 months of galantamine treatment, the increased

rotein level of AChE-R or the ratio of AChE-R variant to theotal AChE protein in CSF positively correlated with percenthanges in both the Attention-DS test (r = 0.87, p < 0.008,= 7), the TMA-test time (r = −0.73, p < 0.04, n = 8) and theMB-test time (r = −0.79, p < 0.05, n = 6, Fig. 7C). Interest-

ngly, the inhibition of CSF AChE-R, but not the AChE-Sfter 9–12 months treatment showed a reverse correlationith the changes in the Attention-DS test (r = −0.79, p < 0.04,= 7, Fig. 8), suggesting that the putative neuroprotective rolef the AChE-R variant might depend on the activity level ofhis AChE variant.


of the AChE-R variant in CSF with the improvement of the patients in theonths treatment follow-up.

No significant correlations between the changes in RBC orSF AChE levels and the neuropsychological subset address-

ng episodic memory were observed throughout the study.


ARTICLE INNBA-6658; No. of Pages 17

12 T. Darreh-Shori et al. / Neurobiology

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ig. 8. Inverse correlation between inhibition of the “read-through” AChE-Rariant and changes in the Attention-DS test after 9–12 months of galan-amine treatment.

. Discussion

In this study, we evaluated the changes in the RBC andSF AChE activities and the protein levels of AChE variants

n CSF in response to placebo and galantamine treatment.e also investigated the relationship of the changes observed

n the RBC or CSF AChE activities with the in vivo AChEctivity measured by PET technique in brain regions of theD patients.The RBC AChE inhibition by galantamine was mild com-

ared to the moderate RBC AChE inhibition in AD patientsreated with donepezil (Darreh-Shori et al., 2006a), which isompatible with the fact that galantamine is a moderate andompetitive AChE inhibitor (Woodruff-Pak et al., 2002).

Several long-term studies with the reversible AChEnhibitors, galantamine, donepezil and tacrine consistentlyave shown an increase in the CSF AChE activity (Davidssont al., 2001; Nordberg et al., 1999), while a sustainednhibition is reported following treatment with the pseudo-rreversible inhibitor, rivastigmine (Darreh-Shori et al.,002). It is however not clear whether this apparent upregu-ation of AChE activity in CSF, following treatment with theeversible inhibitors, is due to a feedback response leading toiminished inhibition exerted by the drug and hence develop-ent of tolerance (Kaufer et al., 1999; Nitsch et al., 1998). In a

ecent study, we reported that the increased CSF AChE activ-ty in AD patients treated with donepezil was related to thenhibition level of the enzyme (Darreh-Shori et al., 2006a). Inhe current study, the protein level of the AChE variants washown to increase about 50–80% over the baseline level inhe galantamine group, but not in the placebo group. Direct


olorimetric assessment of the CSF AChE activity suggestedowever only a moderate 25% increase in the enzyme activitygain basically in the galantamine group. By normalizing theSF AChE activity to the protein level of the CSF AChE vari-

tbor


nts, we found that the activity of the synaptic AChE-S variantas inhibited by 30–36% in CSF. Interestingly, the inhibi-

ion of the CSF AChE-S variant was almost identical to andtrongly correlated with the in vivo AChE inhibition, whichas observed in the majority of the brain regions, in particular

he cortical area. In addition, strong positive correlations werebserved between the plasma or CSF galantamine concentra-ion and the RBC AChE inhibition, the increased CSF AChEctivity, the increased protein level of CSF AChE splice-ariants, the estimated inhibition of AChE variants and then vivo AChE inhibition.

The current report provides evidence that supports the pre-ious findings that CSF AChEs are originated from CNSeurons (Chubb et al., 1976) and that the changes in CSFChE are related to the in vivo AChE expression (Darreh-hori et al., 2002, 2004). Furthermore, the strong correlationsbserved between the CSF and brain AChE inhibition, esti-ated by PET in the same AD patients, provide additional andore direct evidence that supports our earlier findings in the

onepezil-treated AD patients (Darreh-Shori et al., 2006a).hus, the elevations of AChE activity or protein level in CSFf the treated AD patients mainly mirror the in vivo AChEnhibition in the brain regions.

Due to its localization (covalent anchorage) at the synap-ic cleft, the AChE-S variant is putatively regarded as the

ajor AChE variant in CNS responsible for the regulation ofholinergic neurotransmission. This entails that the estimatednhibition level of the AChE-S variant in the current study,ather than an unspecific overall AChE, should be more rele-ant for evaluating the cholinergic neurotransmission in CNS.n this regard, the overall observations suggest that despiten increased expression of the AChE variants, the inhibitionevel of the synaptic AChE variant in the CSF or brain ofhe AD patients was maintained after galantamine treatment.hese findings are important and contradict development of

olerance against reversible ChEIs particularly during theubchronic follow-up (up to 3 months). On the contrary, theass increased expression of AChE variants, in particular theChE-R variant in the treated AD patients might indicate aeneral neuronal plasticity or synaptic remodeling triggeredy (i) the enhanced cholinergic neurotransmission and (ii)he distinct morphogenic properties of AChE variants and/orheir related C-terminal peptides (Day and Greenfield, 2004;ori et al., 2005; Dori and Soreq, 2006; Greenfield, 2005;eshorer and Soreq, 2006). In support of the above notions,e observed positive correlations between the AChE inhibi-

ion and improved performance in neuropsychological testsssociated with attention, such as the digit symbol test andhe trailmaking A and B tests after 3 weeks and 3 monthsf galantamine treatment. In addition, the convergent pat-erns deduced by these observations suggest that the treatmentffect of ChEI therapy involves improvement of the atten-


ion domain of cognition. A similar pattern of relationshipetween inhibition level of cholinesterases and performancef AD patients in specific attention tests is reported followingivastigmine treatment (Almkvist et al., 2004; Darreh-



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The major AChE splice variant in human CSF is theread-through” AChE-R (Darreh-Shori et al., 2004), con-rmed by the three-fold higher level of its protein compared

o the synaptic AChE-S variant observed in our current andrevious studies (Darreh-Shori et al., 2006a). Perrier et al.ave shown however that the AChE-R protein or mRNAevels may be very low compared to the AChE-S proteinr mRNA in different brain regions of mice (Perrier et al.,005). Several explanations may address these discrepan-ies. A possible species related difference. Our preliminaryn situ hybridization analysis on slides from cortical regionsf AD and control brain, using AChE-R and AChE-S specificRNA probes, suggested that although the AChE-R mRNA

evel was less than its synaptic counterpart, it was not anxtremely rare mRNA species (Darreh-Shori, 2006), as isbserved in mice brain (Perrier et al., 2005). Immunoblot-ing analysis of CSF and brain homogenate from AD andontrol subjects also suggest that the AChE-R protein is lown the brain, while it dominates in the CSF (Darreh-Shorit al., 2004), possibly reflecting its secretion into interstitialuid and accumulation in CSF. Furthermore, by combiningucrose density gradient and subsequent immunoblot analy-is of the sucrose fractions corresponding to G4 and G2 + G1ChE protein (Darreh-Shori et al., 2004), we have shown

hat despite ten times higher AChE activity in the G4 than the2 + G1 fractions the protein level of AChE was much less

n the G4 than the G2 + G1 fractions, which mainly containedChE isoform carrying the ARP polypeptide recognized by

he anti-AChE-R antibody (Darreh-Shori et al., 2004). Thisbservation hence implies that either the G4 molecular assem-ly confers a non-linear increase in catalytic efficiency ofhe AChE heavy complexes compared to the lighter isoformsr the lighter isoforms may be in an inactivated state (pos-ibly due to an interaction with extracellular partners, asholinesterases belong to a superfamily of �/� hydrolaseshat includes some adhesion proteins, such as neuroligin andeurotactin (Botti et al., 1998)) or both. Thus, electrophoreticeparation of AChE isoforms under native gel condition andsing the innate activity of the enzyme as the detecting sys-em, as been done in the above study by Perrier et al. (2005)

ight provide misleading results. In addition, the lack of theysteine residue in the C-terminal of AChE-R variant is notconclusive evidence for regarding the AChE-R variant sub-nit as a solely monomeric isoform because we and othersave shown that the C-terminal cysteine residue is not nec-ssary for assembly of G1 subunits into heavy G4 complexesDarreh-Shori et al., 2004; Liao et al., 1993; Perrier et al.,002). Finally, we have shown that an strong detergent suchs SDS in the buffer system of non-reducing electrophore-is is insufficient for eliminating the molecular interactions


etween the AChE subunits observed in the G2 + G1 sucroseractions (Darreh-Shori et al., 2004). Hence it is likely thatddition of a detergent like Triton in the buffer system of theative electrophoretic procedure as has been used in the study

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y Perrier et al. may as well be insufficient to separate AChE-subunits incorporated in G3 or G4 complexes of AChE.

herefore, the lack or presence of a weak G1 activity-stainedignal band under the native gel electrophoretic condition isot a conclusive evidence to presume a low level of AChE-Rrotein in mice brain homogenate. However, it is impor-ant to point out here that for calculating the normalizedctivities of the AChE variants in our current and previoustudies we assumed that all AChE variants and their molecu-ar isoforms have equivalent catalytic activity per unit protein,hich should be regarded with caution, because as mentioned

bove multimerization of the subunits may increase the cat-lytic efficiency of the complex enzyme exponentially ratherhan linearly (Darreh-Shori et al., 2004).

Nonetheless, differential changes in the expression ofChE variants are observed in untreated AD patients andfter treatment with rivastigmine or tacrine (Darreh-Shori etl., 2004). The expression of AChE-R selectively declinesith time in untreated AD patients whereas the protein levelf the G2 AChE-S isoform increases (Darreh-Shori et al.,004). In contrast, acute stress or exposure to ChE inhibitorseems to favor the transcription of the AChE-R variantDarreh-Shori et al., 2004, 2006a; Kaufer et al., 1998; Perriert al., 2005; Shapira et al., 2000). A high inhibition of theynaptic AChE by donepezil is shown to trigger the expres-ion of the AChE-R protein (Darreh-Shori et al., 2006a) mostikely at the expense of the AChE-S variant (Darreh-Shori etl., 2004; Kaufer et al., 1998; Shapira et al., 2000), in partic-lar the G2 isoform (Darreh-Shori et al., 2004). In turn, anncreased ratio of the AChE-R in CSF is shown to correlateositively with a sustained cognition assessed by MMSE testn tacrine-, rivastigmine- or donepezil-treated AD patientsDarreh-Shori et al., 2004, 2006a).

The current findings support these preceding reportsDarreh-Shori et al., 2004; Kaufer et al., 1998; Shapira et al.,000), since we observed 13–27% decline of CSF AChE-

protein expression in the majority of the placebo group,lthough a 3-month period seems to be a too short time toetect disease deterioration in all the patients. The reductionn the placebo treated AD patients was reversed to an increasefter receiving galantamine treatment. This happened appar-ntly without a substantial negative impact on the inhibitionevel of the synaptic AChE variant. In addition, a strongerncrease in AChE-R level was positively associated with theatient’s performance in the clock drawing test, the attentionigit symbol test and the trailmaking A and B tests followingoth subchronic and chronic galantamine treatment. Prin-ipally, these findings support the putative neuroprotectiveole of the “read-through” splice variant of AChE (Darreh-hori et al., 2004; Kaufer et al., 1998; Sternfeld et al., 2000).hough, the nature and mechanism of this favorable role of

he AChE-R variant in the preservation of AD symptoms are


ot fully elucidated. We know that both acute stress and AChEnhibitors elicit a phase of enhanced neuronal excitability,eading to the relatively selective and long-lasting upregula-ion of AChE-R protein (Kaufer et al., 1998). Furthermore, an



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ARTICLE4 T. Darreh-Shori et al. / Neuro

ncreased AChE-R expression, but not its inhibition, is showno positively correlate with the cognitive performance of theatients as assessed by MMSE after 1–2 years of treatmentith donepezil (Darreh-Shori et al., 2006a). In the current

tudy, we also found that the inhibition of AChE-R negativelyorrelated with the patients’ performance in the attention-DSest. Thus, the relative activity level of the AChE-R variant

ay entail neuroprotection. Consequently, the overall obser-ations in the current study and the previous reports give thempression that a positive cognitive response to ChEI therapy

ay not only be dependent on an optimal AChE inhibition butlso the level of upregulation of the AChE-R variant in the ADatients. A plausible mechanistic explanation might be thathe ChEIs increase the persistence of ACh at the synaptic cleftue to the sustained inhibition of the membrane-anchoredynaptic AChE-S variant. This in turn leads to upregulationf the highly secretory AChE-R variant which may prevent arolonged hyperexcitation of the vulnerable neurons (Darreh-hori et al., 2004; Sternfeld et al., 2000) or limits the diffusionange of ACh to within the interstitial fluid or both, whichtherwise could disturb the synchronized neuronal activityf adjacent cholinoceptive neurons.

Evidence suggests that the marked cognitive impairmenteen in AD results from impaired cholinergic neurotransmis-ion owing to selective damage of specific neuronal circuits inhe neocortex, hippocampus, and basal forebrain cholinergicystem (Davies and Maloney, 1976; Rossor, 1983). Along-ide the ongoing neuropathological processes that continue toistress other neuronal networks in the AD brain, the selec-ive decline in the expression level of the AChE-R variantn CSF of untreated AD patients, reported here and in ourrevious studies, may point at disparities in the alternativeChE mRNA splicing as a result of the impaired cholinergiceurotransmission, which may in turn halt neuroregenera-ive mechanisms in the adult brain as both catalytic andon-catalytic activities of AChE variants seem to affect cellroliferation in the ventricular zone and neuronal migrationo their cortical destinations (Dori et al., 2005).

Additionally or alternatively, the widespread reductions inhe activities of choline acetyltransferase and AChE in ADrain in particular in the projectory cholinergic neurons of theasal forebrain (Davies and Maloney, 1976; Rossor, 1983)ower the overall availability of ACh and choline in the brain.equentially, changes in the ACh level may result in aberrantegulation of immune system and inflammatory processesMetz and Tracey, 2005; Pavlov et al., 2006) via �7 nAChRsresent on glial cells in CNS (Saeed et al., 2005; Wang etl., 2003), leading to (astro)gliosis and neurodegeneration.ndeed, recent micro-array analysis indicate that the mainategories of up regulated genes in brains of incipient ADases are those involved in cell proliferation, differentiation,ell adhesions, compliment activation and prostaglandin syn-


hesis (Blalock et al., 2004). In line of the above explanation,e have shown that the disease or the ChEIs change the tran-

cription, alternative splicing and inter-protein interactionsf AChE variants in conjunction with the disease progres-

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ion or the long-term efficacy of the therapeutic treatmentDarreh-Shori, 2006). These clues hence link the long-termherapeutic efficacies of the ChEIs with modulation of AChE

RNA splicing, augmented basal levels of acetylcholine,holine and AChE activities, i.e. enhanced cholinergic neu-otransmission in the brain, which according to accumulatingvidences may improve regulation of the inflammatory pro-esses at work in AD brain (Fukuyama et al., 2001; Metz andracey, 2005; Pavlov et al., 2006; Saas et al., 1999; Sternfeldt al., 2000). Further studies will be required to test the aboveossibilities.

We have recently hypothesized that CSF BuChE mayary inversely with BuChE in cortical amyloid plaques,nd hence a decrease in circulating BuChE molecules in aatient’s CSF may denote ongoing incorporation of BuChEn neuritic plaques and progression of central neurodegener-tion (Darreh-Shori et al., 2006b). Intriguing, we observedreduction in the BuChE level in the CSF sample of the

lacebo-treated patients compared to both their own baselineevels and the galantamine-treated patients. This finding sup-ort our suggestion that monitoring the BuChE level in CSFould have mechanistic and prognostic implications for ADathogenesis (Darreh-Shori et al., 2006b).

We found positive correlations between galantamine con-entration in plasma or CSF and the changes in the RBC andSF AChE activity after 3 weeks and 3 months of galan-

amine treatment but not after the 9 months extension phasef the study. This discrepancy might be reflecting the 3-onth lag-time for initiation of the active treatment in the

lacebo group during the extension phase, i.e. 9 months ofctive treatment in the placebo group compare to the 12onths in the galantamine group at the end of the study.

n fact, larger clinical trials with a similar “Randomized Startesign” like the current study have demonstrated that patients

nitially receiving placebo lost cognitive function that wasot regained after starting the active treatment in the exten-ion phase (Doody et al., 2001; Farlow et al., 2000; Raskindt al., 2000), which is regarded as a clinical sign for possi-le disease-modifying outcome of ChEI therapy (Mori et al.,006). Thus, it is conceivable that the association of the drugith underlying neurochemical events follow a parallel pat-

ern. In addition, galantamine is not a pure AChEIs, but it maylso exercise APL (allosteric-potentiating-ligand) activity onAChRs (Maelicke, 2000). Such a dual AChE-inhibitory andPL actions may in turn add a time-dependent factor to the

omplexity of the pharmacodynamic responses of neurons tohe treatment. Indeed, the changes in the 11C-nicotine bindingy PET in these patients (unpublished experiment), indicatedhat short- and long-term responses to galantamine was com-lex and showed particularly a time-dependent relationshipetween changes in the nicotinic binding sites in the brain andhe activity of AChE in blood and CSF and their association


ith galantamine concentration.A comparison between the increased expression of the

SF AChE variants measured after chronic treatment withonepezil (Darreh-Shori et al., 2006a) and galantamine sug-



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ests several differences between these two reversible ChEIs.he increased expression of CSF AChE variants was less pro-ounced in the galantamine-treated AD patients (50–80%)han in the donepezil-treated AD patients (115–165% in theverall patients group), which might be expected consideringhat donepezil is a much stronger AChEI than galantamine.urthermore, the increased expression of the CSF AChE-variant in the donepezil-treated patients was 50% lower

han the corresponding increase in the AChE-S expressionDarreh-Shori et al., 2006a) whereas in the galantamine-reated AD patients, it was 16% higher than the AChE-Sariant at the subchronic 3-month follow-up, but similar afterhe chronic treatment (9–12 months).

However, it should be noted that in the current galantaminetudy the number of subjects was much fewer than thosencluded in the donepezil study, and the clinical settings forhese two studies were quite different (Darreh-Shori et al.,006a). Thus, the described differences above between thesewo reversible ChEIs should be regarded with caution.

An important limitation of the present study is the rela-ively large number of statistical analyses performed on theognitive tests in relation to the changes in the RBC and CSFChE levels, which may render some of the results obtained

o be by chance. For this reason, result patterns rather thansolated findings were taken into consideration. Another lim-tation of the study is the relatively small number of subjects.n addition, due to the invasive nature of CSF sampling byhe lumbar puncture procedure, several CSF samples were notvailable from the patients who completed the study. Theseeatures make it necessary to be cautious when interpretinghe data since a small sample size may result in a lack oftatistical power, although this might make the significantesults even more interesting. In this regard, the relativelymall number of available CSF samples compared to bloodamples might explain the lack of correlation between theSF AChE-S inhibition and those neuropsychological tests

hat correlated with the RBC AChE inhibition.In summary, by combining two quite simple methods, we

ere able to estimate a 30–36% inhibition of the synapticChE variant in CSF, which was highly consistent with the0–37% in vivo AChE inhibition measured by PET tech-ique in the majority of the brain regions of the same patients.ue to the presence of a placebo group, we showed that the

hanges in the AChE activity or protein levels were specif-cally related to the effect of galantamine. The increasedxpression of AChE variants without any apparent effect onhe inhibition level of the AChE-S in the CSF or brain of theD patients seems to contradict development of a tolerancehenomenon. The main cognitive domain, which was asso-iated with both AChE inhibition and the expression level ofChE-R variant, was the attention domain. The strong pos-

tive correlations between the in vivo AChE activity and the


hanges in the AChE variants in CSF throughout the studyighlights the reliability and accuracy of the combined directolorimetric-ELISA-like assays for evaluating the changesn expression of AChE variants in CSF as a surrogate for

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easuring the in vivo changes of AChE in the brain of ADatients.

isclosure

This study was partially financed by Janssen-Cilag, USA.he authors T. Darreh-Shori, A. Kadir, O. Almkvist, M. Grut,. Wall, G. Blomquist, and B. Langstrom have reported no

onflicts of interest. The author, B. Eriksson is a currentmployee of Janssen-Cilag, Sweden; Agneta Nordberg haseceived honoraria from Janssen-Cilag for giving lectures atcientific meetings as well as for participation in advisoryoard meetings about nicotinic receptors and the treatmentf AD.

cknowledgements

The authors would like to thank Birgitta Strandberg forxcellent assistance. This research was sponsored by theedical Research Council (project no. 05817), Stiftelsen foramla Tjanarinnor, KI foundations, Stohne’s foundation, and

anssen-Cilag (Sweden and USA).

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at doi:10.1016/j.neurobiolaging.006.09.020.

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