targeting acetylcholinesterase and …...targeting acetylcholinesterase and butyrylcholinesterase in...

Targeting acetylcholinesterase andbutyrylcholinesterase in dementia

Roger M. Lane1, Steven G. Potkin2 and Albert Enz3

1 Novartis Neuroscience, Novartis Pharmaceuticals Corporation, NJ, USA2 Department of Psychiatry and Human Behavior, University of California at Irvine Medical Center, Orange, CA, USA3 Novartis Pharma AG, Basel, Switzerland

Abstract

The cholinesterase inhibitors (ChE-Is) attenuate the cholinergic deficit underlying the cognitive and neuro-

psychiatric dysfunctions in patients with AD. Inhibition of brain acetylcholinesterase (AChE) has been the

major therapeutic target of ChE-I treatment strategies for Alzheimer’s disease (AD). AChE-positive

neurons project diffusely to the cortex, modulating cortical processing and responses to new and relevant

stimuli. Butyrylcholinesterase (BuChE)-positive neurons project specifically to the frontal cortex, and may

have roles in attention, executive function, emotional memory and behaviour. Furthermore, BuChE

activity progressively increases as the severity of dementia advances, while AChE activity declines.

Therefore, inhibition of BuChE may provide additional benefits. The two cholinesterase (ChE) enzymes

that metabolize acetylcholine (ACh) differ significantly in substrate specificity, enzyme kinetics,

expression and activity in different brain regions, and complexity of gene regulation. In addition, recent

evidence suggests that AChE and BuChE may have roles beyond ‘classical’ co-regulatory esterase func-

tions in terminating ACh-mediated neurotransmission. ‘Non-classical’ roles in modulating the activity of

other proteins, regional cerebral blood flow, tau phosphorylation, and the amyloid cascade may affect

rates of AD progression. If these additional mechanisms are demonstrated to underlie clinically mean-

ingful effects, modification of the over-simplistic cholinergic hypothesis in AD that is limited to sympto-

matic treatment, ignoring the potential of cholinergic therapies to modify the disease process, may be

appropriate. The specificity of ChE inhibitory activity, up-regulation of AChE activity and changes in the

composition of AChE molecular forms over time, selectivity for AD-relevant ChE molecular forms, brain

vs. peripheral selectivity, and pharmacokinetic profile may be important determinants of the acute and

long-term efficacy, safety and tolerability profiles of the different ChE-Is. This review focuses on new

evidence for the roles of BuChE and AChE in symptom generation and rate of underlying disease

progression in dementia, and argues that it may be appropriate to re-evaluate the place of ChE-Is in the

treatment of dementia.

Received 20 June 2004 ; Reviewed 28 September 2004 ; Revised 28 March 2005 ; Accepted 31 March 2005

Key words : Acetylcholinesterase, Alzheimer’s disease, butyrylcholinesterase, cholinergic, disease

progression.

Introduction

Alzheimer’s disease (AD) is characterized by a loss of

cholinergic neurons and their cortical projections from

the nucleus basalis and associated areas in the basal

forebrain. Cholinergic synaptic function appears to

be particularly susceptible to beta-amyloid (Ab)

peptide toxicity, and loss of synaptic vesicles on axon

terminals may precede cholinergic neuronal loss

(Small et al., 2001). Progressive deterioration of

the widespread and dense cholinergic innervation of

the human cerebral cortex contributes to the salient

cognitive and behavioural disturbances in AD, and

is associated with decreased levels of the neuro-

transmitter, acetylcholine (ACh), choline acetyl-

transferase (ChAT) – the rate-limiting enzyme for

ACh synthesis, and acetylcholinesterase (AChE) – the

ACh-hydrolysing enzyme. Remarkably, brain levels

of another enzyme that hydrolyses ACh, butyryl-

cholinesterase (BuChE), show progressive and

Address for correspondence : S. G. Potkin, M.D., University of

California Irvine, Irvine Hall, Room 166, Irvine, CA 92697-3960, USA.

Tel. : +1 (949) 824 6567 Fax : +1 (949) 824 7873

E-mail : [email protected]

International Journal of Neuropsychopharmacology (2006), 9, 101–124. Copyright f 2005 CINPdoi:10.1017/S1461145705005833

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significant increases in AD (Atack et al., 1986 ; Perry

et al., 1978).

The ‘cholinergic hypothesis ’ was the basis for the

development of presynaptic, synaptic and post-

synaptic treatment approaches designed to maintain

and facilitate the activity of the surviving cholinergic

system. Synaptic cholinesterase (ChE) inhibition has

proved preferable to direct receptor agonist therapy,

as cholinesterase inhibitors (ChE-Is) amplify the natu-

ral spatial and temporal pattern of ACh release, rather

than tonically or globally stimulating either nicotinic

or muscarinic ACh receptors. ChE-Is are approved by

the Food and Drug Administration (FDA) for the

symptomatic treatment of AD. These drugs improve

cognitive and neuropsychiatric symptoms, and stabil-

ize functioning over at least 6 months during clinical

trials in patients with mild to moderate AD (Farlow,

2002). Although of the same drug class, ChE-Is are

structurally diverse (Brufani and Filocamo, 2000;

Table 1). As a result, donepezil and galantamine

possess relative selectivity for AChE, whereas tacrine

and rivastigmine co-inhibit both AChE and BuChE.

AChE and BuChE appear to be simultaneously active

in the synaptic hydrolysis of ACh, terminating its

neurotransmitter action, and co-regulating levels of

ACh (Mesulam, 2003).

Molecular biology of AChE and BuChE

Enzyme structure and function

At the molecular level, AChE and BuChE share 65%

amino-acid sequence homology and are encoded by

different genes on human chromosomes 7 (7q22) and 3

(3q26) respectively (Soreq and Zakut, 1993). Both

AChE and BuChE have a primarily hydrophobic ac-

tive gorge, shown by X-ray crystallography to be 20 A

deep for AChE (Sussman et al., 1991), into which ACh

diffuses and is cleaved. Once ACh enters this active

site, it binds at two locations – a catalytic region near

the base of the gorge and a choline-binding site

midway up it. ACh is hydrolysed by ChE enzymes to

choline and acetate, thereby terminating its neuro-

transmitter function.

Structural features of the two ChE enzymes confer

differences in their substrate specificity. AChE is

highly selective for ACh hydrolysis, while BuChE is

able to metabolize several different molecules includ-

ing various neuroactive peptides (Taylor and Radic,

1994). The reason for substrate diversity between these

enzymes is the variation of several amino acids in the

sequence determining the three-dimensional size and

shape of their active site gorge. It has been proposed

that the efficiency with which AChE and BuChE

hydrolyse ACh is dependent on the substrate concen-

tration. AChE has greater catalytic activity at low ACh

concentrations, resulting in substrate inhibition at

higher doses (Soreq and Zakut, 1993 ; Taylor and

Radic, 1994). However, BuChE is more efficient at high

substrate concentrations.

The peripheral anionic site (PAS) of AChE, the

subsite outside of the active site gorge, mediates

substrate inhibition of AChE (Ordentlich et al., 1995;

Radic et al., 1993). Three aromatic amino acids located

at the entrance to the active site gorge are the key

residues of this subsite. The PAS of BuChE, which is

different from that of AChE, has weaker affinity than

AChE for typical PAS ligands and mediates substrate

activation. The three aromatic residues of the AChE

PAS are missing in the PAS of BuChE, which is formed

by two amino acid residues (Asp70 and Tyr332) in

the human enzyme. A polymorphism of one of the

residues forming the PAS of BuChE is found in the

‘atypical’ BuChE variant (Asp70 mutated to Gly70).

This mutant binds succinylcholine weakly, may

produce prolonged anaesthesia (Neville et al., 1990),

and does not show substrate activation (Masson et al.,

1997). The differences in the enzyme kinetic properties

and locations of brain AChE and BuChE have led to

the suggestion that, in the normal brain, AChE is the

main enzyme responsible for ACh hydrolysis, while

BuChE plays a supportive, functional role.

Molecular forms of ChE

For both AChE and BuChE, a single gene gives rise to

different protein products by alternative splicing in

the coding region of the original transcript. For both

ChEs, all mRNAs that derive from the original

transcript contain a core that is common to each and is

essential for catalytic activity of the enzyme, but

possess dissimilar carboxyl-terminal sequences that

account for their different multimerization, attach-

ment to cell membranes, or existence as soluble

proteins (Massoulie et al., 1999). This provides a series

of diverse but related molecular forms of AChE and

BuChE that have similar catalytic properties but

different cellular and extracellular distributions and

non-catalytic activities.

AChE exists in asymmetric forms (A forms) con-

taining catalytic units attached to a membrane-

anchored protein tail, and three globular forms

(G forms) containing one, two or four catalytic subunits

(monomeric G1, dimeric G2, and tetrameric G4). G1

AChE (usually encoded for by AChE-R mRNA

species – see below) exists solely as a soluble entity,

102 R. M. Lane et al.



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whereas G4 AChE (usually encoded for, as other

multimers, by AChE-S mRNA species – see below)

exists in both soluble and membrane-bound forms

(Massoulie et al., 1999). In the human brain, AChE is

present in G1 and G4 forms, the proportions of which

vary in different brain regions (Atack et al., 1986).

BuChE also exists in G1, G2, and G4 molecular forms

but, similar to AChE, G4 is the predominant isoform in

the mature brain (Arendt et al., 1992 ; Darvesh et al.,

2003a).

In the brains of patients with AD, the level of the

membrane-bound G4 form of AChE is selectively

reduced by 90% or more in certain regions, probably

due to the loss of presynaptic terminals. However,

levels of G1 AChE remain largely unchanged (Siek

et al., 1990). It has been suggested that changes in the

expression of AChE molecular forms may be a direct

consequence of Ab (Saez-Valero et al., 2002). The G1

form of BuChE shows a 30–60% increase in the AD

brain, probably due to an increase in the number of

BuChE-positive glia, while the G4 form decreases or

remains the same as in the normal brain. An analogous

reduction in neuronal AChE and an increase in

neuronal BuChE may accompany peripheral neuro-

degeneration (Moral-Naranjo et al., 2002). The

potencies of ChE enzyme inhibition by the various

ChE-Is have been shown to be significantly different

from brain region to brain region (Zhao and Tang,

2002) with respect to selectivity for different molecular

forms (Enz et al., 1993 ; Rakonczay, 2003), and in AD

relative to the non-AD brain (Guillozet et al., 2003).

Taken together, these findings suggest that the use

of ChE-Is in the treatment of AD should consider

both molecular form-specific and region-specific

Table 1. Overview of pharmacological characteristics of cholinesterase (ChE) inhibitors (adapted from Poirier, 2002)

Donepezil Rivastigmine Galantamine

Chemical class Piperidine Carbamate Tertiary alkaloid

Inhibition of ChE enzymes Non-competitive, rapidly

reversible (<1 ms) of AChE

Non-competitive, very

slowly-reversible (y6–8 h)

of both BuChE and AChE

Competitive, rapidly

reversible (<1 ms) of

AChE

Site on inhibition of target

enzyme

Covers catalytic gorge and

peripheral anionic site,

binding to choline anionic

site

Catalytic binding site Choline anionic site and

catalytic binding site

Increased activity/levels of

target enzymes during

long-term treatmenta,b

5–10 mg/d: y3-fold

increase in AChE levels

over 6–12 monthsa

3–12 mg/d – sustained

decrease in AChE and

BuChE activity over 12

monthsb

24–32 mg/d: y2-fold

increase in AChE levels

over 6 monthsa

Brain vs. peripheral

selectivity

Uncertain Yes None

Preferential isoform

selectivityc,d,eNone in most studiesc,d,

Gl in oneeG1 (or AChE-R)c,d Noned

Allosteric modulation of

nicotinic receptorfNo No Particular subtypes

(e.g. a4/b2) in vitro

Metabolism CYP2D6 and 3A4 AChE and BuChE CYP2D6 and 3A4

Plasma protein binding y96% y40% y20%

Recommended dose 5–10 mg/d, once daily 6–12 mg/d, twice daily

with food

16–24 mg/d, twice

daily with food

Formulations Tablets

Oral solution

(in development)

Capsules

Oral solution

Transdermal patch

(in development)

Tablets

Oral solution

Once daily controlled-

release

AChE, Acetylcholinesterase ; BuChE, butyrylcholinesterase ; nAChR, nicotinic ACh receptor ; CYP450, cytochrome P450.a Davidsson et al. (2001) ; b Darreh-Shori et al. (2002) ; c Enz et al. (1993) ; d Rakonczay (2003) ; e Zhao and Tang (2002) ; f Samochoki

et al. (2000).

Acetylcholinesterase and butyrylcholinesterase in dementia 103



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characteristics of AChE and BuChE inhibition (Zhao

and Tang, 2002).

ChEs in pathophysiological stress

AChEmay have a role in the neurobiology of reactions

to stress, which may alter the splicing of the AChE

gene to yield different transcripts coding for different

molecular forms (Nachon et al., 2003 ; Soreq and

Seidman, 2001). Under normal conditions, much more

AChE-S [the ‘synaptic’ variant, usually existing in a

multimeric (G4), membrane-bound form] is produced

compared with AChE-R [the ‘read-through’ variant,

usually existing in a soluble, monomeric (G1) form].

However, under conditions of acute stress, such as

following head injury (Shohami et al., 2000), anti-

AChE intoxication (Shapira et al., 2000), or during

psychological stress (Kaufer et al., 1998), expression of

the AChE-R form is greatly increased due to alterna-

tive mRNA splicing. AChE-R might contribute to the

acute attenuation of neurodeterioration (Sternfeld

et al., 2000). For example, transgenic animals that

are unable to respond to stress with AChE-R over-

production are exceptionally vulnerable to closed-

head injury (Shohami et al., 2000).

It is presumed that stressful stimuli induce

excitation through ACh release, and that a feedback

response, inducing overexpression of AChE, acts to

return excessive cholinergic neurotransmission to

normal levels (Kaufer et al., 1998). This may be of

importance not only for cholinergic neurotransmission,

but also for neurotransmission modulated by ACh,

such as glutamate-mediated neurotransmission in

the hippocampus (Gray et al., 1996), and for actions

mediated by the non-cholinergic function of ACh,

such as in the regulation of neuronal cytoarchitecture

(Mattson, 1988). AChE mediates other stress-related

responses such as the myeloid and megakaryocytic

expansion that are characteristic of acute and chronic

stress responses (Soreq and Glick, 2000), through the

released C-terminal peptide of AChE-R (Grisaru et al.,

2001). AChE-derived C-terminal peptide fragments

may possess distinct structures and biological activi-

ties (Bon et al., 2004).

However, such acutely beneficial stress responses

may be harmful in the long term. Transgenic mice

overexpressing AChE show progressive cognitive

impairments and neuropathological markers, includ-

ing loss of cortical dendrites, reminiscent of AD (Beeri

et al., 1997) and neuromotor deficits (Andres et al.,

1997). Although absolute levels of AChE appear

important in mediating these effects, it is unclear if the

ratio of AChE-S/AChE-R is also important. Massive

accumulation of both AChE-S and AChE-R in animal

models induces erratic behaviour associated with

impaired working memory under conditions of mild

stress (Cohen et al., 2002), as well as neurodeteriora-

tion (Sternfeld et al., 2000). However, AChE-R ac-

cumulation alone protected neurons from damage

associated with prolonged conflict behaviour (Birikh

et al., 2003). Thus, there may be an optimal AChE-R/

AChE-S ratio for the maintenance of optimal physio-

logical function, which can rapidly alter in response to

stressors in order to maintain homeostasis. However,

chronic increases in, and imbalances of, the ratio of

molecular forms of AChE appear to result in cholin-

ergically mediated dysfunctions.

ChE gene regulation: implications for treatment

Changes in the protein levels of cerebrospinal fluid

(CSF) AChE variants may be reliably used as a surro-

gate marker of changes in the expression of AChE

molecular forms in cholinergic neurons in the brain

(Chubb et al., 1976 ; Tomkins et al., 2001). However,

whereas the G4 activity in the CSF appears to reflect

activity in the brain, the activity of CSF G1 and G2

forms in the CSF appear to be considerably less than in

the brain (Darreh-Shori et al., 2004). The ChE-Is appear

to reverse the AD-induced decrease over time in AChE

levels (Darreh-Shori et al., 2002, 2004 ; Davidsson et al.,

2001). The ability to induce increases in AChE levels

varies greatly amongst ChE-Is. Marked elevations in

CSF levels and activity of AChE are seen following

long-term treatment with tacrine, donepezil and gal-

antamine (Amici et al., 2001 ; Davidsson et al., 2001;

Nordberg et al., 1999 ; Parnetti et al., 2002) (Table 1).

The expression of AChE-R appears to be more sen-

sitive to the effect of treatment or lack of treatment

than that of AChE-S (Darreh-Shori et al., 2004). ChE-I

therapy may increase the R/S ratio, and the ability to

do this may also vary amongst ChE-Is. For example,

rivastigmine was shown to induce greater increases in

the R/S ratio than tacrine (Darreh-Shori et al., 2004).

The R/S ratio has been highly correlated with sus-

tained cognitive improvement over 1 yr of treatment

(Darreh-Shori et al., 2004). Increases in total CSF AChE

levels have been correlated with cognitive response in

AD patients treated with galantamine and donepezil

(Davidsson et al., 2001), but changes in the R/S ratio

were not assessed. ChE-Is may also differentially affect

CSF G2 AChE protein levels. This largely inactive

dimer was reduced in rivastigmine-treated AD

patients, but increased over 2-fold in tacrine-treated

and untreated patients (Darreh-Shori et al., 2004).

Thus, the various ChE-Is may also differentially affect

the inter-protein interactions and multimerization of




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AChE variants in addition to their differing potentials

to induce increases of AChE and changes in the

composition of AChE variants.

Increases in AChE levels could have important

consequences if the findings in experimental animals

are applicable to humans (Grisaru et al., 1999; Soreq

and Seidman, 2001). The amelioration of the symp-

toms of dementia by ChE-Is, if accompanied by sig-

nificant increases in AChE expression, may be both

limited and temporary (Soreq and Glick, 2000). In ad-

dition to tolerance to the symptomatic effects of ChE-I

treatment, the potential roles for AChE in disease

progression (discussed later in this review) may mean

that increased AChE levels accelerate the course of the

disease. Increases in AChE levels may also explain

certain treatment-emergent adverse effects, and the

‘crashes’ or dramatic worsening of symptoms resem-

bling cholinergic delirium that are observed in some

patients when chronic donepezil treatment is dis-

continued (Bhanji and Gauthier, 2003 ; Grossberg,

2003 ; Singh and Dudley, 2003).

The increases in AChE during treatment with

particular ChE-Is may be due to the up-regulation

ofAChEgene expression (Kaufer et al., 1999), reflecting

feedback mechanisms via muscarinic ACh receptors

(Nitsch et al., 1998). Polymerase chain reaction studies

to assess mRNA expression are required before it may

be definitively stated that increased AChE levels in

patients receiving ChE-Is are due to up-regulation of

gene expression. Alternatively, the elevation of AChE

levels in treated patients may be indicative of

improved functioning of cholinergic neurons due to

stimulation (Swaab et al., 1994), or increased regional

cerebral blood flow (rCBF) (Venneri et al., 2002).

Changes in AChE variant composition may reflect

modulation of alternative splicing (Meshorer et al.,

2002).

Overall increases in AChE or BuChE activity during

long-term treatment are not seen with the slowly re-

versible dual inhibitor rivastigmine (Darreh-Shoreh

et al., 2002 ; Table 1). Rivastigmine binds directly to the

catalytic site and forms a relatively stable acyl-enzyme

intermediate that does not undergo hydrolysis for

many hours (Bar-On et al., 2002). In fact, significant

decreases in AChE and BuChE activity are sustained

for at least 12 months of rivastigmine treatment (Amici

et al., 2001 ; Darreh-Shori et al., 2002 ; Parnetti et al.,

2002). A regulatory role for BuChE on the expression

of AChE has been suggested (Layer et al., 1992). After

the in vitro addition of the specific BuChE-inhibitor

(iso-OMPA), not only BuChE activity was completely

suppressed but significant influence on AChE activity

was also detectable. Although the rapidly reversible

dual inhibitor tacrine may up-regulate AChE activity,

increases in AChE activity do not appear to be as

marked as with selective AChE inhibitors (Nordberg

et al., 1998, 1999). Increases in BuChE activity are not

observed in CSF following long-term treatment with

tacrine or rivastigmine. Thus, increased gene ex-

pression may be specific to AChE and could reflect

different regulatory pathways for the AChE and

BuChE genes (Nordberg et al., 2003).

Interestingly, there is evidence that the irreversible

AChE and BuChE inhibitor, metrifonate, also may not

produce up-regulation of AChE. In contrast to tacrine,

metrifonate maintained red blood cell AChE inhi-

bition over 3 months in patients with AD (Cummings

et al., 1998 ; Pettigrew et al., 1998), confirming findings

of no apparent AChE up-regulation by metrifonate in

animal models (Hinz et al., 1998). Similar to tacrine,

donepezil and galantamine are very rapidly revers-

ible inhibitors. Donepezil induces a strong dose-

dependent up-regulation of AChE activity, possibly

due to its non-competitive inhibitory action. Gal-

antamine, a competitive inhibitor which depends

more on the substrate concentration than on the

absolute concentration of the drug, induces less

marked elevations of AChE activity. These differential

findings amongst ChE-Is support the hypothesis

that chemical diversity within the same drug class

provides varied pharmacological profiles among the

individual agents within the class.

Until very recently, the only established function of

AChE was termination of cholinergic neurotransmis-

sion, and the use of AChE inhibitors for the treatment

of the cholinergic imbalances in AD represented a

rational approach. AChE inhibition could be evaluated

in terms of the efficiency of inhibition of cholinesterase

activity, and the consequences of AChE inhibition

assessed by observing cholinergically mediated pro-

cesses such as cognition. The chronic effects of ChE-Is

on AChE activity and changes in the composition of

AChE variants requires further evaluation, as any

potential clinical impact is currently unknown. Inves-

tigation may yield information referring to treatment

effects such as the development of tolerance and treat-

ment withdrawal reactions. Furthermore, now that

additional functions of AChE have been identified, the

effects of ChE-Is must be more broadly considered.

Neuroanatomy of AChE and BuChE expression in

the central nervous system

Neuronal locations

The majority of ChE in the human brain is AChE.

However, it is now known that BuChE has more




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widespread distribution than previously thought

(Mesulam et al., 2002a). In the normal brain, BuChE

activity has been located in all regions that receive

cholinergic innervation. It is mainly found in glial cells

and in endothelial cells, whereas AChE is in neurons

and axons (Mesulam et al., 2002a). Although AChE-

containing neurons are widely distributed and more

numerous than BuChE neurons, there is a particularly

high expression of BuChE immunopositive neurons in

the hippocampus, thalamus and amygdala (Darvesh

et al., 1998, 2003a; Mesulam, 2000). In these structures,

both AChE and BuChE have widespread but different

distributions (Darvesh and Hopkins, 2003). For

example, thalamic nuclei (anteroventral, mediodorsal,

ventral anterior, lateral, and pulvinar) show particu-

larly high BuChE expression ; 90% or more neurons

show intense immunostaining for BuChE (Darvesh

and Hopkins, 2003).

Thalamic nuclei have both specific and non-specific

projections to the cortex. The specific projections are

usually from nuclei that show intense BuChE activity.

As would be expected from the structures to which

they project, pathological states of the subcortical

nuclei in which BuChE-positive neurons are found

have been implicated in working memory, attention,

executive function and behaviour deficits (Darvesh

and Hopkins, 2003). For example, the mediodorsal

nucleus, which is rich in BuChE-positive neurons,

projects mainly to prefrontal and cingulate cortices

(Kievit and Kuypers, 1977), and this nucleus has a vital

role in working memory, planning, and sequencing

complex behaviours, all universal deficits in AD. In

addition, the pulvinar sub-nuclei are involved in

visual attention, a function that may be affected early

in AD (Greenwood et al., 2000). Other thalamic nuclei

that project to the frontal cortex and that, again, almost

exclusively express BuChE include the anteroventral

and lateral dorsal nuclei (Darvesh and Hopkins, 2003).

The role of glial cells

In the non-AD human brain, glial cells such as astro-

cytes, microglia and oligodendrocytes release AChE

and BuChE into the extracellular space when acti-

vated. In patients with AD, intense BuChE and AChE

activity is found in the vicinity of amyloid plaques,

amyloid angiopathy, and neurofibrillary tangles

(NFTs) (Mesulam et al., 1992 ; Wright et al., 1993), and

these activities may stem from glia. In non-AD and AD

brains, AChE-positive glia are widely distributed

throughout the cortical layers and subcortical white

matter, whereas BuChE-positive glia reach high den-

sities only in the deep cortical layers and subcortical

white matter (Wright et al., 1993). In the non-AD brain,

the ratio of BuChE- to AChE-positive glia is higher in

the entorhinal and inferotemporal cortex (two regions

with a high susceptibility to AD pathology), than in

the somatosensory and visual cortex (two regions with

a relatively low susceptibility to the disease) (Wright

et al., 1993). Glial BuChE/AChE ratios are increased in

AD in the entorhinal and inferotemporal cortex, but

not in other brain areas.

Glial cells produce a variety of trophic and neuro-

toxic factors, and regulate levels of synaptic neuro-

transmitters such as glutamate. In patients with AD

both astroglia and microglia proliferate, particularly

around plaques, and the profile of receptors that they

express changes markedly (Meda et al., 2001). It is

generally assumed that reactive gliosis is secondary to

plaque formation, but a direct role for glia in plaque

formation has also been proposed (Schubert et al.,

2001). For example, Ab activates glial cells to act as

pro-inflammatory cells that secrete pro-inflammatory

cytokines, which induce further Ab deposition.

Further studies to determine the relationship of BuChE

and AChE with changes in receptor expression, cyto-

kine release, and function in glial cells are needed to

determine how BuChE and AChE may impact upon

plaque maturation and disease progression. As glial

BuChE may co-regulate ACh, elevated levels of

BuChE in AD may exacerbate the reductions in chol-

inergic neurotransmission by further decreasing ACh

levels.

Symptoms of cholinergic deficiency in dementia

A substantial cortical cholinergic denervation is a

major aspect of advanced AD that is most severe in the

temporal lobes and in adjacent limbic and paralimbic

areas. Cholinergic innervation of the thalamus, orig-

inating in the brainstem, and cholinergic innervation

of the striatum, originating from striatal interneurons,

remain relatively intact. Therefore, the cholinergic

lesion in AD is not generalized but selective.

Moreover, recent studies indicate that loss of cholin-

ergic markers are not detected in individuals with

mild AD, and is a relatively late event during the

course of AD (Davis et al., 1999 ; Minger et al., 2001;

Rinne et al., 2003). Despite the absence of manifest

cholinergic deficits, there is evidence that ChE-Is may

be associated with symptomatic benefits in early AD

(Peterson et al., 2005 ; Salloway et al., 2004).

Cholinergic deficits in the early stages of AD may

relate to defects in cholinergic signal transduction

(Haring et al., 1998 ; Mufson et al., 2000). Abmay act as

a physiological modulator of cholinergic function




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(Auld et al., 1998) and low concentrations of soluble

Ab insufficient to induce neurotoxicity can inhibit

ACh synthesis and release, resulting in cholinergic

hypofunction, decreased neural efficiency and cogni-

tive impairment. These effects seem to appear in the

hippocampus and cortex but not in other brain areas

(Kar et al., 1998). Basal forebrain cholinergic neurons,

but not striatal or brainstem cholinergic neurons,

are among the most sensitive cells in the brain to

age-related neurofibrillary degeneration (Sassin et al.,

2000). Thus, Ab may be implicated in impaired basal

forebrain cholinergic neurotransmission before levels

of Ab have achieved neurotoxic levels.

Ascending cholinergic pathways have an excitatory

neuromodulatory effect on the cholinoceptive neurons

of the cerebral cortex, making them more susceptible

to other incoming excitatory inputs (McCormick,

1990). Mesulam (2004) summarizes evidence that the

neuroanatomy of cortical cholinergic innervation

suggests crucial roles in the ‘modulation of attention

(i.e. the on-line holding and dynamic enhancement of

neural responses to salient events) and memory

(i.e. the off-line encoding, retention, and retrieval of

past events and contingencies) ’. The formermay reflect

widespread cortical innervation and excitatory neuro-

modulation, while the latter may reflect a high level of

cholinergic innervation of limbic areas, including

the hippocampus, where the role of cholinergic inner-

vation in modulating experience-dependent neuro-

plasticity may be of particular relevance (Mesulam,

1999). Although, cortical cholinergic pathways could

potentially influence all aspects of cognition and

behaviour, impaired cholinergic neurotransmission

alone cannot fully account for the clinicopathological

presentation of AD. The highly heterogeneous de-

generation of multiple neurotransmitter pathways,

along with multiple other mutually reinforcing neuro-

degenerative mechanisms, also underlies many of the

symptoms of AD. It is, therefore, difficult to define the

symptom profile of cholinergic deficit in dementia

patients.

It has been proposed that cholinergic deficiency in

the central nervous system is characterized by loss

of attention/concentration and reduced capacity to

detect and select relevant stimuli (Lemstra et al., 2003).

These deficits can result in patients becoming restless,

anxious and confused. Delusions and hallucinations

may be a consequence of patients’ impaired percep-

tion of reality. Neurobiologically based models of

human cognition distinguish between instrumental

functions such as language, perception and praxis,

and fundamental functions such as set shifting, atten-

tion/concentration and rate of information processing

(Albert, 1978 ; Cummings, 1990). It has been suggested

that effects on cholinergically mediated fundamental

functions are responsible for the improvements in AD

symptoms during treatment with ChE-Is (Lemstra

et al., 2003). Instead of serving specific instrumental

cortical functions, the diffusely organized system of

cholinergic input to the cortex serves more funda-

mental roles of detection, selection, discriminating and

processing of sensory stimuli and higher processes

(Sarter and Bruno, 1997 ; Selden et al., 1998). A

deficient cholinergic input system impairs efficient

cortical processing and response to new and relevant

stimuli. Fundamental factors such as motivation and

attention/concentration modulate performance on

instrumental neuropsychological tasks requiring per-

ception, language and praxis. The fact that many

patients with AD suffer mainly from impairments of

instrumental functioning as a result of extensive cor-

tical damage, and have relatively intact fundamental

functioning, may explain the poor response to ChE

inhibition seen in a significant proportion of AD

patients. Assessments of selective and sustained

attention and behaviour have rarely been conducted in

randomized clinical trials in AD.

A diagnosis of AD or ‘dementia’ may be a poor

descriptor of the target for ChE-I therapy. Rather than

a nosological disease category, it may be better to

identify ChE-I treatment with a ‘cholinergic deficiency

syndrome’ of cholinergically mediated fundamental

functions that are irrespective of the precise cause of

neurodegeneration (Lemstra et al., 2003). The role of

cholinergic projections from the basal forebrain in

mediating cortical arousal via ACh release, enhancing

cell responsiveness and increasing signal-to-noise ratio,

shifts the cortical electroencephalogram (EEG) from

the classical slow-wave pattern of high-amplitude,

low-frequency oscillations to the desynchronized,

higher-frequency, low-amplitude waveforms of wake-

fulness and arousal. In AD and related dementias,

there is an increase in slow-wave (theta and delta) ac-

tivity, a decrease in faster frequencies (alpha and beta),

and a prolonged auditory P300 latency on the quanti-

tative electroencephalogram (qEEG) – changes which

are related to deficits in cholinergic neurotransmission

(Adler et al., 2003 ; Braverman and Blum, 2003;

Fogelson et al., 2003; Frodl-Bauch et al., 1999).

ChE-I treatment has been shown to enhance qEEG

coherence with decreased slow-wave activity and in-

creased faster frequencies reflecting increased cortical

arousal, and providing improvements in concen-

tration, sensory processing, and learning and memory

(Adler and Brassen, 2001 ; Brassen and Adler, 2003 ;

Fogelson et al., 2003). On an individual level, it has




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been suggested that such qEEG changes may have a

positive predictive value for therapeutic response

(Adler et al., 2004).

Another objective tool for the evaluation of cogni-

tive response to ChE-Is in demented patients is the

latency of P300 event-related potentials (Werber et al.,

2003). P300 is a cognition-related brain potential that

may reflect cholinergically mediated mental processes,

mainly associated with short-term memory and

attention, occurring after a target stimulus (usually

auditory). The P300 latency is correlated negatively

with cognitive performance and is increased by age,

the administration of scopolamine, and prolonged in

dementia (Braverman and Blum, 2003; Hammond

et al., 1987 ; Polich, 1996 ; Polich et al., 1990). The

administration of ChE-Is in patients with AD reduces

the latencies of P300 potentials (Figure 1) and does so

proportionately to neuropsychological test improve-

ments (Thomas et al., 2001).

Enhancing cholinergic function with ChE

inhibition

Cholinergic neurotransmission

AD involves selective loss of cholinergic neurons in

the brain. The inhibition of AChE and BuChE increases

and maintains the synaptic concentration of ACh and

allows greater interaction with receptors. In the

presynaptic cholinergic neuron, ChAT catalyses the

synthesis of ACh from choline and acetyl-coenzyme

A. ACh is then packaged in synaptic vesicles for

extracellular export. Action potentials arriving at the

presynaptic nerve terminal trigger the release of ACh

into the synaptic cleft, where ACh can interact with

muscarinic and nicotinic receptors located on the pre-

and post-synaptic membrane. Muscarinic M2 receptors

on the presynaptic membrane regulate ACh release

via a negative feedback response. At the post-synaptic

site,muscarinicM1 receptors transduce signals through

pathways in the post-synaptic neuron. ACh is hydro-

lysed in the synaptic cleft by membrane-bound tetra-

meric G4 AChE, or by soluble monomeric G1 AChE.

BuChE is also present in synapses and in the

neuromuscular junction (Mesulam et al., 2002b),

where it binds to the same structural synaptic mem-

brane unit as AChE-S (Krejci et al., 1997). Thus, at the

end of each episode of presynaptic secretion of ACh,

binding of ACh to post-synaptic ACh receptors, and

initiation of a post-synaptic action potential, BuChE

may serve the same biological functions as AChE in

the hydrolysis of ACh (Perrier et al., 2002). A high

affinity choline-uptake mechanism returns choline to

the pre-synaptic neuron. The close spatial relationship

between glial cell protoplasm and the synaptic gap

means that diffusing, extracellular ACh entering glial

cells may be effectively hydrolysed by glial BuChE.

This is analogous to the role of glia in glutaminergic

neurotransmission, where they have a role in metab-

olizing excess glutamate.

The wide distribution of the central cholinergic

pathways means that enhanced cholinergic neuro-

transmission can have broad effects on central behav-

iours and functions, including endocrine effects due

to innervation of the hypothalamus. Both muscarinic

and nicotinic receptors are present not only on cholin-

ergic nerve terminals but also on monoaminergic

nerve terminals. Systemic administration of ChE-Is in

animal models has been demonstrated to significantly

increase levels of norepinephrine, dopamine and

serotonin (Cuadra et al., 1994 ; Giacobini et al., 1996;

Mori et al., 1995 ; Warpman et al., 1996) that may be of

clinical significance in ameliorating a variety of mood

and anxiety symptoms in AD. Again, the effects on

neurotransmitter levels may differ amongst the ChE-I

group and in different brain regions (Trabace et al.,

2001 ; Zhang et al., 2004). In addition to directly

improving the cholinergically mediated symptoms of

AD and indirect effects on multiple other neuro-

transmitters and neuroendocrine functions, enhanced

cholinergic neurotransmission may mediate other

important effects, including changes in CBF and

amyloidogenesis (discussed later in this review).

–30

Improvement

Baseline

Decline

–25

–20

–15

–10

–5

0

Ch

ang

e fr

om

bas

elin

e in

P30

0 la

ten

cies

(m

s)

5

10

15 p < 0.001 vs.donepezil andrivastigmine

Figure 1. Change in P300 latencies (¡standard error) at

6 months in an open randomized study of vitamin E (&,

n=320), 5–10 mg/d donepezil ( , n=20), and 1.5–12 mg/d

rivastigmine (%, n=20) in 60 patients with mild to moderate

dementia. Vitamin Exbaseline latency=381.5¡3.4 ;

Mini-Mental State Examination (MMSE)=16.0¡0.5 ;

donzepilxbaseline latency=382.7¡3.4 ; MMSE=16.0¡0.5 ;

rivastigminexbaseline latency=382.3¡3.4 ; MMSE=16.0¡0.5. Shorter P300 latencies were associated with higher

Wechsler Adult Intelligence Scale scores and lower

Alzheimer Disease Assessment Scale – cognitive subscale

(ADAS-cog) scores (R=0.72). (Adapted with permission from

Thomas et al., 2001.)




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The role of BuChE

Selective BuChE inhibition has been shown to elevate

extracellular ACh levels and to improve learning in

elderly rats performing maze trials (N. Greig, personal

communication ; Giacobini, 2004 ; Giacobini et al.,

1996). Mice with no AChE activity (AChE knockout

mice), are not only able to survive, but also exhibit

structural integrity of central and peripheral choliner-

gic pathways, indicating that BuChE is capable of

compensating for some functions of AChE (Mesulam

et al., 2002b). Studies have also confirmed that BuChE

is able to hydrolyse ACh in the non-AD human brain,

and that it plays an important role in cholinergic

neurotransmission (Mesulam et al. 2002a). Selective

inhibitors of BuChE are not yet available for human

testing. However, there is an increasing appreciation

of the potential importance of BuChE in dementia

(Giacobini, 2004 ; Greig et al., 2003).

The extent of BuChE inhibition in AD patients has

been correlated with cognitive improvement (Darreh-

Shori et al., 2002 ; Giacobini et al., 2002), particularly

with improvements in verbal, spatial, memory, reac-

tion time and speed tasks. The parallel influence of

AChE and BuChE upon cholinergic neurotransmis-

sion explains why mice nullizygous for AChE and

humans with enzymatically silent or greatly reduced

BuChE can escape the potentially disastrous effects of

cholinergic over-activity (Mesulam, 2003). Moreover,

since AChE activity decreases and BuChE activity

increases as AD progresses, the inhibition of

BuChE may become an increasingly important thera-

peutic target over time (Arendt et al., 1992 ; Perry et al.,

1978).

BuChE polymorphic variants

The presence of BuChE variants (K-variant homo-

zygotes and A-variant heterozygotes) has been corre-

lated with preserved attentional performance in a

group of 58 patients with Lewy Body Dementia (DLB)

(McKeith et al., 2003 ; O’Brien et al., 2003 ; Figure 2).

Moreover, these patients showed no significant

improvements in attentional performance relative to

placebo after 20 wk of treatment with the dual ChE

inhibitor, rivastigmine. In contrast, rivastigmine treat-

ment of individuals with one or two wild-type BuChE

alleles showed significant improvements in attentional

performance relative to placebo.

Particular polymorphisms of the gene for BuChE

may alter the activity or the expression of the enzyme.

Attentional performance in patients with BuChE var-

iants was less impaired at baseline and demonstrated

less response to rivastigmine, possibly due to these

patients being close to the ceiling of their attentional

performance at baseline, and having less BuChE

activity to inhibit (Figure 2). K-variant BuChE has

been proposed as a risk factor for AD (Lehmann

et al., 2001), and the consequent dementia phenotype

may have different symptom and ChE-I response

characteristics compared with K-variant non-carriers

with dementia. Evidence of deficits and treatment re-

sponses for symptoms other than attention in patients

with dementia carrying BuChE polymorphisms is

lacking, and care should be taken not to extrapolate

this result to other cognitive and neuropsychiatric

symptom domains. Reductions in attentional per-

formance in individuals with BuChE variants were not

seen in non-demented individuals with mild cognitive

impairment (O’Brien et al., 2003), suggesting that the

involvement of BuChE in attentional performance is

limited to patients with dementia.

The basal forebrain cholinergic neurons project to

all cortical areas, but only receive cortical inputs from

the limbic system. Therefore, cortical cholinergic in-

nervation may preferentially promote the allocation of

attentional resources to events that are of emotional

and motivational significance. Thus the potential

involvement of BuChE in attentional processes corre-

lates well with the neuroanatomical concentration of

BuChE-positive neurons in the amygdala and hippo-

campus (Darvesh et al., 1998).

1600

1400

1200

1000

Mea

n S

RT

scor

e at

bas

elin

e (m

s)

800

600

400

200

0wt/wt (n = 30) wt/K (n = 18)

Genotype

p = 0.03vs. wt/wt

K/K, wt/A (n = 8)

Figure 2. Attention deficit [single reaction time (SRT) in

milliseconds (ms)¡standard error of the mean (S.E.)] in DLB

patients by BuChE genotypes : ‘normal’ (wild-type)

heterozygotes, K-variant heterozygotes, and a combined

group of K-variant homozygotes and atypical heterozygotes.

wt, Wild-type BuChE variant, normal enzyme activity ; K,

K-variant BuChE, reduced enzyme activity ; A, atypical

BuChE variant, reduced enzyme activity. (Adapted with

permission from McKeith et al., 2003.)




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ChE inhibition

Differences in ChE inhibition between agents

The profiles of ChE inhibition differ markedly

amongst the ChE-Is (Table 1). In-vitro studies of

potency and selectivity of ChE-Is are not a reliable in-

dication of the situation in either the AD or non-AD

brain (Poirier, 2002). Positron emission tomography

(PET) ligand binding and CSF studies are required.

The CSF bathes the brain and the spinal cord and,

therefore, biochemical changes that occur in the brain

are likely to be reflected in the CSF. Furthermore,

AChE and BuChE activity in CSF primarily derives

from brain (Atack et al., 1986 ; Chubb et al., 1976;

Giacobini et al., 2002). A 6-wk CSF study in AD

patients indicated approximately equivalent inhi-

bition (y60%) of AChE and BuChE by 2–12 mg/d

rivastigmine (Giacobini et al., 2002). In another study,

after 12 months of 3–12 mg/d rivastigmine in AD

patients, inhibition of CSF AChE and BuChE activity

were 45% and 58% respectively (Darreh-Shori et al.,

2002).

AChE PET ligand-binding studies of donepezil

(5–10 mg/d) in AD patients have shown 27–39% in-

hibition after 4–6 wk (Kuhl et al., 2000 ; Shinotoh et al.,

2001). An AChE PET ligand-binding study with

10 mg/d donepezil or 9 mg/d rivastigmine in AD

patients demonstrated AChE inhibition of y40% in

the frontal cortex andy30% in the temporal cortex for

both drugs over 3–5 months (Kaasinen et al., 2002).

This regional difference was possibly related to a

reduced AChE activity at baseline in the temporal

cortex. A BuChE PET ligand-binding study demon-

strated BuChE inhibition of y50% in the hippo-

campus and cortex with rivastigmine and negligible

inhibition by 10 mg/d donepezil (Rinne et al., 2004).

Galantamine (50 mg/d) for 3–6 months in healthy

volunteers induced appreciable CSF inhibition of

AChE but had no effect on BuChE activity (Thomsen

and Kewitz, 1990).

Influence on safety and tolerability

In addition to alleviating the cognitive and beha-

vioural deficits associated with deficits in cholinergic

transmission, ChE inhibition may induce adverse

effects. These may be acute symptoms associated with

initiating therapy, or chronic symptoms associated

with continued therapy. Although recent speculation

on the clinical effects of BuChE inhibition has im-

plicated it in acute adverse events such as nausea and

vomiting (Wilkinson et al., 2002), data do not support

this view (Darvesh et al., 2003b; Grossberg, 2003 ; Jhee

et al., 2002). Rather, it is the rapidity and maximal in-

creases in brain levels of ACh and brain-compensating

mechanisms that increase dopamine, and not a specific

BuChE inhibition, that are responsible for acute

adverse events (Darvesh et al., 2003b; Grossberg, 2003).

For example, both physostigmine and metrifonate

inhibit AChE and BuChE. Physostigmine, which is not

well tolerated, inactivates both cholinesterases y1000

times faster than metrifonate, the latter drug being

well tolerated (Darvesh et al., 2003b). Peripheral

dopamine blockade and peripheral anticholinergics

do not influence tolerability. Only centrally acting

dopamine-blocking anti-emetics appear to be effective

at relieving ChE-I induced nausea and vomiting (Jhee

et al., 2002).

Adverse effects, other than centrally mediated

nausea and vomiting, that may lead to treatment dis-

continuation of ChE-Is during longer term therapy

may be associated with a lack of central vs. peripheral

selectivity (e.g. muscle cramps) or brain regional

selectivity (e.g. sleep disturbances, extrapyramidal

symptoms), due to a lack of preferential activity on

disease-relevant ChE molecular forms. The distri-

bution of AChE isoforms may vary between brain

regions and in the periphery. For example, relative to

other brain regions there are greater amounts of the G1

molecular form of AChE located in the cortex, amyg-

dala and hippocampus, and G1 AChE is also associ-

ated with neuritic plaques. The G4 molecular form of

AChE is the predominant form in the neuromuscular

endplate, and is co-localized with a variable pro-

portion of G2 AChE.

Donepezil may be associated with a relatively high

incidence of insomnia, vivid dreams, hypnotic use,

muscle cramps, rhinitis, and urinary incontinence

(Grossberg, 2003 ; Salloway et al., 2004; Stahl et al.,

2003 ; Wilkinson et al., 2003). In addition, although

relatively rare in clinical practice bradycardia may

develop with donepezil (Bordier et al., 2003 ; Calvo-

Romero and Ramos-Salado, 1999), that in overdose

this may be protracted (Shepherd et al., 1999), and

donepezil may adversely influence cardiovascular

autonomic control with reductions in heart rate varia-

bility (McLaren et al., 2003; Masuda and Kawamura,

2003). Rivastigmine is preferentially selective for the

G1 form of AChE and does not induce overall

increases in AChE activity (Darreh-Shori et al., 2002;

Enz, 1993 ; Rakonczay, 2003). Galantamine does not

appear to target particular molecular forms of AChE

(Rakonczay, 2003), but its competitive inhibition of

AChE may confer a reduced potential for inhibition

of neuromuscular or brainstemAChE in the absence of

ACh deficiency.




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Hypothalamic cholinergic neurotransmission plays

a major role in the regulation of growth hormone (GH)

secretion, and blockade of muscarinic receptors blunts

the release of GH (Diez et al., 2000). The secretion of

GH and insulin-like growth factor 1 (IGF-1) decline

by 14% with each decade of adult life. Senile GH

deficiency, termed somatopause, is manifested by loss

of muscle, bone mass and cardiac function, and an

increased prevalence of atherosclerosis (Rehman and

Masson, 2001). Acute administration of ChE-Is can

decrease hypothalamic somatostatinergic tone, release

GH, and enhance the GH response to GH-releasing

hormone (GHRH) (Obermayr et al., 2003). GH and

GHRH may improve memory function (Deijen et al.,

1998 ; Schneider-Rivas et al., 1995), and IGF-1 may

improve perceptual motor and processing speed

(Aleman et al., 1999). It has, therefore, been suggested

that some of the cognitive effects of ChE-Is may be

mediated by increased concentrations of GH or IGF-1

(Obermayr et al., 2003). Future investigations are

necessary to find out whether ChE-Is can restore the

senile decline of circadian GH secretion in the long

term, and to assess potential new implications for

patient treatment (Obermayr et al., 2003).

Disease progression: possible mechanisms of

ChE-Is

It is important to emphasize the difference between

‘slowing’ and ‘delaying’ disease progression. All

ChE-Is have demonstrated short-term symptomatic

effects which provide actively treated patients with a

higher baseline from which they subsequently decline.

This effect results in transiently delaying patient

decline. However, ideally, agents should also slow

decline – demonstrated by a widening gap over time

between the courses of treated and untreated patients.

It is the latter effect only that provides evidence of

disease modification. Such effects have only been

suggested in post-hoc analyses (Doraiswamy et al.,

2002 ; Farlow et al., 2000, 2003), and there is, as yet, no

definitive evidence that any of the ChE-Is modify the

underlying disease process.

There are a number of mechanisms whereby ChE-I

might potentially produce disease- modifying effects.

It has been suggested that activation of nerve cells

leads to maintenance of neurons during ageing and in

AD. This ‘use it or lose it ’ principle suggests that

neuronal activation might provide a means of pro-

longing optimal neuronal function, the survival of

remaining neurons, and modification of the course of

the disease. Classical cholinergic signal transduction

pathways may protect against neuronal degeneration

by modifying the formation of amyloidogenic com-

pounds and decreasing tau phosphorylation (Fisher

et al., 2003; Hellstrom-Lindahl, 2000), reducing

neuronal vulnerability to Ab toxicity (Kihara et al.,

2001), promoting neurotrophin release (Isacson et al.,

2002), and reversing the changes in rCBF and glucose

metabolism that occur very early in the course of the

disease (Kirkpatrick et al., 2003 ; Stefanova et al., 2003).

Non-classical effects of AChE and BuChE include the

induction of Ab fibrilization (Inestrosa et al., 1996),

plaque maturation (Darvesh et al., 2003b ; Geula and

Mesulam, 1995), activation of the cytokine cascade

(Reale et al., 2004), and increased neurite outgrowth

(Whyte and Greenfield, 2003). In addition, preliminary

evidence indicates that ChE-Is may modulate gluta-

mate and other biogenic amine neurotransmission

in the brain, and this could represent an additional

mechanism of action in AD (Trabace et al., 2001 ;

Zhang et al., 2004). It is not known which, if

any, of these mechanisms are most likely to con-

tribute to a clinically meaningful disease-modifying

effect or whether these effects would be seen with

dosages of cholinesterase inhibitors used in clinical

practice.

Cholinergic neurotransmission and the amyloid

cascade and tau phosphorylation

Cholinergic neurotransmission, and both AChE and

BuChE, may have important interactions with the

amyloid cascade. For example, the enhancement of

cholinergic mechanisms favours non-amyloidogenic

processing of amyloid precursor protein (APP) (Beach

et al., 2001 ; Fisher et al., 2002 ; Greig et al., 2001 ;

Guillozet et al., 1997 ; Soreq and Seidman, 2001).

In-vitro and in-vivo studies have consistently demon-

strated a link between cholinergic activation and APP

metabolism (Giacobini, 2003). For instance, exper-

imentally or pathologically (e.g. in AD) reduced

cholinergic neurotransmission leads to amyloidogenic

metabolism (Wallace et al., 1991, 1995). Furthermore,

Ab neurotoxicity is attenuated by muscarinic agonists

(Emmerling et al., 1997). Chronic administration of

anti-muscarinic drugs may accelerate the course of AD

(Lu and Tune, 2003), and increase the burden of AD-

type pathology, such as Ab deposits and NFTs (Perry

et al., 2003a). Lesions of cholinergic nuclei cause a

rapid increase in cortical and CSF levels of APP and

this effect can be reversed by ChE-I treatment

(Haroutunian et al., 1997).

Muscarinic agonists such as ACh, which stimu-

late the M1 receptor promote non-amyloidogenic

APP-processing pathways and decrease tau protein




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phosphorylation (Fisher et al., 2000; Nitsch et al.,

1998). Hyperphosphorylation of tau microtubule-

associated protein may represent a link between the

cholinergic signal transduction systems, disruption of

the neuronal cytoskeleton in AD, and the genesis of

NFTs (Sadot et al., 1996). M1 andM3 mAChR-mediated

signal transduction activate protein kinase C (PKC),

and the mitogen-activated protein kinase (MAPK)-

dependent pathways, reducing Ab production by

stimulating a-secretase cleavage of APP and the

secretion of non-amyloidogenic soluble APP (sAPPa)

(Beach et al., 2001 ; Fisher et al., 2003 ; Haring et al.,

1998), which is neuroprotective and neurotrophic

(Hooper and Turner, 2002). In the alternative,

amyloidogenic pathway, b-secretase cleaves APP

releasing sAPPb and a C-terminal fragment (CTFb),

which can be further cleaved by c-secretase to form

Ab, which is released into the extracellular space.

A decrease in a-secretase and a large increase in

b-secretase activity have been reported in the temporal

cortex in patients with AD (Tyler et al., 2002). PKC

may also inhibit glycogen synthase kinase-3 (GSK-3b)

(Forlenza et al., 2000). This may decrease tau phos-

phorylation, further decrease amyloidogenesis, and

decrease the neurotoxic effects and apoptosis induced

by Ab (Planel et al., 2002).

Stimulation of nicotinic cholinergic receptors

(nAChRs) may also influence APP processing to-

wards reduced production of amyloidogenic sAPPc

(Utsuki et al., 2002). Furthermore, althoughmuscarinic

receptor density is little changed in AD, functional

studies have shown impairment of M1 AChR receptor-

mediated signal transduction. The M1 AChR is the

major post-synaptic receptor of the cerebral cortex.

Low concentrations of Ab may disrupt signal trans-

duction through M1 mAChR receptors (Kelly et al.,

1996), and this uncoupling may lead to a decrease

in sAPPa secretion, generation of more Ab, and a

‘vicious cycle’ of further cholinergic deficiency (Fisher

et al., 2002).

Evidence for ChE-I-induced increases in cholinergic

neurotransmission affecting tau and amyloid pathol-

ogy in AD patients is limited. However, in a recent

study, no change was seen in CSF tau levels after 1 yr

in rivastigmine-treated AD patients, while significant

increases were seen in untreated AD patients

(Stefanova et al., 2003). If clinical correlates were to

be demonstrated, the involvement of cholinergic

receptor-mediated signal transduction pathways in

amyloidogenesis and tau phosphorylation might

prompt revision of the cholinergic hypothesis in AD,

which currently ignores the potential of cholinergic

therapies as disease-modifying agents.

Allosteric nAChR modulation

The ChE-I, galantamine, has been shown in vitro to act

as an allosterically potentiating ligand on particular

subtypes of the human nAChR, such as the a4/b2

subtype (Samochocki et al., 2000). The clinical

relevance of this finding is unclear. Loss of nAChRs

occurs early in AD, particularly in patients with APOE

e4 alleles. The established efficacy of galantamine in

subgroups of patients with moderately severe AD, and

in AD patients with APOE e4 alleles is probably due to

AChE inhibition alone (Aerssens et al., 2001 ; Blesa

et al., 2003). All ChE-Is indirectly interact with

nAChRs through the elevation of ACh.

The marked loss of nAChRs that occurs in the AD

brain (Kellar et al., 1987) has been shown to be pre-

vented over the course of 1 yr or more in patients with

mild AD treated with tacrine, which has no apparent

ability to allosterically modulate nAChRs (Nordberg

et al., 1998). AD patients showed increased nicotinic

receptor binding in the temporal cortex, as measured

by PET, after 3 months of treatment with rivastigmine

and to a lesser extent also with tacrine (Nordberg,

2004). The lesser effect of tacrine may relate to its

demonstrated ability to behave as an open channel

blocker of nicotinic receptors (Prince et al., 2002).

Recent data have also shown that donepezil may block

or desensitise nAChRs through a direct interaction of

donepezil with nicotinic receptors (Di Angelantonio

et al., 2004).

Amino acidergic transmission

There has been considerable interest in excitatory

amino-acid theories of neurodegenerative disease

such as AD (Nakanishi et al., 1998). Glutamate is the

major excitatory neurotransmitter in the CNS and can

induce excitotoxicity. In fact, in AD, cortical and hip-

pocampal pyramidal neurons, which are particularly

affected by NFTs and neuronal degeneration, use

glutamate or aspartate as neurotransmitters (Maragos

et al., 1987). Moreover, it has been shown that the

distribution of senile plaques in the AD brain corre-

lates with the distribution of glutaminergic synapses

(Rogers and Morrison, 1985). Glutamate signal trans-

duction at the post-synaptic terminals is initiated

by stimulation of glutamate receptors, especially of the

N-methyl-D-aspartate (NMDA) subtype. Ab can en-

hance the release of glutamate, prevent its uptake by

synaptosomes and glia, and increase neuronal and

glial cell sensitivity to glutamate-induced excito-

toxicity. A degenerative feedback cycle may be gener-

ated of excess glutamate, induction of NMDA

receptor-mediated depolarization and intracellular




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Ca2+ increases, with further glutamate release, and

loss of neuronal function. Excessive intracellular Ca2+

accumulation leads to an abnormal activation of Ca2+-

dependent enzymes, such as neuronal nitric oxide

synthase (nNOS). This enzyme catalyses the formation

of nitric oxide (NO) and citrulline from L-arginine.

Taken together, these data suggest a role for amino

acids in the clinical manifestation and pathogenesis of

AD (Nakanishi et al., 1998) as an alternative, not

necessarily exclusive, to the cholinergic hypothesis.

The effects of oral rivastigmine, an inhibitor of

AChE and BuChE, and CHF2819, a selective inhibitor

of AChE, on extracellular concentrations of amino

acids in the rat hippocampus, were evaluated using in-

vivo microdialysis (Trabace et al., 2001). Rivastigmine,

but not CHF2819, significantly decreased glutamate,

arginine and citrulline levels, without affecting aspar-

tate concentrations. In addition, 3- and 21-d treatment

with rivastigmine increased the expression of the

neuronal glutamate transporter in the rat hippo-

campus but not in cortical areas (Andin et al., 2004).

These results suggest that the modulation of the

glutaminergic system could represent an additional

mechanism of action in AD for ChE-Is.

Ab fibrilization

It has been observed that in AD, brain cortical AChE

activity is associated predominantly with the amyloid

core of mature senile plaques (Alvarez et al., 1998).

Therefore, AChE might have a crucial role in amyloid

deposition at an early stage of the disease. One of the

central events in the AD pathogenesis is the Ab

fibrilization, and both BuChE and AChE may have

roles in the aggregation of Ab. AChE may influence

the in-vitro and in-vivo aggregation state of Ab, per-

haps through the non-catalytic mechanisms related to

the PAS subsite (Bartolini et al., 2003 ; De Ferrari et al.,

2001 ; Inestrosa et al., 1996 ; Lahiri et al., 2000). AChE

may constitute an important co-factor in Ab fibrillo-

genesis, being able to induce a conformational

change of Ab in solution and thus, having a direct role

in fibril formation. Moreover, AChE-Ab complexes

have been shown to be more neurotoxic than Ab pep-

tides alone (Alvarez et al., 1998; Opazo and Inestrosa,

1998), and may be involved in the pathogenesis of AD

(Opazo and Inestrosa, 1998). Thus, both in-vitro and

in-vivo data suggest that AChE behaves as a potent

amyloid-promoting factor and modulates the toxicity

of amyloid fibrils. AChE-induced aggregation was

found to be inhibited by peripheral anionic site ligands

such as propidium, and only partially inhibited by

molecules binding both to the catalytic site and to the

peripheral site such as physostigmine and donepezil

(Bartolini et al., 2003 ; Piazzi et al., 2003).

Pathological amino- orN-terminal-truncated species

of Ab42 may be involved in the earliest stages of

amyloidogenesis in humans (Sergeant et al., 2003).

N-terminal-truncated pyroglutamyl-ended forms of

Ab are present in senile plaques and vascular walls in

individuals with AD (Kuo et al., 1997 ; Saido et al.,

1995). Pyroglutamyl-ended Ab peptides (Ab pE) form

b-sheet structures more readily than the correspond-

ing full-length Ab peptides (He and Barrow, 1999).

Interestingly, experimental data suggest that ChE-Is

may avoid the formation of Ab pE deposition through

the activation of pyrrolidone carboxyl peptidase (Pcp).

The activity of this enzyme is reduced in the plasma of

sporadic AD (Kuda et al., 1997). Acute administration

of rivastigmine increased Pcp activity in frontal cortex

synaptosomes from mice in a dose-dependent manner

(Ramirez-Exposito et al., 2001).

Plaque maturation

The factors that contribute to the transformation of a

relatively inert diffuse plaque to a neuritic or patho-

genic one may involve interactions with additional

plaque constituents (Guillozet et al., 1997). Such con-

stituents include the G1 isoforms of both AChE and

BuChE (Geula and Mesulam, 1995). The diffuse

amyloid plaques of normal ageing tend not to display

BuChE activity, whereas the vast majority of compact

neuritic plaques do (Geula and Mesulam, 1995). The

AChE and BuChE in these pathological structures has

altered sensitivity to ChE-Is and to proteolytic en-

zymes (Geula and Mesulam, 1995; Wright et al., 1993).

BuChE and AChE may interact with other proteins to

form complexes that could serve a regulatory role

(Darvesh et al., 2001, 2003b). It has been suggested that

AChE might have a crucial role in amyloid deposition

at an early stage of the disease (Alvarez et al., 1998),

and based upon these observations, it seems that

BuChE may play a role in the transformation of

benign plaques to a malignant form associated with

neuritic tissue degeneration and clinical dementia.

Furthermore, the substantial increase in BuChE

activity in more pathogenic forms of Ab deposits

raises the possibility that the in-vivo imaging of

BuChE may have potential as a diagnostic tool.

There is an emerging concept that the net biological

response of pro- and anti-inflammatory cytokines

affects the outcome of certain diseases including

neurodegenerative disorders (O’Shea et al., 2002).

Transcription and production of pro-inflammatory

cytokines are markedly enhanced in AD (Reale et al.,




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2004). Pro-inflammatory cytokines released by Ab-

activated glial cells that proliferate around plaques

may promote further Ab deposition (Meda et al., 2001).

ChE-Is may reverse cytokine imbalances observed in

AD (Reale et al., 2004). The putative role for ChE-Is in

the regulation of immune response through reduced

production of inflammatory cytokines could relate

to their therapeutic effects, and requires further

investigation.

Clinical evidence for a role of BuChE in disease

progression

The increases in BuChE in key brain areas may also

provide a possible explanation for the accelerated

rates of atrophy seen in patients with more severe AD.

Perry and colleagues (2003b) reported a significant

correlation between grey-matter BuChE levels in the

temporal cortex and annual cognitive decline on the

MMSE in a prospectively studied autopsy-confirmed

DLB case series. If BuChE variants can affect attentional

performance, possibly through the effect of BuChE on

levels of available ACh, it is possible that BuChE var-

iants may also have an effect upon disease progression

as measured by the rate of cognitive decline.

O’Brien and colleagues (2003) recently presented

data from a study of patients with AD or DLB, show-

ing that the presence of one or more BuChE-K alleles

was associated with a significantly less rapid rate of

cognitive decline over a 2- to 3-yr period than that

found in individuals who were wild type (wt) for

the BuChE gene. Cognitive decline was 9.6¡11.6

CAMCOG points in patients with a K/wt or K/K

phenotype, compared with 16.5¡11.8 points in wt/wt

patients (p=0.04). In a linear regression analysis

evaluating the impact of diagnosis (DLB vs. AD),

the BuChE genotype remained independently and

statistically significantly associated with the rate of

cognitive decline. A further study in patients with AD

has confirmed these findings (Holmes et al., In Press).

These data support the hypothesis that BuChE may

be an important moderator of AD progression (Ballard

and Perry, 2003). The finding that carriers of a

BuChE-K allele have differential rates of disease pro-

gression than non-carriers is not incompatible with the

suggestion that BuChE-K is a risk factor for AD in

males with an APOE e4 allele (Lehmann et al., 2001),

and protective in female non-APOE e4 carriers

(Alvarez-Arcaya et al., 2000). BuChE-K may interact

with APOE e4 to increase the risk of dementia, but

once AD is established carriers of a BuChE-K allele

generally show less rapid progression of dementia

compared to non-carriers (Holmes et al., In Press ;

O’Brien et al., 2003). Thus, evidence suggests that

BuChE activity may have a role in disease progression

in AD.

The influence of ChEs on cerebrovascular responses

Cholinergic neurotransmission and rCBF

The integrity of the cerebral vasculature is crucial for

the maintenance of cognitive function. Cerebro-

vascular function declines with age due to micro-

vascular pathology, including amyloid deposition

(Kalaria and Ballard, 1999) and are more evident

in AD. In addition, cortical microvessels and NO-

synthesizing interneurons in the AD brain exhibit

severe cholinergic denervation (Tong and Hamel,

1999). Cholinergic neurons may be particularly sus-

ceptible to hypoxia, and chronic vascular hypo-

perfusion has been implicated in the pathogenesis and

accelerated course of AD (Aliev et al., 2003).

Cholinergic neurons projecting from both para-

sympathetic ganglia and basal forebrain innervate

cerebral blood vessels (Toda and Okamura, 2003).

Inhibition of ChE increases rCBF through the dilation

of intracortical microvessels (Kasa et al., 2000), and

ablation of basal forebrain cholinergic neurons

reduces rCBF and increases cortical vascular Ab de-

position (Roher et al., 2000). The cerebral vasodilatory

effects of cholinergic activation may be mediated

through cholinergic nerve terminals in contact with

glial cells surrounding microvessels (Chedotal et al.,

1994). In addition, many cholinergic neurons syn-

thesize and release the potent vasodilator NO, and

nitrergic neurons may receive inputs from cholinergic

neurons (Toda and Okamura, 2003). It has been sug-

gested that the cholinergic system plays an important

role in the coupling mechanism between neuronal

activity and functional responses of rCBF (Ogawa

et al., 1994), and may also have a role in ensuring that

the cortex and hippocampus have a sufficient blood

flow during exercise (Kimura et al., 1994 ; Nakajima

et al., 2003). Thus, cholinergic andnitrergic innervation,

and the interaction between these systems, play a

major role in the regulation of cerebrovascular tone

(Toda and Okamura, 2003).

Recent evidence suggests that abnormalities in

rCBF and energy metabolism are early changes in AD

and may precede plaques, NFTs, and the appearance

of neurodegeneration in AD (Gibson, 2002 ; Salmon

et al., 1996). For example, decreases in brain metab-

olism are seen many years before the development of

symptoms in patients predisposed to AD (Small et al.,

2000), and this may reflect a very early dysfunction in




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synaptic connectivity. In contrast to the lack of mani-

fest evidence of deficits in cholinergic markers in early

AD, hypometabolism in entorhinal and posterior

cingulate cortices with FDG-PET can identify early

AD, and perhaps distinguish amongst normal ageing,

mild cognitive impairment and mild AD (de Leon

et al., 2001 ; Silverman et al., 2001). ChEIs have been

shown to increase rCBF and glucose metabolism in

relevant brain areas such as the hippocampus and

cortex in animal models and in man (Ebmeier et al.,

1992 ; Harkins et al., 1997 ; Minthon et al., 1993 ; Potkin

et al., 2001). AD patients who respond to ChE-I treat-

ment show significant dose-dependent increases in

rCBF (Lojkowska et al., 2003 ; Venneri et al., 2002), or

glucose metabolism (Potkin et al., 2001 ; Stefanova

et al., 2003) in temporal, parietal, and frontal cortices

with related improvements in a range of psychometric

tests including working memory and attention for

up to 12 months. Non-responders and untreated

AD patients show significant decreases in rCBF,

glucose metabolism and psychometric performance. A

normalization of the coupling mechanism between

neuronal activity and rCBF or metabolic response

could delay the progressive decline in cognitive

function and possibly reduce the formation of Ab

and the onset of AD if initiated early enough in the

development of the disease.

Chronic hypoxia due to cerebrovascular in-

sufficiency, by harming the cholinergic system, may

induce a vicious circle of further impairment to

cholinergically mediated control of rCBF, resulting in

further cerebrovascular insufficiency. ACh has a major

role in the control of cerebrovascular tone and self-

propagating cycles can be induced by ACh imbalances

that negatively impact brain vascular function and

homeostasis.

Hypoxia can impair mental functions such as

attention and memory, and such impairments are

related to deficits in cholinergic function that may be

reversed by ChE-Is (Gibson, 2002). In animal models,

ChE-Is induce vascular relaxation and improve CBF in

the ischaemic brain (Sadoshima et al., 1995 ; Tanaka

et al., 1994, 1995 ; Tsujimoto et al., 1993). Furthermore,

treatment with ChE-Is may prevent cholinergic

deficits and reduce pyramidal cell loss in ischaemic

hippocampus (Tanaka et al., 1994, 1995). Improved

rCBF or metabolism may improve the functional

potential of neurons in the subcortical region impaired

by ischaemic vascular disease. Improvements in

attention, executive dysfunction and behaviour have

been demonstrated in patients with ischaemic

subcortical vascular dementia treated with ChE-Is

(Moretti et al., 2002, 2004).

Conclusion

The classical role of ChE is in the termination of

ACh-mediated neurotransmission. Cholinergic neuro-

transmission is reduced in AD and it may be enhanced

by ChE-I-induced increases in the synaptic availabil-

ity of ACh. AChE inhibition may result in general

improvements of cortical instrumental functions in

patients with AD, as a result of improved underlying

fundamental functions mediated by the widespread

input of the AChE-positive neurons to the cortex.

BuChE inhibition may result in additional specific

input into frontal cortical structures due to the more

focused projections of BuChE-positive thalamic

neurons to frontal brain areas and the predominant

neuroanatomical localization of BuChE in the limbic

system and subcortical regions. The clinical expression

of the differences between AChE-specific and dual

ChE inhibitors will only be fully elucidated in well-

designed comparative studies that assess improve-

ments in underlying fundamental functions with

sensitive instruments, and in patient populations

with significant fundamental functional deficits.

Determination of the effects of BuChE-specific

inhibition awaits the assessment of BuChE-specific

inhibitors in AD patients.

Relative potencies and ratios of AChE/BuChE

inhibitory activity are not the only determinants of

individual ChE-I clinical profiles. Pharmacokinetic

characteristics, mechanisms of enzyme inhibition,

brain selectivity, and differential selectivity for various

molecular forms of each ChE may also be important

factors. There are marked differences amongst ChE-Is

in their potential to up-regulate AChE gene expression

and in their modulation of alternative splicing of the

AChE gene to produce changes in the composition of

AChE molecular forms. Furthermore, as the severity

of dementia advances, BuChE activity progressively

increases while AChE activity declines. Long-term

clinical studies, ideally comparing agents that inhibit

BuChE alone, both BuChE and AChE, and AChE alone

are needed to ascertain whether these differences

amongst ChE-Is are reflected in differences in symp-

tom control at various disease stages.

Further investigation is required to clarify the

potential mechanisms whereby the non-classical roles

of BuChE and AChE may affect disease progression in

dementia. Many recent studies concern the regulation

of APP processing and modulation of tau phosphory-

lation by ACh receptor stimulation, and how cholin-

ergic deficits and Ab production may be related to one

another. In addition, AChE may have important roles

in amplifying Ab toxicity and in Ab fibrilization.




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Specific expression of AChE and BuChE in neurons,

glia and endothelial cells suggests that, in addition

to co-regulation of cholinergic neurotransmission,

BuChE and AChE may also influence cholinergically

and non-cholinergically mediated cerebral vascular

responses. Normalization of the disrupted coupling

mechanism between neuronal activity and rCBF

observed in AD could delay the progressive decline

in cognitive function, and might possibly reduce the

formation of Ab.

Neuroimaging studies, transgenic animal models

and autopsy studies of amyloid burden are required to

determine the differential effects of BuChE and AChE

inhibition on the neuronal and non-neuronal cholin-

ergic systems. The physiological roles of the different

splice variants or molecular forms of BuChE and

AChE also require further clarification. Screening for

protein partners that interact with various molecular

forms of AChE and BuChE may identify possible

biochemical pathways in which they participate.

These promising leads on the non-classical and non-

synaptic roles of AChE and BuChE in the modulation

of AD and related dementias clearly require further

investigation and offer the potential for improved

treatments.

Acknowledgements

None.

Statement of Interest

Roger M. Lane is an employee of Novartis Pharma-

ceuticals, New Jersey, USA, and Albert Enz is an

employee of Novartis Pharma AG, Basel, Switzerland.

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