M. R. Farlow
INTRODUCTION
Acetylcholine is a neurotransmitter with major roles in learning and memory in both mice and men. Deficits in cognitive functioning are a major feature in Alzheimer’s disease (AD).
Up to 90% of cholinergic neurones may be lost as the illness progresses [1]. The most suc- cessful symptomatic approach to treating cognitive symptoms in AD is focused on raising levels of acetylcholine, primarily by blocking the enzymes (cholinesterases) that metabolize this neurotransmitter.
Two major forms of cholinesterase are found in the brain and spinal cord. The most com- mon form, acetylcholinesterase (AChE), is responsible for 90–95% of activity in the central nervous system in normal individuals, while butyrylcholinesterase (BuChE) is responsible for almost all of the remainder [2]. Butylcholine is not a substrate normally present in mam- malian brain, but rather a synthetic chemical that allows initial differentiation of BuChE.
AChE and BuChE share 65% sequence homology, but the encoding genes are at entirely dif- ferent sites on chromosomes 7 (7q22) and 3 (3q26), respectively [3]. Both AChE and BuChE are very efficient at hydrolysing acetylcholine and are both thought to have parallel func- tions in hydrolysing this neurotransmitter at the synapse [4]. It has been suggested that the primary physiological role of BuChE is to hydrolyse excess acetylcholine [5]. Support for this hypothesis comes from experiments demonstrating that BuChE in brain clearly hydro- lyses acetylthiocholine (a surrogate for acetylcholine that can be easily measured) in the presence of an acetycholine-specific inhibitor [4]. AChE has greater catalytic activity at low concentrations of acetylcholine and the activity of BuChE is greater at much higher concen- trations of this neurotransmitter [3, 6]. Both of these enzymes have a deep hydrophobic gorge where acetycholine is thought to enter by diffusion and to be cleaved. Differences in several amino acids in this gorge are thought to underlie the ability of BuChE to hydrolyse several different molecules beyond acetylcholine [7]. A peripheral anionic site also exists for both enzymes and weaker affinity of this site for many ligands may contribute to the greater diversity of molecules that are hydrolysed by BuChE. AChE is also more specific for acetyl- choline than BuChE, but BuChE also metabolizes a number of other neuroactive peptides [7]. Classically AChE is thought to be produced primarily by neurones and to be active chiefly at the synapse, playing a major role in regulating neurotransmission.
BuChE is synthesized in many tissues including heart, lungs, liver and brain. In the brain it is predominantly produced by glial cells and it has been demonstrated to have other less well-known actions including roles in modulating lipid metabolism, myelin maintenance, inflammation, and in regulation of amyloid precursor protein (APP) processing [8–12]. It has become clear from recent neuropathological studies that substantial amounts of BuChE activity Martin R. Farlow, MD, Professor and Vice Chairman for Research, Department of Neurology, Indiana University School of Medicine, Indianapolis, Indiana, USA
©Atlas Medical Publishing Ltd 2007
are present in the hippocampus, amygdala and other neocortical areas which are known to receive cholinergic input. Much of the enzyme is located in glia, but it diffuses to the synapse where it appears to be enzymatically active and capable of hydrolysing acetylcholine.
CHANGES IN BuChE WITH AGEING AND DEMENTIA
BuChE activity in the brain progressively increases past the age of 60 years in an age- dependent manner [5].
Alternative splicing for both AChE and BuChE, each of which is encoded by a different single gene, gives multiple forms including; an asymmetric form of AChE with tail that anchors to cell membranes and globular forms G1, G2, and G4 with one, two, and four active catalytic sites, respectively [13]. In an analogous manner, BuChE is also found in G1, G2 and G4 forms [2]. The G4 form of AChE which is predominantly at the synapse in the normal brain may be reduced in AD by as much as 90% in some areas of the brain. This reduction is primarily due to loss of the G4 form at presynaptic sites of cholinergic synapses in the brain [14]. Overall AChE levels in AD progressively decline with worsening disease stage by 50–67% in different regions of the brain (Figure 11.1) [2, 15]. The G4 form of BuChE which is of glial origin, maintains stable levels or is mildly reduced, while the G1 form increases by as much as 30–60% with the percentages going up with worsening disease stage [2, 14]. In areas of the cortex affected by AD, the ratio of BuChE to AChE has been shown to increase from 0.5 to as much as 11 [16]. This increase may relate to proliferation of glial cells. These changes suggest BuChE may become a more significant target for drug therapy as the illness worsens, particularly in older patients and in those entering the mod- erate to severe stages of AD.
In both normal and AD brains, AChE-positive glia are found widely throughout the cor- tex and white matter, while BuChE is localized to capillary endothelial cells and glia. These glia are found predominantly in the deep cortical layers and in the subcortical white matter [4, 17]. In normal brains the ratio of BuChE-positive glia to AChE-positive glia are highest in the entorhinal and in frontotemporal cortices (two areas very susceptible to AD) [18]. In AD brain, glial BuChE/AChE ratios are increased in these areas, but this increase does not occur in other areas of the brain. Most investigators believe reactive gliosis occurs around plaques, but it is also possible that glia in AD exacerbate amyloid ! (A!) deposition by their secretion of cytokines [18, 19]. Certainly, increased secretion of BuChE from glia may worsen cholinergic transmission by further driving down levels of acetylcholine.
Although AChE-positive neurones are predominant, BuChE-positive neurones are located in all brain regions that receive cholinergic innervation with particularly large num- bers of these neurones being located in amygdala, hippocampus, and thalamus [6, 20–22].
P"0.01
P"0.01
0 2 4 6 8 10 12 14 16 18 20
AChE BuChE
Cholinesterase activities (nmol/ml # min)
Healthy human brain Human Alzheimer brain
Figure 11.1 AChE activity decreases, and relative BuChE activity increases in the AD brain. With permission from [2].
ANATOMICAL–BEHAVIOURAL CORRELATIONS
Neuronal losses in the specific distributions of BuChE-staining neurones, particularly in subcortical nuclei and the regions they project, to have previously been associated with specific cognitive and or behavioural deficits. The variable presence of BuChE at high levels in greater numbers of neurones in specific areas of the brain and its greater activity in the presence of high concentrations of acetylcholine as compared to AChE, also suggests that it may have dampening or modulating effects on specific cognitive or behavioural modalities.
For example, BuChE-staining neurones are found in particularly high numbers in thalamic nuclei that project to the prefrontal cortex [22]. These nuclei include the medial dorsal nucleus which projects to both prefrontal and cingulate cortices and is involved in working memory and planning, and the pulvinar nucleus which is involved in visual attention.
These deficits are central to the AD process.
Though hypothetical, the specific geographical distribution where BuChE-staining in the brain neurones are located suggests specific aspects of cognitive and behavioural function- ing in AD may be more benefited by BuChE inhibition than others, a concept supported by preliminary data in following sections.
BuChE AND PLAQUE FORMATION
In the brains of patients with AD, there is evidence that BuChE decorates the cortical and subcortical plaques, which are composed predominantly of A!protein, a hallmark feature of the illness. BuChE is also found in close association with neurofibrillary tangles, the other major neuropathological feature which defines AD (Figure 11.2) [23]. The presence of BuChE, as detected by immunostaining, increases both with the numbers of plaques and
(a)
(b)
Figure 11.2 BuChE activity increases in the AD brain, compared with healthy controls. (a) BuChE staining in the temporal cortex of a 71-year-old patient with AD. BuChE is found in plaques ( ), tangles ( ), dystrophic neuritis ( ), and glia ( ) (b) BuChE staining in an 89-year-old non-demented individual.
BuChE staining is limited to the glia ( ). With permission from [23].
with the clinical disease stage [23]. Studies investigating plaque maturation suggest little BuChE is present initially associated with diffuse deposits of A!(early stage plaques), but BuChE staining becomes more prominent as the plaques become more mature exhibiting a compact amyloid core [24].
Histochemistry comparing adjacent brain tissue sections, looking at both BuChE and the A!protein, has suggested that in diffuse or primitive plaques, most of the BuChE is located over plasma membranes of healthy-appearing cellular processes. In more extensive plaques, BuChE begins to decorate amyloid filaments [23]. BuChE-containing plaques almost all bind thioflavin and 93% of all thioflavin-staining plaques were found to contain BuChE. In classic neuronal plaques, BuChE is found colocalized with the amyloid filaments as well as the plaque core suggesting that BuChE may play a role in plaque maturation (Figure 11.3) [2, 25].
The predominant structural form of cholinesterase for both AChE and BuChE that aggregates with A!plaques is the G1 form [2, 23]. The level of G1 BuChE increases in a manner that correlates directly with the deposition of amyloid deposition in neocortical plaques (Figure 11.4) [2, 23].
In tissue culture studies, where A!protein is in the medium when BuChE is added, the neurotoxic effects of the A!protein are amplified. This provides further evidence suggest- ing that BuChE plays a role in the toxic cascade of events that underlies the neuropatholog- ical features of AD and the potential for a BuChE inhibitor to have therapeutically useful effects in reducing neurodegeneration [26].
ANIMAL STUDIES AND BuChE INHIBITION
Supporting evidence that strongly suggests BuChE plays a significant role in cholinergic neurotransmission comes from studies in mice where AChE has been selectively knocked out. These mice have functional cholinergic systems with BuChE substituting for the actions of AChE [27–29].
In these knockout mice, staining for the enzyme, choline acetyltransferase (ChAT), was present in the same distributions as in normal mice, suggesting that AChE is not necessary Figure 11.3 BuChE/AChE in the life cycle of the amyloid plaques. 1. BuChE/AChE co-localise with A!and may accelerate !-amyloid formation and deposition in AD brain; 2. In turn, !-amyloid protein regulates ChE expression, assembly and glycosylation; 3. Increased levels of BuChE as disease progresses, augmenting the cycle; 4. Inhibition by ChE-Is may influence !-amyloid deposition and may influence APP processing. Adapted with permission from [25].
3
4
APP processing
ChE expression glycosylation
ChE-1 1
2 BuChE AChE G1
ChE-!-amyloid complex
for the maintenance of the cholinergic system and that BuChE can substitute for its func- tions at the synapse throughout the brain allowing relatively normal functioning.
Despite low levels of BuChE in the rat brain, cortical perfusion with a selective inhibitor for BuChE has led to a 15-fold increase in acetylcholine, further suggesting this enzyme may have greater influence on cortical neurotransmission than generally recognized [17].
BuChE-specific inhibitors in mouse studies have been demonstrated to improve cogni- tive functioning of the animals in several specific tasks or learning paradigms [30].
Figure 11.4 Correlation between G1 BuChE activity and neocortical amyloid deposition. Adapted with permission from [2].
Density of amyloid deposition in neocortical area 8 (mm$3) Cerebral G1 form of BuChE activity (nmol # min$1# mg$3 protein)
r % 0.86 P " 0.01 0
0.50 0.75 1.00 1.25 1.50
50 100 150 200
Figure 11.5 Mean (&SEM) number of errors made (P"0.05 for PEC 0.5, BNC 0.25, 0.5 mg/kg vs. control) in BNC- and PEC-treated groups of aged rats relative to controls, during acquisition training in the 14-unit T-maze. With permission from [30].
0 50 100 150 250
200
1 2 3
Blocks (four trials per block)
Mean runtime per block (s)
4 Control (n%53) BNC 0.25mg/kg (n%8) BNC 0.5mg/kg (n%10) PEC 0.25mg/kg (n%9) PEC 0.5mg/kg (n%10)
It is of interest that treatment with the BuChE inhibitor, bisnorcymserine, resulted in fewer errors in aged rats running a 14-unit maze. These animals had increased levels of acetylcholine despite the fact that far fewer neurones in rat brain are positive for BuChE [30]
(Figure 11.5). In brain slices from rats, treatment with a BuChE inhibitor improves long-term potentiation. In summary, mouse and rat studies strongly support that BuChE may play a larger role than previously thought in cholinergic functioning and that BuChE inhibitors may have a greater ability to increase levels of acetylcholine than would be predicted from the low levels of BuChE present in these animals.
NON-SYNAPTIC ACTIONS OF BuChE
Both AChE and BuChE appear to have a number of non-synaptic mechanisms of action including: effects on A!processing, inflammation, lipid metabolism, as well as tau phos- phorylation and even regional cerebral blood flow [5, 12, 31, 32].
Cerebral vasculature has extensive cholinergic innervation that plays a role in regulating cerebral blood flow [33]. Both inhibition of AChE and BuChE have been demonstrated to increase blood flow in the cortex and there is also evidence that metabolism of glucose is increased by cholinesterase inhibitors [34–38]. These improvements in blood flow and potentially even metabolism are another method by which these drugs may improve cogni- tive symptoms in AD [30, 39].
BuChE may play a role in neurofibrillary tangle formation. In the temporal cortex obtained after death from 30 prospectively studied patients with either AD or diffuse Lewy body dementia and genotyped for the K variant of BuChE, individuals with one or more K alleles (associated with 30% less BuChE activity) had 42% less phosphorylated tau in the temporal cortex [40]. These data suggest a relationship between BuChE activity and tau phosphorylation, and further that the reduced levels of activity found in patients with the K-variant polymorphism, or inhibition of BuChE by targeted drugs may slow or reduce rates of disease progression.
Finally, in transgenic mice (Swedish'Presenilin 1), treatment with the BuChE inhibitor, phenylethylcymserine, significantly decreased both A!1–40 and A!1–42
Figure 11.6 Reductions in brain levels of A!40and A!42in Tg mice overexpressing human A!, after daily treatment with PEC or vehicle control (P"0.05 PEC vs. control, Student’s ttest). With permission from [30].
0 250 500
Protein concentration (pM)
750 1000
Control PEC
$54%
$47%
Control PBC
A!40 A!42
levels, actions that theoretically could reduce long-term rates of A!deposition and AD (Figure 11.6) [30].
BuChE AND THE CHOLINESTERASE INHIBITORS
Available cholinesterase inhibitors that are currently in clinical use, or that have been tested in humans, are listed in Table 11.1 [41, 47]. Rivastigmine has the most inhibitory activity for BuChE of the drugs currently used in clinical practice, though tacrine (a drug no longer much used because of short half-life and hepatotoxicity) had approximately equivalent lev- els of inhibition for BuChE. Several new selective drugs for BuChE inhibition have been developed (e.g. cymserine and phenylethylcymserine) but whether they have clinical effi- cacy in AD when given as monotherapy and/or whether their use in combination with AChE inhibitors provides any additional benefits is unknown [30].
The characteristics of cholinesterase inhibitors available and currently used in clinical practice differ markedly in their selectivity, or lack thereof, for AChE and BuChE as well as the different forms of these enzymes (Table 11.2) [48]. Because of differences in metabolism, central nervous system (CNS) penetration, plasma binding etc., in vitrostudies of selectivity may not give an accurate representation of these drugs’ in vivoselectivity and potency in the human brain.
Two potential methods to overcome these obstacles are to obtain cerebrospinal fluid (CSF) before and after dosing and/or performing brain neuroimaging using positron emis- sion tomography (PET) ligands for AChE or BuChE as will be further detailed in the next two sections.
CHOLINESTERASE AND CSF STUDIES
It has been reported that CSF AChE activity increases with age in normal subjects, while it remains stable in AD patients not taking cholinesterase inhibitors over 12 months [49].
Other investigators have suggested that AChE activity declines while that of BuChE increases in patients with AD [2].
In general, CSF measurements of BuChE and AChE activities and their inhibition have certainly demonstrated that activities of these enzymes are being affected by different cholinesterase inhibitors, but with wide variability in the methodologies employed, includ- ing sampling times after dose, measurement of levels of enzyme vs. functional activity, and finally differences in the mechanisms of action of these drugs (i.e. donepezil and galanta- mine ionically bind to cholinesterases while rivastigmine binds covalently), as well as fac- tors such as diurnal effects, all are confounds that have made it difficult to interpret CSF BuChE- and AChE-inhibition data with confidence.
BRAIN CHOLINESTERASE AND NEUROIMAGING STUDIES
The effects of cholinesterase inhibition can be measured in the living human brain using PET/MRI by measuring the uptake of a radiolabelled BuChE substrate analogue, N-[11C]methyl-piperidyl-n-butyrate [50]. In normal individuals studies with this compound have recently demonstrated that most BuChE activity is in cerebellum, striatum, thalamus and hippocampus. When rivastigmine is administered in patients with AD, this ligand showed a marked decrease in BuChE activity consistent with inhibition not only in the hippocampus but also temporal, frontal and parietal regions [50].
A BuChE PET ligand study in subjects with AD treated with rivastigmine, which inhibits both cholinesterases, showed 50% inhibition of BuChE in both the hippocampus and cortex, while inhibition of binding by donepezil as expected was negligible [50]. Further studies with such ligands may help to clarify the longer term implications of BuChE as well as the consequences of long-term inhibition of this enzyme in disease progression.
Therapeutic Strategies in Dementia
Donepezil Rivastigmine Galantamine
Chemical class Piperidine Carbamate Tertiary alkaloid
Inhibition of ChE Non-competitive, rapidly Non-competitive, very Competitive, rapidly enzymes reversible ("1 ms) of AChE slowly reversible (!6–8 h) reversible ("1 ms) of AChE
of both BuChE and AChE
Site of inhibition of Covers catalytic gorge and Catalytic binding site Choline anionic site and target
enzyme peripheral anionic site, catabolic binding site
binding to choline anionic site
Increased activity/levels of 5–10 mg/day:!3-fold 3–12 mg/day – sustained 24–32 mg/day: !2-fold target enzymes during increase in AChE levels decrease in AChE and increase in AChE levels long-term treatment1,2 over 6–12 months1 BuChE activity over 12 months2 over 6 months1
Brain vs. peripheral Uncertain Yes None
selectivity
Preferential isoform None in most studies3,4, G1 (or AChE-R)3,4 None4
selectivity3,4,5 G1 in one5
Allosteric modulation of No No Particular subtypes
nicotinic receptor6 (e.g. (4/!2) in vitro
Metabolism CYP2D6 and 3A4 AChE and BuChE CYP2D6 and 3A4
Plasma protein binding !96% !40% !20%
AChE%acetylcholinesterase; BuChE%butyrylcholinesterase; CYP450%cytochrome P450; nAChR%nicotinic Ach receptor.
1[42], 2[43], 3[44], 4[45], 5[46], 6[47].
Table 11.1 Overview of pharmacological characteristics of cholinesterase (ChE) inhibitors (adapted with permission from [41, 48])
6:34 PM Page 140
GENETICS OF BuChE
The gene for BuChE is located on chromosome 3 at q26.1–q26.2 and has several polymorphisms that include silent alleles, atypical (dibacaine-resistant, fluoride-1 and -2(fluoride-resistant) and the K, J and H variants. The last three variants reduce activity of the encoded-for enzyme. The atypical allele is found in approximately 4% of Caucasians and is associated with hypersensitivity to succinylcholine [51].
The BuChE-K polymorphism is a single point mutation with substitution of the amino acid alanine with threonine at position 539 of this 574 amino acid protein that acts genet- ically as a recessive gene. Approximately one-third of the Caucasian population carries this polymorphism and when an individual is homozygous, there is 30% reduction in BuChE activity [49, 51]. The BuChE-A polymorphism occurs in approximately 6% of the population and consists of a single point modification with substitution of asparagine for glycine at position 70 and as this polymorphism is determinant, individuals inheriting either one or two copies have a 30–40% reduction in enzymic activity [49].
The possible role of the BuChE variants in AD, such as protecting function in particular clinical domains, delaying disease progression and or differentially predicting response to cholinesterase inhibition therapy, are still being determined. Data from available studies are discussed in the next section.
CLINICAL ASSOCIATIONS OF BuChE-K AND -A
Several recent studies have suggested that BuChE-K and -A may be associated with differ- ent patterns of cognitive deterioration, greater risk for disease and or lesser rates of disease progression.
Compound (IC50) AChE1 BuChE2 BuChE/AChE3 Clinical dose (mg/day)
BW 284 C51 18.8 48 000 2553
Huperzine A 47 30 000 638 0.15–0.8
Donepezil 22 4150 189 5–10
Phenserine 22 1560 71
Metrifonate 800 18 000 22.5 25–80
Galantamine 800 7300 9 30
Rivastigmine 48 000 54 000 1.1 6–12
Physostigmine 28 16 0.6 36
Tacrine 190 47 0.25 80–160
Eptastigmine 20 5 0.25 45–60
Cymserine 758 50 0.07
Iso-ompa 34 000 980 0.03
Hetopropazine 260 000 300 0.001
Phenylethylcymserine 30 000 6 0.0002
Bambuterol 30 000 3 0.0001
MF-8622 100 000 9 0.00009
AChE%acetylcholinesterase; BuChE%butyrylcholinesterase; IC50%concentration of drug required to inhibit enzyme activity by 50%
1In human erythrocytes.
2In human plasma.
3The higher the ratio the higher the selectivity of the drug for AChE.
Table 11.2 In vitro selectivity (IC50; nmol/l) cholinesterase inhibitors in humans (adapted with permission from [16])