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J. Biomedical Science and Engineering, 2008, 1, 178-181
Published Online November 2008 in SciRes. http://www.srpublishing.org/journal/jbise JBiSE
Enhanced apoptosis and electrostatic acetylcholi-
nesterase activity of abnormally hydrophobic envi-
ronment in alzheimer’s plaques
Rakesh Sharma1 and Soonjo Kwon2
1Department of Radiology and Molecular Biology, Columbia University, New York 10032. 2Department of Biological Engineering, Utah State University, Logan,
Utah 84322. Correspondence should be addressed to Rakesh Sharma (firstname.lastname@example.org).
Received October 6, 2008; revised October 22, 2008; accepted October 22, 2008
Alzheimer’s disease (AD) is considered a slow
neuronal dysfunction process through hypoxia,
ischemia and leads to apoptosis mediated senile
plaques and neurofibrillary tangles (NFTs). Due to
non-invasive approach of plaque characterization,
computational techniques based on Brownian
dynamics simulation are unique to speculate the
electrostatic and kinetic properties of Acetylcho-
linesterase (AChE). Typically the MRI spectros-
copy high choline peak and enzyme specific to
Alzheimer’s Disease (specificity constant (kcat/Km)
of AChE) appeared associated with apoptosis and
hypoxia. A simple display between synergy of
cytokines, apoptosis, elevated AChE and choline
is postulated as initial events. The events may be
distributed heterogeneously within the senile
plaques and neurofibrillary tangles (NFTs) of
Alzheimer’s Disease (AD). The role of decreased
brain AChE and synergy was associated with
specific Magnetic Resonance Spectroscopic
(MRS) pattern profiles in AD. These findings
suggest that that the altered AChE and early
apoptosis events in AD may be associated with
specific MR spectral peak patterns. This study
opens the possibility of reduced AChE levels
causing high choline and reduced N-acetyl ace-
tate (NAA) neurotransmitter by MRS after initial
apoptosis and/or inflammation to make amyloid
plaques in the cerebral tissue of Alzheimer’s
disease (AD) patients. These results can be useful
in clinical trials on AD lesions.
Keywords: Alzheimer’s Disease, Acetylcholi-
nesterase, Electrostatics, Dielectic effect, Ionic
effect, Brownian dynamics, Apoptosis
1.1. Alzheimer’s Disease
Azheimer’s disease (AD) has manifestations of senile
plaques and neurofibrillary tangles (NFTs) in the cerebral
cortex involving hippocampus of Alzheimer’s brains.
Many studies have shown that the levels of the reduced
ACh neurotransmitter in AD brain had been curiosity in
recent past. In this direction, promising success is claimed
in drug therapeutic trials based on AChE inhibitors. In AD
presumably five neurological cell groups are commonly
seen around the cortex rich with AD lesions associated
with the low AChE enzyme. Other important problem of
AD therapeutics was solved by use of AChE inhibitor
higher concentrations such as, tacrine, physostigmine, and
BW284C51 to inhibit AChE within AD lesions. Simulta-
neously, elevated levels of the ACh substrate also inhibit
AChE activity. Serotonin, 5-hydroxytryptophan, car-
boxypeptidase inhibitor, and bacitracin, had been good
choice to effectively inhibit cholinesterase activity within
plaques and tangles, but fail to alter the AChE activity in
normal tissue at standard physiological conditions. The
initial stages of amyloid plaque formation are not known if
they are the result of metabolic defect leading to pathology.
The mechanisms were reviewed by Lu et al. 2003 .
The process of inflammation in AD was described by
Bamberger et al. 2002 . There is continuous hunt of
biomarkers useful in AD reported by Ankarcrona et al.
2002 . However, the sequence of these events remains
unknown. There are several reports showing that neurons
die partly by apoptosis in the AD brain. Drugs blocking
apoptosis could therefore be potentially useful for early
prevention of neuronal cell death. Biomarkers for apop-
tosis should be important tools in the evaluation of drug
effects and in the diagnostics of AD. Future strategies are
more likely to modify the course of the disease. The most
widely accepted hypothesis on the etiopathogenesis of AD
proposes that aggregates of beta amyloid (Abeta) form in
the brain. Under normal conditions, the predominant amy-
loid peptide secreted is Abeta(1-40) with about 10-15%
being the longer 1-42 form. In AD, there appears to be an
increase in the longer more toxic form which is proposed
to trigger tau hyperphosphorylation and neural degenera-
tion. Neurotoxicity is thought to be due to altered calcium
regulation, mitochondrial damage and/or immune stimula-
tion. One strategy for treating AD is the prevention of
Abeta release or the blockade of it neurotoxic activity re-
SciRes Copyright © 2008
R. Sharma / J. Biomedical Science and Engineering 1 (2008) 178-181 179
SciRes Copyright © 2008 JBiSE
ported by Lu 2003 .
Present paper explains the electrostatics of lowered
AChE catalytic activity in AD brain tissue over normal
tissue with possibility of the protein-rich deposits associ-
ated with the onset of AD. The paper further, illustrates the
possibility of reduced AChE associated with high choline
and reduced NAA peaks by MRS and initial events trig-
gered by cytokines, apoptosis and inflammation to synthe-
size amyloid protein as NFT plaques However, the high
concentrations of protein-rich deposits in plaques and
NFTs such as βAP, heparan and dermatan sulfate pro-
teoglycans, serum amyloid P component, complement
factors, and protein kinase C, had been active research
over the increased the hydrophobicity of AChE in AD le-
sions and abnormally reduced dielectric constant reported
by Giambarella et al. 1997 . The AChE catalysis has
been explained as electrostatic steering mechanism where
altered dielectric conditions seen by βAP deposition. Pos-
sibly, dielectric constant shift in AD tissue also allows
Coulombic interactions to permeate longer distances re-
sulting with enhanced enzymatic activity and simultane-
ously decreased ACh levels. MR spectral pattern of en-
hanced choline also supports association with decreased
ACh levels in AD.
The acetylcholinesterase enzyme (AChE) has 537 amino
acid long polypeptide in the postsynaptic neural mem-
branes of central nervous system and neuromuscular junc-
tions by a glycosylphosphatidylinositol linkage. AChE
catalyzes the hydrolysis of the acetylcholine (ACh) sub-
strate neurotransmitter at cholinergic synapses. AChE hy-
drolysis results in the termination of impulse transmission.
The determination of the three-dimensional structure of
AChE dimer enzyme comprises 12-stranded mixed β-sheet
surrounded by 14 α-helices. These subunits assemble
through disulfide linkage and hydrophobic interactions.
The enzyme structure shows structural characteristic of
AChE as a deep (~20Å), narrow active site making en-
zyme’s catalytic site Ser200, His440, and Glu327 at its base.
The walls of this entity are lined with 14 highly conserved
aromatic amino acids of active site. Positively charged
ACh substrate toward the active site caused low-affinity
cation-π interactions. Further, amino acid charge distribu-
tion over AChE creates an electric field around the enzyme
contributing to its enzymatic activity (electrostatic steering
mechanism) involving its substrate, ACh . Authors
determined that the negative field drives the posi-
tively-charged ACh substrate molecule toward the en-
trance of its active site moiety and increases the catalytic
rate of AChE.
1.3. Cytokines, Inflammation, Apoptosis, and
Serum AChE Relationship in AD
Inflammatory processes play a role in disease progression
and pathology of AD, which involves the deposition of
amyloid in the brain and the extensive loss of neurons.
Amyloid plaque deposition is accompanied by the associa-
tion of microglia with the senile plaque, and this interac-
tion stimulates these cells to undergo phenotypic activa-
tion and the subsequent expression of proinflammatory
cytokines and neurotoxic products . Inflammation has
been reported in numerous neurodegenerative disorders
such as Parkinson's disease, stroke and Alzheimer's disease
(AD). In AD, the inflammatory response is mainly located
to the vicinity of amyloid plaques. Cytokines, such as In-
terleukin-1 (IL-1), Interleukin-6 (IL-6), Tumor Necrosis
Factor alpha (TNF-α) and Transforming Growth Factor
beta (TGF-β) have been clearly involved in this inflam-
matory process. Although their expression is induced by
the presence of amyloid-beta peptide, these cytokines are
also able to promote the accumulation of amyloid beta
peptide. Altogether, IL-1, IL-6, TNF-α and TGF-β should
be considered as key players of a vicious circle leading to
the progression of the disease reported by Cacquevel et al.,
2004 . Inflammatory stimuli also induce nitric oxide
production, resulting in oxygen deficiency (hypoxia) and
stimulating adenylate cyclase activity. Under these condi-
tions, the rarte of apoptosis increases. Neuron dysfunction
is partly due to apoptosis in the AD brain (Figure 1).
2. RESULTS AND DISCUSSION
2.1. Ionic Strength
Computed rate constants of Torpedo californica AChE as
model enzyme at various ionic strengths are given in Fig-
ure 2. These values are compared with experimental bi-
molecular association constants (kcat/Km) and enzymatic
specificity constants (kcat/Km) of a related Electrophorus
electricus AChE enzyme as reported by Nolte et al., 1980
. Since the association constant considers the binding
event of the reaction and the specificity constant describes
both binding and subsequent catalytic turnover, k1 is the
theoretical maximum value for the calculated diffu-
sion-controlled rate constant, while kcat/Km sets the lower
limit on these second-order reactions. As seen in Figure 2,
the calculated rate constants found in this work lie be-
tween these two extremes throughout the range of ionic
strengths tested. This provides encouraging support for the
ionic screening approximations used in this work. Fur-
thermore, the decrease in the rate of AChE catalysis with
increasing ionic strength provides strong evidence that an
electrostatic steering mechanism plays a role in AChE
kinetics. The similarity in the negative slope observed for
both association and specificity constants indicates that the
ligand binding step of the reaction is dependent upon sol-
vent salt concentration.
2.2. Substrate Radius
To demonstrate the limited accessibility of this enzyme’s
active site structure, simulations were performed using
various substrate radii (Figure 2). It is reasonable to pre-
dict increased rate constant values with a reduced substrate
radius since the probability of a smaller substrate pene-
trating the active site gorge and reacting with AChE is
higher. The results shown in Figure 3 show this expected
180 R. Sharma / J. Biomedical Science and Engineering 1 (2008) 178-181
SciRes Copyright © 2008 JBiSE
NF-κBATP cAMP + 2 Pi
Protein Kinase C
Figure 1. A postulated schematic sketch of the relation among inflammatory cytokines, adenylate cyclase stimulation, AChE and cytotoxicity
events in AD. (Solid lines represent stimulation effect and dotted lines represent inhibition effect of different effectors. Abbreviation: IKK; IB
kinase kinase, NF-B; nuclear factor B, NF -B -IB; nuclear factor B -inhibitor B complex, NO; nitric oxide, iNOS; inducible nitric
oxide synthase, SOD; superoxide dismutase, PKC; protein kinase C, GTP; guanosine triphosphate, GITP; guanylyl imidotriphosphate, ACh;
acetylcholine, AChE; acetylcholinesterase enzyme. Possible sequence: Low OxygenÆ1. Low ATP (oxidative Phosphorylation); 2. Low Py-
ruvate (low N-Aceto-Acetate by glycolysis) ÆHigh Choline (Reduced AChE). Cytokines trigger adenylate cyclase to inflammation and amy-
loid protein synthesis ?? (mechanism is unknown).)
Table 1. Effect of Dielectric on Activity of wt AChE. 8000 trajecto-
ries were simulated at 300 K, an ionic strength of 5 mM, a solvent
density of 996.5 kg/m3, and a pH of 7.0.
M-1s-1 Standard Error
78 1.3 x 109 ± 0.267 x 109
60 4.7 x 109 ± 0.615 x 109
trend. These computed rate constants also comparable
well with the approximated specificity constant of 4.2 x
109 M-1s-1 for Electrophorus electricus AChE at zero ionic
It has been hypothesized that the active site gorge of
AChE undergoes conformational fluctuations to allow
substrate entry . Further simulation calculations in this
work have used a smaller substrate radius (2 Å) than the
actual hydrodynamic radius of ACh (4.23 Å) to approxi-
mately account for this protein flexibility.
2.3. Reduced Dielectric in Plaques and NFTs
Simulations of the AChE-ACh reaction at both aqueous
(ε=78) and hydrophobic (ε=60) conditions demonstrate
the substantial effect of the AChE environment on its
catalytic activity. The results of this study in Table 1 re-
veal that the abnormally low dielectric medium of the
enzyme increases its catalytic rate by a factor of 3.6.
These results are experimentally supported by Nolte et al.
1980, who found a rate constant of 1.6 x 109 M-1s-1 for
Electrophorus electricus AChE under similar conditions.
These findings are complemented by the graphical
visualization of an intensified electric field near the en-
trance of the AChE active site gorge in the reduced di-
electric environment. This observation can be made by
Figure 2. Experimental and Calculated Rate Constants versus
Ionic Strength. (Calculations for preliminary ionic strength study
used 1000 trajectories at a temperature of 298 K, a dielectric of 78,
a solvent density of 997.0 kg/m3, and a pH of 7.0.)
comparing the electric field contours around the perimeter
of AChE at dielectrics of 78 and 60 (Figure 2). These
contour plots illustrate an electrostatic gradient emerging
from the gorge entrance and extending along the en-
zyme’s surface. This effectively enlarges the active site
target area, resulting in increased enzyme-substrate asso-
ciation. These electric field calculations along the pro-
tein’s surface are consistent with the results of Anto-
siewicz et al. 1995b , who have suggested that electro-
static steering is limited to operation near the surface of
the enzyme. The contour plots found in this work also
offer a plausible explanation for the catalytic rate constant
enhancement at lower dielectrics and further suggest that
electrostatic attraction is an important component of the
AChE mechanism and ultimately its physiological role in
the human nervous system.
The 3D MP-RAGE at TR/TI/TE=10/250/4 ms, flip an-
R. Sharma / J. Biomedical Science and Engineering 1 (2008) 178-181 181
SciRes Copyright © 2008 JBiSE
A B C D
Figure 3. A representative 1.5 Tesla MRI T1 axial image of AD brain (panel A) with post-segmentation (panel B) showin neurofi-
brillary tangle with arrow.(The scan setting were: 3D MP-RAGE were TR/TI/TE=10/250/4 ms, flip angle 15,1.0 x 1.0 mm2 reso-
lution, and 1.4 mm thick partitions showing VOI and spectroscopic voxels and selective Choline and NAA spectral peaks from
right and left sides to compare metabolites in cortical and ventricular regions (Choline peak on right panel D). For details see
reference (Sharma 2005).)
gle 15, 1.0 x 1.0 mm2 resolution, and 1.4 mm thick parti-
tions in our previous report showed NAA/Cr, NAA/Cho
were significantly reduced (p < 0.02 and p value < 0.03
respectively) in AD compared with elderly controls due to
reductions of NAA after NAA correction in AD. Fur-
thermore, the difference of hippocampal NAA between
the groups without atrophy correction (which reflects
both NAA and volume changes) was about 40% larger
than with correction of atrophy as shown in Figure 3. The
major elevated peak was choline at 3.00 ppm. These
finding suggest a possibility of reduced glycolysis leading
to low N-acetyl acetate formation and choline accumula-
tion indirectly reducing TCA cycle to generate enough
ATP in localized tissue. Low ATP and oxygen are well
understood to lead inflammation and amyloid plaque
Abnormal Magnetic Resonance spectral NAA, choline
peak patterns are associated with low AChE enzyme ac-
tivity in AD with possible enhanced ACh breakdown and
surrounding electrostatic field of this enzyme. However,
the association of cytokines, apoptosis to lead hypoxia
and inflammation in amyloid plaque in AD formation is
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