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Amyloid Precursor Protein in Platelets of Patients With Alzheimer Disease
Effect of Acetylcholinesterase Inhibitor Treatment
Barbara Borroni, MD;
Francesca Colciaghi, PhD;
Lucia Pastorino, PhD;
Carla Pettenati, MD;
Elisabetta Cottini, MD;
Luca Rozzini, MD;
Roberto Monastero, MD;
Gian Luigi Lenzi, MD;
Flaminio Cattabeni, PhD;
Monica Di Luca, PhD;
Alessandro Padovani, MD
Arch Neurol. 2001;58:442-446.
ABSTRACT
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Background Amyloid precursor protein (APP) forms with apparent molecular weights
of 130, 110, and 106 kd are present in human platelets. It has been demonstrated
that Alzheimer disease (AD) is specifically associated with a decreased APP
forms ratio in platelets.
Objective To investigate whether acetylcholinesterase (AChE) inhibitor treatment
modifies the ratio of platelet APP forms in patients with AD.
Patients and Methods From a large sample of patients with probable AD, 30 with mild to moderate
AD were selected. Each patient underwent a clinical evaluation including the
Mini-Mental State Examination (MMSE) and platelet APP forms analysis at baseline
and after 30 days. During this interval, 20 of 30 patients with AD were treated
with donepezil hydrochloride (5 mg/d), a piperidine phosphate–based
cholinesterase inhibitor. Platelets were subjected to Western blot analysis
using monoclonal antibody (22C11). The ratio between the immunoreactivity
of the higher-molecular-weight APP form (130 kd) and the lower forms (106
and 110 kd) was measured.
Results All patients taking donepezil completed the 30 days of treatment without
adverse effects. The platelet APP forms ratio at baseline did not differ between
the 2 AD groups (mean ± SD optical density ratio: untreated AD, 0.47
± 0.12; treated AD, 0.38 ± 0.18), whereas a significant difference
was found at follow-up (mean ± SD optical density ratio: untreated
AD, 0.45 ± 0.17; treated AD, 0.77 ± 0.29; P<.001). A significant improvement in MMSE scores in treated AD
patients was observed from baseline (16.9 ± 3.8) to 30 days (18.9 ±
4.42) (P<.009, 30 days vs baseline), but no significant
correlation was found in treated AD patients between MMSE score improvement
and APP forms/ratio increase (P = .09).
Conclusions Administration of AChE inhibitors increases the ratio of APP forms in
platelets of patients with AD, suggesting a potential effect of AChE inhibitors
on APP trafficking or processing in a peripheral cell.
INTRODUCTION
ALZHEIMER DISEASE (AD) is a neurodegenerative disorder characterized
by progressive loss of memory and cognition. The main neuropathologic changes
associated with AD are senile plaques, neurofibrillary tangles, and amyloid
angiopathy. The major proteinaceous component of senile plaques is a self-assembling
peptide, known as amyloid ß peptide, directly implicated in the pathogenesis
of AD.1 Amyloid ß peptide originates from
a larger precursor, the amyloid precursor protein (APP),2, 3
by proteolytic processing mediated by the action of ß-secretase, a recently
cloned aspartic peptidase.4, 5, 6, 7
Amyloid precursor protein is an integral transmembrane cell surface protein
present as numerous alternatively spliced isoforms derived from a single gene
localized on human chromosome 21.8 This protein
is expressed in normal cells and in peripheral tissues, ie, muscle, epithelial,
and circulating cells; among these, platelets represent an important peripheral
source of APP9, 10 and contain
large amounts (>95%) of the circulating APP.11
Previous studies12, 13, 14, 15
have demonstrated that patients with AD show a specific alteration in levels
of platelet APP forms. In particular, a marked decrease in the ratio of 130-kd
APP to the lower (106- and 110-kd) APP forms was found in platelets of patients
with AD compared with control subjects and patients affected by other neurodegenerative
disorders associated with dementia.16
Furthermore, AD has long been referred to as a cholinergic syndrome
given the selective loss of presynaptic cholinergic function in the brain,
particularly in the nucleus basalis.17 Cholinergic
hypofunction and acetylcholinesterase (AChE) hyperactivity have been implicated
as an explanation for the early memory impairment.18
On this ground, AChE inhibitors were introduced as therapeutic tools to restore
the amount of acetylcholine available in the synaptic cleft.19
Evidence that the use of AChE inhibitors produces a significant improvement
in cognitive performance and global functioning has been demonstrated in a
preliminary double-blind trial and confirmed in 2 recent phase 3 trials of
15 weeks'20 and 24 weeks'21
duration.
Recent investigations have claimed that AChE also plays a prominent
role in ß-amyloid fibrillogenesis22 and
modulates APP metabolism,23 thus arguing for
a strict interrelation between APP processing and AChE activity. In addition,
these findings suggest that AChE inhibitors might exert a neuroprotective
role by modulating acetylcholine receptors and in turn enzymes responsible
for APP metabolism.24 However, most of these
results have been derived through short-term treatment in vitro or from animals
treated long term. In fact, in vivo data on human cells are lacking, leaving
still unanswered the question about the real efficacy to modify in vivo APP
metabolism through AChE inhibitor use.25
The aim of this study was to evaluate the effect of AChE inhibitor drug
therapy on APP metabolism in vivo using platelets as a peripheral model. We
longitudinally investigated the APP forms ratio in platelets of patients with
AD treated for 1 month with donepezil hydrochloride (5 mg/d) and untreated
patients with AD, and we compared these groups with the platelet APP forms
ratio in control subjects.
SUBJECTS AND METHODS
SUBJECTS
Patients with probable AD and controls were recruited from the Neurological
Clinic of Brescia, Brescia, Italy, and from the Centro Alzheimer of Passirana-Rho,
Milan, Italy. The study was conducted in accordance with local clinical research
regulations. Written informed consent was obtained from the patient and the
caregiver. All participants underwent medical, epidemiologic, and neuropsychologic
assessments. Additional diagnostic testing included neuroimaging (computed
tomography or magnetic resonance imaging), blood tests, and other evaluations
as needed. A diagnosis of dementia was made according to Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition
(DSM-IV)26 criteria.
A diagnosis of probable AD was based on National Institute of Neurological
and Communicative Disorders and Stroke–Alzheimer's Disease and Related
Disorders Association criteria. Patients were followed up for at least 1 year
before being included in the study. Dementia severity was measured through
the Clinical Dementia Rating Scale. Mini-Mental State Examination (MMSE) scores
at the time of sampling were also recorded.
The following exclusionary criteria for the AD group were designed to
ensure that participants had probable AD as the cause of their dementia: (1)
major depressive disorder, bipolar disorder, schizophrenia, substance use
disorder, or mental retardation according to the criteria of the DSM-IV; (2) cerebrovascular disorders, hydrocephalus, and intracranial
mass, documented by computed tomography or magnetic resonance imaging within
the past 12 months; (3) abnormalities in serum folate and vitamin B12 levels, syphilis serologic findings, or thyroid hormone levels; (4)
a history of traumatic brain injury or another neurologic disease (eg, Parkinson
disease, Huntington disease, or seizure disorders); and (5) significant medical
problems (eg, poorly controlled diabetes or hypertension; cancer within the
past 5 years; or clinically significant hepatic, renal, cardiac, or pulmonary
disorders).
To avoid potential pharmacologic confounding effects on platelet physiologic
findings, patients and controls taking psychotropic agents, nootropic drugs,
antiplatelet agents, anticoagulants, corticosteroids, and serotoninergic drugs
entered the study only after being drug free for at least 14 days before blood
sample collection and platelet preparation. Concomitant treatment with these
drugs was not allowed during the study.
STUDY DESIGN
This was a 4-week, longitudinal, open study conducted in 2 medical centers.
Of 30 consecutive patients with AD, 20 received no drug treatment (AD-n) and
20 were treated with donepezil, 5 mg/d (AD-d). Treatment group status was
assigned by patient eligibility to receive AChE inhibitors based on the presence
of well-known contraindications (ie, supraventricular cardiac conditions,
ulcer disease, history of seizure, history of asthma, or obstructive pulmonary
disease). Patients in the AD-d group received a single dose of donepezil each
evening. All patients were investigated at baseline and after 30 days. At
each session, AD-n and AD-d patients and 10 controls were subjected to a clinical
evaluation, including an MMSE and a venipuncture for platelet sample collection.
PLATELET PREPARATION
Blood samples were drawn from fasting participants between 9 and 10
AM. Patient information and case diagnoses were unknown to the laboratory
investigators who received and analyzed the samples.
A blood sample (27 mL) was taken, with the tourniquet carefully released
immediately after its application, from a vein in the antecubital fossa using
a 19-gauge needle and collected into 3 mL of 3.8% sodium citrate (in the presence
of glucose, 136 mmol/L). Each sample was mixed gently and centrifuged at 200g for 10 minutes to separate platelet-rich plasma within
30 minutes of blood drawing. Platelet-rich plasma was separated from the blood
pellet by means of a plastic pipette, with aspiration of the buffy coat avoided.
Platelets were then collected by further centrifugation at 500g for 20 minutes and washed, and the platelet pellet was stored at -80°C
until used.
Immunoblot experiments were performed with monoclonal antibody 22C11
as described elsewhere.12, 16 Results
are expressed as the ratio between the optical density of the upper (130-kd)
and lower (106- and 110-kd) 22C11 immunoreactive bands. The ratio was determined
for each individual from at least 3 replications.
STATISTICAL ANALYSIS
Quantitative Western blot analysis was performed by means of computer-assisted
imaging (Imaging System-Quantity One; Bio-Rad, Hercules, Calif).
Results were averaged and are expressed as mean ± SD. Clinical
and laboratory results before and after treatment were assessed using paired t tests. Differences were considered statistically significant
at P<.05 (2-tailed).
RESULTS
BASELINE
Demographic and clinical characteristics of the 2 patient groups (AD-n
and AD-d) and the control group are shown in Table 1.
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Demographic and Clinical Characteristics and APP Forms Ratios of the
Sample*
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Whole platelet homogenates from each participant were processed for
Western blot analysis by means of monoclonal antibody 22C11 raised against
the N-terminal domain of APP, thereby recognizing all APP forms present in
the samples. After the optical density of the bands at 106, 110, and 130 kd
was measured by image analysis, the ratio between the highest and the lower
bands was determined for each individual from at least 3 replications.
Mean APP forms ratios were significantly decreased in both AD groups
compared with controls (optical density: AD-d, 0.38 ± 0.18; AD-n, 0.47
± 0.12; and controls, 0.93 ± 0.37) (AD-d vs controls and AD-n
vs controls; P<.001); no significant difference
in the APP forms ratio was found between the 2 AD groups at the beginning
of the study (P = .83) (Table 1).
Both AD patient groups had significantly impaired MMSE scores (AD-d,
16.9 ± 4.1; AD-n, 17.4 ± 6.1) at baseline compared with controls
(28.3 ± 1.6; AD-d vs controls and AD-n vs controls; P<.001).
FOLLOW-UP
All participants completed the study. No serious adverse effects were
reported.
Figure 1 shows a representative
Western blot analysis performed with 22C11 on total platelet lysate from an
AD-n patient, an AD-d patient, and a control subject at baseline and after
30 days.
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Figure 1. Representative Western blot analysis
performed with 22C11 on whole platelet homogenate showing amyloid precursor
protein forms at baseline (T = 0) and after 30 days (T = 30) obtained from
a patient with untreated Alzheimer disease (AD-n; lanes 1 and 2), a patient
with treated AD (AD-d; lanes 3 and 4), and a control subject (lanes 5 and
6). The arrows indicate the position of the amyloid precursor protein forms
and their apparent molecular weight.
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The three 22C11 immunoreactive bands expected in platelet lysate are
clearly visible at 130, 110, and 106 kd.11, 14
No significant differences were found in the immunostaining of the upper band
between AD-n patients at baseline and after 30 days, whereas a significant
increase in the immunoreactivity of the 130-kd band was consistently found
after 30 days of donepezil intake.
The ratio between the upper (130-kd) and lower (110- and 106-kd) APP
forms was measured again for all the experimental groups after 30 days: the
AD-n group, 0.45 ± 0.17; the AD-d group, 0.77 ± 0.29; and the
control group, 0.89 ± 0.4.
The cumulative quantitative analysis is shown in Figure 2. Donepezil treatment determined a 2-fold increase in the
APP forms ratio in the AD-d group (P<.001), whereas
the APP forms ratio remained unchanged from baseline in the AD-n group.
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Figure 2. Quantitative analysis of the ratio
of platelet amyloid precursor protein (APP) forms in control subjects and
patients with treated and untreated Alzheimer disease (AD) at baseline (T
= 0) and after 30 days (T = 30). Error bars represent SD.
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A significant improvement in MMSE scores was observed from baseline
(16.9 ± 3.8) to 30 days (18.9 ± 4.42) (P<.009,
30 days vs baseline) in the AD-d group but not in the AD-n group (17.4 ±
6.1 and 18.1 ± 5.2, respectively; P = .2,
30 days vs baseline). However, no significant correlation was found between
the improvement in MMSE scores and the APP forms ratio changes in the AD-d
group.
COMMENT
In the present study, the effect of 30 days of donepezil treatment,
a piperidine-based cholinesterase inhibitor, on human platelet APP forms was
demonstrated. In particular, a 5-mg donepezil intake daily for 30 days determined
an increase in the platelet APP forms ratio in patients with AD vs controls.
A marked decrease in the ratio of 130-kd APP to the lower (106- and
110-kd) APP forms was found at baseline in platelet samples from patients
with mild to moderate AD compared with control subjects, confirming previous
observations.16 At 30 days of follow-up, no
changes were found in the APP forms ratio of controls and AD-n, although in
a longer time range, a decrease in the platelet APP forms ratio can be observed
in AD.27 On the other hand, a significant increase
in the APP forms ratio was found at follow-up in platelet samples from the
AD-d group, with a 2-fold increase in their baseline values. This study is
an open study because patients were not randomly assigned to each experimental
group, but rather by taking into consideration whether a patient could receive
the pharmacologic treatment. Nevertheless, results were consistent and statistically
significant, although the number of control subjects included in the study
was limited to 10. In addition, AD-d patients showed a significant improvement
in MMSE scores at follow-up. This study, however, did not demonstrate a significant
correlation between changes in MMSE score and changes in the platelet APP
forms ratio.
These results, to our knowledge, are the first in vivo demonstration
of a direct pharmacologic effect of AChE inhibitor therapy on APP levels on
patients with AD, thus confirming results of previous studies arguing for
a complex relation between the cholinergic system and APP metabolism. In 1984,
Smith and Cuello28 suggested that a common
feature shared by the different neuronal populations affected in AD is the
presence of AChE. Accordingly, it has been shown that AChE is prominent in
amyloid plaques and dystrophic neuritis29, 30
and promotes in vitro aggregation of amyloid ß peptide, suggesting a
direct role in amyloid deposition and senile plaque formation.22, 28
Furthermore, it has been demonstrated23, 31
recently that the stimulation of protein kinase C–coupled M1/M3 muscarinic
receptors increases the soluble metabolite sAPP secretion through the -secretase–mediated
pathway of APP processing. In many cell types, the increase of sAPP
is paralleled by a reduction of ß-amyloid release,32
thus suggesting that cortical cholinergic hypoactivity might produce a shift
to the amyloidogenic pathway, leading to an increase of amyloid ß peptide.
In agreement, modulation of APP processing by cholinergic activity has been
reported31, 33 in animal models
of reduced cortical cholinergic innervation. A cholinergic effect on APP metabolism
also has been demonstrated recently for cholinesterase inhibitors. In fact,
in superfused rat cortical brain slices, cholinesterase inhibitors have been
shown to increase sAPP release and to induce APP release from brain
slices and cultured neuroblastoma cells with a pattern correlated to the level
of AChE inhibition.25, 34 Despite
all these in vitro examples of AChE inhibitor treatment as a molecular mechanism
that might be of relevance for the pathogenesis of the disease, results of
clinical trials performed with different compounds suggest that such treatment
does not affect the natural history of AD but solely pharmacologically affects
cognitive functions in patients.
Results of our study, however, in agreement with experimental data mostly
derived by in vitro experiments or animal models, suggest that AChE inhibitors,
at a dosage commonly administered in clinical practice as therapeutic for
AD, might modify the concentration of APP forms in human platelets, rescuing
the values of the ratio of APP forms to control levels. Whether this effect
directly affects a fundamental feature of AD pathogenesis is still a matter
of study.
The significant effect on the APP forms ratio exerted by donepezil therapy
suggests that this peripheral marker might be useful to monitor not only disease
progression27 but also pharmacologic manipulations.
Our data, in addition, strongly support the evidence that sample selection
needs to be strictly defined because peripheral markers are susceptible to
biological manipulation at different levels.
Thirty days of donepezil treatment improved MMSE scores in patients
with AD. However, there was no relation between cognitive and APP forms ratio
changes. Such negative findings might be due to the small patient sample and
the short interval of evaluation; pharmacologic effect on cognitive function
is also associated with different factors, such as level of cholinergic damage,
genetic factors, or sex, whose effect on biological variables is less likely.
Understanding of the molecular mechanism responsible for rescuing platelet
APP levels in patients with AD after donepezil treatment still needs further
investigation.
Although it is known that platelets express the repertoire of enzymes
necessary for APP processing35 and AChE,36 it is difficult to ascribe the donepezil effect to
a generic increase in the concentration of acetylcholine in biological fluids,
which might in turn activate through its receptor(s) a biochemical cascade
capable of affecting APP processing.
More likely, the effect of donepezil involves a direct link between
AChE and -secretase or APP in the peripheral compartment. Recently,
it was suggested24 that AChE and -secretase
might cluster in plasma membrane. Thus, the interaction of donepezil with
AChE might prime a conformational modification of the enzyme that reflects
a modification in -secretase activity. This might restore a correct
balancing in metabolism and redistribution of APP forms in the membrane. Alternatively,
donepezil treatment might indirectly affect APP trafficking either in platelets
or in megakaryocytes, thus making the protein more prone to membrane insertion
and to -secretase activity. Indeed, donepezil might affect glycosylation
of APP forms that, in turn, might affect its processing.37
In addition, to further clarify this in vivo effect of donepezil on platelet
APP forms, it would be of interest to examine different time courses of the
pharmacologic treatment to determine whether donepezil is affecting platelets
or megakaryocytes and to establish the stability and the maximum effect. Experiments
are in progress in our laboratory to elucidate this aspect.
In conclusion, the results of the present study demonstrate that use
of AChE inhibitors such as donepezil modifies APP processing in the platelets
of patients with AD. The platelet APP forms ratio therefore holds the potential
to be a peripheral marker that might be helpful as a tool for studying mechanisms
underlying APP metabolism and for the assessment of pharmacologic effect.
AUTHOR INFORMATION
Accepted for publication September 7, 2000.
This study was supported by a fellowship from the European Neurological
Society, Basel, Switzerland (Dr Monastero).
From the Department of Neurology, University of Brescia, Brescia (Drs
Borroni, Cottini, Rozzini, Monastero, and Padovani); the Institute of Pharmacological
Sciences, University of Milan, Milan (Drs Colciaghi, Pastorino, Cattabeni,
and Di Luca); the Alzheimer Center, Passirana-Rho (Dr Pettenati); and the
Department of Neurology, "La Sapienza" University of Rome, Rome (Dr Lenzi),
Italy.
Corresponding author and reprints: Monica Di Luca, PhD, Institute
of Pharmacological Sciences, University of Milano, Via Balzaretti 9, 20133
Milano, Italy (e-mail: Monica.Diluca{at}unimi.it).
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