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 Previous Article | Next Article 
The Journal of Neuroscience, May 1, 1998, 18(9):3213-3223
Stable Complexes Involving Acetylcholinesterase and Amyloid-
Peptide Change the Biochemical Properties of the Enzyme and Increase
the Neurotoxicity of Alzheimer's Fibrils
Alejandra
Alvarez1,
Rodrigo
Alarcón1,
Carlos
Opazo1,
Eliseo O.
Campos1,
Francisco José
Muñoz1,
Frances H.
Calderón1,
Federico
Dajas2,
Mary K.
Gentry3,
Bhupendra P.
Doctor3,
Fernando G.
De
Mello4, and
Nibaldo C.
Inestrosa1
1 Departamento de Biología Celular y Molecular,
Facultad de Ciencias Biológicas, Pontificia Universidad
Católica de Chile, Santiago, Chile, 2 División
de Neuroquímica, Instituto de Investigaciones Biológicas
Clemente Estable, Montevideo, Uruguay, 3 Division of
Biochemistry, Walter Reed Army Institute of Research, Washington, DC,
20307-5100, and 4 Instituto de Biofisica, Universidade
Federal do Rio de Janeiro, Rio de Janeiro, Brazil
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ABSTRACT |
Brain acetylcholinesterase (AChE) forms stable complexes with
amyloid- peptide (A) during its assembly into filaments, in agreement with its colocalization with the A deposits of
Alzheimer's brain. The association of the enzyme with nascent A
aggregates occurs as early as after 30 min of incubation. Analysis of
the catalytic activity of the AChE incorporated into these complexes shows an anomalous behavior reminiscent of the AChE associated with
senile plaques, which includes a resistance to low pH, high substrate
concentrations, and lower sensitivity to AChE inhibitors. Furthermore,
the toxicity of the AChE-amyloid complexes is higher than that of the
A aggregates alone. Thus, in addition to its possible role as a
heterogeneous nucleator during amyloid formation, AChE, by forming such
stable complexes, may increase the neurotoxicity of A fibrils and
thus may determine the selective neuronal loss observed in Alzheimer's
brain.
Key words:
AChE; A-amyloid fibrils; AChE-A-amyloid fibril
complexes; amyloid formation; Alzheimer's disease; neurotoxicity
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INTRODUCTION |
Alzheimer's disease (AD), the most
common form of dementia in adults, is a neurodegenerative disorder
characterized by selective neuronal loss and the presence of two
different types of fibril deposits: amyloid plaques and neurofibrillary
tangles (Soto et al., 1994 ; Selkoe, 1996). Recent advances in the
molecular genetics of AD indicate that it is a complex disorder with
mutations in many genes, and the apolipoprotein E genotype is a risk
factor (Selkoe, 1997). However, at least 60% of AD patients do not
have a family history of the disease. Therefore, there is a need to search for the mechanisms responsible for the progressive cognitive decline observed in most of the sporadic cases, which correspond to the
great majority of Alzheimer's patients (Katzman and Kawas, 1994;
Inestrosa et al., 1996a; Van Leeuwen et al., 1998). The fact that
neurodegenerative changes occur around senile plaques (Mann and Esiri,
1989), and that the neurotoxicity of A depends on its aggregation
into amyloid fibrils (Lorenzo and Yankner, 1994), indicates that
amyloid fibrils are the primary pathogenic mechanism in AD (Yankner,
1996). It has also been suggested that endogenous factors that modulate
A fibrillogenesis and deposition could play a significant role in
the pathogenesis of the disease (Inestrosa et al., 1996b; Harper and
Lansbury, 1997). In this context, several studies have revealed
numerous other proteins associated with the amyloid plaque deposits.
These proteins include apolipoprotein E (apoE) (Namba et al., 1991),
 1-anti-chymotrypsin (Abraham et al., 1988), heparan
sulfate proteoglycans (Snow et al., 1988), and acetylcholinesterase
(AChE) (Ulrich et al., 1990). AChE plays a key role in cholinergic
transmission in the CNS of mammals (Inestrosa and Perelman, 1989, 1990;
Taylor, 1991) and may also participate in noncholinergic mechanisms
(Massoulié et al., 1993; Small et al., 1996). Most of the
cortical AChE activity present in Alzheimer's brain is predominantly
associated with the amyloid core of senile plaques rather than with the
neuritic component found at the periphery (Ulrich et al., 1990;
Gómez-Ramos et al., 1992; Morán et al., 1993).
Histochemical studies have demonstrated that the AChE associated with
senile plaques differs enzymatically from the AChE associated with
normal fibers and neurons with respect to optimum pH, inhibitor
sensitivity, and inhibition by excess substrate (Mesulam et al., 1987;
Geula and Mesulam, 1989; Schätz et al., 1990; Wright et al.,
1993). AChE directly promotes the assembly of A peptide into amyloid
fibrils (Alvarez et al., 1995; Inestrosa et al., 1996b), and recent
studies from our laboratory have shown that AChE forms complexes with small A peptide fragments (Alvarez et al., 1997). It was therefore our aim to investigate whether the characteristics of AChE observed in
the amyloid plaques of Alzheimer's brain could be reproduced under
in vitro conditions, using complexes obtained with whole A1-40 peptide. We report here that the incorporation of AChE into Alzheimer's amyloid aggregates results in the formation of
stable complexes that change the biochemical and pharmacological properties of the enzyme and cause an increase in the neurotoxicity of
the amyloid- fibrils, suggesting that AChE could play a pathogenic role in AD by influencing the process leading to amyloid toxicity and
the appearance of AD.
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MATERIALS AND METHODS |
Materials. Synthetic peptide A1-40,
corresponding to residues 1-40 of the human A sequence, was
purchased from Chiron Corporation (Emeryville CA). The following
inhibitors were purchased from Sigma (St. Louis, MO): edrophonium
chloride, tetrahydro-aminoacridine hydrochloride (Tacrine), and
propidium iodide. Fasciculin was purified as described previously
(Dajas et al., 1987; Karlsson et al., 1984), and the monoclonal
antibody 25B1 directed against fetal bovine serum AChE was obtained as
described by Gentry et al. (1995).
Purification of brain AChE. The tetrameric G4
AChE form (sedimentation coefficient, 10.7 S) was purified from bovine
caudate nucleus, using acridine affinity chromatography, as described previously (Inestrosa et al., 1987). Both specific activities (6000 U/mg protein), and staining intensities after SDS-PAGE (Laemmli, 1970)
(a single band of 66 kDa) were used to verify purity.
AChE assay. AChE activity was determined by the method of
Ellman et al. (1961).
Preparation of 125I-Tyr derivatives of AChE and
A1-40. For the iodination of AChE and the A
peptide, the iodo-bead method was used (Markwell, 1982); 100 µg of
AChE was labeled with 500 µCi 125I-Na, and 100 µg of
A1-40 peptide was labeled with 1000 µCi
125I-Na (Comisión de Energía Nuclear de
Chile). A single bead was used in each case and incubated in 200 µl
of 0.1 M phosphate buffer, pH 7.0, for 15 min. The
iodinated enzyme was separated from the unreacted 125I on a
Sephadex G-25 column and submitted to 12% SDS-PAGE (Laemmli, 1970).
Autoradiography showed a main band (at 66 kDa) coincident with the
electrophoretic migration of native AChE. The specific activity of the
standard 125I-AChE used in our experiments was estimated on
the order of 2.4 mCi/mmol. For the iodinated A peptide, a Sepharose
Bio-Gel P-2 column was used to separate the peptide from free
125I, and the labeled peptide fraction was submitted to
Tris-Tricine 16% SDS-PAGE (Schagger and von Jagow, 1987).
Autoradiography showed a single band (at 4 kDa) coincident with the
electrophoretic pattern of the native A1-40 peptide.
The specific activity of the standard 125I-A peptide
used in our experiments was estimated on the order of 0.6 mCi/mmol, and
the peptide was used immediately after radioiodination.
Generation of AChE-A complexes in vitro. To obtain
AChE-A complexes, A1-40 peptide was incubated with
or without AChE in a stirred aggregation assay performed as described
previously (Jarrett et al., 1993; Evans et al., 1995). Briefly, stock
solutions were prepared by dissolving lyophilized aliquots of the A
peptide in dimethyl sulfoxide (DMSO) at 15 mg/ml (3.5 mM).
Aliquots of peptide stock solution (70 nmol in  20 µl of DMSO) were
added to PBS, pH 7.4, for a final volume of 725 µl. For the
aggregation experiments performed with AChE, peptide stock (70 nmol in
DMSO) was added to 680 µl of PBS buffer containing AChE (100 nM; 25 µg of the enzyme in ~25 µl of buffer: 20 mM sodium phosphate, 1 mM EDTA, 0.1% Triton
X-100, and 10 mM caproic acid, pH 7.4). The solutions were
stirred continuously (210 rpm), and aggregation was measured versus
buffer blank by turbidity at 405 nm. After 6 hr of incubation with
stirring, the mixtures were incubated without stirring for 4-5 d after
which the aggregates formed were analyzed.
Congo red (CR) assay. CR binding to amyloid aggregates was
used as described previously (Klunk et al., 1989). Aliquots (40 µl)
of the aggregation mixtures were added to 960 µl of a solution of 25 µM CR, 100 mM phosphate buffer, pH 7.4, and
150 mM NaCl and incubated for 30 min. Absorbance was
measured at 480 and 540 nm. The CR binding was estimated by CR(M) = (A540/25,295)  (A480/46,306).
AChE activity in amyloid fibrils. Four hundred-microliter
aliquots of the A1-40 fibrils formed in the presence of
AChE in aggregation assays (5 d of incubation) were centrifuged at 14,000 rpm for 30 min. The supernatants were removed, and the AChE-A complexes were resuspended in 400 µl of PBS. To remove noncomplexed AChE, the aggregates were washed three times by
centrifugation, and resuspension in 400 µl of PBS was accomplished by
vortex stirring. The final pellet was resuspended in 100 µl of PBS.
To quantify the fraction of AChE present in the AChE-A complexes,
the enzymatic activity in the final pellet and supernatants was
determined by the assay of Ellman et al. (1961). In addition,
125I-AChE was used to form AChE-A complexes, and the
radioactivity in the same fractions was also quantified. Furthermore,
AChE activity associated with the amyloid fibrils in vitro
was detected by histochemical staining of the washed aggregates
(Karnovsky and Roots, 1964). The amyloid character of the
A1-40 fibrils was evidenced by the binding of
thioflavine-T (LeVine, 1993) and detected under a fluorescence
microscope, as described previously (Alvarez et al., 1997). Control
samples in which A was incubated alone were subjected to the same
procedures.
Dissociation studies of the AChE-A complexes. A 10 µl
aliquot of the 125I-AChE-A complexes (final pellet) was
added to 290 µl of each of the agents listed below. The complexes
were incubated in polypropylene tubes with each agent for 1 hr with
gentle stirring. The aggregates were then centrifuged at 14,000 rpm for
30 min, and both the AChE activity and radioactivity present in the
pellet and the supernatant were determined. The agents used were 1%
SDS, 6 M guanidine-HCl, 6 M guanidine
isothiocyanate, 8 M urea, PBS-1% Tween 20, PBS-1% Triton
X-100, 1 M NaCl, and 2 M MgCl2.
Velocity sedimentation analysis. AChE (G4 form)
alone or the AChE-125I-A complexes produced in the
stirred kinetic assays were submitted to velocity sedimentation
analysis in 5-20% sucrose gradients, containing a 70% sucrose
cushion at the bottom of the centrifuge tube. A Combi-Sorvall
ultracentrifuge was used. Fractions were collected from the bottom of
the gradient as described previously (Inestrosa et al., 1996b).
Immunogold labeling of the AChE-A complexes. For
immunogold staining, AChE-A complexes were adsorbed onto nickel
grids covered by a carbon-stabilized Formvar film and air-dried. After
washing in buffer (0.05 M Tris-HCl, pH 7.4) for 2 min,
nonspecific binding was blocked by incubation in 0.05 M
Tris-HCl, pH 7.4, with 1% ovalbumin for 1 hr. The grids were then
placed on a droplet of anti-AChE polyclonal antibody diluted 1:50 in
0.05 M Tris-HCl, pH 7.4, with 1% ovalbumin and incubated
at 4°C overnight. These were passed under five droplets of washing
solution (0.05 M Tris-HCl, pH 7.4, with 0.05% Tween 20)
for 5 min each time, placed on a droplet of anti-rabbit IgG conjugated
to 10 nm colloidal gold particles for 1 hr (Sigma; diluted 1:20 in 0.05 M Tris-HCl, pH 7.4, with 1% ovalbumin), and passed under
another five droplets of washing solution (Naslund et al., 1995).
Before the final examination under a JEOL 100-B electron microscope,
the specimens were negatively stained with 2% uranyl acetate in
water.
Determination of kinetic parameters. The kinetics of AChE
activity were determined using appropriate dilutions of
acetylthiocholine in the activity assay; the concentrations used were
in the range of 0.03-1.0 mM, and separate blanks were used
for each substrate concentration. The reaction was started by the
addition of enzyme or AChE-A complex. The Km
and Vmax values for acetylthiocholine were
calculated by regression analysis of the linear portions of the
Lineweaver-Burk plots (1/V vs 1/S). The
substrate inhibition constants (Kss) were
determined from a plot of 1/V versus [S] for
[S] > 1.0 mM (Radic et al., 1991).
Inhibition of AChE activity. The IC50 values for
edrophonium, Tacrine, and propidium (where IC50 is the
inhibitor concentration required for 50% inhibition of AChE activity)
were determined by incubating AChE and AChE-A complexes with
varying concentrations of each inhibitor. The reaction was started by
the addition of the substrate mixture, with the acetylthiocholine
concentration fixed at 0.75 mM, and incubated for 30 min at
37°C. IC50 values were calculated by logarithmic plot
analysis. Inhibition coefficient values
(Ki) were calculated from the equation
Ki = IC50/1 + (S/Km), as described by
Hobbiger and Peck (1969) where S represents the concentration of acetylthiocholine (0.75 mM), and
Km represents the Michaelis-Menten constant.
pH dependence. To study the pH dependence of the enzymatic
activity of AChE and the AChE-A complexes, activity assays were performed at different pH values. The optimal pH was determined over a
range of 4.0-9.0 in PBS buffer. pH values were measured with a pH
meter standardized at 25°C to ±0.01 pH unit. The buffer was titrated
to the required pH by the addition of HCl for pH values <5.0 and by
NaOH for pH values >8.0.
Cytotoxicity assays in PC12 cells. Rat pheochromocytoma
(PC12) cells (Greene and Tischler, 1976; Inestrosa et al., 1981) were cultured in DMEM, 10% fetal calf serum (FCS), 5% horse serum, 100 U/ml penicillin/streptomycin, and 2 mM L-Gln.
For the cytotoxicity assays, cells were seeded in 96-well plates in
serum-free medium with 2 µM insulin at a density of
4 × 103 cells/100 µl per well (Solomon et
al., 1997). AChE-A complexes, amyloid fibrils, or PBS (control)
were added to the wells at different concentrations in a final volume
of 10 µl. The cells were incubated for 48 hr at 37°C, after which
cell viability was measured by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
method (Mosmann, 1983). This involves determining the mitochondrial dehydrogenase activity in intact cells by incubating for 4 hr at 37°C
with 1 mg/ml MTT. The reaction was stopped by the addition of cell
lysis buffer (50% dimethylformamide and 20% SDS, pH 7.4), and the
plates were incubated overnight, after which MTT reduction was
determined in a Uniskan microplate spectrophotometer at 540 and 650 nm.
Results are expressed as the percentage of control values.
Avian retina cell cultures. Primary cultures of retina cells
were prepared following the protocol described previously (De Mello et
al., 1976; Vogel and Nirenberg, 1976; Puro et al., 1977). Briefly,
8-d-old chick embryo retinas were dissected under sterile conditions,
cleared from pigment epithelium, and placed in a
Ca2+- and Mg2+-free salt-balanced
medium (Sheffield and Moscona, 1970). Trypsin, to a final concentration
of 0.05%, was added to the medium and incubated at 37°C for 8 min.
The trypsinized tissue was then centrifuged at 500 × g
for 1 min, and the supernatant was discarded. The pellet was
resuspended in 5 ml of Eagle's basal medium plus 5% FCS and mechanically dissociated by pipetting the tissue 10 times with a
5 ml pipette (Paes de Carvalho and De Mello, 1982). The number of cells
per 35 mm plastic dish was ~30 × 106 in 2 ml
of medium. The plates were then transferred to a humidified atmosphere
of 95% air and 5% CO2 at 37°C. The medium was changed every other day.
Cytotoxicity assays in retina cells. For the cytotoxicity
assays, 6 d retina cell cultures were treated with either
AChE-A complexes or -amyloid fibrils, previously aged for 5 d and added to the wells at a final A peptide concentration of 2.5 µM and incubated for 24 hr at 37°C (Campos et al.,
1997). Then retina cells were analyzed by phase-contrast microscopy
(20× magnification) using an Olympus inverted microscope.
Phase-contrast images were photographed with Kodak (Rochester, NY)
Ektachrome 64T 35 mm film.
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RESULTS |
AChE Forms a stable complex with the amyloid fibrils
Because we have previously shown that AChE is able to promote the
aggregation of the A peptide (Inestrosa et al., 1996b), in the
present work we study the characteristics of the amyloid fibrils formed
in the presence of AChE. To obtain amyloid fibrils, we performed
kinetic stirred aggregation assays, following A aggregation by
turbidity (Jarrett et al., 1993; Evans et al., 1995), in the presence
or absence of AChE. After incubation for 5 hr, A1-40
peptide without AChE aggregated to a lesser extent (twofold less) than
the peptide incubated with AChE (Fig. 1A). Fibrils formed in
the presence of AChE were separated from soluble enzyme by velocity
sedimentation in 5-20% sucrose gradients (Fig. 1B),
using a 70% sucrose cushion at the bottom of the tubes to isolate the
high molecular weight amyloid-AChE complexes. Figure 1B1 shows the sedimentation profile of native brain
AChE (10.7 S), and Figure 1B2 shows a high molecular
weight AChE aggregate at the 20-70% sucrose interface. Using
125I-A peptide these AChE aggregates were shown to
co-sediment with a labeled high molecular weight amyloid aggregate
(Fig. 1B3). The radioactivity detected in the
low-density side of the gradient probably reflects the distribution of
soluble A or small A aggregates. At least 58% of the AChE
activity present in these gradients appeared associated with the A
fibrils, suggesting that AChE is able to bind and interact with the
amyloid aggregates forming a macromolecular complex. The fraction of
AChE incorporated into the AChE-A complexes was established by
extensive washing to remove noncomplexed AChE. Table
1 shows that ~78% of AChE activity
remains associated with the complexes. When 125I-AChE was
used to estimate the amount of enzyme incorporated into the fibrils, at
least 50% of the initial radioactivity was found associated with the
complexes. These results indicate that AChE forms a complex with the
A fibrils in vitro, with an average of 60% of the AChE
becoming incorporated into the amyloid fibrils. The stoichiometric
ratio of AChE/A in the final complexes was ~1:1000.
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Figure 1.
The AChE is associated with A amyloid fibrils.
A, A peptide aggregation assay. A peptide (97 µM in PBS, pH 7.4) was incubated in a kinetic stirred
assay, with ( ) or without ( ) AChE (0.1 µM). A
aggregation was followed by turbidity at 405 nm. B,
Sedimentation velocity analysis. Native brain AChE (10.7 S) as control
and 125I-A amyloid aggregates produced in a stirred
kinetic assay with AChE were submitted to velocity sedimentation
analysis in 5-20% sucrose gradients containing a 70% sucrose cushion
at the bottom of the centrifuge tube. A Combi-Sorval ultracentrifuge
was used. Fractions were collected from the bottom of the gradient as
described previously (Inestrosa et al., 1994). AChE activity and
radioactivity in the corresponding fractions were determined.
1, Control G4 AChE; 2,
AChE-A aggregates; 3, 125I-A amyloid
aggregates.
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The stability of the complexes was analyzed by performing incubations
in the presence of strong dissociating agents, some of which had
previously been shown to dissociate A polymers obtained from AD
brain (Masters et al., 1985). AChE was followed by enzymatic activity
or by following 125I-AChE radioactivity. The interaction of
the enzyme with the A fibrils was resistant to high ionic strength
conditions, including 1 M NaCl and 2 M
MgCl2 (Table 2), and was only
partially affected by PBS-Tween 20 and PBS-Triton X-100. These
detergent buffers were able to remove ~10 and 20%, respectively, of
the total AChE activity associated with the amyloid aggregates. Urea (8 M) released ~12% of the AChE activity from the A
fibrils, and the chaotropic agent 6 M guanidine-HCl
released 57%. On the other hand, SDS and guanidine isothiocyanate
released 84 and 82%, respectively, of the enzyme present in the
complexes. These results are consistent with the notion that stable
AChE-A complexes are formed when AChE is used to accelerate the
formation of amyloid aggregates.
The association of AChE with the amyloid aggregates is an early
event during fibril formation
To investigate how early during the A-AChE complex formation
the enzyme becomes associated with the fibrils, 40 µl aliquots were
taken from turbidity assays at different incubation times, and the AChE
activity present in the pellets was determined. Figure 2 shows that the AChE activity present in
the incubation mixture became rapidly associated with the A
aggregates. In fact, as early as 30 min after mixing, most of the AChE
added was present in the precipitate fraction containing amyloid
aggregates (Fig. 2, ). Concomitant with this, most of the free
enzyme was removed from the soluble fraction (Fig. 2, ). The amyloid
character of the A aggregates formed in the presence of AChE was
determined by Congo red assays (Fig. 2, inset). This result
indicates that the formation of AChE-A complexes is an early event
during the A aggregation process.
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Figure 2.
Temporal dependence of AChE-A fibril complex
formation. Turbidimetric stirred assays of A with AChE were
incubated for 90 min. Then aliquots of 40 µl were taken at different
incubation times, diluted in 400 µl of PBS, and centrifuged, and AChE
activity was determined in the fibril pellet () and the supernatant
fraction () by the assay of Ellman et al. (1961). The amyloid
character of the aggregates formed in the presence of AChE was
determined by Congo red assays (inset) in the pellet
fraction.
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Morphological examination of the AChE-A complexes
The structure of the AChE-A complexes was examined under
fluorescence microscope after staining with thioflavine-T; the
aggregates showed a strong green fluorescence, indicating their amyloid
character (Fig. 3A,B) (Roher
et al., 1986). When these aggregates were histochemically stained for
AChE using the method of Karnovsky and Roots (1964), they showed the
typical brown of this staining reaction (Fig. 3C,D),
confirming the presence of AChE activity in the AChE-A complexes.
Amyloid aggregates generated in the absence of AChE did not show such
histochemical staining (data not shown). Finally, we examined the
amyloid filaments to establish whether the filaments were indeed
complexed to AChE. Using immunogold electron microscopy with antibodies
directed against AChE, small specks could be seen in the AChE-A
complexes (Fig. 4A) and
AChE-A fibers (Fig. 4B). A complexes alone did
not show specific labeling (Fig. 4C). The electron
microscopic examination of the amyloid filaments demonstrated the
existence of the AChE-A complex.
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Figure 3.
Thioflavine-T fluorescence and AChE histochemical
staining of the AChE-A complexes. A, B, Amyloid
aggregates formed in a kinetic stirred assay were washed three times
with PBS and then incubated for 4 min in a solution containing an
excess of thioflavine-T (5 mg/ml). Samples were examined under an
Axioplan fluorescence microscope (D-7082; Zeiss, Oberkochen, Germany)
at 400× magnification. C, D, Amyloid aggregates from a
kinetic stirred assay were washed three times with PBS, after which the
fibrils were stained for AChE with the histochemical staining reaction
of Karnovsky and Roots (1964).
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Figure 4.
Immunogold labeling of the AChE-A complexes.
A, AChE-A complexes were labeled with a polyclonal
antibody raised against AChE, followed by an anti-IgG antibody
conjugated to 10 nm colloidal gold particles, and were visualized by
negative staining. B, Select example of an amyloid fiber
labeled with anti-AChE conjugated to gold particles. C,
Control sample of high molecular mass A aggregates without AChE that
were treated with the same specific antibody as above. In each case, 10 µl aliquots taken from an A peptide kinetic stirred aggregation
assay, performed either in the presence or absence of AChE, were
adsorbed onto 300-mesh Formvar-coated grids, negative-stained with 2%
uranyl acetate, and viewed for fibrils with a Philips electron
microscope. Scale bar: A, C, 90 nm;
B, 80 nm.
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Enzymatic properties of the AChE present in the
AChE-A complexes
There are previous studies indicating that the AChE associated
with the amyloid deposits of AD brains appears to have unique properties (Geula and Mesulam, 1989; Kalaria et al., 1992). Therefore, a thorough biochemical and pharmacological analysis of the
amyloid-associated AChE was performed. Kinetic studies were performed,
and the effects of different inhibitors, excess substrate, and low pH
were evaluated. Figure 5A
shows the Lineweaver-Burk plot (1/V vs 1/S) obtained for
soluble AChE and A-complexed AChE. It is apparent that the kinetic
parameters change, and that the Km and
Vmax values for the amyloid were higher than for
the soluble AChE (Table 3). When the
AChE-A complexes were incubated under different pH conditions, it
became clear that the enzyme associated with amyloid was more resistant
to low-pH conditions than the free enzyme (Fig. 5B). When
the incubations were performed in the presence of increasing substrate
concentrations, the A-associated AChE was more resistant to
inhibition by excess acetylthiocholine (Fig. 5C). A shift
toward the right is observed on the rising side of the curve (in the lower concentration range), whereas on the descending side of the curve
(high concentrations), the data are most coincident (although not
identical) for both AChE and AChE-A. Moreover, the activity peak
for AChE-A is sharper and narrower than that for AChE alone. This
behavior probably indicates that once the substrate has gained access
(in the higher concentration range), the effects of excess substrate on
both AChE and AChE-A are very similar if not identical. However, at
lower concentrations, it is more difficult for the substrate to bind
complexed AChE than free AChE for reasons of physical hindrance.
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Figure 5.
Biochemical characterization of the AChE activity
associated with amyloid- fibrils. AChE-A complexes were washed
exhaustively with PBS using four cycles of centrifugation and
resuspension to remove noncomplexed AChE, and then amyloid-associated
AChE activity was studied (). Soluble native brain AChE was used as
control (). A, Lineweaver-Burk (double-reciprocal)
plots. AChE activity, free and associated with amyloid- fibrils, was
measured over a range of substrate concentrations. B, pH
dependence. Optimal pH for AChE and AChE-A complex activity was
determined over a pH range of 4.0-9.0 in phosphate buffer.
C, Activity of AChE as a function of substrate
concentration. The rate of hydrolysis is plotted as a log function of
acetylthiocholine substrate concentration. The bell-shaped
curves show that both free AChE and the AChE-A complexes
are inhibited by excess substrate. However, the AChE-A complexes
require higher acetylthiocholine concentrations for optimal activity
than the free enzyme.
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Table 3.
Comparison of the kinetic parameters of acetylthiocholine
hydrolysis and effects of the specific inhibitors of AChE
(IC50) on the free AChE and that associated with
amyloid- fibrils
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Finally, the AChE associated with amyloid fibrils appeared more
resistant to inhibition by anti-cholinesterase agents (Table 3). In
particular, when fasciculin (a 61 amino acid peptide isolated from
mamba venom (Rodriguez Ithurralde et al., 1983; Dajas et al., 1987) was
used, a much higher concentration (micromolar) was required to inhibit
the amyloid-associated AChE than the free soluble enzyme (nanomolar)
(Fig. 6A). A similar
trend was observed when monoclonal antibody 25B1 directed against AChE
(Gentry et al., 1995), which also blocks the amyloid formation elicited
by the enzyme, (Reyes et al., 1997) was used (Fig.
6B). The Ki values obtained for
the other anti-cholinesterase agents, Tacrine, edrophonium, and
propidium, were 16-, 14-, and 9-fold higher, respectively, for the
complexed AChE than for the soluble enzyme (Table 3). In all cases, a
~5- to 10-fold increase in the substrate or inhibitor concentrations
was required to reach the same level of AChE inhibition when AChE was
associated with A aggregates. That is to say, the effects were
independent of the structure or size of the inhibitor molecules under
consideration. As such, these results are consistent with a physical
phenomenon, i.e., the location or occlusion of the enzyme enmeshed
within a fibrillar environment. In a broad context, our results are
consistent with the idea that the association of AChE with A fibrils
determines a change in its enzymatic properties, as occurs in the
senile plaques of Alzheimer's brain.
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Figure 6.
Inhibition of the AChE-A complex by fasciculin
and the monoclonal antibody 25-B1 (mAb 25-B1), two
peripheral anionic binding site ligands. The activity is plotted as a
function of fasciculin (A) and mAb 25-B1
(B) concentrations. Free AChE () and
AChE-A complexes () are both inhibited by fasciculin and mAb
25-B1. However, the AChE-A complexes require higher concentrations
of both compounds to reach the same level of inhibition as free AChE.
The IC50 values for free AChE were 249 ± 15 pM for fasciculin and 19.0 ± 0.5 pM for
mAb 25-B1. The corresponding values for the AChE-A complexes were
2746 ± 28 pM for fasciculin and >100 pM
for mAb 25-B1. The IC50 values represent the mean ± SD of three identical samples run in separate experiments.
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AChE-A complexes exhibit increased neurotoxicity in relation to
amyloid fibrils alone
Its has been demonstrated that amyloid fibrils are toxic to
neuronal cells in culture (Lorenzo and Yankner, 1994; Yankner, 1996),
and several studies have shown a strong correlation between A
neurotoxicity and the aggregation state of the A peptide (Busciglio et al., 1992; Mattson et al., 1992; Pike et al., 1993). Hence we
hypothesized that the fibrils of the AChE-A complexes would be
neurotoxic (Inestrosa et al., 1997), and to evaluate this point, we
performed experiments using AChE-A complexes versus A aggregates in neuronal cell cultures. First, rat pheochromocytoma (PC12) cell
cultures (Greene and Tischler, 1976) were treated with fibrillar A
and AChE-A complexes. Cell viability was assessed by a decrease in
their ability to reduce MTT (Mosmann, 1983). As shown in Figure 7, A neurotoxicity was dose-dependent
in the range of 1-25 µM, and cell toxicity decreased to
~27% of control cells measured in the absence of A peptide.
However, when the PC12 cells were treated with A fibrils formed in
the presence of AChE, a major decrease in MTT reduction was apparent in
comparison with A fibrils alone. This effect was dependent on the
concentration of both the A peptide and the AChE used in the
complexes; the highest concentration of AChE added to the cells in
complexed form was 25 nM, in comparison with 25 µM A peptide. At this concentration, cell survival was
minimal, and the cytotoxic effect of the fibrillar A was enhanced
almost twofold, whereas AChE added alone, at 25 nM, did not
show a toxic effect (data not shown). Further experiments were
performed using primary neuronal cultures to discard any possibility of
a specific susceptibility of PC12 cells to the AChE-amyloid
aggregates. The effect of the AChE-A complexes was studied in chick
primary cultures of retina cells (Vogel and Nirenberg, 1976; Puro et
al., 1977; Paes de Carvalho and De Mello, 1982). Figure
8A shows a control
6 d neuronal cell culture. These cells are known to display
abundant neurite outgrowth and present several types of
neurotransmitter receptors, including GABA, dopamine, and acetylcholine
of both nicotinic and muscarinic sort (Enna and Snyder, 1976; Vogel et
al., 1976; Sugiyama et al., 1977; Makman et al., 1980). After 24 hr of
treatment with -amyloid fibrils (2.5 µM), the retina
cells showed a clear loss of neurites and a decrease in cell density
(Fig. 8B). However, when the neuronal cultures were
exposed to AChE-A complexes, aged for 5 d, dramatic changes
were observed. Only a minimal number of cells were seen to present
neurites, and most cells were dead after 24 hr of culture (Fig.
8C). Summarizing, studies with two very different cell
culture systems indicate that the AChE-A complex is more toxic than
A fibrils alone, suggesting that the incorporation of AChE into Alzheimer's amyloid fibrils could lead to an increase in their neurotoxic properties. The AChE associated with the -amyloid deposits present in Alzheimer's brain may indeed be a relevant factor
in triggering the neurodegeneration induced by senile plaques in
specific regions of the brain.
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Figure 7.
AChE-A complexes are more toxic than A
fibrils alone in PC12 cells. Rat pheochromocytoma PC12 cells were
treated with A amyloid fibrils as a control (white
bars) or with AChE-A amyloid fibrils (black
bars) for 48 hr at different A concentrations. The cell
viability after treatment was measured by the MTT reduction assay.
Significantly different from control: *p < 0.05;
**p < 0.001 by nonpaired Student's
t test; n.s., not significant.
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Figure 8.
AChE-A complexes are more toxic than A
fibrils alone in primary cultures of retina cells. Six-day chick retina
cell cultures were incubated for 24 hr in a medium supplemented with
PBS as a control (A), -amyloid fibrils (2.5 µM) (B), and AChE-A complexes
(2.5 µM for the A peptide) (C).
The retina cell cultures were observed by phase contrast microscopy
using 40× magnification under an Olympus inverted microscope.
Representative photographs are shown.
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| |
DISCUSSION |
The interaction of AChE with A aggregates results in the
formation of a stable AChE-A complex
In the present study, the ability of purified AChE to form an
AChE-A1-40 complex was demonstrated. AChE was able to bind and interact with amyloid aggregates forming stable macromolecular complexes. In fact, such complexes were joined by strong intermolecular bonds because they were partially disrupted only by chaotropic agents
(6 M guanidine-HCl and 6 M guanidine
isothiocyanate), agents known to be able to solubilize amyloid cores
from Alzheimer's brain (Masters et al., 1985) and to disrupt amyloid
fibrils (Naiki et al., 1991), and by SDS detergent, able to disassemble
amyloid fibrils formed by the A1-40 peptide (Soreghan
et al., 1994). High ionic strength buffers were unable to dissociate
the enzyme from these complexes. A very high percentage of the initial
AChE present during fibrillogenesis remained bound to the A fibrils, as estimated by the incorporation of 125I-AChE or by
enzymatic activity measurements. Immunogold labeling confirmed that
AChE was tightly bound to the amyloid fibrils.
It would appear that aggregation with AChE is not a promiscuous process
but, rather, thermodynamically favored, because only a small amount of
AChE is required to promote aggregation (Fig. 1). In fact, the
stoichiometric ratio of AChE/A in the final complexes was ~1:1000,
and it is possible that this ratio represents the successive
incorporation of A monomers onto an initial A fibril containing
an AChE-A complex. A morphological examination of such complexes
showed that their structural properties were typical of amyloid; i.e.,
they displayed fluorescent staining with thioflavine-T and a fine
structure similar to that of amyloid A fibrils, as determined by
negative staining under the electron microscope. Finally, our
biochemical data indicated that the incorporation of AChE into the
amyloid complex was an early event during the polymerization and growth
of the fibrils. This suggests a role for the AChE-A complex at the
beginning of the amyloidogenic process, whereby AChE may act as a
heterogeneous nucleus, increasing the rate of fibrillogenesis and
stabilizing the growing amyloid fibrils. Consistent with this
possibility is the finding that when AChE was incubated with partially
formed amyloid fibrils, only a small proportion of the enzyme (2%) was
able to bind to the fibrils (data not shown) (R. Alarcón and
N. C. Inestrosa, unpublished results). Such behavior can be
expected from heteronucleators in a polymerization process, which act
during the initial nucleation phase of filament formation (Ferrone et
al., 1985).
The formation of an AChE-A complex in vitro is
reminiscent of the formation of apoE-A complexes observed in
vivo (Naslund et al., 1995; Castaño et al., 1995). In this
case, the existence of the apoE-A complex together with genetic
evidence has been used to support a role for apoE as a risk factor in
AD (Selkoe, 1996).
The AChE-A complex changes the biochemical properties of
the enzyme
The formation of an AChE-A complex is entirely consistent with
the fact that AChE and butyrylcholinesterase have been identified within A deposits, such as those found in preamyloid diffuse and
mature senile plaques and cerebral blood vessels (Morán et al.,
1993; Geula and Mesulam, 1994), and emphasizes the potential relevance
of AChE in AD. Most of the AChE found in senile plaques is associated
with the amyloid cores, and only a small portion is associated with the
neuritic components at the periphery of the plaques (Ulrich et al.,
1990; Carson et al., 1991). Histochemical studies have demonstrated
that the AChE associated with senile plaques differs from the enzyme
found in normal fibers and neurons with respect to optimal pH,
inhibition by excess substrate, and inhibitor sensitivity (Mesulam et
al., 1987; Geula and Mesulam, 1989; Schätz et al., 1989; Wright
et al., 1993). Furthermore, biochemical studies have indicated that
senile plaque-associated AChE is only partially extracted using
collagenase digestions (Nakamura et al., 1990), heparan sulfate
extractions (Kalaria et al., 1992), or high-salt buffers plus detergent
(Mimori et al., 1997). Interestingly, the AChE present in the
AChE-A complexes reported here showed unique properties similar to
those of senile plaque-associated AChE. In fact, the complexed enzyme
(1) presented Km and Vmax
values higher than those of the free enzyme and was more resistant to
(2) incubation under low pH conditions, (3) inhibition by
anti-cholinesterase agents, and (4) inhibition by excess
acetylthiocholine. Regarding the latter, the shift toward the right
seen in the lower concentration side of the curve (Fig. 5C)
suggests that the fibrils establish a physical barrier that hinders
access of the substrate to the enzyme active site. This would explain
why higher concentrations of acetylthiocholine are required and would
also account for the reactions obtained with small organic inhibitors
as well as larger ones. This physical trapping of the enzyme is also
consistent with our observation (Fig. 2) that formation of the
AChE-A complex is an early rather than late event in the
fibrillogenic process. Maturation of the fibrils inhibits or precludes
occlusion of the enzyme within A, whereas fibrils still in their
initial phases retain the freedom to trap the enzyme. In addition, as
indicated in Table 2, such a mechanism is distinct from the hydrophobic
interactions typical of globular proteins and allows for the
observation that not all denaturants, certainly not high-salt media,
are able to solubilize the fibril components. Physical trapping of the
enzyme could also account for the general observation that
butyrylcholinesterase, which has no peripheral anionic binding site and
displays different kinetic properties to AChE, is also found amid
amyloid fibrils. This theory is quite interesting, in that a structural
gene defect in AChE, butyrylcholinesterase, or any other enzyme species
caught midst the amyloid fibrils is not a requisite.
AChE-A complexes increase the neurotoxicity of
Alzheimer's fibrils
The neurotoxicity of A fibrils, together with the genetic
evidence linking the amyloid precursor protein (APP) with AD,
constitute the most relevant evidence to support a central role for the
A peptide in the pathogenesis of AD (Selkoe, 1996; Yankner, 1996). The observation, in AD brain studies, that neurodegenerative changes occur around senile plaques (Mann and Esiri, 1989) and the
demonstration that A together with various C-terminal fragments of
APP containing the A sequence is neurotoxic in cell cultures gave
rise to the hypothesis that A may be the primary cause of neuronal
degeneration in AD (Yankner et al., 1989; Yankner, 1996). The
neurotoxicity of A is dependent on its aggregation state (Busciglio
et al., 1992; Pike et al., 1993); it requires the assembly of A into amyloid fibrils, whereas nonfibrillar, amorphous aggregates of A are
not neurotoxic (Lorenzo and Yankner, 1994; Busciglio et al., 1995;
Howllett et al., 1995). Our studies showing that the AChE-A complex
presented a stronger cytotoxic effect than A fibrils alone in both
PC12 cells and primary retina cells indicate that the incorporation of
AChE into amyloid fibrils changes their cytotoxic properties. This
result is interesting because, although A is a primary factor in the
pathogenesis of AD (Selkoe, 1997), there are several reports suggesting
that A deposition is not the sole determinant of disease
progression. For example, substantial numbers of amyloid plaques
occasionally appear in the brain in nondemented individuals, suggesting
that A deposition does not invariably lead to AD (Dickson et al.,
1991). It is possible that this may be in part attributable to
differences in the protein composition and structure of the plaques in
demented and nondemented individuals (Barcikowska et al., 1989; Cras et
al., 1991; Mesulam and Geula, 1994). Besides the capacity of AChE to
modulate the toxicity of amyloid fibrils in vitro, other
macromolecules such as apoE and apoJ have also been shown to regulate
the toxicity of these fibrils. However, both of these elements were
found to decrease amyloid toxicity in culture (Oda et al., 1995; Boggs et al., 1996; Puttfarcken et al., 1997), suggesting that an adequate balance between toxic elements and the microenvironment defines the
final toxicity of A fibrils. In this context, it is interesting to
mention that the potential role of AChE in AD is further supported by
the fact that AChE systems, in particular those more vulnerable to AD
such as the lightly stained neurons located in the entorhinal cortex,
the CA1 and subiculum of the hippocampus, and the amygdala (Shen, 1994,
Kasa et al., 1997), are the first to be affected in the pathological
process of AD. Although the cellular mechanism of action of A is not
precisely understood, several processes have been proposed. In brief,
A has been shown to alter Ca2+ homeostasis
(Mattson et al., 1992; Arispe et al., 1993), to enhance excitotoxic
mechanisms (Mattson et al., 1992), and to generate free radicals
(Hensley et al., 1994), although some controversy arises in the
latter case (Sayre et al., 1997). Thus, AChE could affect the toxic
signaling of A at several points. An alternative possibility is that
AChE per se activates neuronal cell death (Calderón et al., 1996,
1998). In any case, further experiments are required to fully
understand the mechanisms by which the AChE-A complex is more toxic
for neuronal cells in culture than A fibrils alone.
| |
FOOTNOTES |
Received Dec. 3, 1997; revised Feb. 12, 1998; accepted Feb. 17, 1998.
This research was supported by Fondo Nacional de Desarrollo
Científico y Technológico Grant 1971240 to N.C.I.,
Comisión Nacional de Investigación Científica y
Technológica PhD Thesis Award 2960052 to A.A., and a predoctoral
fellowship from Dirección de Investigación y Postgrado,
Pontificia Universidad Católica de Chile to C.O. N.C.I. is a
recipient of a Presidential Chair in Science from the Chilean
Government. F.J.M. is on leave from Hospital La Princesa (Madrid,
Spain).
Correspondence should be addressed to Dr. Nibaldo C. Inestrosa,
Molecular Neurobiology Unit, Catholic University of Chile, P.O. Box
114-D, Santiago, Chile.
| |
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Abstract
Introduction
