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A Recessive Mutation in the APP Gene with Dominant-Negative
Effect on Amyloidogenesis
Giuseppe Di Fede1, Marcella Catania1, Michela Morbin1, Giacomina Rossi1, Silvia Suardi1,
Giulia Mazzoleni1, Marco Merlin1, Anna Rita Giovagnoli1, Sara Prioni1, Alessandra
Erbetta2, Chiara Falcone3, Marco Gobbi4, Laura Colombo4, Antonio Bastone4, Marten
Beeg4, Claudia Manzoni4, Bruna Francescucci5, Alberto Spagnoli5, Laura Cantù6, Elena Del
Favero6, Efrat Levy7, Mario Salmona4, and Fabrizio Tagliavini1,*
1Division of Neurology and Neuropathology, “Carlo Besta” National Neurological Institute, 20133
Milan, Italy.
2Division of Neuroradiology, “Carlo Besta” National Neurological Institute, 20133 Milan, Italy.
3Division of Neuroepidemiology, “Carlo Besta” National Neurological Institute, 20133 Milan, Italy.
4Department of Molecular Biochemistry and Pharmachology, Istituto di Ricerche Farmacologiche
“Mario Negri,” 20156 Milan, Italy.
5Division of Cognitive Disorders, Centro Sant’Ambrogio Fatebenefratelli, Cernusco sul Naviglio,
20063 Milan, Italy.
6Department of Medical Chemistry, Biochemistry, and Biotechnology, University of Milan, Segrate,
20090 Milan, Italy.
7Departments of Pharmacology and Psychiatry, New York University School of Medicine, and
Nathan S. Kline Institute, Orangeburg, NY 10962, USA.
Abstract
β-Amyloid precursor protein (APP) mutations cause familial Alzheimer’s disease with nearly
complete penetrance. We found an APP mutation [alanine-673→valine-673 (A673V)] that causes
disease only in the homozygous state, whereas heterozygous carriers were unaffected, consistent
with a recessive Mendelian trait of inheritance. The A673V mutation affected APP processing,
resulting in enhanced β-amyloid (Aβ) production and formation of amyloid fibrils in vitro. Co-
incubation of mutated and wild-type peptides conferred instability on Aβ aggregates and inhibited
amyloidogenesis and neurotoxicity. The highly amyloidogenic effect of the A673V mutation in the
homozygous state and its anti-amyloidogenic effect in the heterozygous state account for the
autosomal recessive pattern of inheritance and have implications for genetic screening and the
potential treatment of Alzheimer’s disease.
Acentral pathological feature of Alzheimer’s disease (AD) is the accumulation of β-Aβ in the
form of oligomers and amyloid fibrils in the brain (1). Aβ is generated by sequential cleavage
of the APP by β- and γ-secretases and exists as short and long isoforms, Aβ1-40 and Aβ1-42
(2). Aβ1-42 is especially prone to misfolding and builds up aggregates that are thought to be
the primary neurotoxic species involved in AD pathogenesis (2,3). AD is usually sporadic, but
*To whom correspondence should be addressed. E-mail: ftagliavini@istituto-besta.it.
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Author Manuscript
Science. Author manuscript; available in PMC 2010 March 13.
Published in final edited form as:
Science. 2009 March 13; 323(5920): 1473–1477. doi:10.1126/science.1168979.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
a small fraction of cases is familial (4). The familial forms show an autosomal dominant pattern
of inheritance with virtually complete penetrance and are linked to mutations in the APP,
presenilin 1, and presenilin 2 genes (5). The APP mutations close to the sites of β- or γ-secretase
cleavage flanking the Aβ sequence overproduce total Aβ or only Aβ1-42, respectively, whereas
those that alter amino acids within Aβ result in greater propensity to aggregation in vitro (6,
7).
We have identified an APP mutation [Ala673→Val673 (A673V)] that causes disease only in
the homozygous state. The mutation consists of a C-to-T transition that results in an alanine-
to-valine substitution at position 673 (APP770 numbering) corresponding to position 2 of Aβ
(Fig. 1A and fig. S1) (8). The genetic defect was found in a patient with early-onset dementia
and in his younger sister, who now shows multiple-domain mild cognitive impairment (MCI)
(9). Six relatives aged between 21 and 88 years, from both parental lineages, who carry the
A673V mutation in the heterozygous state were not affected, as deduced by formal
neuropsychological assessment [supporting online material (SOM) text, fig. S2, and table S1],
consistent with a recessive Mendelian trait of inheritance. The A673V mutation was not found
in 200 healthy individuals and 100 sporadic AD patients. Both mutated and wild-type APP
mRNA were expressed in heterozygous carriers (8).
In the patient, the disease presented with behavioral changes and cognitive deficits at the age
of 36 years and evolved toward severe dementia with spastic tetraparesis, leading to complete
loss of autonomy in about 8 years (SOM text). Serial magnetic resonance imaging showed
progressive cortico-subcortical atrophy (fig. S3). Cerebrospinal fluid analysis evidenced
decreased Aβ1-42 and increased total and 181T-phosphorylated tau compared with that of
nondemented controls and similarly to AD subjects (table S2 and fig. S4) (8). In the plasma of
the patient and his A673V homozygous sister, Aβ1-40 and Aβ1-42 were higher than those in
nondemented controls, whereas the six A673V heterozygous carriers had intermediate amounts
(table S2 and fig. S4).
In conditioned media of fibroblasts prepared from skin biopsies (8), Aβ1-40 and Aβ1-42 were
2.1- and 1.7-fold higher in the patient than in four age-matched controls with no change in
Aβ1-42:Aβ1-40 ratio (table S2 and fig. S4), suggesting that the A673V variant alters APP
processing, which promotes an increase in Aβ formation. To confirm this, we transiently
transfected Chinese hamster ovary (CHO) and COS-7 cells with either mutant or wild-type
APP cDNA and measured Aβ in conditioned media by enzyme-linked immunosorbent assay
(ELISA) (8). Cells expressing A673V APP had significantly higher amounts of both Aβ1-40
and Aβ1-42 than did cells transfected with wild-type APP, with no change in Aβ1-42:Aβ1-40
ratio (table S2). CHO and COS-7 cells with the A673V mutation also had increased secretion
of amino-terminally truncated Aβ species, including Aβ11-40, Aβ11-42, and AβN3pE-42
(table S2). These differences were paralleled by differences in the production of soluble forms
of APP (sAPPβ and sAPPα) and of APP carboxy-terminal fragments (C99 and C83) that
derived from the amyloidogenic β-secretase or nonamyloidogenic α-secretase processing (8).
Fibroblasts of the patient showed increased secretion of sAPPβ and 2.5-fold increase in
sAPPβ:sAPPα ratio (mean of three determinations: 0.5) compared with those of four age-
matched controls [0.2 ± 0.01 (SD)] as deduced by ELISA. Similarly, the sAPPβ:sAPPα ratio
was significantly higher in media from CHO cells expressing the A673V mutation (0.4 ± 0.1)
than in media from control cells (0.1 ± 0.03, P =0.03) (Fig. 1B). Immunoblot analysis of cell
lysates with an antibody to the carboxy-terminal region of APP (8) showed a 1.9 ± 0.2 increase
in C99:C83 ratio in patient’s fibroblasts (mean of three determinations: 0.67) compared with
that in control fibroblasts (0.35 ± 0.02) and 2.5 ± 0.2 increase in mutated CHO cells (0.52 ±
0.10) compared with that of control cells (0.21 ± 0.05, P = 0.0001) (Fig. 1, C and D).
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We then investigated the effects of the A673V mutation on the aggregation and amyloidogenic
properties of Aβ by using synthetic peptides homologous to residues 1 to 40 with and without
the A-to-V substitution at position 2 (Aβ1-40mut and Aβ1-40wt) (8). Laser light scattering
measurements over short periods (first 24 hours after sample preparation) showed that the
aggregation kinetics was faster for Aβ1-40mut than for Aβ1-40wt and that the time constants
of the exponential increase were 1.3 hours and 5.8 hours, respectively (Fig. 2A). Furthermore,
although the initial size distribution of particles generated by the two peptides was similar,
after 24 hours Aβ1-40mut assemblies were much larger than Aβ1-40wt aggregates (Fig. 2B).
Polarized-light and electron microscopy (EM) showed that Aβ1-40mut aggregates with the
tinctorial properties of amyloid (i.e., birefringence after Congo red staining) ultrastructurally
formed by straight, unbranched, 8-nm-diameter fibrils were already apparent after 4 hours.
Amyloid progressively increased up to 5 days, when the samples contained only fibrils
organized in dense meshwork (Fig. 3, B, E, H, and K). Aβ1-40wt followed a qualitatively
similar assembly path but with much slower kinetics. The 8-nm-diameter amyloid fibrils were
first observed after 72 hours, mingled with oligomers and protofibrils (Fig. 3, A and G), and
the size and density of congophilic aggregates reached a plateau only after 20 days (Fig. 3, D
and J). Similar differences were observed between wild-type and mutated peptides homologous
to residues 1 to 42 of Aβ (Fig. 3, M and N), although the aggregation kinetics was faster
compared with that of Aβ1-40.
The finding that the A673V mutation strongly boosts Aβ production and fibrillogenesis raises
the question of why heterozygous carriers do not develop disease, so we analyzed the effects
of the interaction between Aβ1-40mut and Aβ1-40wt. Laser light scattering showed that the
time constant of aggregate formation of equimolar mixtures of wild-type and mutated peptides
was higher (8.3 hours) than the time to aggregate for either Aβ1-40mut (1.3 hours) or
Aβ1-40wt alone (5.8 hours) (Fig. 2A) and that the size distribution of particles was lowest both
at time 0 and after 24 hours (Fig. 2B). Furthermore, the aggregates formed by peptide mixtures
were far more unstable than those generated by either Aβ1-40wt or Aβ1-40mut after dilution
with buffer, with a characteristic dissolution time of 8 min. At the same time, no dissolution
kinetics was observed for samples of Aβ1-40wt and Aβ1-40mut alone (Fig. 2C). This was
confirmed by urea denaturation studies of peptide aggregates (8). Size exclusion
chromatography showed that the elution profiles of Aβ1-40wt and Aβ1-40mut were marked by
a single peak corresponding to the dimer, whereas the mixture gave a smaller peak area
corresponding to the dimer and a second small peak corresponding to the monomer (Fig. 4A).
Polarized light and EM showed that the peptide mixture built up much fewer congophilic
aggregates than not only Aβ1-40mut but also Aβ1-40wt (Fig. 3, C, F, I, and L). Similar results
were observed with Aβ1-42 peptides (Fig. 3, M to O). Amyloid formation was also inhibited
when Aβ1-40wt was incubated with a hexapeptide homologous to residues 1 to 6 containing
the A-to-V substitution in position 2 (Aβ1-6mut) at 1:4 molar ratio (fig. S5).
We analyzed the binding of Aβ peptides with and without the A673V mutation to Aβ1-40wt
by using surface plasmon resonance (8). In addition to Aβ1-40wt and Aβ1-40mut, we used the
hexa-peptides Aβ1-6wt and Aβ1-6mut to evaluate the independent contribution of the amino-
terminal sequence containing the mutation. No difference in binding to immobilized
Aβ1-40wt fibrils was observed between Aβ1-40wt and Aβ1-40mut, consistent with the finding
that Aβ aggregation is primarily driven by hydrophobic stretches in the central and carboxy-
terminal parts of the peptide (Fig. 4B) (10). However, the amino-terminal fragment
Aβ1-6mut showed greater ability to bind to wild-type Aβ1-40 than did Aβ1-6wt (Fig. 4C),
indicating that the A-to-V substitution at position 2 favors the interaction between mutant and
wild-type Aβ.
Lastly, we treated human neuroblastoma SH-SY5Y cells with Aβ1-42wt, Aβ1-42mut, or
mixtures thereof at 5 μM for 24 hours and assessed cell viability by 3-(4,5-dimethylthiazol-2-
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yl)-2,5-diphenyl tetrasodium bromide (8): Aβ1-42mut was more toxic than Aβ1-42wt, and the
mixture was significantly less toxic than either peptide alone (Fig. 4D).
We have identified a mutation in the APP gene showing a recessive Mendelian trait of
inheritance. Recently, a homozygous APP mutation (A693Δ) was detected in three AD patients
from two Japanese pedigrees (11). Because one out of four heterozygous individuals had MCI,
in the absence of experimental studies mimicking the situation in heterozygotes, it is hard to
establish whether A693Δ is a recessive mutation or a dominant APP variant with incomplete
penetrance.
The A673V APP mutation has two pathogenic effects: it (i) shifts APP processing toward the
amyloidogenic pathway and (ii) enhances the aggregation and fibrillogenic properties of Aβ.
However, the interaction between mutant and wild-type Aβ, favored by the A-to-V substitution
at position 2, interferes with nucleation or nucleation-dependent polymerization, or both,
hindering amyloidogenesis and neurotoxicity and thus protecting the heterozygous carriers.
Until recently, the importance of the aminoterminal sequence of Aβ in misfolding and disease
was underestimated because this region is highly disordered in the fibrillar form of the peptide
(12). However, the amino-terminal domain of Aβ is selectively perturbed in amyloidogenesis,
and, most importantly, changes in its primary sequence trigger peptide assembly and fibril
formation (13,14). The importance of this domain is further supported by the finding that
antibodies against it are optimal for plaque clearance in animal models (15). A previous study
reported a distinct heterozygous APP mutation at codon 673 [Ala673→Tyr673 (A673T)] in a
participant without clinical signs of dementia (16). Histological analysis did not detect amyloid
deposits in the brain. However, when the A673T mutation was introduced in a synthetic
Aβ1-40 peptide, it increased the propensity to aggregate, with a much shorter lag phase than
that of the wild-type peptide (17). These observations, together with our results, suggest that
mutations at position 2 of Aβ confer amyloidogenic properties that lead to AD only in the
homozygous state. The finding that the interaction between A673V-mutated and wild-type
Aβ hinders amyloidogenesis, and especially the anti-amyloidogenic properties of the mutated
six-residue peptide, may offer grounds for the development of therapeutic strategies based on
modified Aβ peptides or peptido-mimetic compounds (18,19) for both sporadic and familial
AD.
The present data highlight the importance of screening demented and nondemented human
populations for mutations of the Aβ encoding region of APP. Genetic variants that could be
regarded as normal polymorphisms may turn out to be pathogenic in homozygous individuals.
The identification of such mutations would help to prevent the occurrence of the disease in
their carriers.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
This work was supported by grants from the Italian Ministry of Health (533F/Q/1 to F.T. and M.S., and 71.6/2006
and RFPS 2007/02 to F.T.), CARIPLO Foundation (Guard) to F.T. and M.S., ERA-Net Neuron (nEUROsyn) to F.T.,
Negri-Weizmann Foundation to M.S., the National Institute of Neurological Disorders and Stroke (NS42029) to E.L.,
and the American Heart Association (0040102N) to E.L. A patent application related to this work has been filed by
Fondazione IRCCS Istituto Nazionale Neurologico “Carlo Besta.”
References and Notes
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Fig. 1.
Analysis of APP gene and APP processing. (A) APP gene analysis by restriction fragment
length polymorphism of 169—base pair (bp) polymerase chain reaction (PCR) products
amplified from homozygous (III-16), heterozygous (II-11), and control subjects. In the absence
of the A673V mutation, the enzyme HpyCH4V generates two fragments of 91 and 78 bp. The
mutation abolishes the restriction site, and the PCR product remains uncut. (B)
sAPPβ:sAPPα ratio in conditioned media from CHO cells transfected with wild-type or
A673V-mutated APP and fibroblasts of the proband and four controls. Error bars represent
means ± SD. (C) APP carboxy-terminal fragments C99 and C83 (arrowheads) in CHO cells
transfected with wild-type (lane 2) or A673V-mutated (lane 3) APP and fibroblasts from a
control (lane 4) and the proband (lane 5), as shown by immunoblot analysis. Lane 1 corresponds
to from nontransfected CHO cells. (D) Densitometric analysis of immunoblots, showing a
significant increase in the C99:C83 ratio (P = 0.0001) in cells carrying the A673V mutation.
Error bars represent means ± SD.
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Fig. 2.
Short-time kinetics of Aβ assembly and disassembly, determined by laser light scattering.
(A) Course of the light intensity scattered by solutions of Aβ1-40wt (blue), Aβ1-40mut (red),
and their equimolar mixture (green). The corresponding exponential fits are indicated by full
lines. (B) Particle size distribution of Aβ1-40wt (blue), Aβ1-40mut (red), and the peptide mixture
(green) immediately after sample preparation (time 0) and after 24 hours. (C) Short-time
dissolution kinetics of 48-hour-aged peptide aggregates after fivefold dilution with buffer. a.u.,
arbitrary units.
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Fig. 3.
Aggregation properties of mutated and wild-type Aβ peptides. (A to F) Electron micrographs
of aggregates generated by Aβ1-40wt, Aβ1-40mut, and equimolar mixtures after 72 hours [(A)
to (C), negative staining] and 20 days incubation [(D) to (F), positive staining]. (G to L)
Polarized light microscopy of Aβ aggregates stained with Congo red after 72 hours [(G) to (I)]
and 20 days [(J) to (L)]. (M to O) Electron micrographs of negatively stained aggregates
generated by Aβ1-42wt (M), Aβ1-42mut (N), and equimolar mixtures (O) after 5 days
incubation. The peptide mixture contains mainly amorphous material (O), whereas wild-type
and mutated Aβ1-42 are assembled in fibrillary structures. Scale bars indicate 250 nm [(A) to
(F)], 50 μm [(G) to (L)], and 125 nm [(M) to (O)].
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Fig. 4.
Physicochemical and biological properties of mutated and wild-type Aβ peptides. (A) Size
exclusion chromatograms of Aβ1-40wt, Aβ1-40mut, and equimolar peptide mixture aggregates
after treatment with 1 M urea for 24 hours. Monomeric species (arrow) are only seen in the
peptide mixture. (B and C) Binding of wild-type and mutated Aβ1-40 (B) or Aβ1-6 (C) to
amyloid fibrils of Aβ1-40wt determined by surface plasmon resonance. Solutions of Aβ1-40
(1 μM) or Aβ1-6 (500 μM) were injected onto Aβ1-40wt fibrils immobilized on the sensor chip
for the time indicated by the bars. (D) Viability of human neuroblastoma cells after 24 hours
exposure to 5 μM Aβ1-42wt, Aβ1-42mut, and the equimolar mixture. Error bars represent SD
of the mean of eight replicates. *P = 0.026, **P < 0.001.
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