Transthyretin protects Alzheimer’s mice from the
behavioral and biochemical effects of A? toxicity
Joel N. Buxbaum*†, Zhengyi Ye*, Nata `lia Reixach*, Linsey Friske*, Coree Levy‡, Pritam Das§, Todd Golde§,
Eliezer Masliah¶, Amanda R. Roberts‡, and Tamas Bartfai‡
*Division of Rheumatology Research, W. M. Keck Autoimmune Disease Center, and Department of Molecular and Experimental Medicine, and‡Department
of Molecular and Integrative Neurosciences, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037;§Department of
Neurosciences, Mayo Clinic College of Medicine, Birdsall 210, 4500 San Pablo Road, Jacksonville, FL 32224; and¶Department of Pathology, University of
California at San Diego, La Jolla, CA 92093
Communicated by Ernest Beutler, The Scripps Research Institute, La Jolla, CA, December 24, 2007 (received for review October 3, 2007)
Cells that have evolved to produce large quantities of secreted
proteins to serve the integrated functions of complex multicellular
organisms are equipped to compensate for protein misfolding.
Hepatocytes and plasma cells have well developed chaperone and
proteasome systems to ensure that secreted proteins transit the
cell efficiently. The number of neurodegenerative disorders asso-
ciated with protein misfolding suggests that neurons are particu-
larly sensitive to the pathogenic effects of aggregates of misfolded
molecules because those systems are less well developed in this
lineage. Aggregates of the amyloidogenic (A?1–42) peptide play a
major role in the pathogenesis of Alzheimer’s disease (AD), al-
though the precise mechanism is unclear. In genetic studies exam-
ining protein–protein interactions that could constitute native
mechanisms of neuroprotection in vivo, overexpression of a WT
human transthyretin (TTR) transgene was ameliorative in the
APP23 transgenic murine model of human AD. Targeted silencing
of the endogenous TTR gene accelerated the development of the
neuropathologic phenotype. Intraneuronal TTR was seen in the
brains of normal humans and mice and in AD patients and APP23
mice. The APP23 brains showed colocalization of extracellular TTR
with A? in plaques. Using surface plasmon resonance we obtained
in vitro evidence of direct protein–protein interaction between TTR
and A? aggregates. These findings suggest that TTR is protective
because of its capacity to bind toxic or pretoxic A? aggregates in
both the intracellular and extracellular environment in a chaper-
one-like manner. The interaction may represent a unique normal
host defense mechanism, enhancement of which could be thera-
protein interaction ? protein misfolding ? amyloidosis ? dementia
reports of physical interaction between amyloidogenic (A?)
peptides and TTR proteins in vitro, prevention of A? aggrega-
tion in Caenorhabditis elegans transgenic for both mutant A? and
AD models, immunohistochemically detectable TTR in the
vicinity of A? plaques in A? transgenic mice, and more aggres-
sive histologic disease in such mice after local treatment with
anti-TTR antibody (1–5). However, none of those studies dem-
onstrated functional effects of the putative TTR–A? interaction.
We performed genetic experiments designed to determine
whether TTR has an effect on the development of the neuro-
pathologic and behavioral phenotypes in a well characterized
murine model of human AD.
relationship between Alzheimer’s disease (AD) and tran-
sthyretin (TTR) has been hypothesized on the basis of
APP23 mice, carrying the Swedish autosomal dominant AD
mutation and displaying the neuropathologic (Congophilic
plaques, gliosis, neuronal death, Congophilic angiopathy) and
behavioral (defined cognitive deficits) features associated with
human AD, were mated with mice overexpressing WT human
TTR (hTTR) and animals in which both copies of the endog-
enous TTR gene had been silenced by targeted disruption (6–8).
The transgenics carried ?90 copies of the human gene with
serum concentrations of hTTR between 1 and 3 mg/ml and
cerebrospinal fluid concentrations between 0.007 and 0.019
Results of Barnes maze testing (to assess cognitive function and
spatial learning) of 15-month-old APP23, APP23 overexpressing
APP23/hTTR and control mice the number of errors decreased
across blocks [F (2,64) ? 5.2, P ? 0.01], demonstrating the effect
of training. There was a significant effect of group (genotype) on
the number of errors across the three blocks [(F(2,32) ? 4.1, P ?
0.05] caused by group differences in blocks 2 [F(2,32) ? 3.0, P ?
in these blocks relative to control mice (P ? 0.05). APP23/ hTTR
mice were not significantly different from age-matched WT con-
trols, whereas the performance of APP23/hTTR animals was
superior to that of APP23 mice lacking the hTTR transgene (P ?
0.05). The percentage of trials in each block in which mice used a
[F(2,64) ? 17.7, P ? 0.001], but no significant group by block
interaction. Spatial strategy utilization increased across blocks.
Both control and APP23/ hTTR mice used this strategy more than
APP23 mice in block 3 (P ? 0.05). There was a significant effect of
APP23 on numbers of errors made and strategy utilization in the
15-month-old animals. The presence of hTTR resulted in virtually
normal performance in both measures, indicating amelioration of
the APP23 behavioral phenotype.
There were no significant group differences in anxiety-like
behavior, rearing, or locomotor activity. There were trends for the
older APP23 mice to show increased anxiety-like behavior and
decreased activity (horizontal and vertical). Visual cliff analysis
are consistent with previous analyses of APP23 mice (9).
To determine whether absence of the endogenous murine
TTR (mTTR) gene would have an effect on the development of
disease in APP23 mice we carried out behavioral studies on
5.5-month-old mice expressing the APP23 gene in the presence
(APP23) or absence of mTTR (APP23/mTTR?/?) and in mice
gene (mTTR?/?). The number of errors is shown in Fig. 1c and
the percent spatial strategy is shown in Fig. 1d. Because we had
behavior of APP23 mice, we performed three general analyses.
Author contributions: J.N.B. and T.B. designed research; Z.Y., L.F., C.L., P.D., T.G., E.M., and
A.R.R. performed research; N.R. contributed new reagents/analytic tools; J.N.B., Z.Y., L.F.,
T.G., E.M., A.R.R., and T.B. analyzed data; and J.N.B. wrote the paper.
The authors declare no conflict of interest.
†To whom correspondence should be addressed. E-mail: email@example.com.
© 2008 by The National Academy of Sciences of the USA
February 19, 2008 ?
vol. 105 ?
no. 7 ?
We first examined whether APP23 influenced the measures of
behavior. There was no effect of APP23 on errors [F(1,23) ?
0.152, P ? 0.05], but there was an impact on percent spatial
strategy [F(1,23) ? 7.1, P ? 0.05], suggesting that at a younger
age differences do exist, albeit they are less global. Second,
performance was examined in mice not carrying the hTTR
transgene (WT murine genotype) to determine whether
mTTR ? APP23 behaved differently. There were significant
5.6; P ? 0.01] and percentage spatial strategy [F(2,92) ? 3.5;
P ? 0.05], suggesting a mild learning delay in mice lacking
mTTR. The mTTR decrement was more prominent in WT mice
lacking the human AD gene, as revealed by an APP23 by mTTR
interaction on percentage spatial strategy [F(1,47) ? 5.2;
of APP23 were examined in mTTR?/?mice (WT genotype).
APP23 reduces the percentage spatial strategy utilization and
there was as a complex three-way interaction of blocks, APP, and
hTTR [F(2,72) ? 4.2; P ? 0.05]. Further analysis revealed a
reversal of the APP-associated deficits in spatial strategy utili-
zation by hTTR. This result is consistent with the findings in the
APP23/mTTR?/?mice were more active than APP23 mice
with an intact TTR gene (P ? 0.05), suggesting an effect of
mTTR silencing on activity in the presence of the APP23 gene;
however, examination of the Barnes maze behavior (data not
shown) revealed no difference between the groups in the dis-
tances traveled across the trials, indicating that the cognitive
deficits were independent of the differences in activity. Thus in
the progressive use of spatial strategy and a general slowing of
learning in this test in mTTR null mutant mice.
As in the older animals, results of the light/dark transfer test
of anxiety-like behavior showed no significant effect of group,
nor did the genotype have an effect on the rearing often
correlated with anxiety (data not shown), and the mice were not
Despite the absence of significant differences in behavior
between mTTR?/?mice with or without a human AD gene,
comparison of immunohistochemical staining of the frontal
cortex and the hippocampus of the 5.5-month-old APP23 mice
with an active or silenced mTTR gene revealed that 7 of the 11
animals without TTR had cortical or hippocampal A? deposi-
tion (Table 1 and Fig. 2 A and D). In the presence of the mTTR
gene only one animal had detectable deposits. The quantitation
of SDS and formic acid soluble A?1–40and A?1–42showed no
significant differences in the concentrations or absolute amounts
of the peptides in the extracted hemi-brains of the two groups,
although the concentrations of formic acid-extractable peptides
in the mTTR?/?mice were higher with a trend toward statistical
significance. These findings suggested that immunohistochem-
istry was the more sensitive of the two methods.
Examination of the cortex and hippocampus of 15- to 16-
month-old APP23 mice with an antibody to human A? showed
staining in both the cortex and hippocampus (Table 1 and Fig.
2E). Quantitative analysis of the images revealed that APP23/
hTTR mice had significantly less A? staining in the cortex and
hippocampus (Fig. 2F and Table 1). Staining with the anti-TTR
antibody showed reactivity in neuronal cell bodies of all of the
mice except the TTR knockouts (mTTR?/?) (data not shown).
APP23 mice showed anti-TTR reactivity in neuronal cell bodies
as well as extracellularly in areas of A? deposition (Fig. 2 P–R).
Colocalization of TTR and A? is revealed in Fig. 2 L and O with
lesser amounts of A? staining and smaller plaques in the
APP23/hTTR brains. Using the same antibody brains from
age-matched control animals, bearing neither the human A? nor
the hTTR construct showed staining (as in the 5-month-old
animals; data not shown). Determination of SDS and formic
acid-soluble A?1–40 and A?1–42 showed significantly lower
amounts of A? peptides in the hemi-brains of the APP23/hTTR
animals than in the APP23 mice (Table 1) (10).
To determine whether we could demonstrate a direct inter-
action between TTR and A? we elected to use surface plasmon
resonance (SPR), a methodology that did not involve interfer-
ence with, or acceleration of, fibril formation. Previous liquid-
phase studies using that approach were complicated by the
fibril-forming properties of both components and the possible
effects of preformed aggregates on the measurements. In our
SPR experiments binding to aggregated A? was always better
WT, two male, five female; mTTR?/?, three male, five female; hTTR?, two male, four female; APP23, nine male, nine female; APP23/mTTR?/?, nine male, eight
female; APP23/hTTR?, five male, four female. For older mice, group sizes were: control WT, seven male, seven female; APP23, six male, six female; APP23 httr,
of errors per session and strategy used to locate the escape tunnel were recorded. Errors included nose pokes and head deflections over any hole not having
the tunnel beneath it. Search strategies were divided into operationally defined categories: (i) random, localized hole searches separated by crossings through
the maze center, (ii) serial, systematic hole searches (every or every other hole) in a clockwise or counterclockwise direction, or (iii) spatial, reaching the escape
tunnel with both error and distance (number of holes between the first hole visited and the escape tunnel) scores of 3 or less. Data were analyzed in four session
blocks using two-way ANOVA with genotype and blocks as variables.
Results of behavioral testing of control and APP23 mice. Separate mixed-sex groups of mice were tested. For younger mice, group sizes were: control
www.pnas.org?cgi?doi?10.1073?pnas.0712197105 Buxbaum et al.
species of origin (Fig. 3). The interaction was always stronger for
A?1–42than for A?1–40,although it was not possible to determine
an accurate KDfor the interactions because of the size hetero-
geneity in the A? fibril preparations. Interactions among any
combination of TTR and A? molecular species, i.e., TTR
monomers, TTR tetramers, A?1–40 or A?1–42 monomers, or
fibrils were greater at 37°C than 25°C (data not shown). The
mTTR molecule appeared to have much greater affinity for any
of the A? forms than did the human protein.
We demonstrate markedly improved cognitive function of older
APP23 transgenic mice when they carry multiple copies of the
human WT TTR gene. The effect of endogenous TTR was
confirmed when the APP23 gene was crossed onto a mTTR?/?
background. Those mice showed immunohistochemical evi-
an active endogenous TTR gene. The improved behavioral
performance of older APP23 animals carrying the hTTR gene
was associated with less severe Alzheimer-like brain pathology
and smaller amounts of extractable A? than in the brains of
control APP23 carriers.
Our use of the Barnes maze, rather than the more frequently
reported Morris water maze, reflects current thinking that the
Barnes test eliminates the swimming aspect of the behavior and
that the murine anxiety response to swimming may reduce the
sensitivity of the Morris assay in mice (11, 12).
Antibody staining of brain sections appears to have greater
sensitivity than immunochemical quantitation of soluble A?,
presumably because of its capacity to recognize localized de-
posits. The extraction procedures, although more quantitative,
may be less sensitive because they pool affected and nonaffected
regions of the brain, thus they are subject to dilutional effects,
particularly in younger animals when deposits are smaller and
A recent report (13) indicated that hemizygous mTTR knock-
outs crossed with APPswe/PS1?E9 AD model mice had signif-
icantly greater amounts of A? deposition in the cortex and
hippocampus after 7 months of age. In those studies, at 5 months
of age the animals did not show A? staining or statistically
significant differences in the amounts of extractable A? pep-
tides. Hence detectable AD-like pathology appears earlier in the
homozygous TTR knockouts, suggesting that mTTR demon-
strates a gene dose-dependent protective effect.
Young animals lacking TTR gene expression displayed a
defect in spatial learning even in the absence of the human
AD-associated gene, confirming the recently reported observa-
tion that mTTR has a behavioral function independent of its
interaction with A? (14).
The immunohistochemical findings in both the young and old
animals argue against a primary role for the genetic background of
the carrier strains on the effect. The APP23 animals had been
extensively back-crossed to B6, and hTTR transgenics are carried
knockouts were crossed with the APP23’s for six generations.
The SPR analyses show that both hTTR and mTTR bind
A?1–40and A?1–42.Binding of mTTR to any form of A? has not
been previously investigated. The murine protein has much
greater affinity for A? aggregates, fibrils, and monomers than
does the human protein. Both hTTR and mTTR bind the
aggregates better than they bind soluble monomers, and the
genetic experiments show that both have an effect on A?
toxicity. The SPR experiments indicate that the affinity of both
TTRs for monomeric A? is low. We were unable to define the
precise molecular nature of the TTR-binding species of A? by
using this method. Light-scattering experiments have indicated
that tetrameric hTTR binds A?1–40aggregates but not fibrils,
and it has been suggested that the findings are most consistent
with TTR preventing protofibril elongation and subsequent
lateral association (15). It is likely, given the molecular hetero-
geneity of our fibril preparations, that the binding activity is a
property of subfibrillar A? aggregates present in the incubated
samples rather than in mature fibrils. It has also been reported
that mTTR coimmunoprecipitates with A? in brain extracts
Table 1. A? deposition in brains of APP23 transgenic animals
ParameterAPP23 mTTR APP23 without mTTRAPP23 ? hTTR
Animals at 5.5 months
No. animals with deposits
Mean deposition cortex, %
SDS soluble A?1–42, Pmol/g
Formic acid-soluble A?1–42,
SDS-soluble A?1–40, Pmol/g
Formic acid-soluble A?1–40,
Animals at 16 months
No. animals with deposits
Mean deposition cortex, %
SDS-soluble A?1–42, Pmol/g
Formic acid-soluble A?1–42,
SDS-soluble A?1–40, Pmol/g
Formic acid-soluble A?1–40,
0.033 ? 0.1
0.02 ? 0.08
0.09 ? 0.1
0.11 ? 0.11
6.35 ? 2.5
2.8 ? 0.31
5.5 ? 2.4
3.5 ? 3
22 ? 11.6
5.1 ? 0.7
20 ? 9.4
9.9 ? 17
1.8 ? 1.3
1.1 ? 0.8
4.4 ? 3.5
3.0 ? 3.6
198 ? 121
28 ? 25
53 ? 60
14 ? 14
343 ? 168
429 ? 273
129 ? 154
199 ? 158
Immunohistochemically the mean amounts of A? deposition were measured as percentage of neuropil in the
cortex and hippocampus replaced by A?-stained material.
*Frequencies analyzed by Fisher Exact Test. Means compared by Mann–Whitney.
Buxbaum et al.
February 19, 2008 ?
vol. 105 ?
no. 7 ?
from AD model mice, but the molecular species of A? was not
examined in those experiments (16).
The simplest interpretation of our findings, and one consistent
with a physical interaction between TTR and A?1–40, A?1–42, is
that TTR binds A? in a manner that prevents both toxicity and
plaque formation, presumably by interfering with aggregation of
some A? species larger than monomers (15). The results are
consistent with previous reports of interactions between hTTR
and various A? preparations (1, 2). The greater affinity of the
murine protein for human A? was unexpected, but the marked
physiologic effect of both proteins in the two experiments, i.e.,
suppression of the APP23 phenotype by the human protein,
albeit in relatively high copy number transgenic animals, and
acceleration of deposition in the absence of mouse protein were
unequivocal. The human and mouse proteins are 80% homol-
ogous in amino acid sequence but have different biophysical
properties, the murine protein being much more kinetically
stable (17). That difference per se does not explain the higher
affinity of mTTR for the A? aggregates.
Previous immunopathologic analyses of human AD brains
with antisera for TTR have yielded conflicting results (18, 19).
Our studies clearly show intraneuronal staining for TTR in both
human and murine brains with no staining in the TTR knockout
animals, TTR staining of the plaques in the mouse brains but
predominant vascular staining in human AD brain (Fig. 2 P–S).
These observations by themselves are consistent with either
neuronal TTR synthesis or uptake of TTR synthesized else-
where, presumably the choroid plexus (20, 21). The notion of
neuronal synthesis of TTR, in addition to the well documented
production of the protein in choroid plexus and leptomeningeal
epithelial cells, is supported by the Allen brain atlas and other
microarray studies that show TTR mRNA in other areas of
carefully dissected mouse brains (22, 23). The question of
whether the intraneuronal TTR is synthesized in neurons or only
in the choroid plexus and taken up by neurons is not answered
by our observations. However, the intraneuronal localization of
the protein suggests that hTTR and mTTR are available to bind
A? intraneuronally and/or extraneuronally.
A behavioral, presumably neuronal function for TTR was
proposed on the basis of studies of TTR knockouts that showed
‘‘reduced signs of depressive activity and increased exploratory
activity’’ (24). Our data with respect to activity levels in the
animals are consistent with those findings. Others have sug-
gested that the TTR effect on the behavioral phenotype is
mediated through its role in thyroid or retinoic acid function, an
interpretation not consistent with earlier physiologic studies
(25–27). The SPR interaction studies also argue that the effect
polyclonal antibody identifying mTTR and hTTR or antibody to A? (4G8), and imaged with the confocal microscope (31). (A and B) No amyloid plaques were
detected in nontransgenic mice (A) or mTTR?/?mice (B). (C) Five-month-old APP23 mice with an intact TTR gene show no plaques. (D) In APP23, mTTR?/?mice
of the same age plaques are readily detectable. (E and F) Fifteen-month-old APP23 animals (mTTR?/?) show large and abundant amyloid plaques (E), which are
reduced in APP23 mice expressing hTTR (F). (G–O) Brain sections of APP23 transgenic mice double labeled with antibodies against A? (green) and TTR (red) were
in APP23 (mTTR?/?) transgenics (J–L), which is increased in APP23, hTTR transgenics (M–O). (P and Q) There is mild TTR immunolabeling in neuronal cytoplasm
TTR localization in neuronal cell bodies. (S) In the AD brains TTR immunoreactivity was predominantly in vascular amyloid but also seen in neuronal cell bodies.
A? deposition in AD, APP23, and TTR mouse models. Vibratome sections of frontal cortex were treated with formic acid, immunostained with a
chip surface and exposed to either purified monomer A?1–40or A?1–42or
aggregates formed from the same peptides over 7 days before analysis (31).
The flow rate was 5 ml/min at 37°C. Ligands were Tetramer TTR Surface
(human 4658RU; mouse 5122RU). Analytes were 20 ml of 20 mM A?1–42.In
reverse experiments the A? preparations were applied to the chip and either
mTTR or hTTR was allowed to interact in the flow cell. ■, A? fibrils on mTTR;
Œ, A? fibrils on hTTR; ?, A? monomer on mTTR; ‚, A? monomer on hTTR.
Purified recombinant hTTR or mTTR was applied to the Biacore M5
www.pnas.org?cgi?doi?10.1073?pnas.0712197105Buxbaum et al.
is direct; however, it is possible that the impact of TTR on
behavior is bimodal.
The morphologic studies show differences in TTR localization
between human AD and transgenic mouse model AD brains. In
the hTTR/APP23 mice TTR is seen in proximity to the plaques,
whereas in the human brains there is little TTR immunoreac-
tivity in the plaques. The most prominent TTR staining in the
human brains was in vessels showing Congophilic angiopathy
(Fig. 2S). These observations may reflect differences in patho-
genesis in the two species. Alternatively, in the double transgenic
mice TTR and A? are overproduced and available to interact
throughout life, which may be critical in reducing plaque density.
Perhaps in the human brain plaque formation only occurs when
there is insufficient TTR available to inhibit aggregation, i.e.,
late in life, and the presence of plaques is a marker for
insufficient protective TTR. This would not be the case in blood
vessels where the codeposited TTR could originate in the serum.
It is likely that the transgenic overproduction of the hTTR is
sufficient to overcome the relatively weak binding seen in vitro.
Perhaps endogenously produced mTTR is sufficient to inhibit
A? aggregation when the fragment is generated from endoge-
nous murine or a low copy number human A? transgene but
cannot be produced in sufficient quantities to overcome its
production by A? transgenes integrated in high copy numbers
and transcribed and translated to produce large quantities of
In studies of the regulation of TTR synthesis evidence sug-
gesting transcription in the cerebral cortex was dismissed as
artifactual (28). It is possible that the observation of cortical
expression was not an artifact and that regulation in the cortex
is similar to that in the liver and may actually decline in the face
of cytokine expression. Local cytokine production has been
shown in AD brain (29). If TTR regulation in the brain proper,
to that in the liver, the local inflammatory response to A?
aggregates could suppress TTR transcription. Hence with aging
TTR production may decrease, A?1–40and A?1–42production
may increase, or both. The observed increase in TTR transcrip-
tion in murine AD model brains argues in favor of exhaustion or
inadequate production rather than suppression.
These data provide genetic, immunohistochemical, and bio-
chemical evidence for an interaction between TTR and A?
playing a role in resistance to the development of the neuro-
pathologic and behavioral manifestations in a murine model of
AD and suggest that a similar relationship may exist in the
human brain. It appears that the interaction is physical and that
TTR may behave in a chaperone-like manner for molecular
species of A? larger than monomers. The observations support
the novel notion that increasing cerebral TTR synthesis is a
potential therapeutic/prophylactic approach to human AD.
Materials and Methods
Behavioral. Mice were group-housed in a temperature-controlled room in
which the lights were on a 12-h light/dark cycle (lights off at 6 a.m.). All
behavioral testing was performed in The Scripps Research Institute mouse
behavior assessment core facility during the dark (active) phase. Food and
with the guidelines established by the Department of Agriculture and the
National Institutes of Health in the Guide for the Care and Use of Laboratory
The Scripps Research Institute.
elevated 58 cm above the floor by a tripod. Twenty holes, 5 cm in diameter,
were located 5 cm from the perimeter, and a black Plexiglas escape box (19 ?
day of testing, a training session was performed, which consisted of placing
the mouse in the escape box and leaving it there for 1 min. One minute later,
the first session was started. At the beginning of each session, the mouse was
placed in the middle of the maze in a 10-cm-high cylindrical black start
chamber. After 10 s the start chamber was removed, a buzzer (80 dB) and a
The session ended when the mouse entered the escape tunnel or after 5 min
elapsed. When the mouse entered the escape tunnel, the buzzer was turned
off and the mouse was allowed to remain in the dark for 1 min. When the
the spatial environment), which was randomly determined for each mouse.
The platform and escape box were cleaned with 70% ethanol between mice.
Additional behaviors, including activity levels, anxiety states, and visual
acuity, that may influence findings in Barnes maze testing, were also exam-
ined. Locomotor activity was measured in polycarbonate cages (42 ? 22 ? 20
cm) placed into frames (25.5 ? 47 cm) mounted with two levels of photocell
beams at 2 and 7 cm above the bottom of the cage (San Diego Instruments).
These two sets of beams allowed for the recording of both horizontal (loco-
motion) and vertical (rearing) behavior. A thin layer of bedding material was
applied to the bottom of the cage. Mice were tested for 30 min.
The light/dark transfer procedure has been used to assess anxiety-like
is a rectangular box made of Plexiglas divided by a partition into two envi-
ronments. One compartment (14.5 ? 27 ? 26.5 cm) is dark (8–16 lux), and the
other compartment (28.5 ? 27 ? 26.5 cm) is highly illuminated (400–600 lux)
by a 60-W light source located above it. The compartments are connected by
an opening (7.5 ? 7.5 cm) located at floor level in the center of the partition.
The time spent in the light compartment was used as a predictor of anxiety-
like behavior, i.e., a greater amount of time in the light compartment was
indicative of decreased anxiety-like behavior. The mice were placed in the
a camera mounted above the apparatus. The test duration was 5 min.
The visual cliff test provides a measure of visual acuity. It evaluates the
ability of the animal to see a drop-off at the edge of a horizontal surface. In
onto the ‘‘safe’’ side (the horizontal checkered surface) in most trials. A blind
animal will just as often step down onto the ‘‘negative’’ side (the vertical
appearing surface), i.e., making 50% correct and 50% incorrect choices. Each
mouse was placed onto the center ridge, and the side onto which the animal
stepped down was recorded. Six consecutive trials were used for each mouse,
and the percentage of correct choices was calculated for the individual
isofluthane, the skull was opened, and the brain was removed intact. The
hemispheres were separated with one half being placed in 4% paraformal-
dehyde, and the other snap-frozen and stored at ?80°C for biochemical
Immunohistochemistry. All brains were examined for A? and TTR with antibod-
4°C with the mouse mAb 4G8 (1:600; Senetek), which specifically recognizes
A? or the DAKO anti-TTR antibody, which recognizes both mTTR and hTTR.
Two methods were used to detect primary antibody binding. We used either
a Vector ABC Elite kit and DAB/H2O2 or FITC-conjugated anti-mouse IgG
?2.5 objective for the Olympic Vanox light microscope. The percentage area
of the hippocampus or cortex covered by 4G8-immunoreactive material was
assessed with a Quantimet 570C microscope (Leica). Digitized images were
analyzed with the NIH Image 1.43 program to determine the number of
analyzed per region per mouse, and the average of individual measurements
was used to calculate group means.
Brain extraction to determine A? content. Hemi-brains were dounce-
homogenized in the cold in 2% SDS in water containing 0.7 mg/ml Pepstatin
added to part of each hemi-brain to fully solubilize fibrils that were present.
g for 1 h. The supernates were analyzed by a capture ELISA with antibodies
specific for A?1–40and A?1–42(10).
Biacore experiments to analyze the interaction between TTR and A?1–40and A?1–42.
A?1–40and A?1–42were obtained from Quality Controlled Biochemicals. The
lyophilized powder was dissolved in aqueous NaOH to yield a final concen-
aqueous NaOH (100 mM). The solution was sonicated (20 min, 25°C), then
filtered sequentially through 0.2 ?m and 10-kDa cutoff filters (14,000 ? g, 30
Buxbaum et al.
February 19, 2008 ?
vol. 105 ?
no. 7 ?
min) (31). The concentration of protein was determined by UV absorption at
with aqueous NaOH (pH 10.5) and used immediately. We noted that approx-
imately half of the A? sample was retained on the 10-kDa filter. After
pretreatment, the final peptide solution was seed-free by atomic force mi-
croscopy analysis. A? fibrils were formed by incubating the monomer in PBS
buffer for 7 days at 37°C with constant shaking.
hTTR and mTTR were overexpressed in Escherichia coli and purified as
described (17, 30).
ACKNOWLEDGMENTS. This work was supported by National Institute on
Aging Grants R01 AG15916 (to J.N.B.) and R01 AG18440 (to E.M.), the W. M.
Keck Foundation (J.N.B.), The Skaggs Foundation (Z.Y.), the Fidelity Founda-
tion (J.N.B.), and The Stein Fund.
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