Formulation of a Medical Food Cocktail for Alzheimer’s
Disease: Beneficial Effects on Cognition and
Neuropathology in a Mouse Model of the Disease
Anna Parachikova1, Kim N. Green1, Curt Hendrix2, Frank M. LaFerla1*
1Department of Neurobiology and Behavior, Institute for Memory Impairments and Neurological Disorders, University of California Irvine, Irvine, California, United States
of America, 2Akeso Health Sciences L.L.C., Westlake Village, California, United States of America
Background: Dietary supplements have been extensively studied for their beneficial effects on cognition and AD
neuropathology. The current study examines the effect of a medical food cocktail consisting of the dietary supplements
curcumin, piperine, epigallocatechin gallate, a-lipoic acid, N-acetylcysteine, B vitaminsvitamin C, and folate on cognitive
functioning and the AD hallmark features and amyloid-beta (Ab) in the Tg2576 mouse model of the disease.
Principal Findings: The study found that administering the medical food cocktail for 6 months improved cortical- and
hippocampal- dependent learning in the transgenic mice, rendering their performance indistinguishable from non-
transgenic controls. Coinciding with this improvement in learning and memory, we found that treatment resulted in
decreased soluble Ab, including Ab oligomers, previously found to be linked to cognitive functioning.
Conclusion: In conclusion, the current study demonstrates that combination diet consisting of natural dietary supplements
improves cognitive functioning while decreasing AD neuropathology and may thus represent a safe, natural treatment for
Citation: Parachikova A, Green KN, Hendrix C, LaFerla FM (2010) Formulation of a Medical Food Cocktail for Alzheimer’s Disease: Beneficial Effects on Cognition
and Neuropathology in a Mouse Model of the Disease. PLoS ONE 5(11): e14015. doi:10.1371/journal.pone.0014015
Editor: Colin Combs, University of North Dakota, United States of America
Received February 19, 2010; Accepted September 30, 2010; Published November 17, 2010
Copyright: ? 2010 Parachikova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by grants from the National Institutes of Health (AG-021982 to FML, and Small Business Innovation Research grant
R43AT003025-1 to CH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: CH is an employee of Akeso, and as such stands to gain from the publication of these results. However, he had no role in the analyses or
collecting of data and was therefore unable to influence the outcome. There are patents and products in development based on this research (see end). This does
not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. U.S Patent Application No. 12/325,842, filed 12/1/2008 and published
as U.S. Patent Application Publication No. 2009-0143433A1, published 6/4/2009 entitled ‘‘COCKTAIL FOR MODULATION OF ALZHEIMER’S DISEASE.’’
* E-mail: email@example.com
Alzheimer’s disease (AD) is a disease of the elderly marked by
characterized by the accumulation of beta-amyloid (Ab) protein to
form plaques and tau phosphorylation resulting in tangle formation.
AD is primarily an idiopathic disease with the exception of some
rare (,5%) early onset autosomal-dominant familial cases .
Dementia is best correlated to synaptic and neuronal loss, rather
than directly to pathological burden, and so much interest has been
focused on understanding the pathways that lead firstly to the
formation of pathology, and then from pathology to synaptic
damage, loss and then neuronal death. Strong genetic evidence
suggests that it is the aberrant accumulation of Ab which lies
upstream, and that it is this accumulation which leads to
downstream pathologies such as tangle formation , extensive
inflammation , oxidative damage to lipids, proteins and DNA
, and glycation of proteins . Recent studies have highlighted
the importance of soluble oligomeric species of Ab, in particular the
56 kD molecule Ab*56, in learning and memory .
The cause of sporadic AD remains poorly understood but a
number of risk factors have been identified, and include many
lifestyle and dietary choices, in addition to genetic susceptibility
genes, such as the presence of the apoE4 allele . With the
identification of these risk factors many studies have focused on
understanding how disease progression is impacted using transgenic
mouse models of the disease. For example, we have recently shown,
using the triple transgenic (3xTg-AD) mouse model of the disease,
that stress hormones , or nicotine  intake increase the severity
of AD pathologies, whereas increased dietary intake of omega-3
fatty acids , nicotinamide , or increased cognitive
stimulation , can protect against formation of AD pathologies.
Dietary intake is particularly important, to general well being, as
well as to progression into AD dementia, as highlighted by us and
many other groups. Given the complexity and widespread
distribution of the pathology, multi-faceted approaches will be
required to effectively treat or prevent AD. To that end, we
formulated a medical food cocktail comprising agents that are
known to reduce Ab production, but that also show potent anti-
oxidant and anti-inflammatory properties, or reduce glycation of
proteins. All of these processes have been implicated in AD, and
likely contribute to the synaptic and neuronal loss seen in the
food cocktail ingredients have beneficial effects on APP processing,
PLoS ONE | www.plosone.org1November 2010 | Volume 5 | Issue 11 | e14015
have anti-oxidant, anti-inflammatory or anti-glycation properties.
One of the primary components is curcurmin, a polyphenol that
comprises the active ingredient of the spice turmeric. Curcurmin is
known for its strong anti-oxidant and anti-inflammatory properties,
long history of safe use, and low side-effect profile [13,14]. It has
previously been shown to decreased levels of oxidized proteins and
interleukin-1 beta in an AD transgenic mice, in addition to a 43–
50% decrease in insoluble Ab, soluble Ab and amyloid plaque
burden . Alongside these results, curcurmin has been shown to
inhibit both the formation and growth of Ab fibrils from Ab in a
dose-dependent manner . To increase the bioavailability of
curcumin, and also EGCG, we also included piperine, which is a
component of the spice black pepper [16,17]. Piperine also exhibits
significant antioxidant activity of its own, as well as significant
chemopreventative and immunomodulary effects [18,19,20].
EGCG is a polyphenol that is an active ingredient of green tea. It
exhibits potent antioxidant and anti-inflammatoryproperties as well
as confers neuroprotection in AD mouse models .
a-Lipoic Acid is a naturally occurring disulfide molecule with
antioxidant and anti-inflammatory properties. In a small study of
elderly patients with dementia, dietary supplementation with a-
lipoic acid stabilized cognitive function, as evidenced by no change
in score on 2 neuropsychological tests over more than a 10-month
study period . N-Acetylcysteine is an antioxidant, which has
been given to AD patients who then demonstrated greater
cognitive function than the placebo group after both 3 and 6
months of treatment . Together these ingredients, combined
with B-vitamin’s (B1, B6, B12 and Folate) and the antioxidants
Vitamin C and E provide protection against oxidative damage,
which is known to occur in the AD brain.
The medical food cocktail contains the standardized herbal extracts,
hypothesized that these ingredients would work synergistically to
order to formulate a safe, natural treatment for AD.
To investigate the effects of our medical food cocktail treatment
on cognition and AD pathology, we treated 6-month-old Tg2576
and non-transgenic (NonTg) mice with either 1) low dose diet
(184 mg/kg; n=10), 2) high dose diet (553 mg/kg; n=10) or 3)
control diet (n=10) for a period of 6 months. The well-
characterized Tg2576 mouse model of AD exhibits age related
accumulation of Ab plaque pathology starting at 12 months as well
as behavioral deficits evident as early as 3 months of age .
Following 6 months of treatment, animals were tested on behavioral
tasks to examine primarily hippocampus and cortex dependent
memory. Treatment continued throughout the behavioral tasks.
Medical food cocktail treatment restores memory deficits
in the Tg2576 mouse model of AD
Morris water maze is a spatial task found to be primarily
dependent on the hippocampus . The MWM protocol used
was adapted from . Tg2576 and NonTg mice treated with 1)
low dose medical food cocktail, 2) high dose medical food cocktail
or 3) control diet were tested. Mice were first trained on the MWM
to learn the location of a submerged platform for 4 trials per day
until criterion was reached (escape latency ,25 s). At the start of
the test, all groups performed identically as assessed by their
performance on trials 1 and 2 on day 1 depicted in Figure 1B. The
average latency to the target platform for all groups during the 7
days of training is represented in Figure 1A. As previously
demonstrated NonTg on the control diet performed better on the
acquisition phase of the test as compared to age-matched Tg2576
mice. NonTg mice on the control diet demonstrated learning of
the behavioral task and performed significantly better on the last as
compared to the first day of training. Tg2576 mice on the control
diet also exhibited learning during training but were unable to
reach criterion after 7 days of training. Notably, Tg2576 mice
treated with either low or high doses of the medical food cocktail
diets were undistinguishable from NonTg mice, showing that their
spatial learning abilities were restored to NonTg levels. Treated
Tg2576 mice exhibited improvement in escape latency as
compared to Tg2576 on the control diet. 1.5- and 24-hours
following the 7 days of training, animals were exposed to probe
trial testing sessions, to reflect short-term and long-term memory
respectively. During testing, the hidden platform is removed and a
number of parameters are measured that are known to reflect
aspects of primarily hippocampal-dependent memory. Examining
Table 1. Components of high and low nutrient combination diets added to AIN-17 rodent chow.
Diet (mg/kg chow)
mouse – High
(mg/kg body wt/day)
Diet (mg/kg chow)
mouse – Low (mg/kg
R-a-Lipoic Acid9.15 50.6912.6716.904.23
Vitamin B1 (benfotiamine)3.0416.834.215.611.40
Vitamin C0.301.670.420.56 0.14
Combination Diet for AD
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the latency to the target location at 1.5- and 24-hours following
training, we find again that transgenic mice on the control diet
perform significantly worse than NonTg mice, and that treatment
with the medical food cocktail (either low or high dose)
significantly improves memory at both time points in the
transgenic mice (Figure 1C). Analysis of the number of platform
crosses revealed that Tg2576 mice on the control diet cross the
region where the platform was located significantly less than
NonTg mice. In contrast, medical food cocktail diet treatments in
the Tg2576 mice results in increased number of platform crosses
(both high or low doses of the medical food cocktail), compared to
untreated Tg2576 mice. In addition we also measured the amount
of time animals spent in the target quadrant as compared to the
opposite quadrant of where the platform used to be located
(Figure 1E, F). Notably, these results show that the medical food
cocktail treatment restores both acquisition and memory deficits in
Tg2576 mice back to non-transgenic levels.
Novel object recognition is based on animals’ inherent preference
to explore a novel object more than a familiar object. Animals are
exposed to two identical objects and 1.5 and 24 hours later they are
presented with one familial and one novel object. The amount of
time animals spend exploring the novel object is noted and the
recognition index is generated. NonTg mice spent more time
exploring the novel object as indicated by a recognition index of
.0.5 (Figure 2). In contrast, Tg2576 mice on the control diet spent
approximately the same amount of time exploringthe novel and the
familiar object, suggestive of memory deficits. On both the 1.5- and
24-hour retention tests, we found that medical food cocktail treated
Tg2576 mice performed significantly better than Tg2576 mice on
performing at a level similar to, or even exceeding, non transgenic
mice which do not develop AD pathology. Notably, NonTg mice
treated with the medical food cocktail also performed better than
NonTg mice on the control diet at the 24 hour retention test,
suggesting that this medical food cocktail could improve certain
types of memory in non pathological animals.
These behavior data suggest that 6-month treatment of Tg2576
mice with a low or high dose of medical food cocktail diet
improved primarily cortex and hippocampus dependent memory.
Medical food cocktail treatment reduces Ab levels and
Brain homogenates from both the low and high doses of medical
food cocktail treated and control treated Tg2576 mice were
assessed for Ab levels by sandwich ELISA. We found a significant
decrease in both Ab40and the more amyloidogenic Ab42species in
the detergent soluble fraction with both low and high diet
(Figure 3A). Analysis of the detergent-insoluble fraction revealed a
statistically significant reduction in Ab40levels with medical food
cocktail dietary treatment in the Tg2576 transgenic mice
(Figure 3B). Insoluble Ab42levels were unchanged with treatment.
Next we examined APP processing in treated and untreated
Tg2576 mice. APP can be cleaved either via a non-amyloidogenic
or an amyloidogenic pathway. The non-amyloidogenic pathway
cleaves APP with a-secretase yielding sAPPa and the C83
fragment, which can then be further cleaved via c-secretase. In
contrast, the amyloidogenic pathway results in Ab production via
the sequential cleavage with b- followed by c-secretase. b-secretase
(BACE) cleavage of APP results in the generation of sAPPb and
C99, which is then further cleaved by c-secretase to yield Ab.
Examining the levels of the APP holoprotein and the cleaved
fragments C83 and C99, we found that combination diet
treatment resulted in a significant decreased in both C83 and
C99 levels (Figure 3C, D).
In addition to examining Ab levels and APP processing as a
result of treatment with combination diet in Tg2576 mice, we
further studied the aggregation state of Ab, using conformation-
specific antibodies. We found a significant decrease in the levels of
soluble oligomers (,18–250 kDa species ) using A11 antibody,
but no difference in soluble fibrils as assessed via OC with low
concentration diet (Figure 3E, F). These findings are in line with
reduced Ab in the detergent soluble fraction (Figure 3A). These
data suggest that combination diet treatment results in improved
cognitive functioning coinciding with reduced levels of soluble Ab
species, including oligomeric species.
The current study provides evidence that a combination diet of
dietary supplements, individually known to be beneficial, can not
only improve cognitive functioning in a transgenic mouse models
of AD but also decreases Ab levels and oligomerization. As yet
there is an unmet need for effective treatments and preventative
strategies for AD, and the fastest route to human patients involves
the use of either existing medications, or the formulation of known
safe remedies. Given that human AD is far more complex than we
can effectively model in mice, which develop AD related pathology
and cognitive decline but lack extensive neuronal loss, we must
formulate treatments that attack not just the symptoms seen in
these mice, but also those which we predict will show benefits
downstream of pathology that occur in humans. Our formulation
here has been designed to alter APP processing through reductions
in both Ab production, as well as aggregation, but also to prevent
downstream pathologies such as excessive oxidative damage and
inflammation. It is our hope that such a strategy will slow disease
progression in humans.
Our rationale is that the individual components of the medical
food cocktail work synergistically to produce cognitive and
pathological benefits, and together have larger effects than any
single component alone. In order to take the step from formulation
to human administration we have tested the medical food cocktail
in a well-described transgenic mouse model of AD. Serving as a
proof of principal we saw cognitive recovery, as well as reduction
of Ab. Our results here show that combination approaches to the
treatment of AD are effective in mouse models of AD, and have
high translation potential for the human disorder.
Materials and Methods
The study used 6-month old Tg (HuApp695.K670-M671L)
2576 transgenic and age-matched C57Bl6/SJL non-transgenic
control mice. Mice were obtained from Jackson Labs and a colony
was established. Tg2576 transgenic mice over-express human APP
with the double Swedish mutation . Tg2756 and control mice
were treated for a period of 6 months with either 1) low dose diet
(184 mg/kg; n=10), 2) high dose diet (553 mg/kg; n=10) or 3)
control diet (n=10). The contents of the medical food cocktail
were supplemented to the standard AIN-17 rodent chow.
After treatment, the animals were sacrificed and the brains
removed. The brains were immediately dissected in half along the
coronal line and one-half frozen for biochemical analysis and the
other half fixed in 4% paraformaldehyde.
Animal Treatments and Ethics
All rodent experiments were performed in accordance with
animal protocols approved by the Institutional Animal Care and
Use Committee at the University of California, Irvine (UCI).
Combination Diet for AD
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Figure 1. Prevention of hippocampal spatial memory deficits with medical food cocktail diet in Tg2576 mice. Medical food cocktail
was given to Tg2576 and non-transgenic control (nonTg) mice at 6 months of age, at a low and high (3x higher) dose. After 5 months of treatment,
mice were tested for cognitive functioning on hippocampal and cortical dependent tasks. Mice were trained and tested on the spatial memory
version of the Morris water maze (MWM; n=10 per group). A) Acquisition curves shown for the 7 days of training on the MWM. Non-transgenic mice
perform better as compared to Tg2576 starting day 2 of training (Genotype main effect day 2 F(1,45) 3.586, p=0.0599, day 3 F(1, 45) 4.611 p=0.0332,
day 4 F(1, 45) 4.832, p=0.0519, day 5 F(1, 45) 13.812, p=0.003, day 6 F(1, 45) 4.021, p=0.0465 and day 7 F(1, 45) 15.821, p=0.0001). NonTg mice on
the control diet perform significantly better on day 7 as compared to day 1 of training (p,0.0001). Tg2576 mice on the control diet also exhibited
learning during the acquisition phase of the test (day 1 compared to day 7 p,0.05) but were unable to reach the 25 sec criterion after 7 days of
training. Medical food cocktail improved the spatial learning of Tg2576 during training (low diet day 5 p,0.05, day 6 p,0.01, day 7 p=0.1034; high
diet day 5 p,0.01, day 6 p,0.001, day 7 p,0.01) with mice reaching criterion by day 7 of training. B) All mice started at the same level as shown by
the average of trials 1 and 2 on the 1stday of training (Genotype main effect F(1, 104) 0.094, p=0.9107, treatment main effect F(2, 104) 0.233,
p=0.7928). C–F) Mice were given a memory probe with the platform removed at 1.5-h or 24-h following the last training trial. C) Tg2576 mice took
significantly longer to reach the platform location as compared to non-transgenic mice on the control diet (p,0.05 for both 1.5 and 24-hour probe
trials). Tg2576 mice treated with medical food cocktail exhibited significantly decreased latencies to cross the platform location compared to vehicle-
treated mice (p,0.05 for both 1.5 and 24-hour probe trials for both low and high diet treatments). D) The deficits in memory were also evident in the
Combination Diet for AD
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Morris water maze.
test for spatial memory (i.e. hippocampus dependent) and cued
learning (i.e. non-hippocampal) in rodents. Many studies in the last
two decades have used this test as a reliable measure of
transgenic models [28,30]. All animals were handled briefly on
the 4 days prior to each block of training, and all animals were
tested on motor (gait and stepping) tasks and given a general health
assessment evaluating coat, eye and nose condition, and hind leg
clasping. Hidden and cued platform Morris water maze (MWM)
training and testing were conducted as described previously .
Mice were trained to swim to a 14-cm diameter circular clear
Plexiglass platform submerged 1.5 cm beneath the surface of the
water. The platform location was selected randomly for each
mouse, but was kept constant for each individual mouse
throughout training at each age. On each trial, the mouse was
placed into the tank at one of four designated start locations and
allowed to find and escape onto the platform. If a mouse failed to
find the platform within 60 s, it was manually guided to the
platform and allowed to remain there for 5 s. After this, each
mouse was placed into a holding cage under a warming lamp for
25 s until the start of the next trial.
Retention of the spatial training was assessed 1.5 hr and again
24 hr after the last training trial. Both probe trials consisted of a
60 s free swim in the pool without the platform. Mice were
monitored by a camera and all trials were scored during the probe
trial and again after the probe trial for verification. There were no
The Morris Water Maze (MWM) is a
significant differences between any genotypes in the swim speeds,
degree of thigmotaxis or floating. The parameters measured
during the probe trial included (1) initial latency to cross the
platform location; (2) number of platform location crosses; and (3)
time spent in the quadrant opposite to the target quadrant.
Novel object recognition.
spontaneous tendency of rodents to explore a novel object more
often than a familiar object . Perirhinal cortex lesions and
studies of neuronal activation and responses in rats suggest that it is
cortical and not hippocampal neurons that are involved in the
object recognition task [32,33]. Hippocampal involvement in this
task has also been suggested [34,35,36] and this task is widely used
to study memory impairments in AD models [37,38]. For the
novel object task, mice were familiarized with an empty open field
for a period of 10 minutes. On the following day, mice were
subjected to a 5 minute exploration session in the same context
with two identical objects (Object A; e.g. two identical balls or two
identical dice) placed in symmetrical locations in the open field. 90
minutes and 24 hours later, animals were subjected to a 3 minute
retention phase test where they were exposed to one Object A and
also to a novel object, Object B (for the 90 min time point) and
Object C (for the 24 h time point) placed in the same, symmetrical
locations in the open field.
The time spent exploring the familiar object and the novel
object were calculated where exploration equals touching the
object with nose or paws, or sniffing within 1.5 cm of the object.
Time spent with the novel object as compared to time spent with
both objects was used as memory index.
This task is based on the
Figure 2. Prevention of cortical memory deficits with medical food cocktail diet in Tg2576 mice. We evaluated treated and untreated
Tg2576 and nonTg mice in performance of the primarily perirhinal cortex-dependent contextual task, novel object recognition. Tg2576 mice treated
with medical food cocktail had significantly improved recognition indexes at both the 1.5- and 24-h probe trials, compared to vehicle Tg2576 mice
(1.5 hour probe trial (low diet p,0.05, high diet p,0.01; 24 hour probe trial (p,0.01 for both low and high combination diets). Notably, nonTg mice
treated with medical food cocktail showed improved recognition indexes at the 24-h probe compared to vehicle nonTg mice (p,0.05 for both low
and high combination diets). Error bars indicate SEM. * indicates significance for control Tg2576 mice vs. medical food cocktail treated Tg2576 mice,
** indicates significance for control nonTg mice vs. medical food cocktail nonTg mice.
number of crosses of Tg2576 as compared to the control diet treated non-transgenic mice (p,0. 05 at both 1.5 and 24 hours). Tg2576 mice treated
with medical food cocktail made significantly more platform crosses at both short- and long-term probes than vehicle-treated mice (low diet (p,0.05
at both 1.5 and 24 hours; high diet p,0.01 at 1.5 and p,0.05 at 24 hours). E) Control diet treated Tg2576 mice also spent less time in the target
quadrant (p,0.05 for both 1.5 and 24-hour probe trials). Tg2576 mice treated with medical food cocktail spent significantly more time in the target
quadrant than vehicle-treated transgenic mice (low diet (p,0.05 at 1.5 hours; high diet p,0.05 at both 1.5 and 24 hours). F) In support of the target
quadrant data, time spent in the opposite quadrant was significantly more for Tg2576 as compared to non-transgenic mice on the control diet
(p,0.01 for both 1.5 and 24-hour probe trials). Tg2576 mice treated with medical food cocktail spent significantly less time in the opposite quadrant
than vehicle-treated mice (low diet (p,0.01 at 1.5 and p,0.05 at 24 hours; high diet p,0.01 at both 1.5 and 24 hours). Error bars indicate SEM.
* indicates significance for control Tg2576 mice vs. medical food cocktail treated Tg2576 mice.
Combination Diet for AD
PLoS ONE | www.plosone.org5November 2010 | Volume 5 | Issue 11 | e14015
Protein extracts were prepared from whole brain samples by
homogenizing in T-per (Pierce Biotechnology, Rockford, Il, USA)
extraction buffer and Complete Mini Protease Inhibitor Tablets
(Roche, Indianapolis, IN, USA) followed by high-speed centrifu-
gation at 100,000 g for 1 h. The supernatant was taken as the
protein extract. Protein concentrations were determined by the
Bradford method. Equal amounts of protein (20 mg–50 mg
Figure 3. Medical food cocktail diet reduces Ab levels and decreases aggregation. Soluble (A) and insoluble (B) Ab40and Ab42levels were
measured from Tg2576 whole brain homogenates from animals treated for 6 months with medical food cocktail or vehicle. A) Soluble Ab levels were
significantly reduced with medical food cocktail diet in Tg2576 mice. B) Insoluble Ab40 levels were also significantly reduced with medical food
cocktail diet in Tg2576 mice. C) Western blot analyses of protein extracts from whole-brain homogenates of Tg2576 mice treated for 6 months with
either high dose medical food cocktail or vehicle shown as alternating lanes. Steady state levels of APP were unaffected by treatment, but APP CTF’s
C83 and C99 were reduced by medical food cocktail treatment. D) Quantification of (C) normalized to b-actin levels as a loading control. E) Dot blot
analyses of brain homogenates from Tg2576 mice treated for 6 months with either high dose medical food cocktail or vehicle for Ab oligomers or Ab
fibrils, using conformation specific antibodies, or western blot analyses for Ab*56 using 6E10, shown as alternating lanes. Reductions in both Ab
oligomers and Ab*56 were seen with treatment. F) Quantification of (E). Error bars indicate SEM. * indicates significance (p,0.05) for control Tg2576
mice vs. high dose medical food cocktail treated Tg2576 mice. To assess levels of low molecular weight oligomeric and soluble fibril Ab species we
used the conformation specific antibodies A11 and OC respectively. Dot blot analysis showed a 20% reduction of soluble oligomers in the brains of
Tg2576 animals treated with the high combination diet (Figure 2E, F), but no differences in soluble fibrils.
Combination Diet for AD
PLoS ONE | www.plosone.org6November 2010 | Volume 5 | Issue 11 | e14015
depending on protein of interest) were separated by SDS/PAGE
on a 10% Bis/Tris gel (Invitrogen, Carlsbad, CA, USA),
transferred to 0.45 mM nitrocellulose membranes, blocked for
1 hour in 5% (vol/vol) nonfat milk in Tris-buffered saline (pH 7.5)
supplemented with 0.2% Tween20, and processed as described.
Antibodies and dilutions used in this study include 6E10 (1:1000
Signet, Dedham, MA, USA), CT20 (1:5,000; Calbiochem, San
Diego, CA, USA) for C99 and C83, and aActin (1:10,000; Sigma-
Aldrich, USA). Quantitative densiometric analyses were per-
formed on digitised images of immunoblots with Scion Image 4.0
(Scion Corporation, Frederick, MD, USA).
Ten mg of protein was made up to 10 ml in H20 and pipetted
onto 0.45 mM nitrocellulose membrane (Pierce Biotechnology)
and allowed to dry. The membrane was blocked for 45 minutes in
5% powder milk in TBS-T and then incubated in A11, or OC
(generous gifts from Charlie Glabe, UCI) at 1:1000 overnight at
4uC. The membrane was then washed 5 times in TBS-T and
incubated for 1 hour in HRP goat-anti Rabbit antibody (1:10000,
Sigma-Aldrich). Following an additional 5 washes the membrane
was coated with ECL plus (Amersham) and then developed on
photographic film. Quantitative densiometric analyses were
performed on digitized images of immunoblots using Scion Image
4.0 software (Scion Corporation).
Ab1–40and Ab1–42were measured using a sensitive sandwich
ELISA system. Soluble and insoluble Ab was isolated from whole
brain homogenates using T-per Extraction Buffer (Pierce Biotech-
nology, Rockford, Il, USA) and 70% formic acid (FA) respectively.
Soluble fractions were loaded directly onto ELISA plates and FA
fractions were diluted 1:20 in neutralization buffer (1 M Tris base;
0.5 M NaH4PO4) prior to loading. Secreted Ab was measured
from in vitro assays by direct addition of the cell incubated media
onto the ELISA plates. MaxiSorp immunoplates (Nunc, Roches-
ter, NY, USA) were coated with mAB20.1 (William Van
Nostrand, Stony Brook University, NY) antibody at a concentra-
tion of 25 mg/ml in Coating Buffer (0.1 M NaCO3 buffer,
pH 9.6), and blocked with 3% BSA. Standards of both Ab40
and 42 were made in Antigen Capture Buffer (ACB; 20 mM
NaH2PO4; 2 mM EDTA, 0.4 M NaCl; 0.5 g CHAPS; 1% BSA,
pH 7.0), and loaded onto ELISA plates in duplicate. Samples were
then loaded in duplicate and incubated overnight at 4uC. Plates
were washed and then probed with either HRP-conjugated anti-
Ab 35-40 (C49, for Ab1–40(David Cribbs, University of California,
Irvine)) or anti-Ab 35-42 (D32, for Ab1–42 (David Cribbs,
University of California, Irvine)) overnight at 4uC. 3,39,5,59-
tetramethylbenzidine was used as the chromagen, and the reaction
stopped by 30% O-phosphoric acid, and read at 450 nm on a
Molecular Dynamics plate reader.
Behavioral scores were analyzed using a multifactor or repeated
measures ANOVA including genotype or treatment as indepen-
dent variables, and escape latencies during training and probe trial
measures as dependent variables. To dissect complex interactions
between factors, post-hoc Scheffe tests and Bonferroni corrections
were used to determine individual differences between groups.
Biochemical data was analyzed using planned Students T-tests.
For individual planned comparisons, results were reported as
significant only when P,0.05.
Conceived and designed the experiments: KNG FML. Performed the
experiments: AP. Analyzed the data: KNG. Contributed reagents/
materials/analysis tools: CH. Wrote the paper: AP KNG FML.
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