Transient pharmacologic lowering of Aβ production prior to deposition results in sustained reduction of amyloid plaque p...

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Title:
Transient pharmacologic lowering of Aβ production prior to deposition results in sustained reduction of amyloid plaque pathology
Physical Description:
Mixed Material
Language:
English
Creator:
Das, Pritam
Verbeeck, Christophe
Minter, Lisa
Chakrabarty, Paramita
Felsenstein, Kevin
Kukar, Thomas
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BioMed Central
Publication Date:

Notes

Abstract:
Background: Alzheimer’s disease (AD) is the leading cause of dementia among the elderly. Disease modifying therapies targeting Aβ that are in development have been proposed to be more effective if treatment was initiated prior to significant accumulation of Aβ in the brain, but optimal timing of treatment initiation has not been clearly established in the clinic. We compared the efficacy of transient pharmacologic reduction of brain Aβ with a γ-secretase inhibitor (GSI ) for 1–3 months (M) treatment windows in APP Tg2576 mice and subsequent aging of the mice to either 15M or 18M. Results: These data show that reducing Aβ production in a 2-3M windows both initiated and discontinued before detectable Aβ deposition has the most significant impact on Aβ loads up to 11M after treatment discontinuation. In contrast, initiation of treatment for 3M windows from 7-10M or 12-15M shows progressively decreasing efficacy. Conclusions: These data have major implications for clinical testing of therapeutics aimed at lowering Aβ production, indicating that; i) these therapies may have little efficacy unless tested as prophylactics or in the earliest preclinical stage of AD where there is no or minimal Aβ accumulation and ii) lowering Aβ production transiently during a critical pre-deposition window potentially provides long-lasting efficacy after discontinuation of the treatment. Keywords: Aβ, Alzheimer’s disease, APP, Therapeutics, γ-secretase inhibition
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Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository at the University of Florida community, with research, news, outreach, and educational materials.
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doi:10.1186/1750-1326-7-39 Cite this article as: Das et al.: Transient pharmacologic lowering of Aβ production prior to deposition results in sustained reduction of amyloid plaque pathology. Molecular Neurodegeneration 2012 7:39.
General Note:
Das et al. Molecular Neurodegeneration 2012, 7:39 http://www.molecularneurodegeneration.com/content/7/1/39; Pgs. 1-9

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Full Text


Das et al. Molecular Neurodegeneration 2012, 7:39
http://www.molecularneurodegeneration.com/content/7/1/39


MOLECULAR
NEURODEGENERATION


Transient pharmacologic lowering of A3

production prior to deposition results in

sustained reduction of amyloid plaque pathology

Pritam Das*, Christophe Verbeeck,, Lisa Minter Paramita Chakrabarty3, Kevin Felsenstein3, Thomas Kukar4,
Ghulam Maharvil, Abdul Fauqc, Barbara A Osborne2 and Todd E Golde3*


Abstract
Background: Alzheimer's disease (AD) is the leading cause of dementia among the elderly. Disease modifying
therapies targeting AI3 that are in development have been proposed to be more effective if treatment was initiated
prior to significant accumulation of A3 in the brain, but optimal timing of treatment initiation has not been clearly
established in the clinic. We compared the efficacy of transient pharmacologic reduction of brain AI3 with a
y secretase inhibitor (GSI ) for 1 3 months (M) treatment windows in APP Tg2576 mice and subsequent aging of
the mice to either 15M or 18M.
Results: These data show that reducing AI3 production in a 2-3M windows both initiated and discontinued before
detectable AI3 deposition has the most significant impact on AI3 loads up to 11M after treatment discontinuation.
In contrast, initiation of treatment for 3M windows from 7-10M or 12-15M shows progressively decreasing efficacy.
Conclusions: These data have major implications for clinical testing of therapeutics aimed at lowering AI3
production, indicating that; i) these therapies may have little efficacy unless tested as prophylactics or in the earliest
preclinical stage of AD where there is no or minimal AI3 accumulation and ii) lowering AI3 production transiently
during a critical pre-deposition window potentially provides long-lasting efficacy after discontinuation of the
treatment.
Keywords: AI3, Alzheimer's disease, APP, Therapeutics, y secretase inhibition


Background
Alzheimer's disease (AD) is the leading cause of demen-
tia among the elderly. Numerous studies in humans have
demonstrated the sequential development of various
pathological features that characterize AD and the rela-
tionship of these pathologies to diagnosis of dementia
[1]. These studies support the amyloid cascade hypoth-
esis which posits that accumulation of AP aggregates
triggers a series of downstream pathologies that results
in clinical AD [2]. AD has recently been proposed to
have three pre-clinical stages, characterized in sequence

* Correspondence das pritam@mayo edu; tgolde@ufl edu
Department of Neuroscience, Mayo Clinic College of Medicine, 4500 San
Pablo Rd S, Jacksonville, FL 32224, USA
Center for Translational Research in Neurodegenerative Disease, Department
of Neuroscence, McKnight Brain Institute, College of Medicine, University of
Florida, 1600 SW Archer Road, Gainesville, FL 32608, USA
Full list of author information is available at the end of the article


by the development of AP pathology (stage 1), the pres-
ence of plaque pathology, neurofibrillary pathology and
initial evidence for neurodegeneration (stage 2), and fi-
nally by the presence of stage 2, pathologies plus evi-
dence for subtle cognitive decline (stage 3). This
progression likely occurs over a 10-20 year timeline [3].
The amyloid cascade hypothesis predicts that attenuat-
ing or preventing AP aggregation and accumulation will
attenuate or prevent the subsequent development of AD.
Strong experimental support for the amyloid hypothesis
has supported the rationale for many therapeutic
approaches targeting Ap. Major investments have been
made to develop inhibitors or modulators of AP produc-
tion [4]. y-secretase inhibitors (GSIs), p-secretase inhibi-
tors (BSIs) and y-secretase modulators (GSMs) have
been or are currently being tested in human AD trials.
Both the GSM tarenflurbil (R-flurbiporfren), and the GSI
semagacestat, failed to show efficacy in phase III human


S 2012 Das et al., licensee BioMed Central Ltd. This is an Open Access article distributed under the term of the Creative
Biole iled Central Common Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.






Das et al Molecular Neurodegeneration 2012, 7:39
http://www.molecularneurodegeneration.com/content/7/1/39



trials [4,5]. Several other GSMs, GSIs, and BSIs are cur-
rently in early stage trials. Based on the clinical failures
of tarenflurbil and semagacestat as well as other anti-Ap
therapies not targeting A3 production but its aggrega-
tion or clearance, we and others have begun to question
whether targeting of AP in symptomatic AD may be a
futile endeavor [6].
Given the framework provided by the amyloid cascade
hypothesis, disappointing results from completed trials
of anti-Ap therapies in patients with AD, and several but
limited preclinical studies supporting the concept that
when targeting AP "earlier is better" [7-9], we explored
the relationship between long-term efficacy with respect
to reducing amyloid loads and timing and duration of
AP lowering. In designing these studies, we were struck
by the I,:: -. !.- between AP accumulation in the mouse
brain and in vitro studies of AP aggregation. In vitro, AP
aggregates in a nucleation dependent polymerization re-
action, which exhibits three phases: a lag phase in which
nucleation occurs but not fibril formation, an exponen-
tial growth phase in which fibril formation and growth
occur ii;.11. and a plateau phase where fibril growth
and formation slows [10]. Except for being more
extended in terms of time, studies in APP Tg2576 trans-
genic mice show that AP aggregation and accumulation
follows a similar course in the brain [11]. There is an ini-
tial lag phase where no AP aggregation and accumula-
tion occurs, followed by an exponential accumulation
phase, and finally when amyloid loads become AD-like,
the growth slows or plateaus (see Figure 1A). Given
these :. !. ili. I. we tested the efficacy of transient lower-
ing of AP production in 1-3M treatment "windows" cor-
responding to these various phases of 1. i,' ;p;.. using
the GSI LY-411,575 (LY) in APP Tg2576 mice. These
data show that GSI treatment during the ...* 1 i... I.. -
window (4-7M) before the early exponential phase of de-
position (7-10M) or late exponential phase (12-15M) has
by far the most efficacy in terms of reduction of plaque
loads at 15M of age.

Results and discussion
In vitro and in vivo dosing studies with Ly-411,575
Ly-411,575 is potent orally bioavailable y-secretase in-
hibitor [12]. We synthesized large quantities for these
studies and then .:- *F. i both in vitro and in vivo
studies to validate Ly-411,575 potency. The IC50 in cell
culture for LY-411,575 was between 1-3 nm (see Add-
itional file 1: Figure S1A). Six hours after a single IP
dose, .'' i6 levels are reduced by ~ ". in the brain of
non-depositing Tg2576 mice (Additional file 1: Figure
S1B). Oral administration (10 ..o. I .i : administered
as a single dose reduces brain AP40 levels 24 hours
after the final dose (7 days) by -65% (Additional file 1:
Figure SIC) and plasma AP40 by -95% (Additional file 1:


Page 2 of 9


Figure S1D). Plasma AP levels are reduced more quickly
than brain levels following oral dosing with maximal in-
hibition seen after 3 days ( 'i.iil.. ii file 1: Figure S1D).
For long term in vivo mouse studies, we tested adminis-
tration of Ly-411,575 using various dosing strategies
and time points (Table 1). Oral administration (formu-
lated in rodent chow) continually for 2 weeks with either
- 1 .. _. I : I, or 2.5 5 :... I: .: ," reduced brain AP40
levels by -50% and plasma AP40 by -80%, without overt
toxicities ( Table 1). However, continuous oral dosing
(>2 weeks) using the higher dose of 5 m. I -- -. .. or
10 :. I : .1 resulted in overt toxicities in mice
(Table 1). Therefore, in all long term studies presented
in this manuscript, we have used the 2.5 .." : i;- dose
formulated in rodent chow.

Transient AP3 reduction dramatically reduces the
subsequent accumulation of amyloid plaques
For these experiments, we transiently dosed Tg2576
mice with LY from 4-7M, 7-10M or 12-15M and aged
treated mice to 15M (see Figure 1B for experimental de-
sign). To confirm efficacy of LY in all groups, we mea-
sured plasma AP levels immediately after the last day of
treatment in sentinel mice and showed a similar reduc-
tion in plasma AP levels in all cohorts (Table 2). Follow-
ing sacrifice at 15M, levels of AP in the brain were
examined. The 4-7M LY treatment .'n,.t, ih. II: reduced
AP .:1._i:..-;;.,.. plaque burden in the frontal cortex and
hippocampus was decreased by -68% (Figure 1C) and
FA-solubilized brain AP levels were reduced by -60%
(Figure ID). The 7-10M LY treatment non-significantly
decreased AP plaque burden by -19% compared to con-
trols (Figure 1C), whereas FA-solubilized A3 levels were
reduced by -34% (Figure 1D). The 12-15M GSI treat-
ment group had no I. I- .. llt effect on either plaque
burden or FA-solubilized AP levels (Figure 1C, D). We
further evaluated effect of the 4-7M LY treatment on
cored amyloid plaques and cerebral amyloid angiopathy
(CAA). Compared to untreated control mice, there were
significant reductions both in cored plaques in the
frontal cortex and hippocampus (-47% reduction) and
CAA in the leptomeninges (-41% reduction, Figure 2A,
B). To further determine whether the magnitude of the
effect diminished with further aging, we performed an
additional experiment, where we aged 4-7M LY treated
mice to 18M, and again we observed significantly
reduced AP plaque burden and I ... hii. ..i AP levels
(Figure 2C), demonstrating that the suppression of de-
position was maintained even up 11 months after treat-
ment was halted.
Because AP deposition in mice follows an exponential
course, we sought to determine how the observed reduc-
tions in AP accumulation correlate with AP accumula-
tion to untreated mice. We compared AP levels in the








Das et al. Molecular Neurodegeneration 2012, 7:39
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4000


1 = Laglseeding phase
2 = Early Exponential phase 4
3 = Late Exponential phase
4= Plateau


0 2 4 6 8 10 12 14 16 18 20 22 24

Age in Months


D
2000.

a 1500.

- 1000.

co 500.


0 .


GSI dosing (in months)


Control


GSI dosing (in months)


GSI Rx 4-7m


*..
C. *'~**
14


GSI Rx 7-10m


GSI Rx 12-15m

*



r *. 2 '
'* *-


Figure 1 Effects of LY window therapy on amyloid deposition. A. Time course of AB accumulation in Tg2576 mice. FA solubilized brain
AB42+AP40 levels measured by ELISA are shown (n- 3-5 mice/age group). B. Schematic of the various transient LY dosing strategies. Dotted
ines represent the transient LY treatment time points. C. AB plaque burden analysis of treated cohorts compared to controls (*p < 0.05, ** ns,
ANOVA). D. Biochemical analyses (FA solubilized AB42+AP40 levels) by ELISA showing A levels. (*p < 0.05**ns, ANOVA). E. Representative brai
sections stained with anti-A3 mAb showing AB plaques at 15M of age in the LY treated cohorts. Scale bar- 100 pm.


brains of untreated Tg2576 mice from various ages ran-
ging from 10-18 months and plotted the natural loga-
rithm transformed value of these against AP levels from
the 4-7M LY treated mice, aged to 15M and 18M
(Figure 2D). To minimize variance, we assayed all of the
samples in a single set of ELISA studies. This analysis
showed that the 4 -7M GSI treatment time frame "shifts"


amyloid depositing by approximately 3 months, essen-
tially delaying AP accumulation by a time roughly
equivalent to the treatment window.
We next attempted to determine whether this 4-7M
narrow therapeutic "window" can be further refined. To
do this, we transiently dosed Tg2576 mice with LY for 1,
2 or 3M intervals between the 4-7M age time frame and


Page 3 of 9


I 10 4 7 10 12 15

Rx 4-7 months

Rx 7-10 months

Rx 12-15 months

I Onet of Ap depolition A-I II- AP de-posIn


(434%)
*


(+64


.' ,
*^ *"


'"





.,
.4 o.


%







Das et al Molecular Neurodegeneration 2012, 7:39
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Table 1 Brain and plasma Ap40 levels following in vivo GSI treatment in Tg2576 mice
Compound Strain Route Dose Length of % Ap40
of Mice Treatment Reduction
Brain
LY-411, 575 Tg2576 Intraperitoneal 5 mg/kg 6 hrs 75%
10 mg/kg 6 hrs 77%
25 ma/ka 6 hrs 78%


-411, 575 Tg2576 Oral (Suspension



-411, 575 Tq2576 Oral (Formulated


mol-Aid) 10 mg/kg/day 1 Day 47%
3 Days 65%
7 days 75%
dent Chow) 1 mg/kg/day 2 weeks 36%
2.5 mg/kg/day 2 weeks 51%
5 mg/kg/day 2 weeks 78%
10 mg/kg/day 2 weeks 83%


Tg2576 mice (3 months old) were dosed with GSI LY-411,575 using various routes, time points and indicated doses. After GSI treatment, groups of mice
(n =3/group) were sacrificed and mouse brains were harvested and extracted in 2% SDS. Brain and plasma Ap40 levels were then measured by ELISA.
*Diarrhea and hair loss.


again aged the mice to 15M. AP levels were again 'I.1F!
cantly reduced in this second 4-7M LY cohort (-59%
reduction) at 15M of age compared to controls
I' ...... 2E). Similarly, i) the 4-6M LY cohort showed a
significant reduction in AP levels (-51% reduction) and;
ii) the 4-5M LY group showed a modest but non-
significant reduction in AP levels (- 24% reduction)
iT .... 2E). However, in the remaining 5-6M, 6-7M or
5-7M LY cohorts tested, none showed any significant
reductions in AP levels at 15M compared to controls
(Figure 2E). Since we show that the 4-7M LY "shifts"
amyloid deposition equivalently to the duration of
treatment, we wondered whether we will see a similar
shift in amyloid deposition in the shorter treatment
intervals examined that showed ".. ,.;. e.g., in the 4-
5M and 4-6M cohorts. Again, we compared AP levels
in the brains of untreated T.. '76 mice from various
ages ranging from 10-15 months against A3 levels
from the 4-5M, 4-6M, and 4-7M LY treated cohorts
aged to 15M (Figure 2F). This analysis seems to show
that the 4-5M GSI treatment "shifts" amyloid depositing


Table 2 Plasma Ap40 levels following transient GSI
treatment in Tg2576 mice
Treatment Time Treatment Groups Plasma Ap40 (pM)
4-7 Month Ly-411,575 341.3 20.1 (81%)
Control 1742.6 103.5
7-10 Month Ly-411,575 327.5 18.5 (79%)
Control 1520.7 100.8
12-15 Month Ly-411,575 427.3 31.4 (70%)
Control 1388.4 223.2
Tg2576 mice were transiently dosed with GSI LY-411,575(2.5 mg/kg/day) at
indicated time points. After GSI treatment was discontinued (on last day of
treatment), plasma from groups of mice (n =3/group) was collected and
plasma A340 levels were measured by ELISA.
-Numbers in parenthesis represent percent reduction in Ap3 levels.


by approximately 1 month, however, both the 4-6M and
4-7M treatment intervals essentially show a similar shift
in AP accumulation, namely by roughly 3 months
(Figure 2F). These data would ".. at least within
the 4-7M time frame that we have studied, that the
4-6M age interval may play a critical role in the early
seeding phases of amyloid deposition. Additional studies,
using either earlier treatment times (e.g. starting at 3M
of age) or longer treatment intervals may be necessary to
fully appreciate the usefulness of this early "therapeutic
window" to achieve the best possible outcome on subse-
quent amyloid deposition.

Transient GSI does not permanently alter APP levels or
processing
To determine if the 4-7M LY treatment had long-
lasting effects on AP production or APP levels and
processing, we conducted a number of studies. We
measured plasma AP levels at termination of GSI treat-
ment and then determined how long before steady
state levels of AP were .....1.. i, ..i Plasma AP levels
were significantly lower immediately after treatment,
but returned to control levels between with 1-2 weeks
after treatment was halted I'T,- ,. 3A). We also mea-
sured levels of full-length APP and APP C-terminal
fragments (CTFs) in the brain at the end of the 4-7M
treatment and at 15M. Immediately f.il... ;"i discon-
tinuation of treatment, APP levels were unchanged but
APP CTFs were increased T.3.,... 3B,D). However, at
15 months, there were no -...-c.r'. ::.,,T .-:.... in full
length APP or CTFs (Figure 3C,D). F -.II;, as GSI
treatment has been shown to affect peripheral lympho-
cytes [13], we performed FACS analysis and .p11 ..i..i. ,i
both B and T cell numbers in the spleen after the 4-
7M LY transient treatment and at 15M. B and T cell
numbers were not ,II, .: (Figure 4A). However, when


Page 4 of 9


% Ap40
Reduction
Plasma
92%
96%
92%


Overt
Toxicity

No
No
No


95%








Das et al. Molecular Neurodegeneration 2012, 7:39
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2 D
D 8.5

8.0


- u 7.0
0. 0
S6.5
6.0
5.5


F


2000


GSI dosing (in months)


* Untreated
SGSI Rx 4-7m


9 10 11 12 13 14 15 16 17 18 19
Age in Months


-- Untreated
SGSI Rx (4-7m)
GSIRx(4-6m)
GSIRx(4-5m)




.. s9 .. ..
op 14f
^ ^

Age In Months


Figure 2 Effects of LY window therapy on amyloid deposition. A. Quantitative burden analysis of cored amyloid deposits and CAA in the
4-7M LY treated group compared to controls. (p < 0.05, Student's t test). B. Representative brain sections stained with mAb 13.1.1 showing cored
plaque and CAA immune-reactivity at 15M in the brains of Control and 4-7M LY treated Tg2576 mice. Arrows indicate CAA in meningeal vessels.
Scale bar-80 pm. C. Biochemical analyses of FA solubilized AB levels and plaque burden analysis from Tg2576 mice transiently dosed with LY
(2.5mpk/day) from 4-7M, an aaged to 18M. (p <0.05, Student's t test). Tg2576 mice (n 5-6/group) were used, data represented is from one
independent experiment. D. Natural logarithm transformed AB values of untreated Tg2576 mice (at indicated ages) and AB levels from 4-7M LY
treatment measured at 15M and 18M of age. E. Biochemical analyses of FA solubilized A levels of Tg2576 mice transiently dosed with LY
(2.5mpk/day) at 1, 2, and 3M intervals and then aged till 15M. (p <0.05, ANOVA). Tg2576 mice (n=5-8/group) were used, data represented is
from one independent experiment. F. Graph representing AB values of untreated Tg2576 mice (from indicated ages) and AB levels from 4-5M,
4-6M and 4-7M LY transient treatment measured at 15M. FA solubilized brain A342+A340 levels measured by ELISA are shown.


we analyzed Thl and Th2 cytokine profiles, splenic
CD4+ T cells from the 4-7M LY treated cohort showed
a bias towards Thl polarization with increased IFN-y
secretion (a canonical Thi cytokine) and concurrent
decreases in IL-4 levels (a signature Th2 cytokine),
which persisted even after the treatment was halted for
8 months (Figure 4B, C,). The significance of this long-
lasting effect on peripheral CD4 + T cell immune
responses is not clear, but certainly warrants further
investigation.


Conclusion
Our current data show that transiently lowering A3 pro-
duction in Tg2576 mice during the pre-deposition "seed-
ing" phase has a major impact on subsequent A3
accumulation with the effect persisting for at least 11M.
Later treatment windows showed decreasing efficacy of
treatment. These current data have major implications
for trial design targeting A3 production in humans. In-
deed, they would suggest that efficacy in terms of plaque
reduction is likely to be maximal during seeding phase,


Page 5 of 9


B Control

C -


S.


-0.75 0

-050 !
-000

-ooo


GSI Rx4-7m


I 'r
'a








Das et al. Molecular Neurodegeneration 2012, 7:39
http://www.molecularneurodegeneration.com/content/7/1/39


---f----- ------

S-GSI Rx
-*- Control


/
cc


/*

Co


GSI 4-7M Control kDa


CTFD 15
CTFa
50
Actin ll 50
Tested at 7M


GSI 4-7M Control kDa
FL-APP-- -100
u --15
S10
Actin 50

Tested at 15M


_a
n m

4) -

a 4-1



aC,
0 r


GSI Rx(4-7m) f
E Control


*
2



l~l0
S0 1' 0


GSI Rx 4-7m tstat 7m GSI R 4-7m testedat 7m R4Zesat 15m


and rapidly decline if treatment is initiated during the
exponential phase of deposition. A recent report [14]
using passive immunotherapy provided more limited evi-
dence that a similar effect may be observed with anti-Ap
passive immunotherapy initiated during the early depos-
ition phase persisting for 3 months after discontinuation


0) 2000.

0 1500.

a 1000.
E
C 500*
F-


Page 6 of 9


-.-


Figure 3 Long term LY treatment does not permanently affect
APP levels or processing. A. Plasma Ap40 levels by ELISA after
the end of 4-7M LY treatment, (7M), 1 week after termination
of treatment, and 2 weeks after termination of treatment. B.
Representative anti-APP CT20 immunoblot shows APP levels and
accumulation of CTFs after 4-7M LY treatment (tested at 7M). C.
Representative anti-APP CT20 immunoblot showing full length APP
levels and CTFs after transient 4-7M LY treatment (tested at 15M). D.
Quantitative intensity analysis of anti-CT20 immuno-reactive ful
length APP and APP CTF protein levels normalized to actin.



of treatment. Given the plethora of anti-Ap therapies
currently in development, it will be essential to deter-
mine for each modality whether efficacy is both main-
tained after discontinuation and similarly influenced by
timing of the treatment.
The near equivalence of the delay in subsequent de-
position to the length of the treatment window during a
pre-deposition phase suggests that the GSI treatment
from 4-7M alters or reverses seeding of Ap. Indeed,
there are no plaques forming during this period of time.
Future studies will be necessary to determine the exact
mechanisms by which lowering AP in this window alters
subsequent deposition. As even in vitro detection of AP
nucleation relies on indirect assays that have limited
sensitivities, these studies will likely require a better
understanding of nucleation and better assays to detect
it. Developing such assays will be critical as direct trans-
lation of these studies would be facilitated by defining
the equivalent window in humans. Regardless of mech-
anism, these preclinical window therapy studies have
major implications regarding the potential of prophylac-
tic therapy targeting AP production in humans. Not only
do these data suggest that prophylactic therapy in the
appropriate window will be more efficacious then initiat-
ing therapy in individuals with any preexisting AP path-
ology, but they suggest that such therapy, even if
discontinued might delay subsequent onset AD by a
time roughly equivalent to the time of treatment.

Methods
Tg2576 mice
APP Tg2576 mice were generated, maintained and geno-
typed as described previously [7]. All animal husbandry
procedures performed were approved by the Mayo
Clinic Institutional Animal Care and Use Committee in
accordance with National Institutes of Health guidelines.
All animals were housed three to five to a cage and
maintained on ad libitum food and water with a 12 h
light/dark cycle.

LY-411, 575
LY-411, 575 (LY) was synthesized at the Mayo Clinic
Chemical Core as previously described [15]. LY at a


0
+
#







Das et al. Molecular Neurodegeneration 2012, 7:39
http://www.molecularneurodegeneration.com/content/7/1/39


L control 4
-0



80 -40 T
-20
60- ::: III I 1, -60
S0


20- i20 0



GSI Rx 4-7m. tested at 7m GS/ Rx 4-7m. tested at 15m
Cell Surface Marker

lGSI Rx CD4+ T cells
IE] Control CD4+ T cells


[In


In I


,40 -
z
,30 -i
-20
,10


Polarizing Conditions


concentration of 16.75 mg per kg of diet was homoge-
nously incorporated into Harlan Teklad 7012 kibble
chow by Research Diets, Inc, New Jersey). Based upon
dietary consumption at this age, these diets were
designed to deliver 2.5 mg of LY on average per day.
Tg2575 mice (n= 7-15/group/experiment) were transi-
ently treated with LY chow at 1, 2 or 3 months interval
(between 4-7 months of age), from 7-10 months of age
and from 12-15 months of age. Untreated control mice
from each age group were as aged and used for analysis.
Groups of mice (n =3) were sacrificed immediately after
the end of each treatment time-point to access efficacy
of LY inhibition. After treatment was halted, mice were
then aged till 15 months or 18 months of age and then
sacrificed for AP analysis. Chow consumption, general
health and body weight were monitored on a weekly
basis in all treatment groups.


Biochemical AP ELISA assay
Snap-frozen hemi-forebrains from Tg2576 mice were
two-step 'p .... li) extracted in 2% SDS buffer
followed by 70% formic acid (FA) as described pre-
viously [16]. A3 levels from brain lysates or plasma
were the measured using sandwich ELISA techniques
as described previously [16] with end specific mAbs
2.1.3 (human A3x-42 specific, Mayo) and mAb 13.1.1
(human Apx-40 specific, Mayo) for capture and HRP-
conjugated mAb Ab5 (human Apl-16 specific, Mayo)
for detection.


l GSI Rx CD4+ T cells
E=Control CD4+ T cells


GSRx4-7m, tested at 15m


Polarizing Conditions
Figure 4 Effect of LY treatment on peripheral immune
Distribution of splenic B cell (IgD, CD 9) and I ce is (CD4 a
from control and Tg2576 mice treated with LY (4-7M). Perc
positive cells are shown (n=4 mice/group). B and C. FN p
(B) and I -4 production (C) by CD4+ T cells from LY (4-7M)
Tg2576 mice and untreated control mice were measured b
Equal numbers of purified, splenic CD4+ T cells (pooled fro
mice/group) were used. ND= not determined.


-1200 Immunohistological analysis
L li Ji sections of :' fi'. embedded sections were
-900 F used for analysis. Immunohistochemical staining was
-00 done using pan-A3 antibody (mAb 33.1.1; Mayo), mAb
9 13.1.1 (human Apx-40 specific, binds cored plaques and
-300 CAA; Mayo) as previously .i1 .. [17]. Immunohisto-
chemically stained sections were captured using the
Scanscope XT image scanner (Aperio, Vista, CA, USA)
and analyzed using the I! ,.: *-._. .p._. program. AP plaque
burden was then calculated using the Positive Pixel
Count program (Aperio). At least 5 sections/brain
30 tpm apart, were used and averaged by a blinded obser-
cells. A.
ni C8) ver to calculate plaque burden.
eni
rodiucion
treated Western blotting
y ELISA. 2% SDS solubilized brain lysate samples were separated
m n 4 on Bis-Tris 12% XT gels (Bio-Rad, Hercules, CA, USA)
and probed with the antibody CT20 (anti-APP C-
terminal 20 amino acid; 1:1000; P.D) and anti-actin
(1:1000; Sigma, St. Louis, MO, USA). Band intensity was
i': i ti;; .1 using ImageJ software (National Institutes of
Health, Bethesda, MD, USA).


Page 7 of 9


1200-


I-
.....m ND


GSI Rx 4-7mestedat7m







Das et al. Molecular Neurodegeneration 2012, 7:39
http://www.molecularneurodegeneration.com/content/7/1/39


Cell isolation, in vitro polarization and
Immuno-phenotyping
-ii. 1. cell suspensions were prepared :,1 ii; from
spleens from control or GSI -treated mice. CD4+ T cells
were positively selected using the BD-IMAG system and
CD4+ DM particles (BD Biosciences, San Diego, CA),
according to the manufacturer's directions. Twelve-well
tissue culture plates were precoated with anti-hamster Ig
(Sigma), then with antibodies specific for CD3c and
CD28. Equal numbers of cells (3x106) were added to
each II For TH1 polarizing conditions, anti-IL4
(10pg/ml) plus IL-12 (1 ng/ml) was added to cul-
ture for TH2 polarizing conditions, anti-IFNy
(10pg/ml) plus IL-4 (1 ng/ml) was added to culture
wells. No additional reagents were added to cells cul-
tured under neutral conditions. After 24 hours, all wells
were pulsed with IL-2 (10 ng/ml). After 96 hours, super-
natants were collected and frozen at -80C until analyzed
by ELISA for cytokine secretion. IFNy and IL-4 cytokine
secretion was measured using ELISA kits per manu-
factures instructions (BD fi ',,"..._. 'I For cell sur-
face i .;:;..- approximately 1x106 cells (each antibody)
were stained with the :ii ... 11 ...i ..l. i. .i ..!, l;
IgD-FITC, biotinylated anti-CD19 plus strep-PE, CD4-
FITC, CD8-PE (BD I ....- ",' Data were analyzed
using an LSRII flow cytometer (BD Biosciences) and
FACSDiva software for acquisition and analysis.


Statistics
All statistics were done using Graphpad Prism (Version
5.0). Comparisons of multiple groups were done by one-
way ANOVA followed by Tukey's post hoc testing. Com-
parisons between two groups were done by : !.i;. ,.
t test with Welch's correction for unequal variances
where appropriate.


Additional file

Additional file 1: Figure S1A Wild type APP overexpressing CHO cell
were treated with indicated concentrations of LY411,575 overnight
Conditioned media were then assayed for secreted A1340 by sandwich
ELSA Figure 51B. Acute LY 411,575 treatment reduces A340 levels in
brains of mice Tg2576 mice (3 month od) were injected w:th LY-411,575
(5 mg/kg) intraperitonealy Mice were sacrificed 6 hrs after and brain (2%
SDS solubiiized) A340 levels were measured ELISA
(n=3 mice/group) Figure S1C, D. Tg2576 mice were dosed orally
(suspended in Koo: Aid) with Y 411,575 (10 mg/kg) for indicated times
and brain (2% SDS soiubilized) A340 levels (C) and plasma A040 levels
(D) were measured by ELISA (n-3 mice/group)


Competing interests
The authors have no competing interest,


Authors' contributions
PD and TEG designed, analyzed and interpreted all aspects of the
co-wrote the manuscript CV pedfrmed all animal dosing studies,
and harvestingg of tissues and E iSA analysis PC performed western
techniques, tissue staniing and quatirficatior of plaque burdens K


;tudy and
sacking
betting
F assisted


with conceptualization of the study and edited the manuscript TK
performed in vitro cell culture analysis of LY-411, 575 GM and AF synthesized
LY 411, 575 used in this study LM and BAO performed immune cell
isolation, in vitro polarization and immune phenotyping studies All authors
read and approved the final manuscript


Acknowledgements
The authors thank Monica Castanedes-Casey, Virginia PhiiDs, and Linda
Rousseau for assistance with nistopathology Supported by the Nationa
istitules of He(lth/Nalion(al institute on Aging grant (P01AG25531 to BA5,
[EG)

Author details
Department of Neuroscience, Mayo Clinic College of Medicine, 4500 San
Pablo Rd S, Jacksonville, FL 32224, USA 2Department of Veterinary & Anima
Sciences, University of Massachusetts, 661 N Pleasant St, Amherst, MA
01003, USA Center for Translationa Research in Neurodegenerative Disease,
Department of Neuroscience, McKnight Brain Institute, College of Medicine,
University of Florida, 1600 SW Archer Road, Gainesville, FL 32608, USA
4Department of Pharmacology and Neurology, Emory University School of
Medicine, 1510 Clifton Rd NE, 5123 Rollins Research Center, Atlanta, GA
30322, USA

Received: 22 June 2012 Accepted: 6 August 2012
Published: 14 August 2012


References
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4 Golde TE, Petrucelli L, Lewis J Targeting Abeta and tau in Alzheimer's
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8 Abiamowski D, et. a!' Dynamics of Abeta turnover and deposition in
different beta-amyloid precursor protein transgenic mouse models
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11 Kawarabayashi T, et al Age-Dependent Changes in Brain, CSF, and Plasma
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12 Dovey H, Varghese J, Andeison JP' Functional gamma-secretase inhibitors
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13 Wong GT, er ac Chronic treatment with the gamma-secretase inhibitor
LY-411,575 inhibits beta-amyloid peptide production and alters
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14 Karlnoski RA, et ao Suppression of amyloid deposition leads to long-term
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15 Fauq Al et a! A multigram chemical synthesis of the gamma-secretase
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http://www.molecularneurodegeneration.com/content/7/1/39




16 Chakrabarty P, et al Massive gliosis induced by interleukin-6 suppresses
Abeta deposition in vivo: evidence against inflammation as a driving
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17 Chakrabarty P, et al IFN-gamma promotes complement expression and
attenuates amyloid plaque deposition in amyloid beta precursor protein
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doi:10.1186/1750-1326-7-39
Cite this article as: Das et al Transient pharmacologic lowering of AI
production prior to deposition results in sustained reduction of amyloid
plaque pathology. Molecular Neurodegeneration 2012 7'39


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Supplementary Figure 1


A

300-



g 200-



100-



0










C
100"




60
O-










40
C20
20
20


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Control 1 Day 3 Days 7 Days


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Iog[Ly411575] M


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3 Days 7 Days






Supplementary Figure 1 Legend.


A. Wild type APP overexpressing CHO cells were treated with indicated concentrations of LY-411,575
overnight. Conditioned media were then assayed for secreted A340 by sandwich ELISA.
B. Acute LY-411,575 treatment reduces A340 levels in brains of mice. Tg2576 mice (3 month old) were
injected with LY-411,575 (5 mg/kg) intraperitoneally. Mice were sacrificed 6 hrs later and brain (2% SDS
solubilized) A340 levels were measured ELISA. (n=3 mice/group)
C, D. Tg2576 mice were dosed orally (suspended in Kool-Aid) with LY-411,575 (10 mg/kg) for indicated times
and brain (2% SDS solubilized) A340 levels (C) and plasma A340 levels (D) were measured by ELISA (n=3
mice/group).




Full Text

PAGE 1

-13 -12 -11 -10 -9 -8 -7 -6 0 100 200 300Alog[Ly411575] MA 40 pM Control LY-411, 5 75 (5mg/kg) 0 25 50 75 100BA 40 pM A 40 (% Control) 0 20 40 60 80 100 Control1 Day3 Days7 DaysC D A 40 (% Control) 0 20 40 60 80 100 Control1 Day3 Days7 DaysSupplementary Figure 1

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Supplementary Figure 1 Legend. A. Wild type APP overexpressing CHO cells were tr eated with indicated conc entrations of LY-411,575 overnight. Conditioned media were then assayed for secreted A 40 by sandwich ELISA. B. Acute LY-411,575 treatment reduces A 40 levels in brains of mice. Tg2576 mice (3 month old) were injected with LY-411,575 (5 mg/kg) intraperitoneally. Mice were sacrifi ced 6 hrs later and brain (2% SDS solubilized) A 40 levels were measured ELISA. ( n =3 mice/group) C, D. Tg2576 mice were dosed orally (suspended in Kool -Aid) with LY-411,575 (10 mg/kg) for indicated times and brain (2% SDS solubilized) A 40 levels (C) and plasma A 40 levels (D) were measured by ELISA ( n =3 mice/group).



PAGE 1

RESEARCHARTICLEOpenAccessTransientpharmacologicloweringofA productionpriortodepositionresultsin sustainedreductionofamyloidplaquepathologyPritamDas1*,ChristopheVerbeeck1,LisaMinter2,ParamitaChakrabarty3,KevinFelsenstein3,ThomasKukar4, GhulamMaharvi1,AbdulFauq1,BarbaraAOsborne2andToddEGolde3*AbstractBackground: Alzheimer ’ sdisease(AD)istheleadingcauseofdementiaamongtheelderly.Diseasemodifying therapiestargetingA thatareindevelopmenthavebeenproposedtobemoreeffectiveiftreatmentwasinitiated priortosignificantaccumulationofA inthebrain,butoptimaltimingoftreatmentinitiationhasnotbeenclearly establishedintheclinic.WecomparedtheefficacyoftransientpharmacologicreductionofbrainA witha -secretaseinhibitor(GSI)for1 – 3months(M)treatmentwindowsinAPPTg2576miceandsubsequentagingof themicetoeither15Mor18M. Results: ThesedatashowthatreducingA productionina2-3Mwindowsbothinitiatedanddiscontinuedbefore detectableA depositionhasthemostsignificantimpactonA loadsupto11Maftertreatmentdiscontinuation. Incontrast,initiationoftreatmentfor3Mwindowsfrom7-10Mor12-15Mshowsprogressivelydecreasingefficacy. Conclusions: ThesedatahavemajorimplicationsforclinicaltestingoftherapeuticsaimedatloweringA production,indicatingthat;i)thesetherapiesmayhavelittleefficacyunlesstestedasprophylacticsorintheearliest preclinicalstageofADwherethereisnoorminimalA accumulationandii)loweringA productiontransiently duringacriticalpre-depositionwindowpotentiallyprovideslong-lastingefficacyafterdiscontinuationofthe treatment. Keywords: A ,Alzheimer ’ sdisease,APP,Therapeutics, -secretaseinhibitionBackgroundAlzheimer ’ sdisease(AD)istheleadingcauseofdementiaamongtheelderly.Numerousstudiesinhumanshave demonstratedthesequentialdevelopmentofvarious pathologicalfeaturesthatcharacterizeADandtherelationshipofthesepathologiestodiagnosisofdementia [1].ThesestudiessupporttheamyloidcascadehypothesiswhichpositsthataccumulationofA aggregates triggersaseriesofdownstreampathologiesthatresults inclinicalAD[2].ADhasrecentlybeenproposedto havethreepre-clinicalstages,characterizedinsequence bythedevelopmentofA pathology(stage1),thepresenceofplaquepathology,neurofibrillarypathologyand initialevidenceforneurodegeneration(stage2),andfinallybythepresenceofstage2,pathologiesplusevidenceforsubtlecognitivedecline(stage3).This progressionlikelyoccursovera10 – 20yeartimeline[3]. TheamyloidcascadehypothesispredictsthatattenuatingorpreventingA aggregationandaccumulationwill attenuateorpreventthesubsequentdevelopmentofAD. Strongexperimentalsupportfortheamyloidhypothesis hassupportedtherationaleformanytherapeutic approachestargetingA .Majorinvestmentshavebeen madetodevelopinhibitorsormodulatorsofA production[4]. -secretaseinhibitors(GSIs), -secretaseinhibitors(BSIs)and -secretasemodulators(GSMs)have beenorarecurrentlybeingtestedinhumanADtrials. BoththeGSMtarenflurbil(R-flurbiporfren),andtheGSI semagacestat,failedtoshowefficacyinphaseIIIhuman *Correspondence: das.pritam@mayo.edu ; tgolde@ufl.edu1DepartmentofNeuroscience,MayoClinicCollegeofMedicine,4500San PabloRdS,Jacksonville,FL32224,USA3CenterforTranslationalResearchinNeurodegenerativeDisease,Department ofNeuroscience,McKnightBrainInstitute,CollegeofMedicine,Universityof Florida,1600SWArcherRoad,Gainesville,FL32608,USA Fulllistofauthorinformationisavailableattheendofthearticle 2012Dasetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.Das etal.MolecularNeurodegeneration 2012, 7 :39 http://www.molecularneurodegeneration.com/content/7/1/39

PAGE 2

trials[4,5].SeveralotherGSMs,GSIs,andBSIsarecurrentlyinearlystagetrials.Basedontheclinicalfailures oftarenflurbilandsemagacestataswellasotheranti-A therapiesnottargetingA productionbutitsaggregationorclearance,weandothershavebeguntoquestion whethertargetingofA insymptomaticADmaybea futileendeavor[6]. Giventheframeworkprovidedbytheamyloidcascade hypothesis,disappointingresultsfromcompletedtrials ofanti-A therapiesinpatientswithAD,andseveralbut limitedpreclinicalstudiessupportingtheconceptthat whentargetingA “ earlierisbetter ” [7-9],weexplored therelationshipbetweenlong-termefficacywithrespect toreducingamyloidloadsandtiminganddurationof A lowering.Indesigningthesestudies,wewerestruck bytheparallelsbetweenA accumulationinthemouse brainand invitro studiesofA aggregation. Invitro ,A aggregatesinanucleationdependentpolymerizationreaction,whichexhibitsthreephases:alagphaseinwhich nucleationoccursbutnotfibrilformation,anexponentialgrowthphaseinwhichfibrilformationandgrowth occurrapidly,andaplateauphasewherefibrilgrowth andformationslows[10].Exceptforbeingmore extendedintermsoftime,studiesinAPPTg2576transgenicmiceshowthatA aggregationandaccumulation followsasimilarcourseinthebrain[11].ThereisaninitiallagphasewherenoA aggregationandaccumulationoccurs,followedbyanexponentialaccumulation phase,andfinallywhenamyloidloadsbecomeAD-like, thegrowthslowsorplateaus(seeFigure1A).Given theseparallels,wetestedtheefficacyoftransientloweringofA productionin1-3Mtreatment “ windows ” correspondingtothesevariousphasesofdepositionusing theGSILY-411,575(LY)inAPPTg2576mice.These datashowthatGSItreatmentduringthepre-deposition window(4-7M)beforetheearlyexponentialphaseofdeposition(7-10M)orlateexponentialphase(12-15M)has byfarthemostefficacyintermsofreductionofplaque loadsat15Mofage.ResultsanddiscussionInvitroandinvivo dosingstudieswithLy-411,575Ly-411,575ispotentorallybioavailable -secretaseinhibitor[12].Wesynthesizedlargequantitiesforthese studiesandthenperformedboth invitro and invivo studiestovalidateLy-411,575potency.TheIC50incell cultureforLY-411,575wasbetween1 – 3nm(seeAdditionalfile1:FigureS1A).SixhoursafterasingleIP dose,A 40levelsarereducedby~75%inthebrainof non-depositingTg2576mice(Additionalfile1:Figure S1B).Oraladministration(10mg/kg/day)administered asasingledosereducesbrainA 40levels24hours afterthefinaldose(7days)by~65%(Additionalfile1: FigureS1C)andplasmaA 40by~95%(Additionalfile1: FigureS1D).PlasmaA levelsarereducedmorequickly thanbrainlevelsfollowingoraldosingwithmaximalinhibitionseenafter3days(Additionalfile1:FigureS1D). Forlongterm invivo mousestudies,wetestedadministrationofLy-411,575usingvariousdosingstrategies andtimepoints(Table1).Oraladministration(formulatedinrodentchow)continuallyfor2weekswitheither -1mg/kg/dayor2.55mg/kg/dayreducedbrainA 40 levelsby~50%andplasmaA 40by~80%,withoutovert toxicities(Table1).However,continuousoraldosing (>2weeks)usingthehigherdoseof5mg/kg/dayor 10mg/kg/dayresultedinoverttoxicitiesinmice (Table1).Therefore,inalllongtermstudiespresented inthismanuscript,wehaveusedthe2.5mg/kg/daydose formulatedinrodentchow.TransientA reductiondramaticallyreducesthe subsequentaccumulationofamyloidplaquesFortheseexperiments,wetransientlydosedTg2576 micewithLYfrom4-7M,7-10Mor12-15Mandaged treatedmiceto15M(seeFigure1Bforexperimentaldesign).ToconfirmefficacyofLYinallgroups,wemeasuredplasmaA levelsimmediatelyafterthelastdayof treatmentinsentinelmiceandshowedasimilarreductioninplasmaA levelsinallcohorts(Table2).Followingsacrificeat15M,levelsofA inthebrainwere examined.The4-7MLYtreatmentsignificantlyreduced A deposition;plaqueburdeninthefrontalcortexand hippocampuswasdecreasedby~68%(Figure1C)and FA-solubilizedbrainA levelswerereducedby~60% (Figure1D).The7-10MLYtreatmentnon-significantly decreasedA plaqueburdenby~19%comparedtocontrols(Figure1C),whereasFA-solubilizedA levelswere reducedby~34%(Figure1D).The12-15MGSItreatmentgrouphadnosignificanteffectoneitherplaque burdenorFA-solubilizedA levels(Figure1C,D).We furtherevaluatedeffectofthe4-7MLYtreatmenton coredamyloidplaquesandcerebralamyloidangiopathy (CAA).Comparedtountreatedcontrolmice,therewere significantreductionsbothincoredplaquesinthe frontalcortexandhippocampus(~47%reduction)and CAAintheleptomeninges(~41%reduction,Figure2A, B).Tofurtherdeterminewhetherthemagnitudeofthe effectdiminishedwithfurtheraging,weperformedan additionalexperiment,whereweaged4-7MLYtreated miceto18M,andagainweobservedsignificantly reducedA plaqueburdenandFA-solubilizedA levels (Figure2C),demonstratingthatthesuppressionofdepositionwasmaintainedevenup11monthsaftertreatmentwashalted. BecauseA depositioninmicefollowsanexponential course,wesoughttodeterminehowtheobservedreductionsinA accumulationcorrelatewithA accumulationtountreatedmice.WecomparedA levelsintheDas etal.MolecularNeurodegeneration 2012, 7 :39 Page2of9 http://www.molecularneurodegeneration.com/content/7/1/39

PAGE 3

brainsofuntreatedTg2576micefromvariousagesrangingfrom10 – 18monthsandplottedthenaturallogarithmtransformedvalueoftheseagainstA levelsfrom the4-7MLYtreatedmice,agedto15Mand18M (Figure2D).Tominimizevariance,weassayedallofthe samplesinasinglesetofELISAstudies.Thisanalysis showedthatthe4-7MGSItreatmenttimeframe “ shifts ” amyloiddepositingbyapproximately3months,essentiallydelayingA accumulationbyatimeroughly equivalenttothetreatmentwindow. Wenextattemptedtodeterminewhetherthis4-7M narrowtherapeutic “ window ” canbefurtherrefined.To dothis,wetransientlydosedTg2576micewithLYfor1, 2or3Mintervalsbetweenthe4-7Magetimeframeand Figure1 EffectsofLYwindowtherapyonamyloiddeposition.A .TimecourseofA accumulationinTg2576mice.FAsolubilizedbrain A 42+A 40levelsmeasuredbyELISAareshown( n =3 – 5mice/agegroup) .B .SchematicofthevarioustransientLYdosingstrategies Dotted linesrepresentthetransientLYtreatmenttimepoints. C .A plaqueburdenanalysisoftreatedcohortscomparedtocontrols(* p <0.05,**ns, ANOVA). D .Biochemicalanalyses(FAsolubilizedA 42+A 40levels)byELISAshowingA levels.(* p <0.05,**ns,ANOVA). E .Representativebrain sectionsstainedwithanti-A mAbshowingA plaquesat15MofageintheLYtreatedcohorts.Scalebar=100 m. Das etal.MolecularNeurodegeneration 2012, 7 :39 Page3of9 http://www.molecularneurodegeneration.com/content/7/1/39

PAGE 4

againagedthemiceto15M.A levelswereagainsignificantlyreducedinthissecond4-7MLYcohort(~59% reduction)at15Mofagecomparedtocontrols (Figure2E).Similarly,i)the4-6MLYcohortshoweda significantreductioninA levels(~51%reduction)and; ii)the4-5MLYgroupshowedamodestbutnonsignificantreductioninA levels(~24%reduction) (Figure2E).However,intheremaining5-6M,6-7Mor 5-7MLYcohortstested,noneshowedanysignificant reductionsinA levelsat15Mcomparedtocontrols (Figure2E).Sinceweshowthatthe4-7MLY “ shifts ” amyloiddepositionequivalentlytothedurationof treatment,wewonderedwhetherwewillseeasimilar shiftinamyloiddepositionintheshortertreatment intervalsexaminedthatshowedefficacy,e.g.,inthe45Mand4-6Mcohorts.Again,wecomparedA levels inthebrainsofuntreatedTg2576micefromvarious agesrangingfrom10 – 15monthsagainstA levels fromthe4-5M,4-6M,and4-7MLYtreatedcohorts agedto15M(Figure2F).Thisanalysisseemstoshow thatthe4-5MGSItreatment “ shifts ” amyloiddepositing byapproximately1month,however,boththe4-6Mand 4-7Mtreatmentintervalsessentiallyshowasimilarshift inA accumulation,namelybyroughly3months (Figure2F).Thesedatawouldsuggest,atleastwithin the4-7Mtimeframethatwehavestudied,thatthe 4-6Mageintervalmayplayacriticalroleintheearly seedingphasesofamyloiddeposition.Additionalstudies, usingeitherearliertreatmenttimes(e.g.startingat3M ofage)orlongertreatmentintervalsmaybenecessaryto fullyappreciatetheusefulnessofthisearly “ therapeutic window ” toachievethebestpossibleoutcomeonsubsequentamyloiddeposition.TransientGSIdoesnotpermanentlyalterAPPlevelsor processingTodetermineifthe4-7MLYtreatmenthadlonglastingeffectsonA productionorAPPlevelsand processing,weconductedanumberofstudies.We measuredplasmaA levelsatterminationofGSItreatmentandthendeterminedhowlongbeforesteady statelevelsofA werenormalized.PlasmaA levels weresignificantlylowerimmediatelyaftertreatment, butreturnedtocontrollevelsbetweenwith1 – 2weeks aftertreatmentwashalted(Figure3A).Wealsomeasuredlevelsoffull-lengthAPPandAPPC-terminal fragments(CTFs)inthebrainattheendofthe4-7M treatmentandat15M.Immediatelyfollowingdiscontinuationoftreatment,APPlevelswereunchangedbut APPCTFswereincreased(Figure3B,D).However,at 15months,therewerenosignificantdifferencesinfull lengthAPPorCTFs(Figure3C,D).Finally,asGSI treatmenthasbeenshowntoaffectperipherallymphocytes[13],weperformedFACSanalysisandquantified bothBandTcellnumbersinthespleenafterthe4 – 7MLYtransienttreatmentandat15M.BandTcell numberswerenotaffected(Figure4A).However,when Table1BrainandplasmaA 40levelsfollowing invivo GSItreatmentinTg2576miceCompoundStrain ofMice RouteDoseLengthof Treatment %A 40 Reduction Brain %A 40 Reduction Plasma Overt Toxicity LY-411,575Tg2576Intraperitoneal5mg/kg6hrs75%92%No 10mg/kg6hrs77%96%No 25mg/kg6hrs78%92%No LY-411,575Tg2576Oral(SuspensioninKool-Aid)10mg/kg/day1Day47%70%No 3Days65%86%No 7days75%95%No LY-411,575Tg2576Oral(FormulatedInRodentChow)1mg/kg/day2weeks36%67%No 2.5mg/kg/day2weeks51%78%No 5mg/kg/day2weeks78%91%Yes* 10mg/kg/day2weeks83%95%Yes*Tg2576mice(3monthsold)weredosedwithGSILY-411,575usingvariousroutes,timepointsandindicateddoses.AfterGSItreatment,groupsofmice ( n =3/group)weresacrificedandmousebrainswereharvestedandextractedin2%SDS.BrainandplasmaA 40levelswerethenmeasuredbyELISA. *Diarrheaandhairloss. Table2PlasmaA 40levelsfollowingtransientGSI treatmentinTg2576miceTreatmentTimeTreatmentGroupsPlasmaA 40(pM) 4-7Month Ly-411,575341.320.1 (81%) Control1742.6103.5 7-10Month Ly-411,575327.518.5 (79%) Control1520.7100.8 12-15Month Ly-411,575427.331.4 (70%) Control1388.4223.2Tg2576miceweretransientlydosedwithGSILY-411,575(2.5mg/kg/day)at indicatedtimepoints.AfterGSItreatmentwasdiscontinued(onlastdayof treatment),plasmafromgroupsofmice( n =3/group)wascollectedand plasmaA 40levelsweremeasuredbyELISA. -NumbersinparenthesisrepresentpercentreductioninA levels.Das etal.MolecularNeurodegeneration 2012, 7 :39 Page4of9 http://www.molecularneurodegeneration.com/content/7/1/39

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weanalyzedTh1andTh2cytokineprofiles,splenic CD4+Tcellsfromthe4-7MLYtreatedcohortshowed abiastowardsTh1polarizationwithincreasedIFNsecretion(acanonicalTh1cytokine)andconcurrent decreasesinIL-4levels(asignatureTh2cytokine), whichpersistedevenafterthetreatmentwashaltedfor 8months(Figure4B,C,).ThesignificanceofthislonglastingeffectonperipheralCD4+Tcellimmune responsesisnotclear,butcertainlywarrantsfurther investigation.ConclusionOurcurrentdatashowthattransientlyloweringA productioninTg2576miceduringthepre-deposition “ seeding ” phasehasamajorimpactonsubsequentA accumulationwiththeeffectpersistingforatleast11M. Latertreatmentwindowsshoweddecreasingefficacyof treatment.Thesecurrentdatahavemajorimplications fortrialdesigntargetingA productioninhumans.Indeed,theywouldsuggestthatefficacyintermsofplaque reductionislikelytobemaximalduringseedingphase, Figure2 EffectsofLYwindowtherapyonamyloiddeposition.A .QuantitativeburdenanalysisofcoredamyloiddepositsandCAAinthe 4-7MLYtreatedgroupcomparedtocontrols.(* p <0.05,Student ’ s t test). B .RepresentativebrainsectionsstainedwithmAb13.1.1showingcored plaqueandCAAimmune-reactivityat15MinthebrainsofControland4-7MLYtreatedTg2576mice.ArrowsindicateCAAinmeningealvessels. Scalebar=80 m. C .BiochemicalanalysesofFAsolubilizedA levelsandplaqueburdenanalysisfromTg2576micetransientlydosedwithLY (2.5mpk/day)from4-7M,andagedto 18M.(* p <0.05,Student ’ s t test).Tg2576mice( n =5-6/group)wereused,datarepresentedisfromone independentexperiment. D .NaturallogarithmtransformedA valuesofuntreatedTg2576mice(atindicatedages)andA levelsfrom4-7MLY treatmentmeasuredat15Mand18Mofage. E .BiochemicalanalysesofFAsolubilizedA levelsofTg2576micetransientlydosedwithLY (2.5mpk/day)at1,2,and3Mintervalsandthenagedtill15M.(* p <0.05,ANOVA).Tg2576mice( n =5-8/group)wereused,datarepresentedis fromoneindependentexperiment. F .GraphrepresentingA valuesofuntreatedTg2576mice(fromindicatedages)andA levelsfrom4-5M, 4-6Mand4-7MLYtransienttreatmentmeasuredat15M.FAsolubilizedbrainA 42+A 40levelsmeasuredbyELISAareshown. Das etal.MolecularNeurodegeneration 2012, 7 :39 Page5of9 http://www.molecularneurodegeneration.com/content/7/1/39

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andrapidlydeclineiftreatmentisinitiatedduringthe exponentialphaseofdeposition.Arecentreport[14] usingpassiveimmunotherapyprovidedmorelimitedevidencethatasimilareffectmaybeobservedwithanti-A passiveimmunotherapyinitiatedduringtheearlydepositionphasepersistingfor3monthsafterdiscontinuation oftreatment.Giventheplethoraofanti-A therapies currentlyindevelopment,itwillbeessentialtodetermineforeachmodalitywhetherefficacyisbothmaintainedafterdiscontinuationandsimilarlyinfluencedby timingofthetreatment. Thenearequivalenceofthedelayinsubsequentdepositiontothelengthofthetreatmentwindowduringa pre-depositionphasesuggeststhattheGSItreatment from4-7MaltersorreversesseedingofA .Indeed, therearenoplaquesformingduringthisperiodoftime. Futurestudieswillbenecessarytodeterminetheexact mechanismsbywhichloweringA inthiswindowalters subsequentdeposition.Aseven invitro detectionofA nucleationreliesonindirectassaysthathavelimited sensitivities,thesestudieswilllikelyrequireabetter understandingofnucleationandbetterassaystodetect it.Developingsuchassayswillbecriticalasdirecttranslationofthesestudieswouldbefacilitatedbydefining theequivalentwindowinhumans.Regardlessofmechanism,thesepreclinicalwindowtherapystudieshave majorimplicationsregardingthepotentialofprophylactictherapytargetingA productioninhumans.Notonly dothesedatasuggestthatprophylactictherapyinthe appropriatewindowwillbemoreefficacioustheninitiatingtherapyinindividualswithanypreexistingA pathology,buttheysuggestthatsuchtherapy,evenif discontinuedmightdelaysubsequentonsetADbya timeroughlyequivalenttothetimeoftreatment.MethodsTg2576miceAPPTg2576miceweregenerated,maintainedandgenotypedasdescribedpreviously[7].Allanimalhusbandry proceduresperformedwereapprovedbytheMayo ClinicInstitutionalAnimalCareandUseCommitteein accordancewithNationalInstitutesofHealthguidelines. Allanimalswerehousedthreetofivetoacageand maintainedon adlibitum foodandwaterwitha12h light/darkcycle.LY-411,575LY-411,575(LY)wassynthesizedattheMayoClinic ChemicalCoreaspreviouslydescribed[15].LYata GSI R x ( 47 m) 1w e e kPo st R x 2 we e k s Po s t R x 0 500 1000 1500 2000GSI Rx Control Plasma A levels (pM)A B C D AP P C T F C T F A PP CTF CTF 0 1 2GSI Rx (4-7m) Control GSI Rx 4-7m, tested at 7m GSI Rx 4-7m, tested at 15m 4 6 8 0 1 2 4 6 8 Relative Protein LevelsRelative Protein Levels Figure3 LongtermLYtreatmentdoesnotpermanentlyaffect APPlevelsorprocessing.A .PlasmaA 40levelsbyELISAafter theendof4 – 7MLYtreatment,(7M),1weekaftertermination oftreatment,and2weeksafterterminationoftreatment. B Representativeanti-APPCT20immunoblotshowsAPPlevelsand accumulationofCTFsafter4-7MLYtreatment(testedat7M). C Representativeanti-APPCT20immunoblotshowingfulllengthAPP levelsandCTFsaftertransient4-7MLYtreatment(testedat15M). D Quantitativeintensityanalysisofanti-CT20immuno-reactivefull lengthAPPandAPPCTFproteinlevelsnormalizedtoactin. Das etal.MolecularNeurodegeneration 2012, 7 :39 Page6of9 http://www.molecularneurodegeneration.com/content/7/1/39

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concentrationof16.75mgperkgofdietwashomogenouslyincorporatedintoHarlanTeklad7012kibble chowbyResearchDiets,Inc,NewJersey).Basedupon dietaryconsumptionatthisage,thesedietswere designedtodeliver2.5mgofLYonaverageperday. Tg2575mice(n=7-15/group/experiment)weretransientlytreatedwithLYchowat1,2or3monthsinterval (between4 – 7monthsofage),from7 – 10monthsofage andfrom12 – 15monthsofage.Untreatedcontrolmice fromeachagegroupwereasagedandusedforanalysis. Groupsofmice(n=3)weresacrificedimmediatelyafter theendofeachtreatmenttime-pointtoaccessefficacy ofLYinhibition.Aftertreatmentwashalted,micewere thenagedtill15monthsor18monthsofageandthen sacrificedforA analysis.Chowconsumption,general healthandbodyweightweremonitoredonaweekly basisinalltreatmentgroups.BiochemicalA ELISAassaySnap-frozenhemi-forebrainsfromTg2576micewere two-stepsequentiallyextractedin2%SDSbuffer followedby70%formicacid(FA)asdescribedpreviously[16].A levelsfrombrainlysatesorplasma werethemeasuredusingsandwichELISAtechniques asdescribedpreviously[16]withendspecificmAbs 2.1.3(humanA x-42specific,Mayo)andmAb13.1.1 (humanA x-40specific,Mayo)forcaptureandHRPconjugatedmAbAb5(humanA 1 – 16specific,Mayo) fordetection.ImmunohistologicalanalysisSagittalsectionsofparaffinembeddedsectionswere usedforanalysis.Immunohistochemicalstainingwas doneusingpan-A antibody(mAb33.1.1;Mayo),mAb 13.1.1(humanA x-40specific,bindscoredplaquesand CAA;Mayo)aspreviouslydescribed[17].Immunohistochemicallystainedsectionswerecapturedusingthe ScanscopeXTimagescanner(Aperio,Vista,CA,USA) andanalyzedusingtheImageScopeprogram.A plaque burdenwasthencalculatedusingthePositivePixel Countprogram(Aperio).Atleast5sections/brain 30 mapart,wereusedandaveragedbyablindedobservertocalculateplaqueburden.Westernblotting2%SDSsolubilizedbrainlysatesampleswereseparated onBis-Tris12%XTgels(Bio-Rad,Hercules,CA,USA) andprobedwiththeantibodyCT20(anti-APPCterminal20aminoacid;1:1000;P.D)andanti-actin (1:1000;Sigma,St.Louis,MO,USA).Bandintensitywas quantifiedusingImageJsoftware(NationalInstitutesof Health,Bethesda,MD,USA). Ig D CD19 CD4 C D 8 Ig D C D1 9 C D 4 C D 8 0 20 40 60 80 100GSI Rx (4-7m) Control GSI Rx 4-7m, tested at 7m GSI Rx 4-7m, tested at 15m 0 20 40 60 80 100Cell Surface Marker% Cell Population% Cell Population N e u t ral TH1 T H2 Ne u t ra l TH 1 TH2 0 25 50 75 100 125 150 175GSI Rx CD4+ T cells Control CD4+ T cells GSI Rx 4-7m, tested at 7m GSI Rx 4-7m, tested at 15m 0 10 20 30 40 50NDPolarizing ConditionsIFN (ng/ml)IFN (ng/ml) Neutral TH1 TH2 N eu tral T H 1 TH 2 0 300 600 900 1200 GSI Rx CD4+ T cells Control CD4+ T cells GSI Rx 4-7m, tested at 7m GSI Rx 4-7m, tested at 15m 0 300 600 900 1200NDPolarizing ConditionsIL-4 (pg/ml)IL-4 (pg/ml)B C A Figure4 EffectofLYtreatmentonperipheralimmunecells.A DistributionofsplenicBcell(IgD,CD19)andTcells(CD4andCD8) fromcontrolandTg2576micetreatedwithLY(4-7M).Percent positivecellsareshown( n =4mice/group). BandC .IFNproduction ( B )andIL-4production( C )byCD4+TcellsfromLY(4-7M)treated Tg2576miceanduntreatedcontrolmiceweremeasuredbyELISA. Equalnumbersofpurified,splenicCD4+Tcells(pooledfromn=4 mice/group)wereused.ND=notdetermined. Das etal.MolecularNeurodegeneration 2012, 7 :39 Page7of9 http://www.molecularneurodegeneration.com/content/7/1/39

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Cellisolation, invitro polarizationand Immuno-phenotypingSinglecellsuspensionswerepreparedasepticallyfrom spleensfromcontrolorGSI-treatedmice.CD4+Tcells werepositivelyselectedusingtheBD-IMAGsystemand CD4+DMparticles(BDBiosciences,SanDiego,CA), accordingtothemanufacturer ’ sdirections.Twelve-well tissuecultureplateswereprecoatedwithanti-hamsterIg (Sigma),thenwithantibodiesspecificforCD3 E and CD28.Equalnumbersofcells(3x106)wereaddedto eachwell.ForTH1polarizingconditions,anti-IL4 (10 g/ml)plusIL-12(1ng/ml)wasaddedtoculturewells;forTH2polarizingconditions,anti-IFN (10 g/ml)plusIL-4(1ng/ml)wasaddedtoculture wells.Noadditionalreagentswereaddedtocellsculturedunderneutralconditions.After24hours,allwells werepulsedwithIL-2(10ng/ml).After96hours,supernatantswerecollectedandfrozenat-80Cuntilanalyzed byELISAforcytokinesecretion.IFN andIL-4cytokine secretionwasmeasuredusingELISAkitspermanufacturesinstructions(BDPharmingen).Forcellsurfacestaining,approximately1x106cells(eachantibody) werestainedwiththefollowingantibodiesindividually: IgD-FITC,biotinylatedanti-CD19plusstrep-PE,CD4FITC,CD8-PE(BDPharmingen).Datawereanalyzed usinganLSRIIflowcytometer(BDBiosciences)and FACSDivasoftwareforacquisitionandanalysis.StatisticsAllstatisticsweredoneusingGraphpadPrism(Version 5.0).ComparisonsofmultiplegroupsweredonebyonewayANOVAfollowedbyTukey'sposthoctesting.ComparisonsbetweentwogroupsweredonebyStudent's t testwithWelch'scorrectionforunequalvariances whereappropriate.AdditionalfileAdditionalfile1: FigureS1A WildtypeAPPoverexpressingCHOcells weretreatedwithindicatedconcentrationsofLY-411,575overnight. ConditionedmediawerethenassayedforsecretedA 40bysandwich ELISA. FigureS1B. AcuteLY-411,575treatmentreducesA 40levelsin brainsofmice.Tg2576mice(3monthold)wereinjectedwithLY-411,575 (5mg/kg)intraperitoneally.Miceweresacrificed6hrslaterandbrain(2% SDSsolubilized)A 40levelsweremeasuredELISA. ( n =3mice/group) FigureS1C,D. Tg2576miceweredosedorally (suspendedinKool-Aid)withLY-411,575(10mg/kg)forindicatedtimes andbrain(2%SDSsolubilized)A 40levels (C) andplasmaA 40levels (D) weremeasuredbyELISA( n =3mice/group). Competinginterests Theauthorshavenocompetinginterests. Authors ’ contributions PDandTEGdesigned,analyzedandinterpretedallaspectsofthestudyand co-wrotethemanuscript.CVperformedallanimaldosingstudies,sacking andharvestingoftissuesandELISAanalysis.PCperformedwesternblotting techniques,tissuestainingandquantificationofplaqueburdens.KFassisted withconceptualizationofthestudyandeditedthemanuscript.TK performed invitro cellcultureanalysisofLY-411,575.GMandAFsynthesized LY-411,575usedinthisstudy.LMandBAOperformedimmunecell isolation, invitro polarizationandimmuno-phenotypingstudies.Allauthors readandapprovedthefinalmanuscript. Acknowledgements TheauthorsthankMonicaCastanedes-Casey,VirginiaPhillips,andLinda Rousseauforassistancewithhistopathology.SupportedbytheNational InstitutesofHealth/NationalInstituteonAginggrant(P01AG25531toBAO, TEG). Authordetails1DepartmentofNeuroscience,MayoClinicCollegeofMedicine,4500San PabloRdS,Jacksonville,FL32224,USA.2DepartmentofVeterinary&Animal Sciences,UniversityofMassachusetts,661N.PleasantSt.,Amherst,MA 01003,USA.3CenterforTranslationalResearchinNeurodegenerativeDisease, DepartmentofNeuroscience,McKnightBrainInstitute,CollegeofMedicine, UniversityofFlorida,1600SWArcherRoad,Gainesville,FL32608,USA.4DepartmentofPharmacologyandNeurology,EmoryUniversitySchoolof Medicine,1510CliftonRdNE,5123RollinsResearchCenter,Atlanta,GA 30322,USA. Received:22June2012Accepted:6August2012 Published:14August2012 References1.JackCRJr, etal : Hypotheticalmodelofdynamicbiomarkersofthe Alzheimer'spathologicalcascade. LancetNeurol 2010, 9 (1):119 – 128. 2.HardyJ,SelkoeDJ: TheamyloidhypothesisofAlzheimer'sdisease: progressandproblemsontheroadtotherapeutics. Science 2002, 297 (5580):353 – 356. 3.DuboisB, etal : RevisingthedefinitionofAlzheimer'sdisease:anew lexicon. LancetNeurol 2010, 9 (11):1118 – 1127. 4.GoldeTE,PetrucelliL,LewisJ: TargetingAbetaandtauinAlzheimer's disease,anearlyinterimreport. ExpNeurol 2010, 223 (2):252 – 266. 5.ImbimboBP,GiardinaGA: gamma-secretaseinhibitorsandmodulatorsfor thetreatmentofAlzheimer'sdisease:disappointmentsandhopes. Curr TopMedChem 2011, 11 (12):1555 – 1570. 6.GoldeTE,SchneiderLS,KooEH: Anti-abetatherapeuticsinAlzheimer's disease:theneedforaparadigmshift. Neuron 2011, 69 (2):203 – 213. 7.DasP, etal : ReducedeffectivenessofAbeta1-42immunizationinAPP transgenicmicewithsignificantamyloiddeposition. NeurobiolAging 2001, 22 (5):721 – 727. 8.AbramowskiD, etal : DynamicsofAbetaturnoveranddepositionin differentbeta-amyloidprecursorproteintransgenicmousemodels followinggamma-secretaseinhibition. JPharmacolExpTher 2008, 327 (2):411 – 424. 9.LevitesY, etal : Anti-Abeta42-andanti-Abeta40-specificmAbsattenuate amyloiddepositioninanAlzheimerdiseasemousemodel. JClinInvest 2006, 116 (1):193 – 201. 10.JarrettJT,LansburyPTJr: Seeding"one-dimensionalcrystallization"of amyloid:apathogenicmechanisminAlzheimer'sdiseaseandscrapie? Cell 1993, 73 (6):1055 – 1058. 11.KawarabayashiT,etal : Age-DependentChangesinBrain,CSF,andPlasma AmyloidProteinintheTg2576TransgenicMouseModelofAlzheimer's Disease. JNeurosci 2001, 21: 372 – 381. 12.DoveyH,VargheseJ,AndersonJP: Functionalgamma-secretaseinhibitors reducebeta-amyloidpeptidelevelsinthebrain. JNeurochem 2000, 76: 1 – 10. 13.WongGT, etal : Chronictreatmentwiththegamma-secretaseinhibitor LY-411,575inhibitsbeta-amyloidpeptideproductionandalters lymphopoiesisandintestinalcelldifferentiation. JBiolChem 2004, 279 (13):12876 – 12882. 14.KarlnoskiRA, etal : Suppressionofamyloiddepositionleadstolong-term reductionsinAlzheimer'spathologiesinTg2576mice. JNeurosci 2009, 29 (15):4964 – 4971. 15.FauqAH, etal : Amultigramchemicalsynthesisofthegamma-secretase inhibitorLY411575anditsdiastereoisomers. BioorgMedChemLett 2007, 17 (22):6392 – 6395.Das etal.MolecularNeurodegeneration 2012, 7 :39 Page8of9 http://www.molecularneurodegeneration.com/content/7/1/39

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16.ChakrabartyP, etal : Massivegliosisinducedbyinterleukin-6suppresses Abetadeposition invivo :evidenceagainstinflammationasadriving forceforamyloiddeposition. FASEBJ 2010, 24 (2):548 – 559. 17.ChakrabartyP, etal : IFN-gammapromotescomplementexpressionand attenuatesamyloidplaquedepositioninamyloidbetaprecursorprotein transgenicmice. JImmunol 2010, 184 (9):5333 – 5343.doi:10.1186/1750-1326-7-39 Citethisarticleas: Das etal. : TransientpharmacologicloweringofA productionpriortodepositionresultsinsustainedreductionofamyloid plaquepathology. MolecularNeurodegeneration 2012 7 :39. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Das etal.MolecularNeurodegeneration 2012, 7 :39 Page9of9 http://www.molecularneurodegeneration.com/content/7/1/39


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title
p Transient pharmacologic lowering of Aβ production prior to deposition results in sustained reduction of amyloid plaque pathology
aug
au id A1 ca yes snm Dasfnm Pritaminsr iid I1 email das.pritam@mayo.edu
A2 VerbeeckChristopheverbeeck.christophe@mayo.edu
A3 MinterLisaI2 lminter@vasci.umass.edu
A4 ChakrabartyParamitaI3 pchakrabarty@ufl.edu
A5 FelsensteinKevinkfelsenstein0@ufl.edu
A6 KukarThomasI4 thomas.kukar@emory.edu
A7 MaharviGhulamghulam.maharvi@mayo.edu
A8 FauqAbdulfauq.abdul@mayo.edu
A9 Osbornemi ABarbaraosborne@vasci.umass.edu
A10 GoldeEToddtgolde@ufl.edu
insg
ins Department of Neuroscience, Mayo Clinic College of Medicine, 4500 San Pablo Rd S, Jacksonville, FL, 32224, USA
Department of Veterinary & Animal Sciences, University of Massachusetts, 661 N. Pleasant St., Amherst, MA, 01003, USA
Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, McKnight Brain Institute, College of Medicine, University of Florida, 1600 SW Archer Road, Gainesville, FL, 32608, USA
Department of Pharmacology and Neurology, Emory University School of Medicine, 1510 Clifton Rd NE, 5123 Rollins Research Center, Atlanta, GA, 30322, USA
source Molecular Neurodegeneration
issn 1750-1326
pubdate 2012
volume 7
issue 1
fpage 39
url http://www.molecularneurodegeneration.com/content/7/1/39
xrefbib pubidlist pubid idtype doi 10.1186/1750-1326-7-39pmpid 22892055
history rec date day 22month 6year 2012acc 682012pub 1482012
cpyrt 2012collab Das et al.; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg
kwd Aβ
Alzheimer’s disease
APP
Therapeutics
γ-secretase inhibition
abs
sec
st
Abstract
Background
Alzheimer’s disease (AD) is the leading cause of dementia among the elderly. Disease modifying therapies targeting Aβ that are in development have been proposed to be more effective if treatment was initiated prior to significant accumulation of Aβ in the brain, but optimal timing of treatment initiation has not been clearly established in the clinic. We compared the efficacy of transient pharmacologic reduction of brain Aβ with a γ-secretase inhibitor (GSI ) for 1–3 months (M) treatment windows in APP Tg2576 mice and subsequent aging of the mice to either 15M or 18M.
Results
These data show that reducing Aβ production in a 2-3M windows both initiated and discontinued before detectable Aβ deposition has the most significant impact on Aβ loads up to 11M after treatment discontinuation. In contrast, initiation of treatment for 3M windows from 7-10M or 12-15M shows progressively decreasing efficacy.
Conclusions
These data have major implications for clinical testing of therapeutics aimed at lowering Aβ production, indicating that; i) these therapies may have little efficacy unless tested as prophylactics or in the earliest preclinical stage of AD where there is no or minimal Aβ accumulation and ii) lowering Aβ production transiently during a critical pre-deposition window potentially provides long-lasting efficacy after discontinuation of the treatment.
bdy
Background
Alzheimer’s disease (AD) is the leading cause of dementia among the elderly. Numerous studies in humans have demonstrated the sequential development of various pathological features that characterize AD and the relationship of these pathologies to diagnosis of dementia
abbrgrp
abbr bid B1 1
. These studies support the amyloid cascade hypothesis which posits that accumulation of Aβ aggregates triggers a series of downstream pathologies that results in clinical AD
B2 2
. AD has recently been proposed to have three pre-clinical stages, characterized in sequence by the development of Aβ pathology (stage 1), the presence of plaque pathology, neurofibrillary pathology and initial evidence for neurodegeneration (stage 2), and finally by the presence of stage 2, pathologies plus evidence for subtle cognitive decline (stage 3). This progression likely occurs over a 10–20 year timeline
B3 3
.
The amyloid cascade hypothesis predicts that attenuating or preventing Aβ aggregation and accumulation will attenuate or prevent the subsequent development of AD. Strong experimental support for the amyloid hypothesis has supported the rationale for many therapeutic approaches targeting Aβ. Major investments have been made to develop inhibitors or modulators of Aβ production
B4 4
. γ-secretase inhibitors (GSIs), β-secretase inhibitors (BSIs) and γ-secretase modulators (GSMs) have been or are currently being tested in human AD trials. Both the GSM tarenflurbil (R-flurbiporfren), and the GSI semagacestat, failed to show efficacy in phase III human trials
4
B5 5
. Several other GSMs, GSIs, and BSIs are currently in early stage trials. Based on the clinical failures of tarenflurbil and semagacestat as well as other anti-Aβ therapies not targeting Aβ production but its aggregation or clearance, we and others have begun to question whether targeting of Aβ in symptomatic AD may be a futile endeavor
B6 6
.
Given the framework provided by the amyloid cascade hypothesis, disappointing results from completed trials of anti-Aβ therapies in patients with AD, and several but limited preclinical studies supporting the concept that when targeting Aβ “earlier is better”
B7 7
B8 8
B9 9
, we explored the relationship between long-term efficacy with respect to reducing amyloid loads and timing and duration of Aβ lowering. In designing these studies, we were struck by the parallels between Aβ accumulation in the mouse brain and it in vitro studies of Aβ aggregation. In vitro, Aβ aggregates in a nucleation dependent polymerization reaction, which exhibits three phases: a lag phase in which nucleation occurs but not fibril formation, an exponential growth phase in which fibril formation and growth occur rapidly, and a plateau phase where fibril growth and formation slows
B10 10
. Except for being more extended in terms of time, studies in APP Tg2576 transgenic mice show that Aβ aggregation and accumulation follows a similar course in the brain
B11 11
. There is an initial lag phase where no Aβ aggregation and accumulation occurs, followed by an exponential accumulation phase, and finally when amyloid loads become AD-like, the growth slows or plateaus (see Figure
figr fid F1 1A). Given these parallels, we tested the efficacy of transient lowering of Aβ production in 1-3M treatment “windows” corresponding to these various phases of deposition using the GSI LY-411,575 (LY) in APP Tg2576 mice. These data show that GSI treatment during the pre-deposition window (4-7M) before the early exponential phase of deposition (7-10M) or late exponential phase (12-15M) has by far the most efficacy in terms of reduction of plaque loads at 15M of age.
fig Figure 1caption Effects of LY window therapy on amyloid depositiontext
b Effects of LY window therapy on amyloid deposition. A. Time course of Aβ accumulation in Tg2576 mice. FA solubilized brain Aβ42 + Aβ40 levels measured by ELISA are shown (n = 3–5 mice/age group). B. Schematic of the various transient LY dosing strategies. Dotted lines represent the transient LY treatment time points. C. Aβ plaque burden analysis of treated cohorts compared to controls (*p < 0.05, ** ns, ANOVA). D. Biochemical analyses (FA solubilized Aβ42 + Aβ40 levels) by ELISA showing Aβ levels. (*p < 0.05, **ns, ANOVA). E. Representative brain sections stained with anti-Aβ mAb showing Aβ plaques at 15M of age in the LY treated cohorts. Scale bar = 100 μm.
graphic file 1750-1326-7-39-1
Results and discussion
In vitro and in vivo dosing studies with Ly-411,575
Ly-411,575 is potent orally bioavailable γ-secretase inhibitor
B12 12
. We synthesized large quantities for these studies and then performed both in vitro and in vivo studies to validate Ly-411,575 potency. The IC50 in cell culture for LY-411,575 was between 1–3 nm (see Additional file
supplr sid S1 1: Figure S1A). Six hours after a single IP dose, Aβ40 levels are reduced by ~75% in the brain of non-depositing Tg2576 mice (Additional file
1: Figure S1B). Oral administration (10 mg/kg/day) administered as a single dose reduces brain Aβ40 levels 24 hours after the final dose (7 days) by ~65% (Additional file
1: Figure S1C) and plasma Aβ40 by ~95% (Additional file
1: Figure S1D). Plasma Aβ levels are reduced more quickly than brain levels following oral dosing with maximal inhibition seen after 3 days (Additional file
1: Figure S1D). For long term in vivo mouse studies, we tested administration of Ly-411,575 using various dosing strategies and time points (Table
tblr tid T1 1). Oral administration (formulated in rodent chow) continually for 2 weeks with either 1 mg/kg/day or 2.5 5 mg/kg/day reduced brain Aβ40 levels by ~50% and plasma Aβ40 by ~80%, without overt toxicities ( Table
1). However, continuous oral dosing (>2 weeks) using the higher dose of 5 mg/kg/day or 10 mg/kg/day resulted in overt toxicities in mice (Table
1). Therefore, in all long term studies presented in this manuscript, we have used the 2.5 mg/kg/day dose formulated in rodent chow.
suppl
Additional file 1
Figure S1A Wild type APP overexpressing CHO cells were treated with indicated concentrations of LY-411,575 overnight. Conditioned media were then assayed for secreted Aβ40 by sandwich ELISA. Figure S1B. Acute LY-411,575 treatment reduces Aβ40 levels in brains of mice. Tg2576 mice (3 month old) were injected with LY-411,575 (5 mg/kg) intraperitoneally. Mice were sacrificed 6 hrs later and brain (2% SDS solubilized) Aβ40 levels were measured ELISA. (n=3 mice/group) Figure S1C, D. Tg2576 mice were dosed orally (suspended in Kool-Aid) with LY-411,575 (10 mg/kg) for indicated times and brain (2% SDS solubilized) Aβ40 levels (C) and plasma Aβ40 levels (D) were measured by ELISA (n=3 mice/group).
name 1750-1326-7-39-S1.pdf
Click here for file
table
Table 1
Brain and plasma Aβ40 levels following
in vivo
GSI treatment in Tg2576 mice
tgroup align left cols 8
colspec colname c1 colnum 1 colwidth 1*
c2 2
c3 3
c4 4
c5 5
c6 6
c7 7
c8
thead valign top
row rowsep
entry
Compound
Strain of Mice
Route
Dose
Length of Treatment
% Aβ40 Reduction
Brain
% Aβ40 Reduction
Plasma
Overt Toxicity
tfoot
Tg2576 mice (3 months old) were dosed with GSI LY-411,575 using various routes, time points and indicated doses. After GSI treatment, groups of mice (n =3/group) were sacrificed and mouse brains were harvested and extracted in 2% SDS. Brain and plasma Aβ40 levels were then measured by ELISA.
*Diarrhea and hair loss.
tbody
morerows
LY-411, 575
Tg2576
Intraperitoneal
5 mg/kg
6 hrs
75%
92%
No
10 mg/kg
6 hrs
77%
96%
No
25 mg/kg
6 hrs
78%
92%
No
LY-411, 575
Tg2576
Oral (Suspension in Kool-Aid)
10 mg/kg/day
1 Day
47%
70%
No
3 Days
65%
86%
No
7 days
75%
95%
No
LY-411, 575
Tg2576
Oral (Formulated In Rodent Chow)
1 mg/kg/day
2 weeks
36%
67%
No
2.5 mg/kg/day
2 weeks
51%
78%
No
5 mg/kg/day
2 weeks
78%
91%
Yes*
10 mg/kg/day
2 weeks
83%
95%
Yes*
Transient Aβ reduction dramatically reduces the subsequent accumulation of amyloid plaques
For these experiments, we transiently dosed Tg2576 mice with LY from 4-7M, 7-10M or 12-15M and aged treated mice to 15M (see Figure
1B for experimental design). To confirm efficacy of LY in all groups, we measured plasma Aβ levels immediately after the last day of treatment in sentinel mice and showed a similar reduction in plasma Aβ levels in all cohorts (Table
T2 2). Following sacrifice at 15M, levels of Aβ in the brain were examined. The 4-7M LY treatment significantly reduced Aβ deposition; plaque burden in the frontal cortex and hippocampus was decreased by ~68% (Figure
1C) and FA-solubilized brain Aβ levels were reduced by ~60% (Figure
1D). The 7-10M LY treatment non-significantly decreased Aβ plaque burden by ~19% compared to controls (Figure
1C), whereas FA-solubilized Aβ levels were reduced by ~34% (Figure
1D). The 12-15M GSI treatment group had no significant effect on either plaque burden or FA-solubilized Aβ levels (Figure
1C, D). We further evaluated effect of the 4-7M LY treatment on cored amyloid plaques and cerebral amyloid angiopathy (CAA). Compared to untreated control mice, there were significant reductions both in cored plaques in the frontal cortex and hippocampus (~47% reduction) and CAA in the leptomeninges (~41% reduction, Figure
F2 2A, B). To further determine whether the magnitude of the effect diminished with further aging, we performed an additional experiment, where we aged 4-7M LY treated mice to 18M, and again we observed significantly reduced Aβ plaque burden and FA-solubilized Aβ levels (Figure
2C), demonstrating that the suppression of deposition was maintained even up 11 months after treatment was halted.
Table 2
Plasma Aβ40 levels following transient GSI treatment in Tg2576 mice
center
Treatment Time
Treatment Groups
Plasma Aβ40 (pM)
Tg2576 mice were transiently dosed with GSI LY-411,575(2.5 mg/kg/day) at indicated time points. After GSI treatment was discontinued (on last day of treatment), plasma from groups of mice (n =3/group) was collected and plasma Aβ40 levels were measured by ELISA.
-Numbers in parenthesis represent percent reduction in Aβ levels.
4-7 Month
Ly-411,575
char ±
341.3 ± 20.1
(81%)
Control
1742.6 ± 103.5
7-10 Month
Ly-411,575
327.5 ± 18.5
(79%)
Control
1520.7 ± 100.8
12-15 Month
Ly-411,575
427.3 ± 31.4
(70%)
Control
1388.4 ± 223.2
Figure 2Effects of LY window therapy on amyloid deposition
Effects of LY window therapy on amyloid deposition. A. Quantitative burden analysis of cored amyloid deposits and CAA in the 4-7M LY treated group compared to controls. (*p < 0.05, Student’s t test). B. Representative brain sections stained with mAb 13.1.1 showing cored plaque and CAA immune-reactivity at 15M in the brains of Control and 4-7M LY treated Tg2576 mice. Arrows indicate CAA in meningeal vessels. Scale bar = 80 μm. C. Biochemical analyses of FA solubilized Aβ levels and plaque burden analysis from Tg2576 mice transiently dosed with LY (2.5mpk/day) from 4-7M, and aged to ul 18M. (*p < 0.05, Student’s t test). Tg2576 mice (n = 5-6/group) were used, data represented is from one independent experiment. D. Natural logarithm transformed Aβ values of untreated Tg2576 mice (at indicated ages) and Aβ levels from 4-7M LY treatment measured at 15M and 18M of age. E. Biochemical analyses of FA solubilized Aβ levels of Tg2576 mice transiently dosed with LY (2.5mpk/day) at 1, 2, and 3M intervals and then aged till 15M. (*p < 0.05, ANOVA). Tg2576 mice (n = 5-8/group) were used, data represented is from one independent experiment. F. Graph representing Aβ values of untreated Tg2576 mice (from indicated ages) and Aβ levels from 4-5M, 4-6M and 4-7M LY transient treatment measured at 15M. FA solubilized brain Aβ42 + Aβ40 levels measured by ELISA are shown.
1750-1326-7-39-2
Because Aβ deposition in mice follows an exponential course, we sought to determine how the observed reductions in Aβ accumulation correlate with Aβ accumulation to untreated mice. We compared Aβ levels in the brains of untreated Tg2576 mice from various ages ranging from 10–18 months and plotted the natural logarithm transformed value of these against Aβ levels from the 4-7M LY treated mice, aged to 15M and 18M (Figure
2D). To minimize variance, we assayed all of the samples in a single set of ELISA studies. This analysis showed that the 4 -7M GSI treatment time frame “shifts” amyloid depositing by approximately 3 months, essentially delaying Aβ accumulation by a time roughly equivalent to the treatment window.
We next attempted to determine whether this 4-7M narrow therapeutic “window” can be further refined. To do this, we transiently dosed Tg2576 mice with LY for 1, 2 or 3M intervals between the 4-7M age time frame and again aged the mice to 15M. Aβ levels were again significantly reduced in this second 4-7M LY cohort (~59% reduction) at 15M of age compared to controls (Figure
2E). Similarly, i) the 4-6M LY cohort showed a significant reduction in Aβ levels (~51% reduction) and; ii) the 4-5M LY group showed a modest but non-significant reduction in Aβ levels (~ 24% reduction) (Figure
2E). However, in the remaining 5-6M, 6-7M or 5-7M LY cohorts tested, none showed any significant reductions in Aβ levels at 15M compared to controls (Figure
2E). Since we show that the 4-7M LY “shifts” amyloid deposition equivalently to the duration of treatment, we wondered whether we will see a similar shift in amyloid deposition in the shorter treatment intervals examined that showed efficacy, e.g., in the 4-5M and 4-6M cohorts. Again, we compared Aβ levels in the brains of untreated Tg2576 mice from various ages ranging from 10–15 months against Aβ levels from the 4-5M, 4-6M, and 4-7M LY treated cohorts aged to 15M (Figure
2F). This analysis seems to show that the 4-5M GSI treatment “shifts” amyloid depositing by approximately 1 month, however, both the 4-6M and 4-7M treatment intervals essentially show a similar shift in Aβ accumulation, namely by roughly 3 months (Figure
2F). These data would suggest, at least within the 4-7M time frame that we have studied, that the 4-6M age interval may play a critical role in the early seeding phases of amyloid deposition. Additional studies, using either earlier treatment times (e.g. starting at 3M of age) or longer treatment intervals may be necessary to fully appreciate the usefulness of this early “therapeutic window” to achieve the best possible outcome on subsequent amyloid deposition.
Transient GSI does not permanently alter APP levels or processing
To determine if the 4-7M LY treatment had long-lasting effects on Aβ production or APP levels and processing, we conducted a number of studies. We measured plasma Aβ levels at termination of GSI treatment and then determined how long before steady state levels of Aβ were normalized. Plasma Aβ levels were significantly lower immediately after treatment, but returned to control levels between with 1–2 weeks after treatment was halted (Figure
F3 3A). We also measured levels of full-length APP and APP C-terminal fragments (CTFs) in the brain at the end of the 4-7M treatment and at 15M. Immediately following discontinuation of treatment, APP levels were unchanged but APP CTFs were increased (Figure
3B,D). However, at 15 months, there were no significant differences in full length APP or CTFs (Figure
3C,D). Finally, as GSI treatment has been shown to affect peripheral lymphocytes
B13 13
, we performed FACS analysis and quantified both B and T cell numbers in the spleen after the 4–7M LY transient treatment and at 15M. B and T cell numbers were not affected (Figure
F4 4A). However, when we analyzed Th1 and Th2 cytokine profiles, splenic CD4+ T cells from the 4-7M LY treated cohort showed a bias towards Th1 polarization with increased IFN-γ secretion (a canonical Th1 cytokine) and concurrent decreases in IL-4 levels (a signature Th2 cytokine), which persisted even after the treatment was halted for 8 months (Figure
4B, C,). The significance of this long-lasting effect on peripheral CD4 + T cell immune responses is not clear, but certainly warrants further investigation.
Figure 3Long term LY treatment does not permanently affect APP levels or processing
Long term LY treatment does not permanently affect APP levels or processing. A. Plasma Aβ40 levels by ELISA after the end of 4–7M LY treatment, (7M), 1 week after termination of treatment, and 2 weeks after termination of treatment. B. Representative anti-APP CT20 immunoblot shows APP levels and accumulation of CTFs after 4-7M LY treatment (tested at 7M). C. Representative anti-APP CT20 immunoblot showing full length APP levels and CTFs after transient 4-7M LY treatment (tested at 15M). D. Quantitative intensity analysis of anti-CT20 immuno-reactive full length APP and APP CTF protein levels normalized to actin.
1750-1326-7-39-3
Figure 4Effect of LY treatment on peripheral immune cells
Effect of LY treatment on peripheral immune cells.A. Distribution of splenic B cell (IgD, CD19) and T cells (CD4 and CD8) from control and Tg2576 mice treated with LY (4-7M). Percent positive cells are shown (n = 4 mice/group). B and C. IFN production (B) and IL-4 production (C) by CD4+ T cells from LY (4-7M) treated Tg2576 mice and untreated control mice were measured by ELISA. Equal numbers of purified, splenic CD4+ T cells (pooled from n =4 mice/group) were used. ND = not determined.
1750-1326-7-39-4
Conclusion
Our current data show that transiently lowering Aβ production in Tg2576 mice during the pre-deposition “seeding” phase has a major impact on subsequent Aβ accumulation with the effect persisting for at least 11M. Later treatment windows showed decreasing efficacy of treatment. These current data have major implications for trial design targeting Aβ production in humans. Indeed, they would suggest that efficacy in terms of plaque reduction is likely to be maximal during seeding phase, and rapidly decline if treatment is initiated during the exponential phase of deposition. A recent report
B14 14
using passive immunotherapy provided more limited evidence that a similar effect may be observed with anti-Aβ passive immunotherapy initiated during the early deposition phase persisting for 3 months after discontinuation of treatment. Given the plethora of anti-Aβ therapies currently in development, it will be essential to determine for each modality whether efficacy is both maintained after discontinuation and similarly influenced by timing of the treatment.
The near equivalence of the delay in subsequent deposition to the length of the treatment window during a pre-deposition phase suggests that the GSI treatment from 4-7M alters or reverses seeding of Aβ. Indeed, there are no plaques forming during this period of time. Future studies will be necessary to determine the exact mechanisms by which lowering Aβ in this window alters subsequent deposition. As even in vitro detection of Aβ nucleation relies on indirect assays that have limited sensitivities, these studies will likely require a better understanding of nucleation and better assays to detect it. Developing such assays will be critical as direct translation of these studies would be facilitated by defining the equivalent window in humans. Regardless of mechanism, these preclinical window therapy studies have major implications regarding the potential of prophylactic therapy targeting Aβ production in humans. Not only do these data suggest that prophylactic therapy in the appropriate window will be more efficacious then initiating therapy in individuals with any preexisting Aβ pathology, but they suggest that such therapy, even if discontinued might delay subsequent onset AD by a time roughly equivalent to the time of treatment.
Methods
Tg2576 mice
APP Tg2576 mice were generated, maintained and genotyped as described previously
7
. All animal husbandry procedures performed were approved by the Mayo Clinic Institutional Animal Care and Use Committee in accordance with National Institutes of Health guidelines. All animals were housed three to five to a cage and maintained on ad libitum food and water with a 12 h light/dark cycle.
LY-411, 575
LY-411, 575 (LY) was synthesized at the Mayo Clinic Chemical Core as previously described
B15 15
. LY at a concentration of 16.75 mg per kg of diet was homogenously incorporated into Harlan Teklad 7012 kibble chow by Research Diets, Inc, New Jersey). Based upon dietary consumption at this age, these diets were designed to deliver 2.5 mg of LY on average per day. Tg2575 mice (n = 7-15/group/experiment) were transiently treated with LY chow at 1, 2 or 3 months interval (between 4–7 months of age), from 7–10 months of age and from 12–15 months of age. Untreated control mice from each age group were as aged and used for analysis. Groups of mice (n =3) were sacrificed immediately after the end of each treatment time-point to access efficacy of LY inhibition. After treatment was halted, mice were then aged till 15 months or 18 months of age and then sacrificed for Aβ analysis. Chow consumption, general health and body weight were monitored on a weekly basis in all treatment groups.
Biochemical Aβ ELISA assay
Snap-frozen hemi-forebrains from Tg2576 mice were two-step sequentially extracted in 2% SDS buffer followed by 70% formic acid (FA) as described previously
B16 16
. Aβ levels from brain lysates or plasma were the measured using sandwich ELISA techniques as described previously
16
with end specific mAbs 2.1.3 (human Aβx-42 specific, Mayo) and mAb 13.1.1 (human Aβx-40 specific, Mayo) for capture and HRP-conjugated mAb Ab5 (human Aβ1–16 specific, Mayo) for detection.
Immunohistological analysis
Sagittal sections of paraffin embedded sections were used for analysis. Immunohistochemical staining was done using pan-Aβ antibody (mAb 33.1.1; Mayo), mAb 13.1.1 (human Aβx-40 specific, binds cored plaques and CAA; Mayo) as previously described
B17 17
. Immunohistochemically stained sections were captured using the Scanscope XT image scanner (Aperio, Vista, CA, USA) and analyzed using the ImageScope program. Aβ plaque burden was then calculated using the Positive Pixel Count program (Aperio). At least 5 sections/brain 30 μm apart, were used and averaged by a blinded observer to calculate plaque burden.
Western blotting
2% SDS solubilized brain lysate samples were separated on Bis-Tris 12% XT gels (Bio-Rad, Hercules, CA, USA) and probed with the antibody CT20 (anti-APP C-terminal 20 amino acid; 1:1000; P.D) and anti-actin (1:1000; Sigma, St. Louis, MO, USA). Band intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Cell isolation, in vitro polarization and Immuno-phenotyping
Single cell suspensions were prepared aseptically from spleens from control or GSI -treated mice. CD4+ T cells were positively selected using the BD-IMAG system and CD4 + DM particles (BD Biosciences, San Diego, CA), according to the manufacturer’s directions. Twelve-well tissue culture plates were precoated with anti-hamster Ig (Sigma), then with antibodies specific for CD3ε and CD28. Equal numbers of cells (3x10sup 6) were added to each well. For TH1 polarizing conditions, anti-IL4 (10μg/ml) plus IL-12 (1 ng/ml) was added to culture wells; for TH2 polarizing conditions, anti-IFNγ (10μg/ml) plus IL-4 (1 ng/ml) was added to culture wells. No additional reagents were added to cells cultured under neutral conditions. After 24 hours, all wells were pulsed with IL-2 (10 ng/ml). After 96 hours, supernatants were collected and frozen at -80C until analyzed by ELISA for cytokine secretion. IFNγ and IL-4 cytokine secretion was measured using ELISA kits per manufactures instructions (BD Pharmingen). For cell surface staining, approximately 1x106 cells (each antibody) were stained with the following antibodies individually: IgD-FITC, biotinylated anti-CD19 plus strep-PE, CD4-FITC, CD8-PE (BD Pharmingen). Data were analyzed using an LSRII flow cytometer (BD Biosciences) and FACSDiva software for acquisition and analysis.
Statistics
All statistics were done using Graphpad Prism (Version 5.0). Comparisons of multiple groups were done by one-way ANOVA followed by Tukey's post hoc testing. Comparisons between two groups were done by Student's t test with Welch's correction for unequal variances where appropriate.
Competing interests
The authors have no competing interests.
Authors’ contributions
PD and TEG designed, analyzed and interpreted all aspects of the study and co-wrote the manuscript. CV performed all animal dosing studies, sacking and harvesting of tissues and ELISA analysis. PC performed western blotting techniques, tissue staining and quantification of plaque burdens. KF assisted with conceptualization of the study and edited the manuscript. TK performed in vitro cell culture analysis of LY-411, 575. GM and AF synthesized LY-411, 575 used in this study. LM and BAO performed immune cell isolation, in vitro polarization and immuno-phenotyping studies. All authors read and approved the final manuscript.
bm
ack
Acknowledgements
The authors thank Monica Castanedes-Casey, Virginia Phillips, and Linda Rousseau for assistance with histopathology. Supported by the National Institutes of Health/National Institute on Aging grant (P01AG25531 to BAO, TEG).
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epdcx:valueString Transient pharmacologic lowering of Aß production prior to deposition results in sustained reduction of amyloid plaque pathology
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Abstract
Background
Alzheimer’s disease (AD) is the leading cause of dementia among the elderly. Disease modifying therapies targeting Aβ that are in development have been proposed to be more effective if treatment was initiated prior to significant accumulation of Aβ in the brain, but optimal timing of treatment initiation has not been clearly established in the clinic. We compared the efficacy of transient pharmacologic reduction of brain Aβ with a γ-secretase inhibitor (GSI ) for 1–3 months (M) treatment windows in APP Tg2576 mice and subsequent aging of the mice to either 15M or 18M.
Results
These data show that reducing Aβ production in a 2-3M windows both initiated and discontinued before detectable Aβ deposition has the most significant impact on Aβ loads up to 11M after treatment discontinuation. In contrast, initiation of treatment for 3M windows from 7-10M or 12-15M shows progressively decreasing efficacy.
Conclusions
These data have major implications for clinical testing of therapeutics aimed at lowering Aβ production, indicating that; i) these therapies may have little efficacy unless tested as prophylactics or in the earliest preclinical stage of AD where there is no or minimal Aβ accumulation and ii) lowering Aβ production transiently during a critical pre-deposition window potentially provides long-lasting efficacy after discontinuation of the treatment.
http:purl.orgdcelements1.1creator
Das, Pritam
Verbeeck, Christophe
Minter, Lisa
Chakrabarty, Paramita
Felsenstein, Kevin
Kukar, Thomas
Maharvi, Ghulam
Fauq, Abdul
Osborne, Barbara A
Golde, Todd E
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BioMed Central Ltd
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Pritam Das et al.; licensee BioMed Central Ltd.
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Molecular Neurodegeneration. 2012 Aug 14;7(1):39
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