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Orally available compound prevents deficits in memory caused by the Alzheimer amyloid- oligomers.

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Orally Available Compound Prevents
Deficits in Memory Caused by the
Alzheimer Amyloid-␤ Oligomers
Matthew Townsend, PhD,1* James P. Cleary, PhD,2,3 Tapan Mehta, BS,1 Jacki Hofmeister, VT,2
Sylvain Lesne, PhD,4 Eugene O’Hare, PhD,5 Dominic M. Walsh, PhD,6 and Dennis J. Selkoe, MD1
Objective: Despite progress in defining a pathogenic role for amyloid ␤ protein (A␤) in Alzheimer’s disease, orally bioavailable
compounds that prevent its effects on hippocampal synaptic plasticity and cognitive function have not yet emerged. A particularly attractive therapeutic strategy is to selectively neutralize small, soluble A␤ oligomers that have recently been shown to
mediate synaptic dysfunction.
Methods: Using electrophysiological, biochemical, and behavioral assays, we studied how scyllo-inositol (AZD-103; molecular
weight, 180) neutralizes the acutely toxic effects of A␤ on synaptic function and memory recall.
Results: Scyllo-inositol, but not its stereoisomer, chiro-inositol, dose-dependently rescued long-term potentiation in mouse hippocampus from the inhibitory effects of soluble oligomers of cell-derived human A␤. Cerebroventricular injection into rats of the
soluble A␤ oligomers interfered with learned performance on a complex lever-pressing task, but administration of scyllo-inositol
via the drinking water fully prevented oligomer-induced errors.
Interpretation: A small, orally available natural product penetrates into the brain in vivo to rescue the memory impairment
produced by soluble A␤ oligomers through a mechanism that restores hippocampal synaptic plasticity.
Ann Neurol 2006;60:668 – 676
The large, insoluble deposits (amyloid plaques) of aggregated amyloid ␤-protein (A␤) that occur abundantly in the limbic and association cortices of Alzheimer’s disease (AD) patients are in equilibrium with
small, diffusible oligomers of the peptide that appear
capable of interfering with hippocampal synaptic function and memory.1 Secreted forms of soluble A␤ oligomers can be generated by cultured cells that express
human ␤-amyloid precursor protein (APP).2 These
low-n oligomers are present in the conditioned medium (CM) at low-nanomolar to subnanomolar concentrations similar to those of A␤ in human cerebrospinal fluid (CSF) and brain.3,4 Radiosequencing,
immunoprecipitation with various A␤-specific antibodies, and separation by size-exclusion chromatography
under nondenaturing conditions have all established
that these dimers, trimers, and tetramers are composed
of N- and C-terminally heterogeneous human A␤ peptides, including the A␤1-40 and A␤1-42 species that occur in human brain and extracellular fluids.2,4 – 6 In
normal adult rats, the oligomers have been found to
potently inhibit hippocampal long-term potentiation
(LTP) in vivo,7 and this synaptic effect can be prevented by A␤ antibodies in vivo via passive infusion or
active A␤ vaccination.8 Moreover, intracerebroventricular (ICV) injection of the secreted oligomers interferes
potently but transiently with the memory of a complex
task in rats.9
Given the enormous personal and societal burdens
that AD inflicts, agents that interfere with the adverse
effects of A␤ on neuronal function are being sought
intensively. McLaurin and colleagues10,11 identified
myo-inositol, the head group of the naturally occurring glycolipid phosphatidylinositol, and certain ste-
From the 1Center for Neurologic Diseases, Harvard Medical School
and Brigham and Women’s Hospital, Boston, MA; 2Geriatric Research, Education and Clinical Center, Minneapolis Veterans Affairs
Medical Center; Departments of 3Psychology and 4Neurology, University of Minnesota, Minneapolis, MN; 5School of Psychology,
Queen’s University, Belfast, United Kingdom; 6Laboratory for Neurodegenerative Research, Conway Institute of Biomedical and Biomolecular Research, University College Dublin, Dublin, Republic of
Ireland.
M.T. and J.P.C. contributed equally to this work.
*Current address: Merck Research Laboratories—Boston, Boston,
MA 02115
This article includes supplementary materials available via the
Internet at http://www.interscience.wiley.com/jpages/0364-5134/
suppmat
Published online Dec 22, 2006 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.21051
Address correspondence to Dr Selkoe, Center for Neurologic Diseases, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA 02115. E-mail: dselkoe@rics.bwh.harvard.edu
Received Aug 7, 2006, and in revised form Oct 30. Accepted for
publication Nov 6.
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Published 2006 by Wiley-Liss, Inc., through Wiley Subscription Services
reoisomers as small-molecule inhibitors of synthetic
A␤42 aggregation and neurotoxicity in vitro. These
investigators showed that, among inositol stereoisomers, myo-inositol, scyllo-inositol (also called scyllocyclohexanehexol or AZD-103), and epi-inositol, but
not chiro-inositol, interacted with A␤42 peptide in
vitro to promote its conformational change from random coil to ␤-sheet structure and stabilized it in
small, nonfibrillar complexes.11 These stabilized complexes were significantly less toxic to NGFdifferentiated PC-12 cells and primary human neuronal cultures than were untreated A␤42 or chiroinositol–treated A␤42.11 The authors observed that
the active inositol stereoisomers attenuated neurotoxicity, at least in part by interfering with the ability of
A␤42 to interact with the neuronal membrane.11
They recently reported that scyllo-inositol reduces
plaque burden in TgCRND8 mice and improves performance on memory tasks such as the Morris water
maze.12
Many features of scyllo-inositol, including its small
size, neutral pH, stability, stereo-selectivity, and favorable toxicity profile, make it a highly attractive
candidate to test for therapeutic benefit in AD. Here,
we demonstrate that this small, orally available compound can successfully neutralize the inhibitory ef-
Fig 1. Cyclohexanehexols rescue long-term potentiation (LTP)
from the inhibitory effects of soluble amyloid ␤ protein (A␤)
oligomers. (A) Perfusion of wild-type mouse hippocampal slices
with conditioned media (CM) collected from control CHO
cells had no effect on LTP. The CM was applied to a recirculating perfusion for 20 minutes before delivering 4 highfrequency stimuli (arrows; 100Hz over 1 second). In contrast,
the CM of 7PA2 cells readily inhibited LTP at about 1 hour
after HFS and thereafter (7PA2 CM ⫺ LTP at 60 minutes ⫽ 133.2 ⫾ 5.4 standard error of the mean [SEM]
[n ⫽ 26] and CHO⫺ CM LTP at 60 minutes ⫽ 213.4 ⫾
12.0 [n ⫽ 20]; Student’s t test, p ⬍ 0.01). (B) A 15
-minute preincubation of 7PA2 CM with 1.25 ␮M AZD103 (scyllo-cyclohexanehexol) before perfusion over brain slices
was sufficient to rescue LTP (LTP at 60 minutes ⫽ 205.5 ⫾
14.1; n ⫽ 11). Two enantiomers of AZD-103 were similarly
tested. Epi-cyclohexanehexol also restored LTP to control levels
(LTP at 60 minutes ⫽ 184.2 ⫾ 13.8; n ⫽ 7), whereas
chiro-cyclohexanehexol was not significantly different from
7PA2 alone (127.9 ⫾ 6.3; n ⫽ 8) (analysis of variance
[ANOVA] Tukey’s post hoc test, p ⬍ 0.05 for epi vs chiro
and p ⬍ 0.01 for scyllo vs chiro). (C) Basal synaptic transmission was unaffected by AZD-103 (scyllo alone, no HFS),
and LTP induced in the presence of CHO⫺ CM was similarly unaltered by AZD-103 (LTP at 60 minutes ⫽ 219.2 ⫾
19.3; n ⫽ 7). The addition of 1.25 ␮M AZD-103 to the
perfusion 20 minutes after the addition of 7PA2 CM (postperfusion) did not rescue the inhibition of LTP (LTP at 60
minutes ⫽ 138.3 ⫾ 8.3; n ⫽ 5; ANOVA Tukey’s post hoc
test, p ⬎ 0.05).
fects of pre-existing A␤ oligomers on hippocampal
LTP and rescue memory function in awake, behaving
animals.
Results
Scyllo-inositol Prevents the Inhibition of Long-term
Potentiation by Soluble Amyloid ␤ Protein Oligomers
Field potential recordings were made in the CA1 region of wild-type mouse hippocampal slices. As in our
previous work,5,7 a brief (20-minute) treatment of hippocampal neurons with A␤-rich CM from CHO cells
stably expressing human APPV717F (7PA2 cells) caused
a strong and statistically significant inhibition of LTP
measured at 60 minutes after high-frequency stimulation, compared with CM from the untransfected parental line (designated CHO⫺) (Fig 1A). Lowmolecular-weight oligomers of A␤, particularly trimers,
‹
Townsend et al: A␤ Neutralized by AZD-103
669
isolated from the 7PA2 CM by nondenaturing size exclusion chromatography (SEC) have been shown to be
sufficient to mediate this inhibition of LTP, whereas
the simultaneously isolated A␤ monomer is without effect.5,13
To determine whether scyllo-inositol could neutralize
the effect of soluble A␤ oligomers on this form of hippocampal synaptic plasticity, we preincubated 7PA2
CM in vitro for 15 minutes with 1.25 ␮M scylloinositol before perfusion over brain slices. This low micromolar concentration of scyllo-inositol fully rescued
LTP from the inhibitory effects of 7PA2 CM (see Fig
1B). Two enantiomers of scyllo-inositol (see Suppl. Fig.
3) were tested to establish whether the rescue of LTP
was stereospecific within this family of compounds.
Epi-inositol (epi-cyclohexanehexol), which protects
neurons from synthetic A␤42 cytotoxicity,11 also rescued
LTP,
whereas
chiro-inositol
(chirocyclohexanehexol), which is inactive in assays of synthetic A␤42 neurotoxicity,11 was without effect (see Fig
1B). Thus, two cyclohexanehexol enantiomers previously shown to stabilize synthetic A␤ as a small, nontoxic conformer effectively neutralized the inhibitory
effects of secreted, soluble A␤ oligomers on LTP. Because scyllo-inositol (AZD-103) proved to be more effective at protecting PC-12 cells than epi-inositol,11 all
further studies were performed with the former compound.
We performed additional controls to test AZD-103
for nonspecific effects on LTP. AZD-103 alone did not
alter baseline synaptic transmission (no high-frequency
stimulation), nor did it affect the normal LTP induced
in the presence of control CHO⫺ CM (see Fig 1C). A
20-minute application of 1.25 ␮M AZD-103 after perfusing slices with 7PA2 CM did not confer any protection on LTP compared with 7PA2 alone (see Fig
1C). These results demonstrate that although AZD103 can neutralize the acute synaptic effects of A␤ before the soluble oligomers encounter hippocampal tissue, it did not reverse the effects of A␤ under these
experimental conditions.
Dose and Time Dependence of the AZD-103 Effects
The rescue of LTP by AZD-103 was found to be dose
dependent. The final concentration of AZD-103 added
to 7PA2 CM (“postconditioning”) was varied between
0.125 and 5 ␮M and then tested in LTP experiments,
as done previously (Fig 2A). The 1.25 ␮M dose was
found to be the lowest tested concentration that significantly improved LTP. The 7PA2 CM prepared for
this set of experiments was slightly more potent than
that shown in Figure 1, and thus should be compared
with the internal controls within this experiment (see
Fig 2A). A curve fit applied to the data set suggested an
inhibitory concentration of 50% (IC50) of approximately 1.0 ␮M.
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Fig 2. Dose- and time-dependent responses of AZD-103 on
hippocampal long-term potentiation (LTP). (A) The concentration of AZD-103 added to 7PA2 conditioned media (CM)
was varied between 0.125 and 5.0␮M before testing on LTP.
In these postconditioning experiments (diamonds), 1.25␮M
AZD-103 was the lowest concentration that provided significant protection against the inhibitory effects of A␤ on LTP at
60 minutes after HFS (5.0␮M AZD-103 LTP at 60 minutes ⫽ 201.3 ⫾ 12.9, n ⫽ 5; 1.25␮M AZD-103 LTP at
60 minutes ⫽ 183.4 ⫾ 10.4, n ⫽ 9; 0.5␮M AZD-103
LTP at 60 minutes ⫽ 134.1 ⫾ 6.0, n ⫽ 13; and
0.125␮M AZD-103 LTP at 60 minutes ⫽ 120.8 ⫾ 10.6,
n ⫽ 4; analysis of variance [ANOVA] Tukey’s post hoc test, p
⬍ 0.05). Based on these data, the inhibitory concentration
50% (IC50) is predicted to be approximately 1␮M under
these experimental conditions. In preconditioning experiments
(circles), AZD-103 was added directly to the 7PA2 cells during the conditioning period, resulting in almost complete rescue
of LTP at 0.125␮M (0.5␮M AZD-103 LTP at 60 minutes ⫽ 191.9 ⫾ 12.6, n ⫽ 7; and 0.125␮M AZD-103
LTP at 60 minutes ⫽ 184.7 ⫾ 10.7, n ⫽ 6; ANOVA
Tukey’s post hoc test, p ⬍ 0.05). The controls for this experiment are: 1.25␮M AZD-103 ⫹ CHO⫺ (⫻), LTP at 60
minutes ⫽ 201.0 ⫾ 13.2, n ⫽ 7; 7PA2 alone (‚), LTP at
60 minutes ⫽ 112.8 ⫾ 12.4, n ⫽ 4; 7PA2 no HFS, LTP
at 60 minutes ⫽ 88.4 ⫾ 7.7, n ⫽ 3 䊐). (B) The duration
of the AZD-103 (0.5␮M) coincubation with 7PA2 CM was
varied between 30 minutes and 4 hours (light gray bar, 30
minutes; gray bar, 2 hours; dark gray bar, 4 hours). (Compare to 15 min co-incubation at this dose shown in Fig. 2A.)
No improvement in the small partial rescue of LTP at this
low dose occurred with longer coincubation (ANOVA Tukey’s
post hoc test, p ⬎ 0.05).
We next determined whether application of AZD103 directly to 7PA2 cells during their production and
secretion of the oligomers (referred to as “preconditioning”) would similarly rescue the effects of the secreted A␤. AZD-103 was added to 7PA2 cells at the
beginning of the 15-hour conditioning period, after
which the CM was collected (prepared as described in
Methods) and perfused over brain slices as in Figure 1.
Intriguingly, when applied directly to the A␤-secreting
7PA2 cells, AZD-103 proved more effective at rescuing
LTP, achieving almost full rescue at the lowest concentrations tested (0.125␮M) (see Fig 2A, circles). This
could not be attributed to toxicity to the 7PA2 cells,
because they continued to robustly secrete a range of
proteins (including A␤) into the medium at the same
levels as untreated cells (see later). Therefore, AZD-103
not only neutralized pre-existing A␤ oligomers, but
provided an additional benefit when applied directly to
cells during oligomer generation and secretion.
A time course was established to determine whether
coincubating 7PA2 CM with AZD-103 for longer periods would affect the rescue of LTP. A dose of 0.5␮M
AZD-103, which had a modest effect after a 15-minute
postconditioning incubation with 7PA2 CM (see Fig
2A), was chosen to test whether extending the coincubation from 30 minutes to 4 hours would improve the
effectiveness of the compound. As shown in Figure 2B,
there was no additional benefit of incubations longer
than 15 minutes.
AZD-103 Binds to but Does Not Depolymerize CellDerived, Low-n Amyloid ␤ Protein Oligomers
AZD-103 has been found to bind to and alter the conformation of pure, synthetic A␤ in vitro11 and to inhibit endogenous A␤ aggregation in vivo.12 To test for
depolymerization of the oligomers by AZD-103, we
fractionated the 7PA2 CM using SEC. As previously
reported, this technique effectively separates soluble A␤
species by molecular weight.5 The cell-secreted oligomers were highly stable during overnight incubations
at 37°C and did not spontaneously aggregate or disaggregate.4 Treating the oligomer-containing SEC fractions with AZD-103 (5␮M) for 14 hours did not significantly alter the pattern or intensity of the A␤
species (Fig 3A). We conclude that AZD-103 does not
decrease the stability of low-n oligomers.
Although AZD-103 did not alter the electrophoretic
pattern of the SEC-fractionated oligomers on sodium
dodecyl sulfate polyacrylamide gel electrophoresis, the
compound did directly neutralize the synaptic effects of
these isolated, low-n oligomers. SEC fractions containing A␤ dimers, trimers, and tetramers were pretreated
with AZD-103 and tested in LTP experiments.
Whereas untreated SEC fractions that contained these
oligomers readily inhibited LTP as expected, pretreating equivalent SEC fractions with 1.25␮M AZD-103
fully restored normal LTP (see Fig 3B), confirming
that AZD-103 specifically neutralizes the inhibitory effects of isolated, low-n oligomers of human A␤ on this
form of synaptic plasticity.
Because AZD-103 did not destabilize low-n oligomers yet did neutralize their effects on LTP, we hypothesized that perhaps AZD-103 binds to A␤ oligomers in their native conformation and masks one or
more epitopes that are essential for their biological effects. If so, we reasoned that AZD-103 may compete
with anti-A␤ antibodies for binding to A␤ in immunoprecipitation experiments. Although multiple antibodies to distinct epitopes within A␤ were tested
(R1282, 6E10, m266, 4G8, and 12F4; see Supplemental Figs 1A and D), we observed no significant difference in the immunoprecipitation profile of 7PA2 CM
pretreated with AZD-103 compared with that of untreated 7PA2 CM, as exemplified with R1282 in Figure 3C. Even high AZD-103 concentrations (100␮M)
did not alter the ability to immunoprecipitate the A␤
species and detect them by Western blotting (see Supplemental Fig 1A). We conclude that AZD-103 neither
destabilizes A␤ oligomers nor interferes with epitope
recognition by multiple anti-A␤ antibodies.
Because submicromolar concentrations of AZD-103
were sufficient to rescue LTP when applied directly to
the 7PA2 cells (but not when applied to the 7PA2 CM
post-conditioning), we investigated whether this difference could be attributed to changes in the pattern of
oligomers found in the CM. However, no differences
were observed in the oligomer/monomer patterns after
direct treatment of the 7PA2 cells with AZD-103
(“preconditioning”) compared with the addition of
AZD-103 to the collected CM (“postconditioning”)
(see Fig 3C). Thus, the available data suggest that although AZD-103 does not interfere with A␤ synthesis
or oligomerization in our system, it does have a more
potent ability to bind and neutralize the oligomers
when AZD-103 is present at the time of oligomer formation. Notably, these results also indicate that the
health of the 7PA2 cells was not compromised by incubation with AZD-103.
Epoxy-activated Sepharose has been shown to bind
to myo-inositols similar to AZD-103.14 We therefore
coupled AZD-103 or its inactive stereoisomer, chiroinositol, to this resin to determine whether AZD-103
would pull down A␤ species from 7PA2 CM (see Fig
3D and Supplemental Fig 1E). The chiro/epoxy resin
preferentially pulled down monomeric A␤, whereas the
AZD-103/epoxy resin preferentially pulled down A␤
trimers (n ⫽ 5) relative to beads alone. Notably, only
the active stereoisomer, AZD-103, bound trimeric A␤
above background levels, and we have shown the trimer to be a particularly potent inhibitor of LTP.13
These results demonstrate that AZD-103 binds directly
Townsend et al: A␤ Neutralized by AZD-103
671
Fig 3. AZD-103 neutralizes the synaptotoxic activity of isolated low-n amyloid ␤ protein (A␤) oligomers but does not alter their
stability. (A) Size exclusion chromatography (SEC) was used to separate A␤ species as described previously.5 The fractions were incubated for 14 hours with or without 5.0 ␮M AZD-103 at 37°C, and then subjected to Western blotting. Treatment with AZD103 did not cause a significant change in the size distribution of any A␤ species (blot is representative of three independent SEC
runs). (B) Nevertheless, AZD-103 neutralized the synaptotoxic effects of SEC fractions 7 and 8 in LTP experiments. SEC fractions
were preincubated with AZD-103 at 37°C for 20 minutes before delivering a HFS (as in Fig 1) (SEC frac 7 and 8 LTP at 60
minutes ⫽ 131.1 ⫾ 14.3, n ⫽ 7; SEC frac 7 and 8 ⫹ 1.25␮M AZD-103 LTP at 60 minutes ⫽ 222.0 ⫾ 26.47, n ⫽ 5;
Student’s t test, p ⬍ 0.01). (C) Similar to the SEC results in the western blots in (A), immunoprecipitation of A␤ from whole
7PA2 conditioned media (CM) showed no significant effects of AZD-103 on A␤ oligomers. AZD-103 was applied directly to 7PA2
cells during the conditioning period (pre-cond) or else to the CM (post-cond) and immunoprecipitated with a polyclonal anti-A␤
antibody (R1282). There was no clear dose-dependent effect of AZD-103 as detected by blotting with anti-A␤ monodoxal antibody
6E10. (D) AZD-103, coupled to epoxy-agarose beads, precipitated A␤ oligomers from 7PA2 CM. Low levels of A␤ adhered nonspecifically to the beads alone. Nevertheless, AZD-103/epoxy-agarose preferentially bound trimeric A␤ (n ⫽ 5), which we have reported to be a potent inhibitor of LTP, whereas the inactive isomer chiro-inositol preferentially bound monomeric A␤. The standard pattern of A␤ bands in the 7PA2 cm is shown after R1282 IP in the right lane. An additional experiment using stronger
blocking conditions is shown in Supplemental Figure 1. MW ⫽ molecular weight.
to and neutralizes secreted A␤ trimers that inhibit hippocampal LTP.
AZD-103 Prevents Reference Memory Errors Induced
by Amyloid ␤ Protein Oligomers In Vivo
We previously reported that the SEC-isolated A␤ oligomers from 7PA2 CM significantly increase errors
during recall of a complex learned behavior in normal
adult rats, whereas the A␤ monomer fraction from the
same chromatography run and at greater concentrations has no effect.9 Similar results were also obtained
with whole 7PA2 CM compared with control CHO⫺
CM.9 We therefore used the same learned task, the Alternating Lever Cyclic Ratio (ALCR) assay, to determine whether AZD-103 could mitigate the detrimental
effects of soluble A␤ oligomers on cognitive behavior
in normal adult rats. ALCR, a test of delayed alternation and complex reference memory, has proved sensi-
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tive to direct experimental administration of A␤ in several previous studies.9,15,16 In brief, rats learn a
complex sequence of lever-pressing requirements to
earn food reinforcement in a two-lever experimental
chamber. The exact number of lever presses required
for each reward changes by first increasing in 7 unequal steps from 2 lever presses per food pellet up to
56 presses per pellet, and then decreasing back to 2
required presses per reward (ie, the sequence 2, 6, 12,
20, 30, 42, 56, 56, 42, 30, 20, 12, 6, 2 presses). Subjects must also alternate between levers after each food
reward. This “cycle” of response requirements repeats
six times during each daily assessment session. Each
failure to alternate levers and each premature switch
from a correct lever choice is recorded as a “Switching
Error.” If the subject perseverates in responding on an
incorrect lever choice, these are recorded as “Perseveration Errors.”
To assess the effect of AZD-103 on soluble
oligomer-induced errors, we trained 35 rats under
ALCR until relatively low baseline error rates demonstrated mastery of the complex reference memory rules
associated with the task. Each rat then received a single
permanent indwelling cannula placed in either the
right or left lateral ventricle and was allowed to recover
for 5 days.9 During the next week, baseline error rates
were established under ICV injections of saline (0.9%)
or “sham” injections during which the entire injection
procedure was performed but no injectant was administered. All ICV injections were 20␮l over a 5-minute
period given to awake, freely moving rats 2 hours before the ALCR behavioral testing session. As a positive
control to test the sensitivity of the ALCR paradigm to
disruption of reference memory and learned behavior,
we administered the prototypical anticholinergic amnestic agent, scopolamine. Compared with baseline error rates (mean of three previous ALCR sessions), ICV
scopolamine injections significantly increased Switching Errors to 400% ( p ⬍ 0.001) of baseline rates and
increased Perseveration Errors to 611% ( p ⬍ 0.001) of
baseline rates (all tests ⫽ Bonferroni-corrected Student’s t test). When 7PA2 CM containing soluble A␤
oligomers and monomers was injected ICV (20␮l) (see
Supplemental Fig 2), mean Switching Error rates increased significantly to 120% of their baseline levels
( p ⫽ 0.044), and mean Perseveration Error rates increased to 135% ( p ⫽ 0.026) (Fig 4A). Injections of
A␤-free CHO⫺ CM did not increase either type of
error, as described previously.9 To determine whether
AZD-103 could mitigate the error increases caused by
A␤ oligomers, we incubated either 7PA2 CM or
CHO⫺ CM with 5.0␮M AZD-103 at 37°C for 2
hours before ICV injection. Preincubation of 7PA2
CM with AZD-103 completely rescued both types of
ALCR errors from the detrimental effect of the ICVadministered oligomeric A␤ (see Fig 4A). AZD-103 incubated with CHO⫺ CM had no effect on error rates
(see Fig 4A).
Compounds that can be administered orally and
readily penetrate to brain sites of action offer significant clinical advantages. To assess the effectiveness of
oral AZD-103 administration in preventing cognitive
deficits produced by soluble A␤ oligomers, we administered AZD-103 to rats in their drinking water. Three
ascending doses of 30, 100, and 300mg/kg/day were
given for at least 3 days at each dose level (5 days for
the initial dose of 30mg/kg/day). To achieve these
daily oral doses, we adjusted concentrations of AZD103 based on average daily drinking water consumption. Rats received sham or 0.9% saline ICV injections
and were tested daily under ALCR during the entire
oral dosing regimen. After at least 3 days on each
AZD-103 oral dose, we administered 7PA2 CM ICV
as described earlier and assessed the rats’ performance
Fig 4. ICV or oral AZD-103 returned 7PA2 conditioned
media (CM)–induced error increases to baseline levels. (A)
AZD-103 (5.0 ␮M) was incubated with 7PA2 CM for 2
hours before intracerebroventricular (ICV) injection. When
compared with the mean error rate from the three previous
sessions, 7PA2 CM alone resulted in significantly increased
Switching/Approach Errors (blue bars; 120%, corrected p ⫽
0.044) and Perseveration Errors (red bars; 135%, corrected p
⫽ 0.026). After AZD-103 pre-incubation with 7PA2 CM,
error rates returned to baseline levels (dotted lines). AZD incubated with CHO⫺ CM, (containing no A␤ oligomers), did
not significantly affect either type of error. (B) When AZD103 was given in drinking water at doses of 30, 100, or 300
mg/kg/day, Switching Errors were reduced from 130% of baseline after ICV 7PA2 CM alone (p ⫽ 0.01) to levels (112,
100, and 113%, respectively) that were not significantly different than baseline (dotted lines). Perseveration Errors under
oral AZD were reduced from 169% of baseline error rate
under 7PA2 CM alone to error rates not different from baseline levels (124, 120, and 102% at the same respective doses).
under ALCR. Thereafter, the effects of 7PA2 CM were
reassessed under ALCR after a 5-day washout period
during which normal tap water was reinstated for
drinking.
As expected, A␤ oligomers in 7PA2 CM significantly increased Switching Errors to 130% of the baseline error rate ( p ⫽ 0.01, Bonferroni t test) and increased Perseveration Errors to 169% of baseline ( p ⫽
0.004) (see Fig 4B). However, under all three oral
doses of AZD-103, both types of errors were restored
essentially to baseline levels (see Fig 4B). No dose of
Townsend et al: A␤ Neutralized by AZD-103
673
oral AZD-103 administered alone (without 7PA2 CM)
had a significant effect on Switching Errors or Perseveration Errors (data not shown). Importantly, no
AZD-103 dose either affected how quickly rats completed the required cycles or reduced the number of
food reinforcers they received (data not shown). When
7PA2 CM was injected ICV into the same rats after
the 5-day washout period (ie, on normal drinking water), the rats again showed significantly elevated error
rates under the ALCR test (data not shown). CSF collected from the fourth ventricle of age-matched rats
treated exactly as described earlier under the oral dosing regimen showed mean AZD-103 CSF levels of
3.86, 6.32, and 10.08␮g/ml (21.4, 35.1, and 56.0␮M)
after the respective oral doses of 30, 100 and 300mg/
kg/day. Histological examination of the rat brain hemispheres contralateral to the cannula placement was entirely normal after either oral or ICV administration of
AZD-103 (data not shown).
Discussion
Here, we report that a small (molecular weight, 180)
organic molecule previously found to alter the conformation of synthetic A␤42 and inhibit its aggregation
into neurotoxic assemblies10 –12 can penetrate the brain
after oral administration and can fully prevent the adverse effects of secreted oligomers of human A␤ on
both cognitive performance and hippocampal LTP.
AZD-103 significantly improved LTP at lowmicromolar to submicromolar concentrations when
added to 7PA2 CM containing pre-existing A␤ oligomers (postconditioning) and proved even more effective when incubated with the 7PA2 cells during
the period of oligomer formation and secretion (preconditioning). The molecular specificity of its effects
is supported by the finding that the epi-enantiomer of
AZD-103 was almost as potent as AZD-103 itself,
whereas the chiro-enantiomer was without activity in
the LTP assay.
That AZD-103 specifically neutralizes low-n A␤ oligomers was established using SEC to separate these oligomers from monomers and the much larger soluble
fragments of secreted APPs. We found that AZD-103
coupled to epoxy-agarose can pull down A␤ trimers,
which we have shown to inhibit LTP. We therefore
hypothesize that AZD-103 interacts directly with A␤
to prevent its subsequent binding to neuronal target
molecules in the hippocampus that mediate its toxicity.
Far larger quantities of purified cell-derived A␤ oligomers would be required to test whether AZD-103
alters the conformation of A␤ oligomers using biophysical methods such as circular dichroism or nuclear
magnetic resonance, which is not currently feasible.
We have previously reported that learned behavior
and reference memory under the ALCR test are adversely affected by physiologically relevant levels of sol-
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December 2006
uble A␤ oligomers that are present in 7PA2 CM. We
now establish that when AZD-103 and 7PA2 CM are
incubated together before ICV injection, A␤ oligomer–
induced errors in behavioral performance and reference
memory are entirely prevented. These cognitive results
mirror the ability of AZD-103 at similar concentrations to block the synaptic effects of A␤ oligomers on
hippocampal LTP.
Oral administration of AZD-103 in the drinking
water at dose levels that could readily be administered
to humans blocked A␤-induced cognitive errors at all
doses tested. Washout of the AZD-103 reversed the
protection and allowed A␤-induced errors to recur.
AZD-103 levels found in CSF from the fourth ventricle of treated rats varied directly with the dose of orally
administered AZD-103, demonstrating the absorptive
bioavailability and brain penetration of the compound.
At even the lowest dose tested (30mg/kg/day), there
was an essentially complete rescue of the oligomerinduced deficits in cognitive performance. Although a
daily dose of 30mg/kg/day was sufficient to block the
adverse behavioral effects of A␤ oligomers, appreciably
greater doses of AZD-103 (300mg/kg/day) had no discernible adverse effects on behavior under ALCR or
any clinical or neuropathological effects throughout the
study. Consistent with these observations, AZD-103 by
itself was not found to have any effects on hippocampal synaptic function. These observations suggest that
the safe dose range of AZD-103 is significantly greater
than its effective dose.
After the completion of our studies, McLaurin and
colleagues12 recently reported beneficial effects of sustained oral administration of AZD-103 on cerebral A␤
burden, A␤-associated cytopathology, and Morris water maze performance in APP transgenic mice. Our
study corroborates and extends these findings and provides further insights into mechanism. First, we show
that AZD-103 does not depolymerize synaptotoxic
low-n oligomers A␤, but rather appears to bind and
neutralize them, fully preventing their biological effects
on hippocampal LTP. Second, our experimental approach allows one to specifically ascribe the rescue of
both hippocampal LTP and the memory of a novel
learned behavior distinct from the water maze to a biochemically defined species of soluble A␤, low
n-oligomers, in the absence of monomers, protofibrils,
or fibrils. Third, we find that the compound is most
potent in neutralizing the synpatotoxicity of low-n oligomers if it is applied to cells during the generation of
the oligomers, but it also successfully neutralizes preexisting oligomers. Fourth, we show that AZD-103
does not prevent the loss of synaptic plasticity if it is
preapplied to hippocampal slices before the addition of
oligomers, suggesting that the compound needs to bind
A␤ before A␤ encounters its neuronal targets. Fifth, we
show that the compound has acute, rapid effects on
hippocampal synapses and can provide short-term relief
of oligomer-compromised memory in vivo, in addition
to the effects seen with chronic administration by
McLaurin and colleagues.12 Sixth, we establish a dosedependent increase in brain levels of AZD-103 after
oral administration and rapid reversibility of benefit after cessation of dosing. Seventh, we confirm the lack of
any detectable neurotoxicity in vivo. Eighth, although
the compound can decrease high-molecular-weight oligomers in brain and enhance trimer levels,12 our data
indicate that although AZD-103 does not further depolymerize trimers and related low-n oligomers, it prevents their synaptotoxic effects on LTP and on at least
one form of cognition.
In conclusion, we report that scyllo-cyclohexanehexol
(AZD-103) has many of the properties sought in a
disease-modifying therapeutic for AD. It is a small,
orally bioavailable compound with enantiomeric specificity. It addresses a well-documented target, the soluble assemblies of secreted A␤ that have been extensively
confirmed as interfering with hippocampal synaptic
function in a variety of animal models of AD (reviewed
in Walsh and Selkoe17). Although side effects unrelated
to its mechanism of action cannot be excluded, initial
in vivo toxicity data appear benign. Moreover, the
compound is soluble, can be readily administered
orally, and penetrates into the brain in quantities sufficient to prevent cognitive deficits produced by levels
of A␤ similar to those that occur in human CSF.
Taken together, our findings provide evidence that
AZD-103 neutralizes the neurotoxic activity of soluble
A␤ oligomers.
Experimental Procedures
Cyclohexanehexols were provided by Transition Therapeutics (Toronto, Ontario,
Canada). The structures of these molecules are provided in Supplemental Figure 3A.
CYCLOHEXANEHEXOLS.
7PA2 cell CM113 was collected and
prepared as described previously.5
A〉 PREPARATION.
ANTIBODIES. Antibodies directed against A␤ included
monoclonals 6E10 and 4G8 (Signet), 2G3, 21F12,
m266, and 3D6 (Elan) and the rabbit polyclonal
R1282. Supplemental Figure 1D details the regions of
A␤ that are recognized by these antibodies.
IMMUNOPRECIPITATION. Precipitation of A␤ from
8ml CM prepared as described earlier was performed as
documented previously.5 Immunoprecipitations with
epoxy-agarose were performed using a protocol published by Pharmacia (Gaithersburg, MD). In brief,
5␮M AZD-103 (or its chiro-enantiomer) were coupled
to 0.25gm rehydrated epoxy-agarose resin in 50mM
phosphate buffer, pH 9.0. Excess ligand was sequentially washed off with water, 0.1M bicarbonate, pH
8.0, and 0.1M acetate buffer, pH 4.0. The beads were
then blocked with 1M ethanolamine; washed with
0.1M acetate, 0.5M NaCl, and 0.1M borate; and
stored in 0.5 M NaCl. To reduce nonspecific binding
in subsequent experiments, we also blocked beads with
0.5% bovine serum albumin (see Supplemental Fig 1).
SIZE EXCLUSION CHROMATOGRAPHY. SEC was performed as Walsh and colleagues5 described.
Samples were electrophoresed on 10
to 20% Tricine gels (Invitrogen, La Jolla, CA, or BioRad, Richmond, CA), and the proteins transferred to
0.2␮m Optitran nitrocellulose. The membranes were
boiled in phosphate-buffered saline (to enhance the exposure of A␤ epitopes) and blocked for 1 hour in 50%
Odyssey blocking buffer diluted in phosphate-buffered
saline. Blots were probed with the monoclonal antibody 6E10. Immunoreactive bands were detected and
quantified using a Licor Odyssey imaging system.
WESTERN BLOTS.
Field potential recordings were
made from coronal sections of postnatal days 16 to 28
male and female Swiss Webster mice, as described previously,13 in compliance with Harvard University’s Animal Resources and Comparative Medicine policies for
use of laboratory animals. Field potential recordings
were made at room temperature. Electrodes were specifically placed just below the surface of the slice to
maximize the exposure to circulating A␤. The intensity
of the stimulus was set to 20 to 30% of the maximum
evoked excitatory postsynaptic potential or until a population spike was elicited. Slices were perfused for 20
minutes in ACSF to establish a steady baseline. During
this interval, 1ml 15⫻ concentrated CM was thawed,
mixed with AZD-103 (for postconditioning experiments), and incubated at 37°C for 15 minutes. This
mixture was then added to 14ml circulating media,
giving a final concentration of 1.25␮M AZD-103, unless otherwise indicated. This 1⫻ CM/ACSF/AZD103 solution was recirculated over the slice at 2.5 to
3ml/min whereas being continuously aerated with 95%
oxygen. LTP was induced 20 minutes later by delivering four 100Hz stimuli every 5 minutes. The slope of
the EPSP was monitored for 1 hour after the last highfrequency stimulation.
ELECTROPHYSIOLOGY.
BEHAVIORAL TESTING, SURGERY, AND DRUG ADMINISTRATION. The ALCR procedure was used to assess cog-
nitive performance and reference memory. This task
involves progressively increasing and decreasing response requirements as described in Results. The two
types of errors generated under this procedure have
proved sensitive to ICV injections of A␤ oligomers.9
Townsend et al: A␤ Neutralized by AZD-103
675
Under general anesthesia, a single permanent indwelling cannula was aimed at either the right or left
lateral ventricle and permanently affixed to the skull.
Rats were allowed to recover for 5 days, and then stable response and error rates on ALCR were reestablished. Cannula placement was confirmed by histological examination of the brain after the last experimental
session.
Behavioral testing begun 2 hours after ICV injection
of A␤ oligomers. All ICV injections were 20␮l and
were slowly administered over a 5-minute period in
freely moving animals. For ex vivo assessments, AZD103, at a concentration of 5.0␮M, was pre-incubated
with either 7PA2 CM or CHO⫺ CM for 2 hours at
37°C. For oral dosing, AZD-103 was mixed in the
rats’ drinking water and was available ad libitum for at
least 3 days before behavioral testing after ICV A␤ oligomer administration. Daily doses were determined by
adjusting the concentration of AZD-103 in drinking
water based on each rat’s average daily water consumption (minus spillage) to achieve doses of 30, 100, and
300mg/kg/day AZD-103. Doses of oral AZD-103 were
studied in an ascending order over sequential independent experiments.
Typical comparisons were done using
Student’s t test or analysis of variance with Tukey–
Kramer post hoc tests. For the behavioral studies, analysis was done using a Bonferroni test. Error bars indicate the standard error of the mean.
STATISTICS.
This research was supported by the NIH (AG027443, D.J.S.; T32
NS07484, M.T.).
We thank J. McLaurin and P. St George-Hyslop for advice on this
project. J. Copeman at Transition Therapeutics provided us with
inositol reagents and contributed to the critical review of the manuscript. Transition Therapeutics also contributed small amounts of
supplemental research funds to both J.P.C.’s and D.J.S.’s laboratories to defray some of the costs of these experiments, as did K. Ashe.
S. Narayanan and M. LaVoie made valuable contributions to experimental design.
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