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Current Medicinal Chemistry, 2014, 21, ????-???? 1
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Effects of Tetrahydrohyperforin in Mouse Hippocampal Slices: Neuropro-
tection, Long-term Potentiation and TRPC Channels
C. Montecinos-Oliva
1
, A. Schller
2,3
, J. Parodi
1
, F. Melo
2,3
and N.C. Inestrosa*
,1,4,5

1
Centro de Envejecimiento y Regeneracin (CARE), Departamento de Biologa Celular y Molecular; Facultad de
Ciencias Biolgicas, Pontificia Universidad Catlica de Chile, Santiago, Chile;
2
Molecular Bioinformatics Laboratory,
Millennium Institute on Immunology and Immunotherapy;
3
Departamento de Gentica Molecular y Microbiologa;
Facultad de Ciencias Biolgicas, Pontificia Universidad Catlica de Chile, Santiago, Chile;
4
Center for Healthy Brain
Ageing, School of Psychiatry, Faculty of Medicine, University of New South Wales, Sydney, Australia;
5
Centro de
Excelencia en Biomedicina de Magallanes (CEBIMA), Universidad de Magallanes, Punta Arenas, Chile
Abstract: Tetrahydrohyperforin (IDN5706) is a semi-synthetic compound derived from hyperforin (IDN5522) and is the
main active principle of St. Johns Wort. IDN5706 has shown numerous beneficial effects when administered to wild-type
and double transgenic (APPswe/PSEN1E9) mice that model Alzheimers disease. However, its mechanism of action is
currently unknown. Toward this end, we analysed field excitatory postsynaptic potentials (fEPSPs) in mouse hippocampal
slices incubated with IDN5706 and in the presence of the TRPC3/6/7 activator 1-oleoyl-2-acetyl-sn-glycerol (OAG), the
TRPC channel blocker SKF96365, and neurotoxic amyloid -protein (A) oligomers. To study spatial memory, Morris
water maze (MWM) behavioural tests were conducted on wild-type mice treated with IDN5706 and SKF96365. In silico
studies were conducted to predict a potential pharmacophore. IDN5706 and OAG had a similar stimulating effect on
fEPSPs, which was inhibited by SKF96365. IDN5706 protected from reduced fEPSPs induced by A oligomers.
IDN5706 improved spatial memory in wild-type mice, an effect that was counteracted by co-administration of SKF96365.
Our in silico studies suggest strong pharmacophore similarity of IDN5706 and other reported TRPC6 activators
(IDN5522, OAG and Hyp9). We propose that the effect of IDN5706 is mediated through activation of the TRPC3/6/7
channel subfamily. The unveiling of the drugs mechanism of action is a necessary step toward the clinical use of
IDN5706 in Alzheimers disease.
Keywords: A oligomers, Alzheimer's Disease, neuroprotection, hippocampus, tetrahydrohyperforin, TRPC channels.
INTRODUCTION
Alzheimers disease (AD) is characterised by a progres-
sive loss of cognitive abilities, eventually leading to the
death of the individual [1]. Accumulation of the amyloid -
protein (A), a product of the processing of the amyloid pre-
cursor protein (APP), is believed to play a key role in the
cognitive deficits observed in AD [2]. The mechanisms in-
volved in the pathogenic changes triggered by A oligomers

are not clearly understood. A oligomers trigger neuronal
dysfunction and cytoskeletal alterations, early manifestations
that lead to aberrant remodelling of dendrites and axons,
synaptic loss [3], and eventually, a progressive loss of neu-
ronal populations [4]. Synaptic failure is correlated with a
reduction in synaptic proteins and alterations in synaptic
function [2, 5].
Hyperforin is a prenylated phloroglucinol derivative and
the primary active molecule responsible for the anti-
depressant activity of St. Johns Wort (Hypericum perfora-
tum)[6]. It has been used for centuries in Chinese traditional
medicine as a sedative, antimalarial and diuretic substance

*Address correspondence to this author at the CARE Biomedical Center,
Pontificia Universidad Catlica de Chile, Av. Alameda 340, Santiago,
Chile; Tel: + (56)-26862724; Fax: + (56)-2-6862959;
E-mail: ninestrosa@bio.puc.cl
[7]. Hyperforin has been suggested to enhance memory in
rodents [8] and may have additional anti-inflammatory, anti-
bacterial, antiangiogenic and antitumoral effects. Accord-
ingly, we have previously shown that Hyperforin reduces the
behavioural alterations induced by intra-hippocampal injec-
tion of A fibrils in an acute rat model of AD [9]. Hyper-
forin, a natural compound, is chemically instable, easily oxi-
dised, sensitive to heat and light, and degrades quickly as
well as its bioactivity may be rapidly lost during storage [10,
11]. Tetrahydrohyperforin (IDN5706) is a semi-synthetic
derivative of Hyperforin that has higher stability and in-
creased oral bioavailability [12], while maintaining its neu-
roprotective properties [13].
So far, the mechanism of action of IDN5706 that ex-
plains its memory and behaviour altering effects is not
known. Studies indicate that the canonical transient receptor
potential 6 (TRPC6) channel, a tetrameric, non-selective
cation channel, is specifically activated by Hyperforin [14].
TRPC6 channels have also been found to promote dendritic
growth [15] and play a role in the formation of excitatory
synapses [16]. Here, we report that IDN5706-mediated acti-
vation of TRPC channels improves the synaptic response
measured by a reversible increase in field excitatory post-
synaptic potential (fEPSP), has neuroprotective effects on
A oligomers, and improves spatial memory in wild-type
2 Current Medicinal Chemistry, 2014, Vol. 21, No. 1 Montecinos-Oliva et al.
mice. All of these effects might be related to the mechanism
of action of IDN5706 in the mammalian central nervous sys-
tem.
MATERIALS AND METHODS
Reagents
Tetrahydrohyperforin (IDN5706) and Solutol were a gift
from Indena SpA, Milan, Italy. Tetrahydrohyperforin is a
semi-synthetic derivative of Hyperforin (WO 03/091194 A1;
WO 2004/106275A2). SKF-96365 (1-[2-(4-Methoxyphenyl)
-2-[3-(4-methoxyphenyl)propoxy] ethyl]imidazole) was ob-
tained from Cayman Chemical (Ann Harbor, MI) and a 2.4
mM stock solution was prepared in a 25% Solutol aqueous
solution. OAG (1-Oleoyl-2-acetyl-sn-glycerol) was obtained
from Sigma (St. Louis, MO), 3 mM stock solution was pre-
pared in ethanol (20 mg mL
-1
).
A

oligomer Preparation
A
1-42
was obtained from Genemed Biotechnologies, Inc.
(South San Francisco, CA, USA). A lyophilised stock pep-
tide was resuspended in anhydrous sterile dimethyl sulfoxide
(DMSO) to form 5 mM aliquots that were immediately fro-
zen. Aliquots were diluted in PBS, pH 7.4 to a final concen-
tration of 100 M and stirred continuously at approximately
1350 rpm for 1 h at room temperature. Final concentrations
for electrophysiology studies were 1 M A oligomers and
0.02% DMSO. Data on the ratio of monomers to oligomeric
tetramers (low molecular weight) present after following this
protocol are reported in our previous publications [17].
Animal Management
C57Bl/6 mice were kept in the University Animal Facil-
ity in accordance with the Bioethical Committee of the Pon-
tificia Universidad Catolica de Chile with ad libitum access
to food and water in a 12:12 hour light/dark cycle. Constant
monitoring of general health and behaviour was performed
during the injections and test periods, in accordance with the
Guide for the Care and Use of Laboratory Animals published
by the National Academy of Science, National Academy
Press, Washington, D.C. for experiments involving animals.
Slice Preparation and Electrophysiology
Hippocampal slices from C57Bl/6 mice were prepared
with ice-cold artificial cerebrospinal fluid (ACSF) containing
124 mM NaCl, 2.7 mM KCl, 1.25 mM KH
2
PO
4
, 2 mM
Mg
2
SO
4
, 26 mM NaHCO
3
, 2.5 mM CaCl
2
, and 10 mM D-
glucose bubbled with 95%/5% O
2
/CO
2
gas, according to
standard procedures previously described by our laboratory
[18]. In every case, 100 M picrotoxin, (PTX, Sigma-
Aldrich, P1675) a non-competitive GABA
A
antagonist, was
used to inhibit GABAergic activity, and no epileptiform ac-
tivity was detected. All protocols were conducted by stimu-
lating pyramidal cells and recording in the stratium radiatum
within the CA1 area of the hippocampus. To generate long-
term potentiation (LTP), we employed high frequency stimu-
lation (HFS), three trains of 500 ms stimuli at 100 Hz with a
20 s interval and theta burst stimulation (TBS), five trains of
10 bursts at 5 Hz each train having 4 pulses of 100 Hz, with
a 20-s interval. To discriminate LTP generation, we deter-
mined a threshold of 30-40% potentiation. Paired pulse fa-
cilitation was measured as the slope ratio between two con-
secutive responses (R2/R1) to two stimulation pulses with a
100 ms interval. Presynaptic volley was measured in order to
establish any changes in the number of fibres stimulated dur-
ing the experiment. Bar charts were obtained by calculating
the average amplitude reached within 20-30 min of treatment
for field potential and peak slope reached between 5-10 min
after LTP induction. In every case, drugs were diluted in
ACSF and the control used was ACSF. Recordings were
filtered at 2.0-3.0 kHz, sampled at 4.0 kHz using an A/D
converter, and stored with pClamp 10 (Molecular Devices).
Evoked postsynaptic responses were analysed off-line, using
analysis software (pClampfit, Molecular Devices) that al-
lowed visual detection of events, computing only those
events that exceeded an arbitrary threshold. In every case, an
average of 4 responses per min was plotted. To avoid the
analysis of population spikes in LTP protocols, the slope was
plotted instead of the amplitude.
Immunoblot
We treated 350 m hippocampal slices from two-month-
old male mice for 30 min in ACSF with different solutions.
Bath temperature was maintained at 37C during treatment.
Next, cortical and hippocampal tissue was dissected in ice
cold ACSF solution. Samples were lysed and 40 g of pro-
tein were loaded onto a 10% SDS-PAGE gel and transferred
to a PVDF membrane. Primary antibodies used were as fol-
lows: PSD-95 (clone K28/43 UC Davis/NIH Neuromab Fa-
cility), vGlut1 (clone N59/36, UC Davis/NIH Neuromab
Facility), Syp (sc-7568, Santa Cruz Biotechnology, Inc.), -
tubulin (ab7751, Abcam). Secondary antibodies were anti-
rabbit or anti-goat conjugated to IgG peroxidase and blots
were developed using an ECL kit (Western Lighting Plus
ECL, PerkinElmer).
Administration of Drugs
From a total of 20 male 5-month-old mice, five were in-
jected intraperitoneally (i.p.) with 6 mg kg
-1
IDN5706 three
days a week (Monday, Wednesday and Friday) for 10 weeks
and with 6 mg kg
-1
solutol (vehicle solution for IDN5706
and SKF96365 preparation, LD
50
is 8.74 g kg
-1
) 2 h before
the water maze training session (group 1). Five mice were
injected i.p. with IDN5706, on the same schedule as group 1,
and then 20 mg kg
-1
SKF96365 injections 2 h before the
training session (group 2). The third group of five animals
received 6 mg kg
-1
solutol injections for 10 weeks and 20 mg
kg
-1
SKF96365 injections 2 h before the training session
(group 3). The last group received 6 mg kg
-1
solutol injec-
tions for 10 weeks and also before the training sessions
(group 4). The animals received SKF96365 two hours before
testing because TRPC6 dysfunction results in podocyte fail-
ure. We did not want to expose animals to a dose that would
affect the kidneys or the general health of the mice. Within 2
h of injection, needle injection wound had completely healed
the wound, which is particularly important to avoid infec-
tions as animals are introduced into a pool. During the entire
treatment period, the mice were subjected to a supervision
protocol to track their weight, behaviour and general health.
All treatment groups were chosen randomly.
Tetrahydrohyperforin Effects in Hippocampal Slices Current Medicinal Chemistry, 2014, Vol. 21, No. 1 3
Morris Water Maze Behavioural Task
After drug administration, a total of twenty wild-type
mice (8 months old) were subjected to 5 days of training
followed by a 2-day resting period and a final three days
of training. A circular white pool was made opaque with
non-toxic white paint, and a platform was hidden (diame-
ter: 9 cm) in quadrant four. The water temperature was
kept between 18-20 C. Testing criteria were achieved
when the animal reached the platform within 60 sec and
stayed on it for a minimum of 3 sec. When finished, the
animals were returned to their cages, following protocols
previously established by our group [19]. The data were
gathered and analysed with a video tracking system (HVS
Imagen, UK).
Molecular Docking
The crystal structure of the human pregnane X receptor
(PXR; PDB entry 1m13) [20] was downloaded from the Pro-
tein Data Bank and prepared for docking with the Molecular
Operating Environment (MOE) version 2011.10 (Chemical
Computing Group, Montreal, Canada). Hydrogen atoms
were added, and the complex was subjected to restrained
energy minimisation (AMBER10 force field with
parm@frosst small molecules parameters and Generalised
Born solvation model) until the RMS gradient fell below
0.05 kcal (mol )
-1
. Molecular docking was performed with
GOLD version 5.1 (The Cambridge Crystallographic Data
Centre, Cambridge, UK) [21]. Residues within 8 from the
co-crystallised ligand Hyperforin were defined as the binding
pocket. CHEMPLP scoring functions and automatic search
parameters (200% efficiency) were selected. Hydrogen
bonds were constrained to favour interaction with residues
Ser-247, Gln-285 and His-407. Water molecules and the co-
crystallised ligand Hyperforin were removed, and ten dock-
ing poses were generated. The root mean squared deviation
(RMSD) of co-ordinates of equivalent atoms was calculated
for the docking poses with Hyperforin as the reference
ligand.
Pharmacophore Alignment
Up to 10,000 conformers were generated for each ana-
lysed compound with the LowModeMD search method of
MOE, which employs a short molecular dynamics simulation
utilising velocities with low kinetic energy on the high-
frequency vibrational modes [22]. Then, an exhaustive
search for all pharmacophore queries that showed good
structural overlay with the conformers was performed. The
pharmacophore queries were restricted to include a minimum
of one H-bond donor and two H-bond acceptors, with
spherical projection sites of radius 1 .
Statistical Analysis
Data analysis was carried out with Prism software
(GraphPad Software Inc., La Jolla, CA). The results were
expressed as the means S.E. For statistical analysis, nor-
mally distributed data were analysed by one-way ANOVA
with a posteriori tests performed using Tukeys test. Non-
normally distributed data were analysed by the Kruskal
Wallis test with post hoc tests performed using Dunns test.
RESULTS
IDN5706 increases the amplitude of fEPSP and LTP
in hippocampal slices from wild-type mice. First, we ex-
plored the effectiveness of IDN5706 in mouse hippocampal
slices by measuring the field excitatory postsynaptic poten-
tial (fEPSP) and long-term potentiation (LTP). We measured
fEPSPs at increasing concentrations of IDN5706 and deter-
mined a concentration-dependent rise in the amplitude of
fEPSPs, with an EC
50
of 0.5 g mL
-1
(corresponding to ~1
M) (Fig. 1A). Then, LTP was generated using high-
frequency stimulation (HFS; 100 Hz, 500 ms, three stimula-
tion trains). LTP induction was stronger in slices exposed to
1 M IDN5706, compared to the control ACSF solution
(2.96 0.17 r.u. vs. 2.16 0.19 r.u., N=3); in both condi-
tions, LTP was stable for at least 1 hour after stimulation
(Fig. 1B). These data suggest that IDN5706 alters basal neu-
ronal activity, facilitates LTP induction, and positively alters

Fig. (1). IDN5706 improves LTP in hippocampal slices from wild-type animals. A) Dose-response curve for fEPSP amplitude in hippo-
campal slices from 2-month-old mice in the presence of increasing concentrations of IDN5706. An EC
50
of 0.5 g mL
-1
(~1 M) was deter-
mined. B) LTP analysis in hippocampal slices of wild-type mice, stimulated through high frequency stimulation (HFS) at time point zero
(arrow). Slices were treated with 1 M IDN5706 (filled triangles) for 20 min (horizontal line) or bathed in ACSF (control; empty circles).
Representative traces for each group are shown in the inset.
4 Current Medicinal Chemistry, 2014, Vol. 21, No. 1 Montecinos-Oliva et al.
fEPSPs in a dose-dependent manner in hippocampal slices of
wild-type mice.
Activation of TRPC channels with OAG increases the
amplitude of fEPSPs. To determine whether our experimen-
tal model is sensitive to a TRPC channel agonist, 1-oleoyl-2-
acetyl-sn-glycerol (OAG), we performed electrophysiologi-
cal studies in CA1 mouse hippocampal slices. We observed
that OAG increased fEPSP amplitude (Fig. 2A-B) at a
maximum concentration of 100 M, similar to previous re-
ports [23]. OAG is an analogue of naturally occurring dia-
cylglycerols, a group of endogenous compounds that activate
TRPC3/6/7 channels [23]. When we added the nonspecific
cation channel blocker lanthanum (30 M La
3+
, known to
block TRP channels), we observed an inhibition of the OAG
effect (Fig. 2C). Quantification of fEPSPs indicated a reduc-
tion in the peak amplitude of over 40% after simultaneous
treatment with OAG and lanthanum (2.46 0.16 r.u. vs. 1.44
0.23 r.u., N=3). Treatment with lanthanum alone did not
alter the basal fEPSP amplitude (Fig. 2D). These results con-
firmed the presence of OAG-sensitive channels in the CA1
area and indicated that we are able to modulate TRPC3/6/7
channels, inducing changes in fEPSP amplitude similar to
the effect of IDN5706 for LTP (Fig. 1).
The effect of IDN5706 on synaptic activity is blocked
by the TRPC channel blockers lanthanum and
SKF96365. To determine whether IDN5706 acts on TRPC
channels, we evaluated its effects in the presence of the
TRPC channel antagonists lanthanum and SKF96365.
SKF96365 is a broad range inhibitor of TRP channels spe-
cific to the canonical type (TRPC) [24]. Measurements of
fEPSP amplitude in slices treated with IDN5706 (1 M)
were similar to those observed with OAG. The fEPSP ampli-
tude increased two-fold over basal recordings with ACSF
(2.31 0.12 r.u. vs. 1.02 0.07 r.u.). This increase was par-
tially blocked by 20 M SKF96365 (2.31 0.12 r.u. vs. 1.78
0.10 r.u.) and completely inhibited by 30 M lanthanum
(2.31 0.12 r.u. vs. 1.02 0.12 r.u.) (Fig. 3A). Quantifica-
tion of the peak amplitudes revealed that lanthanum was able
to block the stimulating effect of IDN5706 by almost 100%,
whereas 40% inhibition was observed with SKF96365 (Fig.
3B). Then, we compared the effect of 1 M Hyperforin
(IDN5522), the compound from which tetrahydrohyperforin
is derived, to determine whether it produces a similar elec-
trophysiological effect on fEPSPs in hippocampal slices as 1
M IDN5706. No difference in fEPSPs was observed; in
fact, each treatment resulted in a two-fold increased peak

Fig. (2). OAG enhances the fEPSP amplitude of hippocampal slices, and lanthanum inhibits this effect. A) Mouse hippocampal slices
were exposed to different concentrations of OAG (1, 50 and 100 M; filled circles, shaded triangles and filled triangles, respectively) for a
period of 30 min (horizontal bar), after which the slices were washed with ACSF. Untreated slices were bathed in ACSF (empty circles).
fEPSP amplitude increased during exposure to OAG. B) The maximum effect was reached with 100 M OAG. A significant difference is
observed with concentrations 50 M, compared to ACSF. The inset shows representative traces of each treatment. C) Lanthanum (5 M)
blocks the effect of 100 mM OAG (filled triangles) without affecting the basal membrane potential (filled circles), as previously shown for
OAG (empty triangles). ACSF controls are also shown (empty circles). D) Quantification of fEPSP peak slope under different conditions.
Inset shows representative traces for each treatment. Mean values SEM were plotted for 6 different experiments from a minimum of 3 ani-
mals *P< 0.05, **P<0.01.
Tetrahydrohyperforin Effects in Hippocampal Slices Current Medicinal Chemistry, 2014, Vol. 21, No. 1 5
amplitude of fEPSPs (Table 1, Supplementary Fig. 1). Addi-
tionally, Paired Pulse Facilitation (PPF) was not affected
during treatment with OAG (the activator of TRPC3/6/7
channels; data not shown), IDN5706, SKF96365 or the co-
administration of IDN5706 and SKF96365 (Fig. 3C, D).
These results indicate that the alterations observed in fEPSPs
are not caused by presynaptic changes but rather by postsyn-
aptic modifications [25, 26]. This finding is consistent with
the fact that TRPC channels are mainly located at the excita-
tory postsynaptic region [16]. SKF96365 by itself causes a
small increase in fEPSP slope, an effect that has not been
widely described before (data not shown). These results sug-
gest that TRPC channels are involved in the effects of
IDN5706 on the fEPSP amplitude.
Synaptic protein levels are not significantly affected
after 30 min treatment with IDN5706 or SKF96365. To
determine whether the effects observed in electrophysiologi-
cal recordings were a product of a change in synaptic archi-
tecture, we studied the effects of acute treatments (30 min
exposure) with IDN5706 (1 M) and SKF96365 (20 M) in
ACSF on hippocampal slices. Because SKF96365 is a more
specific blocker of TRPC channels than La
3+
[24], the latter
condition was not included in all following experiments. The
evaluated proteins include the following: PSD95, the main
scaffolding protein located in the postsynaptic side of gluta-
matergic synapses [27]; vGluT1, a vesicular transporter of
glutamate present in releasing vesicles on the presynaptic
side of glutamatergic synapses; and Syp, which is fundamen-
tal to the release of neurotransmitters in glutamatergic and
GABAergic synapses. Syp was studied to characterise the
overall effect on neurotransmitter release. Protein levels
were compared to those in slices treated only with ACSF. As
a control, a different group treated only with solutol (1 M)
was evaluated (Fig. 4A). Data quantification in (Fig. 4B)
shows there were no significant differences in protein levels
after 30 min of treatment, which is the exposure time used in
our electrophysiology studies. These data indicate that the
changes observed are most likely due to rapid ion influx
rather than synaptic protein up-regulation, supporting our
hypothesis regarding the involvement of TRPC channels in
the mechanism of action of IDN5706.
IDN5706 prevents the fEPSP reduction triggered by
A oligomers: dependence on TRPC channel activation.
Our group has previously reported that both in hippocampal
slices and cultured neurons, A oligomers reduce synaptic
activity (i.e., fEPSPs) in paired pulse stimulation and LTP
[28-31]. Specifically, treatment of hippocampal slices with
A oligomers reduced fEPSP amplitude, whereas this reduc-
tion was not observed following coincubation with A

oli-
gomers and IDN5706 [28]. To test whether these neuropro-

Fig. (3). IDN5706 increases fEPSP amplitude, an effect blocked by lanthanum and SKF96365 in mouse hippocampal slices. A) Field
recordings of hippocampal slices from two-month-old mice incubated with IDN5706 (1 M, 30 min., horizontal bar) in the presence (filled
circles) or absence (filled triangles) of 5 M lanthanum or 20 M SKF96365 (empty triangles). Untreated slices were bathed in ACSF
(empty circles). B) Quantification of fEPSP peak slope under different conditions. The inset shows representative traces. C) Facilitation in-
dex (R2/R1) was calculated from the experiments in A). D) Quantification of the average facilitation obtained in C) during treatment (from 0
to 30 min). Mean values SEM were plotted for 6 different experiments from a minimum of 3 animals *P < 0.05, ** P < 0.01.
6 Current Medicinal Chemistry, 2014, Vol. 21, No. 1 Montecinos-Oliva et al.
tective effects of IDN5706 are affected by TRPC blockage,
we evaluated fEPSPs after treatment with A oligomers,
IDN5706 and SKF96365. The addition of 20 M SKF96365
to hippocampal slices treated with 1 M IDN5706 and 1 M
A oligomers resulted in reduced fEPSP amplitudes (Fig.
5A). Quantification of the fEPSP peak amplitudes suggested
that the inhibition of TRPC channels prevented the neuropro-
tection provided by IDN5706 (Fig. 5B). A summary of the
effects of IDN5706 and SKF96365 on A

oligomers-induced

neurotoxicity is shown in (Table 2). Next, we evaluated LTP
generation in hippocampal slices exposed to A oligomers in
the presence or absence of IDN5706. LTP was weakly in-
duced in slices exposed to A oligomers (Fig. 5C)
.
In com-
parison, LTP was robustly induced in slices incubated with
A oligomers in the presence of IDN5706 (1.91 0.05 r.u.
vs. 2.22 0.04 r.u., N=4). In addition, there was an 83% de-
crease in fEPSPs when slices were incubated with A oli-
gomers

(1.29 0.05 r.u. vs. 2.22 0.04 r.u., N=4). Signifi-
cant differences are found between all treatments (Fig. 5D).
These results suggest that IDN5706 facilitates LTP induction
and protects against A oligomers-induced

neurotoxicity.
Next, we tested the effect of SKF96365 on LTP generation
induced by theta-burst stimulation (TBS) by perfusing
mouse hippocampal slices with 1 M IDN5706 and 20 M
SKF96365 for 20 min (10 min prior and 10 min after stimu-
lation). As shown in (Fig. 5E), simultaneous treatment of
IDN5706 and SKF96365 inhibited the generation and main-
tenance of LTP in the CA1 field. Quantification of the LTP
data (Fig. 5F) shows that treatments with IDN5706 alone
and IDN5706 plus SKF96365 were significantly different
from the ACSF control experiment (2.85 0.26 r.u., N=3 vs.
1.71 0.43 r.u., N=4 and 1.18 0.11 r.u., N=3 vs. 1.71
0.43 r.u., N=4, respectively) as well as significantly different
from each other. These results indicate that the neuroprotec-
tive effects of IDN5706 are abolished after co-incubation
with SKF96365, which is a TRPC blocker.
Table 1. Peak fEPSP amplitude of hyperforin and tetra-
hydrohyperforin
Condition
Amplitude Peak fEPSP
(Relative Units)
Control 1 0.1
IDN5522 2.2 0.42**
IDN5706 2.4 0.31 ***
Peak fEPSP amplitude of mouse hippocampal slices treated with IDN5706 (Tetrahy-
drohyperforin, 1 M) and IDN5522 (Hyperforin, 1 M), given as mean SEM of 4
different experiments per treatment. ** P < 0.01, *** P < 0.001.
SKF96365 prevents the improved performance of
wild-type mice treated with IDN5706 in the Morris water
maze. Because IDN5706 has a significant effect on synaptic
activity in the CA1 area of the hippocampus, a zone widely
studied for its role in spatial memory [32], we performed the
Morris water maze test on mice treated with IDN5706 and
SKF96365. A total of 20 animals were i.p. injected with dif-
ferent drugs, resulting in a total of four groups of five ani-
mals each (for details, see Materials and Methods). Injec-
tions of SKF96365 (20 mg kg
-1
) occurred only on training
days (in order to avoid any systemic damage due to TRPC
inhibition in podocytes [33]). Groups 1 and 2 were treated
with IDN5706. Injection of SKF96365 into mice treated with
IDN5706 from group 2 resulted in increased escape latency
compared with animals that were treated with IDN5706
alone (group 1) during the training sessions (Fig. 6A). Injec-
tion of SKF96365 into the solutol control animals (group 3)
produced a small but not significant increase in their escape
latencies, compared with animals that were injected with
solutol alone (group 4) and with animals that did not receive
SKF96365 (Fig. 6B). The escape latencies on training day 5
from all four treatment groups are plotted in (Fig. 6C). It is
evident that the group treated only with IDN5706 performed
better in the Morris water maze than animals treated with
solutol alone or IDN5706 plus SKF96365, reflected in sig-
nificantly diminished escape latencies (7.6 0.95 s, N=5 vs.
18.08 1.01 s, N=5, and 19.74 4.19 s, N=5). Representa-
tive swimming trajectories of the four different treatments
are shown (Fig. 6D) to exemplify the difference in spatial
memory. Animals treated with IDN5706 performed better
than the three other treatment groups, reflecting improved
spatial memory. Because velocity (Fig. 6E) was not signifi-
cantly different between the four groups (24.69 1.35 cm s
-
1
, 15.01 0.48 cm s
-1
, 16.42 1.19 cm s
-1
, 14.33 0.72 cm
s
-1
; in same order as in the bar chart, N=5 for each group),
and the general health and weight of each animal was nor-
mal, any motor impairment caused by SKF96365 injections
was disregarded. Swimming distance (Fig. 6F) was higher in
animals that had increased escape latency (solutol, solutol
plus SKF96365 and IDN5706 plus SKF96365, 376.25
44.47 cm, 210.39 66.70 cm and 313.43 61.94 cm, re-
spectively, with N=5 for each group) and was reduced in the
group with lower escape latency values (IDN5706, 126.60
28.58 cm, N=5). Therefore, we infer that IDN5706 improved
spatial memory in wild-type mice and that this improvement
is counteracted by the TRPC channel blocker SKF96365.
In silico conformational analysis suggests a similar
binding mechanism for IDN5706 and other reported
TRPC activators. To evaluate whether tetrahydrohyperforin
(IDN5706) is able to interact with its target channel in a
similar way to other potential TRPC activators (Hyper-
forin/IDN5522, Hyp9, and OAG), we performed molecular
docking and pharmacophore analysis. IDN5706 is a chemi-
cally closely related derivative of Hyperforin [34] but has a
modified molecular geometry caused by two additional ste-
reo centres introduced by chemical reduction of two car-
bonyl groups (Fig. 7A). Due to the current lack of a high-
resolution structure for TRPC, we relied on the crystal struc-
ture of the human pregnane X receptor (PXR) in complex
with Hyperforin (PDB entry 1m13) [20], as suggested by
[35]. Under the assumption that Hyperforin is bound to PXR
in a bioactive form, this complex can be utilised to define the
potential pharmacophore for the interaction with TRPC [35].
Molecular docking of IDN5706 predicted a similar binding
mode (RMSD = 1.02 ) compared to Hyperforin (Fig. 7B).
A common pattern of hydrogen bonds to Ser-247, Gln-285,
and His-407 was predicted. However, rotation of the isobutyl
alcohol side chain of IDN5706 was required to facilitate the
hydrogen bond to Ser-247. Docking of Hyp9 and OAG pro-
duced docking poses that interacted with the same residues
(not shown). We next analysed the conformational space and
pharmacophoric properties of all four potential TRPC6 acti-
Tetrahydrohyperforin Effects in Hippocampal Slices Current Medicinal Chemistry, 2014, Vol. 21, No. 1 7
vators: IDN5706, Hyperforin, Hyp9, and OAG. We chose a
ligand-based method to avoid the bias of an unrelated crystal
structure. An average of 1036 conformations were generated
by short molecular dynamics runs for each compound, fol-
lowed by pharmacophore alignment. A similar alignment
was predicted for the four activators, which is in agreement
with our docking results (Fig. 7C). The pattern of three po-
tential hydrogen bonds was reproduced using the receptor-
free method. We conclude that IDN5706 is potentially able
to interact with its target channel on a similar molecular ba-
sis as other TRPC channel activators.
DISCUSSION
In this work, we demonstrated that 1) IDN5706 has a
neuroprotective effect on fEPSPs and synaptic function in
mouse hippocampal slices exposed to A

oligomers, 2) ap-
plication of IDN5706 increased the amplitude of LTP, 3)
treatment of hippocampal slices with IDN5706 or OAG, a
known TRPC3/6/7 activator [35], induced a similar increase
in fEPSP amplitude, 4) the stimulating effect of both com-
pounds was blocked by the nonspecific cation channel
blocker lanthanum, and the TRPC broad range inhibitor
SKF96365, 5) the improvement in memory described in
IDN5706 treated mice was blocked by TRPC inhibition and
finally 6) IDN5706 shares a common pharmacophore with
other TRPC activators.
IDN5706 increased the synaptic response recorded in ba-
sal (ACSF) conditions in response to paired pulse stimula-
tion, as evidenced by a dose-dependent increase in fEPSP
amplitude (Fig. 1A). We explored the functional conse-
quences of the observed change in the fEPSP amplitude and
found an improvement in synaptic plasticity responses (Fig.
1B). It is worth noting that the success rate for LTP induc-
tion (i.e., the number of slices that were induced by HFS or
TBS that actually generated LTP) was approximately 50%
for control and 66% for IDN5706 treatment. We noted that
IDN5706 increased the slope of fEPSP prior to LTP induc-
tion and kept increasing fEPSPs, even without TBS stimula-
tion, as observed in (Supplementary Fig. 1). This correlated
with rapid intracellular calcium elevation, making it difficult
to obtain a steady state baseline during the LTP experiments
(see Fig. 1B and 4C, -10 to 0 min). In general, IDN5706
exerted a positive effect on synaptic efficacy in hippocampal
slices of wild-type mice.
Hyperforin, the compound from which IDN5706 was de-
rived, is a specific activator of the TRPC6 channel [35] and
was shown to have various neurobiological effects (for re-
view, see [17]). Drugs acting as channel agonists may allow
the influx of calcium ions and LTP generation [36]. TRPC
channels are non-selective cation channels [37] and have
been shown to be important for the regulation of the forma-
tion of excitatory synapses and the improvement of spatial
memory [16]. In this context, these receptors may have an
important role in the modulation of LTP [38, 39]. Therefore,
we investigated their relevance to the effects of IDN5706.
OAG, a diacylglycerol analogue and a TRPC3/6/7 chan-
nel modulator, is able to cross the plasma membrane and
intracellularly activate the channels [24, 40]. Here, OAG
increased the amplitude of fEPSPs in a dose-dependent man-
ner (Fig. 2A, B), which was in agreement with concentra-
tions established by other groups [23] and confirmed its abil-
ity to modulate TRPC3/6/7 channels in our experimental
model. These data and the observation that this effect could
be completely blocked by lanthanum and partially blocked
by SKF96365 (Fig. 3) allowed us to confine the effect of
OAG to the TRPC channel family. As a different measure of
synaptic function, we calculated the PPF ratio and discov-
ered that neither IDN5706, SKF96365, nor the co-
administration of both drugs affected PPF; these findings
imply that the effects observed in our experiments have a
postsynaptic, not presynaptic, explanation, as has been
widely described for the PPF ratio in the hippocampus [25,
26]. SKF96365 is a TRPC inhibitor commonly employed to
study TRPC6 channels [24]. A specific TRPC6 inhibitor is
currently not available. SKF96365 is the drug most com-
monly employed for this purpose [24, 41-44]. For that


Fig. (4). Synaptic proteins are not significantly affected by 30 min of treatment with IDN5706. A) Immunoblot of synaptic proteins
shown in duplicate for ACSF and triplicates for other treatments. Each lane represents a hippocampal sample from a different animal. B)
Quantification of A) indicates there are no significant differences in protein levels after 30 min of treatment in the hippocampus. PSD-95,
post-synaptic density 95; Syp, synaptophysin; vGlut1, vesicular glutamate transporter 1 and III-tubulin. Proteins were standardised against
-tubulin levels and relative protein levels against the ACSF condition.
8 Current Medicinal Chemistry, 2014, Vol. 21, No. 1 Montecinos-Oliva et al.

Fig. (5). The protective effect of IDN5706 on A oligomers is partially blocked by SKF96365, which prevents LTP generation. A) The
effect of A

oligomers (1 M) on fEPSP amplitude was prevented by co-administration of IDN5706 (Table 2). When a triple treatment of
SKF96365 (20 M), A

oligomers and IDN5706 (1 M) was administered (filled circles), the recovery of the fEPSP amplitudes is prevented,
resulting in a similar response to A oligomers alone. Co-treatment with IDN5706 and SKF96365 (20 M, empty triangles) partially blocked
the effect of IDN5706 (filled triangles). The horizontal line represents the time of exposure to each treatment. Control slices where bathed in
ACSF (empty circles). B) Quantification of A), in relative units compared with basal levels. Representative traces of each treatment are
shown in the inset. C) LTP in hippocampal slices from two-month-old mice incubated with 1 M A oligomers in the presence (filled trian-
gles) or absence (empty triangles) of IDN5706 (1 M) for 40 min (horizontal line). Control slices were bathed in ACSF (empty circles). TBS
was applied at time point zero (arrow). Mean values SEM were plotted for 6 different experiments. D) Average fEPSP reached for each
treatment in C) between 50-60 min after TBS E) LTP induced by a theta-burst stimulation (TBS). Slices were incubated with IDN5706 (filled
circles) and co-incubated with IDN5706 and SFK96365 (filled triangles) for 10 min before and after TBS (horizontal bar). Control slices were
bathed in ACSF (empty circles). F) Average slopes for each treatment in E) between 50 and 60 min after TBS. Mean values SEM were
plotted for 6 different experiments from a minimum of 3 animals *P < 0.05, ** P < 0.01, *** P < 0.001.
Tetrahydrohyperforin Effects in Hippocampal Slices Current Medicinal Chemistry, 2014, Vol. 21, No. 1 9
Table 2. Summary, effects of IDN5706 and SKF96365 on A
o
.
Condition
Average fEPSP Amplitude
(Relative Units)

ACSF 1.014 0.07 see Fig. 5A-B
IDN5706 2.491 0.05 see Fig. 5A-B
A oligomers 0.718 0.06 see ref. [27]
IDN5706 + Ao 1.068 0.05 not graphed
IDN5706 + Ao+SKF96365 0.073 0.05 see Fig. 5A--B
Average amplitude obtained after treatment of IDN5706 (Tetrahydrohyperforin, 1 M) and SKF96365 (20 M) on neurotoxicity caused by A oligomers. Notice that the value for A
oligomers alone and IDN5706 + A oligomers are a result from experimental data not graphed in this work. Ao is concordant with previous publications from our group [27].


Fig. (6). Improved spatial memory by IDN5706 treatment is affected by SKF96365. Morris water maze escape latencies of wild-type
mice injected i.p. for 10 weeks with A) 1 M IDN5706 and co-injected with 6 mg kg
-1
solutol (group 1, filled circles) or 20 M SKF96365
(group 2, empty circles), 2 hours before training. B) Solutol solution and co-injected with 20 M SKF96365 (group 3, empty circles), or with
6 mg kg
-1
solutol alone (group 4, filled circles) 2 hours before training. At day 5 of training, C) quantification of escape latencies for each
experimental group, D) representative swimming tracks for each treatment E) quantification of velocities under different conditions, and F)
quantification of total swimming distance under different treatments are shown. Both velocities and swimming paths were monitored during
the entire experiment and measured at day 5. Mean values SEM were plotted for 6 different experiments from a minimum of 3 animals per
treatment group. *P< 0.05, **P< 0.01.
reason, once the coarse-grained effect of La
3+
was estab-
lished, a more fine-grained approach was chosen using
SKF96365.
Toxic A oligomers act as drivers of neurodegeneration
in Alzheimers disease. They negatively modulate synaptic
plasticity and memory [13, 30] and damage the synaptic
cleft [45]. Previously, we and others have shown that A
oligomers generated a synaptotoxic effect in hippocampal
neurons and slices, reducing synaptic efficacy and impair-
ing synaptic transmission [9, 28, 46]. IDN5706 increased
fEPSPs and LTP, even in the presence of A oligomers.
IDN5706, therefore, prevented the toxic effects of A oli-
gomers and was allowed neurons to generate a LTP after
TBS in the presence of A oligomers, which did not occur
in the presence of A oligomers alone (Fig. 5C, D and Ta-
ble 2). The results presented in (Table 2) are from several
independent experiments where different batches of A
oligomers with varying oligomer composition were used.
With our preparation protocol, dimer and trimer species are
the most common, but we often observe different toxicity
levels, although A oligomers always exerts evident toxic
effects (i.e., fEPSP decreases). Because the experiments
10 Current Medicinal Chemistry, 2014, Vol. 21, No. 1 Montecinos-Oliva et al.
shown here were performed in wild-type mice, there is no
direct comparison with plaque formation. Further studies
are necessary to clarify the molecular mechanisms involved
in the reduction of A oligomer aggregation by IDN5706.
Finally, when A oligomers plus IDN5706 were adminis-
tered in the presence of SKF96365, the protective effect of
IDN5706 was completely abolished (Fig. 5A, B). This indi-
cates that active TRPC channels are required for IDN5706 to
exert its neuroprotective effects. LTP was not induced in the
presence of IDN5706 plus SKF96365 (Fig. 5E-F). This
could be explained by the role of TRPC in neuronal depo-
larisation due to the increase in calcium at the postsynaptic
site (consistent with results in Fig. 3C). It is important to
emphasise that SKF96365 has been reported to be involved
in the inhibition of low-voltage-activated T-type calcium
channels [42], which is why the concentration of the inhibi-
tor is critical. Accordingly, we used 20 M in an attempt to
avoid this effect, which could also be responsible for the
partial blockade of IDN5706 observed in (Fig. 3A). Because
synaptic protein levels were not significantly affected after
30 min of treatment with IDN5706 (Fig. 4) but there was an
evident electrophysiological response, we conclude that the
effects of SKF96365 on fEPSPs are the product of changes
in ionic conductance and not protein synthesis. We did not
evaluate protein levels after longer periods of exposure, and
it is possible that significant changes may exist due to in-
creased protein synthesis. There is also a chance we did not
evaluated the specific proteins that were affected. However,
the proteins examined represent significant proteins at the
glutamatergic synapse in the CA1-CA3 circuitry of the hip-
pocampus, and therefore support our hypothesis that
IDN5706 activates channel opening in short time frames.
There is evidence that hyperforin not only activates TRPC6
channels but also inhibits the degradation after 24 h [47]. We
did not examine the protein levels after long exposures to
IDN5706 because we were aiming to determine early (30
min) effects to understand the electrophysiology results ob-
tained. Nevertheless, in previous studies from our group,
different protein levels were examined after the same injec-
tion protocol used here [48].

Fig. (7). In silico conformational analysis suggests a similar binding mode for IDN5706 and other reported TRPC activators. A)
Chemical structures of IDN5522 (dicyclohexylammonium salt of hyperforin), IDN5706 (tetrahydrohyperforin), Hyp9 (a 2,4-
diacylphloroglucinol derivative [35], and OAG (1-oleoyl-2-acetyl-sn-glycerol). B) Binding pose of IDN5706 (orange sticks) generated by
molecular docking into the binding pocket of the human pregnane X receptor (PXR; PDB entry 1m13). Co-crystallised hyperforin and inter-
acting PXR residues are shown in white sticks. Hydrogen bonds are indicated by dashed lines and oxygen-hydrogen distances are given in
angstroms. The bound water molecules were excluded from docking. C) Pharmacophore alignment of IDN5706 (orange), hyperforin (white),
and the synthetic TRPC6 activator Hyp9 (blue). Conformers of each compound were generated by short runs of molecular dynamics simula-
tion and were subsequently aligned to maximise structural and pharmacophoric overlay. OAG was omitted for clarity. Aligned pharma-
cophore features are labelled.
Tetrahydrohyperforin Effects in Hippocampal Slices Current Medicinal Chemistry, 2014, Vol. 21, No. 1 11
We performed Morris water maze experiments with 5-
month-old, wild-type mice, injected with 20 mg kg
-1

SKF96365 [49] for 2 weeks, in order to prevent any toxic
effect of this channel blocker. Our results indicated that
SKF96365 did not alter the general health or motor capacity
of the animals; body weight, behaviour and velocity in the
Morris water maze were unchanged (data not shown). This is
an important fact because TRPC channels are ubiquitously
found in diverse tissues, including podocytes [33] and the
brain [50, 51]. The same rationale was followed when we
decided to inject animals only during the ten days of the
Morris water maze and not throughout the entire injection
protocol (10 weeks). Overall, our maze performance data
allowed us to infer that SKF96365 was counteracting the
reported effect of IDN5706 on spatial memory in mice [48],
but it did not affect the escape latency of mice treated only
with SKF96365 (group 3), showing no toxic effects. The
increased escape latency observed on the last 3 days (after a
2-day resting period) is expected because the animals were
tested for memory recall on those days, not memory acquisi-
tion as in the first 5 days. This finding is consistent with our
electrophysiological evidence. Velocity and distance were
measured in all mice. No significant difference was found in
velocity, either within or between groups, which again sup-
ports the lack of any toxic effect of IDN5706 and/or
SKF96365 (Fig. 6E, F). The procedure was in agreement
with the protocols suggested by other authors [52]. There is
evidence that IDN5706 is able to cross the blood brain bar-
rier, leading to low concentrations of tetrahydrohyperforin in
brain tissue in studies where animals were given IDN5706
orally [12].
TRPC channels can be modulated in the hippocampus by
OAG, lanthanum, SKF96365 in a similar manner to
IDN5706, causing an increase in fEPSPs in paired pulse and
LTP protocols, and generating neuroprotection against A
oligomers. Moreover, the positive effects in LTP induction
correlate with increased memory and learning performance.
Analysis of IDN5706 by molecular docking to the bind-
ing pocket of PXR predicted a binding mode involving a
conserved three-residue hydrogen bonding pattern, which
was also observed in the PXR-Hyperforin crystal structure.
We obtained similar results with a ligand-based (receptor-
free) method of pharmacophore alignment. The three re-
ported TRPC activators, IDN5522, Hyp9, and OAG (two of
which were used in this research), as well as IDN5706
aligned well and shared a common potential pharmacophore
of two hydrogen bond acceptors and one donor. They may
thus interact in a similar way with their biological target
channel. When IDN5706 and IDN5522 were independently
administered to mouse hippocampal slices, the increase in
the fEPSP amplitude was comparable and no significant dif-
ferences were observed (Table 2 and Supplementary Fig. 1).
This observation is consistent with the idea that both com-
pounds share a similar mechanisms of action and is in
agreement with our in silico analysis.
It was recently shown that hyperforin-related phloroglu-
cinols such as Hyp9 neither activate nor antagonise PXR
[53]. However, here we employed a PXR-hyperforin co-
crystal structure to model a potential receptor-bound bioac-
tive pharmacophore of hyperforin and IDN5706. Hyperforin
was indeed shown to activate PXR [53].
To develop IDN5706 into an effective and safe treatment
of Alzheimers Disease, we must first unveil the mechanism
of action. Taking into account our results and those reported
in the literature, we conclude that IDN5706 causes neuropro-
tection in hippocampal slices by activating TRPC channels.
CONFLICT OF INTEREST
The author(s) confirm that this article content has no con-
flicts of interest.
ABBREVIATIONS
A = Amyloid -protein
DAG = 1,2-diacyl-sn-glycerol
fEPSP = Field excitatory postsynaptic potential
IDN5522 = Hyperforin
IDN5706 = Tetrahydrohyperforin
LTP = Long Term Potentiation
OAG = 1-oleoyl-2-acetyl-sn-glycerol
SKF96365 = 1-[2-(4-Methoxyphenyl)-2-[3-(4 methoxy-
phenyl) propoxy] ethyl]imidazole
TRPC6 = Transient Receptor Potential Canonical
channel subfamily 6
ACKNOWLEDGEMENTS
This work was supported by grants from FONDEF (N
D07I1052); FONDECYT (1120156 to NCI); the Basal Cen-
ter of Excellence in Aging and Regeneration (CONICYT-
PFB12/2007) to NCI; and the ICM (Iniciativa Cientfica
Milenio, Chile; No. P09-016-F) to FM. AS. is grateful for a
FONDECYT postdoctoral research grant (N 3110009).
SUPPLEMENTARY MATERIALS
Supplementary material is available on the publishers
web site along with the published article.
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Received: ??????????? Revised: ??????????? Accepted: ???????????