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Current Medicinal Chemistry, 2014, 21, ????-????

0929-8673/14 $58.00+.00 2014 Bentham Science Publishers

Effects of Tetrahydrohyperforin in Mouse Hippocampal Slices: Neuropro-tection, Long-term Potentiation and TRPC Channels

C. Montecinos-Oliva1, A. Schller

2,3, J. Parodi

1, F. Melo

2,3and N.C. Inestrosa*

,1,4,5

1Centro de Envejecimiento y Regeneracin (CARE), Departamento de Biologa Celular y Molecular; Facultad de

Ciencias Biolgicas, Pontificia Universidad Catlica de Chile, Santiago, Chile; 2Molecular Bioinformatics Laboratory,

Millennium Institute on Immunology and Immunotherapy;3Departamento de Gentica Molecular y Microbiologa;

Facultad de Ciencias Biolgicas, Pontificia Universidad Catlica de Chile, Santiago, Chile;4Center for Healthy Brain

Ageing, School of Psychiatry, Faculty of Medicine, University of New South Wales, Sydney, Australia;5Centro 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, Morriswater 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:Aoligomers, 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 thedeath 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 thecognitive deficits observed in AD [2]. The mechanisms in-volved in the pathogenic changes triggered by Aoligomersare not clearly understood. A oligomers trigger neuronaldysfunction and cytoskeletal alterations, early manifestationsthat 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 areduction in synaptic proteins and alterations in synapticfunction [2, 5].

Hyperforin is a prenylated phloroglucinol derivative andthe primary active molecule responsible for the anti-depressant activity of St. Johns Wort (Hypericum perfora-tum)[6]. Ithas been used for centuries in Chinese traditionalmedicine 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: [email protected]

[7]. Hyperforin has been suggested to enhance memory inrodents [8] and may have additional anti-inflammatory, anti

bacterial, antiangiogenic and antitumoral effects. Accordingly, we have previously shown that Hyperforin reduces thebehavioural alterations induced by intra-hippocampal injection 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 awell as its bioactivity may be rapidly lost during storage [1011]. Tetrahydrohyperforin (IDN5706) is a semi-syntheticderivative of Hyperforin that has higher stability and increased oral bioavailability [12], while maintaining its neuroprotective properties [13].

So far, the mechanism of action of IDN5706 that explains its memory and behaviour altering effects is noknown. Studies indicate that the canonical transient receptopotential 6 (TRPC6) channel, a tetrameric, non-selectivecation channel, is specifically activated by Hyperforin [14]TRPC6 channels have also been found to promote dendriticgrowth [15] and play a role in the formation of excitatorysynapses [16]. Here, we report that IDN5706-mediated activation of TRPC channels improves the synaptic responsemeasured by a reversible increase in field excitatory postsynaptic potential (fEPSP), has neuroprotective effects onA oligomers, and improves spatial memory in wild-type


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2 Current Medicinal Chemistry, 2014, Vol. 21, No. 1 Montecinos-Oliva et a

mice. All of these effects might be related to the mechanismof 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 asemi-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 2.4mM stock solution was prepared in a 25% Solutol aqueoussolution. OAG (1-Oleoyl-2-acetyl-sn-glycerol) was obtainedfrom Sigma (St. Louis, MO), 3 mM stock solution was pre-pared in ethanol (20 mg mL-1).

Aoligomer Preparation

A1-42was 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 approximately1350 rpm for 1 h at room temperature. Final concentrationsfor electrophysiology studies were 1 M A oligomers and0.02% DMSO. Data on the ratio of monomers to oligomerictetramers (low molecular weight) present after following thisprotocol 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. Constantmonitoring of general health and behaviour was performedduring the injections and test periods, in accordance with theGuide for the Care and Use of Laboratory Animals publishedby the National Academy of Science, National AcademyPress, Washington, D.C. for experiments involving animals.

Slice Preparation and Electrophysiology

Hippocampal slices from C57Bl/6 mice were preparedwith ice-cold artificial cerebrospinal fluid (ACSF) containing124 mM NaCl, 2.7 mM KCl, 1.25 mM KH2PO4, 2 mMMg2SO4, 26 mM NaHCO3, 2.5 mM CaCl2, and 10 mM D-glucose bubbled with 95%/5% O2/CO2 gas, according to

standard procedures previously described by our laboratory[18]. In every case, 100 M picrotoxin, (PTX, Sigma-Aldrich, P1675) a non-competitive GABAA antagonist, wasused 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 radiatumwithin 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 a20 s interval and theta burst stimulation (TBS), five trains of10 bursts at 5 Hz each train having 4 pulses of 100 Hz, witha 20-s interval. To discriminate LTP generation, we deter-

mined a threshold of 30-40% potentiation. Paired pulse facilitation was measured as the slope ratio between two con-secutive responses (R2/R1) to two stimulation pulses with a100 ms interval. Presynaptic volley was measured in order toestablish any changes in the number of fibres stimulated dur-ing the experiment. Bar charts were obtained by calculatingthe average amplitude reached within 20-30 min of treatmenfor field potential and peak slope reached between 5-10 min

after LTP induction. In every case, drugs were diluted inACSF and the control used was ACSF. Recordings werefiltered at 2.0-3.0 kHz, sampled at 4.0 kHz using an A/Dconverter, and stored with pClamp 10 (Molecular Devices)Evoked postsynaptic responses were analysed off-line, usinganalysis software (pClampfit, Molecular Devices) that allowed visual detection of events, computing only thoseevents that exceeded an arbitrary threshold. In every case, anaverage of 4 responses per min was plotted. To avoid theanalysis of population spikes in LTP protocols, the slope waplotted instead of the amplitude.

Immunoblot

We treated 350 m hippocampal slices from two-monthold male mice for 30 min in ACSF with different solutionsBath temperature was maintained at 37C during treatmentNext, cortical and hippocampal tissue was dissected in icecold ACSF solution. Samples were lysed and 40 g of protein were loaded onto a 10% SDS-PAGE gel and transferredto a PVDF membrane. Primary antibodies used were as follows: PSD-95 (clone K28/43 UC Davis/NIH Neuromab Facility), vGlut1 (clone N59/36, UC Davis/NIH NeuromabFacility), Syp (sc-7568, Santa Cruz Biotechnology, Inc.), tubulin (ab7751, Abcam). Secondary antibodies were antirabbit or anti-goat conjugated to IgG peroxidase and blotswere developed using an ECL kit (Western Lighting PlusECL, PerkinElmer).

Administration of Drugs

From a total of 20male 5-month-old mice, five were injected intraperitoneally (i.p.) with 6 mg kg

-1 IDN5706 three

days a week (Monday, Wednesday and Friday) for 10 weeksand with 6 mg kg-1 solutol (vehicle solution for IDN5706and SKF96365 preparation, LD50 is 8.74 g kg

-1) 2 h before

the water maze training session (group 1). Five mice wereinjected i.p. with IDN5706, on the same schedule as group 1and then 20 mg kg-1 SKF96365 injections 2 h before thetraining session (group 2). The third group of five animalsreceived 6 mg kg

-1solutol 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 session(group 4). The animals received SKF96365 two hours beforetesting because TRPC6 dysfunction results in podocyte failure. We did not want to expose animals to a dose that wouldaffect the kidneys or the general health of the mice. Within 2h of injection, needle injection wound had completely healedthe wound, which is particularly important to avoid infections as animals are introduced into a pool. During the entiretreatment period, the mice were subjected to a supervisionprotocol to track their weight, behaviour and general healthAll treatment groups were chosen randomly.


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Tetrahydrohyperforin Effects in Hippocampal Slices Current Medicinal Chemistry, 2014, Vol. 21, No. 1

Morris Water Maze Behavioural Task

After drug administration, a total of twenty wild-typemice (8 months old) were subjected to 5 days of trainingfollowed by a 2-day resting period and a final three daysof training. A circular white pool was made opaque withnon-toxic white paint, and a platform was hidden (diame-ter: 9 cm) in quadrant four. The water temperature waskept between 18-20 C. Testing criteria were achievedwhen the animal reached the platform within 60 sec andstayed on it for a minimum of 3 sec. When finished, theanimals were returned to their cages, following protocolspreviously established by our group [19]. The data weregathered and analysed with a video tracking system (HVSImagen, 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 MolecularOperating Environment (MOE) version 2011.10 (ChemicalComputing Group, Montreal, Canada). Hydrogen atoms

were added, and the complex was subjected to restrainedenergy minimisation (AMBER10 force field withparm@frosst small molecules parameters and GeneralisedBorn solvation model) until the RMS gradient fell below0.05 kcal (mol )-1. Molecular docking was performed withGOLD version 5.1 (The Cambridge Crystallographic DataCentre, Cambridge, UK) [21]. Residues within 8 from theco-crystallised ligand Hyperforin were defined as the bindingpocket. CHEMPLP scoring functions and automatic searchparameters (200% efficiency) were selected. Hydrogenbonds were constrained to favour interaction with residuesSer-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 calculatedfor the docking poses with Hyperforin as the referenceligand.

Pharmacophore Alignment

Up to 10,000 conformers were generated for each ana-lysed compound with the LowModeMD search method oMOE, which employs a short molecular dynamics simulationutilising velocities with low kinetic energy on the high-frequency vibrational modes [22]. Then, an exhaustivesearch for all pharmacophore queries that showed goodstructural overlay with the conformers was performed. Thepharmacophore queries were restricted to include a minimumof one H-bond donor and two H-bond acceptors, withspherical projection sites of radius 1 .

Statistical Analysis

Data analysis was carried out with Prism software(GraphPad Software Inc., La Jolla, CA). The results wereexpressed as the means S.E. For statistical analysis, normally distributed data were analysed by one-way ANOVAwith a posteriori tests performed using Tukeys test. Nonnormally distributed data were analysed by the KruskalWallis test with post hoc tests performed using Dunns test.

RESULTSIDN5706 increases the amplitude of fEPSP and LTP

in hippocampal slices from wild-type mice. First, we explored the effectiveness of IDN5706 in mouse hippocampaslices by measuring the field excitatory postsynaptic potential (fEPSP) and long-term potentiation (LTP). We measuredfEPSPs at increasing concentrations of IDN5706 and determined a concentration-dependent rise in the amplitude ofEPSPs, with an EC50of 0.5 g mL

-1 (corresponding to ~1M) (Fig. 1A). Then, LTP was generated using highfrequency stimulation (HFS; 100 Hz, 500 ms, three stimulation trains). LTP induction was stronger in slices exposed to1 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 neuronal activity, facilitates LTP induction, and positively alter

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 50of 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.


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fEPSPs in a dose-dependent manner in hippocampal slices ofwild-type mice.

Activation of TRPC channels with OAG increases theamplitude 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 observedthat 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 activateTRPC3/6/7 channels [23]. When we added the nonspecificcation channel blocker lanthanum (30 M La

3+, known toblock TRP channels), we observed an inhibition of the OAGeffect (Fig. 2C). Quantification of fEPSPs indicated a reduc-tion in the peak amplitude of over 40% after simultaneoustreatment with OAG and lanthanum (2.46 0.16 r.u. vs. 1.44 0.23 r.u., N=3). Treatment with lanthanum alone did notalter the basal fEPSP amplitude (Fig. 2D). These results con-firmed the presence of OAG-sensitive channels in the CA1area and indicated that we are able to modulate TRPC3/6/7channels, 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 andSKF96365. To determine whether IDN5706 acts on TRPCchannels, we evaluated its effects in the presence of theTRPC channel antagonists lanthanum and SKF96365SKF96365 is a broad range inhibitor of TRP channels specific to the canonical type (TRPC) [24]. Measurements ofEPSP amplitude in slices treated with IDN5706 (1 M

were similar to those observed with OAG. The fEPSP amplitude increased two-fold over basal recordings with ACSF(2.31 0.12 r.u. vs. 1.02 0.07 r.u.). This increase was partially 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). Quantification of the peak amplitudes revealed that lanthanum was ableto block the stimulating effect of IDN5706 by almost 100%whereas 40% inhibition was observed with SKF96365 (Fig3B). Then, we compared the effect of 1 M Hyperforin(IDN5522), the compound from which tetrahydrohyperforinis derived, to determine whether it produces a similar electrophysiological effect on fEPSPs in hippocampal slices as 1M 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


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amplitude of fEPSPs (Table 1, Supplementary Fig. 1). Addi-tionally, Paired Pulse Facilitation (PPF) was not affectedduring treatment with OAG (the activator of TRPC3/6/7channels; data not shown), IDN5706, SKF96365 or the co-administration of IDN5706 and SKF96365 (Fig. 3C, D).These results indicate that the alterations observed in fEPSPsare not caused by presynaptic changes but rather by postsyn-aptic modifications [25, 26]. This finding is consistent withthe fact that TRPC channels are mainly located at the excita-tory postsynaptic region [16]. SKF96365 by itself causes asmall increase in fEPSP slope, an effect that has not beenwidely 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. Todetermine 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 minexposure) with IDN5706 (1 M) and SKF96365 (20 M) inACSF on hippocampal slices. Because SKF96365 is a morespecific blocker of TRPC channels than La3+ [24], the lattercondition was not included in all following experiments. Theevaluated proteins include the following: PSD95, the mainscaffolding protein located in the postsynaptic side of gluta-

matergic synapses [27]; vGluT1, a vesicular transporter oglutamate present in releasing vesicles on the presynapticside of glutamatergic synapses; and Syp, which is fundamental to the release of neurotransmitters in glutamatergic andGABAergic synapses. Syp was studied to characterise theoverall effect on neurotransmitter release. Protein levelwere compared to those in slices treated only with ACSF.Aa control, a different group treated only with solutol (1 Mwas evaluated (Fig. 4A). Data quantification in (Fig. 4Bshows there were no significant differences in protein levelafter 30 min of treatment, which is the exposure time used inour electrophysiology studies. These data indicate that thechanges observed are most likely due to rapid ion influx

rather than synaptic protein up-regulation, supporting ouhypothesis regarding the involvement of TRPC channels inthe mechanism of action of IDN5706.

IDN5706 prevents the fEPSP reduction triggered by

A oligomers: dependence on TRPC channel activationOur group has previously reported that both in hippocampaslices and cultured neurons, A oligomers reduce synapticactivity (i.e., fEPSPs) in paired pulse stimulation and LTP[28-31]. Specifically, treatment of hippocampal slices withAoligomers reduced fEPSP amplitude, whereas this reduction was not observed following coincubation with A oligomers 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.


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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 SKF96365to hippocampal slices treated with 1 M IDN5706 and 1 MA oligomers resulted in reduced fEPSP amplitudes (Fig.5A). Quantification of the fEPSP peak amplitudes suggestedthat the inhibition of TRPC channels prevented the neuropro-tection provided by IDN5706 (Fig. 5B). A summary of the

effects of IDN5706 and SKF96365 on Aoligomers-inducedneurotoxicity is shown in (Table 2). Next, we evaluated LTPgeneration in hippocampal slices exposed to Aoligomers inthe presence or absence of IDN5706. LTP was weakly in-duced in slices exposed to Aoligomers (Fig. 5C). In com-parison, LTP was robustly induced in slices incubated withAoligomers 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 inductionand protects against A oligomers-induced neurotoxicity.Next, we tested the effect of SKF96365 on LTP generation

induced by theta-burst stimulation (TBS) by perfusingmouse hippocampal slices with 1 M IDN5706 and 20 MSKF96365 for 20 min (10 min prior and 10 min after stimu-lation). As shown in (Fig. 5E), simultaneous treatment ofIDN5706 and SKF96365 inhibited the generation and main-tenance of LTP in the CA1 field. Quantification of the LTPdata (Fig. 5F) shows that treatments with IDN5706 aloneand IDN5706 plus SKF96365 were significantly differentfrom 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 differentfrom each other. These results indicate that the neuroprotec-tive effects of IDN5706 are abolished after co-incubationwith SKF96365, which is a TRPC blocker.

Table 1. Peak fEPSP amplitude of hyperforin and tetra-

hydrohyperforin

ConditionAmplitude 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 ofwild-type mice treated with IDN5706 in the Morris water

maze. Because IDN5706 has a significant effect on synapticactivity in the CA1 area of the hippocampus, a zone widelystudied for its role in spatial memory [32], we performed theMorris water maze test on mice treated with IDN5706 andSKF96365. 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 trainingdays (in order to avoid any systemic damage due to TRPC

inhibition in podocytes [33]). Groups 1 and 2 were treatedwith IDN5706. Injection of SKF96365 into mice treated withIDN5706 from group 2 resulted in increased escape latencycompared with animals that were treated with IDN5706alone (group 1) during the training sessions (Fig. 6A). Injection of SKF96365 into the solutol control animals (group 3produced a small but not significant increase in their escapelatencies, compared with animals that were injected with

solutol alone (group 4) and with animals that did not receiveSKF96365 (Fig. 6B). The escape latencies on training day 5from all four treatment groups are plotted in (Fig. 6C). It ievident that the group treated only with IDN5706 performedbetter in the Morris water maze than animals treated withsolutol alone or IDN5706 plus SKF96365, reflected in significantly diminished escape latencies (7.6 0.95 s, N=5 vs18.08 1.01 s, N=5, and 19.74 4.19 s, N=5). Representa-tive swimming trajectories of the four different treatmentare shown (Fig. 6D) to exemplify the difference in spatiamemory. Animals treated with IDN5706 performed betterthan the three other treatment groups, reflecting improvedspatial memory. Because velocity (Fig. 6E) was not significantly different between the four groups (24.69 1.35 cm s1

, 15.01 0.48 cm s-1

, 16.42 1.19 cm s-1

, 14.33 0.72 cms-1; in same order as in the bar chart, N=5 for each group)and the general health and weight of each animal was normal, any motor impairment caused by SKF96365 injectionwas disregarded. Swimming distance (Fig. 6F) was higher inanimals that had increased escape latency (solutol, solutoplus SKF96365 and IDN5706 plus SKF96365, 376.25 44.47 cm, 210.39 66.70 cm and 313.43 61.94 cm, respectively, with N=5 for each group) and was reduced in thegroup with lower escape latency values (IDN5706, 126.60 28.58 cm, N=5). Therefore, we infer that IDN5706 improvedspatial memory in wild-type mice and that this improvemenis counteracted by the TRPC channel blocker SKF96365.

In silico conformational analysis suggests a similar

binding mechanism for IDN5706 and other reportedTRPC activators. To evaluate whether tetrahydrohyperforin(IDN5706) is able to interact with its target channel in asimilar way to other potential TRPC activators (Hyperforin/IDN5522, Hyp9, and OAG), we performed moleculadocking and pharmacophore analysis. IDN5706 is a chemically closely related derivative of Hyperforin [34] but has amodified molecular geometry caused by two additional stereo centres introduced by chemical reduction of two carbonyl groups (Fig. 7A). Due to the current lack of a highresolution structure for TRPC, we relied on the crystal structure of the human pregnane X receptor (PXR) in complexwith Hyperforin (PDB entry 1m13) [20], as suggested by

[35]. Under the assumption that Hyperforin is bound to PXRin a bioactive form, this complex can be utilised to define thepotential pharmacophore for the interaction with TRPC [35]Molecular docking of IDN5706 predicted a similar bindingmode (RMSD = 1.02 ) compared to Hyperforin (Fig. 7B)A common pattern of hydrogen bonds to Ser-247, Gln-285and His-407 was predicted. However, rotation of the isobutyalcohol side chain of IDN5706 was required to facilitate thehydrogen bond to Ser-247. Docking of Hyp9 and OAG produced docking poses that interacted with the same residues(not shown). We next analysed the conformational space andpharmacophoric properties of all four potential TRPC6 acti


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vators: IDN5706, Hyperforin, Hyp9, and OAG. We chose aligand-based method to avoid the bias of an unrelated crystalstructure. An average of 1036 conformations were generatedby short molecular dynamics runs for each compound, fol-lowed by pharmacophore alignment. A similar alignmentwas predicted for the four activators, which is in agreementwith 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 ableto 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 aneuroprotective effect on fEPSPs and synaptic function inmouse 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, aknown TRPC3/6/7 activator [35], induced a similar increasein fEPSP amplitude, 4) the stimulating effect of both com-pounds was blocked by the nonspecific cation channelblocker lanthanum, and the TRPC broad range inhibitorSKF96365, 5) the improvement in memory described inIDN5706 treated mice was blocked by TRPC inhibition andfinally 6) IDN5706 shares a common pharmacophore withother 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 fEPSPamplitude (Fig. 1A). We explored the functional conse-quences of the observed change in the fEPSP amplitude andfound 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 orTBS that actually generated LTP) was approximately 50%for control and 66% for IDN5706 treatment. We noted thatIDN5706 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 correlatedwith rapid intracellular calcium elevation, making it difficulto obtain a steady state baseline during the LTP experiments(see Fig. 1B and 4C, -10 to 0 min). In general, IDN5706exerted a positive effect on synaptic efficacy in hippocampaslices of wild-type mice.

Hyperforin, the compound from which IDN5706 was de-rived, is a specific activator of the TRPC6 channel [35] andwas shown to have various neurobiological effects (for review, see [17]). Drugs acting as channel agonists may allowthe influx of calcium ions and LTP generation [36]. TRPCchannels are non-selective cation channels [37] and havebeen shown to be important for the regulation of the forma

tion of excitatory synapses and the improvement of spatiamemory [16]. In this context, these receptors may have animportant role in the modulation of LTP [38, 39]. Thereforewe investigated their relevance to the effects of IDN5706.

OAG, a diacylglycerol analogue and a TRPC3/6/7 channel 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 manner (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 experimentamodel. 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 oOAG to the TRPC channel family. As a different measure o

synaptic function, we calculated the PPF ratio and discov

ered that neither IDN5706, SKF96365, nor the coadministration of both drugs affected PPF; these findings

imply that the effects observed in our experiments have a

postsynaptic, not presynaptic, explanation, as has beenwidely 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 iscurrently not available. SKF96365 is the drug most com

monly employed for this purpose [24, 41-44]. For tha

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.


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

effect of Aoligomers (1 M) on fEPSP amplitude was prevented by co-administration of IDN5706 (Table 2). When a triple treatment o

SKF96365 (20 M), Aoligomers and IDN5706 (1 M) was administered (filled circles), the recovery of the fEPSP amplitudes is prevented

resulting in a similar response to Aoligomers 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 ar

shown in the inset. C) LTP in hippocampal slices from two-month-old mice incubated with 1 M Aoligomers 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 TBSE) 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 wer

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.


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Table 2. Summary, effects of IDN5706 and SKF96365 on Ao.

ConditionAverage fEPSP Amplitude

(Relative Units)

ACSF 1.014 0.07 see Fig. 5A-B

IDN5706 2.491 0.05 see Fig. 5A-B

Aoligomers 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 Aoligomers. Notice that the value for A

oligomersalone and IDN5706 + Aoligomers are a result from experimental data not graphed in this work. Aois 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 La3+

was estab-lished, a more fine-grained approach was chosen usingSKF96365.

Toxic Aoligomers act as drivers of neurodegenerationin Alzheimers disease. They negatively modulate synapticplasticity and memory [13, 30] and damage the synapticcleft [45]. Previously, we and others have shown that Aoligomers generated a synaptotoxic effect in hippocampalneurons and slices, reducing synaptic efficacy and impair-ing synaptic transmission [9, 28, 46]. IDN5706 increasedfEPSPs and LTP, even in the presence of A oligomers.

IDN5706, therefore, prevented the toxic effects of Aoligomers and was allowed neurons to generate a LTP afteTBS in the presence of Aoligomers, which did not occuin the presence of Aoligomers alone (Fig. 5C, Dand Table 2). The results presented in (Table 2) are from severaindependent experiments where different batches of Aoligomers with varying oligomer composition were usedWith our preparation protocol, dimer and trimer species arethe most common, but we often observe different toxicitylevels, although Aoligomers always exerts evident toxiceffects (i.e., fEPSP decreases). Because the experiments


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shown here were performed in wild-type mice, there is nodirect comparison with plaque formation. Further studiesare necessary to clarify the molecular mechanisms involvedin the reduction of Aoligomer aggregation by IDN5706.

Finally, when Aoligomers plus IDN5706 were adminis-tered in the presence of SKF96365, the protective effect ofIDN5706 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 thepresence of IDN5706 plus SKF96365 (Fig. 5E-F). Thiscould be explained by the role of TRPC in neuronal depo-larisation due to the increase in calcium at the postsynapticsite (consistent with results in Fig. 3C). It is important toemphasise that SKF96365 has been reported to be involvedin the inhibition of low-voltage-activated T-type calciumchannels [42], which is why the concentration of the inhibi-tor is critical. Accordingly, we used 20 M in an attempt toavoid this effect, which could also be responsible for thepartial blockade of IDN5706 observed in (Fig. 3A). Becausesynaptic protein levels were not significantly affected after

30 min of treatment with IDN5706 (Fig. 4) but there was anevident electrophysiological response, we conclude that theeffects of SKF96365 on fEPSPs are the product of changein ionic conductance and not protein synthesis. We did noevaluate protein levels after longer periods of exposure, andit is possible that significant changes may exist due to increased protein synthesis. There is also a chance we did noevaluated the specific proteins that were affected. Howeverthe proteins examined represent significant proteins at the

glutamatergic synapse in the CA1-CA3 circuitry of the hippocampus, and therefore support our hypothesis thaIDN5706 activates channel opening in short time framesThere is evidence that hyperforin not only activates TRPC6channels but also inhibits the degradation after 24 h [47]. Wedid not examine the protein levels after long exposures toIDN5706 because we were aiming to determine early (30min) effects to understand the electrophysiology results obtained. Nevertheless, in previous studies from our groupdifferent protein levels were examined after the same injection 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.


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Tetrahydrohyperforin Effects in Hippocampal Slices Current Medicinal Chemistry, 2014, Vol. 21, No. 1 1

We performed Morris water maze experiments with 5-month-old, wild-type mice, injected with 20 mg kg-1SKF96365 [49] for 2 weeks, in order to prevent any toxiceffect of this channel blocker. Our results indicated thatSKF96365 did not alter the general health or motor capacityof the animals; body weight, behaviour and velocity in theMorris water maze were unchanged (data not shown). This isan important fact because TRPC channels are ubiquitously

found in diverse tissues, including podocytes [33] and thebrain [50, 51]. The same rationale was followed when wedecided to inject animals only during the ten days of theMorris water maze and not throughout the entire injectionprotocol (10 weeks). Overall, our maze performance dataallowed 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 onlywith SKF96365 (group 3), showing no toxic effects. Theincreased escape latency observed on the last 3 days (after a2-day resting period) is expected because the animals weretested for memory recall on those days, not memory acquisi-tion as in the first 5 days. This finding is consistent with ourelectrophysiological evidence. Velocity and distance were

measured in all mice. No significant difference was found invelocity, either within or between groups, which again sup-ports the lack of any toxic effect of IDN5706 and/orSKF96365 (Fig. 6E, F). The procedure was in agreementwith the protocols suggested by other authors [52]. There isevidence that IDN5706 is able to cross the blood brain bar-

rier, leading to low concentrations of tetrahydrohyperforin inbrain tissue in studies where animals were given IDN5706orally [12].

TRPC channels can be modulated in the hippocampus byOAG, lanthanum, SKF96365 in a similar manner toIDN5706, causing an increase in fEPSPs in paired pulse andLTP protocols, and generating neuroprotection against A

oligomers. Moreover, the positive effects in LTP inductioncorrelate with increased memory and learning performance.

Analysis of IDN5706 by molecular docking to the bind-ing pocket of PXR predicted a binding mode involving aconserved three-residue hydrogen bonding pattern, whichwas 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 ofwhich were used in this research), as well as IDN5706aligned well and shared a common potential pharmacophoreof two hydrogen bond acceptors and one donor. They maythus interact in a similar way with their biological target

channel. When IDN5706 and IDN5522 were independentlyadministered to mouse hippocampal slices, the increase inthe fEPSP amplitude was comparable and no significant dif-ferences were observed (Table 2and Supplementary Fig. 1).

This observation is consistent with the idea that both com-pounds share a similar mechanisms of action and is inagreement 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. Hyperforinwas indeed shown to activate PXR [53].

To develop IDN5706 into an effective and safe treatmenof Alzheimers Disease, we must first unveil the mechanismof action. Taking into account our results and those reportedin the literature, we conclude that IDN5706 causes neuroprotection in hippocampal slices by activating TRPC channels.

CONFLICT OF INTEREST

The author(s) confirm that this article content has no conflicts of interest.

ABBREVIATIONS

A = Amyloid -protein

DAG = 1,2-diacyl-sn-glycerol

fEPSP = Field excitatory postsynaptic potential

IDN5522 = Hyperforin

IDN5706 = Tetrahydrohyperforin

LTP = Long Term PotentiationOAG = 1-oleoyl-2-acetyl-sn-glycerol

SKF96365 = 1-[2-(4-Methoxyphenyl)-2-[3-(4 methoxyphenyl) propoxy] ethyl]imidazole

TRPC6 = Transient Receptor Potential Canonicachannel subfamily 6

ACKNOWLEDGEMENTS

This work was supported by grants from FONDEF (ND07I1052); FONDECYT (1120156 to NCI); the Basal Center of Excellence in Aging and Regeneration (CONICYTPFB12/2007) to NCI; and the ICM (Iniciativa Cientfica

Milenio, Chile; No. P09-016-F) to FM. AS. is grateful for aFONDECYT postdoctoral research grant (N 3110009).

SUPPLEMENTARY MATERIALS

Supplementary material is available on the publishersweb site along with the published article.

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Received: ??????????? Revised: ??????????? Accepted: ???????????

Documents

Montecinos TRPC6 2014.pdf