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

C. Montecinos-Oliva1, A. Schüller2,3, J. Parodi1, F. Melo2,3 and N.C. Inestrosa*,1,4,5

1Centro de Envejecimiento y Regeneración (CARE), Departamento de Biología Celular y Molecular; Facultad de

Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile; 2Molecular Bioinformatics Laboratory,

Millennium Institute on Immunology and Immunotherapy; 3Departamento de Genética Molecular y Microbiología;

Facultad de Ciencias Biológicas, Pontificia Universidad Católica 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. John’s Wort. IDN5706 has shown numerous beneficial effects when administered to wild-type and double transgenic (APPswe/PSEN1 E9) mice that model Alzheimer’s 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 drug’s mechanism of action is a necessary step toward the clinical use of IDN5706 in Alzheimer’s disease.

Keywords: A oligomers, Alzheimer's Disease, neuroprotection, hippocampus, tetrahydrohyperforin, TRPC channels.

INTRODUCTION

Alzheimer’s 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. John’s 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 Católica 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 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

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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 μM and stirred continuously at approximately 1350 rpm for 1 h at room temperature. Final concentrations for electrophysiology studies were 1 μM 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 KH2PO4, 2 mM Mg2SO4, 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, 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 μm hippocampal slices from two-month-old male mice for 30 min in ACSF with different solutions. Bath temperature was maintained at 37°C during treatment. Next, cortical and hippocampal tissue was dissected in ice cold ACSF solution. Samples were lysed and 40 μg 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, LD50 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 Å)-1. Molecular docking was performed with GOLD version 5.1 (The Cambridge Crystallographic Data Centre, Cambridge, UK) [21]. Residues within 8 Å 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 Å.

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 Tukey’s test. Non-normally distributed data were analysed by the Kruskal–Wallis test with post hoc tests performed using Dunn’s 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 EC50 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 EC50 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.


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 μM, 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 μM La3+, 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 μM) 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 μM SKF96365 (2.31 ± 0.12 r.u. vs. 1.78 ± 0.10 r.u.) and completely inhibited by 30 μM 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 μM 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 μM 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 μM; 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 μM OAG. A significant difference is observed with concentrations 50 μM, compared to ACSF. The inset shows representative traces of each treatment. C) Lanthanum (5 μM) 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.


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 μM) and SKF96365 (20 μM) in ACSF on hippocampal slices. Because SKF96365 is a more specific blocker of TRPC channels than La3+ [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 μM) 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 μM, 30 min., horizontal bar) in the presence (filled circles) or absence (filled triangles) of 5 μM lanthanum or 20 μM 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.


tective effects of IDN5706 are affected by TRPC blockage, we evaluated fEPSPs after treatment with A oligomers, IDN5706 and SKF96365. The addition of 20 μM 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 μM IDN5706 and 20 μM 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 μM) and IDN5522 (Hyperforin, 1 μM), 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 Å) 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-


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.


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 μM), A oligomers and IDN5706 (1 μM) 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 μM, 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.


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 + A o 1.068 ± 0.05 not graphed

IDN5706 + A o+SKF96365 0.073 ± 0.05 see Fig. 5A--B

Average amplitude obtained after treatment of IDN5706 (Tetrahydrohyperforin, 1 μM) and SKF96365 (20 μM) 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. A o 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 μM IDN5706 and co-injected with 6 mg kg-1 solutol (group 1, filled circles) or 20 μM SKF96365 (group 2, empty circles), 2 hours before training. B) Solutol solution and co-injected with 20 μM 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 using SKF96365.

Toxic A oligomers act as drivers of neurodegeneration in Alzheimer’s 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


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 μM 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.


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 Alzheimer’s 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 (Nº D07I1052); FONDECYT (1120156 to NCI); the Basal Cen-ter of Excellence in Aging and Regeneration (CONICYT-PFB12/2007) to NCI; and the ICM (Iniciativa Científica 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 publisher’s web site along with the published article.

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

Articulo TRPC6

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