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Jul 25, 2006 - acylphloroglucinol compound isolated from Hypericum perforatum (St John's Wort), on. Ab-induced spatial memory impairments and on Ab ...

Molecular Psychiatry (2006) 11, 1032–1048 & 2006 Nature Publishing Group All rights reserved 1359-4184/06 $30.00 www.nature.com/mp

ORIGINAL ARTICLE

Hyperforin prevents b-amyloid neurotoxicity and spatial memory impairments by disaggregation of Alzheimer’s amyloid-b-deposits MC Dinamarca1, W Cerpa1, J Garrido2, JL Hancke3 and NC Inestrosa1 1 Centro de Regulacio´n Celular y Patologı´a ‘Joaquı´n V. Luco’ (CRCP), MIFAB, Santiago, Chile; 2Departamento de Biologı´a Celular y Molecular Facultad de Ciencias Biolo´gicas, Pontificia Universidad Cato´lica de Chile, Santiago, Chile and 3Instituto de Farmacologı´a, Universidad Austral, Valdivia, Chile

The major protein constituent of amyloid deposits in Alzheimer’s disease (AD) is the amyloid b-peptide (Ab). In the present work, we have determined the effect of hyperforin an acylphloroglucinol compound isolated from Hypericum perforatum (St John’s Wort), on Ab-induced spatial memory impairments and on Ab neurotoxicity. We report here that hyperforin: (1) decreases amyloid deposit formation in rats injected with amyloid fibrils in the hippocampus; (2) decreases the neuropathological changes and behavioral impairments in a rat model of amyloidosis; (3) prevents Ab-induced neurotoxicity in hippocampal neurons both from amyloid fibrils and Ab oligomers, avoiding the increase in reactive oxidative species associated with amyloid toxicity. Both effects could be explained by the capacity of hyperforin to disaggregate amyloid deposits in a dose and time-dependent manner and to decrease Ab aggregation and amyloid formation. Altogether these evidences suggest that hyperforin may be useful to decrease amyloid burden and toxicity in AD patients, and may be a putative therapeutic agent to fight the disease. Molecular Psychiatry (2006) 11, 1032–1048. doi:10.1038/sj.mp.4001866; published online 25 July 2006 Keywords: amyloid-b-peptide; hyperforin; neurotoxicity; spatial learning; disaggregation

Introduction Alzheimer’s disease (AD) is one of the most common neurodegenerative dementias, characterized by a progressive deterioration of cognitive functions.1 The neurotoxicity of the amyloid-b-peptide (Ab) is highly dependent on its conformation, quaternary structure and morphology of the bundles formed.2–4 It has been proposed that Ab aggregation would require inherent depolymerization mechanisms in order to explain the morphology and the stable size of the senile plaques observed in AD,5 which indicates that amyloidogenesis is a continuous process of polymerization and depolymerization.6 As Ab is toxic to neurons,7 the main targets for therapeutic intervention of the Ab cascade include the inhibition of Ab production, the inhibition of Ab aggregation and fibril formation, in addition to the inhibition of the consequent inflammatory responses caused by the Ab deposition. In this context, several substances are known to inhibit Ab fibrillogenesis in vitro, including laminin,6,8 melatonin,9 nordihydroguaiaretic acid,10 polyphenols,11 site-directed monoclonal antibodies,12 Correspondence: Dr NC Inestrosa, CRCP Biomedical Center P., Catholic University of Chile, PO Box 114-D, Santiago, Chile. E-mail: [email protected] Received 19 January 2006; revised 1 May 2006; accepted 23 May 2006; published online 25 July 2006

a1-antichymotrypsin,13 Ginkgo biloba extract,14 type IV collagen15 and b-sheet breaker peptides.16 Nevertheless, an effective therapeutic approach that interferes directly with the neurodegenerative process in AD is eagerly awaited. On the other hand, the progressive deterioration of memory and learning causes that AD patients commonly exhibit symptoms of depression in the early stages of the disease, since they realize that their cognitive functions are getting worse. Hyperforin (HYP) is an acylphloroglucinol, a photosensitive and natural derivative from Hypericum perforatum, also known as the St John’s Wort. The natural product is a complex mixture of compounds comprising several natural derivatives and HYP was identified as the molecule responsible for the antidepressant activity by a mechanism involving the inhibition of uptake of monoamines and other neurotransmitters.17 Besides its antidepressive activity, it has been suggested that HYP possesses memory-enhancing properties in rodents.18 These antecedents drove us to investigate whether hyperforin derivatives would be able to reduce both the b-amyloid deposition and improve spatial learning acquisition. We used a rat model consisting of an intrahippocampal stereotaxic bilateral injection of preformed Ab fibrils. The injections were administered into the hippocampus to induce the formation of b-amyloid deposits and the

Hyperforin affects Ab deposits in vitro and protects from its neurotoxicity in vivo MC Dinamarca et al

spatial learning acquisition was evaluated using the Morris water maze protocol.16 After the behavioral studies were finished, a histological analysis of the hippocampal region was performed, to evaluate amyloid deposition, the reactive astrocytes and microglia around the injection site. We report here that HYP treatment partially induces b-amyloid hippocampal burden fragmentation and decreased of astroglial and microglial reaction and both events can be related to a significant improvement of the spatial learning acquisition. HYP prevents both Ab fibrils as well as Ab oligomer neurotoxicity, reducing the reactive oxygen species generated by them. Moreover, we present direct evidence that HYP induces disaggregation of the b-amyloid in a dose and time-dependent manner in vitro, and that fibrils disaggregates into protofibrils, which are the intermediate species in this phenomenon in vitro. Our results open the possibility that HYP could be of potential use as a new therapeutic agent for AD.

Materials and methods Synthetic peptides and reagents Ab140 and Ab142 E22G peptides corresponding to the human sequence (Bachem Inc., Torrance, CA, USA, lot no. T-20964amd and Genemed Synthesis Inc., South San Francisco, CA, USA) were dissolved in dimethyl sulphoxide (DMSO) at a concentration of 15 mg/ml and immediately stored in aliquots at 201C before assaying. INDENA (Milan, Italy) provided diciclohexyl ammonium salt of Hyperforin C35H51O14C12H24N (HYP), octahydroperforinate lithium salt C35H59O4Li (OHP-Li) and octahydroperforinate of galanthamine C35H51O14C17H22NO3 (OHPGal). The compounds were dissolved in DMSO at 2 mg/ml and kept protected from light at 201C. AntiGFAP polyclonal antibody was obtained from DAKO (DAKO, Carpinteria, CA, USA). OX-42 a monoclonal antibody anti-CD11b protein from the histocompatibility complex class 2 was obtained from Serotec, Oxford, UK.

Surgical and injection protocol Male Sprague–Dawley rats (300 g; 3 months old) were anesthetized with Equitesin (3.5 ml/kg i.p.) and injected bilaterally into the dorsal hippocampus (3.5 mm AP, 72.0 mm ML and 2.7 mm DV, according to Bregma) stereotaxically with a 10 ml Hamilton syringe with 27G stainless-steel. The animals were injected in the hippocampus bilaterally with 3 ml (at rate of 0.5 ml/min) of 40 mg Ab fibrils formed as described previously.19 Mock animals were injected with the same volume of vehicle alone (artificial Cerebrospinal Fluid (aCSF)). To observe the effect of HYP on neurotoxicity and behavior rats were coinjected with amyloid fibrils and 6 mM HYP. At 4 days after the intracerebral injection, rats were trained in the Morris Water Maze for 2 weeks and the perfor-

mances of the different groups were recorded. After training, the animals were fixed by intracardiac perfusion to carry out the histochemical procedures.

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Behavioral test All animals were trained in a circular water maze (1.6 m diameter and 75 cm deep, painted black)20 using a two trial per day regimen. The platform (9 cm diameter) is located in the center of northwest quadrant (hidden platform quadrant). Data were gathered with a video tracking system for water maze (HVS Imagen, Hampton, UK). Briefly, the rats were trained with two trials a day, for 5 consecutive days, followed by 2 days off, and then trained for five additional days. Each trial began when the rats were allowed to swim. A trial continued until the animal was put onto the platform for 5 s and returned to its cage. Upon completion of the trials the rats were removed from the maze, dried and returned to its cage. Water (50 cm deep) was maintained at 19–211C, for details see Chacon et al.16 Perfusion and fixation Animals were anesthetized with Equitesin (3.5 ml/kg i.p.) and injected with heparin (4 USP/kg, i.p.) before perfusion. Rats were perfused through the heart with saline (0.9% NaCl) followed by fixation with 4% paraformaldehyde in 0.1 M phosphate-buffer saline (PBS). Brains were removed from their skulls and post-fixed in the same fixative for 3 h at room temperature, followed by 20% sucrose in PBS at 41C overnight. After fixation, the brains were coded to ensure unbiased processing and analysis. The brains were then cut into 30 mm coronal sections with a cryostat (Leitzx 1900) at 201C, from Bregma 1.8 mm to Bregma 4.8 mm.21 Sections from the same brain were divided in groups for analysis by the following procedures: Nissl staining (0.3% cresyl violet) and immunohistochemical staining of glial fibrillary acidic protein (GFAP). Immunohistochemical staining Free-floating immunohistochemical procedure was performed as previously described.22 Washing and dilution of immunoreagents was performed with 0.01 M phosphate-buffered saline (PBS) with 0.2% Triton X-100 (PBS-T) throughout the experiments, and two PBS-T washes were performed between each antibody incubation. Sections were pretreated with 0.5% H2O2 for 30 min to reduce endogenous peroxidase activity followed by treatment with 3% bovine serum albumin (BSA) at room temperature for 1 h to avoid non-specific binding. GFAP detection was performed using rabbit anti-GFAP (1:500) polyclonal antibody incubated overnight at 41C. A horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG second antibody (1:600) was used for GFAP detection, incubated for 1 h at room temperature. The staining was developed by incubating for 15 min with 0.6% diaminobenzidine followed by the addition of H2O2 (0.01% final concentration) and incubation for 4 min. Molecular Psychiatry

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After immunostaining, all sections were mounted on gelatin-coated slides, air-dried and dehydrated by serial rinses in graded ethanol solutions, cleared with xylene and cover-slipped with Canada balsam (Merck, Darmstadt, Germany). Immunofluorescence staining At 3 weeks after the intrahippocampal injection, the brain sections were fixed and mounted in slides washed with PBS and PBS-T, and followed by glycine 0.15 M and NaBH4 (10 mg/ml) incubations to diminish the background. Sections were washed again with PBS and PBS-T and then followed by treatment with 3% BSA at room temperature for 1.5 h to avoid nonspecific binding. Activated microglial cells detection was performed using OX-42 (1:100) an anti-type 3-complement receptor (CD11B) antibody (Serotec, Oxford, UK) incubated overnight at 41C. After washing with PBS-T containing 0.5 mg/ml BSA, sections were incubated for 2 h at room temperature with an anti-mouse IgG-FITC second antibody (1:500). The sections were washed with PBS-T in 0.5% BSA, PBS and finally water. The slides were cover-slipped with fluorescent mounting medium (DAKO). Nissl staining Mounted sections were defatted in xylene and hydrated in ethyl alcohol and water series. Nissl staining (cresyl violet) was performed as previously described.23 Congo red staining Mounted sections were defatted in xylene and hydrated in ethyl alcohol and water series. Congo red (CR) staining using ‘alkaline CR methods’ was performed as previously described.24,25 Image analysis The ROS fluorescence was imaged with a LSM 5 Pascal Zeiss confocal microscope, and analyzed by Sigman Scan Pro software.26 The fluorescence intensity quantification was carried out as the difference between the final fluorescence emitted by the neurons in the presence of the respective treatment and the background fluorescence of the cells without treatment of 10 pictures of three independent experiments. The number of neurons in vivo experiments was calculated with the Image analysis (Image-Pro Express) in  40 magnification coronal brain sections stained with Nissl. Digital quantification of positive stained areas, sections stained for nitrotyrosine, sections stained with CR and Th-S fluorescence positive deposits were measured with Sigma Scan Pro software, using three 40  pictures of the injection site per treatment.23 Results from this analysis revealed the average of total area of the deposit. Primary neuronal hippocampal cell cultures and cell viability assay Hippocampal neurons were dissociated by trypsinization as described previously26 and seeded at a density of 120  103 cells per well of 48-well micro-

Molecular Psychiatry

titer plates precoated with (50 mg/ml) polylysine, in 100 ml of Neurobasal medium supplemented with B-27 and maintained at 371C with an atmosphere 8% CO2. After 2 h, the culture media was changed. All experiments were performed on the seventh day of culture using Neurobasal medium without B-27 supplement. The hippocampal neurons were incubated for 24 h with Ab in the absence or presence of HYP. Briefly, cells were washed two times with 100 ml of PBS and then placed in Neurobasal medium (100 ml). To assess cytotoxicity, we used the modified 3-[4,5-dimethylthiazol-2-yl]-2,5-dipheniltetrazolium bromide (MTT) assay as an index of mitochondrial activity.27 The MTT assay was carried out by adding 10 ml of a 5 mg/ml MTT solution to each well (100 ml) and the multiwell plate was incubated for 30 min, after which the reaction was stopped with 100 ml of a MTT solubilization solution containing 10% Trito´n X-100 in acidic isopropanol, 0.1 N HCl (pH 4.8). Absorption values at 550–650 nm were determined using an automatic microtiter plate reader (Uniskan, Helsinki, Finland) and MTT reduction results were expressed as a percentage of control (untreated) cells.26 Measurement of intracellular peroxides Relative levels of cellular peroxides were quantified by confocal laser microscope image analysis of cultured cells loaded with 2,7-dichlorofluorescein diacetate (DCF) as detailed previously.28 Briefly, cells grown on glass coverslip pre-coated with polylysine, under the same condition described above. After 7 days in culture, the neurons were treated in Neurobasal medium without B-27 supplement with 5 mM Ab in the presence or absence of 1 mM HYP for 12 h. The cells were loaded for 30 min (371C) with 200 mM DCF in KRH-glc containing 0.02% pluronic acid. Coverslips were washed three times and left in KRHglucose for 20–40 min until cell fluorescence had reach plateau. Ab disaggregation assay Ab peptide (250 mM) in PBS buffer pH 7.2 was stirred for 24 h at room temperature to form amyloid fibrils. Then, Ab fibrils were incubated with HYP, added at final concentrations as indicated in the corresponding experiments. Time course studies were designed to measure relative changes in fibril concentration, determined semiquantitatively by (Thioflavine-T) ThT fluorescence and qualitatively on the transmission electron microscope. The samples were stirred continuously at room temperature for 6 h at 1300 r.p.m. and sterile aliquots were taken at different time points for analysis. The measurement of the relative changes in fibril concentrations was determined at different time intervals by ThT fluorescence and transmission electron microscopy. ThT-based fluorometric assay Aliquots of Ab peptide at the indicated concentrations were incubated at different times in PBS pH 7.2

Hyperforin affects Ab deposits in vitro and protects from its neurotoxicity in vivo MC Dinamarca et al

at room temperature. For coincubation experiments, HYP derivatives were added in different concentrations. To quantify amyloid formation, the ThT fluorescence method was used.29 ThT binds specifically to amyloid, such binding produces a shift in its emission spectrum, and an increase in the fluorescent signal, which is proportional to the amount of amyloid formed.30 Following incubation, Ab alone, or in the presence of the HYP derivatives, 50 mM sodium phosphate buffer pH 6.0 and 0.1 mM ThT in a final volume of 2 ml were added. Fluorescence was monitored at excitation 450 nm and emission 485 nm using a Shimadzu spectrofluorometer, as described previously.31 We confirmed that HYP did not quench ThT fluorescence at the used concentrations. Electron microscopy Fresh aliquots of samples were diluted 1:3 in water and 5 ml were placed on Parlodion/carbon coated 300-mesh copper grids for 1 min. Excess sample was removed and 15 ml of 2% aqueous uranyl acetate was placed onto the grid for 30 s, followed by removal of excess staining solution with filter paper and airdrying. Observations were carried out using Philips Tecnai 12 electron microscope, as described previously.6 The quantification of amyloid aggregates and fibrils was made using Sigma Scan Pro software.16 Statistical analysis Data from the image analysis were exported to a Sigma Plot file for statistical analysis. Results were expressed as mean7standard error. Student’s t-test was performed to analyze the image data.

Results HYP protects from the neuropathological changes induced by Ab in vivo To evaluate the potential neuroprotective effect of HYP derivatives against the Ab-neurotoxicity in vivo, male Sprague–Dawley rats were injected stereotaxically into the dorsal hippocampus, with 80 mg of Ab fibrils in the absence or presence of 6 mM of each compound.23 In order to study the histopathological changes observed after the treatments, the astrocytic response and neuronal damage at the injection site was analyzed (Figure 1). The presence of reactive astrocytes is an early event in AD pathology and size, quantity and density increase in response to brain injury.32 The GFAP immunoreactivity was used to visualize the astrocyte response after the intrahippocampal injection. The animals injected with Ab fibrils showed an important glial reaction (Figure 1c) in contrast to the basal GFAP levels of the control rats injected with aCSF (Figure 1a). Animals injected with amyloid fibrils plus HYP showed a decrease of the GFAP-staining comparable to control (Figure 1e). The injection of Ab fibrils induced astrocyte proliferation, an increase in astrocyte density, soma size and GFAP staining in the astrocytes present around the injection site (Table 1). Contrary, coinjection of HYP and Ab

reduced the reactive astrocyte proliferation induced by Ab, decreased the intensity of GFAP staining in the astrocyte soma and abolished completely the enlargement of the astrocyte perikaryon (Table 1). The HYP derivatives, OHP-Gal and OHP-Li, also decrease the astrogliosis reaction but not as well as in the case of HYP (Figure 1g and i, Table 1). We analyzed whether HYP and HYP derivatives have positive effect over the neuronal cell loss generated by Ab injection using a specific neuronal stain (Nissl stain), which did not stain glial cells.22 Sections of the dorsal hippocampal region stained with Nissl showed that the injection of Ab fibrils produced an important neuronal damage (Figure 1d), and HYP protect against the loss of granular neurons present mainly in the upper leaf of the dentate gyrus near the injection site (Figure 1f, see the inset). Animals injected with Ab fibrils and OHP-Gal and OHP-Li showed some protection but the effect was clearly smaller than HYP (Figure 1h and i, see the inset). The quantification of neuronal loss appears similar in animals treated with HYP and aCSF (Table 1), however, the neuron levels in animals treated with Ab decreased almost 70% respect to the control and this reduction was specific prevented by the concomitant injection of HYP (Table 1) and only partially affected by HYP derivatives.

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HYP avoids the formation of CR-positive amyloid deposits induced by Ab injection With the aim to detect whether the protection against astrogliosis and Ab-neurotoxicity generated by HYP and HYP derivatives correlated with a reduction of amyloid deposits, specific amyloid staining was used. As observed in Figure 2a and b, control animals injected with Ab fibrils showed the presence of strong birefringence staining with CR in the upper leaf of the dentate gyrus, respectively. In contrast, amyloid deposits found in rats coinjected with Ab and HYP were clearly reduced, showing only small fragmented amyloid deposits by staining with CR (Figure 2c–d). The image analysis of coronal brain sections analyzing the amyloid deposit area and birefringence stained with CR revealed a 90 and 82% reduction of amyloid burden in animals injected with HYP compared to controls (Figure 2i), respectively. We also analyzed the HYP effect on the amyloid burden formation using another marker for Ab, the Thioflavin-S dye. These results showed than HYP reduced a 79% of the amyloid burden formation (data not shown) consistent with the results observed with CR staining. The HYP derivative OHP-Gal showed some effect on the prevention of the amyloid deposits formation (Figure 2e and f), decreasing the area of amyloid deposits in a 50% and the birefringence in a 65% (Figure 2i). On the other hand, the OHP-Li had no effect in prevented the amyloid deposits formation (Figure 2g and h). Therefore, the decrease of the neuronal damage and astroglial response induced by Ab fibrils injection of the rats treated with HYP, and Molecular Psychiatry

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Figure 1 HYP protects from hippocampal morphological and astrogliosis reaction generated by amyloid fibril injection. (a–i) GFAP immunodetection on coronal sections of hippocampus hypertrophic astrocytes. (b–j) Nissl staining shows cellular layers of hippocampus. (a, b) aCSF; (c, d) amyloid fibril (e, f) coinjection of amyloid fibril and HYP; (g, h) coinjection of amyloid fibril and OHP-Gal; (i, j) coinjection of amyloid fibril and OHP-Li. GFAP staining shows  40 magnification and Nissl staining, panels show  4 magnification, bar correspond to 250 mm, inset shows  40 magnification, bar correspond to 25 mm.

Molecular Psychiatry

Hyperforin affects Ab deposits in vitro and protects from its neurotoxicity in vivo MC Dinamarca et al

Table 1

Compound

aCSF Ab HYP OHP-Gal OHP-Li

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Astrocytic response and neuronal damage generated by Ab are prevent by HYP

Density of reactive astrocytes (GFAP þ cell/4  103 mm2)

GFAP intensity of astrocytic soma (arbitrary units)

Size of the astrocytic soma (arbitrary units)

Number of neurons in the dentate gyrus (cells/4  103 mm2)

1275 4078 2374* 2676* 2774*

135712 260710 151720* 180715* 163713*

102719 311717 10679* 112713* 11678*

200720 6078 162715** 10579* 101711*

Fibrillar b-amyloid was injected stereotaxically in the hippocampus alone or coinjected with HYP, OHP-Gal or OHP-Li. The quantification of the GFAP inmunostain and Nissl stain of coronal brain sections around the injection site was realized using the Sigma Scan Pro program. *P < 0.05; **P < 0.01. The bold characters emphasize the hyperforin action over the others natural compounds.

Figure 2 HYP decreases the amyloid deposition in a rat model. (a, b) CR stain and birefringence at polarized light of control rats injected with Ab fibril. (c, d) Coinjection of amyloid fibril and HYP; (e, f) coinjection of amyloid fibril and OHP-Gal; (g, h) coinjection of amyloid fibril and OHP-Li. (i) Graph represents a digital quantification of the birefringence intensity of the amyloid deposits (black bars) and amyloid deposits CR-positives area (gray bars). Each image was quantified with the Sigma Scan Pro software. Panels show  40 magnification, bar correspond to 25 mm. Each bar represents the average7s.e.m. **P < 0.005 and *P < 0.05 (t-student test).

in a minor order the OHP-Gal, correlates with the decrease of amyloid burdens by this compound. HYP protects from the spatial memory loss induced by Ab deposits Based on these observations, we then analyzed whether HYP and HYP derivatives prevent the

behavioral impairment caused by Ab injection, evaluated by the Morris water maze test20 (Figure 3). The analysis of the behavioral performance showed that animals injected with Ab had the highest latency values (black circles), in accordance with the neurotoxic effects of Ab.16 The animals injected with Ab and HYP present significantly lower escape latency Molecular Psychiatry

Hyperforin affects Ab deposits in vitro and protects from its neurotoxicity in vivo MC Dinamarca et al

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values than animals injected with Ab (Figure 3a), indicating that HYP was able to reduce the cognitive impairment on spatial memory performance induced by Ab. In contrast, animals injected with aCSF or HYP did not show significant difference in their escape latency values mainly during the second week of training (Figures 3a and 4a), suggesting that HYP neither induces spatial learning impairments nor enhances the cognitive status of the rat is also the

case for the animals injected with OHP-Gal or OHP-Li (Figure 3c and e). When we analyzed the effect of both compounds over the escape latency in the animals injected with Ab fibrils, we did not observe positive effects, therefore OHP-Gal (Figure 3c) and OHP-Li (Figure 3e) did not protect from the behavioral impaired induced by Ab. Spatial acuity is a more sensitive parameter to determine spatial learning, representing the probability to find the rat in a

Figure 3 HYP protects the spatial memory acquisition from amyloid fibril-neurotoxicity. Behavioral performances in Morris water maze evaluated by the escape latency showed along the 2 weeks of training, aCSF (blue circles, n = 6), HYP alone (a), OHP-Gal alone (c) and OHP-Li alone (d) (red squares n = 5), amyloid fibrils (black circles, n = 6), and green squares (n = 5) amyloid fibril coinjected with HYP (a), OHP-Gal (c) or OHP-Li (d). (c, d and f) Spatial acuity displayed by rats injected as described in (a, b or c). *P < 0.05 (two-way ANOVA test). Molecular Psychiatry

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Figure 4 HYP protects from the spatial memory impairment induced by amyloid fibril-injection. (a) Escape latency values of representative days of training of rats injected with aCSF (white bars), HYP alone (gray bars), amyloid fibrils (black bars) and HYP plus amyloid fibrils (dash bars). (b) Average time in the four quadrant (platform quadrant) of all days of training. (c) Swimming speed scores of the different groups, showing no motor alterations by the injection procedure. (d) Representative swimming paths at day 8 of training. Each bar represents the average7s.e.m. *P < 0.05 (two-way ANOVA test).

specific region around the hidden platform. In Figure 3b, the relationship between spatial acuity and the escape latency average for the animals of the different groups treated with HYP is shown. The graph shows that animals injected with Ab were localized in a region of the graph with high escape latency values and low spatial acuity scores, reflecting the impaired spatial memory of these animals. In contrast, rats injected with Ab and HYP showed low escape latency values (similar to control rats) and spatial acuity scores close to those of control animals, supporting the protecting effect of HYP on memory impairment generated by Ab neurotoxicity. On the other hand, the animals injected with OHP-Gal (Figure 3d) or OHP-Li (Figure 3f) were localized in a region of the graph with high escape latency values and low spatial acuity scores, similar to the rats injected with Ab. With the aim to further study the positive effects of HYP over the spatial memory loss induced by Ab, we analyzed others behavior parameters. First, we analyzed the escape latency correspond to the days 8, 9

and 10 of training, where we appreciated the most significant differences in the spatial learning. The Figure 4a shows that the rats injected with Ab already showed high escape latency values contrary to the animals injected with Ab and HYP which showed an escape latency values similar to the animal controls injected with aCSF or HYP. Therefore, the presence of HYP prevented the spatial learning impairment generated for the amyloid deposits. An additional parameter analyzed was the permanence in the Quadrant 4 (Q4), an imaginary annulus where the platform is located (Figure 4b). As observed in the graph, animals injected with Ab and HYP showed higher permanence time in Q4, similar to control rats. In contrast, those rats injected with Ab showed lower time of permanence. To rule out that both procedures affect the locomotion performance, the swimming speed average (cm/s) was recorded (Figure 4c), no difference among the studied groups was observed. Representative navigation paths at day 8 of training show the notorious impaired spatial learning acquisition of animals injected with Ab fibril in Molecular Psychiatry

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Figure 5 HYP prevents the nitrotyrosine proteins formation and reduce the inflammatory process associated to Amyloid deposits in the rat hippocampus. (a) aCSF; (b) HYP alone; (c) Amyloid fibrils; (d) HYP plus amyloid fibrils. (e) OX-42 positives microglia cells were quantifications with the Sigma Scan Pro software. Nitrotyrosine immunofluorescence of coronal brain sections of rats injected with: (e) aCSF; (f) HYP alone; (g) Amyloid fibrils; (h) HYP plus amyloid fibrils. (j) antinitrotyrosine staining quantifications performed with the Sigma Scan Pro software. Each bar represents the average7s.e.m. *P < 0.05 (two-way ANOVA test).

comparison with those coinjected with HYP, which display a navigation pattern similar to control animals (Figure 4d). HYP prevents the inflammatory process and protein tyrosine nitration induced by amyloid As only HYP reduces significant the astrocyte reaction, the neuronal death and the spatial memory impairment triggered by Ab fibril injection, we analyzed more deeply its property. We first analyzed its effect over the inflammatory process, since one of the faculty described for St John’s wort is its antiinflammatory properties.33 The reactive microglia is

associated with the brains of AD patients and is particularly concentrated in the areas of senile plaque formation.34 We performed immunohistochemical procedures using the antibody OX-42 as a microglial reaction marker in the injection site.35 The injection of aCSF shows the basal fluorescence (Figure 5a). Injection of HYP alone shows a weak signal, similar to the basal levels of rat control (Figure 5b). On the other hand, amyloid fibrils injection induced a sixfold increase in the activated microglia density (Figure 5c). The amyloid fibril and HYP coinjection prevents the activation of microglial cells (Figure 5d) to a level close to animals injected with HYP. Thus,

Figure 6 HYP protects from Ab neurotoxicity in vitro. (a) Hippocampal neuron viability assay using MTT reduction method with increasing HYP concentrations. (b) Hippocampal culture treated with 5 mM final product from Ab aggregation assay in the absence or presence of increasing HYP concentrations (0.01–1.0 mM) for 24 h. Hippocampal neurons were treated and then loaded with 2,3-DCF probe during 30 min and observed at confocal microscope in vivo to evaluate the ROS production. (c) Confocal pictures of neurons without treatment showing the background fluorescence. Cells were incubated with 5 mM Ab (d), with 1 mM HYP (e) or with 5 mM Ab and 1 mM HYP (f) for 12 h, (g) Quantification of the fluorescence for the neurons treated with Ab, HYP and Ab with HYP. Each bar represents the fluorescence intensity average 7 the s.e.m. (h) Electron microscope showed the material corresponding to Ab-oligomers. The picture shows the amylospheroids with a diameter of 9–12 nm. (i) Neuronal viability was measured by MTT method. Hippocampal culture treated with HYP and Ab-oligomers. The soluble Ab fraction was added to the neurons at the concentrations of 10 mM. *P < 0.01 and **P < 0.001. Molecular Psychiatry

Hyperforin affects Ab deposits in vitro and protects from its neurotoxicity in vivo MC Dinamarca et al

HYP reduces the appearance of active microglial induced by Ab, preventing the inflammatory process associated to the Ab neurotoxicity.

We next evaluated the HYP effect on the generation of radical oxygen species (ROS) induced by the injection of Ab. Evidence described showed that

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HYP have antioxidant properties,36 so to determine whether the neuroprotective effect of HYP against Ab peptide neurotoxicity is mediated by an antioxidant pathway, we performed a nitrotyrosine immunod etection as an oxidative marker in the injection site of the animals, because it is well known that protein tyrosine nitration is an important marker of oxidative stress.37 The anti-nitrotyrosine staining was carried out 3 weeks after the intrahippocampal injection. The basal level of fluorescence is showed in the Figure 5e. Injection of HYP alone showed a weak signal (Figure 5f). On the other hand, amyloid fibril injection induced a clear signal for protein tyrosine nitration (Figure 5g). The section of animals with coinjection of amyloid and HYP presents low levels of nitrotyrosine-positive signal, similar to the control levels (Figure 5h). These data shows that the formation of nitrated-proteins is a consequence of the Ab injection, which is abolished by the presence of the HYP (Figure 5h). HYP protected from Ab-neurotoxicity in vitro To study the more detailing the HYP effects, we used the hippocampal neuron cultures. The hippocampal cells were treated with increasing HYP concentrations to evaluate the possible drug toxicity (Figure 6a), using the MTT reduction assay, an indicator of neuronal death. At low HYP concentrations the neuronal viability was not altered (0.01–1.0 mM), however, at very high concentrations (100 mM) neuronal cell death is clearly detected. Then, the neurons were treated with increasing HYP concentrations in the presence of Ab fibrils (Figure 6b). Previously, we had determined that 5 mM Ab generated around 50% of neuronal death after 24 h incubation.38 Under these conditions, the amyloid aggregates caused around 47% of the neuronal cell death (Figure 6b), however, the Ab-neurotoxicity in the presence of increasing HYP concentrations was significantly lower than Ab in the absence of the drug, therefore, HYP protected against the Ab-neurotoxicity in hippocampal neurons (Figure 6b). In addition, the reactive oxygen species (ROS) have been implicated in many aspects of aging and neurodegenerative diseases such as AD.39–41 The HYP effect on the intracellular production of ROS induced by the Ab on hippocampal neurons was evaluated using the fluorescence of the 2,3-DCF probe.40 Neurons were treated for 12 h with Ab, HYP and Ab in the presence of HYP, and then neurons were observed under the confocal microscope (Figure 6c–f). Figure 6c shows the basal levels of fluorescence in the neurons without treatment. The neurons treated with Ab, had an important increase in the ROS fluorescence levels (Figure 6d) respect to the control neurons. Neurons treated with HYP alone, only show a slight increase in the fluorescence (Figure 6e). However, HYP reduces severely the intracellular ROS levels induced by Ab treatment (Figure 6f). The fluorescence intensity levels were quantified in the Figure 6g, the graph showed a significantly decreases of peroxides generated by Ab

Molecular Psychiatry

when the cells were coincubated with HYP. These results suggest that HYP prevents the Ab-neurotoxicity in vitro through the reduction of the oxidative damage generated by Ab and also in vivo as we showed in Figure 6h. Recent studies suggest that the toxicity of Ab and other amyloidogenic proteins lies not in the insoluble fibrils that accumulate but rather in the soluble oligomeric intermediates.42 These soluble oligomers include spherical structures 3–20 nm in diameter that appear to represent strings of the spherical particles43 Ab is produced at discrete sites within cells as a monomer,44 and it rapidly enters into equilibrium with dimers and trimers,45 a process similar to that described for synthetic Ab.46 It appears that a fraction of these oligomers is highly stable (via either strong hydrophobic interactions or covalent cross-links), and a portion of these is subsequently secreted.45 These secreted oligomers of Ab can interact with neurons, altering their electrical activity and normal physiology.45 In view of these considerations, we generated Ab oligomers from the arctic mutant Ab142 E22G 47 (Figure 6h). Then, the toxicity in hippocampal neurons was measured. Ab oligomers (10 mM) produced almost 80% of neuronal cell death in 24 h, however, HYP was able to prevent its toxicity to values similar to the control neurons (Figure 6i), suggesting that HYP not only protects from Ab fibrils, but it was also capable to prevent neurotoxicity induced by the Ab oligomers. HYP disassembles the preformed amyloid fibrils To understand the effect of HYP on the amyloidogenic process, we designed experiments in which amyloid fibrils were incubated with increasing concentrations of HYP and the amount of remaining amyloid was measured at defined intervals of time. A concentration- and time-dependence effect of HYP on the disassembly of the fibers followed by the ThT assay was observed (Figure 7a). Previous to the experiments, the amyloid fibrils were formed at room temperature under continuous stirring for 24 h. We confirmed the presence of fibrils in the aggregates by electron microscopy before the addition of HYP. The fluorescence of ThT was almost unchanged during the incubation of 250 mM Ab fibrils at room temperature and continuous stirring for 6 h. Higher HYP concentrations were able to decrease the fluorescence and they disassemble the amyloid fibrils almost instantaneously. The final remnant percentage of amyloid aggregated was only 20% (Figure 7a). Lower HYP concentrations showed a gradual decrease in fluorescence. The ThT fluorescence decreased immediately after addition of HYP in a concentration-dependent manner. Electron microscopic analyses of the time course experiments present above were carried out. Aliquots of 250 mM fibrils incubated alone (Figure 7b–e) or in the presence of HYP (46 nM) (Figure 7f–i) were processed for electron microscopy at 0, 1, 3 and 6 h after the addition of HYP. The starting material was

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Figure 7 Effect of HYP on the depolymerization of amyloid fibrils. Kinetics of 250 mM Ab fibril disaggregation was followed by ThT fluorescence under constant stirring (a); symbols represent the different HYP concentration points in the assay: 0 (K); 0.046 (J); 0.46 (.); 4.6 (,); 46 (’); and 100 mM (&). (a) Electron microscopy. Aliquots took at different times (0, 1, 3 and 6 h). Pictures (b–e) correspond to 250 mM amyloid fibrils alone and (f–i) to amyloid fibrils with 46 nM HYP. The arrowheads indicate the amorphous material. The arrows in (i) indicate the protofibrils (bar = 225 nm). (j) Shows a fourfold increase in the magnification of (i), the arrows indicate the protofibrillar material (bar represents 900 nm). The histogram (k) shows the quantification of the fibrillar material area from samples obtained in three separate experiments. Each point represents the mean values 7s.e.m. with *P < 0.005 and **P < 0.001.

characterized by large dense masses formed by numerous sheared amyloid fibrils as depicted in Figure 7b and f; they did not change consistently

throughout the experiment in the absence of HYP (Figure 7b–e). When the starting material was mixed with HYP (46 nM) (Figure 7f–i) the large aggregates of Molecular Psychiatry

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fibrils were reduced markedly and small amorphous aggregates (see arrowheads in Figure 7h) were occasionally observed along with the appearance of smaller and denser aggregates of 5 nm diameter protofibrils (see arrows in Figure 7i and j).48,49 The relative abundance of amyloid fibrils was quantified (Figure 7k). The black bars show the amyloid fibrils without HYP and the gray bars show the amyloid fibrils plus HYP at 0, 1, 3 and 6 h of incubation. HYP cause a significant decrease of approximately 20% in the amyloid fibrils (Figure 7k). Our data clearly suggests that under the conditions tested, HYP induces a significant reversion of the amyloid aggregation process as shown by a decrease in the amount of amyloid fibrils and a replacement of amyloid fibrils by amorphous material and protofibrils.

Discussion Our data shows that HYP protects from behavioural and morphological disturbances generated by Ab in vivo. We also demonstrate that two others HYPderivatives had no anti-amyloidogenic effect, but showed positive effects over the astrogliosis and neuronal damage induced by Ab injection, suggesting that chemical modifications of HYP can be made with the aim to improve its stability and bioavailability for its therapeutic use. In this context, we decided to further study the effect of HYP in order to elucidate a possible mechanism. One of the features present in AD brain analyzed in the present study is the presence of reactive astrocytes associated with senile plaques, which apparently plays a role in the neurodegenerative events of the disease.49 In fact, neuronal processes are attracted to plaques in response to trophic signals emanating from reactive plaque-associated astrocytes.50 In this context, we analyzed the astroglial cells present after HYP and HYP derivatives injection. We showed that HYP clearly diminished the astrogliosis around the amyloid deposit, this effect was lower in animals injected with OHP-Gal or OHP-Li. Different degrees of astrocyte reactivity were observed by GFAP immunostaining, after the animals were injected with Ab fibrils alone, they presented a strong glial reaction with the presence of classical appearance of hypertrophic astrocytes.16 Thus, at least until 18 days after coinjection of HYP plus Ab, the decrease of the reactive astrocytes was correlated with the protection of the granular cell layer loss. On the other hand, the microglial activation was analyzed, with the idea to associate it to a proinflammatory process.50 We found that HYP was able to reduce the activation of microglial cells that surround the Ab deposits, and this is a clear beneficial effect considering that reactive microglia are present around amyloid plaques in AD brains.34,35 Also, we determined that HYP prevented the morphological damage caused by amyloid fibrils in vivo. The Nissl staining showed that the amyloid deposits triggered neuronal cell loss Molecular Psychiatry

in the upper leaf of the dentate gyrus, which was prevented by HYP, these results correlated with the protection on the spatial memory loss induced by Ab (see also16). The neuronal cell loss produced by Ab injection correlates with the behavioural impairment. In this study, we have established that the coinjection of Ab and HYP resulted in an almost complete protection from behavioural disturbances, evaluated by the Morris water maze test, contrary to what was observed in animals injected with OHP-Gal or OHPLi. Also, when we analyzed the amyloid deposits formed by injection of Ab and HYP or HYP derivatives, we observed that only animals coinjected with Ab and HYP showed a reduction of amyloid deposits by CR stain (Figure 2c) and ThS fluorescence (data not shown). Our observations indicated only a partial decrease of hippocampal amyloid deposits induced by Ab injection, suggesting that the amyloid deposits are not the only cause of the behavioral disturbances observed here, similar results were obtained previously using b-sheet breaker peptides.16 We do not know the precise mechanism of action of HYP, but the data showed in this work strongly suggest that a reduction of amyloid deposition observed after HYP treatment could be the result of amyloid fibril depolymerization. Other natural derivatives, like the Curcumin of the yellow Curry spice, reported anti-flammatory and antioxidant effects.51,52 In addition, Curcumin was able to reverse the amyloid-induced neuropathology, along with its capacity to block amyloid polymerization.53 Ginkgo biloba another natural product is largely used as a positive memory modulator54 and it has been reported that shows prevention of age-related spatial memory deficits in a transgenic mouse model of AD.55 Also, certain ingredients of green tea modulate APP cleavage sufficiently to reduce the Ab levels and amyloid plaques in AD mouse models.56 Recently, it has been reported than Resveratrol, a polyphenol from grapes and red wine, reduce the intracellular and secreted amyloid levels.57 The precise mechanisms by which all these compounds exert their effect remain to be elucidated. HYP protects from the neurotoxicity induced by Ab in hippocampal neuron cultures, moreover, we showed that HYP protects against oligomer-mediated neurotoxicity. This result is important, since Ab oligomers (including stable dimers and trimers) can interfere with LTP and thus synaptic plasticity45,47 generating the neurotoxicity observed in triple transgenic mice,58 and ROS are involved in Ab neurotoxicity.26,41,59 Therefore, our results suggest that the protection from the Ab neurotoxicity was partially mediated by the ROS reduction. It has been observed that protofibrils seem to be an important intermediate in Ab fibrilogenesis.60 Our results support the hypothesis that HYP causes the disassembly of fibrils to amorphous material and protofibrils. Therefore, we assumed that amyloid deposits are unstable in the presence of HYP. The HYP exponential concentration dependence for fibril

Hyperforin affects Ab deposits in vitro and protects from its neurotoxicity in vivo MC Dinamarca et al

depolymerization suggests that there is a rather width concentration range requirement and the critic concentration for its effect on amyloid depolymerization is lower than 46 nM. Of practical point of view is the observation that this HYP concentration is lower than the plasma levels of the drug when it is administrated orally to humans.61 The disassembly of preformed fibrils, followed by ThT fluorescence was shown to be a time- and HYP concentration-dependent process, suggesting that the HYP anti-amyloidogenic action may be of therapeutic value for AD patients. Previous studies in behavioral paradigms suggest that H. perforatum extracts show an antidepressive activity, where HYP plays a key role in those effects. Table 2

In this context, the antidepressive activity of HYP has been related to the inhibition of the uptake of neurotransmitters in a nonselective manner by interference with intracellular sodium homeostasis.17 Also, it was observed that HYP has a facilitator action on hippocampus acetylcholine released.62 Moreover, there is evidence that HYP increase the soluble cleavage of APP, reducing the secreted Ab levels.63 There are numerous natural derivatives used to prevent several pathologies. In the case of AD, curcumin and G. biloba extract EGb 761 are the more studied natural compounds. Table 2 shows a summary and a comparison of the neurological and the anti-amyloidogenic properties between these com-

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Effects of natural derivatives on AD neurodegenerative and amyloidogenic changes

General neurological effects

Anti-amyloidogenic effets

Hyperforin

Curcumin

Ginkgo

St John’s wort is an antidepressant approved by the commission E of the German Federal Institute for Drugs and Medical Devices64

NA

NA

Antidepressant by inhibition of serotonin uptake by free intracellular Na þ elevation in mouse brain synaptosomes17

Antidepressant by serotonin and noradrenaline increase levels in rat depression model65

ND

Weak inhibitor of monoamine oxidase-A and -B in vitro66

Inhibition of monoamine oxidase activity in mouse brain53

Inhibition of monoamine oxidase activity in mouse corpus striatum67

Stimulation of Ach release in vivo62

ND

Antagonists of GABAAR, in Xenopus leavis oocytes68

Inhibition of ROS production in cell culture and in vivo69a

Inhibition of ROS production in human cell line culture52

Inhibition of ROS production in neuroblastoma cell line culture70

Anti inflammatory effect71a

Anti-inflammatory effect51

ND

Improve amyloid cognitive deficits in vivoa

ND

Prevent spatial memory deficits in Alzheimertransgenic model in vivo55

Decrease of b-amyloid depositions in vivoa

Decrease of b-amyloid in vivo53

Decrease of b-amyloid in vivo55

Enhancement of secretory APP proteolytic precessing in PC12 cell line culture63

ND

Inhibition of b-amyloid production in aging rats72

Ab protection in hippocampal cell culturea

Ab protection in neuroblastoma cell culture53

Ab protection in hippocampal cell culture73

Inhibition of Ab polymerization and disaggregates Ab in vivoa

Inhibition of Ab polymerization and disaggregates Ab in vivo53

Inhibition of Ab aggregation in neuroblastoma cell line74

ND: non determined. NA: non applicable. a Present manuscript. Molecular Psychiatry

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pounds and hyperforin. The neurological effects evaluated are related to AD: antidepressant properties, effect over neurotransmission and anti-oxidant and anti-inflammatory properties. On the other hand, we also evaluated the effects of these compounds over features related to AD: the cognitive alterations produced by Ab, the Ab burden stability, the amyloid production, the cell-toxicity generated by Ab, the amyloid polymerization and the stability of amyloid fibrils. Concerning the neurological effects of hyperforin, curcumin and ginkgo, it has been described that St John’s wort may be effective for depression. In Europe this herb is widely prescribed and a number of studies have been conducted that support the treatment efficacy of certain St John’s wort extracts. An overview of 23 clinical studies in Europe indicated that this herb might be useful in cases of mild to moderate depression. The Commission E of the German Federal Institute for Drugs and Medical Devices has approved St John’s wort as a nonprescription medicine for ‘psycho-vegetative instability, depressive moods, anxiety and/or nervous disorder’.64 On the other hand, neither curcumin nor ginkgo have been approved for antidepressant treatment, nevertheless in a rat depression model, curcumin showed antidepressant effects, increasing the serotonin and noradrenaline levels in vivo. Another important issue mentioned in Table 2 is the inflammatory properties of these compounds. Hyperforin and curcumin have antiinflammatory effects, such activity has not been described for ginkgo. Also, the three compounds had been described as anti-oxidant, because they reduce the production of ROS. Table 2 also showed an analysis of the specific antiamyloidogenic properties, where the principal differences were found between hyperforin and curcumin. The paper of Yang et al., showed that curcumin inhibits the amyloid b-fibrils polymerization and reduces the amyloid in vivo, no study on the effects of curcumin on cognitive effects was reported.53 In the present work, we demonstrate that HYP inhibits the amyloid b-fibrils polymerization and reduces the amyloid deposits in vivo, similar to curcumin, however, we also showed the beneficial HYP effects over the spatial memory loss induced by Ab accumulation. On the other hand, hyperforin, as ginkgo, has an effect over the APP processing. Ginkgo treatment by lowering circulating free cholesterol decreased APP processing and inhibits the production of Ab.72 Hyperforin is an activator of secretory processing of APP in part by a mechanism of action that affects intracellular pH.63 In conclusion, HYP: (1) decreases amyloid deposit formation, Ab-induced neuropathological changes and behavioral impairments in a rat model of amyloidosis; (2) prevents Ab-induced neurotoxicity in hippocampal neurons, avoiding the increase in reactive oxidative species associated with amyloid toxicity; (3) disaggregates amyloid fibrils in a dose and time-dependent manner, suggesting that in addition to its anti-deppressive action, HYP obtained

Molecular Psychiatry

from the St John Wort, may present advantages for people in risk of AD, and eventually for its treatment.

Acknowledgments We thank Joselyn Mauna and Rocio Artigas for their help with the glial and microglial studies. This research was supported by grants from FONDAP (No 13980001) and the Millennium Institute for Fundamental and Basic Biology (MIFAB).

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