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 1Centro 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 a1-antichymotrypsin,13 Ginkgo biloba extract,14 type IV collagen15 and b-sheet breaker peptides.16 Never- Alzheimer’s disease (AD) is one of the most common theless, an effective therapeutic approach that inter- neurodegenerative dementias, characterized by a 1 feres directly with the neurodegenerative process in progressive deterioration of cognitive functions. AD is eagerly awaited. The neurotoxicity of the amyloid-b-peptide (Ab)is On the other hand, the progressive deterioration of highly dependent on its conformation, quaternary 2–4 memory and learning causes that AD patients com- structure and morphology of the bundles formed. It monly exhibit symptoms of depression in the early has been proposed that Ab aggregation would require stages of the disease, since they realize that their inherent depolymerization mechanisms in order to cognitive functions are getting worse. Hyperforin explain the morphology and the stable size of the 5 (HYP) is an acylphloroglucinol, a photosensitive senile plaques observed in AD, which indicates that and natural derivative from Hypericum perforatum, amyloidogenesis is a continuous process of polymer- also known as the St John’s Wort. The natural product ization and depolymerization.6 As Ab is toxic to 7 is a complex mixture of compounds comprising neurons, the main targets for therapeutic interven- several natural derivatives and HYP was identified tion of the Ab cascade include the inhibition of Ab as the molecule responsible for the antidepressant production, the inhibition of Ab aggregation and fibril activity by a mechanism involving the inhibition of formation, in addition to the inhibition of the uptake of monoamines and other neurotransmitters.17 consequent inflammatory responses caused by the Besides its antidepressive activity, it has been Ab deposition. In this context, several substances are suggested that HYP possesses memory-enhancing known to inhibit Ab fibrillogenesis in vitro, including properties in rodents.18 These antecedents drove us laminin,6,8 melatonin,9 nordihydroguaiaretic acid,10 11 12 to investigate whether hyperforin derivatives would polyphenols, site-directed monoclonal antibodies, be able to reduce both the b-amyloid deposition and improve spatial learning acquisition. We used a rat Correspondence: Dr NC Inestrosa, CRCP Biomedical Center P., model consisting of an intrahippocampal stereotaxic Catholic University of Chile, PO Box 114-D, Santiago, Chile. bilateral injection of preformed Ab fibrils. The E-mail: [email protected] Received 19 January 2006; revised 1 May 2006; accepted 23 May injections were administered into the hippocampus 2006; published online 25 July 2006 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 1033 spatial learning acquisition was evaluated using the mances of the different groups were recorded. After Morris water maze protocol.16 After the behavioral training, the animals were fixed by intracardiac studies were finished, a histological analysis of the perfusion to carry out the histochemical procedures. hippocampal region was performed, to evaluate amyloid deposition, the reactive astrocytes and Behavioral test microglia around the injection site. All animals were trained in a circular water maze We report here that HYP treatment partially (1.6 m diameter and 75 cm deep, painted black)20 induces b-amyloid hippocampal burden fragmenta- using a two trial per day regimen. The platform tion and decreased of astroglial and microglial (9 cm diameter) is located in the center of northwest reaction and both events can be related to a significant quadrant (hidden platform quadrant). Data were improvement of the spatial learning acquisition. HYP gathered with a video tracking system for water maze prevents both Ab fibrils as well as Ab oligomer (HVS Imagen, Hampton, UK). Briefly, the rats were neurotoxicity, reducing the reactive oxygen species trained with two trials a day, for 5 consecutive days, generated by them. Moreover, we present direct followed by 2 days off, and then trained for five evidence that HYP induces disaggregation of the additional days. Each trial began when the rats were b-amyloid in a dose and time-dependent manner allowed to swim. A trial continued until the animal in vitro, and that fibrils disaggregates into protofibrils, was put onto the platform for 5 s and returned to its which are the intermediate species in this phenom- cage. Upon completion of the trials the rats were enon in vitro. Our results open the possibility that removed from the maze, dried and returned to its HYP could be of potential use as a new therapeutic cage. Water (50 cm deep) was maintained at 19–211C, agent for AD. for details see Chacon et al.16 Perfusion and fixation Materials and methods Animals were anesthetized with Equitesin (3.5 ml/kg Synthetic peptides and reagents i.p.) and injected with heparin (4 USP/kg, i.p.) before Ab1À40 and Ab1À42 E22G peptides corresponding to the perfusion. Rats were perfused through the heart with human sequence (Bachem Inc., Torrance, CA, USA, saline (0.9% NaCl) followed by fixation with 4% lot no. T-20964amd and Genemed Synthesis Inc., paraformaldehyde in 0.1 M phosphate-buffer saline South San Francisco, CA, USA) were dissolved in (PBS). Brains were removed from their skulls and dimethyl sulphoxide (DMSO) at a concentration post-fixed in the same fixative for 3 h at room of 15 mg/ml and immediately stored in aliquots at temperature, followed by 20% sucrose in PBS at 41C À201C before assaying. INDENA (Milan, Italy) pro- overnight. After fixation, the brains were coded to vided diciclohexyl ammonium salt of Hyperforin ensure unbiased processing and analysis. The brains C35H51O14C12H24N (HYP), octahydroperforinate were then cut into 30 mm coronal sections with a lithium salt C35H59O4Li (OHP-Li) and octahydroper- cryostat (Leitzx 1900) at À201C, from Bregma 21 forinate of galanthamine C35H51O14C17H22NO3 (OHP- À1.8 mm to Bregma À4.8 mm. Sections from the Gal). The compounds were dissolved in DMSO at same brain were divided in groups for analysis by the 2 mg/ml and kept protected from light at À201C. Anti- following procedures: Nissl staining (0.3% cresyl GFAP polyclonal antibody was obtained from DAKO violet) and immunohistochemical staining of glial (DAKO, Carpinteria, CA, USA). OX-42 a monoclonal fibrillary acidic protein (GFAP). antibody anti-CD11b protein from the histocompat- ibility complex class 2 was obtained from Serotec, Immunohistochemical staining Oxford, UK. Free-floating immunohistochemical procedure was performed as previously described.22 Washing and Surgical and injection protocol dilution of immunoreagents was performed with 0.01 M phosphate-buffered saline (PBS) with 0.2% Male Sprague–Dawley rats (300 g; 3 months old) were Triton X-100 (PBS-T) throughout the experiments, anesthetized with Equitesin (3.5 ml/kg i.p.) and and two PBS-T washes were performed between each injected bilaterally into the dorsal hippocampus antibody incubation. Sections were pretreated with (À3.5 mm AP, 72.0 mm ML and À2.7 mm DV, accord- 0.5% H2O2 for 30 min to reduce endogenous perox- ing to Bregma) stereotaxically with a 10 ml Hamilton idase activity followed by treatment with 3% bovine syringe with 27G stainless-steel. The animals were serum albumin (BSA) at room temperature for 1 h to injected in the hippocampus bilaterally with 3 ml (at avoid non-specific binding. GFAP detection was rate of 0.5 ml/min) of 40 mgAb fibrils formed as performed using rabbit anti-GFAP (1:500) polyclonal