Elizabeth S Yamada

Gesine Respondek

Stefanie Müssner

Anderson C F De Andrade

Matthias Höllerhage

Christel Depienne

Agnès Rastetter

Agathe Tarze

Bertrand Friguet

Mohamed Salama

Mathias Höllerhage

Pierre Champy

Wolfgang H Oertel

Günter U Höglinger
email: guenter.hoeglinger@dzne.de

Annonacin, a natural lipophilic mitochondrial complex I inhibitor

Keywords: environmental neurotoxin, microtubule-associated protein tau, neurodegeneration, tauopathy Abbreviations

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Introduction

The microtubule-associated protein tau (MAPT), normally found in axons, is involved in microtubule polymerization and stabilization [START_REF] Weingarten | A protein factor essential for microtubule assembly[END_REF]. From one MAPT gene on chromosome 17q21 encoding 16 exons, six different tau protein isoforms are generated by alternative splicing in the adult human brain [START_REF] Lee | Neurodegenerative tauopathies[END_REF]. Tauopathies are neurodegenerative diseases defined by the presence of filamentous inclusions of hyperphosphorylated tau in neurons or glial cells [START_REF] Ballatore | Tau-mediated neurodegeneration in Alzheimer's disease and related disorders[END_REF]. Present evidence strongly suggests that the etiological spectrum of tauopathies ranges from monogenetic variants to predominantly environmental forms, with a rather large group of "sporadic" forms occupying the middle spectrum, where a genetic predisposition and hypothetical environmental triggers are probably jointly involved.

An example of an environmentally caused tauopathy is the atypical Parkinson syndrome endemic in Guadeloupe, linked to high consumption of nutritional products of Annonaceae plants [START_REF] Caparros-Lefèbvre | Possible relation of atypical parkinsonism in the French West Indies with consumption of tropical plants: a case-control study. Caribbean Parkinsonism Study Group[END_REF][START_REF] Champy | Quantification of acetogenins in Annona muricata linked to atypical parkinsonism in guadeloupe[END_REF][START_REF] Champy | MALDI-TOF MS profiling of Annonaceous acetogenins in Annona muricata products for human consumption[END_REF][START_REF] Lannuzel | Atypical parkinsonism in Guadeloupe: a common risk factor for two closely related phenotypes?[END_REF]. The implicated neurotoxins are Annonaceous acetogenins, a unique family of potent and lipophilic inhibitors of complex I of the respiratory chain [START_REF] Bermejo | Acetogenins from Annonaceae: recent progress in isolation, synthesis and mechanisms of action[END_REF] showing tropism towards mitochondria [START_REF] Derbré | Annonaceous acetogenins: the hydroxyl groups and THF rings are crucial structural elements for targeting the mitochondria, demonstration with the synthesis of fluorescent squamocin analogues[END_REF][START_REF] Kojima | Convergent synthesis of fluorescence-labeled probes of Annonaceous acetogenins and visualization of their cell distribution[END_REF]. Annonacin, a prototypical representative of this class, causes neuronal loss and somatodendritic accumulation of phospho-tau in vivo and in vitro [START_REF] Lannuzel | The mitochondrial complex I inhibitor annonacin is toxic to mesencephalic dopaminergic neurons by impairment of energy metabolism[END_REF][START_REF] Champy | Annonacin, a lipophilic inhibitor of mitochondrial complex I, induces nigral and striatal neurodegeneration in rats: possible relevance for atypical parkinsonism in Guadeloupe[END_REF][START_REF] Escobar-Khondiker | Annonacin, a natural mitochondrial complex I inhibitor, causes tau pathology in cultured neurons[END_REF]. A systematic investigation of 24 globally distributed lipophilic complex I inhibitors has demonstrated a correlation between their potency to inhibit mitochondrial complex I with their potency to induce somatodendritic redistribution of phosphorylated tau protein in cultured rodent striatal neurons [START_REF] Höllerhage | Natural lipophilic inhibitors of mitochondrial complex I are candidate toxins for sporadic neurodegenerative tau pathologies[END_REF]. Finally, a number of studies in patients with progressive supranuclear palsy, a prototypical tauopathy, using different methodological approaches, point to a primary impairment of mitochondrial energy metabolism [START_REF] Martinelli | Deficit of brain and skeletal muscle bioenergetics in progressive supranuclear palsy shown in vivo by phosphorus magnetic resonance spectroscopy[END_REF][START_REF] Swerdlow | Mitochondrial dysfunction in cybrid lines expressing mitochondrial genes from patients with progressive supranuclear palsy[END_REF][START_REF] Albers | Further evidence for mitochondrial dysfunction in progressive supranuclear palsy[END_REF]Stamelou et al., 2009). Together, these clinical and experimental observations suggest that that complex I inhibition could contribute to pathogenic mechanisms in some sporadic tauopathies.

At the other end of the spectrum, 44 pathogenic MAPT mutations have been associated with a wide range of autosomal dominantly inherited tauopathies, as the frontotemporal dementias with parkinsonism linked to chromosome 17 (FTDP-17-Tau;[START_REF] Foster | Frontotemporal dementia and parkinsonism linked to chromosome 17: a consensus conference[END_REF][START_REF] Lee | Neurodegenerative tauopathies[END_REF]. Typically, the neuropathological findings consist of frontotemporal neuronal loss and gliosis with intraneuronal neurofibrillary tangles (NFTs) and, occasionally, glial tau deposits [START_REF] Van Swieten | Phenotypic variation in hereditary frontotemporal dementia with tau mutations[END_REF][START_REF] Lindquist | Alzheimer disease-like clinical phenotype in a family with FTDP-17 caused by a MAPT R406W mutation[END_REF]. Interestingly, distinct clinical presentations may occur in association with the same mutation [START_REF] Lee | Neurodegenerative tauopathies[END_REF][START_REF] Reed | Phenotypic correlations in FTDP-17[END_REF][START_REF] Baba | The effect of tau genotype on clinical features in FTDP-17[END_REF], even within the same family [START_REF] Bird | A clinical pathological comparison of three families with frontotemporal dementia and identical mutations in the tau gene (P301L)[END_REF], suggesting that additional genetic or environmental modifiers contribute to the determination of the final clinical and neuropathological phenotype.

The R406W mutation is a particularly interesting MAPT missense mutation in exon 13 (Arg 406 to Trp, numbered according to the longest tau isoform), first described in North America (Reed et al., 1997;[START_REF] Hutton | Association of missense and 5'-splicesite mutations in tau with the inherited dementia FTDP-17[END_REF], later in the Netherlands [START_REF] Rizzu | High prevalence of mutations in the microtubule-associated protein tau in a population study of frontotemporal dementia in the Netherlands[END_REF][START_REF] Van Swieten | Phenotypic variation in hereditary frontotemporal dementia with tau mutations[END_REF], Belgium (Rademaker et al., 2003), Sweden [START_REF] Ostojic | The tau R406W mutation causes progressive presenile dementia with bitemporal atrophy[END_REF][START_REF] Passant | Familial presenile dementia with bitemporal atrophy[END_REF], Denmark [START_REF] Lindquist | Alzheimer disease-like clinical phenotype in a family with FTDP-17 caused by a MAPT R406W mutation[END_REF][START_REF] Lindquist | Genetic testing in familial AD and FTD: mutation and phenotype spectrum in a Danish cohort[END_REF] and Japan [START_REF] Ikeuchi | The role of PSEN1 and MAPT R406W mutations[END_REF].

High variability in the clinical manifestation has been described between and within R406W kindreds. Age of onset varied from 45 to 75 years and most studied cases initially resemble Alzheimer disease (AD) with early memory impairment, with frontal lobe symptoms and language problems appearing later (Reed et al., 1997;[START_REF] Lindquist | Alzheimer disease-like clinical phenotype in a family with FTDP-17 caused by a MAPT R406W mutation[END_REF]. However, some patients showed frontal personality changes as the earliest and most striking feature with the presence of parkinsonian symptoms being variable (Reed et al., 1997;[START_REF] Van Swieten | Phenotypic variation in hereditary frontotemporal dementia with tau mutations[END_REF].

The phenotypic variability of inherited tauopathies suggests the presence of environmental modifiers. Potential candidates are toxins already implicated in environmental variants of tauopathies. Searching for gene-environment interactions leading to pathogenic synergy, we investigated therefore the effects of annonacin in mice overexpressing the longest human tau isoform with the R406W mutation [START_REF] Zhang | Retarded axonal transport of R406W mutant tau in transgenic mice with a neurodegenerative tauopathy[END_REF].

Methods

Animals

R406W +/+ transgenic mice breeder pairs were donated by Dr. Virginia M.-Y. Lee, University of Pennsylvania School of Medicine (Philadelphia, USA). Their full description is given at [START_REF] Zhang | Retarded axonal transport of R406W mutant tau in transgenic mice with a neurodegenerative tauopathy[END_REF]. Briefly, the longest human tau isoform containing the R406W mutation was cloned into the mouse prion promoter (MoPrP.Xho) expression vector at the XhoI site, and fertilized in a mixed C57BL6 x C3H (B6C3/F1) strain background.

Homozygous R406W +/+ and non-transgenic wild-type (R406W -/-) mice used were kept in the same B6C3 background. Only males were used in the experiments. The genotype was confirmed by genomic DNA samples isolated from the tails using a peqGold tissue DNA extraction kit (PEQLAB, Erlangen, Germany). Cerebral expression of the mutated human protein was confirmed by Western blots.

Complex I inhibitor

Annonacin was obtained from fruit pulp of Annona muricata, as described (Champy et al., 2005). The purity of this compound was confirmed by analytical reversed-phase HPLC-DAD/ESI-Q-MS.

Annonacin exposure

The animal work was approved by the appropriate governmental authority (Regierungspräsidium Giessen,(1)MR20/15-Nr03/2005) and conducted in accordance to the European Community Council Directive 86/609/EEC. Male R406W -/-and R406W +/+ mice were 16-18 weeks old, weighting 30-40 g at the time of operation. Three to six animals were housed per cage with free access to food and water under a 12-h/12-h light/dark cycle at 26°C. Osmotic mini-pumps (Alzet 1003, 100 µl, 1.04 µl/h; Charles River Laboratory, Sulzfeld, Germany) were filled with solutions containing annonacin or vehicle in equal volumes of dimethylsulfoxide (DMSO) and polyethyleneglycol 400 (PEG) and incubated in sterile 0.9% (wt/vol) NaCl for 4 h prior to implantation. Mice were anaesthetized by an intraperitoneal (i.p.) injection of 10 mL/kg of ketamine:xylazine:saline (1:1:8), corresponding to a dose of 10 mg/kg of ketamine and 20 mg/kg of xylazine. Pumps were implanted under the skin on the back of the mice, at approximately the level of the cervical column. Forty-one R406W -/-and 42 R406W +/+ mice were infused with annonacin at a rate of 0, 6 or 9 mg/kg/day, calculated with respect to their body weight. A higher dose of 12 mg/kg/day provoked 100% mortality within 3 days of exposure and was not further pursued. Animals were weighted before and after the 3-day infusion period.

Tissue preparation

After the 3-day infusion period, mice received a lethal dose of 200 mg/kg of sodium pentobarbital i.p. and were perfused transcardially with ice-cold 0.1 M phosphate-buffered saline (PBS) for 2 minutes. Thereafter, the brains were quickly removed. The hemispheres were separated by a mid-sagittal cut. One hemisphere was post-fixed in 4% (wt/vol) paraformaldehyde in 0.1 M PBS for 24 h, cryoprotected in 10% (wt/vol) sucrose in 0.1 M PBS for 48 h, frozen in isopentane at -30°C for 2 min and stored at -80°C for histology. The other hemisphere was immediately dissected, frozen in -30°C isopentane and stored at -80°C for western blotting, measurement of proteasomal proteolytic activities and quantitative PCR. A minimum of three samples per treatment group was analyzed in each assay.

Immunohistochemistry

Brains were cut into 40-µm sections using a cryostat (Leica, Wetzlar, Germany), collected in 10 regularly spaced series, and stored in 0.1 M PBS containing 0.01% (wt/vol) sodium azide at 4°C. Free-floating sections were incubated successively for 15 min with 0.1% H 2 O 2 in 0.1 mol/L PBS to block endogenous peroxidase activity, for 1 h with 5% (vol/vol) normal donkey serum (NDS) in 0.1 M PBS and 0.2% Triton X-100 to inhibit non-specific binding sites, and for 24 h at 4°C with the mouse monoclonal primary antibodies raised against the following phospho-tau epitopes: AT270 antibody against pT181, AT8 antibody against pS202/pT205, AT100 antibody against pT212/pS214, AT180 antibody against pT231, (all from Thermo Scientific, Rockford, IL, USA; used 1:100), and AD2 antibody against pS396/pS404 (Bio-Rad, Hercules, CA; 1:1000). We used the anti-NeuN mouse monoclonal antibody (Millipore, Billerica, MA, USA; 1:1000) for labeling neurons, and the anti-Iba1 rabbit polyclonal antibody (Wako, Osaka, Japan; 1:1000) for microglia. All antibodies were diluted in 0.1 M PBS with 5% NDS and 0.2% Triton X-100. Sections were then incubated for 2 h at room temperature with the appropriate biotinylated secondary antibody (anti-rabbit or antimouse IgG; Jackson ImmunoResearch, West Grove, PA, USA; 1/200) in 0.1 M PBS. The signal was amplified with the avidin-biotin method (VECTASTAIN Elite ABC Kit, Vector Laboratories, Burlingame, CA, USA; 1:200). Bound antibodies were visualized with 0.5 mg/ml of 3,3'-diamino-benzidine-tetrachloride (DAB, Sigma-Aldrich, Munich, Germany). To exclude non-specific labeling, the primary antibodies were omitted.

Image analysis

Phospho-tau labeled neurons were analyzed in 5-7 animals per group for R406W -/-and 10-12 per group for R406W +/+ . Neu-N labeled neurons and Iba1 labeled microglia were analysed in 3-5 animals per group.

Stereological cell counts were done by an observer blinded to the animals' identity on regularly spaced (1/10) sections (average post-processing thickness 20 µm) under a 40x objective on an Olympus Microphot with the optical fractionator method using the StereoInvestigator software (MicroBrightField, Inc., Williston, VT, USA) in the frontal and parietal cortex of one hemisphere. Frontal cortex was analysed between 1.94 and 0.86 mm anterior and 0 to 2.0 mm lateral from bregma; parietal cortex between 1.1 and 2.3 mm posterior and 1.0 to 3.0 mm lateral from bregma. Coordinates were based on [START_REF] Paxinos | The Mouse Brain in Stereotaxic Coordinates[END_REF]. Our criterion for counting a neuron was the presence of its nucleus either within the counting frame, or touching its right or top limit, but not touching its left or bottom limit. Total cell numbers were estimated by integration along the rostrocaudal extent of the structures. This sampling strategy gave a Schäfer coefficient of error ≤ 0.12.

Because of the small volume of the amygdala, neurons therein were counted in one anatomically matched section per animal, sampling through the basolateral nucleus at approximately 1.34 mm posterior from bregma. The estimate was then normalized to the area of the structure.

Phospho-tau immunoreactivity in the hippocampus was determined on three regularly spaced, anatomically matched sections (from -1.5 mm to -2.3 in relation to bregma; [START_REF] Paxinos | The Mouse Brain in Stereotaxic Coordinates[END_REF]) by taking pictures with an Olympus-E330 camera with a Componon-S 2.8/50 magnification lens, attached to a light table (Copylizer eVision exe.cutive, Kaiser Fototechnik, Buchen, Germany). Pictures were analysed using Image-J software (U.S. National Institutes of Health, MD, USA, http://imagej.nih.gov/ij) to measure the mean grayness of C2/C3 region, from which the background grayness of the molecular layer was subtracted.

Western blots

Proteins were extracted in commercial tissue lysing buffer (T-PER Tissue Protein

Extraction Reagent, Thermo Scientific, Rockford, USA) containing inhibitors of proteases and phosphatases (Halt Protease Inhibitor Cocktail, Halt Phosphatase Inhibitor Cocktail, 1% each, Thermo Scientific). After centrifugation (10,000 x g, 4°C, 5 min), supernatants were recovered and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis.

To prepare sarkosyl-insoluble protein extracts, the remaining pellets were further processed according to [START_REF] Delobel | Analysis of tau phosphorylation and truncation in a mouse model of tauopathy[END_REF] with minor modifications. After homogenization of the pellets in the lysing buffer containing protease-and phosphatase-inhibitors and centrifugation at 4000 x g, 4°C, for 20 min, 1% sarkosyl was added to the supernatants and incubated for 1.5 hours at room temperature, then spun at 80,000 x g for 30 min. The pellets were then resuspended in 10 µl of 50 mmol/L Tris/HCL, pH 7.4 and also separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Proteins were then transferred onto nitrocellulose membranes and incubated with the following primary antibodies: anti-Tau5, 1:10000, anti-α-Synuclein, 1:1000 (Chemicon, Billerica, USA); anti-Tau14, 1:1000 (Zymed Laboratories, San Francisco, USA); anti-HT7, 1:1000, anti-AT8, 1:1000, anti-AT180, 1:1000 (Thermo Scientific, Waltham, USA); antiphospho-SAPK/JNK [Thr 183 /Tyr 185 ], 1:1000, anti-p44/42-MAPK [Erk1/2], 1:1000, phospho-GSK-3β [Ser9], 1:1000 (Cell Signaling, Danvers, USA); anti-phospho-GSK-3β [pY216], 1:500 (BD Transduction Laboratories, Franklin Lakes, USA); anti-GSK-3β, 1:1000, anti-p35, 1:1000, anti-p25, 1:1000, anti-Cdk5, 1:50000, anti-ß-actin, 1:1000 (Santa Cruz, Santa Cruz, USA).

Immunoreactivity was revealed with the chemiluminescent substrate kit (Pierce ECL Western Blotting Substrate, Thermo Scientific) and exposed on x-ray films (Amersham Hyperfilm TM ECL, GE Healthcare).

Proteasomal peptidase activities

Brain samples collected from frontal cortex of R406W -/-and R406W +/+ mice treated with 9 mg/kg/d or vehicle were analyzed. Supernatants were collected after sonication and centrifugation (20,000 x g, 4°C, 20 min). Degradation of 25 μM Suc-Leu-Leu-Val-Tyr-7amido-4-methylcoumarin (chymotrypsin (CT)-like activity), 40 μM N-t-Boc-Leu-Ser-Thr-Arg-7-amino-4-methylcoumarin (trypsin (T)-like activity), and 150 μM N-Cbz-Leu-Leu-Glu-betanaphthylamide (peptidylglutamyl-peptide hydrolase (PGPH) activity) (substrates from Sigma-Aldrich) was monitored for 20 min using 20 μg protein in 200 μl Tris-HCl at 37°C by spectrofluorimetry (excitation/emission wavelengths: 350/440 nm for 7-amino-4methylcoumarin, 333/410 nm for beta-naphtylamine). Peptidase activities were calculated from the slopes of the linear curves in relation to standards generated using pure fluorescent products.

Quantitative PCR

Total RNA from mouse brain was isolated (Qiagen RNeasy Mini kit, Invitrogen, Grand Island, NY, USA) and RNA integrity was controlled with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). First-strand synthesis was performed with 100 ng of total RNA (SuperScript III First-Strand Kit, Invitrogen). Real-time PCR experiments were performed using 5 µl of 1/20 diluted RT-PCR products, 0.4 µM of each primer and 12.5 µl of Sybr Green PCR master mix (Applied Biosystems, Foster City, CA, USA) in a total volume of 25 µl. Two primer pairs, one specific for human MAPT (forward: GCTCCAAAGACACCACCCA; reverse: GCTGTAGCCGCTGCGATC) and one amplifying both human and mouse MAPT cDNA (forward: GGCTCCACTGAGAACCTGAA; reverse: CCGGGACGTGTTTGATATT) were designed using primer Express 1.0. β-actin (forward: CTGTGTGGATCGGTGGCTC, reverse: CTGCTTGCTGATCCACATCTG) was used as control gene. Each sample was run in triplicate on an ABI PRISM 7500 Detection system (Applied Biosystems) and three different experiments were used for final quantifications.

Statistical analysis

For statistical analysis we used GraphPad Prism 5.0 (La Jolla, CA, USA). Results were expressed as mean ± SEM. A p-value < 0.05 was assumed to be statistically significant. The experimental groups were compared with a one-way analysis of variance (ANOVA) with the Newman-Keuls post-hoc test, or two-way ANOVA followed by a post-hoc least significant difference (LSD) test, as appropriate.

Results

Annonacin-induced mortality

Vehicle-treated animals showed no mortality. Both R406W -/-or R406W +/+ mice, treated with doses of 6 and 9 mg/kg/day of annonacin for 3 days, had a mortality rate of 6-13%.

Annonacin increases phospho-tau in neuronal somata in R406W +/+ , but not in R406W -/- mice

Vehicle-treated R406W -/-mice at 16-18 weeks of age had very low detectable levels of phospho-tau staining using the antibodies AT8 (Figure 1) and AT180 (Figure 2) in the cortex, hippocampus and amygdala. In vehicle-treated R406W +/+ mice of the same age, higher levels of AT8 and AT180 immunoreactivity were observed in the cerebral neuropil (Figures 1 and2); in the cortex, many pyramidal neurons were stained in their soma and apical dendrites; in the hippocampus and amygdala phospho-tau labeling was more localized in the soma; in the hippocampus, AT8 and AT180 labeling was more intense in the pyramidal layer of CA3 region, with some animals also exhibiting labeling through CA1 region.

Three days of systemic annonacin exposure did not significantly alter the AT8 and AT180 immunoreactivity in any of the investigated brain regions in R406W -/-mice (Figures 1 and2). However, R406W +/+ mice treated likewise with annonacin had significantly increased numbers of neurons with somatodentritic immunoreactivity for phospho-tau protein in a dosedependent manner in several brain regions, as compared to vehicle-treated R406W +/+ mice (Figures 1 and2). Such an increase was observed for pyramidal neurons in the frontal and parietal cortex labeled for both AT8 (Figure 1A-D) and AT180 (Figure 2A-D) epitopes. In the hippocampus, there was a significant increase in the intensity of AT8 (Figure 1E-F) and AT180 (Figure 2E-F) labeling in CA2/3 layer neurons.

In the amygdala, neuronal somata were already intensely AT8-and AT180immunoreactive in vehicle-treated R406W +/+ mice and annonacin exposure had no significant additional effect on phospho-tau labeling (not shown). Other brain regions examined in R406W +/+ mice, such as striatum, midbrain and cerebellum had no obvious accumulation of phospho-tau in neurons after annonacin-treatment. In no brain region analyzed, we observed AT8 or AT180 immunoreactivity in glial cells.

Immunohistochemistry with the AT100 and AT270 antibodies gave no signal in vehicle-treated R406W -/-mice and labeled neuropil (AT270) or nuclei (AT100) in R406W +/+ mice. However, somatodendritic accumulation of phospho-tau was not detected with the AT100 and AT270 antibodies in vehicle-or annonacin-treated R406W -/-or R406W +/+ mice (not shown). With the AD2 antibody, very low levels of immunoreactivity were detected in R406W +/+ mice, mainly localized in a few neurons in the deeper layers of the neocortex; no significant alteration was observed upon annonacin treatment (not shown).

Annonacin increases tau protein levels in R406W +/+ , but not in R406W -/-mice

In order to check whether an increase in tau protein levels contributes to the observed neuronal accumulation of phospho-tau, we performed Western blot analysis in brain tissue extracted from mice treated with 6 or 9 mg/kg/d of annonacin or vehicle.

T14 and HT7 antibodies, recognizing specifically human tau, gave no signal in the frontal cortex in R406W -/-mice (not shown). Expectedly, both antibodies labeled a single band of human tau (the transgene) at 67-69 kD in R406W +/+ mice. This band showed a significant increase upon annonacin treatment (Figure 3A-B).

The Tau5 antibody, which recognizes both mouse and human tau isoforms, demonstrated that annonacin exposure increased the amount of total tau in R406W +/+ , but not in R406W -/-mice (Fig. 3C-D). With the Tau5 antibody, two bands were strongly labeled at 67-69 kD and 52-54 kD in R406W +/+ mice, while only the lower kD band was intensely labeled in R406W -/-mice (Fig. 3D). Therefore, the longer isoform corresponds mainly to human tau, while the shorter isoform corresponds mainly to mouse tau; annonacin exposure increased both long and short isoforms in R406W +/+ (Figure 3E), but did not increase the short isoform in R406W -/-mice. As control, we checked -synuclein protein levels, which did not change under annonacin treatment in R406W +/+ and R406W -/-mice (Figure 3D,F).

To determine whether the annonacin-induced increase in total tau was due to augmentation of expression at the transcriptional level, tau mRNA levels were measured by quantitative PCR in cortical tissue from annonacin-intoxicated versus vehicle-exposed animals.

The results demonstrated that annonacin does not alter the expression of mouse (Figure 3G) and human tau (Figure 3H). This suggests that the observed increase in tau protein levels is therefore regulated at a posttranscriptional level.

Impairment of tau degradation could contribute to the annonacin-induced accumulation of tau protein by a mechanism involving the inhibition of the ubiquitin-proteasome system, since tau is mainly degraded by this system [START_REF] David | Proteasomal degradation of tau protein[END_REF][START_REF] Goldbaum | Proteasome inhibition stabilizes tau inclusions in oligodendroglial cells that occur after treatment with okadaic acid[END_REF][START_REF] Poppek | Phosphorylation inhibits turnover of the tau protein by the proteasome: influence of RCAN1 and oxidative stress[END_REF]. In order to test this hypothesis, the proteasomal proteolytic activities were measured in cortical tissue from annonacin-intoxicated versus vehicle-exposed animals from both R406W +/+ transgenic and non-transgenic mice lineages. The results showed that annonacin intoxication with 9 mg/kg/d inhibits proteasomal chymotrypsin-like (LLVY) and peptidylglutamyl-peptide hydrolase (LLE) activities in R406W +/+ transgenic, but not in nontransgenic mice (Figure 3I). In summary, annonacin increased both mouse and human transgenic tau protein levels in R406W +/+ , but not in R406W -/-mice, putatively by reduced proteasomal degradation.

Annonacin increases the amount of insoluble and phospho-tau in R406W +/+ , but not in R406W -/-mice

We checked whether annonacin treatment altered the solubility of tau protein. Western blot analysis demonstrated that treatment with annonacin increased the accumulation of Tau5-, HT7-, AT8-, and AT180-immunoreactive tau protein in the sarcosyl-insoluble fraction of brain homogenates of R406W +/+ mice (Figure 4A), but not of R406W -/-mice (not shown).

In order to further characterize the effect of annonacin exposure on tau, we compared the amount of phospho-tau in the soluble fraction of homogenates of cortical tissue extracted from R406W +/+ and R406W -/-mice exposed to annonacin or vehicle. Protein levels of phospho-tau labeled at the AT8 or AT180 epitopes were increased after annonacin exposure in R406W +/+ , but not in R406 -/-mice (Figure 4B,C,F). Both long and short tau isoforms labeled with AT8 (Figure 4D) and AT180 (Figure 4G) were increased in R406W +/+ ; the effect was observed with both the lower (6mg/kg) and the higher dose (9 mg/kg) of annonacin exposure, suggesting that annonacin increased the amount of phosphorylated transgenic human tau and of phosphorylated endogenous mouse tau in R406W +/+ transgenic mice, but not in R406W -/-mice.

The ratio between AT8/Tau5 (Figure 4E) and AT180/Tau5 (Figure 4H) increased significantly with annonacin exposure in R406W +/+ mice, suggesting that annonacin increased phosphorylation of tau levels more than just explained by the increase in total tau protein. As control, we assessed the levels of β-actin in both R406W +/+ and R406W -/-mice which showed no increase after annonacin exposure (Figure 4B, bottom row).

Annonacin exposure alters the levels of tau kinases

An increase in phospho-tau could have resulted from an overactivation of tau kinases. Therefore, we tested the hypothesis whether annonacin intoxication alters tau kinases.

Candidate tau kinases were selected based on their capacity to phosphorylate tau at both the AT8 and AT180 sites [START_REF] Hanger | Tau phosphorylation: the therapeutic challenge for neurodegenerative disease[END_REF]. Western blot analysis in frontal cortex samples were performed for the kinases SAPK, MAPK (Erk1/2), GSK-3β, and Cdk5.

Annonacin intoxication increased the levels of SAPK in both R406W +/+ and R406W -/- mice in a dose-dependent manner (Figure 5A,B). The protein levels of MAPK and GSK-3 increased only in R406W +/+ mice, but only after 9 m/kg annonacin treatment (Figure 5A,C,D). Levels of phosphorylated forms of GSK-3β at both activating and inactivating sites were also increased in R406W +/+ after annonacin treatment, but only with 6 mg/kg (Figure 5A,E,F).

Protein levels of Cdk5 increased in both R406W +/+ and R406W -/-in a dose-dependent manner (Figure 5A,G). Activation of Cdk5 requires its association with p35 or p39; a truncated form of p35, called p25, is responsible for an overactivation of Cdk5 and has been associated with tau hyperphosphorylation [START_REF] Cheung | Cdk5: a multifaceted kinase in neurodegenerative diseases[END_REF]. We observed a striking increase in the p25/p35 ratio, selectively in R406W +/+ after annonacin exposure, 10-fold at low and 20-fold at high toxin doses (Figure 5A, H-J).

Annonacin exposure did not cause neuronal death nor microglial reaction

Histological assessment through quantification of the number of NeuN-immunoreactive neurons demonstrated that a 3-day annonacin intoxication was not sufficient to cause neuronal cell death in R406W +/+ nor in R406W -/-mice in the brain regions analysed (Figure 6). Statistical analysis (2-way ANOVA, post hoc-Bonferroni test) demonstrated significant interaction between mice genotype and annonacin treatment for frontal cortex data (Figure 6 A-B; F=3.84; p=0.046), but not for parietal cortex (Fig. 6 C-D; F=0.003; p=0.996), hippocampus F=0.768;p=0.478), or amygdala (not shown). For all regions, the genotype (R406W -/-versus R406W +/+ ) significantly affected the observed variance, whereas annonacin treatment (0, 6 or 9 mg/kg/d) had no significant influence. These results suggest that R406W +/+ had significantly lower neuronal number in the cortex (frontal and parietal), hippocampus and amygdala compared to R406W -/-mice. Such a difference was mostly independently of annonacin treatment although there was a tendency of lower neuronal number in R406W +/+ treated with 9 mg annonacin compared to R406W vehicle-treated (p=0.09).

Annonacin exposure did not cause significant alteration in the number of microglia or astrocytes in the analysed brain regions (cortex and hippocampus) of either R406W +/+ or R406W -/-mice (Figure 7).

Discussion

The R406W is an FTDP-17 mutation associated with relatively late onset and long disease duration; although the initial clinical setting resembles AD, frontal lobe symptoms may prevail later in the disease [START_REF] Ostojic | The tau R406W mutation causes progressive presenile dementia with bitemporal atrophy[END_REF]. Transgenic mice carrying the human R406W mutation show an age-dependent somatodentritic accumulation of phosphorylated tau in the neocortex, hippocampus, cerebellum and spinal cord, and NTF-like inclusions were detected at 12 months, but not at 6 months of age [START_REF] Zhang | Retarded axonal transport of R406W mutant tau in transgenic mice with a neurodegenerative tauopathy[END_REF]. The R406W +/+ mice used in our study were 16 to 18-week old at the time of sacrifice. It seemed an appropriate window to introduce a toxin to assess its effects on the pathological phenotype, because the animals would not have yet developed intense tau pathology or any discernible behavioral deficits at this time point.

Previous work has shown that low nanomolar concentrations of annonacin cause neuronal cell death and induce tau redistribution from the axon to the cell body of striatal neurons in vitro [START_REF] Escobar-Khondiker | Annonacin, a natural mitochondrial complex I inhibitor, causes tau pathology in cultured neurons[END_REF]. These effects were shown to be associated with annonacin-induced ATP reduction [START_REF] Escobar-Khondiker | Annonacin, a natural mitochondrial complex I inhibitor, causes tau pathology in cultured neurons[END_REF]. [START_REF] Höllerhage | Natural lipophilic inhibitors of mitochondrial complex I are candidate toxins for sporadic neurodegenerative tau pathologies[END_REF] have extended the findings to additional natural complex I inhibitors, demonstrating their capacity to induce redistribution of phospho-tau to the somatodendritic compartment in good correlation with their capacity to reduce ATP levels. These in vitro models employed fetal neurons, in which tau is physiologically more phosphorylated than in adult neurons [START_REF] Goedert | Epitope mapping of monoclonal antibodies to the paired helical filaments of Alzheimer's disease: identification of phosphorylation sites in tau protein[END_REF], thus annonacin did only cause a redistribution of phospho-tau, but not a measurable increase in tau phosphorylation [START_REF] Escobar-Khondiker | Annonacin, a natural mitochondrial complex I inhibitor, causes tau pathology in cultured neurons[END_REF].

In our present study, we have shown that annonacin exposure increased the number of neurons with somatodentritic accumulation of phospho-tau (AT8 and AT180) in R406W +/+ , but not R406W -/-mice. This observed effect could have resulted from mere redistribution of phospho-tau from the axonal to the somatodentritic compartment, as in the above mentioned fetal in vitro models [START_REF] Escobar-Khondiker | Annonacin, a natural mitochondrial complex I inhibitor, causes tau pathology in cultured neurons[END_REF][START_REF] Höllerhage | Natural lipophilic inhibitors of mitochondrial complex I are candidate toxins for sporadic neurodegenerative tau pathologies[END_REF], or else there could have been a contribution of an increase in tau protein levels or an increase in tau phosphorylation in the presently used adult in vivo model. In order to investigate these two latter possibilities, we performed Western blot analyses of brain tissue extracts. Annonacin increased the amount of total (both human and mouse) tau protein in R406W +/+ , but not in R406W -/-mice, without increased mRNA expression. Similarly, in our previous cell culture experiments, annonacin induced an increase in total tau protein levels in absence of increased tau mRNA levels [START_REF] Escobar-Khondiker | Annonacin, a natural mitochondrial complex I inhibitor, causes tau pathology in cultured neurons[END_REF]. Since annonacin reduced proteasomal proteolytic activity in our present in vivo experiments, and since tau can be degraded by the proteasome [START_REF] David | Proteasomal degradation of tau protein[END_REF][START_REF] Goldbaum | Proteasome inhibition stabilizes tau inclusions in oligodendroglial cells that occur after treatment with okadaic acid[END_REF], we suggest that annonacin increases total tau level, apparently by reducing proteasomal degradation, but not by increasing tau expression.

Proteasome inhibition and tau protein increase after annonacin exposure was surprisingly only occurring in R406W +/+ , but not in R406W -/-mice. The excess of proteolytic demand by overexpression a mutated human tau protein in R406W +/+ transgenic mice may already challenge the ubiquitin-proteasome system (UPS) to its limits. There is also evidence that hyperphosphorylation of tau impairs its degradation by the proteasome [START_REF] Poppek | Phosphorylation inhibits turnover of the tau protein by the proteasome: influence of RCAN1 and oxidative stress[END_REF] and the addition of oxidative stress induced by complex I inhibition imposes an additional challenge to the UPS, as shown previously [START_REF] Höglinger | The mitochondrial complex I inhibitor rotenone triggers a cerebral tauopathy[END_REF]. The combination of these factors may overwhelm the clearance capacity of the UPS, leading to tau accumulation. Consistently, we observed no accumulation of the α-synuclein protein, which is predominantly degraded by autophagy rather than the UPS in neuronal cells [START_REF] Vogiatzi | Wild type alpha-synuclein is degraded by chaperone-mediated autophagy and macroautophagy in neuronal cells[END_REF].

There is evidence that the R406W mutation is less phosphorylated than the WT tau, especially at the Ser396 and Ser404 phosphorylation sites, which are in close proximity to the mutation [START_REF] Perez | The FTDP-17-linked mutation R406W abolishes the interaction of phosphorylated tau with microtubules[END_REF][START_REF] Miyasaka | Molecular analysis of mutant and wildtype tau deposited in the brain affected by the FTDP-17 R406W mutation[END_REF][START_REF] Zhang | Retarded axonal transport of R406W mutant tau in transgenic mice with a neurodegenerative tauopathy[END_REF]. Consistently, we have detected very low levels of AD2 immunoreactivity in R406W +/+ mice, and no significant alteration upon annonacin treatment. On the other hand, we have observed that hyperphosphorylation of tau at the AT8 and AT180 sites was increased after annonacin exposure in R406W +/+ mice, and that this increase was not explained by a mere augment in tau protein levels, since the increase in phospho-tau significantly exceeded the increase in total tau. Thus, increased phosphorylation by activation of kinases is likely occurring upon annonacin treatment. We therefore asked which kinases were possibly implicated. We studied candidate kinases known to phosphorylate tau at the AT8 and AT180 site [START_REF] Hanger | Tau phosphorylation: the therapeutic challenge for neurodegenerative disease[END_REF]. The protein levels of SAPK and Erk1/2 were increased by annonacin treatment in both R406W +/+ and R406W -/-mice, and thus unlikely account for the observed tau phosphorylation, which occurred only in R406W +/+ , but not in R406W -/-mice. GSK-3 protein level was increased in R406W +/+ , but not in R406W -/-mice, at the high annonacin dose. This result correlates with the AT180/Tau5 ratio, which was also increased at the high annonacin dose, suggesting that GSK-3 might be implicated in tau phosphorylation at the AT180-reactive T231 epitope. The effect of GSK-3 at the low annonacin dose remained inconclusive, because the GSK-3 protein level remained unaltered, and phosphorylation of GSK-3was increased at both activating and inactivating sites. The Cdk5 protein level was increased in both R406W +/+ and R406W -/-mice at both annonacin concentrations. The ratio p25/p35 (Cdk5 activator/inhibitor), however, was only increased in R406W +/+ mice, 10-fold and 20-fold at the low and high annonacin dose, respectively. This result correlates with AT8/Tau5 ratio suggesting that Cdk5 might be implicated in tau phosphorylation at the AT8-reactive S202/T205 epitope.

Therefore, GSK-3 and Cdk5 are likely candidates for the observed annonacin effects in R406W +/+ mice. These two kinases are proline-directed serine/threonine kinases and are considered to be the major enzymes involved in tau phosphorylation at disease-related sites, especially in abnormal tau hyperphosphorylation in AD [START_REF] Plattner | The roles of cyclin-dependent kinase 5 and glycogen synthase kinase 3 in tau hyperphosphorylation[END_REF][START_REF] Sengupta | Regulation of phosphorylation of tau by cyclin-dependent kinase 5 and glycogen synthase kinase-3 at substrate level[END_REF]. Many studies have revealed intriguing functional associations between GSK-3 and Cdk5. The AT180 site T231, which was highly phosphorylated in our annonacin experiments, is phosphorylated by GSK-3but the phosphorylation was shown to be more intense when tau is pre-phosphorylated by Cdk5 at S235 [START_REF] Sengupta | Potentiation of GSK-3catalyzed Alzheimer-like phosphorylation of human tau by Cdk5[END_REF][START_REF] Cohen | GSK3 inhibitors: development and therapeutic potential[END_REF]. Prephosphorylation of tau by Cdk5 stimulates both the rate and the extent of a subsequent phosphorylation catalyzed by GSK-3 [START_REF] Sengupta | Potentiation of GSK-3catalyzed Alzheimer-like phosphorylation of human tau by Cdk5[END_REF]. In our R406W +/+ mice, the Cdk5 activating ratio p25/p35 was significantly increased at low annonacin doses, whereas both Cdk5 and GSK-3 were activated after high annonacin doses, and consistently there was a significant increase in AT180 labeling.

On the other hand, Cdk5 has also been implicated in a regulatory role onto GSK-3activation [START_REF] Plattner | The roles of cyclin-dependent kinase 5 and glycogen synthase kinase 3 in tau hyperphosphorylation[END_REF][START_REF] Cheung | Cdk5: a multifaceted kinase in neurodegenerative diseases[END_REF]. It has been shown in some studies in vivo and in vitro that over-activation of Cdk5 leads to inhibition of GSK-3, or the reverse, that the decreased activity of Cdk5 parallels the enhancement of GSK-3 activity [START_REF] Hallows | Decreased cyclin-dependent kinase 5 (Cdk5) activity is accompanied by redistribution of Cdk5 and cytoskeletal proteins and increased cytoskeletal protein phosphorylation in p35 null mice[END_REF][START_REF] Vandebroek | Identification and isolation of a hyperphosphorylated, conformationally changed intermediate of human protein tau expressed in yeast[END_REF][START_REF] Plattner | The roles of cyclin-dependent kinase 5 and glycogen synthase kinase 3 in tau hyperphosphorylation[END_REF]. It has been argued that hyperphosphorylation of sites such as AT8 and PHF-1 is not due to a direct action of Cdk5, but is rather a consequence of loss of inhibitory control of GSK3- by Cdk5; such loss was observed in aged mice and associated with increased levels of p25 [START_REF] Plattner | The roles of cyclin-dependent kinase 5 and glycogen synthase kinase 3 in tau hyperphosphorylation[END_REF]. In our experiments the p25/p35 ratio paralleled the AT8/Tau5 ratio suggesting that increased Cdk5 levels might be implicated in this tau phosphorylation. Another possibility would be that the increased levels of p25 impaired the Cdk5 inhibition over GSK-3, and the latter would then directly hyperphosphorylate tau at AT8 site. Additional studies will be necessary to elucidate the mechanism underlying annonacin effects on tau hyperphosphorylation.

In non-exposed R406W +/+ mice, phospho-tau staining exhibited a homogeneous aspect with no intracellular fibrillar deposits. Annonacin exposure increased the number of neurons with somatodentritic accumulation of phospho-tau, but did not affect the aspect of the staining, which remained homogeneous within the cytoplasm. Although we have detected an increase in the sarcosyl-insoluble fraction of total and phospho-tau in brain homogenates of annonacintreated R406W +/+ mice, we have not observed any structures that morphologically resembled NFT in brain sections. Pathological activity of cytosolic non-fibrillized hyperphosphorylated tau has been reported in several animal models that express human tau suggesting that tau fibrillization or the presence of NFT is not necessary for neurodegeneration [START_REF] Wittmann | Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles[END_REF][START_REF] Santacruz | Tau suppression in a neurodegenerative mouse model improves memory function[END_REF]. Although we have not observed neuronal cell death, astroglial or microglial reactions, the brief period of annonacin treatment used in our study was enough to accelerate tau changes expected due to the overexpression of the transgene alone. In this regard, annonacin exposure changed the course of the disease and provoked an effect equivalent to an acceleration of the aging process. Consistently, we have also observed tau alterations in other tau-transgenic mice lines, using other complex 1 inhibitors, and also in different exposure protocols (e.g. more chronic and oral administration), but the presentation of these results would be beyond the scope of the present work.

In summary, we have shown that a brief 3-day exposure with annonacin, a natural lipophilic mitochondrial complex I inhibitor, is sufficient to increase tau protein levels, to alter tau kinases, to increase tau phosphorylation and to increase neuronal somatodendritic accumulation of phospho-tau in transgenic mice overexpressing human R406W-mutant tau, demonstrating that an environmental factor can change the course of the disease determined by a genetic factor. Figure 3. Tau protein level is increased in brains of R406W +/+ , but not R406W -/-mice, after annonacin exposure (0, 6 or 9 mg/kg/d). A,B, Total human (i.e. transgenic) tau levels, identified by Western blots using the antibodies T14 and HT7, are increased in R406W +/+ mice after 6 mg/kg (T14, * p= 0.015; HT7, * p=0.045) and 9 mg/kg (T14,* p=0,002; HT7, * p=0.031) of annonacin treatment in comparison to vehicle. C,D, With the Tau5 antibody, which labels both human and endogenous mouse tau, the effect was also observed in R406W +/+ (6 mg/kg, p=0.049; 9 mg/kg, p=0.012), but not in R406W -/-mice. D,E, The Tau5 antibody recognized in R406W +/+ mice (D) a long isoform (~69 kDa) corresponding to human transgenic R406W-tau, and a short isoform (~55 kDa) corresponding to endogenous mouse tau ; both isoforms were increased after 6 mg/kg long, * p=0.045; short, * p=0.048) and 9 mg/kg (long, * p=0.018; short, * p=0.012) annonacin treatment in comparison to 0 mg/kg in R406W +/+ mice (D,E); in R406W -/-mice, the Tau5 antibody only recognized the short isoform, which was unaltered upon annonacin-treatment (D). D,F, α-synuclein protein levels were not altered in R406W -/-and R406W +/+ mice upon annonacin treatment. A-D, β-actin protein levels are shown as loading controls. G,H, Quantitative PCR showed no significant increase in total (i.e. mouse and human) tau mRNA (G) or human tau mRNA (H) after 9 mg/kg annonacin treatment in neither R406W +/+ nor R406W -/-mice. As expected, R406W +/+ expressed more total tau (G: 0 mg/kg, * p=0.003; 9 mg/kg, * p=0.003) and human tau tau (H: 0 mg/kg, * p=0.001; 9 mg/kg, * p=0.001) than R406W -/-mice. I, However, both the chymotrypsin-like (LLVY) and peptidylglutamyl-peptide hydrolase (LLE) proteasomal proteolytic activities were significantly reduced after 9 mg/kg annonacin exposure in R406W +/+ (LLVY, * p=0.024; LLE, * p=0.016), but not in R406W -/-mice. * p<0.05, ANOVA followed by post hoc LSD test, annonacin vs. vehicle (A-E,I); * p<0.05, R406W +/+ vs. R406W -/-(G-H).

Figure 4.

Insoluble and phosphorylated tau are increased in brain tissue of R406W +/+ , but not R406W -/-mice, after annonacin exposure (0, 6 or 9 mg/kg/d). A, Western blot analysis shows increased human tau (HT7), total tau (Tau5), and phospho-tau (AT8 and AT180) in the sarcosyl-insoluble fraction of brain homogenates after 9 mg/kg annonacin exposure in R406W +/+ mice (A), but not in R406W-/-mice (not shown). B-D, Also in the soluble fraction of R406W +/+ , but not R406W -/-mice, annonacin caused a dose-dependent increase in AT8labeled phospho-tau (B,C, 6 mg/kg, * p=0.004; 9 mg/kg, * p=0.026), affecting both the long (human, 6 mg/kg, * p=0.007; 9 mg/kg, * p=0.010 ) and short (mouse, 6 mg/kg, * p=0.002 ; 9 mg/kg, * p=0.042) isoform (B,D); β-actin protein levels are shown as loading controls. E, The ratio of AT8-reactive phospho-tau to Tau5-reactive total tau was also significantly increased in R406W +/+ (6 mg/kg, * p=0.011; 9 mg/kg, * p=0.002), but not R406W -/-mice. B,F-H, The phospho-tau antibody AT180 yielded similar results. F: 6 mg/kg, * p=0.011; 9 mg/kg, * p=0.001; G: long, 6 mg/kg, * p=0.003; 9 mg/kg, * p=0.001; G: short, 6 mg/kg, * p=0.038; 9 mg/kg, * p=0.002; H: 9 mg/kg, * p=0.007. * p<0.05, ANOVA followed by post hoc LSD test, annonacin vs. vehicle. A,B, SAPK levels were increased after both annonacin doses in both R406W +/+ (6 mg/kg, * p=0.036; 9 mg/kg, * p=0.034) and R406W -/-mice (6 mg/kg, * p=0.012; 9 mg/kg, * p=0.004).

A,C, Erk1/2 levels were increased only in R406W +/+ mice after 9 mg/kg annonacin exposure (* p=0.021). A,D-F, GSK-3β total protein levels were increased only in R406W +/+ mice after 9 mg/kg annonacin exposure (* p=0.046). GSK-3β phosphorylated both at Y216 leading to activation (E) and at S9 leading to inactivation (F) was increased only in R406W +/+ mice after 6 mg/kg annonacin treatment (E: phosphoY216, * p=0.046; F: phosphoS9, p=0.003). A,G, Cdk5 protein levels were increased after both the low and high annonacin doses in both R406 +/+ (6 mg/kg, * p=0.046; 9 mg/kg, * p=0.005) and R406W -/-(6 mg/kg, * p=0.013; 9 mg/kg, * p=0.021) mice. A,H-J, In R406W +/+ , but not in R406W -/-mice, after annonacin exposure, there was a decrease in p35 levels (H: 6 mg/kg, * p=0.02; 9 mg/kg, * p=0.018) along with an increase in p25 levels (I, (6 mg/kg, * p=0.027; 9 mg/kg, * p=0.03), resulting in an increase in the p25/p35 ratio (J, (6 mg/kg, * p=0.049; 9 mg/kg, * p=0.045), reaching approximately 10-fold of normal levels at low and 20-fold at high toxin doses. In R406W -/-

Figure 1 .

 1 Figure 1. Immunohistochemistry with the AT8 antibody shows somatodendritic accumulation

Figure 2 .

 2 Figure 2. Immunohistochemistry with the AT180 antibody shows somatodendritic

Figure 5 .

 5 Figure 5. Protein levels of tau kinases are differentially altered upon annonacin exposure in brain homogenates of R406W +/+ and R406W -/-mice after annonacin exposure (0, 6 or 9

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (HO2402/6-1, HO2402/8-1), the German Ministry of Education and Research (BMBF GEF 10-54), the Centre National de la Recherche Scientifique (CNRS), a Marie Curie Incoming International Fellowship (21996 to E.S.Y.) and the German Academic Exchange Service (DAAD to A.C.F.d.A.). We are very grateful to Dr. Virginia M.-Y. Lee for kindly providing the pair breeders of R406W +/+ transgenic mice. The authors declare no competing financial interests.

Disclosure statement

All authors disclose any actual or potential conflicts of interest that could inappropriately influence (bias) the work submitted. The data submitted in the present paper have not been published or submitted elsewhere. All procedures concerning animal experimentation had former approval from the appropriate ethical committee. All authors have reviewed and approved the contents of the manuscript. mice, 9 mg/kg annonacin resulted in decrease in both p35 (H, * p=0.004) and p25 (I, * p=0.027). * p<0.05, ANOVA followed by post hoc LSD test, annonacin vs. vehicle.


Mohamed Belmeguenai

Sylvana Mercone

Carlo Adamo

Laurence Méchin

Cédric Fur

Philippe Monod

D G Schlom

P Moch

Temperature dependence of magnetic properties of La0.7Sr0.3MnO3/SrTiO3 thin films on silicon substrates

Keywords: number͑s͒: 75.40.Gb, 76.50.ϩg, 75.30.Gw, 75.40.Cx II. SAMPLES AND EXPERIMENTAL SETUPS

come

I. INTRODUCTION

Perovskite manganites ͓͑RE͒ 1-x ͑AE͒ x MnO 3 , where RE = rare earth and AE= alkaline earth͔ form an interesting class of compounds where the interplay between metal-insulator and ferromagnetic transitions results in a variety of fascinating properties such as colossal magnetoresistance. 1 The origin of ferromagnetism in these materials is the doubleexchange interaction 2 which leads to a strong correlation between magnetization and charge transport properties. La 0.7 Sr 0.3 MnO 3 ͑LSMO͒ is one of the perovskite manganites which show a colossal magnetoresistance and is expected to have a spin polarization close to 100% ͑half metal͒ 3 allowing for potential technological applications in spin-sensitive devices.

Integrating La 0.7 Sr 0.3 MnO 3 onto semiconducting materials is a challenging task for potential applications that utilize both information processing and data storage in the same device, such as high-density magnetic memories, sensors, and infrared bolometers. In order to grow an epitaxial quality manganite film on a semiconductor substrate, the filmsubstrate lattice mismatch needs to be minimized and chemical reactions between the substrate and the deposited film should be eliminated. Recent progress on direct integration 4,5 of SrTiO 3 ͑STO͒ on Si has opened the possibility of integrating both STO and La 0.7 Sr 0.3 MnO 3 onto this technologically important semiconductor. Pradhan et al. 6 have succeeded in the fabrication of high-performance La 0.67 Ba 0.33 MnO 3 samples which are ferromagnetic in the vicinity of room temperature. Despite the intense research activity on La 0.7 Sr 0.3 MnO 3 thin films grown on Si substrates, [7][8][9] mainly focused on their structural and static magnetic properties, their dynamic magnetic properties remain unexplored, according to our knowledge. The dynamics of these types of materials within the 1-10 GHz frequency range, which determines the high-speed response, is key for their future technological applications, especially in view of increasing data rates in magnetic storage devices. Therefore, the aim of this paper is to investigate the dynamic magnetic properties of La 0.7 Sr 0.3 MnO 3 thin films deposited on a Si substrate first overlaid with a thin STO buffer.

In this paper, we present static magnetometric measurements, conventional ferromagnetic resonance ͑FMR͒ and, in addition, microstrip line ferromagnetic resonance ͑MS-FMR͒, thus allowing for a full magnetic characterization of La 0.7 Sr 0.3 MnO 3 thin films at different temperatures. In the first part of this work, we describe the samples and the experimental setups ͑Sec. II͒. In Sec. III, we define the model to be used for the analysis of our measurements. The first part of Sec. IV summarizes the main static magnetic characteristics derived from superconducting quantum interference device ͑SQUID͒ data; its second part presents dynamic measurements obtained using FMR and MS-FMR and provides the derived magnetic parameters which satisfactory fit our data. In Sec. V, conclusions are presented. mum in -scan of the 002 STO and the 002 La 0.7 Sr 0.3 MnO 3 peaks are 0.26°and 0.22°, respectively. The pole figure of La 0.7 Sr 0.3 MnO 3 measured around its 110 peak ͑Fig. 1͒ shows full in-plane epitaxy of the La 0.7 Sr 0.3 MnO 3 film with pseudocubic axes ͓100͔ and ͓010͔ along the sample edges ͑which define the x axis and the y axis, respectively͒. Therefore, from the x-ray study, fourfold in-plane anisotropy is expected with one principal axis which can be chosen parallel to ͓110͔. Detailed study of structural properties of these samples will be reported in a forthcoming paper.

B. Experimental setups

The static measurements were carried out using a SQUID sweeping the ͑4-400 K͒ temperature range with a maximum applied field of 5 T. The saturation magnetization M s as a function of temperature was measured. Some conclusions about the anisotropy properties were also obtained from these data.

For the dynamic measurements, 9.5 GHz conventional FMR ͑Ref. 10͒ and MS-FMR ͑Ref. 11͒ were used. The conventional FMR setup in a bipolar X-band Bruker ESR spectrometer with a TE 102 resonant cavity immersed is an Oxford cryostat allowing for exploring the 4-300 K temperature interval. The MS-FMR setup consists of a homemade mounting which, up to now, only works at room temperature. The resonance fields ͑conventional FMR͒ and frequencies ͑MS-FMR͒ are obtained from a fit assuming a Lorentzian derivative shape of the recorded data. In contrast with the conventional FMR, the MS-FMR allows for dynamic measurements over a large frequency range and for the application of low external fields, in order to put in evidence small magnetic anisotropies which cannot be detected using the necessarily high applied magnetic fields used in conventional FMR.

III. MAGNETIC ENERGY-DENSITY MODEL AND EXPRESSIONS FOR THE UNIFORM MAGNETIC MODE

Figure 2 reports the coordinate system used as reference, where M ͑respectively, H ͒ is the in-plane angle between the magnetization M ͑respectively, the external applied field H͒ and the x axis while M ͑respectively, H ͒ is the out-of- plane angle between the magnetization ͑respectively, the external field H͒ and the direction normal to the sample plane ͑z axis͒. We assume fourfold in-plane anisotropy, as expected from the crystallographic orientation ͑see Sec. II͒ and we then define 4 as the angle between one of the anisotropy axes and the x axis.

The free-energy density E is then written as

E =-M s H͓sin M sin H cos͑ M -H ͒ + cos M cos H ͔ -͑2M s 2 -K Ќ ͒sin 2 M - 1 8 K 4 ͓3 + cos 4͑ M -4 ͔͒sin 4 M . ͑1͒
M s is the magnetization at saturation. K Ќ and K 4 are the uniaxial out-of-plane and the in-plane anisotropy constant, respectively. K Ќ and K 4 are effective anisotropy constants resulting from the simultaneous occurrence of a true volume term and of the additional contribution of a surface energy term. More explicitly, K Ќ = K ЌV + ͑K ЌS ͒ / d and K 4 = K 4V + ͑K 4S ͒ / d, where d is the film thickness and where the indices V and S are related to the volume and to the surface contribution, respectively. It is easily proved that, in the case of small thickness values, as considered in this study, this approach using effective volume energy density allows for a satisfactory evaluation of the frequencies of the magnetic modes. 12 The resonance frequency F r of the uniform precession mode is deduced from the energy density from the expression 13 F r

2 = ͩ ␥ 2 ͪ 2 1 M s 2 sin 2 M ͫ ץ 2 E ץ M 2 ץ 2 E ץ M 2 -ͩ ץ 2 E ץ M ץ M ͪ 2 ͬ ,

͑2͒

where ␥ is the gyromagnetic factor ͑␥ = g B , B being the Bohr magneton and g being the Landé factor͒ and where the derivatives are evaluated at the equilibrium position.

Sample plane

FIG. 2. ͑Color online͒ Sketch of the coordinate system used. M ͑respectively, H ͒ is the in-plane angle between the magnetization M ͑respectively, the external field H͒ and the x axis while M ͑re- spectively, H ͒ is the out-of-plane angle between the magnetization ͑respectively, the external field H͒ and the z axis and 4 is the angle of fourfold in-plane anisotropy axis H 4 with the x axis.

H 4 = 4K 4
M s is the fourfold in-plane anisotropy field.

In the case of an in-plane applied magnetic field, the magnetization lies in the plane of the film, M = H = 90°. The in-plane resonance frequency is then given by

F r 2 = ͩ ␥ 2 ͪ 2 ͫ H cos͑ M -H ͒ +4M eff + K 4 2M S ͑3 + cos 4͑ M -4 ͒͒ ͬ ϫ ͫ H cos͑ M -H ͒ + 2K 4 M S cos 4͑ M -4 ͒ ͬ , ͑3͒ where 4M eff =4M s - 2K Ќ M s defines the effective demagnetiz- ing field.
The M value is calculated from the energy minimization at equilibrium. It is a solution of

H sin͑ ⌴ -⌯ ͒ = 1 2 K 4 M s sin 4 ⌴ . ͑4͒
In the case of an out-of-plane applied magnetic field, for H significantly smaller than 90°, high applied fields are needed and, consequently, the fourfold anisotropy contribution can be neglected. It results that the resonance frequency for an out-of-plane applied magnetic field is given by

F out 2 = ͩ ␥ 2 ͪ 2 ͓H cos͑ M -H ͒ -4M eff cos 2 M ͔ ϫ͓H cos͑ M -H ͒ -4M eff cos 2 M ͔͑ 5͒
with

H sin͑ M -H ͒ =2M eff sin 4 M . ͑6͒

IV. RESULTS AND DISCUSSION

A. Static magnetic properties

The saturation magnetizations of the 20 nm and the 50 nm samples were measured using the SQUID up to 400 K. The values obtained for 4M s , under an in-plane applied field of 5000 Oe, at room temperature are 3000 and 2500 Oe, respectively, for the 20-nm-thick and for the 50-nm-thick films, with an error bar which can reach 10%, mostly due to the limited precision concerning the evaluation of the corrections arising from the poorly controllable contribution of the substrate. Typical temperature dependences of 4M s are shown in Fig. 3͑a͒ for the 20-nm-thick and 50-nm-thick films. The measured Curie temperature T c is the same, 330 K, for both films. A theoretical estimation of the low-temperature magnetization at saturation M s0 leads to 4M s0 = 7350 Oe ͑assuming a mean kinetic momentum equal to 11/6 and a Landé factor equal to 2͒. Within the frame of the experimental precision this value fits our measurement for the 50-nm-thick sample ͑7900 Oe͒ but is slightly larger in the case of the 20-nm-thick film ͑6100 Oe͒. Such a reduction in the magnetization in thin films is frequently observed. 9,14 Both samples ͑20 and 50 nm͒ show a saturation magnetization nearer from the theoretically expected one and significantly higher than the previously reported values in comparable situations ͑e.g., La 0.7 Sr 0.3 MnO 3 films grown on a LaAlO 3 substrate 14 ͒.A s shown in Fig. 3͑a͒, a classical fit of the temperature dependence of M s ͑T , H͒ using a Brillouin function B J ͑with J = 1.5 or 2͒ provides a reasonable agreement with the experimental data assuming T c = 330 K. In the case of the 50-nmthick film, the best fit is obtained using the above-mentioned 7350 Oe theoretical value of 4M s0 ; however, a reduced value of 4M s0 ͑5900 Oe͒ has to be used for the 20-nm-thick sample.

The room-temperature hysteresis loop using an in-plane applied magnetic field along the sample diagonal ͓͑110͔ di-rection͒ shows a rectangular shape with a coercive field of about 7 Oe, as illustrated in Fig. 3͑b͒ for the 50-nm-thick sample. This suggests a weak planar anisotropy but the static data do not allow a convincing quantitative determination of in-plane anisotropy parameters which monitor the magnetic energy density. Moreover, hysteresis loops obtained using a magnetic field parallel to the ͓100͔ and to the ͓010͔ axes do not appreciably differ from each other. These loops do not behave in the same way as the observed one applying a magnetic field along the ͓110͔ axis; their shape is smoother and the measured coercive field is smaller. This result qualitatively agrees with a description of the in-plane anisotropy in terms of a fourfold contribution with an easy axis along the ͓110͔ direction ͑and, indeed, a hard axis along the ͓100͔ one͒.

When the applied field is normal to the film, the magnetization loops roughly agree with the usual scheme of a coherent rotation in the hard-axis geometry, as shown in Fig. 3͑c͒. The effective demagnetizing field H eff =4M eff is found to lay around 5000 Oe for the 50-nm-thick film. However, it cannot be derived precisely.

B. Dynamic magnetic properties

In principle, both FMR and MS-FMR can give access to all the coefficients which define the magnetic energy density. However, the g value, which determines the gyromagnetic factor ␥, is precisely accessible only by the MS-FMR technique, through the study of the frequency variation versus the amplitude of an applied magnetic field perpendicular to the film. More generally, MS-FMR provides more accurate evaluations of the magnetic parameters since it allows for experimental studies not only versus the direction of the applied field, as in the case of conventional FMR, but also versus its amplitude. The magnetic parameters where precisely derived at room temperature from MS-FMR measurements. Their variation versus temperature was studied using conventional FMR, except in the case of the g factor which could not be precisely evaluated through this technique. Fortunately, g is expected to be practically independent of the temperature, 15 as assumed in the present work.

Room-temperature (300 K) results

For both films ͑20 and 50 nm͒ we find g = 1.915. This value is slightly smaller than the theoretically expected one ͑Ϸ2͒ which is generally observed in bulk La 0.7 Sr 0.3 MnO 3 . 15 Both films approximately show the same effective demagnetizing field.

For the 50 nm film the best fit obtained from the MS-FMR study under a perpendicular magnetic field corresponds to 4M eff = 4900 Oe. Taking account of the static magnetization reported above it results that K Ќ is negative ͑thus implying an easy-axis uniaxial anisotropy with an anisotropy field

H Ќ = ͉2 K Ќ M s ͉ of about 2000 Oe͒
From the MS-FMR measurements of the frequency dependence versus the direction of an in-plane applied field we deduce that 4 = 45°, as expected, and that this angle is re- lated to the easy in-plane anisotropy axis. For the 50-nmthick sample the corresponding measured anisotropy field

H 4 =4 K 4
M s is equal to 28 Oe. Figures 4 and5 illustrate that the obtained parameters allow for an excellent fit of all the experimental data for fields applied perpendicular ͑Fig. 4͒ as well as parallel to the films ͓Figs. 5͑a͒ and 5͑b͔͒, including conventional FMR results.

Temperature dependence

The variations in the resonance fields versus their orientation for both samples was studied by conventional FMR as a function of the temperature in view of deriving the temperature dependence of the effective demagnetizing field and of the uniaxial and in-plane anisotropy parameters. Figure 6͑a͒ shows a typical spectrum at 120 K for 50-nm-thick film while Fig. 6͑b͒ presents angular variations in the resonance fields in this sample at the same temperature. Two distinct modes noted 1 and 2 are observed. We begin by focusing on the so-called "principal" mode 1 while mode 2 will be discussed in the end of this section.

The derived values of the effective demagnetizing fields of the 20 nm and of the 50 nm films do not appreciably differ from each other. These values can be obtained from in-plane and, when the resonant field does not overpass the experimentally available maximum value ͑16000 Oe͒, from out-ofplane measurements. In the 20-nm-thick and the 50-nm-thick samples, they are decreasing functions of the temperature, varying from about 14000 Oe at 4 K down to 5000 Oe at 300K, as shown in Fig. 7. The evaluation of the perpendicu- (c) FIG. 3. ͑Color online͒͑a͒ Temperature dependences of 4M s in aL a 0.7 Sr 0.3 MnO 3 ͑50 nm͒ / SrTiO 3 ͑20 nm͒ / Si sample and a La 0.7 Sr 0.3 MnO 3 ͑20 nm͒ / SrTiO 3 ͑20 nm͒ / Si film measured under a 5000 Oe in-plane applied field; the full black lines represent the best obtained fit using a simple molecular-field model ͑see text͒. ͑b͒ Room-temperature hysteresis loops for three different orientations of an in-plane applied magnetic field in the La 0.7 Sr 0.3 MnO 3 ͑50 nm͒ / SrTiO 3 ͑20 nm͒ / Si sample. ͑c͒ Roomtemperature hysteresis loops for an applied magnetic field perpendicular to the La 0.7 Sr 0.3 MnO 3 ͑50 nm͒ / SrTiO 3 ͑20 nm͒ / Si sample plane. lar uniaxial anisotropy suffers from a rather a low accuracy, due to the lack of precision concerning the evaluation of the magnetization ͑see the preceding section͒. However, its approximate variation versus temperature is shown in Fig. 8͑a͒; the perpendicular uniaxial easy-axis anisotropy field H Ќ decreases from about 5000 Oe down to 2000 Oe when T increases from 4 to 300 K. As mentioned above the K Ќ coefficient is negative. Figure 8͑b͒ presents the resulting temperature variation in ͉K Ќ ͉; in both samples it approximately linearly decreases above 120 K from about 1.5 ϫ 10 6 erg/ cm 3 down to 2.5ϫ 10 5 erg/ cm 3 at 300 K. Large negative values of K Ќ have been observed previously in La 0.7 Sr 0.3 MnO 3 films grown on a SrTiO 3 substrate. 10 The main source of uniaxial perpendicular anisotropy is generally considered as proceeding from the interfacial strain through the magnetoelastic coupling. 10,14 Notice that its sign depends on the sign of the mismatch between the lattice constants of La 0.7 Sr 0.3 MnO 3 and the substrate; it was found positive in the case of LaAlO 3 ͑compressive strain͒ 14 but it is negative in the case of STO ͑extensive strain͒. 10 The temperature variation in the fourfold in-plane anisotropy field was also studied and it is shown in Fig. 9͑a͒;i n both films the easy axis remains along the ͓110͔ direction ͑i.e., 4 = 45°and H 4 Ͼ 0͒. Within the experimental accuracy the 20-nm-thick and the 50-nm-thick samples show identical values of the coefficient K 4 in the full studied temperature range, as shown in Fig. 9͑b͒; it decreases from about 1.4 ϫ 10 5 erg/ cm 3 at 4 K down to 10 4 erg/ cm 3 at 300 K. This result is in agreement with previously reported observations in La 0.7 Sr 0.3 MnO 3 thin films on STO ͑001͒ substrates 16,17 and with the symmetry put in evidence by our x-ray diffraction spectra and by Kerr microscopy; 18 it suggests that this inplane anisotropy, induced by the ͑001͒ STO seed layer, is of magnetocrystalline nature. The values of K 4 at 100 K reported in Ref. 14 for a large manifold of La 0.7 Sr 0.3 MnO 3 films deposited on different substrates allowing for a fourfold in-plane symmetry lie in the ͓3.9ϫ 10 4 ,13ϫ 10 4 ͔ erg/ cm 3 interval, to compare to our own determination, 8.5 ϫ 10 4 erg/ cm 3 . In addition, they report nearly the same decrease versus temperature as the observed one in the present study. Since the measured values of K Ќ and K 4 do not appreciably differ for the two studied samples with different thicknesses, we deduce that the main contribution to the anisotropy derives from the volume terms and that the surface energy plays a minor role. The variations in the normalized anisotropy constants k Ќ = K Ќ ͑T͒ / K Ќ ͑T 0 ͒͑ with T 0 =4 K͒ and k 4 = K 4 ͑T͒ / K 4 ͑T 0 ͒ versus the normalized saturation magnetization m = M s ͑T͒ / M s ͑T 0 ͒ are reported in Figs. 10͑a͒ and10͑b͒; the observed behavior is approximately given by k Ќ = m 2 and k 4 = m 6 , respectively. As usual, 19 the anisotropy coefficients show a relative decrease versus temperature significantly faster than the observed one for the magnetization. This decrease is more pronounced for the fourfold term K 4 than for the uniaxial one K Ќ .

For simplicity, in the above discussion we omitted to mention the presence of two distinct resonant fields in the thicker film ͑50 nm͒, as illustrated in Fig. 6. This multiplicity is observed in the 140-300 K temperature range when applying a magnetic field perpendicular to the sample, and in the 120-200 K temperature range in the case of an in-plane applied field. In order to interpret the data in this sample we had to identify the principal uniform mode within this multifields temperature interval; it is defined as the mode presenting a continuous variation in the resonance field around the boundaries of this interval. Its resonance field is close to the measured one in the 20-nm-thick film, which argues for the validation of this choice. In the perpendicular geometry the principal uniform mode corresponds to the higher measured field; at low temperature it disappears from the spectra due to the limited available value of the applied field. The failure to observe the secondary resonance outside the 120-200 K temperature range is not yet completely understood. For this extra resonance, the measured field value and its variation versus the angular orientation prevent for its identification in terms of a standing magnetic mode along the direction normal to the sample. We then believe that, in this sample, two different magnetic phases coexist, thus giving rise to two observable uniform modes: the standard principal one ͑mode 1 in Figs. 6 and 7͒ which is always present ͑for an in-plane applied field͒ and the extra "auxiliary" phase ͑mode 2͒ which needs specific thickness conditions in order to be stabilized. The uniform mode related to this auxiliary phase can be studied in order to derive the effective demagnetizing field and the in-plane anisotropy related to this phase as shown in Figs. 7 and9͑a͒. The effective demagnetizing fields which provide the best fit for the spectra obtained with an in-plane applied magnetic field agree with the derived ones in the case of a perpendicular applied field, as seen in Fig. 7. Also notice that the in-plane anisotropy remains fourfold with an easy axis along ͓110͔ but that for the mode 2 the in-plane anisotropy field H 4 is smaller than for the mode 1. It is not possible to evaluate the uniaxial perpendicular anisotropy field H Ќ for this phase since its saturation magnetization cannot be measured separately. Finally, the simultaneous presence of two phases could slightly modify the above determined values of K Ќ and of K 4 related to the principal mode since the SQUID measurements do not allow for separate evaluations of the two involved magnetizations. However, we believe that the apparent adequacy of our classical analysis of the magnetic static measurements argues for the hypothesis of a weak perturbation arising from the auxiliary phase in the calculation of the static magnetic properties of the principal one.

V. CONCLUSION

The temperature dependence of the static and of the dynamic magnetic properties of 20-nm-thick and 50-nm-thick La 0.7 Sr 0.3 MnO 3 films, deposited on Si substrates overlaid by a 20-nm-thick STO buffer layer have been studied, using SQUID and ferromagnetic resonance ͑conventional inside cavity: FMR and microstrip line: MS-FMR͒, respectively. Our x-ray diffraction measurements show an excellent epi-taxial ͑001͒ orientation of the La 0.7 Sr 0.3 MnO 3 films. Their magnetic behavior is interpreted assuming a magnetic energy density characterized by a strong uniaxial anisotropy along the direction normal to the plane of the films and by an in-plane anisotropy showing a fourfold symmetry. The inplane and out-of-plane anisotropies decrease drastically when the temperature increases. A good fit of all the experimental data, using appropriate values of the magnetic coefficients describing the free energy, is obtained. The dependence of the anisotropies on the nature of the buffer layer will be studied in future work.

ACKNOWLEDGMENT

The authors would like to thank T. Chauveau for some x-ray measurements during this study. C.A. and D.G.S. acknowledge the financial support of the National Science Foundation through Grant No. ECCS-0708759.

FIG. 1 .

 1 FIG. 1. ͑Color online͒ 110 pole figure for the La 0.7 Sr 0.3 MnO 3 ͑50 nm͒ / SrTiO 3 ͑20 nm͒ / Si film. Directions x and y correspond to ͓100͔ and ͓010͔, respectively.

FIG. 4 .

 4 FIG.4. MS-FMR resonance frequency dependence on the amplitude of a perpendicular applied magnetic field in the La 0.7 Sr 0.3 MnO 3 ͑50 nm͒ / SrTiO 3 ͑20 nm͒ / Si sample.

FIG. 5 .FIG. 6 .

 56 FIG.5. ͑a͒ MS-FMR in-plane angular dependence of the resonance frequency. ͑b͒ Resonance frequency of the uniform mode as a function of the in-plane applied field ͑parallel to the x axis͒ in the La 0.7 Sr 0.3 MnO 3 ͑50 nm͒ / SrTiO 3 ͑20 nm͒ / Si sample.

  FIG. 7. ͑Color online͒ Temperature dependence of the effective demagnetizing field in the La 0.7 Sr 0.3 MnO 3 ͑50 nm͒ / SrTiO 3 ͑20 nm͒ / Si sample and in the La 0.7 Sr 0.3 MnO 3 ͑20 nm͒ / SrTiO 3 ͑20 nm͒ / Si sample. The results are obtained from the fit of the experimental data using perpendicular and in-plane applied fields.

FIG. 9 .FIG. 10 .

 910 FIG. 9. ͑Color online͒ Temperature dependence ͑a͒ of the inplane fourfold anisotropy field and ͑b͒ the related anisotropy coefficient K 4 in the La 0.7 Sr 0.3 MnO 3 ͑50 nm͒ / SrTiO 3 ͑20 nm͒ / Si sample and in the La 0.7 Sr 0.3 MnO 3 ͑20 nm͒ / SrTiO 3 ͑20 nm͒ / Si sample.