Océane Guillemaud

Kelly Ceyzériat

Thomas Saint-Georges

Karine Cambon

Fanny Petit

Lucile Ben Haim

Maria-Angeles Carrillo-De Sauvage

Martine Guillermier

Sueva Bernier

Anne-Sophie Hérard

Charlène Joséphine

Alexis Pierre Bémelmans

Emmanuel Brouillet

Philippe Hantraye

Gilles Bonvento

Carole Escartin
email: carole.escartin@cea.fr

Complex roles for reactive astrocytes in the triple transgenic mouse model of Alzheimer disease

Keywords: Reactive astrocytes, Alzheimer disease, Tau, JAK-STAT3 pathway, SOCS3, Neuroinflammation conceptualization, data curation, project supervision, funding acquisition

In Alzheimer disease (AD), astrocytes undergo complex changes and become reactive. The consequences of this reaction are still unclear. To evaluate the net impact of reactive astrocytes in AD, we recently developed viral vectors targeting astrocytes that either activate or inhibit the JAK2-STAT3 pathway, a central cascade controlling astrocyte reaction.

We aimed to evaluate whether reactive astrocytes contribute to Tau as well as amyloid pathologies in the hippocampus of 3xTg-AD mice, an AD model that develops Tau hyper-phosphorylation and aggregation in addition to amyloid deposition. JAK2-STAT3 pathway-mediated modulation of reactive astrocytes in 25% of the hippocampus of 3xTg-AD mice, did not significantly influence Tau phosphorylation or amyloid processing and deposition at early, advanced and terminal stage of the disease. Interestingly, inhibition of the JAK2-STAT3 pathway in hippocampal astrocytes did not improve short-term spatial memory in the Y maze but it did reduce anxiety in the elevated plus maze.

Our unique approach to specifically manipulate reactive astrocytes in situ show these cells may impact behavioral outcomes without influencing Tau or amyloid pathology.

INTRODUCTION

Alzheimer disease (AD) is a devastating neurodegenerative disease and the most common form of dementia [START_REF] Querfurth | Alzheimer's disease[END_REF]. It is characterized by extracellular accumulation of amyloid plaques, Tau hyper-phosphorylation, synaptic alterations and neuronal degeneration. The exact mechanisms responsible for AD are still disputed [START_REF] Sala Frigerio | Alzheimer's Disease Mechanisms and Emerging Roads to Novel Therapeutics[END_REF]. The amyloid cascade hypothesis was first to be put forward. Here, successive cleavages of the amyloid precursor protein (APP) by -and -secretases, generate high levels of soluble amyloid beta (A ) peptides.

These peptides then oligomerize and form amyloid plaques in the brain of AD patients and can cause synaptic dysfunction, neurodegeneration and cognitive impairment [START_REF] Hardy | Alzheimer's disease: the amyloid cascade hypothesis[END_REF]. The amyloid hypothesis was later refined to include Tau hyper-phosphorylation as a key pathogenic element [START_REF] Kametani | Reconsideration of Amyloid Hypothesis and Tau Hypothesis in Alzheimer's Disease[END_REF][START_REF] Medina | New perspectives on the role of tau in Alzheimer's disease. Implications for therapy[END_REF]. Hyper-phosphorylated Tau proteins can also oligomerize and form neurofibrillary tangles that inhibit microtubule assembly and alter neuronal functions. Indeed, progression of Tau pathology from the entorhinal cortex to other isocortex regions [START_REF] Braak | Neuropathological stageing of Alzheimer-related changes[END_REF] correlates better than amyloid pathology with cognitive impairment observed in patients [START_REF] Arriagada | Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer's disease[END_REF][START_REF] Masters | Alzheimer's disease[END_REF].

Recently, evidence has emerged showing that AD is not only caused by cell-autonomous processes within neurons, and that glial cells may also play an instrumental role in disease progression [START_REF] Arranz | The role of astroglia in Alzheimer's disease: pathophysiology and clinical implications[END_REF][START_REF] De Strooper | The Cellular Phase of Alzheimer's Disease[END_REF][START_REF] Heneka | Neuroinflammation in Alzheimer's disease[END_REF]. In particular, astrocytes perform a number of key brain functions, including neurotransmitter recycling, ion homeostasis, metabolic and trophic support [START_REF] Verkhratsky | Physiology of Astroglia[END_REF]. In the brain of AD patients or in animal models, astrocytes are reactive and participate in neuroinflammation. Reactive astrocytes are classically characterized by morphological changes (hypertrophy and process reorganization), as well as upregulation of intermediate filament proteins [Glial fibrillary acidic protein (GFAP), vimentin, [START_REF] Hol | Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system[END_REF]]. Much less is known about their functional features and exactly how they impact AD outcomes (Ben Haim et al., 2015a). Several studies have reported alterations in some of the multiple functions operated by astrocytes such as glutamate uptake [START_REF] Masliah | Deficient glutamate transport is associated with neurodegeneration in Alzheimer's disease[END_REF], metabolic supply [START_REF] Allaman | Amyloid-beta aggregates cause alterations of astrocytic metabolic phenotype: impact on neuronal viability[END_REF][START_REF] Sancheti | Hypermetabolic state in the 7-month-old triple transgenic mouse model of Alzheimer's disease and the effect of lipoic acid: a 13C-NMR study[END_REF], production of antioxidant molecules [START_REF] Abeti | Beta-amyloid activates PARP causing astrocytic metabolic failure and neuronal death[END_REF][START_REF] Allaman | Amyloid-beta aggregates cause alterations of astrocytic metabolic phenotype: impact on neuronal viability[END_REF] or regulation of synaptic transmission [START_REF] Delekate | Metabotropic P2Y1 receptor signalling mediates astrocytic hyperactivity in vivo in an Alzheimer's disease mouse model[END_REF][START_REF] Jo | GABA from reactive astrocytes impairs memory in mouse models of Alzheimer's disease[END_REF][START_REF] Lian | NFkappaB-Activated Astroglial Release of Complement C3 Compromises Neuronal Morphology and Function Associated with Alzheimer's Disease[END_REF][START_REF] Wu | Tonic inhibition in dentate gyrus impairs long-term potentiation and memory in an Alzhiemer's disease model[END_REF]; for recent reviews see (Ben Haim et al., 2015a;[START_REF] Chun | Reactive astrocytes in Alzheimer's disease: A double-edged sword[END_REF]. Interestingly, astrocytes may directly participate in APP metabolism and A clearance through various mechanisms, ranging from extracellular cleavage to phagocytosis or drainage through the glymphatic system [START_REF] Ries | Mechanisms of Abeta Clearance and Degradation by Glial Cells[END_REF]. But how these clearance mechanisms are altered when astrocytes acquire reactive features during AD progression is still unknown.

Similarly, the exact impact of reactive astrocytes on Tau hyper-phosphorylation and aggregation remains to be established [START_REF] Kahlson | Glial Tau Pathology in Tauopathies: Functional Consequences[END_REF][START_REF] Leyns | Glial contributions to neurodegeneration in tauopathies[END_REF].

We recently developed a method that efficiently blocks morphological and molecular changes in reactive astrocytes, by inhibiting the Janus kinase -Signal transducer and activator of transcription 3 (JAK-STAT3) pathway, through astrocyte-specific expression of its inhibitor suppressor of cytokine signaling 3 [SOCS3, [START_REF] Ceyzeriat | Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer's disease[END_REF]]. We showed that reactive astrocytes promote amyloid deposition and learning deficits in the APP/PS1dE9 transgenic mouse model of AD, and early synaptic deficits in 3xTg-AD mice, another transgenic model that also develops Tau pathology.

Here, we aimed to evaluate in 3xTg-AD mice, whether reactive astrocytes 1) influence Tau and amyloid pathologies at different disease stages and 2) have long-term effects on cognitive functions.

Unexpectedly, we found that JAK2-STAT3 activation in reactive astrocytes does not affect Tau or amyloid pathology in 3xTg-AD mice at early, advanced and terminal disease stages. In addition, inhibition of reactive astrocytes does not improve working memory but it does reduce anxiety in 3xTg-AD mice.

MATERIAL AND METHODS

Animals.

Triple transgenic AD (here referred to as 3xTg mice) mice express human APPswe and human Tau P301L under a Thy-1 promoter as well as a point mutation on the mouse Psen1 gene (PS1 M146V ), on a mixed C57BL/6J x 129Sv background [START_REF] Oddo | Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction[END_REF]. C57BL/6J x 129Sv mice were used as controls. Only females were used in this study, as they display earlier neuropathology [START_REF] Carroll | Sex differences in beta-amyloid accumulation in 3xTg-AD mice: role of neonatal sex steroid hormone exposure[END_REF][START_REF] Hirata-Fukae | Females exhibit more extensive amyloid, but not tau, pathology in an Alzheimer transgenic model[END_REF]. Breeding pairs were obtained from the Mutant Mouse Regional Resource Centers. All experimental protocols were approved by a local ethics committee (CETEA N°44) and submitted to the French Ministry of Education and Research (Approvals # APAFIS#4565-20 16031711426915 v3 and APAFIS#4503-2016031409023019). They were performed in an authorized facility (#D92-032-02), under the supervision of a veterinarian and in strict accordance with the recommendations of the European Union (2010-63/EEC) for the care and use of laboratory animals. All efforts were made to promote animal welfare. Mice were housed under standard environmental conditions (12-hour light-dark cycle, temperature: 22 ± 1°C and humidity: 50%) with ad libitum access to food and water. Mice of the appropriate genotype were randomly allocated to experimental groups.

Stereotactic injections of viral vectors.

Adeno-associated viruses of serotype 9 (AAV2/9) encoding Gfp, murine Socs3, or a constitutively active form of Jak2 (JAK2 T875N , aka JAK2ca) were produced and titrated at the MIRCen viral vector facility, as described previously [START_REF] Ceyzeriat | Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer's disease[END_REF]. All transgenes were under the control of the gfaABC 1 D promoter, which drives specific expression in astrocytes, including those of the dorsal hippocampus, where the viral vectors were delivered [ [START_REF] Ceyzeriat | Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer's disease[END_REF] Before surgery, mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) and received s.c. injection of lidocaine (7 mg/kg) at the incision site, 10 min before injection.

Mice received paracetamol in drinking water (1.6 mg/ml) for 48 h after surgery. AAV were diluted in 0.1 M phosphate buffer saline (PBS) with 0.001% pluronic acid, at a final total concentration of 2.5 10 9 viral genome/µl. Mice were positioned on a stereotactic apparatus with tooth bar set at 0 mm. They were injected in the CA1 region of the hippocampus (coordinates: -3 mm antero-posterior, +/-3 mm lateral and -1.5 mm ventral, from dura) with 2 µl of AAV suspensions at a rate of 0.2 µl/min.

After 5 min, the cannula was slowly removed and the skin sutured. Mice recovered for at least 2 months before analysis.

Experimental groups.

Four mouse cohorts were analyzed (Supplemental Fig. 1). The number of mice analyzed is mentioned in each figure .   1. Homozygous 3xTg mice were injected at 6 months with AAV-GFP (3xTg-GFP mice) or AAV-SOCS3 + AAV-GFP at the same total viral titer (3xTg-SOCS3 mice), to block reactive astrocytes.

AAV-GFP was co-injected with AAV-SOCS3 to visualize infected cells. WT mice of the same age, gender and genetic background were injected with AAV-GFP (WT-GFP) as control. Mice were tested on the elevated plus maze and the Y maze at 8 and 14-15 months. Mice were euthanized by an overdose of pentobarbital at the age of 16 months. The two brain hemispheres were rapidly dissected on ice. One was drop-fixed in 4% paraformaldehyde (PFA) and used for immunohistochemistry. The other was cut into 1-mm-thick slices on ice and the hippocampal GFP + region was dissected out under a fluorescent macroscope, snap frozen in liquid nitrogen and stored at -80°C until protein extraction.

2. At 3 months, WT mice were injected with AAV-GFP, and 3xTg mice were injected with AAV-GFP or with AAV-JAK2ca + AAV-GFP (same total viral titer, 3xTg-JAK2ca mice), to exacerbate astrocyte reaction. Mice were euthanized as 16 months and analyzed by histological and biochemical approaches as described for the first cohort.

3. At 4 months, WT mice were injected with AAV-GFP, and 3xTg mice were injected with AAV-GFP or with AAV-SOCS3 + AAV-GFP. Mice were euthanized by an overdose of pentobarbital at the age of 9 months. The hippocampus was dissected out, snap frozen in liquid nitrogen and stored at -80°C until protein extraction. 4. At 24 months, WT mice were injected with AAV-GFP, and 3xTg mice were injected with AAV-GFP or with AAV-SOCS3 + AAV-GFP. Mice were analyzed by biochemical approaches at 26 months as described for cohort 3.

The early and mid-stage cohorts (cohorts 1, 2, 3) were all injected at an early stage (3 to 6 months), when there is no detectable amyloid or Tau deposition [START_REF] Carroll | Sex differences in beta-amyloid accumulation in 3xTg-AD mice: role of neonatal sex steroid hormone exposure[END_REF][START_REF] Oddo | Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction[END_REF]. Endstage mice were injected at 24 months to assess whether a late manipulation of the JAK-STAT3 pathway in astrocytes could reverse or reduce advanced amyloid and Tau pathology.

Immunohistology.

Dedicated hemispheres were post-fixed for 24 h in 4% PFA, cryoprotected in a 30% sucrose solution and cut on a freezing microtome into serial 30-µm-thick coronal sections. Series were stored at -20°C in an anti-freeze solution until used for immunostaining.

Immunofluorescence. Sections were rinsed in PBS for 3 x 10 min and blocked in 4.5% normal goat serum (NGS) diluted in PBS with 0.2% Triton X-100 (PBST) for 1h at room temperature (RT).

Sections were incubated overnight at 4°C with anti-GFAP-Cy3 antibody (1:1,000, Sigma, #C9205, Saint Louis, MO) diluted in 3% NGS/PBST. Sections were rinsed 3 x 10 min in PBS and incubated with secondary Alexa Fluor-conjugated antibodies (1:1,000, Invitrogen, Carlsbad, CA) in 3% NGS/PBST for 1h at RT. After 3 washes in PBS, sections were incubated overnight at 4°C with an anti-GFP biotinylated antibody (1:500, Vector Laboratories, #BA-0702, Burlingame, CA) in 3% NGS/PBST. After 3 rinses in PBS, sections were incubated for 1h at RT with Streptavidine-FITC (1:1,000, ThermoFisher Scientific, Waltham, MA) in 3% NGS/PBST and rinsed 3 times with PBS before being mounted on SuperFrost® Plus slides (ThermoFisher Scientific) and coverslipped with Fluorsave™ medium (Calbiochem, Darmstadt, Germany). For Iba1 (1:500, Wako, #191974, Neuss, Germany) and Vimentin (1:750, Abcam, #Ab24525, Cambridge) staining, a standard protocol was performed as described above but endogenous GFP was not amplified and sections were incubated with DAPI (1:2,000, Invitrogen, #D21490) before being coverslipped with Vectashield™ medium (Vector Laboratories, #H1400). For methoxy-XO4 (MXO4, Tocris, Minneapolis, MN) staining, sections were incubated with 33 µg/ml MXO4 in 0.1 M PBS for 30 min at RT under mild agitation.

After 3 rinses with PBS, a standard protocol for immunofluorescence was performed as described above. For A staining with the 4G8 antibody (1:500, Covance, #SIG39240, Princeton, NJ), sections were treated with 70% formic acid for 2 min before incubation with primary biotinylated 4G8 antibody for 48h at 4°C. After 3 rinses with PBS, sections were incubated for 1h at RT with Streptavidine-Cy3 (1:1,000, Sigma) in 3.5% NGS/PBST. Sections were then mounted as previously described.

Immunohistochemistry. Sections were rinsed in PBS for 3 x 10 min and incubated with H 2 O 2 (1:100, Sigma) for 20 min at RT to inhibit peroxidases. For HT7 antibody, sections were pre-treated with 10 mM citrate buffer for 20 min at 90°C before H 2 O 2 treatment. Sections were then blocked in 4.5% NGS/PBST for 1h at RT and incubated overnight at RT or 48h at 4°C in 3% NGS/PBST with the following primary antibodies: anti-A 42 (1:500, Rabbit, Life Technologies, #44-344, Carlsbad, CA), AT8 (1:400, Mouse, ThermoFisher Scientific, #MN1020B), HT7 (1:1,000, Mouse, Innogenetics, #90204, Zwijnaarde, Belgium), anti-phospho-Ser422-Tau (1:400, Rabbit, Abcam, #Ab79415, Cambridge, UK). After 3 x 10 min rinses in PBS, sections were incubated for 1h at RT with biotinylated secondary antibodies (1:4,000, except at 1:1,000 for anti-phospho-Ser422-Tau, Vector Laboratories) in 3% NGS/PBST. They were washed 3 x 10 min and incubated for 1h at RT with avidin/biotin complexes (1:250 in PBST, Vector Laboratories) and after 3 washes with PBS, incubated with VIP (Vector Laboratories). Sections were mounted on SuperFrost Plus slides and dried overnight at RT. Sections were dehydrated in acetone and xylene and coverslipped with Eukitt mounting medium (Chem-Lab, Zedelmgem, Belgium).

Immunostaining quantification.

GFAP staining was quantified on 10x-tiled images of serial brain sections (240 µm between each sections), acquired with an epifluorescence microscope (BM6000B, Leica, Nussloch, Germany).

Virally transduced GFP + regions were manually segmented on each section and the GFAP mean grey value was measured in this GFP + region with Image J. Vimentin was quantified on 10x-tiled images taken from another series of brain sections, acquired with the epifluorescence microscope. An intensity threshold was applied and the area of immunopositive pixels within the manually-segmented GFP + region were automatically extracted on each section. The Vimentin + volume was calculated by the Cavalieri method [START_REF] West | Unbiased stereological estimation of the number of neurons in the human hippocampus[END_REF]) (Volume = t x area; where t is the distance between two 2 sampled sections, here 240 µm). It was then normalized to the total GFP + volume, calculated by the same method. Representative fluorescent images in Fig. 1 and supplemental Fig. 2 were acquired with a 40x objective on a confocal microscope (TCS SP8, Leica).

The number of Iba1 positive cells around each plaque was quantified by scanning the whole slice on the z axis, under the 40x objective of the confocal microscope. An image was acquired at the center of the plaque to measure its cross-sectional area. In each mouse brain, 6-26 plaques within the GFP + hippocampal region were sampled. They were taken from at least 3 fields of view from different sections.

Serial sections stained for HT7, Phospho-Ser422-Tau or AT8 were scanned under bright light, with an Axio scanZ.1 (Zeiss, Oberkochen, Germany) to cover the entire anteroposterior axis of the hippocampus. The immunopositive area within the manually-segmented hippocampus was automatically detected on each section, with an intensity threshold applied with Image J. The immunopositive volume was calculated by the Cavalieri method, normalized to the total volume of the hippocampus, calculated by the same method and expressed as a percentage. Hippocampal volume was not different between groups (data not shown).

The number and surface of all MXO4-labelled plaques were quantified on 10x-tiled fluorescent images of a series of slices covering the anteroposterior axis of the hippocampus, acquired on the Leica epifluorescence microscope. A 42 plaques were quantified on immunostained sections scanned under bright field on the Axio scan. Automatic detection of objects according to intensity and size thresholds was performed on these images, after manual segmentation of the hippocampus on each section, with Image J. To quantify 4G8 immunoreactivity, the subiculum and stratum pyramidale of the CA1 region were manually segmented on 10x-tiled fluorescent images of a series of slices covering the anteroposterior axis of the hippocampus and the mean grey value was measured with Image J. Investigators were partially blinded to the group when analyzing immunostained sections, as the group can be deduced based on the presence of amyloid or Tau staining or GFP levels for example.

Protein extraction.

Hippocampal samples were homogenized by sonication (Labortechnik, Wasserburg, Germany, 6 strokes with cycle = 0.5, 30% amplitude) in lysis buffer [50 mM Tris-HCl pH = 7.4, 150 mM NaCl, 1% Triton X-100 with 1:100 phosphatase inhibitors (Sigma, cocktail 2) and 1x protease inhibitors (Roche, Basel, Switzerland)] and centrifuged at 20,000 g for 20 min at 4°C. The supernatant contains Triton X-100-soluble proteins and was used for western blotting and MSD® ELISA tests. Protein concentration was measured by the BCA test (Pierce, Waltham, MA).

Western blot.

Samples were diluted in loading buffer with DTT (NuPAGE® LDS sample buffer and sample reducing agent, Invitrogen). Ten µg of proteins were loaded on a NuPAGE™ 4-12% Bis-Tris Midi Gel (Life Technologies) and exposed to 200 V for 45 min in NuPAGE™ Running Buffer (Invitrogen).

Proteins were transferred on a nitrocellulose membrane with an iBlot Gel transfer device (Invitrogen).

After 3 x 10 min rinses in Tris buffer saline and 0.1% Tween 20 (TBST), membranes were blocked in 5% milk in TBST for 1h at RT and incubated for 3h at RT, or overnight at 4°C with the following primary antibodies: 6E10 (1:500, Mouse, Covance, #SIG-39320-20), anti-ApoE (1:1,000, Rabbit, Abcam, #Ab20874), anti-BACE1 (1:1,000, Rabbit, Cell signaling, #5606P, Danvers, MA), anti-GFAP (1:5,000, Rabbit, Dako, #Z0334, Troy, MI), anti-IDE (1:400, Rabbit, Abcam, #Ab32216), anti phospho-Ser404-Tau (1:1000, Rabbit, Sigma, #T7444) and anti-Tubulin (1:1,000, Mouse, Sigma, #T5168). After 3 x 10 min washes in TBST, membranes were incubated for 1h at RT with HRPconjugated secondary antibodies (1:5,000, Vector laboratories) diluted in TBST with 5% milk.

Membranes were incubated with the Clarity Western ECL substrate (Bio-Rad, Hercules, CA) and the signal was detected with a Fusion FX7 camera (ThermoFisher Scientific). Band intensity was quantified with ImageJ and normalized to Tubulin . Each antibody was used on at least 2 different membranes. Representative images and quantification are shown.

MSD® ELISA tests.

Triton x-100 soluble proteins were diluted in the diluent provided for the V-PLEX A peptide panel kit (A 38, A 40, A 42, 6E10 antibody, MSD®, Rockville, MD). For the phospho-Thr231-Tau/Total Tau kit (MSD®), samples were diluted in 20 mM Tris HCl pH = 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, with phosphatase and protease inhibitors. For both assays, samples were loaded in triplicate and the manufacturer's protocol was followed. Peptide levels were quantified with a standard curve, using the Discovery Workbench4.0, MSD® software, and normalized to the protein content in each well.

Behavioral analysis.

WT-GFP, 3xTg-GFP and 3xTg-SOCS3 mice were tested at the age of 8 and 14-15 months on the elevated plus maze to evaluate anxiety and after one month on the Y maze to assess working spatial memory. All tests were performed between 8 am and noon. Mice were handled daily for 2 min for 5 d before the first training session. Both mazes were cleaned with 10% ethanol and carefully dried between each phase and each mouse to remove odors. Investigators were blinded to the group when performing experiments.

The elevated plus maze test was formed by four connected arms placed 50 cm above the ground, illuminated at 100 lux in the center (see Fig. 6b). Two arms made of black Plexiglas were closed (nonstressful arms) while the two others were open (stressful arms). Each mouse was placed in the center of the maze and video-tracked for 5 min with the EthoVision XT11.5 software (Noldus IT, The Netherlands). The following parameters were measured: distance traveled and time spent in each arm, frequency of entry into each arm, duration and number of head dipping (counted manually during the experiment). Mice performing fewer than 4 entries into different arms were excluded from the analysis.

The Y maze test was composed of three arms in black Plexiglas, illuminated at 110 lux in the center. Each mouse was placed at the extremity of the "start arm" and left for a training session of 15 min, with one of the two other arms closed with a black Plexiglas door (the closed arm was randomly chosen for each mouse). Then, the mouse was brought back to its cage for 5 min, the maze was cleaned, the closed arm was opened and the mouse placed at the extremity of the start arm for a 5 min test session, under videotracking (see Fig. 6a). The distance traveled and time spent in each arm (start, familiar, and novel arms) were measured and analyzed with EthoVision. Given their natural curiosity, mice are expected to spend more time exploring the new arm. Mice that stayed in the start arm during training or made less than 4 total entries in the 3 arms during testing were excluded.

Statistical analysis.

Results are expressed as mean ± SEM. Statistical analysis were performed with Statistica software (StatSoft, Tulsa, OK). Student t test and one-way ANOVA and Tukey's post hoc test were used to compare two and three groups, respectively. Two-way (group, arm) ANOVA followed by Tuckey's post-hoc test was used for Y maze analysis. For each analysis, normality of variables or residues and homoscedasticity were assessed. If any or both conditions of application were not fulfilled, nonparametric tests were used. Two groups were compared by the Mann-Whitney test and 3 independent groups were compared by a Kruskal-Wallis test followed by a Mann-Whitney test. The significance level was set at p < 0.05. The number of analyzed mice per group is indicated in each bar histogram.

RESULTS

The JAK2-STAT3 pathway efficiently controls reactive features of hippocampal astrocytes in 3xTg mice.

We previously showed that adeno-associated viral (AAV) gene transfer of SOCS3, an inhibitor of the JAK-STAT3 pathway, in hippocampal astrocytes, prevents their acquisition of reactive molecular features, including in 9 and 12-month-old 3xTg mice (Ben Haim et al., 2015b;[START_REF] Ceyzeriat | Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer's disease[END_REF].

We studied whether SOCS3 was still able to block reactive astrocytes at a later stage, when both Tau and amyloid pathologies are present in 16-month-old 3xTg mice.

Six-month-old female 3xTg mice were injected with an AAV2/9 vector encoding Socs3 under the gfaABC 1 D promoter (3xTg-SOCS3 mice). This viral vector allows the selective transduction of astrocytes, in the dorsal hippocampus (Fig. 1a, Supplemental Fig. 2), covering 25% of the whole hippocampus. WT and 3xTg mice were injected with an AAV encoding GFP as control (WT-GFP and 3xTg-GFP mice respectively, see Supplemental Fig. 1). Brain sections from 16-month-old 3xTg-GFP mice displayed 3-fold higher GFAP levels than WT-GFP mice (Fig. 1b,c). In 3xTg-GFP mice, hippocampal astrocytes appear hypertrophic, a typical hallmark of reactive astrocytes. AAV-gene transfer of SOCS3 in astrocytes reduced GFAP levels and normalized astrocyte morphology (Fig. 1b,c).

Mirror experiments were performed on another mouse cohort to further increase the activation of astrocytes by stimulation of the JAK2-STAT3 pathway. Three-month-old female 3xTg mice were injected in the hippocampus with an AAV encoding a constitutively active form of JAK2 (JAK2ca), the upstream kinase of the JAK2-STAT3 pathway, and were studied at 16 months (3xTg-JAK2ca mice). Control groups included WT-GFP and 3xTg-GFP mice injected and analyzed at the same age (Supplemental Fig. 1). 3xTg-JAK2ca mice displayed enhanced astrocyte reaction, visible as a twofold increase in GFAP immunoreactivity and exacerbated enlargement and tortuosity of astrocyte processes, compared with 3xTg-GFP mice (Fig. 1b,c). Modulation of GFAP levels by SOCS3 and JAK2ca was also observed by western blotting on hippocampal samples taken from WT-GFP, 3xTg-GFP, 3xTg-SOCS3 and 3xTg-JAK2ca mice (Fig. 1e). The immunoreactivity for vimentin, another intermediate filament characteristic of reactive astrocytes, was also reduced by more than 50% by SOCS3 in the hippocampus of 3xTg mice, although it did not reach significance (Fig. 1b,d).

Conversely, vimentin immunoreactivity was increased more than 3-fold by JAK2ca (Fig. 1b,d). The number of microglia cells in direct contact with amyloid plaques was not impacted by either SOCS3 or JAK2ca in 3xTg-AD mice (Fig. 2a,b), suggesting that our strategy modulates reactive astrocytes without significantly impacting microglial cells around plaques, as reported in another mouse model of AD [START_REF] Ceyzeriat | Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer's disease[END_REF].

These results show that manipulation of the JAK2-STAT3 pathway in hippocampal astrocytes is an efficient strategy to modulate the reactive state of astrocytes in 3xTg mice. We next evaluated how manipulation of reactive astrocytes by SOCS3 and JAK2ca impacted Tau hyper-phosphorylation and amyloid deposition, two cardinal features of AD.

Modulation of reactive astrocytes does not influence Tau phosphorylation in 3xTg mice.

At the age of 16 months, 3xTg mice display both Tau and amyloid pathologies. Tau can be phosphorylated on multiple epitopes. Three main approaches were used to characterize the effects of reactive astrocytes on Tau phosphorylation: i) immunostainings with antibodies against phospho-Ser422-Tau, phospho-Ser202/Thr205-Tau (AT8 antibody) and total human Tau (HT7 antibody) as a control, ii) ELISA MSD® assay with antibodies against phospho-Thr231-Tau (AT180 antibody) and total Tau and iii) western blotting against phospho-Ser404-Tau.

Positive staining for HT7, phospho-Ser422-Tau and AT8 was observed in 3xTg mice but not in WT mice (Fig. 3a). HT7 immunoreactivity was not changed by SOCS3 or JAK2ca in the hippocampus of 3xTg mice (Fig. 3a,b), suggesting that the expression of the human Tau transgene is not impacted by our experimental manipulations. Immunostaining with phospho-Ser422-Tau and AT8 antibodies revealed dystrophic neurons and numerous ghost tangles in the hippocampus (Fig. 3a).

Immunoreactivity for these two antibodies was similar between the 3xTg-GFP, 3xTg-SOCS3 and 3xTg-JAK2ca groups (Fig. 3b), suggesting that modulation of reactive astrocytes through the JAK2-STAT3 pathway does not impact the level of phosphorylation of these epitopes.

We next measured hippocampal concentrations of phospho-Thr231-Tau and total human Tau by MSD® assays on protein homogenates prepared from the hippocampus of 3xTg-GFP, 3xTg-SOCS3 or 3xTg-JAK2ca. Levels of phospho-Thr231-Tau normalized by total Tau and total Tau were not impacted by SOCS3 or JAK2ca in 16-month-old 3xTg mice (Fig. 3c). To assess whether reactive astrocytes impact Tau phosphorylation at a later stage of the disease, we performed the same measurement in 26-month-old female 3xTg mice that were injected 2 months before, with AAV-GFP or AAV-SOCS3. Tau phosphorylation on Thr231 was also unaffected by SOCS3 in 26-month-old 3xTg mice (Fig. 3d).

Lastly, we analyzed phospho-Ser404-Tau levels by western blotting on hippocampal protein samples from 16-month-old 3xTg-GFP, 3xTg-SOCS3 and 3xTg-JAK2ca. We found no difference between the groups (Supplemental Fig. 3).

Our results show that JAK2-STAT3 pathway modulation of reactive astrocytes in 3xTg mice does not influence Tau phosphorylation on several epitopes, at advanced and terminal stages of the disease.

Modulation of reactive astrocytes does not impact amyloid deposition in 3xTg mice.

Amyloid pathology was assessed by four approaches: i) fluorescent staining with the Congo-red derivative Methoxy-XO4 (MXO4) that binds dense-core amyloid plaques of different size, ii) immunostaining with the A 42 antibody that labels small dense-core plaques [START_REF] Serrano-Pozo | Neuropathological alterations in Alzheimer disease[END_REF] iii) immunofluorescent staining with the 4G8 antibody that labels diffuse plaques, as well as intracellular A , which is prominent in 3xTg mice [START_REF] Oddo | Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction[END_REF], and finally iv) MSD® measurement of Triton X100-soluble A levels in hippocampal homogenates.

In 16-month-old 3xTg mice, numerous MXO4 + plaques were found in the subiculum and CA1 region of the hippocampus (Fig. 4a). Modulation of reactive astrocytes by SOCS3 or JAK2ca had no detectable impact on hippocampal amyloid plaques stained by MXO4 (Fig. 4a). Detection of amyloid plaques by immunostaining with the A 42 antibody also showed no difference in their number between the three groups (Supplemental Fig. 4). The average individual size of MX04 + or Ab42 + plaques was identical between groups (Fig. 4b,c and Supplemental Fig. 4, respectively). Similarly, immunoreactivity in the subiculum and pyramidal layer of the CA1 region for intracellular A labelled by the 4G8 antibody was identical between groups (Supplemental Fig. 4).

We next measured Triton X-100 soluble A levels by MSD® assays on protein homogenates prepared from the hippocampus of 3xTg-GFP, 3xTg-SOCS3 or 3xTg-JAK2ca mice. At the age of 16 months, only A 42 was reliably detected and quantified (A 40 was undetectable in 2 to 4 mice in each group, corresponding to 30% of the two cohorts). Hippocampal A 42 concentrations were not significantly modified by SOCS3 or JAK2ca (Fig. 5a). Accordingly, several proteins involved in APP metabolism were found at similar levels in 16-month-old in 3xTg-GFP, 3xTg-SOCS3 and 3xTg-JAK2ca mice [APP; BACE1, the -secretase that cleaves APP through the amyloïdogenic pathway; insulin degrading enzyme (IDE) that degrades A ; and apolipoprotein E (ApoE) a protein secreted by astrocytes, which binds and clears cholesterol and A (Supplemental Fig. 5)].

To study whether reactive astrocytes impact A levels at an earlier or later stage of the disease, we performed the same measurement of A levels in 9-month-old and 26-month-old female 3xTg-GFP and 3xTg-SOCS3 mice. Again, A 42 concentrations were identical between groups at both ages and A 38 and A 40 only reached detectable levels in 26-month-old 3xTg mice (Fig. 5b,c). The A 42/A 40 ratio was not impacted by SOCS3 but concentrations of A 38, the least toxic form [START_REF] Li | Decoding the synaptic dysfunction of bioactive human AD brain soluble Abeta to inspire novel therapeutic avenues for Alzheimer's disease[END_REF], were significantly decreased by SOCS3 (Fig. 5c).

Overall, our analysis demonstrates that JAK2-STAT3-dependent reactive astrocytes do not overtly contribute to amyloid metabolism and deposition in 3xTg mice, at three different disease stages.

Inhibition of reactive astrocytes improves anxiety but not memory in 3xTg mice.

We previously showed that hippocampal reactive astrocytes contribute to deficits in synaptic transmission and plasticity in 9-month-old 3xTg mice [START_REF] Ceyzeriat | Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer's disease[END_REF]. Here, we analyzed whether synaptic restoration by SOCS3 translates into cognitive improvement in 3xTg mice.

We tested whether SOCS3-mediated inhibition of reactive astrocytes improved spatial memory deficits on the Y maze [START_REF] Carroll | Progesterone and estrogen regulate Alzheimer-like neuropathology in female 3xTg-AD mice[END_REF] and anxiety on the elevated plus maze [START_REF] Blanchard | Pharmacologic reversal of neurogenic and neuroplastic abnormalities and cognitive impairments without affecting Abeta and tau pathologies in 3xTg-AD mice[END_REF]. WT-GFP, 3xTg-GFP mice and 3xTg-SOCS3 were first tested at 8 months. Each mouse was placed on the Y maze to explore two arms (the start and familial arms, one being closed) for 15 min of training (Fig. 6a). After 5 min in their home cage, the mouse was placed again in the start arm of the maze with the three arms opened. In average, WT-GFP, 3xTg-GFP and 3xTg-SOCS3 mice traveled the same total distance during the test session, showing equivalent exploration (Fig. 6a). However, they explored the three arms differently (p < 0.001 for Arm effect, two-way ANOVA, Fig. 6a). WT-GFP mice displayed the expected preference for the novel arm during the test session. On the contrary, 3xTg-GFP and 3xTg-SOCS3 mice did not discriminate the new arm compared to the two other arms, revealing an alteration in working memory (Fig. 6a). These mice were also tested on the elevated plus maze. The time spent in the open arms of the maze was used as a surrogate for the level of anxiety. All three groups explored similarly the open arms, suggesting that 3xTg mice did not display higher anxiety at this age (Fig. 6b).

These mice were tested again at the age of 14 months. At this later stage, WT-GFP mice displayed an even stronger preference for the new arm on the test phase (Fig. 6a, p < 0.001 for Group x Arm effect, two-way ANOVA) whereas 3xTg-GFP mice explored significantly more the start arm than the novel and familiar arms (Fig. 6a). This lack of preference for the novel arm confirms the alteration in working memory, while the preference for the start arm suggests increased anxiety to explore more distant arms. SOCS3 did not improve working spatial memory in 3xTg mice, but it reduced their anxiety, as they did not remain in the start arm and explored the three arms similarly (Fig. 6a).

On the elevated plus maze, 3xTg-GFP mice tended to spend less time and to perform less head dipping in open arms, an indication of higher anxiety. SOCS3 reduced anxiety in 3xTg mice, by significantly increasing the time spent in the stressful open arms (Fig. 6b).

Our data show that inhibition of reactive astrocytes by SOCS3 does not alleviate memory deficits in 3xTg mice but reduces anxiety levels.

DISCUSSION

Our multi-level preclinical analysis shows that modulation of hippocampal reactive astrocytes through the JAK2-STAT3 pathway does not significantly impact Tau or amyloid pathologies, at different stages in 3xTg-AD mice. However, SOCS3-mediated inhibition of reactive astrocytes reduced their anxiety, without restoring short-term spatial memory.

The JAK2-STAT3 pathway and reactive astrocytes.

This study further demonstrates the central role played by the JAK2-STAT3 pathway in the control of reactive astrocytes as previously reported in other mouse models of neurodegenerative diseases (Ben Haim et al., 2015b;[START_REF] Ceyzeriat | Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer's disease[END_REF][START_REF] Reichenbach | Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model[END_REF] and other diseases like ischemia, spinal cord injury or trauma (see, Ceyzériat et al., 2016 for review). Strikingly, we found that expression of SOCS3 or JAK2ca in astrocytes was able to respectively inhibit or exacerbate GFAP expression and morphological changes in hippocampal reactive astrocytes of 3xTg mice. These effects are extremely stable (observed after > 10 months post-injection) and allow analysis of reactive astrocytes at different disease stages. Previously, we have shown that, besides these two hallmarks, SOCS3 was also able to normalize the transcriptome of reactive astrocytes in neurodegenerative disease models (Ben Haim et al., 2015b;[START_REF] Ceyzeriat | Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer's disease[END_REF]. It would be interesting to explore the molecular profile of astrocytes in 3xTg-GFP and 3xTg-SOCS3 mice as well, to assess whether the same genes are dysregulated in these two AD models, and whether they are normalized by SOCS3.

Similar reduction in reactive gene levels was found with the conditional knock-out of STAT3 in astrocytes in APP/PS1dE9 mice [START_REF] Reichenbach | Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model[END_REF] and mice with spinal cord injury [START_REF] Anderson | Astrocyte scar formation aids central nervous system axon regeneration[END_REF]. Thus, activation of the JAK2-STAT3 pathway is necessary for both the induction and persistence of several molecular and morphological features of reactive astrocytes, identifying this cascade as a master regulator of astrocyte reaction in disease.

Reactive astrocytes do not influence molecular hallmarks of AD in 3xTg mice.

We show that JAK2-STAT3-dependent reactive astrocytes do not significantly impact Tau and amyloid pathologies. This was observed at three different stages of the disease (9, 16 and 24 months), ruling out stage-dependent effects. We found that 30% of 3xTg mice did not display detectable A 40 levels at 16 months. In the brain of 3xTg mice, A 40 levels are either reported to be higher than those of A 42 [START_REF] Hirata-Fukae | Females exhibit more extensive amyloid, but not tau, pathology in an Alzheimer transgenic model[END_REF], equivalent [START_REF] Oddo | Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction[END_REF] or lower [START_REF] Goni | Immunomodulation targeting of both Abeta and tau pathological conformers ameliorates Alzheimer's disease pathology in TgSwDI and 3xTg mouse models[END_REF]. A 40 levels are probably dependent on the exact brain region analyzed and the mouse age, as well as the extraction protocol and detection method. A 40 was readily detected in 26-month-old mice but its levels remained lower than those of A 42. We only found a significant reduction in A 38 levels in the hippocampus of end-stage 3xTg-SOCS3 mice. A 40 and A 42 tended to be reduced in this group as well, but it did not reach significance. It is possible that a higher number of mice per group would lead to significant differences between groups but nonetheless, the effects would remain small (20-30 % range). A 38 was shown to be less synaptotoxic than A 40 or A 42 [START_REF] Li | Decoding the synaptic dysfunction of bioactive human AD brain soluble Abeta to inspire novel therapeutic avenues for Alzheimer's disease[END_REF], suggesting that such an end-stage and marginal reduction in A 38 levels may not significantly alleviate neuronal dysfunctions. Therefore, JAK2-STAT3-dependent reactive astrocytes have only a minor role in amyloid processing in this model.

The lack of SOCS3 effects on amyloid deposition is in contrast with our previous study reporting a significantly reduced number of MXO4 + amyloid plaques in APP/PS1dE9 mice with the same AAV-SOCS3 [START_REF] Ceyzeriat | Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer's disease[END_REF]. This AAV also infected 25% of hippocampal astrocytes in these mice, showing that even a partial inhibition of reactive astrocytes in the hippocampus is able to impact amyloid deposition. The present result is also in opposition with two studies reporting lower amyloid deposition after inhibition of reactive astrocytes through the calcineurin-NFAT pathway [START_REF] Furman | Targeting astrocytes ameliorates neurologic changes in a mouse model of Alzheimer's disease[END_REF][START_REF] Sompol | Calcineurin/NFAT Signaling in Activated Astrocytes Drives Network Hyperexcitability in Abeta-Bearing Mice[END_REF] or after STAT3 conditional knockout in astrocytes of APP/PS1dE9 mice [START_REF] Reichenbach | Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model[END_REF]. However these two strategies to interfere with reactive astrocytes could impact a different set of astrocyte molecular and functional features than SOCS3 expression, leading to different effects on amyloid deposition. The fact that the same experimental manipulation of reactive astrocytes (through the JAK2-STAT3 pathway) in two AD mouse models have different effects on the same disease outcome (amyloid plaques), assessed with the same method (MXO4) underlines that astrocyte reaction is highly context-dependent. How can the discrepancies between these two AD models be explained? In 16-month-old 3xTg mice, both amyloid and Tau pathologies are present, whereas only amyloid pathology develops in APP/PS1dE9 mice. This important difference (in addition to other differences such as sex, genetic background, and genetic constructs) could change the mechanisms of amyloid production, accumulation, aggregation and clearance. In addition, mutant Tau itself impacts astrocyte physiology [START_REF] Dabir | Impaired glutamate transport in a mouse model of tau pathology in astrocytes[END_REF][START_REF] Leyns | Glial contributions to neurodegeneration in tauopathies[END_REF] and could directly change their ability to clear amyloid.

Most importantly, accumulating evidence shows that astrocytes and their reactive counterparts are heterogeneous. Reactive astrocytes may adopt different molecular and functional states depending on the pathological context, and they could subsequently influence disease processes differentially [START_REF] Anderson | Heterogeneity of reactive astrocytes[END_REF][START_REF] Escartin | Questions and (some) answers on reactive astrocytes[END_REF]. Therefore, even if the causes for SOCS3 differential impact on amyloid deposition in 3xTg and APP/PS1dE9 mice were not identified, our studies illustrate that reactive astrocytes have complex and subtle effects in vivo, and call for caution in generalizing their roles in disease. In particular, this study was performed on female mice, and the same experiments should be reproduced in males.

Inhibition of reactive astrocytes does not restore spatial memory but reduces anxiety in 3xTg mice.

We previously reported that SOCS3 expression in astrocytes fully restored early synaptic and LTP alterations in 9-month-old 3xTg mice [START_REF] Ceyzeriat | Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer's disease[END_REF]. Here, we explored whether this normalization of synaptic activity translated into improved short-term memory and reduced anxiety in 3xTg mice. We found that 8-month-old 3xTg mice displayed working memory alterations on the Y maze and normal levels of anxiety on the elevated plus maze. SOCS3 did not influence this behavioral deficit at this early stage. At 14 months, 3xTg mice displayed altered spatial memory and increased anxiety. Unexpectedly, blocking reactive astrocytes with SOCS3 reduced anxiety but did not improve working memory.

The absence of SOCS3 effect on working memory could be explained by the fact that this type of memory involves other brain structures besides the targeted hippocampus. For example, it is now admitted that the prefrontal cortex is involved in spatial information processing [START_REF] Funahashi | Working Memory in the Prefrontal Cortex[END_REF].

Accordingly, we found that SOCS3 was able to improve spatial learning but not memory retrieval on the Morris water maze in APP/PS1dE9 mice [START_REF] Ceyzeriat | Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer's disease[END_REF], further stressing the role of complex brain circuits for memory tasks. A strategy targeting the JAK-STAT3 pathway in a larger part of the brain through multiple injections with i.v. delivery of blood brain barrier permeant vectors encoding SOCS3 or pharmacological approaches would help uncover the effects of reactive astrocytes on memory impairment in AD mice.

Conversely, reduced anxiety after AAV-SOCS3 injection in the dorsal hippocampus of 3xTg-SOCS3 mice was unexpected, because anxiety-related behaviours are thought to be controlled by the ventral hippocampus and amygdala [START_REF] Bannerman | Regional dissociations within the hippocampus--memory and anxiety[END_REF][START_REF] Calhoon | Resolving the neural circuits of anxiety[END_REF]. However, recent data describe extensive connections within subfields of the hippocampus and between the hippocampus and other limbic areas [START_REF] Bienkowski | Integration of gene expression and brain-wide connectivity reveals the multiscale organization of mouse hippocampal networks[END_REF]. Therefore, it is possible that targeting only a fraction of the hippocampus can impact larger brain circuits that regulate anxiety. Another nonexclusive possibility is that that gap junction-based astrocyte networks could propagate SOCS3 effects and influence distant hippocampal neurons.

Anxiety-related behaviors were evaluated on the elevated plus maze, as the mouse propensity to explore anxiogenic open arms. Additional tests like the open field would be useful to better delineate anti-anxiogenic effects of SOCS3 in 3xTg mice. Interestingly, we observed that 3xTg mice remained in the start arm of the Y maze, an indication of increased anxiety and this was corrected by SOCS3 as well, further demonstrating its positive effect on anxiety.

When do reactive astrocytes start to play a role in AD pathogenesis? Reactive glial cells (astrocytes and microglia) appear in response to an abnormal environment [e.g. dysfunctional neurons, presence of toxic oligomerized proteins (see, Ben Haim et al., 2015a, for review)]. But, positron emission tomography studies on prodromal AD patients showed that reactive glial cells may be present at early disease stages, before overt signs of amyloid deposition [START_REF] Hamelin | Early and protective microglial activation in Alzheimer's disease: a prospective study using 18F-DPA-714 PET imaging[END_REF][START_REF] Heneka | Focal glial activation coincides with increased BACE1 activation and precedes amyloid plaque deposition in APP[V717I] transgenic mice[END_REF]. In accordance, our results indicate that reactive astrocytes contribute to early synaptic deficits, before amyloid deposition [START_REF] Ceyzeriat | Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer's disease[END_REF] and their inhibition may improve some behavioral outcomes without reducing A and Tau pathologies.

CONCLUSIONS

Overall, the JAK2-STAT3 pathway appears as a core signaling cascade for reactive astrocytes. By modulating this pathway in different models of neurodegenerative diseases, we show that reactive astrocytes are multifaceted cells, with subtle functional effects, depending on the specific disease context. Our vector-based strategy is versatile and efficient to dissect out the roles of JAK2-STAT3dependent reactive astrocytes in different pathological conditions. A better understanding of the molecular changes occurring in reactive astrocytes and their impact on neurons is required to devise efficient therapeutic strategies targeting astrocytes. a, The total distance traveled during the test phase of the Y maze is not different between the three groups, at both 8 and 14 months (8 months: F(2, 19) = 1.789, p = 0.194; 14 months: F(2,13) = 0.156, p = 0.857). At 8 months, WT-GFP mice preferentially explore the novel arm (N) over the start (S) or the familiar arm (F). In contrast, 3xTg-GFP and 3xTg-SOCS3 show no preference for the novel arm, reflecting an altered working memory. Similarly, at 14-months, WT-GFP still explore preferentially the novel arm while 3xTg-GFP mice show no preference for the novel arm. They traveled significantly more distance in the start arm than WT-GFP mice, suggesting increased anxiety. Interestingly, 3xTg-SOCS3 mice did not stay within the start arm and explored all arms similarly, suggesting that SOCS3 reduces mouse anxiety but does not correct short-term memory. Two-way (group, arm) ANOVA and Tuckey post-hoc test [8 months: Arm effect F(2, 57)=17.444 p < 0.001, Group x Arm interaction F(4, 57) = 1.3734, p = 0.254. 14 months: Arm effect F(2, 39) = 6.724, p < 0.01, Group x Arm interaction F(4, 39) = 8.220), p < 0.001]. * p < 0.05, ** p < 0.01, *** p < 0.001 (between arms, within the same group), # p < 0.05 (between groups, for the same arm). b, At 8 months, the three mouse groups

FIGURE LEGENDS

  Fig. 2].

Figure 1 .

 1 Figure 1. Long-term modulation of reactive astrocytes by targeting the JAK2-STAT3

Figure 2 .

 2 Figure 2. Modulation of reactive astrocytes does not impact the number of microglial cells

Figure 3 .

 3 Figure 3. Modulation of reactive astrocytes does not impact Tau pathology in 3xTg mice.

Figure 4 .

 4 Figure 4. Modulation of reactive astrocytes does not impact amyloid load in 16-month-old

Figure 5 .

 5 Figure 5. Modulation of reactive astrocytes does not impact soluble A levels in 3xTg mice.

Figure 6 .

 6 Figure 6. SOCS3-inbition of reactive astrocytes does not correct memory deficits but

  explored similarly the open arm on the elevated plus maze test. Kruskal-Wallis test. At 14 months, 3xTg-GFP mice tend to spend less time and perform less head dipping in open arms than WT-GFP mice. SOCS3 expression in hippocampal astrocytes significantly increases the time spent in open arms. Time in open arm: ANOVA and Tuckey post-hoc test, F(2, 21) = 3.702, * p < 0.05. Head dipping: Kruskal-Wallis test.

Figure1Figure5

  Figure1Click here to download high resolution image

Supplemental figure 1. Experimental design.

Four independent cohorts of female 3xTg mice and age-and gender-matched WT controls were injected and analyzed at the indicated ages.

Supplemental figure 2. AAV infect astrocytes selectively in 3xTg mice.

AAV2/9 encoding GFP under a gfaABC 1 D promoter were injected in the hippocampus of 3xTg-AD mice. All GFP + cells co-express the astrocytic marker GFAP (magenta) and display the typical ramified and bushy morphology of astrocytes. GFP + cells do not colocalize with microglial cells labelled with IBA1 (magenta) or with neurons in the CA1 pyramidal cell layer, visualized with DAPI staining (blue).

Supplemental figure 3. Modulation of reactive astrocytes does not impact phospho-Ser404-

Tau levels in 3xTg mice.

Representative western blotting and quantification of phospho-Ser404-Tau (P-Ser404-Tau) in 3xTg-GFP, 3xTg-SOCS3 and 3xTg-JAK2ca mice. P-Ser404-Tau levels (normalized by tubulin ) are similar between groups. Mann-Whitney test. Quantifications are expressed relatively to the 3xTg-GFP group in each cohort, whose value was set to 1.

Supplemental figure 4. Modulation of reactive astrocytes does not impact amyloid pathology in 3xTg mice.

a, Representative images of amyloid plaques labelled with A 42 antibody in 16-month-old 3xTg-GFP, 3xTg-SOCS3 and 3xTg-JAK2ca mice. b, Quantification of A 42 + plaques in the whole hippocampus.

The number (b) and average individual size (c) of plaques are not impacted by SOCS3 or JAK2ca. N = 9/group. Mann-Whitney test. d, Quantification of 4G8 immunoreactivity in the subiculum and stratum pyramidale of 16-month-old 3xTg-GFP, 3xTg-SOCS3 and 3xTg-JAK2ca mice. There is no difference in 4G8 signal between groups. Mann-Whitney test. Quantifications in d are expressed relatively to the 3xTg-GFP group in each cohort, whose value was set to 1.

Supplemental figure 5. Modulation of reactive astrocytes does not impact proteins involved in A me aboli m or clearance in 3 Tg mice.

a, Representative western blotting and quantification of human APP (hAPP) and key enzymes (BACE1, IDE) or transporter (ApoE) involved in A production or clearance in 16-month-old 3xTg-GFP, 3xTg-SOCS3 and 3xTg-JAK2ca. Protein levels are similar between groups. Mann-Whitney test.

Quantifications are expressed relatively to the 3xTg-GFP group in each cohort, whose value was set to 1.

Acknowledgements

We are grateful to D. Cheramy and Dr. E. Diguet (Servier) for sharing their expertise and equipment for MSD® kits. We thank A. Panatier and S.H.R. Oliet (NeuroCentre Magendie, Bordeaux) for providing 4-month-old 3xTg mice of the 3 rd cohort, as well as D. Gonzales, S. Laumond, J. Tessaire and people at the animal facility of the NeuroCentre Magendie for mouse care and genotyping. We thank L. Vincent, K. Bastide, P. Woodling and J.M. Hélies for their help with mouse housing and transfer. We are very grateful to Dr. J. Martin for his careful reading of the manuscript.

Funding sources


Dima El

Célia Da Costa Pereira

A Cognitive Agent Framework in Information Retrieval : Using User Beliefs to Customize Results

Keywords: Cognitive Agent, Information Retrieval, Knowledge Extraction, Belief Revision

This paper presents a framework for a cognitive agent in information retrieval that customizes the list of returned documents based on what the agent believes about the user knowledge. Throughout the interactions between the agent and the user, the agent builds its beliefs by extracting the content of the documents examined by the user. The agent's belief base consists of "simple beliefs" represented by the document's keywords as well as "contextual rules" that allow the agent to derive new beliefs about the user knowledge. The agent is therefore able to compare its own beliefs about the user knowledge with the knowledge conveyed by a given document, and thus understand if the document really contains useful information for the user or not. Finally, in case of inconsistency, the agent revises its belief base to restore consistency.

Introduction and Motivation

The aim of Information Retrieval (IR) systems is to provide the user with relevant information for a given query. While in the traditional information retrieval approaches, relevance consists of matching the documents with the topics of the user query, more recent systems consider personalizing the returned results according to the user's interests. To this aim, they use, for example, information about query logs, click-through, time spent examining the document, or the explicit and/or implicit feedback from the users. In recent decades, relevance has also been viewed as a multi-faceted concept and several criteria or dimensions have been considered to assess its value [START_REF] Pasi | A multi-criteria content-based filtering system[END_REF][START_REF] Da Costa Pereira | Multidimensional relevance: A new aggregation criterion[END_REF].

We believe that users can be considered as cognitive agents [START_REF] Móra | Bdi models and systems: Bridging the gap[END_REF] having their own beliefs and knowledge about the world. They try to fulfill needs for information by requesting queries and acquire new information by examining the results. Taking into account the cognitive components of the user in information search engines has recently been set as one of the "major challenges" by the IR community [START_REF] Culpepper | Research frontiers in information retrieval: Report from the third strategic workshop on information retrieval in lorne (SWIRL 2018)[END_REF]. However, many years ago, Maron already stressed in [START_REF] Maron | Mechanized documentation: The logic behind a probabilistic interpretation[END_REF], that a document relevance should consider the user's prior knowledge or "cognitive map". He defined the value of a retrieved document as the extent to which its content helps the user fill a gap in his/her cognitive map.

More recently, Cholvy et al. [START_REF] Cholvy | Usefulness of Information for Goal Achievement[END_REF] proposed 3 definitions for measuring the usefulness of information for a cognitive agent. The definitions are based on the fact that a user requesting information needs it to achieve a goal. Therefore, they consider as useful, any piece of information allowing to decrease the information gap to achieve the goal. However, their proposal is adapted to a static situation in which the agent's beliefs do not need to be updated after the arrival of new information.

In this paper, when returning results to the users, we are aiming to consider the cognitive attitudes of the users, in particular their knowledge.We propose a framework for Information Retrieval systems which is run by cognitive rule-based agent. The agent has some beliefs about the user knowledge that are extracted from the content of the documents examined by the user. The content extraction is done using the Rapid Extraction Keyword Extraction (RAKE) [START_REF] Rose | Automatic Keyword Extraction from Individual Documents[END_REF] method. A belief revision operator is incorporated into the model to ensure that the agent's beliefs about the user are consistent and not contradictory.

Rule-Based Agents

A Rule-based agent consists of facts (ground literals) and rules (Horn clauses). Here, rules and facts are "treated" as beliefs. The facts represent information that the agent has currently obtained about its environment. They might change over time as a result of the addition/deletion of other facts from the agent's beliefs due to: (i) new information (dynamic environment) (ii) the rule's reasoning process itself. The rules are relationships between facts, and will be used to derive new beliefs (derived beliefs) from the agent's existing beliefs.

The agent's beliefs are then represented in predicate logic, in the form of literals and Horn clause rules. The rules have the form α 1 &α 2 . . . &α n → β where α 1 , α 2 , . . . , α n (n ≥ 1), β are literals. β is called the derived belief, and each α i is a premise of the rule. The & represents the logical and operator.

Belief Revision

AGM Belief Revision

Belief revision is the process of modifying the belief base to maintain its consistency whenever new information becomes available. The AGM belief revision theory [START_REF] Alchourrón | On the logic of theory change: Partial meet contraction and revision functions[END_REF] defines postulates that a rational agent should satisfy when performing belief revision. In such theory, a belief base is closed under logical consequence. We consider a belief base K and a new piece of information α. K is inconsistent, when both α and ¬α are in Cn(K), or Cn(K) = ⊥, or both α and ¬α are logical consequences of K. Three operators are considered: Expansion K + α: adds a new belief α that does not contradict with the existing beliefs. Contraction K ÷ α: removes a belief α and all other beliefs that logically imply/entail it. Revision K * α: adds a belief α as long as it does not cause contradiction in K. If the addition will cause inconsistencies in K, the revision operation starts by minimal changes in K to make it consistent with α, then adds α. In particular, if the agent has to contract a belief α, it does not contract other beliefs that derived α, as long they are consistent with the remaining beliefs (minimal change)coherence approach [START_REF] Gärdenfors | Belief revision: An introduction, ser[END_REF]. The AGM framework comprises sets of postulates to be respected by these operators to ensure consistent and minimal belief revision.

On another side, the Reason-maintenance belief revision approach considers tracking dependencies between beliefs, so that the reason(s) for believing in a belief α can be traced. When α should be given up, the agent must ensure that α is no longer derivable and give up believing the things that derived it.

Partial Entrenchment Ranking

A belief is gradual and an agent might have beliefs more entrenched (or accepted) than others. Williams [START_REF] Williams | Iterated theory base change: A computational model[END_REF] have proposed a quantitative approach for the AGM framework, by developing finite partial entrenchment rankings to represent epistemic entrenchment -a piece of information is labelled by a degree of confidence denoting how strongly we believe it. The epistemic entrenchment [START_REF] Gärdenfors | Revisions of knowledge systems using epistemic entrenchment[END_REF] captures the notions of significance, firmness, or defeasibility of beliefs.

Epistemic entrenchment relations induce preference orderings of beliefs according to the importance of these beliefs in the face of change. If inconsistency arises during a belief revision operation, the least significant beliefs (i.e., beliefs with the lowest entrenchment degree) are given up in order to restore consistency.

Tracking Beliefs and preferences

We present in this section the approach proposed by Alechina et al. [START_REF] Alechina | Preference-based belief revision for rulebased agents[END_REF] to track beliefs and calculate their preference for rule-based agents. The core of their work was a proposition for belief revision and contraction for such agents which are a synthesis of AGM and reason maintenance styles. The details of their algorithms will not be described here as they do not fit the purpose of the paper. The authors considered preferences on beliefs, assuming that a user might prefer some beliefs over others. Those preferences are used to decide which belief(s) should be removed to restore consistency.

The dependency between beliefs is considered as follows. For every fired rule instance, a Justification J will record: (i) a belief α, which corresponds to the derived belief and (ii) a support list, s, which contains the premises of the rules (contextual beliefs of a plan used to derive α). The dependency information of a belief had the form of two lists: dependencies and justifications. A dependencies list records the justifications of a belief, and a justifications list contains all the Justifications where the belief is a member of a support. They define preferences using a notion of quality associated with justifications, assuming the quality of a justification is represented by non-negative integers in the range 0,. . .,m, where the value of 0 means lowest quality and m means the highest quality. The lower the value, the least the quality. Definition 1. The preference value of a belief α, p(α), is equal to that of its highest quality justification.

p(α) = max{qual(J 0 ), . . . , qual(J n )} (1)
Definition 2. The quality of justification J, qual(J), is equal to the preference of the least preferred belief in its support list.

qual(J) = min{p(α) : α ∈ support of J} (2)
Literals with no supports (or empty support) are assigned the lowest quality. An a priori quality is assigned to each justification with empty support.

Knowledge Extraction from documents -RAKE

The RAKE (Rapid Automatic Keyword Extraction) [START_REF] Rose | Automatic Keyword Extraction from Individual Documents[END_REF] is an unsupervised, domain-independent, and language-independent method used for extracting keywords from documents.

RAKE starts its extraction on a document by parsing the text into a set of candidate keywords. First, the document text is split into an array of words by specified word delimiters. This array is then split into sequences of contiguous words at phrase delimiters and stop word positions. Words within a sequence are assigned the same position in the text and together are considered a candidate keyword. After every candidate keyword is identified and the graph of word cooccurrences is complete, a score is calculated for each candidate keyword. The method evaluates several metrics for calculating word scores, based on the degree and frequency of word vertices in the graph: (1) word frequency freq(w), (2) word degree deg(w), and (3) ratio of degree to frequency deg(w)/freq(w).

RAKE also identifies keywords that contain interior stop words such as axis of evil. To find these, it looks for pairs of keywords that adjoin one another at least twice in the same document and in the same order. A new candidate keyword is then created as a combination of those keywords and their interior stop words. The score for the new keyword is the sum of its member keyword scores. After candidate keywords are scored, the top one-third scoring candidates are selected as keywords for the document.

Proposed Framework

Our framework proposes a rule-based agent for Information Retrieval systems that uses its cognitive abilities to acquire beliefs about its user's knowledge to customise the returned results. When the IR agent has α in its belief base, it believes that the user knows that α is true. If the belief base contains ¬α, then the agent believes the user knows that α is not true. When neither α nor ¬α is in the belief base, the agent believes neither the user knows α is true nor the user knows that α is false.

The user knowledge is extracted from the documents he/she has examined and the rules are acquired through the integration with external entities like semantic entities, ontology or knowledge mining tools.We represent the contextual rules by Horn clause rules and consider them static. The origin of the rules is not discussed in this paper; this is left for future work.

As belief is gradual, we adopt the partial entrenchment approach briefly discussed in Section 3.2 to represent the degrees of the derived beliefs. The entrenchment degree for a derived belief will be calculated based on the premises of the rule that derived it. The degrees of the extracted beliefs instead, will be calculated based on the score of the corresponding keywords in the document. The beliefs can be revised and the relations between them are tracked as proposed by the Alechina's approach and discussed in Section 3.3, i.e. by using the concept of beliefs and justifications with their related preference degree value and quality of justification.

Finally, we employ the agent beliefs to return filtered results to the user.

Model Architecture

In this section, we present a client-side web search agent implementing the methods discussed in the previous sections to personalize search while learning about the user knowledge. The agent can be a browser plugin acting as a proxy for web search engines or a filter built on top of existing search applications. By running the agent on the client-side, no information about the user behaviour and activity is released to the outside as they all reside on his/her computer. Also, in terms of server scalability and efficiency, the model distributes computation and storage among its users rather than centralising them.

The framework has 3 main modules: (1) rule-based module, modeling the beliefs and rules. It performs inference reasoning about the user's knowledge and revise its beliefs if needed, to maintain consistency. (2) Knowledge extractor module, extracting knowledge from the documents examined by the user. (3) Result filtering module; selecting the "useful" documents to be proposed to the user. These modules work together, as follows, for every submitted query: (i) the system sends the query to the search engine/application, (ii) receives the result from the search engine, and (iii) filters them by excluding those that are not useful to the user, according to the user knowledge. After the user has clicked on the results, the agent updates its beliefs about the knowledge supposed to be acquired by the user. Finally, a reasoning cycle is performed to run the applicable rules and revise the beliefs as needed.

Extracting Knowledge and Belief Entrenchment

The agent belief base is composed of the beliefs it learns about its user and of the contextual rules it owns. When the user reads a document d, the agent learns the user has acquired some knowledge about its content; so it uses some knowledge extraction/NLP tool to extract the representation of the document content. We use the RAKE method [START_REF] Rose | Automatic Keyword Extraction from Individual Documents[END_REF] as an easy and understandable method, to extract the keywords representing the document and calculate their related scores. The beliefs resulting from this process are referred to extracted beliefs. As the keywords are associated to the agent beliefs, their related scores are associated to degrees of beliefs. We define the degree of a belief as follows: Definition 3. The degree of a belief α is the degree to which the agent believes the user is knowledgeable about α. It is represented by a decimal ranging between 0 and 1, where 0 means the lowest degree -the agent believes the user has absolutely no knowledge about α, and 1 means the highest degree -the agent believes the user has the maximum knowledge about α.

Let us consider document d = {k 1 , . . . , k n }. We suppose that a keyword k i is associated with an extracted belief b j whose degree is calculated as follows:

degree(b j ) = λ • Rakescore(k i ) max kj ∈d Rakescore(k j ) . (3)
In Equation 3the score of an extracted RAKE keyword is first divided by the highest keyword score of the document: the score is hence normalized. It is then multiplied by an adjustment factor λ ∈ [0, 1]. The adjustment factor is used to weaken the magnitude of the scores: when λ is equal to 1, the "top score" keyword of all examined documents will be assumed to have the maximal degree 1, which means that the agent assumes the user has the maximal knowledge about this keyword. The adjustment factor may vary based on different characteristics such as the source of the document, for example.

A derived belief is the belief resulting from firing the agent's rules. The dependency between the beliefs is tracked by the concept of justification and dependencies lists as per Alechina's approach presented in Section 3.3. Our definition of degree of derived beliefs is inspired by their notion of preference. Indeed, we use Equation 1 to calculate the degree of a derived belief, and Equation 2 to calculate the quality of a justification.

Revising Beliefs

While the agent is acquiring more information about the user, it is adding beliefs to its belief base. The beliefs might be new, already existing, or contradicting with the existing ones; that calls for the need of revising beliefs to ensure the belief base is consistent. The belief revision approach we have adopted here combines the AGM and reason-maintenance styles while tracking the beliefs.

In case of contradiction between a pair of beliefs, we give preference to the more entrenched one, i.e. the belief with the highest degree. Our model does not contract a belief α unless a more preferred contradictory belief ¬α was added. When contracting a belief α, we don't see a need to contract beliefs that derived α: when the rule deriving α tries to add it again, the addition will be discarded because it will be faced by ¬α that is more preferred. In other terms, we contract the belief in question with its related justification(s), without contracting neither the rule's premises nor the rule itself. For example, consider an agent's belief base: B = {astronomy[0.8], astronomy → planet(pluto)}.

The agent reasoning will result in the execution of the rule and therefore adding planet(pluto) with an entrenchment degree of 0.8. Suppose the agent was later informed that the agent believes pluto is not a planet, assuming the degree of entrenchment of the related belief is 0.9. The agent attempts to add ¬planet(pluto)[0.9] in its belief base. Upon revising the beliefs, planet(pluto) is contracted and ¬planet(pluto) is added, since the belief with the higher degree is given priority. However, when planet(pluto) is removed, the fact that the user knows about astronomy does not change: the belief astronomy is not contracted. The related rule remains in the belief base as well.

Document Similarity and Result Filtering

The filtering process will be dependent on the similarity between the agent's current beliefs and the documents to be proposed to the user. We propose a similarity function, Sim(B, d), that calculates this similarity measure based on the degrees of the intersected beliefs and the document knowledge. The formula is inspired by the similarity function proposed by Lau et al. in [START_REF] Lau | Belief revision for adaptive information retrieval[END_REF]. We consider a set of beliefs B = {b 1 , b 2 , . . . , b m } and a document d = {k 1 , k 2 , . . . , k n } characterized by a set of keywords representing the content. Let us consider S, the set of keywords appearing both in d and in B defined by

S = {k i ∈ d : extent(B, k i ) > 0 ∨ extent(B, ¬k i ) > 0}. Sim(B, d) = max{ k i ∈d [extent(B,ki))-extent(B,¬ki)],0} |S| if |S| = 0 0 otherwise. (4)
The extent(B, k) = degree(k), if k ∈ B; and 0 otherwise. The degree(k) is calculated as discussed in Section 5.2. The similarity formula "rewards" the documents containing keywords that are in the set B.

We set a cutoff value γ that allows to decide whether the knowledge inside a document is similar to a set of beliefs or not. The filter is used according to the intended application: when the purpose of framework is to reinforce the user knowledge, then documents that are "near" the agent beliefs will be returned returned. The documents having a similarity score greater than the cutoff will be returned as the final results to the user. Contrarily, when the framework is employed for novelty purposes, the documents having similarity below the cutoff will be returned.

Conclusion and Future Work

In this paper, we proposed an innovative framework for a rule-based information retrieval agent which relies on its cognitive abilities to learn about the user's knowledge, and propose new/relevant documents accordingly.

The components of the framework are: (1) rule-based module, modeling the agent's beliefs and rules. It performs inference reasoning about the user's knowledge and revise its beliefs if needed, to maintain consistency. (2) Knowledge extractor module, extracting knowledge from the documents examined by the user.

(3) Result filtering module, selecting the "useful" documents to be proposed to the user. The process of acquisition of information is session and time independent. Another advantage of the proposed framework is that it can be built on top of existing non-cognitive search engines, applications, or open-source libraries.

As for future work, we plan to take into account the confidence in the sources of the documents, which will probably affect the degree of entrenchment of a belief. Another possible extension could be to integrate some semantic analysis in order to deal with synonyms.


M Salhi

V Kemlin

S Faci

G Baili

A Khadour

S Mostarshedi

ESYCOM A.-L Billabert

Measurement of Brillouin gain spectrum with dual-frequency laser for optical fibre sensor

In this Letter, the authors present a first measurement of Brillouin gain spectrum with dual-frequency solid-state laser. This allows a coherent detection of the Brillouin backscattered spectrum in a frequency range below the hundreds of MHz by fine-tuning of the difference between the two cross-polarised fields emitted by the laser. The photodetection at low-frequency benefits from the balanced detection technique where high dynamic range measurements are largely available. The narrow line-width of the cross-polarised field permits also the direct measurement of the Brillouin spectrum line-width. The system is demonstrated by measuring the Brillouin frequency shift with temperature when standard single-mode fibre-28 (SMF-28) and large effective area fibre (LEAF) are fully heated. This gives temperature coefficients close to theoretical expectations with values of 1.16 MHz/°C for SMF-28 and 1.15 MHz/°C for LEAF.

Introduction:

The phenomenon of spontaneous or stimulated Brillouin scattering applied to optical fibre sensors has been the subject of numerous scientific investigations during last few decades thanks to its high capacities such as (i) high sensitivity to a localised temperature variation and/or deformation in the fibre, (ii) long sensing range and good spatial resolution, and (iii) potential applications for a large-scale structural health monitoring [START_REF] Hartog | An introduction to distributed optical fibre sensors (series in fiber optic sensors)[END_REF]. The performances of these distributed sensors are constantly improved to have the most accurate measurement of the Brillouin frequency shift (BFS) [START_REF] Mao | 1-cm-spatial-Resolution Brillouin optical time-domain analysis based on bright pulse Brillouin gain and complementary code[END_REF][START_REF] Agrawal | Nonlinear fiber optics[END_REF]. There are several conventional systems allowing a distributed measurement of this BFS either in time domain [START_REF] Tu | Strain variation measurement with short-time Fourier transform-based Brillouin optical time-domain reflectometry sensing system[END_REF], in frequency domain [START_REF] Bernini | Frequency-domain approach to distributed fiber-optic Brillouin sensing[END_REF] or in correlation domain [START_REF] Mizuno | Low-cost Brillouin optical correlation-domain reflectometry involving merely one fibre amplifier[END_REF]. Some of these systems use the same laser source as a pump wave for the excitation of Brillouin backscattering and also as a local oscillator (LO) for coherent detection, which requires a high bandwidth photodetector combined with frequency down-conversion electronics [START_REF] Bernini | Self-demodulated heterodyne frequency domain distributed Brillouin fiber sensor[END_REF]. To overcome this constraint, these systems have been upgraded by introducing an optical frequency translation block to shift the pump wave frequency by the Brillouin frequency difference [START_REF] Wang | Performance of Brillouin optical time domain reflectometer with erbium doped fiber amplifier[END_REF]. Another solution consists in using two independent laser sources for pump wave and LO wave with a frequency difference close to the BFS value [START_REF] Galindez-Jamioy | Brillouin distributed fiber sensors: an overview and applications[END_REF]. However, the uncorrelated laser diodes solution presents an inability to have high beat-frequency stability through a simple optical phaselocked loop (OPLL) [START_REF] Rideout | A true time delay beamforming system incorporating a wavelength tunable optical phase-lock loop[END_REF] and a lack of frequency tunability of the frequency difference, making the adjustment of the frequency difference as near as possible to BFS quite difficult, knowing that there are fibres with several BFS. The tuning range of the frequency difference is thus limited by the OPLL parameters. We believe that a new generation of Brillouin sensors that would overcome these systems penalties is greatly needed. For this, we implement the Brillouin sensor with a dual-frequency cross-polarised laser (DFL) which offers the best compromise between compactness and large tunability. Previous work based on such configuration has been initiated in [START_REF] Chaccour | Cross-polarized dualfrequency VECSEL at 1.5 mm for fiber-based sensing applications[END_REF] with a dual-frequency cross-polarised vertical-cavity surface-emitting laser (VECSEL). The advantage of solid-state laser over VECSEL is the stability of lasing modes, particularly with temperature. In this Letter, we present first measurements of a Brillouin-fibre optic gain spectrum based on DFL. The corresponding emitted fields are cross-polarised, which facilitates their spatial separation. Besides, the frequency difference stabilisation could simply be achieved because the two optical frequencies are generated within the same laser cavity. The frequency difference between the two laser eigenstates can be tuned over the full free spectral range of the cavity. This makes it possible to be adjusted at a frequency close to the BFS of silica-based fibres. Using the heterodyne coherent detection scheme, one ends up with a beating frequency lower than 500 MHz, low enough for signal processing, with low noise and high dynamic electronics. Here, we seek to take advantage of these unique features to develop a state-of-the-art Brillouin optical timedomain reflectometer (B-OTDR) sensor.

The Letter is organised as follows: first, a description of the laser source and developed experimental set up is given. Then, we present the static measurement results of the Brillouin gain spectrum of a 40 km single-mode fibre (SMF) and a 10 km large effective area (LEAF). Finally, the measured peak-frequency shifts of the Brillouin spectrum with temperature are given for the used fibres and their respective temperature coefficients are estimated and compared to theoretical values.

Description of experimental set-up and measurement results:

The originality of the proposed Brillouin optical sensor resides in the use of a single laser source generating simultaneously the pump and LO optical signals. It consists of a dual-frequency dual-polarisation laser where the frequency difference between the two laser eigenstates can be tuned to down-convert the Brillouin spectrum to an intermediate frequency through a simple coherent detection. In this section, we describe the laser source, the developed experimental set-up, and present measurement results of a Brillouin gain spectrum.

Dual-frequency dual-polarisation laser description: The schematic diagram of a dual-frequency dual-polarisation laser is shown in Fig. 1a. This laser was initially developed for RADAR clock applications [START_REF] Pillet | Wideband dual-frequency lidarradar for high-resolution ranging, profilometry, and Doppler measurement[END_REF]. It consists of an optically pumped Er/Yb solid-state laser emitting at the telecom wavelength [START_REF] Morvan | Dual-frequency laser at 1.53 mm for generating high-purity optically carried microwave signals up to 20 GHz[END_REF][START_REF] Brunel | Generation of tunable highpurity microwave and terahertz signals by two-frequency solid state lasers[END_REF]. The control of the gain medium and the use of the 1-mm birefringent LiTaO 3 crystal allow the simultaneous emission of two cross-polarised eigenstates. An uncoated silica etalon inserted inside the cavity permits the selection of one optical mode for each polarisation. The tunability of the frequency difference between the two cross-polarised eigenstates is ensured by the two electro-optic and thermo-optic effects of the LiTaO 3 crystal having typical intrinsic coefficients of 1 MHz/V and 1.1 GHz/K, respectively [START_REF] Pillet | Wideband dual-frequency lidarradar for high-resolution ranging, profilometry, and Doppler measurement[END_REF][START_REF] Pillet | Dual-frequency laser at 1.5 mm for optical distribution and generation of high-purity microwave signals[END_REF]. In this laser configuration, the maximum frequency difference between the two fields can reach 30 GHz. Furthermore, since the two modes arise from the same cavity, any thermal drift inside the cavity length or vibration of the mirror mounts induces equal frequency variations in both modes and results in a narrow bandwidth beat note [START_REF] Pillet | Wideband dual-frequency lidarradar for high-resolution ranging, profilometry, and Doppler measurement[END_REF]. At the laser output, the optical beam is collimated and fibre-coupled to a single-mode polarisation maintaining fibre. The BFS n B is typically between 10.5 and 11 GHz for silica-based fibre. Fig. 1b shows the optical spectrum of the two modes of the laser where the frequency difference has been stabilised using the optical phase-locked loop method and external RF reference, as described in [START_REF] Pillet | Wideband dual-frequency lidarradar for high-resolution ranging, profilometry, and Doppler measurement[END_REF][START_REF] Morvan | Dual-frequency laser at 1.53 mm for generating high-purity optically carried microwave signals up to 20 GHz[END_REF][START_REF] Brunel | Generation of tunable highpurity microwave and terahertz signals by two-frequency solid state lasers[END_REF][START_REF] Pillet | Dual-frequency laser at 1.5 mm for optical distribution and generation of high-purity microwave signals[END_REF]. The power of the laser modes depends on the cavity gain profile and can be adjusted by the crystal applied voltage. Then, the two modes are chosen to promote the pump wave and at the same time to prevent the saturation of the photodiode with high LO power. The OPLL has been developed with laboratory instrument solutions where the spectral purity of the resulting beat note has been improved from a typical short-term line-width over 1 kHz in freerunning conditions to a value below 200 mHz when the beat note is locked [START_REF] Pillet | Dual-frequency laser at 1.5 mm for optical distribution and generation of high-purity microwave signals[END_REF]. However, knowing that the use of an external reference is not important since the two laser frequencies are phase correlated. As the purpose of the locking loop is to provide long-term frequency drift compensation with an alternative procedure that does not require an external reference but based on an optical frequency locked loop. This opens the way to a more simplified and compact system compared to conventional ones. The implementation of this technique for DFLs is discussed in [START_REF] Pillet | Wideband dual-frequency lidarradar for high-resolution ranging, profilometry, and Doppler measurement[END_REF][START_REF] Pillet | Dual-frequency laser at 1.5 mm for optical distribution and generation of high-purity microwave signals[END_REF]. In our experiments, it is important to precise that these results were obtained without insulation of the laser cavity compared to the laboratory environment. Thus, we were able to lock the laser at the desired beat frequency for the tenth of minutes and more.

V 2 V 2 V 1 V 1 relative power,
Experimental set-up for Brillouin spectra measurement: The proposed system based on a dual-frequency laser is illustrated in Fig. 2. The DFL frequencies (n 1 and n 2 , n 1 . n 2 ) are synchronised with an OPLL as detailed in [START_REF] Morvan | Dual-frequency laser at 1.53 mm for generating high-purity optically carried microwave signals up to 20 GHz[END_REF] and are separated by a beam polarisation splitter, where the optical frequency n 1 is used as a pump wave to excite the Brillouin scattering process and the optical frequency n 2 is used as an optical LO for coherent heterodyne detection. The pump wave power is controlled by an erbium-doped fibre amplifier followed by a variable attenuator. An optical circulator is used to inject the pump wave into the fibre under test and to drive the backscattered Brillouin Stokes signal (frequency n S ) to the detection block. A 50/50 optical coupler is used to combine Stokes signal and optical LO signal for detection with a lowspeed commercial photodiode. The resulting electrical signal is then amplified with a 50 dB LNA (RF Amp) and filtered with a 825 MHz low pass filter. The amplified electrical signal is analysed with an electrical spectrum analyser in a frequency bandwidth lower than 1 GHz, where the spectrum is acquired directly by processing the fast Fourier transform of the time signal.

DFL RF signal processing Amp PD PD LPF f RF optical path v 2 v 2 ≈ v 1 -v B v AS ≈ v 1 + v B v S ≈ v 1 -v B v 1 electrical path OPLL 90:10 50:50 10% 90% CW, v 1 , v 2 BPS EDFA circulator AV 1 2 3 v S v 1 v 2 v 2 v s v s v 1 v 1 v AS
g B (n) = g B0 Dn B 2 2 (n -n B0 ) 2 + Dn B 2 2 (1)
where g B0 is the Brillouin gain coefficient at the Brillouin frequency peak n B and Dn B is the full width at half maximum (FWHM) of the gain spectrum. The spontaneous Brillouin spectrum of a 40 km SMF-28 and a 10 km LEAF have been characterised in a climate-controlled room for an injected pump power of 20 mW and an optical LO power of 100 m W. Fig. 3 shows the measured and fitted Brillouin spectrum for the 40 km SMF-28 when the DFL beat frequency has been locked at 10.83 GHz. The measured peak frequency of the spectrum is around 120 MHz, which corresponds to a typical BFS value of 10.8 GHz for such fibre. For spectrum characterisation purposes, a Lorentzian fit gives a Brillouin line-width of 9.32 MHz, which is consistent with our expectations [START_REF] Thévenaz | Brillouin distributed time-domain sensing in optical fibers: state of the art and perspectives[END_REF], since the diffusion process is in stimulated mode (or amplified spontaneous diffusion) where the critical power is 3 mW. The Brillouin spectrum of the 10 km length LEAF is shown in Fig. 4. The multi-peak Brillouin spectrum corresponding to the different acoustic modes of the fibre has been measured. As only frequency values of Brillouin peaks are sought in this characterisation, the electrical filter, which influences only on the peak powers, has not been changed. Only three peaks of Brillouin backscattering signal are identified at frequencies of 743.1, 1140, and 1360 MHz. The frequency interval between the first and the second peak is about 400 MHz, which is compliant with typical LEAF values. The second and third peaks are attenuated by the low-pass filter where the fourth peak is filtered and being drowned in the noise floor. The other peaks of the spectrum are linked to signal distortion, probably due to inter-peak coupling. The Lorentzian fit of the first Brillouin peak gives a line-width of 7.76 MHz describing an amplified diffusion process. It is important to notice that the high phase stability of the two laser eigenstates gives a measured beat note line-width of a few kHz in free-running mode [START_REF] Pillet | Wideband dual-frequency lidarradar for high-resolution ranging, profilometry, and Doppler measurement[END_REF]. This allows a precise measurement of the Brillouin gain line-width compared to other state-of-the-art solutions. Thus, direct measurement of the FWHM Brillouin line-width is obtained with a dual-frequency laser based on B-OTDR that can be used to evaluate temperature/strain variation.

BFS temperature dependence: The BFS dependence with temperature and/or strain is the physical parameter that is exploited in distributed fibre sensors. The dependence of this shift is quantified by the associated sensitivity coefficients of the considered fibre

dn B = C T DT + C e De (2)
where C T and C e are, respectively, the sensitivity coefficients of temperature and strain, DT and De are changes in temperature and strain, respectively. For a wavelength around 1550 nm, the coefficients related to BFS are typically C T ≈ 1 MHz/°C and C e ≈ 0.05 MHz/me for SMF-28 [START_REF] Thévenaz | Brillouin distributed time-domain sensing in optical fibers: state of the art and perspectives[END_REF]. For the LEAF, these coefficients are of 1.19 MHz/°C and 48 kHz/me for peak 1 and 1.22 MHz/°C and 50 kHz/me for peak 2 [START_REF] Zhou | Distributed temperature and strain discrimination with stimulated Brillouin scattering and Rayleigh backscatter in an optical fiber[END_REF]. To determine these coefficients with the proposed system, each fibre (SMF-28 and LEAF) has been completely heated in a controlled temperature oven from 24 to 30°C by a step of 1°C. Fig. 5 shows the measurement results for the 40 km length SMF-28 when the beat frequency of the DFL has been stabilised at 10.83 GHz. The linear behaviour of the dependence of the frequency shift with the temperature is observed. As expected, the central frequency of the spectrum shifts towards the high frequencies with the increase of the fibre temperature. The sensitivity coefficient C T has been estimated according to the BFS with temperature. A recording temperature error of 0.4°C is considered, which is linked to the maximum difference between the oven probe and an external probe positioned at the surface of the spooled optical fibres. This means that a relative error of 0.4°C around an external probe measured temperature is considered. This error is represented by horizontal error bars in the figures. Then, the uncertainty on the recorded values is estimated from a triangular distribution function with a 95% confidence interval. This distribution is determined from the minimum and maximum values of the coefficient C T between two consecutive temperature points according to the error bars. The estimated sensitivity coefficient is obtained by considering the extreme error case and is of 1.16 MHz/°C + 65 kHz/°C for SMF-28, which agrees well with the theoretical expectation. Fig. 5 BFS with temperature for SMF-28

For the LEAF characterisation, only the first two peaks are considered and the DFL's beat frequency has been stabilised at 9.83 GHz giving a first photo-detected Brillouin peak frequency close to 740 MHz and the second peak around 1140 MHz. As for SMF-28, the Brillouin multipeak frequency shift related to LEAF has the same behaviour with temperature. Fig. 6 shows a quasi-linear increase of the two first peaks with the increase of the fibre temperature. The estimated sensitivity coefficients for these peaks are, respectively, of 1.15 MHz/°C+ 31kHz/°C and 1.35 MHz/°C + 37 kHz/°C. These coefficients are in good agreement with those presented in [START_REF] Zhou | Distributed temperature and strain discrimination with stimulated Brillouin scattering and Rayleigh backscatter in an optical fiber[END_REF].

Fig. 1

 1 Fig. 1 Dual-frequency dual-polarisation laser a Topology b Output optical spectrum

Fig. 2 Fig. 3

 23 Fig. 2 Experimental setup for characterisation of Brillouin backscattering spectrum based on dual-frequency laser. All parameters are defined in text

Fig. 4

 4 Fig. 4 LEAF multi-peaks frequency Brillouin gain spectrum at room temperature

Fig. 6

 6 Fig.6Multi-peak BFS with temperature for LEAF Conclusions: We characterise the Brillouin gain spectrum using a dualfrequency and dual-polarisation laser for the next-generation of distributed optical sensing systems. The advantages of this system are: (i) the two laser frequencies are phase correlated leading to a low noise sensor and (ii) the wide tunability of the frequency difference allowing to adjust the frequency difference to BFS. The Brillouin spectra of SMF-28 and LEAF have been characterised to validate the proposed system. The averaged spectra have shown a high stability over time and allows the direct measurement of the Brillouin line-width. The BFS with temperature has been characterised for both fibres for which the expected linear relationship has been observed. For SMF-28, the measured temperature coefficient is around a value of 1.16 MHz/°C, while for LEAF, it is 1.15 MHz/°C for the first peak frequency and 1.35 MHz/°C for the second one. These temperature sensitivity coefficients of both fibres are in good agreement with the published data.