Celia Fourrier

Camille Kropp

Agnès Aubert

Julie Sauvant

Carole Vaysse

Jean-Michel Chardigny

Sophie Layé

Corinne Joffre

Dr Nathalie Castanon
email: nathalie.castanon@inra.fr

Célia Fourrier

Rapeseed oil fortified with micronutrients improves cognitive alterations associated with metabolic syndrome

Keywords: n-3 PUFAS, Antioxidants, Metabolic syndrome, Obesity, Inflammation, Anxiety, Memory, Hippocampus, Glutamatergic receptors, db/db mice Highlights

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The prevalence of the metabolic syndrome (MetS) is steadily increasing worldwide to become a major public health concern. It is not only characterized by metabolic disturbances but also by low-grade inflammation [START_REF] Mraz | The role of adipose tissue immune cells in obesity and lowgrade inflammation[END_REF][START_REF] Wensveen | The "Big Bang" in obese fat: Events initiating obesity-induced adipose tissue inflammation[END_REF][START_REF] Cani | Gut microbiota-mediated inflammation in obesity: a link with gastrointestinal cancer[END_REF][START_REF] Cox | Obesity, inflammation, and the gut microbiota[END_REF]. Such a cluster of dysregulations makes the MetS an important risk factor for related severe metabolic and cardiovascular comorbidities [START_REF] Cani | Gut microbiota-mediated inflammation in obesity: a link with gastrointestinal cancer[END_REF]. To worsen the picture, longitudinal, epidemiological and case-control studies also highlight an elevated prevalence of mood symptoms and cognitive alterations in patients with MetS as compared to the general aged-matched population [START_REF] Agusti | Interplay Between the Gut-Brain Axis, Obesity and Cognitive Function[END_REF][START_REF] Castanon | Neuropsychiatric comorbidity in obesity: role of inflammatory processes[END_REF][START_REF] Mansur | Is there a "metabolic-mood syndrome"? A review of the relationship between obesity and mood disorders[END_REF][START_REF] Philippou | The role of metabolic syndrome factors on cognition using latent variable modeling: The neurocognitive study on aging[END_REF]. These neuropsychiatric alterations not only impair their quality of life and medical care [START_REF] Brunault | High preoperative depression, phobic anxiety, and binge eating scores and low medium-term weight loss in sleeve gastrectomy obese patients: a preliminary cohort study[END_REF][START_REF] Hilgendorf | A behavioral rating system predicts weight loss and quality of life after bariatric surgery[END_REF], but also represent important risk factors for other MetS-related comorbidities, which in turn compromise the health outcomes [START_REF] Klakk | Symptoms of depression in young adulthood is associated with unfavorable clinical-and behavioral cardiovascular disease risk factors[END_REF][START_REF] Ramirez | Review of biologic and behavioral risk factors linking depression and peripheral artery disease[END_REF]. This alarming issue promotes heightened interest in identifying new strategies to tackle the development of neuropsychiatric alterations in the context of MetS.

Although pharmacotherapy is a first-line treatment in psychiatry, the remission rates are particularly low in patients with obesity/MetS and they often experience important side effects [START_REF] Pacher | Cardiovascular side effects of new antidepressants and antipsychotics: new drugs, old concerns?[END_REF][START_REF] Rush | Acute and longer-term outcomes in depressed outpatients requiring one or several treatment steps: a STAR*D report[END_REF]. This highlights a need to develop further therapeutic strategies for those individuals. As already shown in other conditions associated with neuropsychiatric symptoms, nutritional interventions aiming to guaranty adequate levels of essential nutrients that are crucial for normal body and brain function, but cannot be synthesized de novo by mammals, may represent a promising strategy [START_REF] Husted | The importance of n-6/n-3 fatty acids ratio in the major depressive disorder[END_REF][START_REF] Thesing | Omega-3 and omega-6 fatty acid levels in depressive and anxiety disorders[END_REF][START_REF] Vauzour | Neuroinflammatory processes in cognitive disorders: Is there a role for flavonoids and n-3 polyunsaturated fatty acids in counteracting their detrimental effects?[END_REF]. In that context, nutrients such as n-3 polyunsaturated fatty acids (n-3 PUFAs) and antioxidants (AO) (e.g. phenolic compounds and vitamins), which are insufficiently consumed in western societies, particularly by obese individuals [START_REF] Simopoulos | Evolutionary aspects of diet and essential fatty acids[END_REF], have been shown to alleviate neuropsychiatric symptomatology [START_REF] Larrieu | Food for Mood: Relevance of Nutritional Omega-3 Fatty Acids for Depression and Anxiety[END_REF][START_REF] Miquel | Poor cognitive ageing: Vulnerabilities, mechanisms and the impact of nutritional interventions[END_REF][START_REF] Sanoobar | Coenzyme Q10 as a treatment for fatigue and depression in multiple sclerosis patients: A double blind randomized clinical trial[END_REF]. For example, n-3 PUFAs and vitamin E co-supplementation reduced depressive and anxious symptoms in women with polycystic ovary syndrome [START_REF] Jamilian | The effects of omega-3 and vitamin E co-supplementation on parameters of mental health and gene expression related to insulin and inflammation in subjects with polycystic ovary syndrome[END_REF].

Moreover, supplementation with eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3, the main n-3 PUFA in the brain) decreased stress-induced anxiety in students [START_REF] Kiecolt-Glaser | Omega-3 supplementation lowers inflammation and anxiety in medical students: a randomized controlled trial[END_REF]. Similarly, depressive-like and anxiety-like behaviors are prevented in rodent models of depression by dietary supplementation with vitamin E or EPA/DHA [START_REF] Larrieu | Nutritional omega-3 modulates neuronal morphology in the prefrontal cortex along with depression-related behaviour through corticosterone secretion[END_REF][START_REF] Manosso | Antidepressant-like effect of alpha-tocopherol in a mouse model of depressive-like behavior induced by TNF-alpha[END_REF]. Similarly, supplementation with both n-3 PUFAs and plant-derived AO reduced memory deficits [START_REF] Bensalem | Polyphenol-rich extract from grape and blueberry attenuates cognitive decline and improves neuronal function in aged mice[END_REF][START_REF] Bensalem | Dietary Polyphenol Supplementation Prevents Alterations of Spatial Navigation in Middle-Aged Mice[END_REF]Delpech et al., 2015a,b;[START_REF] Labrousse | Short-term long chain omega3 diet protects from neuroinflammatory processes and memory impairment in aged mice[END_REF][START_REF] Létondor | EPA/DHA and Vitamin A Supplementation Improves Spatial Memory and Alleviates the Age-related Decrease in Hippocampal RXRγ and Kinase Expression in Rats[END_REF] and depressive-like behavior in aged rodents [START_REF] Moranis | Long term adequate n-3 polyunsaturated fatty acid diet protects from depressive-like behavior but not from working memory disruption and brain cytokine expression in aged mice[END_REF].

Although the impact of n-3 PUFAs and/or AO supplementations on neuropsychiatric comorbidities of obesity/MetS is still poorly known, mounting evidence already reports beneficial properties regarding metabolic and cardiovascular alterations (for reviews, see [START_REF] Lorente-Cebrian | Role of omega-3 fatty acids in obesity, metabolic syndrome, and cardiovascular diseases: a review of the evidence[END_REF][START_REF] Mozaffari | Dietary Total Antioxidant Capacity and Cardiovascular Disease Risk Factors: A Systematic Review of Observational Studies[END_REF]. Besides, because n-3 PUFAs are sensitive to oxidation due to their unsaturated nature, providing AO together with n-3 PUFAs may improve their beneficial impact on health by reducing their oxidation [START_REF] Svennevig | Antioxidants in combination with omega-3 fatty acids protect rat neonatal cardiomyocytes against H2O2-induced oxidative stress[END_REF][START_REF] Van Meeteren | Antioxidants and polyunsaturated fatty acids in multiple sclerosis[END_REF]. For example, dietary administration of n-3 PUFA-rich rapeseed oil fortified with naturally constituting AO (i.e. coenzyme Q10, tocopherol and the phenolic compound canolol) improves obesity-related cardiovascular dysfunction [START_REF] Leger | Dietary canolol protects the heart against the deleterious effects induced by the association of rapeseed oil, vitamin E and coenzyme Q10 in the context of a high-fat diet[END_REF] and alterations of glucose metabolism in rats [START_REF] Capel | Rapeseed oil fortified with micronutrients can reduce glucose intolerance during a high fat challenge in rats[END_REF]. Combined n-3 PUFAs and AO may similarly alleviate MetS-related neuropsychiatric comorbidities, since they are able to modulate the activity of several neurobiological systems that likely participate to their development [START_REF] Larrieu | Food for Mood: Relevance of Nutritional Omega-3 Fatty Acids for Depression and Anxiety[END_REF].

Over the last decades, compelling evidence revealed a key role for dysregulation of peripheral and brain inflammatory processes and resulting alterations of brain function in impairing mood, learning and memory (Capuron and Castanon, 2017;[START_REF] Capuron | Immune system to brain signaling: neuropsychopharmacological implications[END_REF][START_REF] Dantzer | From inflammation to sickness and depression: when the immune system subjugates the brain[END_REF]. In animal models of obesity/MetS, increased production of inflammatory cytokines was reported in brain areas important for memory formation and mood, such as the hippocampus [START_REF] Castanon | Role of neuroinflammation in the emotional and cognitive alterations displayed by animal models of obesity[END_REF][START_REF] Kanoski | Western diet consumption and cognitive impairment: links to hippocampal dysfunction and obesity[END_REF][START_REF] Boitard | Impairment of hippocampal-dependent memory induced by juvenile high-fat diet intake is associated with enhanced hippocampal inflammation in rats[END_REF][START_REF] De Cossio | Impact of prebiotics on metabolic and behavioral alterations in a mouse model of metabolic syndrome[END_REF][START_REF] Dinel | Cognitive and emotional alterations are related to hippocampal inflammation in a mouse model of metabolic syndrome[END_REF][START_REF] Dinel | Lipopolysaccharide-induced brain activation of the indoleamine 2,3-dioxygenase and depressive-like behavior are impaired in a mouse model of metabolic syndrome[END_REF]. This was associated with emotional alterations, memory deficits [START_REF] Dinel | Cognitive and emotional alterations are related to hippocampal inflammation in a mouse model of metabolic syndrome[END_REF][START_REF] Dinel | Lipopolysaccharide-induced brain activation of the indoleamine 2,3-dioxygenase and depressive-like behavior are impaired in a mouse model of metabolic syndrome[END_REF] and synaptic alterations [START_REF] Dinel | Cognitive and emotional alterations are related to hippocampal inflammation in a mouse model of metabolic syndrome[END_REF][START_REF] Dinel | Lipopolysaccharide-induced brain activation of the indoleamine 2,3-dioxygenase and depressive-like behavior are impaired in a mouse model of metabolic syndrome[END_REF][START_REF] Erion | Obesity elicits interleukin 1-mediated deficits in hippocampal synaptic plasticity[END_REF]. Interestingly, n-3 PUFAs and AO can display both immunomodulatory and neuromodulatory properties (Bazinet and Laye, 2014;[START_REF] Fourrier | Docosahexaenoic acid-containing choline phospholipid modulates LPS-induced neuroinflammation in vivo and in microglia in vitro[END_REF][START_REF] Miquel | Poor cognitive ageing: Vulnerabilities, mechanisms and the impact of nutritional interventions[END_REF][START_REF] Rey | Dietary n-3 long chain PUFA supplementation promotes a pro-resolving oxylipin profile in the brain[END_REF][START_REF] Rey | Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro[END_REF][START_REF] Vauzour | Neuroinflammatory processes in cognitive disorders: Is there a role for flavonoids and n-3 polyunsaturated fatty acids in counteracting their detrimental effects?[END_REF]. In the context of MetS, a dietary supplementation with EPA, DHA or their precursor, α-linolenic acid (ALA, 18:3n-3), improved systemic inflammatory alterations both in patients and animal models of the disease [START_REF] Karlsen | Bilberry juice modulates plasma concentration of NF-kappaB related inflammatory markers in subjects at increased risk of CVD[END_REF][START_REF] Kolehmainen | Bilberries reduce low-grade inflammation in individuals with features of metabolic syndrome[END_REF][START_REF] Wang | N-3 Polyunsaturated Fatty Acids and Inflammation in Obesity: Local Effect and Systemic Benefit[END_REF]. Moreover, depressive symptoms are negatively correlated with n-3 PUFAs plasma levels in MetS patients [START_REF] Chalut-Carpentier | Involvement of dietary Fatty acids in multiple biological and psychological functions, in morbidly obese subjects[END_REF]. On the other hand, the AO curcumin reduces anxiety in patients with MetS [START_REF] Esmaily | An investigation of the effects of curcumin on anxiety and depression in obese individuals: A randomized controlled trial[END_REF], and dietary supplementation with polyphenols improves spatial memory, neuroinflammation and neuronal plasticity in murine models of obesity/MetS [START_REF] Liu | Luteolin protects against high fat diet-induced cognitive deficits in obesity mice[END_REF][START_REF] Infante-Garcia | Mango leaf extract improves central pathology and cognitive impairment in a type 2 diabetes mouse model[END_REF]. However, the mechanisms underlying the beneficial effects of n-3 PUFAs and AO on MetS-related neuropsychiatric symptoms remain poorly understood. In addition, it is unknown whether their association could increase their respective efficiency.

In this study, we addressed these questions by assessing for the first time if a supplementation with both ALA and a mix of AO (coenzyme Q10, tocopherol and canolol), that may reduce n-3 PUFAs oxidation and engage multiple molecular targets at once [START_REF] Fairbairn | Combining a high DHA multinutrient supplement with aerobic exercise: Protocol for a randomised controlled study assessing mobility and cognitive function in older women[END_REF], improves emotional and/or cognitive alterations displayed by a murine model of MetS, the db/db mice. In order to identify the mechanisms underlying the expected behavioral improvement, we concomitantly measured the impact of this cosupplementation on the neurobiological systems known to be targeted by n-3 PUFAs and/or AO in other medical conditions with neuropsychiatric comorbidities. The db/db mice, which display classical features of MetS (including obesity, insulin resistance, dyslipidemia and dysregulated glucose homeostasis) because of a spontaneous mutation inactivating the leptin receptor, are notably suitable in the context of this study. Indeed, their metabolic dysfunctions have been associated with emotional and cognitive alterations, along with increased hippocampal inflammation [START_REF] Dinel | Cognitive and emotional alterations are related to hippocampal inflammation in a mouse model of metabolic syndrome[END_REF][START_REF] Dinel | Lipopolysaccharide-induced brain activation of the indoleamine 2,3-dioxygenase and depressive-like behavior are impaired in a mouse model of metabolic syndrome[END_REF][START_REF] Erion | Obesity elicits interleukin 1-mediated deficits in hippocampal synaptic plasticity[END_REF] and decreased local neuronal plasticity [START_REF] Erion | Obesity elicits interleukin 1-mediated deficits in hippocampal synaptic plasticity[END_REF][START_REF] Stranahan | Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons[END_REF]. This innovative experimental design, combining n-3 PUFAs/AO administration with a detailed assessment of MetS-related emotional, cognitive, inflammatory and neurobiological alterations, allowed us to show that this dietary supplementation restored spatial memory performances in db/db mice. Moreover, it provided interesting evidence pointing to the involvement of changes in neuronal plasticityassociated protein expression in the reported cognitive improvement.

Material and methods

Animals and Diets

All experiments were conducted according to the relevant French ( PUFAs/AO Diet) [START_REF] Capel | Rapeseed oil fortified with micronutrients can reduce glucose intolerance during a high fat challenge in rats[END_REF][START_REF] Gladine | Optimized rapeseed oil enriched with healthy micronutrients: a relevant nutritional approach to prevent cardiovascular diseases. Results of the Optim'Oils randomized intervention trial[END_REF][START_REF] Leger | Dietary canolol protects the heart against the deleterious effects induced by the association of rapeseed oil, vitamin E and coenzyme Q10 in the context of a high-fat diet[END_REF]. The palm/rapeseed diet was rich in the plant-derived n-3 PUFA α-linolenic acid (ALA), which decreases the n-6/n-3 ratio from 68 in the control diet to 2 in the n-3 PUFAs/AO diet (see Table 1 for detailed composition). Body weight, as well as food and water intake were measured once a week over a 12-week period. Mice were handled for a few minutes daily for 3 days before the experiment onset to minimize stress reactions to manipulation. They were 10 to 15 week-old at the time of behavioral assessments. Sacrifice occurred after 13 weeks of diet exposure (Figure 1).

Behavioral measures

Three complementary tests (light/dark box, elevated plus-maze, open-field) were used to measure anxiety-like behaviors as previously described [START_REF] De Cossio | Impact of prebiotics on metabolic and behavioral alterations in a mouse model of metabolic syndrome[END_REF]. Spatial learning and memory were then assessed in a Morris water maze [START_REF] Bensalem | Dietary Polyphenol Supplementation Prevents Alterations of Spatial Navigation in Middle-Aged Mice[END_REF].

Experiments were performed in the morning under conditions of dim light and low noise,

with at least a one-week interval between tests. All testing sessions were recorded with a Panlab SMART video tracking system (Bioseb, France). Testing equipment was thoroughly cleaned between each session.

Light/dark box (L/D box).

The L/D box test is based on the innate aversion of rodents for illuminated areas and on their spontaneous exploratory behavior in response to mild stressors, namely novel environment and light. The apparatus was composed of two plexiglass chambers: a first one small and dark (14 x 21 x 21 cm) and a second one large and illuminated (30 x 21 x 21 cm).

Both chambers were connected by a 7 x 5 cm opening. Each mouse was placed individually in the dark chamber and recordings were made over a 10-min period. A reduction of the time spent in the illuminated chamber was considered as an anxiety-like index.

Elevated plus-maze (EPM).

The EPM apparatus was made of a plus shaped acryl maze with two opposing open arms (30 x 8 x 15 cm) connected by a central platform (8 x 8 cm) and elevated 120 cm above the floor. Each mouse was placed individually in the center of the EPM over a 10-min period and the number of arm entries and the percent of time spent in open arms were recorded. A reduction of the percent of time spent in the open arms was considered as an anxiety-like index.

Open-field (OF).

The OF apparatus consisted of a square area (40 x 40 cm) from which escape is prevented by surrounding walls (16 cm high). The square area was virtually divided into a central area, considered as an anxiogenic area, and the periphery defined as the rest of the apparatus. Each mouse was placed randomly in one of the corners and allowed to freely explore the OF for 10 min. A reduction of the percent of time spent and number of entries in the central area was used as an anxiety-like index.

Morris water maze (MWM).

As previously described [START_REF] Bensalem | Dietary Polyphenol Supplementation Prevents Alterations of Spatial Navigation in Middle-Aged Mice[END_REF], mice were first submitted to two familiarization days to familiarize with swimming and to avoid jumping from the platform during training. Twenty-four hours after the familiarization phase, they were tested in a cued task to evaluate potential visuo-motor deficits. Seventy-two hours after the cued task, mice were then trained to locate the submerged platform by using distal extra-maze cues with six trials per day for 4 consecutive days. Latency and distance to find the platform were analyzed by Imetronics videotrack system (France). Spatial memory was evaluated 72 h after the last training session, after the platform was removed, by the percentage of time spent swimming in the quadrant were the platform was located during training (target quadrant) using the Panlab SMART system (Bioseb, France). Day 1 path lengths were not included in the analysis because too many mice did not reach the platform before the 90 s cut-off.

Tissue sampling

After 13 weeks of diet consumption, mice were anesthetized by isoflurane inhalation.

Blood samples were immediately collected through cardiac puncture into citrate-treated tubes. After centrifugation (10 min, 1000 g, 4°C), aliquots of plasma were stored at -80°C for further analysis. Mice were transcardially perfused with cold PBS (pH 7.4) to remove all traces of blood from tissues. Brains were rapidly extracted from the skull and the two hemispheres were separated to allow different neurobiological measures on the same mouse, and consequently minimize the number of animals used. The right hemisphere was immediately immersed into a 4 % paraformaldehyde fixative solution (prepared in PBS, pH 7.4) for 3 weeks and in a sucrose solution for 1 week before being sliced in 50 µm sections on a vibratome (Leica). Brain slices were then stored in a freezing solution at -20°C for further immunochemistry staining. The left hemisphere was rapidly dissected and the hippocampus and the cerebral cortex were removed and stored at -80°C for further analysis of protein expression and fatty acid composition analyses.

Cytokine and corticosterone assays

As previously described, plasma TNF-α, IL-6, IL-1β, IL-10 and IFN-γ were measured with the Mouse Cytokine/Chemokine magnetic bead panel multiplex assay (Millipore, France) following manufacturer's instructions [START_REF] Dinel | Cognitive and emotional alterations are related to hippocampal inflammation in a mouse model of metabolic syndrome[END_REF]. Total plasma corticosterone was measured with an in-house radioimmunoassay using a highly specific antibody provided by H. Vaudry (University of Rouen, France) as previously described [START_REF] Richard | Plasma transcortin influences endocrine and behavioral stress responses in mice[END_REF]. All samples were run in duplicate.

Brain fatty acid content

Brain fatty acid content was only measured in the cortex, but we recently showed that modulating dietary n-3 PUFAs levels induces similar changes in this structure than in the hippocampus [START_REF] Joffre | Modulation of brain PUFA content in different experimental models of mice[END_REF]. Total lipids from the cortex were extracted according to Folch's method [START_REF] Folch | A simple method for the isolation and purification of total lipides from animal tissues[END_REF] and fatty acids transmethylated according to the method of [START_REF] Morrison | Preparation of Fatty Acid Methyl Esters and Dimethylacetals from Lipids with Boron Fluoride--Methanol[END_REF]. Briefly, fatty acid methyl esters were analyzed on a FOCUS GC gas chromatograph (Thermo Electron Corporation), separated using a BPX70-fused silica capillary column (SGE, Courtaboeuf, France) and identified by making a comparison with commercial standards as previously described [START_REF] Fourrier | Docosahexaenoic acid-containing choline phospholipid modulates LPS-induced neuroinflammation in vivo and in microglia in vitro[END_REF][START_REF] Lafourcade | Nutritional omega-3 deficiency abolishes endocannabinoid-mediated neuronal functions[END_REF]. Fatty acid composition was expressed as the percentage of total fatty acids.

RNA and protein extraction

Total RNA was extracted from the hippocampus using Trizol (Invitrogen, Life Technologies). The below phase contained DNA and proteins. After DNA removal, proteins were purified in denaturing conditions.

RT-qPCR

RNA purity and concentration were determined using a Nanodrop spectrophotometer (Nanodrop, Life Technologies) and reverse-transcribed as previously described [START_REF] De Cossio | Impact of prebiotics on metabolic and behavioral alterations in a mouse model of metabolic syndrome[END_REF]. Real-time qPCR was performed using Taqman gene expression assays for sequence-specific primers purchased from Applied Biosytems (Foster City, CA) as previously described [START_REF] De Cossio | Impact of prebiotics on metabolic and behavioral alterations in a mouse model of metabolic syndrome[END_REF][START_REF] Dinel | Cognitive and emotional alterations are related to hippocampal inflammation in a mouse model of metabolic syndrome[END_REF][START_REF] Dinel | Lipopolysaccharide-induced brain activation of the indoleamine 2,3-dioxygenase and depressive-like behavior are impaired in a mouse model of metabolic syndrome[END_REF]. Fluorescence was determined on an ABI PRISM 7500-sequence detection system (Applied Biosystems, Foster, CA). The calculation of relative expression levels was performed according to the methods of [START_REF] Schmittgen | Analyzing real-time PCR data by the comparative C(T) method[END_REF] and plotted as fold change relative to the appropriate control condition.

Western Blot analysis

Protein concentration was assessed by bicinchoninic acid assay (Interchim, Montluçon, France) according to the manufacturer's instructions. Equal amounts of protein (25μg) were loaded and separated on SDS-polyacrylamide gels (8% or 10% depending of protein molecular weight) and transferred onto polyvinyl difluoride membranes (Millipore).

Membranes were saturated with a blocking solution containing 5% non-fat dried milk and 0.05% Tween-20, and incubated overnight in a solution containing 5% BSA with the primary antibodies (for details, see Supplementary Table S1). After washing, membranes were incubated for 1 h with the appropriate peroxidase-conjugated secondary antibody (1:5000, Jackson Immuno Research laboratories, Westgrove, PA, USA). The blots were developed using Western Lighting Chemiluminescence Reagent Plus (PerkinElmer Life Science, Waltham, MA, USA). Chemiluminescence was captured by a ChemiDoc detection system and quantified by Image Lab software. Between each revelation, membranes were incubated for 15 min in Re-Blot Plus Strong Antibody Stripping Solution (Millipore) according to manufacturer's instructions in order to erase the previous antibody.

Immunohistochemistry

Adult hippocampal neurogenesis was assessed in the dentate gyrus (DG) of the hippocampus, a brain region critical for memory encoding which continuously generates new neurons throughout life. Neurogenesis was assessed by counting the number of immature neurons characterized by the marker doublecortin (DCX), a cytoplasmic protein transiently expressed in newborn immature neurons only [START_REF] Brown | Transient expression of doublecortin during adult neurogenesis[END_REF]. Brain sections were first incubated with an anti-DCX antibody (1:1000) (goat monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by a biotinylated donkey anti-goat secondary antibody

(1:200) (Jackson Immuno Research Laboratories Inc, West Grove, PA, USA), and avidinbiotin peroxidase complex (Vector, Burlingame, USA; 1:1000). The presence of peroxidase was then revealed using diaminobenzidine and the number of DCX-positive cells estimated as previously described [START_REF] De Cossio | Impact of prebiotics on metabolic and behavioral alterations in a mouse model of metabolic syndrome[END_REF]. The fraction of DCX positive neurons with large vertical dendrites corresponding to a higher level of differentiation was also counted [START_REF] Touyarot | A mid-life vitamin A supplementation prevents age-related spatial memory deficits and hippocampal neurogenesis alterations through CRABP-I[END_REF].

Data analysis

Results are presented as mean ± SEM and were analyzed using a two-way ANOVA with genotype (db/+ vs. db/db) and diet (control vs. n-3 PUFAs/AO) as between factors, followed by a Bonferroni post-hoc test when appropriate. Probe test comparisons of each group with chance level in the MWM have been performed with a one sample t-test. Spatial learning was analyzed using a 3-way ANOVA with repeated measures (genotype x diet x days) followed by a post-hoc Fisher PLSD test.

Results

Dietary n-3 PUFAs/AO supplementation increased brain n-3 PUFA levels

Because n-3 PUFAs provided through the diet influence brain n-3 and n-6 PUFA levels [START_REF] Wainwright | Dietary essential fatty acids and brain function: a developmental perspective on mechanisms[END_REF], which in turn modulate neurobiological function, brain fatty acid composition was assessed in the cortex after 13 weeks of exposure to control diet or n-3

PUFAs/AO (Table 2). Statistical analysis of the cortical n-6/n-3 PUFA ratio revealed main effects of genotype (F(20,1)=103.04; p<0.001) and diet (F(20,1)=441.22; p<0.001), as well as a significant interaction between both factors (F(20,1)=99.00; p<0.001). Specifically, this ratio was lower in control diet db/db mice than in their db/+ counterparts (t(10)=10.94; p<0.001).

Chronic Indeed, it impaired spatial memory performances in db/+ mice, which displayed normal memory performances when fed the control diet, since this group swam randomly (t(7)=1.42; p=0.20). Altogether, these data showed that in the present conditions n-3 PUFAs/AO improved spatial memory, but not anxiety-like behaviors in db/db mice. The next question was then what biological correlates might underlie these behavioral improvements.

Dietary n-3 PUFAs/AO supplementation reduced neither plasma corticosterone levels nor brain inflammation in db/db mice

Increased activity of the hypothalamo-pituitary adrenal (HPA) axis, as well as peripheral and brain inflammatory processes, especially in the hippocampus, have been shown to regulate learning and memory (Bazinet and Laye, 2014;Delpech et al., 2015b;[START_REF] Vauzour | Neuroinflammatory processes in cognitive disorders: Is there a role for flavonoids and n-3 polyunsaturated fatty acids in counteracting their detrimental effects?[END_REF], including in db/db mice [START_REF] Erion | Obesity elicits interleukin 1-mediated deficits in hippocampal synaptic plasticity[END_REF][START_REF] Stranahan | Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons[END_REF].

Moreover, these processes can be impacted by both n-3 PUFAs and AO (Bazinet and Laye, 2014;[START_REF] Vauzour | Neuroinflammatory processes in cognitive disorders: Is there a role for flavonoids and n-3 polyunsaturated fatty acids in counteracting their detrimental effects?[END_REF]. We therefore measured the effect of n-3 PUFAs/AO on HPA axis activity (as assessed through plasma corticosterone levels and hippocampal glucocorticoid receptor (GR) activation), plasma cytokine levels, and hippocampal expression of different inflammatory mediators. We focused on the hippocampus, as it is strongly involved in emotional regulation, learning and memory [START_REF] Bannerman | Double dissociation of function within the hippocampus: spatial memory and hyponeophagia[END_REF].

Moreover, the MWM learning and probe test protocols used here are hippocampus-dependent [START_REF] Schenk | Dissociation between components of spatial memory in rats after recovery from the effects of retrohippocampal lesions[END_REF][START_REF] Bensalem | Dietary Polyphenol Supplementation Prevents Alterations of Spatial Navigation in Middle-Aged Mice[END_REF]. Akin to previously published data (de [START_REF] De Cossio | Impact of prebiotics on metabolic and behavioral alterations in a mouse model of metabolic syndrome[END_REF][START_REF] Dinel | Cognitive and emotional alterations are related to hippocampal inflammation in a mouse model of metabolic syndrome[END_REF][START_REF] Dinel | Lipopolysaccharide-induced brain activation of the indoleamine 2,3-dioxygenase and depressive-like behavior are impaired in a mouse model of metabolic syndrome[END_REF], plasma corticosterone levels were higher in db/db than db/+ mice (genotype: F(1,45)=21.27; p<0.001) regardless of their diet (p>0.1; Fig. 3A). Consistent with these data, n-3 PUFAs/AO did not change hippocampal GR expression (genotype: F(1,38)=11.71; p<0.01; Fig. 3B), nor its local activation through its phosphorylation, which remained higher in db/db than db/+ mice (genotype: F(1,38)=21.17; p<0.001; Fig. 3C) regardless of their diet. Memory performances were therefore improved in supplemented db/db mice despite persistent overactivation of their HPA axis.

Plasma cytokine concentrations were found to be below the limit of detection in all groups (data not shown). As previously described, hippocampal mRNA expression of the inflammatory cytokine IL-6 (genotype: F(1,43)=20.39; p<0.001, Fig. 4A) and TNF-α To study further the potential impact of the n-3 PUFAs/AO supplementation on hippocampal inflammation, we then assessed the degree of activation of different intracellular signaling pathways known to be induced by cytokines (Fig. 5, Supplementary Table S2). In particular, we measured the activation of the STAT3 pathway, which is activated by both IL- [START_REF] De Cossio | Impact of prebiotics on metabolic and behavioral alterations in a mouse model of metabolic syndrome[END_REF][START_REF] Erion | Obesity elicits interleukin 1-mediated deficits in hippocampal synaptic plasticity[END_REF][START_REF] Stranahan | Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons[END_REF]. In agreement with these findings, db/db mice also displayed reduced neurogenesis compared to db/+ mice in the present study (Fig. 6A), as revealed by reduced number of total DCX-positive cells (genotype: F(1,40)=25.52; p<0.001, Fig. 6B). Moreover, among all the DCX-positive cells, the proportion of ramified cells was lower in db/db mice than in db/+ mice (genotype:

(
F(1,40)=7.947; p<0.01; Fig. 6C), suggesting that less cells are involved in a process of maturation. However, both the total number of DCX-positive cells and the proportion of these cells with ramifications were unaffected by n-3 PUFAs/AO, suggesting no effect of the supplementation on formation and maturation of newborn neurons.

Hippocampal insulin resistance has been suggested to be an important mediator of impaired synaptic plasticity and cognitive deficits in MetS [START_REF] Biessels | Hippocampal insulin resistance and cognitive dysfunction[END_REF].

Moreover, n-3 PUFAs have been shown to modulate the activation of insulin signaling pathway in the hippocampus [START_REF] Sun | Fish oil modulates glycogen synthase kinase-3 signaling pathway in diabetes-induced hippocampal neurons apoptosis[END_REF]. We therefore evaluated whether the cognitive p<0.001), whatever the diet (data not shown). This suggests that the n-3 PUFAs/AO diet did not improve insulin resistance in the hippocampus of db/db mice.

In addition to hippocampal neurogenesis and brain insulin resistance, other processes modulating local synaptic plasticity, such as LTP, have also been shown to be impaired in db/db mice [START_REF] Erion | Obesity elicits interleukin 1-mediated deficits in hippocampal synaptic plasticity[END_REF][START_REF] Wosiski-Kuhn | Glucocorticoid receptor activation impairs hippocampal plasticity by suppressing BDNF expression in obese mice[END_REF]. They may therefore be targeted by n-3 PUFAs/AO to improve spatial memory. In the hippocampus, glutamatergic NMDA and AMPA receptors are respectively constituted of the GluN1, GluN2A and GluN2B subunits for NMDA receptors and GluR1 and GluR2 for AMPA receptors. Changing the subunit composition of these receptors has been shown to modulate synaptic function, especially LTP [START_REF] Keller | Development of a Computational Approach/Model to Explore NMDA Receptors Functions[END_REF]. In addition, n-3 PUFAs and AO are known to modulate NMDA and AMPA subunit composition [START_REF] Calon | Dietary n-3 polyunsaturated fatty acid depletion activates caspases and decreases NMDA receptors in the brain of a transgenic mouse model of Alzheimer's disease[END_REF][START_REF] Schroeter | Epicatechin stimulates ERK-dependent cyclic AMP response element activity and up-regulates GluR2 in cortical neurons[END_REF]. We therefore measured if such a modulation may similarly participate to n-3 PUFAs/AO-induced cognitive improvement in db/db mice. GluN1 protein expression was similar in all groups, whatever the genotype and the diet (Fig. 7, supplementary Table S2).

Discussion

In the present study, we attempted to measure the impact of combined n-3 PUFAs/AO supplementation on both emotional and cognitive alterations in a model of MetS.

Interestingly, we show that this supplementation differentially affected anxiety-like behaviors and memory performances, since it selectively reversed spatial memory deficits displayed by db/db mice, but had no effect on anxiety-like behaviors. Moreover, by testing the potential implication of different neurobiological processes, chosen as candidates because they were known to modulate memory, to be targeted by n-3 PUFAs and/or AO in other conditions associated with memory deficits, and to be impaired in db/db mice, we provide evidence that this memory improvement likely involved modulation of neuronal plasticity, through alterations of glutamatergic receptor composition in the hippocampus.

Our results confirm previous findings reporting cognitive alterations in db/db mice [START_REF] Dinel | Cognitive and emotional alterations are related to hippocampal inflammation in a mouse model of metabolic syndrome[END_REF][START_REF] Dinel | Lipopolysaccharide-induced brain activation of the indoleamine 2,3-dioxygenase and depressive-like behavior are impaired in a mouse model of metabolic syndrome[END_REF][START_REF] Erion | Obesity elicits interleukin 1-mediated deficits in hippocampal synaptic plasticity[END_REF], including in the MWM task [START_REF] Li | Impairment of longterm potentiation and spatial memory in leptin receptor-deficient rodents[END_REF][START_REF] Stranahan | Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons[END_REF]. Of note, some of these studies show deficits in both the learning and retention phases [START_REF] Li | Impairment of longterm potentiation and spatial memory in leptin receptor-deficient rodents[END_REF][START_REF] Stranahan | Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons[END_REF], whereas db/db mice displayed normal learning in the present study. This discrepancy can likely be linked to differences in the experimental protocols respectively used in the different studies. Unlike what was done here, no habituation to the test conditions (i.e. water immersion and swimming) was performed before spatial learning in most studies [START_REF] Stranahan | Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons[END_REF][START_REF] Stranahan | Voluntary exercise and caloric restriction enhance hippocampal dendritic spine density and BDNF levels in diabetic mice[END_REF][START_REF] Zhao | Diabetes-induced central cholinergic neuronal loss and cognitive deficit are attenuated by tacrine and a Chinese herbal prescription, kangen-karyu: elucidation in type 2 diabetes db/db mice[END_REF]. Testing conditions in the MWM are however highly stressful and likely interfere with spatial learning performances. Supporting this assumption, these performances were shown to be improved when mice are handled or submitted to habituation [START_REF] Holscher | Stress impairs performance in spatial water maze learning tasks[END_REF]. Moreover, some of these studies only reported swimming time to reach the platform [START_REF] Chen | Antidiabetic drugs restore abnormal transport of amyloid-beta across the blood-brain barrier and memory impairment in db/db mice[END_REF], whereas path length is more appropriate since swimming speed is decreased in db/db mice [START_REF] Zhao | Chotosan ameliorates cognitive and emotional deficits in an animal model of type 2 diabetes: possible involvement of cholinergic and VEGF/PDGF mechanisms in the brain[END_REF]. An important finding of this study is that n-3

PUFAs/AO supplementation restored spatial memory performances in db/db mice. This is consistent with previous studies showing beneficial impact of n-3 PUFAs or AO supplementation on cognitive deficits associated with obesity [START_REF] Infante-Garcia | Mango leaf extract improves central pathology and cognitive impairment in a type 2 diabetes mouse model[END_REF][START_REF] Liu | Luteolin protects against high fat diet-induced cognitive deficits in obesity mice[END_REF] and other conditions, including aging or Alzheimer's disease [START_REF] Bazinet | Polyunsaturated fatty acids and their metabolites in brain function and disease[END_REF][START_REF] Vauzour | Neuroinflammatory processes in cognitive disorders: Is there a role for flavonoids and n-3 polyunsaturated fatty acids in counteracting their detrimental effects?[END_REF]. In agreement with previously published data [START_REF] Dinel | Cognitive and emotional alterations are related to hippocampal inflammation in a mouse model of metabolic syndrome[END_REF][START_REF] Sharma | Neurobehavioral deficits in db/db diabetic mice[END_REF][START_REF] Zhao | Chotosan ameliorates cognitive and emotional deficits in an animal model of type 2 diabetes: possible involvement of cholinergic and VEGF/PDGF mechanisms in the brain[END_REF], db/db mice also displayed here increased anxiety-like behaviors, as assessed in several complementary and well-validated behavioral tests that model different core symptoms of anxiety [START_REF] Cryan | Animal models of mood disorders: Recent developments[END_REF]. Despite this broad behavioral characterization, anxiety-like behaviors remained higher in db/db mice than in db/+ mice, regardless of their diet. It could be argued that an anxiolytic effect could have been seen after longer exposure to diet supplementation. It is noteworthy however that these findings fit with those reporting a temporal dissociation between the onset of spatial memory alterations and anxiety-like behavior in diet-induced obese models that suggests the involvement of different underlying mechanisms [START_REF] André | Diet-induced obesity progressively alters cognition, anxiety-like behavior and lipopolysaccharide-induced depressive-like behavior: focus on brain indoleamine 2,3-dioxygenase activation[END_REF]. Similarly, our results

suggest that MetS-associated anxiety-like behaviors and memory deficits likely rely on independent mechanisms that are differentially impacted by dietary n-3 PUFAs/AO supplementation.

The hippocampus represents a key area for the control of anxiety and memory [START_REF] Bannerman | Double dissociation of function within the hippocampus: spatial memory and hyponeophagia[END_REF]. Moreover, hippocampal inflammation has been shown to participate in the development of emotional alterations (Capuron and Castanon, 2017;Capuron et al., 2017). In particular, enhancing or blocking brain TNF-α in inflammatory conditions change anxiety-like behavior [START_REF] Camara | Effects of centrally administered etanercept on behavior, microglia, and astrocytes in mice following a peripheral immune challenge[END_REF][START_REF] Klaus | Differential effects of peripheral and brain tumor necrosis factor on inflammation, sickness, emotional behavior and memory in mice[END_REF]. Akin to these findings, we recently showed that increased hippocampal TNF-α mediates the development of these behavioral alterations in db/db mice [START_REF] Fourrier | Brain tumor necrosis factor-alpha mediates anxiety-like behavior in a mouse model of severe obesity[END_REF]. In the present study, we confirmed the association between increased anxiety-like behavior and hippocampal inflammation, in particular increased expression of TNF-α and activation of its intracellular signaling pathways. The lack of impact of the dietary n-3 PUFAs/AO supplementation on hippocampal TNF-α in db/db mice may therefore explain why their anxiety-like behavior remained elevated. Conversely, this suggests that hippocampal TNF-α is unlikely involved in impaired memory performances displayed by db/db mice, although it is known, as other cytokines, to modulate local synaptic plasticity [START_REF] Beattie | Control of synaptic strength by glial TNFalpha[END_REF][START_REF] Prieto | Cytokines and cytokine networks target neurons to modulate long-term potentiation[END_REF][START_REF] Stellwagen | Synaptic scaling mediated by glial TNF-alpha[END_REF]. These findings also support the assumption that 13 weeks of supplementation are sufficient to improve spatial memory deficits in db/db mice, but not to reduce anxiety-like behaviors, even if they were only assessed after 5 to 8 weeks of supplementation. Hippocampal IL-1β has also been shown to play an important role in hippocampal-dependent memory processes [START_REF] Goshen | A dual role for interleukin-1 in hippocampal-dependent memory processes[END_REF] and to contribute to spatial memory deficits in db/db mice [START_REF] Erion | Obesity elicits interleukin 1-mediated deficits in hippocampal synaptic plasticity[END_REF]. Since hippocampal IL-1β expression was normalized by n-3 PUFAs/AO in these mice, it may be assumed that the present dietary supplementation improved spatial memory by targeting hippocampal IL-1β. More studies are however necessary to test this assumption. Although increased IL-1β mRNA expression was already shown to be associated with increased IL-1β protein levels in both db/+ and db/db mice (Kumari et al., 2007), this needs to be confirmed in the present study. This confirmation is particularly relevant since changes in IL-1β mRNA expression were not accompanied by concomitant changes in the activation of the IL-1βassociated p38 and p44/42 signalling pathways. Nonetheless, these findings do not totally exclude a role for IL-1β and/or other inflammatory processes in n-3 PUFAs/AO-induced cognitive improvement in the context of obesity, as previously reported in other inflammatory conditions [START_REF] Joffre | n-3 LCPUFA improves cognition: the young, the old and the sick[END_REF][START_REF] Labrousse | Short-term long chain omega3 diet protects from neuroinflammatory processes and memory impairment in aged mice[END_REF][START_REF] Miquel | Poor cognitive ageing: Vulnerabilities, mechanisms and the impact of nutritional interventions[END_REF]. [START_REF] Smedt-Peyrusse | Docosahexaenoic acid prevents lipopolysaccharide-induced cytokine production in microglial cells by inhibiting lipopolysaccharide receptor presentation but not its membrane subdomain localization[END_REF][START_REF] Layé | Anti-Inflammatory Effects of Omega-3 Fatty Acids in the Brain: Physiological Mechanisms and Relevance to Pharmacology[END_REF]. Moreover, n-3 PUFAs/AO may act on other brain areas than the hippocampus. Supporting this assumption, similar behavioral improvement by long-term dietary supplementation with polyphenols has been associated with reduced microglia activation in the hippocampus and the cortex of db/db mice [START_REF] Infante-Garcia | Mango leaf extract improves central pathology and cognitive impairment in a type 2 diabetes mouse model[END_REF]. However, it is worth mentioning that we reported here no reduced expression of the different inflammatory mediators assessed either in the hippocampus (Fig. 4) or prefrontal cortex (Supplementary Fig. S1). Moreover, hippocampal microglia overactivation in db/db mice has been related to their increased HPA axis activity [START_REF] Dey | Surveillance of adverse events following immunisation in Australia annual report[END_REF], that was not normalized by the n-3 PUFAs/AO supplementation. This suggests that glucocorticoids unlikely participate to memory improvement reported in supplemented db/db mice, and that their microglia likely remained activated. However, more experiments aiming to deeply study in db/db mice the impact of the n-3 PUFAs/AO supplementation on brain inflammatory processes, particularly microglial activation, are necessary to understand further the contribution of inflammation in the reported memory improvement.

Hippocampal neurogenesis and brain insulin resistance are also known to play an important role in spatial memory [START_REF] Biessels | Hippocampal insulin resistance and cognitive dysfunction[END_REF][START_REF] Boitard | Juvenile, but not adult exposure to high-fat diet impairs relational memory and hippocampal neurogenesis in mice[END_REF] and to be targeted by n-3 PUFAs and/or AO [START_REF] Biessels | Hippocampal insulin resistance and cognitive dysfunction[END_REF][START_REF] Shukitt-Hale | Blueberry polyphenols attenuate kainic acid-induced decrements in cognition and alter inflammatory gene expression in rat hippocampus[END_REF][START_REF] Sun | Fish oil modulates glycogen synthase kinase-3 signaling pathway in diabetes-induced hippocampal neurons apoptosis[END_REF]. Although we confirmed that the insulin pathway is impaired in the hippocampus of db/db mice [START_REF] Clodfelder-Miller | Physiological and pathological changes in glucose regulate brain Akt and glycogen synthase kinase-3[END_REF], we showed that the n-3 PUFAs/AO dietinduced cognitive improvement occurred despite any significant alleviation of brain insulin resistance. Extending previously published data that showed a reduction of hippocampal neurogenesis in db/db mice (de Cossio et al., 2017), we reported here that they specifically displayed a decrease in the number of ramified new-born neurons, meaning that their neurons were less involved in processes of maturation and network integration. It may be therefore assumed that n-3 PUFAs and/or AO may improve memory performances in db/db mice by reversing the impairment of their hippocampal neurogenesis. This is however unlikely since these impairments were not reversed in supplemented-db/db mice. On the contrary, other hippocampal neurobiological processes also known to modulate learning and memory, and

shown to be altered in db/db mice, were changed by the n-3 PUFAs/AO supplementation.

This particularly concerned synaptic plasticity. Indeed, we demonstrated for the first time that hippocampal levels of the AMPA subunit GluR2 and the NMDA subunit GluN2B were respectively decreased and increased in db/db mice in comparison to their db/+ controls. The subunit composition of AMPA and NDMA receptors is a key regulator of LTP [START_REF] Luscher | NMDA receptor-dependent long-term potentiation and long-term depression (LTP/LTD)[END_REF][START_REF] Makino | Enhanced synaptic plasticity in mice with phosphomimetic mutation of the GluA1 AMPA receptor[END_REF][START_REF] Paoletti | NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease[END_REF], which is impaired in db/db mice [START_REF] Erion | Obesity elicits interleukin 1-mediated deficits in hippocampal synaptic plasticity[END_REF][START_REF] Li | Impairment of longterm potentiation and spatial memory in leptin receptor-deficient rodents[END_REF][START_REF] Stranahan | Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons[END_REF] and represents one of the main neurobiological substrate of spatial memory [START_REF] Bannerman | Hippocampal synaptic plasticity, spatial memory and anxiety[END_REF][START_REF] Tsien | The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory[END_REF]. In particular, GluR2 expression is enhanced in the hippocampus following hippocampaldependent learning [START_REF] Whitlock | Learning induces long-term potentiation in the hippocampus[END_REF]. Interestingly, n-3 PUFAs/AO supplementation normalized the levels of GluR2 in db/db mice, and decreased GluN2A and GluN2B in the hippocampus of both db/+ and db/db mice. On the other hand, no detectable impact of this dietary supplementation was detected in the prefrontal cortex (Supplementary Fig. S2), pointing to the hippocampus as a prime target for these nutrients. Both n-3 PUFAs and AO supplementations have already been related to changes in NMDA and AMPA receptor composition in the hippocampus [START_REF] Calon | Dietary n-3 polyunsaturated fatty acid depletion activates caspases and decreases NMDA receptors in the brain of a transgenic mouse model of Alzheimer's disease[END_REF][START_REF] Schroeter | Epicatechin stimulates ERK-dependent cyclic AMP response element activity and up-regulates GluR2 in cortical neurons[END_REF], but the potential concomitant impact on cognition and emotional behaviors was not evaluated in these studies.

The modulation of hippocampal LTP, that likely occurs as a consequence of these changes in AMPA/NMDA receptor subunit composition, appears therefore as a particularly suitable candidate to explain the impact of n-3 PUFAs/AO supplementation on memory performances. More studies are needed to test this assumption, which is however already supported by previous experiments showing that changing n-3 PUFAs or AO dietary levels beneficially impacts LTP and cognition in other conditions (Delpech et al., 2015b;[START_REF] Wang | Tetrahydroxystilbene glucoside, a plant-derived cognitive enhancer, promotes hippocampal synaptic plasticity[END_REF]. A limitation of the present study is that it does not allow to decipher further the mechanisms by which n-3 PUFAs and AO act on those neurobiological processes. Moreover, the respective contribution of each nutrient to the reported behavioral improvement cannot be evaluated, since they were provided together. It is worth mentioning however that their combination has been reported to be actually beneficial [START_REF] Fairbairn | Combining a high DHA multinutrient supplement with aerobic exercise: Protocol for a randomised controlled study assessing mobility and cognitive function in older women[END_REF], suggesting that both n-3 PUFAs and AO are likely involved in the overall effect of the supplementation.

In conclusion, we showed here that a dietary supplementation combining n-3 PUFAs and AO improved spatial memory deficits associated with MetS. Moreover, our study provides a detailed description of the neurobiological impact of this supplementation and, by doing so, points to modulation of synaptic plasticity as a likely underlying mechanism of the reported behavioral improvement. It is worth mentioning however that, based on our results, such a dietary supplementation does not seem necessarily appropriate and/or recommended in healthy individuals. In our hands, n-3 PUFAs/AO supplementation indeed impaired memory in lean db/+ mice, suggesting potential interferences of these nutrients with neurobiological systems underlying normal learning and memory. Supporting this assumption, a few studies previously reported a pro-oxidant effect of AO, such as vitamin C and α-tocopherol, especially when the dose administered is higher than classical recommendations [START_REF] Carocho | A review on antioxidants, prooxidants and related controversy: natural and synthetic compounds, screening and analysis methodologies and future perspectives[END_REF][START_REF] Kondakci | Novel pro-oxidant activity assay for polyphenols, vitamins C and E using a modified CUPRAC method[END_REF]. Similarly, n-3 PUFA supplementation could make brain DHA more responsive to beta-oxidation [START_REF] Plourde | Kinetics of 13C-DHA before and during fish-oil supplementation in healthy older individuals[END_REF], which may contribute to the cognitive impairments observed in db/+ mice [START_REF] Hennebelle | Ageing and apoE change DHA homeostasis: relevance to age-related cognitive decline[END_REF]. It is therefore possible that a dietary supplementation with specific nutrients such as the ones used in the present study might be relevant only in subjects who display pre-existing neurobiological and cognitive dysfunctions prior to the supplementation onset. More studies are needed to address this issue, but the present work already highlights the necessity of considering the initial biological profile of people to be supplemented. Meanwhile, by assessing the impact of n-3

PUFAs and AO on a broad panel of neurobiological processes, this study helps understanding how these nutrients may act centrally to modulate behavior, although additional mechanisms not tested here can obviously also participate. Of note, the present findings could be particularly interesting to better understand the mechanisms underlying the positive effects of the Mediterranean diet on aging-related cognitive alterations, since n-3 PUFAs and AO are among the main components of this diet [START_REF] Capel | Rapeseed oil fortified with micronutrients can reduce glucose intolerance during a high fat challenge in rats[END_REF][START_REF] Feart | Mediterranean diet and cognitive health: an update of available knowledge[END_REF][START_REF] Lourida | Mediterranean diet, cognitive function, and dementia: a systematic review[END_REF][START_REF] Radd-Vagenas | Effect of the Mediterranean diet on cognition and brain morphology and function: a systematic review of randomized controlled trials[END_REF]. Altogether, this study points to a dietary combination of n-3 PUFAs and AO as a promising nutritional strategy to improve the management of neuropsychiatric symptoms, not only in patients with MetS and/or severe obesity, but also in other conditions associated with cognitive deficits. When interaction, *p<0.05)

5.

Fig. 2B )

 2B Fig. 2B), and in the illuminated chamber of the L/D box (genotype F(1,44)=47.68; p<0.001;

  improvement observed in db/db mice fed the n-3 PUFAs/AO diet was associated with alleviation of hippocampal insulin resistance by assessing local activation of the insulin signaling pathway. As anticipated, phosphorylation levels of IRS-1 (insulin receptor substrate-1), Akt and GSK3β were increased in db/db mice (genotype effect: P-IRS1/IRS: F(1,38)=23.31; p<0.001; P-Akt/Akt: F(1,40)=6.65; p<0.05; P-GSK3β/GSK3β: F(1,38)=23.61;

Indeed, n- 3

 3 PUFAs and AO may affect other signaling pathways and/or cytokines. They may also target post-transcriptional and/or post-translational mechanisms that modulate production and actions of brain cytokines. Alternatively, these nutrients may act through indirect mechanisms to reduce neuroinflammation. For example, changes in glial cell membrane fluidity due to the incorporation of n-3 PUFAs, particularly DHA, have previously been shown to impair the availability of inflammatory receptors for their ligands (De Smedt-

Figure 1 .

 1 Figure 1. Schematic view of the study protocol.

Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .

 234567 Figure 2. Effect of n-3 PUFAs/AO supplementation on anxiety-like behaviors and spatial

Dietary n-3 PUFAs/AO supplementation improved spatial memory deficits, but not anxiety-like behaviors in db/db mice

  consumption of n-3 PUFAs/AO decreased n-6/n-3 PUFA ratio in supplemented db/+ PUFAs levels in both db/+ and db/db mice. Of note, it did not change total body weight gain and food consumption, as well as fasted blood glucose levels that remained higher in db/db mice than db/+ mice, regardless of their diet (data not shown).We and others previously reported increased anxiety-like behavior and altered hippocampus-dependent spatial memory in db/db mice[START_REF] De Cossio | Impact of prebiotics on metabolic and behavioral alterations in a mouse model of metabolic syndrome[END_REF][START_REF] Dinel | Cognitive and emotional alterations are related to hippocampal inflammation in a mouse model of metabolic syndrome[END_REF][START_REF] Dinel | Lipopolysaccharide-induced brain activation of the indoleamine 2,3-dioxygenase and depressive-like behavior are impaired in a mouse model of metabolic syndrome[END_REF][START_REF] Erion | Obesity elicits interleukin 1-mediated deficits in hippocampal synaptic plasticity[END_REF][START_REF] Stranahan | Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons[END_REF]. Previous findings from our laboratory also demonstrated a beneficial effect of n-3 PUFAs or AO supplementation on mood[START_REF] Larrieu | Nutritional omega-3 modulates neuronal morphology in the prefrontal cortex along with depression-related behaviour through corticosterone secretion[END_REF][START_REF] Moranis | Long term adequate n-3 polyunsaturated fatty acid diet protects from depressive-like behavior but not from working memory disruption and brain cytokine expression in aged mice[END_REF] and cognitive alterations[START_REF] Bensalem | Dietary Polyphenol Supplementation Prevents Alterations of Spatial Navigation in Middle-Aged Mice[END_REF] Delpech et

	supplemented mice displayed reduced cortical levels of arachidonic acid (AA; 20:4 n-6), the second most prominent PUFA in the brain (diet: F(1,20)=47.69; p<0.001). Overall, n-3 PUFAs/AO consumption for 13 weeks was sufficient to increase brain n-3 PUFAs and decrease n-6 3.2

(t(10)=31.70; p<0.001) and db/db mice (t(10)=6.33; p<0.001) as compared to their respective controls. As expected, this was associated with an increase in n-3 PUFA levels (diet: F(20,1)=104.04; p<0.001) in both genotypes (controls vs. supplemented db/+ and db/db mice: t(10)=8.57; p<0.001 and t(10)=5.53; p<0.001 respectively), although this effect was stronger in db/+ mice (genotype x diet interaction: F(20,1)=19.08; p<0.001). Interestingly, both the genotype (F(1,20)=68.86; p<0.001) and n-3 PUFAs/AO (diet: F(1,20)=75.98; p<0.001; genotype x diet interaction: F(20,1)=16.56; p<0.001) notably impacted levels of DHA (22:6 n-3), the most prominent n-3 PUFA incorporated in the brain. Indeed, its levels were higher in control diet db/db mice than db/+ mice (t(10)=7.43; p<0.001), in lines with the hyperlipidemia and alterations of lipid metabolism already reported in the liver of db/db mice

[START_REF] Gotoh | Effects of three different highly purified n-3 series highly unsaturated fatty acids on lipid metabolism in C57BL/KsJ-db/db mice[END_REF]

, and significantly increased in both supplemented db/+ and db/db mice.

Conversely, al., 2015a;[START_REF] Labrousse | Short-term long chain omega3 diet protects from neuroinflammatory processes and memory impairment in aged mice[END_REF][START_REF] Létondor | EPA/DHA and Vitamin A Supplementation Improves Spatial Memory and Alleviates the Age-related Decrease in Hippocampal RXRγ and Kinase Expression in Rats[END_REF]

associated with aging, stress or an immune challenge. In line with these findings, we tested here if n-3 PUFAs/AO supplementation may similarly improve behavioral alterations displayed by db/db mice. Akin to our previous studies, db/db mice displayed increased anxiety-like behaviors in comparison to db/+ mice, since they visited less often (genotype F(1,43)=77.49; p<0.001; Fig. 2A, left) and spent proportionally less time (genotype F(1,43)=41.95; p<0.001; Fig. 2A, right) in the anxiogenic central area of the OF. Similarly, when compared to db/+ mice, all db/db mice spent proportionally less time in the open arms of the EPM (genotype F(1,42)=9.81; p<0.01;

4 Dietary n-3 PUFAs/AO supplementation modulated subunit composition of hippocampal AMPA and NMDA receptors, but did not restore neurogenesis in db/db mice Experimentally

	10 and IL-6, and MAPK pathways (including p44/42 and p38 pathways), which are rather
	targeted by IL-1β and TNF-α. In agreement with increased cytokine expression in the
	hippocampus, local levels of phosphorylated STAT3 were found to be higher in db/db mice
	than in db/+ mice (genotype: F(1,41)=18.52; p<0.001), regardless of the diet. Hippocampal
	levels of phosphorylated p44/42 (genotype: F(1,39)=4.23; p<0.05) and p38 proteins (genotype:

F(1,37)=22.38; p<0.001) were also higher in db/db than db/+ mice, whatever the diet. Hence, db/db mice displayed increased activation of these signaling pathways in the hippocampus, in agreement with local inflammation, but it was not reduced by n-3 PUFAs/AO.

3.changing n-3 PUFAs and/or AO intakes in rodents is known to modulate neurobiological processes underlying memory. These processes include neurogenesis

[START_REF] Shukitt-Hale | Blueberry polyphenols attenuate kainic acid-induced decrements in cognition and alter inflammatory gene expression in rat hippocampus[END_REF]

and synaptic plasticity

(Delpech et al., 2015a;[START_REF] Lafourcade | Nutritional omega-3 deficiency abolishes endocannabinoid-mediated neuronal functions[END_REF]

, which have both been shown to be impaired in db/db mice (de

Table 1 .

 1 legends Detailed nutritional content of the control and n-3 PUFAs/AO diets.

Table 2 .

 2 Effect of n-3 PUFAs/AO supplementation on brain fatty acid composition (n=6/group. *p<0.05; **p<0,01 ***p<0.001. When interaction, ### p<0.001 vs control diet same genotype; *p<0.05; ***p<0.001 vs db/+ same diet). SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; AO: antioxidants.

ACKNOWLEDGEMENTS

This work was performed, in partnership with the SAS PIVERT, within the frame of the French Institute for the Energy Transition (Institut pour la Transition Energétique, ITE) P.I.V.E.R.T. (www.institut-pivert.com) selected as an Investment for the Future ("Investissements d'Avenir"). This work was supported, as part of the Investments for the Future, by the French Government under the reference ANR-001-01. It was also financially supported by the Institut National de la Recherche Agronomique (INRA). C.F. was supported by a doctoral fellowship from the Région Aquitaine and the INRA (Département de Nutrition Humaine; 22000763). The Western blot analysis was done in the Biochemistry and Biophysics Platform of the Bordeaux Neurocampus at the Bordeaux University funded by the LABEX BRAIN. The authors thank M. Cadet, C. Tridon, S. Delbary and B. Pere for taking care of the animals.

DECLARATION OF INTEREST

Declaration of interest: none.


Sarah Atallah

Beatrice Berçot

Valérie Laurence

Caroline Hoffmann
email: caroline.hoffmann@curie.fr

ASSOCIATION OF MYCOPLASMA HOMINIS AND HEAD AND NECK CANCER WITH UNKNOWN PRIMARY

Introduction

Beside HPV infection, there is currently no evidence of association between head and neck squamous cell carcinomas and microbial infections. We report the case of a cervical squamous cell carcinoma by Mycoplasma hominis.

Case summary

A 20-years-old woman, consulted for a swelling on the left cervical side. Clinical examination found a large fixed mass. Biological tests found no evidence of infection. Biopsies of the cervical lesion diagnosed an HPV negative squamous cell carcinoma. Microbiological tests of 16sRNA identification showed the presence of Mycoplasma hominis in the 3 specimens. The patient was treated by induction chemotherapy associated to antibiotherapy, followed by chemo-radiotherapy.

Discussion

The present case suggests that oropharyngeal infection by Mycoplasma hominis might be more frequent than expected, that 16sRNA is an efficient technic to isolate this pathogen and finally that further studies are required to document its potential oncogenic role in head and neck squamous cell carcinomas.

Keywords:

Mycoplasm, infection, squamous cell carcinoma, cervical lymphadenopathy, 16S RNA

Introduction

Beside Human papilloma virus (HPV) infection implicated as an oncogenic factor in oropharyngeal head and neck squamous cell carcinomas (HNSCC) [START_REF] Gillison | Evidence for a causal association between human papillomavirus and a subset of head and neck cancers[END_REF], there is currently no evidence of association between HNSCC and microbial infections. To our knowledge, we report the first case of a cervical SCC node with unknown primary, which was HPV negative, but colonized by Mycoplasma hominis (Mh).

Case report

A 19-years-old woman, with no history of smoking or alcohol use, consulted for a swelling of the left side of her neck. Clinical examination found a large painless fixed mass and no fever. Biological tests found no evidence of infection. Computerized tomography scan (CT scan) revealed a 10cm mass, with a hypodense core and a peripheral enhancement, invading the cervical muscles and surrounding the primary carotid artery. Ultrasound-guided biopsies (3 samples) of the unresectable cervical lesion diagnosed an HPV negative (p16 and Polymerase Chain Reaction (PCR)) squamous cell carcinoma. Because of the young age of the patient, an infection was still suspected and microbial tests were performed on the same biopsies. The 16S ribosomal RNA (16s rRNA) microbiological testing showed the presence of Mh in all the specimens: PCR product's sequence showed 100% homology with the gene coding for Mh 16S rRNA (GenBank accession number AF443616.3, BLAST search). The serologies performed, HIV, B and C hepatitis were negative. PCR technique associated to mass spectrometry (ESI-MS) found no Mh in an oropharyngeal biopsy performed secondarily.

Cultures from genital tract samples were negative for Mh.

An MRI, a TEP-CT and a left tonsillectomy were performed, but were unable to identify the primary carcinoma (Fig. 1).

The patient was treated by induction chemotherapy (Docetaxel-Cisplatin-5FU) associated to fifteen days of antibiotherapy (josamycin) and followed by chemo-radiotherapy. After 13 months of follow-up, there is no evidence of recurrence (Fig. 1). The diagnosis of Fanconi anemia was also excluded by clinical examination, blood test and skin biopsy.

Discussion

Carcinoma of unknown primary involving cervical nodes in level II to IV are usually related to an oropharyngeal primary tumor [START_REF] Chernock | Approach to metastatic carcinoma of unknown primary in the head and neck: squamous cell carcinoma and beyond[END_REF]. When imaging is not able to identify the primary tumor, tonsillectomy is recommended in order to identify small tumors hidden inside the tonsil, together with HPV testing. In the present case involving a 19-year old patient, additionally to the histological analyses, microbial tests were also performed because the first hypothesis after clinical examination was an infection and not a cancer. These microbial tests identified Mh, which is a common commensal bacterium of the genitourinary tract. Sackel et al. reported that Mh, Ureaplasma urealyticum and Neisseria gonorrhoeae were found in 14.3%, 14.8% and 4,9% of the pharyngeal samples obtained from 1044 men and women attending clinics in Boston, USA, respectively [START_REF] Sackel | Orogenital contact and the isolation of Neisseria gonorrhoeae, Mycoplasma hominis, and Ureaplasma urealyticum from the pharynx[END_REF]. These results were obtained by cultures. Additionally, they showed that colonization alone was not necessarily associated with sore throat or other upper respiratory diseases [START_REF] Sackel | Orogenital contact and the isolation of Neisseria gonorrhoeae, Mycoplasma hominis, and Ureaplasma urealyticum from the pharynx[END_REF]. Beyond colonization of the oropharyngeal tract by this genital pathogen, two reports described an association with infection. One study showed that the rate of mycoplasma isolation was significantly higher in children undergoing tonsillectomy for recurrent adenotonsillitis (34.5%) than in the cases of tonsillectomy for sleep apnea (20%) [START_REF] Charnock | Recurrent tonsillitis. The role of Chlamydia and Mycoplasma[END_REF]. Mh infections, particularly extra-genital infections, are probably underdiagnosed for 3 main reasons: (i) the test is not performed, (ii) the culture technique is long and difficult, and (iii) 16sRNA is not systematically used.

The 16S rRNA technique is a recent, reliable and reproducible tool for bacteria identification [START_REF] Woo | Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories[END_REF], now routinely used for pathogen identification, particularly in cases of slow-growing, unusual, fastidious bacteria and for bacteria that are poorly differentiated by conventional methods. This technique consists of amplification and sequencing the RRS gene coding for 16S rRNA, a chromosomal gene of about 1500 base pairs, present in all bacterial species (universal gene). The 5 'and 3' ends are preserved in all bacteria and are used to link the two complementary universal primers that initiate the PCR reaction. The gene's sequence is specific to each species and allows their precise identification by sequencing the amplification product. For each sequence obtained, homologous sequences are searched in databases via the BLASTN programs of the National Center for Biotechnology Information (NCBI) [START_REF] Patel | 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory[END_REF]. Contamination is one limit of this technique. In the present case, this bias has been excluded by using a standard hybridation technique for Mh identification, and by the repeated biopsies confirming the colonization.

The role of mycoplasma infections in carcinogenesis remains a matter of debate.

Recent epidemiologic, genetic, and molecular studies suggest that mycoplasma infection and inflammation may initiate prostate cancer. Namiki and al. showed that Mycoplasma chronic infection (19 weeks of culture with benign human prostate cells) promoted malignant transformation resulting from a dysregulation of genes such as Ras, Myc or P53 [START_REF] Namiki | Persistent exposure to Mycoplasma induces malignant transformation of human prostate cells[END_REF]. Logunov and al., in a model of rat fibroblasts infected by Mycoplasma arininis, showed that the bacteria suppressed the transcriptional activity of p53 and activated the Nf-kB pathway, eventually inducing a Ras-dependent malignant transformation. This finding indicates that mycoplasma infection can act as an equivalent of p53-suppressing oncogene [START_REF] Logunov | Mycoplasma infection suppresses p53, activates NF-kappaB and cooperates with oncogenic Ras in rodent fibroblast transformation[END_REF] (Fig. 2).

Conclusion

This report describes an original case of association between Mh and cervical SCC in a young patient. It may suggest that, besides HPV infections, other genital pathogenic agent may be related to HNSCC. Further studies are required to document this association and the hypothesis of a potential oncogenic role.

Disclosure statement

Dr Caroline Hoffmann: consultant and honorary, Nanobiotix. No disclosure related to this publication.

Figure 1 -Figure 2 -

 12 Figure 1 -Cervical MRI before treatment (A) and after 13 months of follow-up (B)

Figure 1 -Figure 2 -

 12 Figure 1 -Cervical MRI before treatment (A) and after 13 months of follow-up (B)