Helen M Melo

Gisele Da

S Seixas Da Silva

Marcella Ramos Sant'ana

Camila Vieira

Ligo Teixeira

Julia R Clarke

Miya Coreixas

Bruno C De Melo

Juliana T S Fortuna

Vivian S Miya Coreixas

Leticia Forny-Germano

José Henrique Ledo

Maíra S Oliveira

Claudia P Figueiredo

Raphaelle Pardossi-Piquard

Frédéric Checler

José María Delgado-García

Agnès Gruart

Licio A Velloso

Marcio L F Balthazar

Dennys E Cintra

Sergio T Ferreira

Fernanda G De Felice

Palmitate is increased in the cerebrospinal fluid of obese humans and induces memory impairment in mice via pro-inflammatory TNF-α

Keywords: palmitate, memory impairment, obesity, brain inflammation, microglia, TNF-α

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Introduction

Worldwide obesity has nearly tripled in the past four decades. In 2016, more than 1.9 billion adults were overweight and over 650 million were obese (WHO, 2018). Overweight and obesity states are defined by abnormal or excessive fat accumulation that may impair health (WHO, 2018), and are frequently associated with metabolic and cardiovascular disorders. Diabetes, dyslipidemia and hypertension often overlap in overweight and obese humans [START_REF] Chan | Obesity, fat distribution, and weight gain as risk factors for clinical diabetes in men[END_REF][START_REF] Guh | The incidence of co-morbidities related to obesity and overweight: a systematic review and meta-analysis[END_REF][START_REF] Hu | Diet, lifestyle, and the risk of type 2 diabetes mellitus in women[END_REF][START_REF] Janssen | Body mass index, waist circumference, and health risk: evidence in support of current National Institutes of Health guidelines[END_REF][START_REF] Mokdad | Prevalence of obesity, diabetes, and obesity-related health risk factors[END_REF][START_REF] Must | The disease burden associated with overweight and obesity[END_REF][START_REF] Wilson | Overweight and obesity as determinants of cardiovascular risk: the Framingham experience[END_REF]. Obesity and/or increased fat intake are linked to cognitive decline and a higher risk of developing dementia [START_REF] Kalmijn | Dietary intake of fatty acids and fish in relation to cognitive performance at middle age[END_REF][START_REF] Kivipelto | Obesity and vascular risk factors at midlife and the risk of dementia and Alzheimer disease[END_REF].

Individuals become overweight and/or obese when there is an imbalance between calories consumed and calories expended, often when an increased intake of unbalanced diets high in fat is combined with decreased physical activity [START_REF] Spiegelman | Obesity and the regulation of energy balance[END_REF]. In obesity-related insulin resistance and type 2 diabetes (T2D), consumption of high-fat meals results in excess serum levels of saturated fatty acids (SFAs) [START_REF] Meek | Insulin regulation of regional free fatty acid metabolism[END_REF][START_REF] Roust | Postprandial free fatty acid kinetics are abnormal in upper body obesity[END_REF] which impair metabolism and cause peripheral insulin resistance [START_REF] Fernández-Real | Insulin resistance, inflammation, and serum fatty acid composition[END_REF][START_REF] Mayer | Usual dietary fat intake and insulin concentrations in healthy women twins[END_REF][START_REF] Parker | Relationship of dietary saturated fatty acids and body habitus to serum insulin concentrations: the Normative Aging Study[END_REF][START_REF] Vessby | Substituting dietary saturated for monounsaturated fat impairs insulin sensitivity in healthy men and women: The KANWU Study[END_REF]. Palmitate is the most abundant SFA present in the circulation [START_REF] Quehenberger | Lipidomics reveals a remarkable diversity of lipids in human plasma1[END_REF], and also in the cerebrospinal fluid (CSF) [START_REF] Pilitsis | Quantification of free fatty acids in human cerebrospinal fluid[END_REF]. Increased brain uptake and accumulation of palmitate was reported in obese patients with metabolic syndrome [START_REF] Karmi | Increased brain fatty acid uptake in metabolic syndrome[END_REF]. In mice, SFAs, including palmitate, have been shown to affect the hypothalamus, a key regulator of peripheral metabolic homeostasis [START_REF] Milanski | Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF][START_REF] Valdearcos | Microglia Dictate the Impact of Saturated Fat Consumption on Hypothalamic Inflammation and Neuronal Function[END_REF]. Palmitate has been related to insulin resistance, endoplasmic reticulum stress and increased pro-inflammatory response in the hypothalamus of mice [START_REF] Benoit | Palmitic acid mediates hypothalamic insulin resistance by altering PKC-theta subcellular localization in rodents[END_REF][START_REF] Cheng | Palmitic acid induces central leptin resistance and impairs hepatic glucose and lipid metabolism in male mice[END_REF][START_REF] Marwarha | Palmitateinduced Endoplasmic Reticulum stress and subsequent C/EBPα Homologous Protein activation attenuates leptin and Insulin-like growth factor 1 expression in the brain[END_REF]. However, it remains unclear whether palmitate is increased in the central nervous system of overweight or obese humans, and if it interferes with mechanisms of synaptic plasticity and memory.

We hypothesized that palmitate levels in the central nervous system could be altered in obesity and that palmitate could affect brain regions involved in learning and memory. We found that palmitate levels were increased in the CSF of overweight and obese humans with amnestic mild cognitive impairment (aMCI). Elevated CSF palmitate levels were associated with decreased cognitive performance in overweight individuals with diabetes, dyslipidemia and/or hypertension. When infused into the lateral brain ventricle of mice, palmitate impaired synaptic plasticity and memory. Palmitate reduced insulin expression and induced phosphorylation of neuronal insulin receptor substrate 1 (IRS-1) at multiple inhibitory serine residues in primary hippocampal cultures. Palmitate further activated astrocytes and microglia in vitro and in the hippocampi of mice, and caused an increase in hippocampal TNF-α levels. Minocycline, a tetracycline antibiotic commonly used to inhibit microglial activation, protected against palmitate-induced cognitive impairment. Palmitate failed to cause cognitive impairment in mice treated with infliximab, a monoclonal TNF-aneutralizing antibody, and in TNF-a receptor knock-out (TNFR1 -/-) mice, indicating that TNF-a signaling underlies the deleterious impact of palmitate on memory. Results establish a mechanism leading to memory deficits triggered by palmitate and provide a rational basis for the increased risk of cognitive decline in obese individuals.

Results

Palmitate is increased in the cerebrospinal fluid (CSF) of overweight and obese humans

Because it has not been demonstrated whether levels of palmitate in the CNS are altered in overweight or obese humans, we initially measured palmitate levels in the CSF from normal weight, overweight and obese individuals with aMCI [START_REF] Teixeira | Relation between aerobic fitness and brain structures in amnestic mild cognitive impairment elderly[END_REF][START_REF] Teixeira | Cognitive and structural cerebral changes in amnestic mild cognitive impairment due to Alzheimer's disease after multicomponent training[END_REF]. We found that palmitate levels were increased in the CSF of overweight and obese humans (Figure 1A; normal weight, N = 14; overweight, N = 26; obese, N = 9; ****p < 0.0001, one-way ANOVA followed by Holm-Sidak's post hoc test). CSF palmitate levels further showed positive correlations with body mass index (BMI) and with abdominal circumference in individuals with aMCI (Figure 1B, C; N = 49 subjects; **** p < 0.0001, linear regression). This correlation was observed in men and women (Figure S1A-D; N = 16 males/33 females; *p < 0.05 (male); ****p < 0.0001 (female), linear regression). We next stratified this cohort into patients affected or not by diabetes, dyslipidemia and/or hypertension, conditions that usually overlap in overweight and obese patients. Only 2 out of 14 normal weight subjects had diabetes, dyslipidemia or hypertension. On the other hand, 6 out of 9 obese subjects had diabetes, dyslipidemia and/or hypertension. Among overweight humans we found a more equilibrate proportion of individuals with (N = 15) and without (N = 11) diabetes, dyslipidemia and/or hypertension (Table S1). We found that overweight individuals with diabetes, dyslipidemia or hypertension exhibited a trend of increase in CSF palmitate compared to overweight individuals not affected by these co-morbid conditions (Figure 1D; individuals with diabetes, dyslipidemia and/or hypertension, N = 15; individuals without diabetes, dyslipidemia and/or hypertension, N = 11; p = 0.095, unpaired t-test).

CSF palmitate correlated inversely with cognitive performance in overweight individuals. We initially found a trend of decrease in mini-mental state examination (MMSE) scores for overweight subjects with diabetes, dyslipidemia and/or hypertension compared to individuals without these conditions (Figure S1E; patients with diabetes, dyslipidemia and/or hypertension, N = 15; patients without diabetes, dyslipidemia and/or hypertension, N = 11; p = 0.099, unpaired t-test). We next observed that CSF palmitate correlated inversely and significantly with MMSE score in overweight subjects affected by diabetes, dyslipidemia and/or hypertension (Figure 1E; N = 14; *p < 0.05, linear regression), but not in overweight subjects without these conditions (Figure 1F; N = 11, no statistical significance, linear regression). Overall, these results indicate that obesity is associated with increased CSF palmitate levels in humans. Results further show that CSF palmitate is elevated in overweight individuals with diabetes, dyslipidemia and/or hypertension and is associated with decreased cognitive performance.

Palmitate inhibits synaptic plasticity and impairs memory in mice

Numerous studies in humans and animal models indicate that obesity and a diet rich in saturated fatty acids are linked to deficits in learning and memory [START_REF] Arnold | High fat diet produces brain insulin resistance, synaptodendritic abnormalities and altered behavior in mice[END_REF][START_REF] Knight | Highfat diet-induced memory impairment in triple-transgenic Alzheimer's disease (3xTgAD) mice is independent of changes in amyloid and tau pathology[END_REF][START_REF] Whitmer | Central obesity and increased risk of dementia more than three decades later[END_REF] No statistically significant differences were observed in average weights between vehicle-and palmitate-infused mice (Figure S2F, N = 6 vehicle-infused/N= 5 palmitateinfused mice). In addition, control measurements of total distance travelled (Figure S2G) and average speed (Figure S2H) during the Barnes maze task training phase, as well as the number of crossings in an open field arena ten days after i.c.v. infusion (Figure S2I), showed that palmitate did not affect locomotor activity of mice. Furthermore, ten days after infusion, no differences in the total amount of time spent exploring the two objects in the NOR test were detected between vehicle-and palmitate-infused mice, indicating no impact on motivation or exploratory behavior (Figure S2J). Altogether, assessment of memory employing various tests indicated that i.c.v. palmitate induced memory impairment in mice.

We next investigated whether palmitate altered synaptic proteins in mice.

Immunoreactivity of the pre-synaptic marker protein, synaptophysin, was reduced in the hippocampus of mice ten days after i.c.v. infusion of palmitate (Figure S3A; vehicle (N = 9), palmitate (N =9), *p < 0.05, unpaired t-test). No differences were observed for the post-synaptic marker protein, PSD-95 10 days after palmitate infusion (Figure S3B; vehicle (N = 5, palmitate (N = 5), no statistically significant difference).

Palmitate inhibits neuronal IRS-1 signaling via microglial activation in vitro

Both in vitro and in vivo studies have indicated that insulin regulates neuronal survival, acts as a growth factor, and regulates circuit function and plasticity [START_REF] Chiu | Insulin receptor signaling regulates synapse number, dendritic plasticity, and circuit function in vivo[END_REF][START_REF] Lee | Insulin promotes dendritic spine and synapse formation by the PI3K/Akt/mTOR and Rac1 signaling pathways[END_REF][START_REF] Zhao | Permissive role of insulin in the expression of long-term potentiation in the hippocampus of immature rats[END_REF]. These findings prompted us to analyze whether palmitate would impact neuronal insulin signaling in primary hippocampal cultures (containing ~ 50 % neurons, 50 % non-neuronal cells, largely astrocytes and microglia). Exposure to palmitate (200 µM for 24 hours) triggered a marked reduction in insulin expression (Figure 3A; N = 3 independent cultures; **p < 0.005, paired t-test) in the absence of changes in cell viability for up to 48 hours (Figure S4A, B; N = 5 independent cultures; *p < 0.05, one-way ANOVA followed by Dunnett's post-hoc test). Previous studies have used similar concentrations of palmitate to induce insulin resistance in hypothalamic cells [START_REF] Mayer | Palmitate attenuates insulin signaling and induces endoplasmic reticulum stress and apoptosis in hypothalamic neurons: rescue of resistance and apoptosis through adenosine 5' monophosphate-activated protein kinase activation[END_REF] and increased cytokine levels in purified astrocyte culture [START_REF] Liu | Palmitate-activated astrocytes via serine palmitoyltransferase increase BACE1 in primary neurons by sphingomyelinases[END_REF].

Hippocampal neurons exposed to palmitate further showed decreased IRS-1 phosphorylation at tyrosine residue 612 (Figure 3B-C; N = 4 independent cultures; *p < 0.05, unpaired t-test) and increased phosphorylation at inhibitory serine residues 636 (IRS-1pSer636), 307 (IRS-1pS307) and 312 (IRS-1pS312) (Figure 3D-I; IRS-1pSer636, N = 4 independent cultures; IRS-1pSer307, N = 3 independent cultures; IRS-1pSer312, N = 3 independent cultures; *p < 0.05, unpaired t-test). Interestingly, no differences were observed in IRS-1pSer636 in hippocampal neurons exposed to oleate (Figure S4 C-D; N = 3 independent cultures).

Palmitate induces hippocampal inflammation

Landmark studies have shown that inflammation is a key mechanism underlying impaired peripheral insulin signaling in obesity and T2D [START_REF] Hotamisligil | Tumor Necrosis Factor : A Key Component of the Obesity-Diabetes Link[END_REF][START_REF] Hotamisligil | Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance[END_REF][START_REF] Hotamisligil | IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alphaand obesity-induced insulin resistance[END_REF]. Specifically, hypothalamic inflammation has been associated with obesity [START_REF] Milanski | Saturated fatty acids produce an inflammatory response predominantly through the activation of TLR4 signaling in hypothalamus: implications for the pathogenesis of obesity[END_REF][START_REF] Thaler | Obesity is associated with hypothalamic injury in rodents and humans[END_REF]. Thus, we next investigated if palmitate could cause hippocampal inflammation. We found that exposure of hippocampal cultures to palmitate (200 µM for 4 hours) induced an increase in GFAP immunoreactivity (Figure 4A-B; N = 3 independent cultures; **p = 0.008, unpaired t-test). Similarly, palmitate caused hippocampal astrogliosis in mice 10 days after i.c.v infusion (Figure 4 C-D; N= 6 per group, *p < 0.05, unpaired t-test).

Using purified microglial cultures, we found that exposure to palmitate (200 µM for 2 hours) induced microglial activation, as indicated by increased Iba-1 immunoreactivity and average cell area (Figure 4 E-G; N = 3 independent cultures, **p < 0.005, *p < 0.05, unpaired t-test). Similarly, palmitate increased the number of Iba-1 positive cells in the hippocampi of palmitate-infused mice (Figure 4 H-I; N = 5 vehicle-infused, N = 6 palmitate-infused mice; ** p = 0.0057, unpaired t-test). Control experiments showed that exposure to oleate had no effect on Iba-1 immunoreactivity or average cell area in microglial cultures and that microglial activation obtained by palmitate treatment was similar to that induced by LPS (Figure S5A-C; N = 3 independent cultures, *p < 0.05, one-way ANOVA followed by Holm-Sidak's post hoc test).

Microglia is implicated in palmitate-induced memory impairment

Microglial activation induced by palmitate was fully blocked by the tetracycline antibiotic, minocycline (Figure 5 A-C; N = 3 independent cultures, *p < 0.05, two-way ANOVA followed by Holm-Sidak's post hoc test). To determine whether palmitate-induced activation of a microglial inflammatory response could lead to increased IRS-1 phosphorylation in neurons, we exposed hippocampal cultures to conditioned medium from purified microglial cultures that had been previously exposed to palmitate (200 µM for 2 hours). Microglial conditioned medium (MCM) induced an increase in neuronal IRS-1pSer636 (Figure 5D,E; N = 3 independent cultures; *p < 0.05, unpaired t-test), and this increase was fully prevented by minocycline in hippocampal neurons exposed to palmitate (Figure 5F, G; N = 3 independent cultures, *p < 0.05; **p < 0.005; two-way ANOVA followed by Holm-Sidak's post hoc test).

Palmitate failed to cause memory impairment in mice treated with minocycline (50 mg/kg/d, IP, for 3 consecutive days ending on the day of vehicle or palmitate i.c.v. infusion) (Figure 5H; vehicle (N = 10), palmitate (N = 7), vehicle plus minocycline (N = 9), palmitate plus minocycline (N = 8), *p < 0.05; **p =0.005; ***p < 0.001 (Two-way ANOVA followed by Holm-Sidak's post hoc test comparing exploration times in the novel object). Moreover, while palmitate increased the number of Iba-1 positive cells in the hippocampi of control mice, no increase was observed when mice that received palmitate were treated with minocycline (Figure 5I-J; vehicle (N = 9), palmitate (N = 11), vehicle plus minocycline (N = 4), palmitate plus minocycline (N = 4), ** p < 0.005, two-way ANOVA followed by Dunnett's post hoc test).

Pro-inflammatory TNF-a mediates palmitate-induced cognitive impairment in mice

Palmitate has been shown to increase the expression of TNF-α in adipocyte and muscle cells [START_REF] Ajuwon | Palmitate Activates the NF-κB Transcription Factor and Induces IL-6 and TNFα Expression in 3T3-L1 Adipocytes[END_REF][START_REF] Jové | Palmitate induces tumor necrosis factor-alpha expression in C2C12 skeletal muscle cells by a mechanism involving protein kinase C and nuclear factor-kappaB activation[END_REF]. We found that exposure of hippocampal cultures to palmitate (200 µM for 4 hours) induced an increase in TNF-a in the medium (Figure 6A; N = 8 independent cultures, significance of pairing: correlation coefficient (r) = 0.94, ***p < 0.0001; **p = 0.006, paired t-test).

We further found that palmitate induced an increase in TNF-α mRNA and protein levels in the hippocampus that could be detected 24 hours after infusion in mice (Figure 6B, C; mRNA: vehicle (N = 11), palmitate (N = 8); protein: vehicle (N = 10), palmitate (N = 14); *p < 0.05, unpaired t-test). TNF-α mRNA (but not protein levels) remained elevated 10 days after infusion of palmitate (Fig. 6D,E; mRNA: vehicle (N = 6), palmitate (N = 4); *p < 0.05 (unpaired t-test); protein: vehicle (N = 9), palmitate (N = 10), no statistically significant difference, unpaired t-test).

Neuronal IRS-1pSer636 induced by palmitate in hippocampal cultures was prevented by infliximab (1 µg/ml), a TNF-a neutralizing monoclonal antibody, suggesting that increased TNF-α secreted by microglia mediates the inhibition of neuronal IRS-1 signaling induced by palmitate (Figure 6 F-G; N = 3 independent cultures, **p < 0.005; two-way ANOVA followed by Holm-Sidak's post hoc test).

We next asked whether a strategy to attenuate abnormally increased TNF-a signaling could alleviate memory impairment induced by palmitate. To test this hypothesis, we carried out an i.c.v. injection of the infliximab (10 ng in 1 µl) 1 hour before the infusion of palmitate in mice. Infliximab completely prevented palmitate-induced NOR memory deficits both 24 hours and 10 days post-infusion, suggesting that TNF-α mediates palmitate-induced cognitive impairment (Figure 6H-I; 24 h: vehicle (N = 10), palmitate (N = 10), vehicle plus infliximab (N = 9), palmitate plus infliximab (N = 10), *p < 0.05, **p < 0.005, two-way ANOVA followed by Holm-Sidak's post hoc test comparing exploration times in the novel object); 10 days: vehicle (N = 9), palmitate (N = 9), vehicle plus infliximab (N = 10), palmitate plus infliximab (N = 10), **p < 0.005, two-way ANOVA followed by Holm-Sidak's post hoc test comparing exploration times in the novel object). Palmitate also did not cause microgliosis in mice treated with infliximab (Figure 6 J-K; vehicle (N = 10), palmitate (N = 12), vehicle plus infliximab (N = 5), palmitate plus infliximab (N = 6), **p < 0.005, two-way ANOVA followed by Dunnett's post hoc test).

We further examined whether infliximab could reverse memory impairment in mice fed a high fat diet (HFD; 35% fat content by weight). Mice fed an HFD for 12 weeks exhibited memory impairment in the NOR test (Figure 7A; normal diet (N = 9), HFD (N = 10), *p < 0.05, unpaired t-test comparing exploration times in the novel object). HFD-fed mice were then divided into two groups. One group continued to receive HFD for 4 additional weeks, while the other group received HFD for 4 additional weeks and were treated i.c.v. with infliximab (10 μg in 1 µl) for 3 consecutive days ending on the day of the NOR test.

While mice that were kept on a HFD up to 16 weeks continued to exhibit memory impairment in the NOR test, short-term treatment with infliximab reversed memory impairment induced by HFD (Figure 7 B; HFD (N = 4), HFD plus infliximab (N = 5), *p < 0.05, unpaired t-test comparing exploration times in the novel object).

TNFR1 deletion prevents memory impairment in mice

To further investigate the role of TNF-α in memory impairment induced by palmitate, we infused palmitate (9 nmol, i.c.v.) in TNFR1 -/-or wild type C57BL/6 mice. Significantly, mice lacking TNFR1 were protected from memory impairment induced by palmitate both 24 hours (Figure 7C) and 8 days (Figure 7D) after infusion (24h: vehicle-infused wild type (N = 9), palmitate-infused wild type (N = 9), vehicle-infused TNFR1 -/-(N = 9), palmitateinfused TNFR1 -/-(N = 10); 8 days: vehicle-infused wild type (N = 10), palmitate-infused wild type (N = 10), vehicle-infused TNFR1 -/-(N = 7), palmitate-infused TNFR1 -/-(N = 8); *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001, two-way ANOVA followed by Holm-Sidak's post hoc test).

Discussion

Obese and T2DM patients show structural brain alterations with reduced cerebral volume, including hippocampal and cortical atrophies [START_REF] Gustafson | Adiposity indices and dementia[END_REF][START_REF] Gustafson | A 24-year follow-up of body mass index and cerebral atrophy[END_REF][START_REF] Ho | Obesity is linked with lower brain volume in 700 AD and MCI patients[END_REF]. Moreover, T2D and obese patients, as well as animal models of diabetes, present cognitive impairment [START_REF] Elias | Lower cognitive function in the presence of obesity and hypertension: the Framingham heart study[END_REF][START_REF] Jeon | Resveratrol attenuates obesity-associated peripheral and central inflammation and improves memory deficit in mice fed a high-fat diet[END_REF][START_REF] Roberts | Association of type 2 diabetes with brain atrophy and cognitive impairment[END_REF][START_REF] Takeda | Diabetes-accelerated memory dysfunction via cerebrovascular inflammation and Abeta deposition in an Alzheimer mouse model with diabetes[END_REF], and T2D favors the development of dementia later in life [START_REF] Ott | Association of diabetes mellitus and dementia: the Rotterdam Study[END_REF].

Consumption of HFDs containing elevated amounts of SFAs induces obesity and T2D, and the association between obesity and dementia is increasingly rising concerns regarding the possible deleterious impact of unhealthy diets in the brain [START_REF] Kalmijn | Dietary intake of fatty acids and fish in relation to cognitive performance at middle age[END_REF][START_REF] Morris | Dietary fats and the risk of incident Alzheimer disease[END_REF]. In animal models of obesity and AD, administration of a HFD induces cognitive impairment and accelerates AD pathology [START_REF] Jeon | Resveratrol attenuates obesity-associated peripheral and central inflammation and improves memory deficit in mice fed a high-fat diet[END_REF][START_REF] Takeda | Diabetes-accelerated memory dysfunction via cerebrovascular inflammation and Abeta deposition in an Alzheimer mouse model with diabetes[END_REF].

Moreover, damage to blood-brain barrier permeability as well as increased palmitate levels in the brain have been observed in mice fed a HFD [START_REF] Spinelli | Brain insulin resistance impairs hippocampal synaptic plasticity and memory by increasing GluA1 palmitoylation through FoxO3a[END_REF][START_REF] Takechi | Blood-Brain Barrier Dysfunction Precedes Cognitive Decline and Neurodegeneration in Diabetic Insulin Resistant Mouse Model: An Implication for Causal Link[END_REF].

Our results established that increased body weight is associated with increased CSF palmitate in humans. Results further showed that CSF palmitate is elevated in overweight individuals with diabetes, dyslipidemia and/or hypertension and, importantly, is associated with decreased cognitive performance. These findings suggest that the brain may sense alterations that occur in obesity.

In peripheral tissues, inflammatory and metabolic stress signaling cascades trigger disruption of insulin signaling [START_REF] Hotamisligil | Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance[END_REF][START_REF] Hotamisligil | IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alphaand obesity-induced insulin resistance[END_REF] and palmitate induces insulin resistance in hypothalamus [START_REF] Mayer | Palmitate attenuates insulin signaling and induces endoplasmic reticulum stress and apoptosis in hypothalamic neurons: rescue of resistance and apoptosis through adenosine 5' monophosphate-activated protein kinase activation[END_REF], liver [START_REF] Nakamura | Palmitate induces insulin resistance in H4IIEC3 hepatocytes through reactive oxygen species produced by mitochondria[END_REF] and muscle [START_REF] Yuzefovych | Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress[END_REF] cells. Our findings in hippocampal cultures indicated that palmitate disrupted insulin expression and signaling, with decreased insulin mRNA levels and IRS-1pTyr612, and increased IRS-1pSer636, pSer307 and pSer312. In line with current findings, mice fed a HFD exhibited increased immunoreactivity for IRS-1pSer616 in the hippocampus and impaired spatial memory [START_REF] Arnold | High fat diet produces brain insulin resistance, synaptodendritic abnormalities and altered behavior in mice[END_REF]. Increased palmitate levels may thus be linked to brain inflammation and impaired insulin signaling in overweight humans with diabetes, dyslipidemia and/or hypertension, contributing to decreased cognitive performance.

Our analysis indicated that palmitate levels in the CSF ranged from 0.8-2.7 µg/ml in normal weight humans to 1.5-4.1 µg/ml and 3.8-5.9 µg/ml in overweight and obese individuals, respectively. An early study reported that healthy humans present ~ 0.64 µg palmitate/ml CSF, similar to the range we have found [START_REF] Pilitsis | Quantification of free fatty acids in human cerebrospinal fluid[END_REF]. More recently, lipid profiles were evaluated in brain and plasma of post-mortem T2D elderly subjects and reported ~ 20% increase in palmitate cholesteryl esters in brain homogenates from T2D in comparison to controls [START_REF] Ginneken | Characterization of the lipid profile post mortem for Type-2 diabetes in human brain and plasma of the elderly with LCMS-techniques: a descriptive approach of diabetic encephalopathy[END_REF]. However, this study did not include a quantitative analysis of palmitate levels. Additional studies aiming at investigating levels of palmitate in the CSF and/or brains in the context of obesity are anticipated in the field.

Palmitate did not induce cognitive impairment in mice that had been treated with either infliximab or minocycline, strategies to neutralize TNF-α or microglia, respectively. These results suggest that palmitate acts on microglial cells, which likely secret soluble factors, including TNF-α, that impair memory. Peripheral inflammation in obesity and T2D involves many components of the classical inflammatory response, including abnormal production of TNFα, IL-1β and IL-6, and activation of a network of inflammatory signaling pathways [START_REF] Gregor | Inflammatory mechanisms in obesity[END_REF][START_REF] Hotamisligil | Inflammation, metaflammation and immunometabolic disorders[END_REF]. Microglia are centrally involved in the immune response in the CNS. When activated, microglia release proinflammatory cytokines and become an antigen presenting cell [START_REF] Hanisch | Microglia: Active sensor and versatile effector cells in the normal and pathologic brain[END_REF]. TNF-α is increased in the serum of obese patients [START_REF] Hotamisligil | Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance[END_REF], mediates insulin resistance in obesity and T2D [START_REF] Hotamisligil | The role of TNFalpha and TNF receptors in obesity and insulin resistance[END_REF], and has been recently implicated in brain dysfunction in trauma and in different neurodegenerative disorders, including AD and Parkinson's disease [START_REF] Heneka | Innate immune activation in neurodegenerative disease[END_REF][START_REF] Mccoy | TNF signaling inhibition in the CNS: Implications for normal brain function and neurodegenerative disease[END_REF][START_REF] Wyss-Coray | Inflammation in neurodegenerative disease--a double-edged sword[END_REF]. Elevated TNF-α has further been linked to cognitive deficits [START_REF] Azevedo | Activated microglia mediate synapse loss and short-term memory deficits in a mouse model of tran[END_REF][START_REF] Lourenco | TNF-α mediates PKR-dependent memory impairment and brain IRS-1 inhibition induced by Alzheimer's βamyloid oligomers in mice and monkeys[END_REF]. In peripheral tissues and hypothalamic cells, is suggested that palmitate activates Toll-like receptors 2/4 leading to increased inflammatory response, insulin resistance and cellular stress [START_REF] Holland | Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acidinduced ceramide biosynthesis in mice[END_REF][START_REF] Shi | TLR4 links innate immunity and fatty acid -induced insulin resistance Find the latest version : TLR4 links innate immunity and fatty acid -induced insulin resistance[END_REF].

In conclusion, current findings indicate that palmitate levels are increased in the CSF of overweight and obese humans. In mice, palmitate induces hippocampal inflammation, and triggers increased TNF-α levels, inhibition of synaptic plasticity and memory impairment.

Collectively, results suggest a mechanism by which excess palmitate derived from unhealthy diets affects brain function and contributes to cognitive impairment in obese and T2D patients. Further, strategies aimed at decreasing brain inflammation in obesity and T2D may help to alleviate the impact of palmitate in the brain. A healthy lifestyle that prevents excessive weight gain may prevent the increase in SFAs in the circulation and in the brain, thus contributing to healthy brain aging. (B-I) Neuronal IRS-1pTyr612 (B-C), pSer636 (D-E), pSer307 (E-F) and pSer312 (G-H) immunoreactivities in cultured hippocampal neurons exposed to vehicle or 200 μM palmitate for 4 hours (10-30 images analyzed per experimental condition per experiment using independent neuronal cultures). Scale bar = 10 μm. Bars represent means ± standard errors and symbols represent independent cultures. *p < 0.05 (unpaired t-test). In representative IRS-1 PTyr612 images, for better viewing, the image color was changed to green using ZEN software. (D, E) Neuronal IRS-1pSer636 immunoreactivities in hippocampal cultures exposed for 4 hours to conditioned medium from purified microglial cultures treated with vehicle or 200 μM palmitate (10-30 images analyzed per experimental condition per experiment using independent hippocampal and microglial cultures). Scale bar = 10 μm. Bars represent means ± standard errors and symbols represent experiments with independent hippocampal and microglial cultures. *p < 0.05 (unpaired t-test).

(F, G) Neuronal IRS-1pSer636 immunoreactivities in hippocampal cultures exposed for 4 hours to vehicle or 200 μM palmitate in the absence or presence of 1 μM minocycline (10-30 images analyzed per experimental condition per experiment using independent cultures). Scale bar = 50 μm; 10 μm (inset). Bars represent means ± standard errors and symbols represent experiments from 3 independent cultures. *p < 0.05; **p < 0.005 (two-way ANOVA followed by Holm-Sidak's post hoc test).

(H) Novel object recognition (NOR) test in male 3-5-month-old mice infused i.c.v. with vehicle or palmitate (3 nmol) and treated or not with minocycline (50 mg/kg/d, ip, for 3 consecutive days ending on the day of vehicle or palmitate infusion). Testing was performed 10 days after infusions. Bars (means ± SEM) represent time spent exploring the familiar (F) or novel (N) objects, and symbols represent individual mice. *p < 0.05; **p =0.005; ***p < 0.001 (two-way ANOVA followed by Holm-Sidak's post hoc test).

(I, J) Representative images of Iba-1 immunolabeling (I) and number of Iba-1 positive cells (J) in the dentate gyrus of the hippocampus from 2-4 month-old mice that received i.c.v. infusions of vehicle or palmitate (3 nmol) and were treated or not with minocycline (50 mg/kg/d, IP, for 3 consecutive days ending on the day of vehicle or palmitate infusion). Animals were perfused 10 days after palmitate infusion. Scale bar = 50 μm; 10 μm (inset). (J) Bars represent means ± standard errors and symbols represent individual animals. **p < 0.005 (two-way ANOVA followed by Dunnett's post hoc test). Part of the vehicle and palmitate data was shown in Figure 4I. (F, G) Neuronal IRS-1pSer636 immunolabeling in hippocampal cultures exposed for 4 hours to vehicle or 200 μM palmitate in the absence or presence of 1 μg/ml infliximab (20-30 images analyzed per experimental condition per experiment using independent cultures). Scale bar = 10 μm. Bars represent means ± SEM and symbols represent independent cultures. **p < 0.005; *p < 0.05 (two-way ANOVA followed by Holm-Sidak's post hoc test).

(H, I) Novel object recognition (NOR) test in male 3-5-month-old mice infused i.c.v. with vehicle or palmitate (3 nmol) and treated or not with infliximab (100 ng, i.c.v., 30 min previously to the infusion of vehicle or palmitate). Testing was performed 24 hours (H) or 10 days (I) after infusions. Bars (means ± SEM) represent time spent exploring the familiar (F) or novel (N) objects, and symbols represent individual mice. *p < 0.05, **p =0.005 (twoway ANOVA followed by Holm-Sidak's post hoc test).

(J, K) Representative images of Iba-1 immunolabeling (J) and number of Iba-1 positive cells (K) in the dentate gyrus of the hippocampus from 2-4 month-old mice that received i.c.v. infusions of vehicle or palmitate (3 nmol) and were treated or not with infliximab (100 ng, i.c.v., 30 min previously to the infusion of vehicle or palmitate). Animals were perfused 10 days after palmitate infusion. Scale bar = 50 μm; 10 μm (inset). (K) Bars represent means ± standard errors and symbols represent individual animals. **p < 0.005 (two-way ANOVA followed by Dunnett's post hoc test). Part of the vehicle and palmitate data was shown in Figure 4I.

STAR Methods

CONTACT FOR REAGENT AND RESOURCE SHARING

This study did not generate new unique reagents. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Fernanda G. De Felice (felice@bioqmed.ufrj.br).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Human subjects: We studied 49 subjects diagnosed as aMCI recruited from the Neuropsychology and Dementia clinic, Hospital de Clínicas, University of Campinas invited to participate in the project in order to check their memory [START_REF] Magalhães | Systemic Inflammation and Multimodal Biomarkers in Amnestic Mild Cognitive Impairment and Alzheimer's Disease[END_REF][START_REF] Rabelo | Is cerebral microbleed prevalence relevant as a biomarker in amnestic mild cognitive impairment and mild Alzheimer's disease?[END_REF][START_REF] Teixeira | Relation between aerobic fitness and brain structures in amnestic mild cognitive impairment elderly[END_REF][START_REF] Teixeira | Cognitive and structural cerebral changes in amnestic mild cognitive impairment due to Alzheimer's disease after multicomponent training[END_REF][START_REF] Weiler | Intranetwork and internetwork connectivity in patients with Alzheimer disease and the association with cerebrospinal fluid biomarker levels[END_REF]. aMCI patients were diagnosed using the core criteria of the National Institute on Aging/Alzheimer's Association for MCI [START_REF] Albert | The diagnosis of mild cognitive impairment due to Alzheimer's disease: Recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease[END_REF][START_REF] Raj | A network diffusion model of disease progression in dementia[END_REF] and had a score of 0.5 (with an obligatory memory score of 0.5), estimated on the basis of a semi structured interview. Exclusion criteria for all participants were history of other neurologic/psychiatric diseases or head injury with loss of consciousness, drug or alcohol addiction, a Hachinski ischemic score 15 above 4 and a Fazekas Scale score16 above 1. The Medical Research Ethics Committee of the University of Campinas (UNICAMP) approved our study, and we obtained written informed consent from all participants (or from their responsible guardians if the participants were incapable of consenting) before study initiation. Pre-diagnostic procedures also comprised of laboratory tests including vitamin B12, folate, and thyroid hormones. CSF was obtained from all participants once, by lumbar puncture of the L3/L4 or L4/L5 intervertebral space, using a 25-gauge needle, and collected via a syringe in 12-mL polypropylene tubes (Sarstedt, Nümbrecht, Germany). CSF samples were centrifuged at 700 rpm for 10 minutes at 4ºC and were subsequently aliquoted into 1 ml microtubes (Eppendorf) and stored at -80ºC until the time of analysis.

BMI was calculated by dividing the person´s weight, in kilograms, by their height, in meters squared, or BMI = weight (in kg)/ height 2 (in m 2 ). BMI was categorized as: normal weight: 18.5-23.9 kg/m 2 ; overweight: 24.0-27.9 Kg/m 2 ; and obesity: ≥ 28 kg/m 2 . Abdominal circumference was measured to the nearest 0.1 cm by a non-elastic flexible tape. We used the method recommended by the World Health Organization (WHO), which consists measuring midway between the lowest rib margin and the iliac crest at the mid-axillary line World Health Organization (WHO Consultation on Obesity, 1999; Geneva, Switzerland) Abdominal obesity was defined as >102cm cm for men and > 88 cm for women [START_REF] Franklin | American association of clinical endocrinologists and American college of endocrinology comprehensive clinical practice guidelines for medical care of patients with obesity[END_REF].

Patients were divided into groups with or without diabetes, dyslipidemia and hypertension based on the use of medication for these diseases.

Intrahippocampal palmitate administration and placement of recording electrodes:

All experiments were conducted in accordance with the "Principles of Laboratory Animal Care" (NIH publication no. 85-23, revised 1996) and with the Guidelines of the European Union (2003/65/CE) for the use of laboratory animals in chronic experiments. Every effort was made to reduce the number of animals used and to minimize their suffering. C57BL/6 male mice (3-5 months-old; 25-35g) were obtained from an official supplier (University of Granada Animal House, Granada, Spain) and housed five per cage until experiments began. Mice were kept on a 12 h light/dark cycle with constant ambient temperature (21.5 ± 1.5 °C) and humidity (55 ± 8%). Food and water were available ad libitum. Mice were anesthetized with 4% chloral hydrate and, prior to electrode implantation, received a single intra-CA2 (1.8 mm lateral, 1.5 mm posterior do bregma and 1.1 mm from the brain surface; Franklin and Paxinos, 2004) infusion of palmitate (0.3 nmol) or vehicle in a final volume of 1 µL using a Hamilton syringe, which was kept in place for an additional 30 seconds after drug administration to avoid backflow. Immediately after infusion, bipolar stimulating electrodes were implanted in the CA3 hippocampal subregion of the dorsal hippocampus (2 mm lateral and 1.5 mm posterior to bregma, and 1-1.5 mm from the brain surface; Franklin and Paxinos, 2004) and a bipolar recording electrode was placed in the ipsilateral stratum radiatum underneath the CA1 area (1.2 mm lateral and 2.2 mm posterior to bregma and 1-1.5 mm from the brain surface; Franklin and Paxinos, 2004). Electrodes were made from 50-μm Teflon-coated tungsten wire (Advent Research Materials, UK). The final location of the recording electrodes in the CA1 area was determined after the field potential depth profile evoked by paired (10-to 500-ms interval) pulses presented to the Schaffer collateral. A bare silver wire was fixed to the skull as ground. The four wires were soldered to a four-pin socket (RS Amidata) that was then fixed to the skull using dental cement [START_REF] Gruart | Involvement of the CA3-CA1 synapse in the acquisition of associative learning in behaving mice[END_REF][START_REF] Madronal | Differential Effects of Long-Term Potentiation Evoked at the CA3 CA1 Synapse before, during, and after the Acquisition of Classical Eyeblink Conditioning in Behaving Mice[END_REF]. After surgery, animals were housed in individual cages until the end of experiment. Only data from animals with correct electrode placement were analyzed.

Intracerebroventricular infusion of palmitate or oleate in Swiss mice: Male Swiss mice were obtained from our animal facility (Federal University of Rio de Janeiro) or from Laboratory Animal Breeding Center (Fundação Oswaldo Cruz -CECAL/Fiocruz, Rio de Janeiro) and were 2-5 months-old at the beginning of experiments. Animals were housed in groups of five per cage with free access to food and water, under a 12 h light/dark cycle with controlled room temperature (21 ± 2 °C). The animals weighed approximately 30 -50 g. Mice were randomly assigned into one of two groups: vehicle or palmitate.

For intracerebroventricular (i.c.v.) infusion of vehicle or palmitate, animals were anesthetized and carefully restrained with 2.5% isoflurane (Cristalia) using a vaporizer system, and a 2.5-mm-long needle was unilaterally inserted 1 mm to the right of the midline point equidistant from each eye and 1 mm posterior to a line drawn through the anterior base of the eyes [START_REF] Clarke | Alzheimer-associated Aβ oligomers impact the central nervous system to induce peripheral metabolic deregulation[END_REF][START_REF] Figueiredo | Memantine rescues transient cognitive impairment caused by high-molecular-weight Aβ oligomers but not the persistent impairment induced by low-molecular-weight oligomers[END_REF][START_REF] Laursen | Intracerebroventricular injections in mice. Some methodological refinements[END_REF][START_REF] Lourenco | TNF-α mediates PKR-dependent memory impairment and brain IRS-1 inhibition induced by Alzheimer's βamyloid oligomers in mice and monkeys[END_REF]. Three nmol of palmitate or oleate (or an equivalent volume of vehicle) were infused in a final volume of 3 µl. When indicated (see Results and Figure legends), one hour before i.c.v. palmitate infusion mice received 10 ng infliximab (or an equivalent volume of phosphate buffered saline -PBS) in a final volume of 1 µl at the same injection site as the fatty acid. In these animals, 3 nmol palmitate (or an equivalent volume of vehicle) were infused in a final volume of 2 µl so that the final volume injected i.c.v. was 3 µl.

Regarding the LTP experiment, we increased the amount injected palmitate in the behavioral experiments to ensure its distribution to different brain regions following icv infusion.

Accurate placement of the needle into the right lateral ventricle was confirmed by macroscopic examination of dissected brains before biochemical analysis. Mice showing any signs of misplaced injections or brain hemorrhage were excluded from further analysis (~ 5% of animals throughout our study). All procedures were performed in the light phase of the daily cycle, followed the "Principles of Laboratory Animal Care" (US National Institutes of Health) and were approved by the Institutional Animal Care and Use Committee of the Federal University of Rio de Janeiro (protocols IBQM045 and IBQM133/15).

Intracerebroventricular infusion of palmitate in TNFR1

-/-and C57BL/6 mice: Male 3-4 months-old TNFRp55 -/-(TNFR1 KO) mice [START_REF] Arruda | Low-Grade Hypothalamic Inflammation Leads to Defective Thermogenesis, Insulin Resistance, and Impaired Insulin Secretion[END_REF][START_REF] Pfeffer | Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection[END_REF] were kindly donated by Dr. Licio A. Velloso from the University of Campinas, São Paulo, Brazil, and male 3-4 months-old C57BL/6 wild type mice from our animal facility (Federal University of Rio de Janeiro) were used as controls. The animals weighed approximately 25-35 g. All animals were housed in groups of five per cage with free access to food and water, under a 12 h light/dark cycle with controlled room temperature (21 ± 2 °C) and were randomly assigned into vehicle or palmitate groups. All procedures were performed in the light phase and followed the "Principles of Laboratory Animal Care" (US National Institutes of Health) and were approved by the Institutional Animal Care and Use Committee of the Federal University of Rio de Janeiro (protocol # IBQM133/15). TNFR1 -/-and controls were randomly assigned into one of two groups: vehicle or palmitate.

Intracerebroventricular infusion of vehicle or palmitate were performed as described above.

Nine nmol palmitate (or an equivalent volume of vehicle) were infused in a final volume of 1.5 µl. We increased the amount injected palmitate in the behavioral experiments to ensure its distribution to different brain regions following i.c.v. infusion.

High fat diet (HFD) administration: Male 3-3.5 months-old C57BL/6 wild type from our animal facility (Federal University of Rio de Janeiro) were randomly divided into two groups fed either standard rodent chow or HFD (35% fat content by weight -PragSoluções®; [START_REF] Razolli | TLR4 Expression in Bone Marrow-Derived Cells Is Both Necessary and Sufficient to Produce the Insulin Resistance Phenotype in Diet-Induced Obesity[END_REF] for 16 weeks. For NOR experiments, HFD mice were treated with infliximab (10 µg) or PBS for 3 days before memory testing, with the last injection in the day of NOR task. Animals were housed in groups of five per cage with free access to food and water, under a 12 h light/dark cycle with controlled room temperature (21 ± 2 °C). All procedures were performed in the light phase and followed the "Principles of Laboratory Animal Care" (US National Institutes of Health) and were approved by the Institutional Animal Care and Use Committee of the Federal University of Rio de Janeiro (protocol # IBQM133/15).

Mature hippocampal cultures:

Primary rat hippocampal cultures were prepared according to established procedures [START_REF] De Felice | Protection of synapses against Alzheimer's-linked toxins: insulin signaling prevents the pathogenic binding of Abeta oligomers[END_REF] and adapted from the original protocol described by Brewer [START_REF] Brewer | Isolation and culture of adult rat hippocampal neurons[END_REF]. Hippocampi from 18-day-old embryos were dissected and the meninges were removed. Tissue was dissociated in PBS 0.05% trypsin/EDTA (Invitrogen) solution at 37 °C for 5-10 minutes under occasional gentle shaking followed by mechanical dissociation with glass Pasteur pipettes in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) medium supplemented with 10% horse-serum (Invitrogen) and antibiotics (100 U/ml penicillin/streptomycin) (Invitrogen). Cells in suspension were counted in a Neubauer chamber and were plated on culture plaques or glass coverslips previously coated with 10 μg/ml or 1 mg/ml poly-L-lysine, respectively. The density of culture was 50.000 cells/well (260 cells/mm 2 ) on coverslips for immunocytochemistry experiments and 30.000 cells/well (645 cells/mm 2 ) for the cell viability experiments. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 for 60 minutes and DMEM was replaced by Neurobasal medium supplemented with 2% B27 supplement (Invitrogen), 0.5 mM Glutamax (Invitrogen), 100 U/ml penicillin/streptomycin (Invitrogen) and 10 μg/ml amphotericin-B (Invitrogen). Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2 for 18-21 days prior to use. Our cultures contain, on average, 50% neurons and 50% non-neuronal cells. The objective is to have a mixed culture to investigate the interplay between neuronal and non-neuronal cells. Sex of 18-day-old embryos could not be determined at this embryonic age and the embryos were not weighed.

Cultures were exposed at 37 °C to 50-500 µM palmitate for 4, 24 or 48 hours. When present, infliximab (1 μg/ml) or minocycline (1 μM) were added to cultures 30 minutes or 1 hour before application of vehicle or palmitate (200 μM), respectively.

Primary cultures and conditioned media from mouse microglia: Primary microglial cultures were prepared as described previously [START_REF] Azevedo | Activated microglia mediate synapse loss and short-term memory deficits in a mouse model of tran[END_REF][START_REF] Ledo | Amyloid-β oligomers link depressive-like behavior and cognitive deficits in mice[END_REF][START_REF] Ledo | Cross Talk Between Brain Innate Immunity and Serotonin Signaling Underlies Depressive-Like Behavior Induced by Alzheimer's Amyloid-β Oligomers in Mice[END_REF].

Cortices from neonatal Swiss mice were dissociated, and the resulting cells were plated on poly-L-lysine-coated 75-cm 2 flasks. The cells were maintained in DMEM with F12 medium (1:1 ratio), 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin for 2 weeks at 37 °C in a humidified atmosphere containing 5% CO2. The medium was changed after 1 week and before cell harvesting. After 14 days, microglia were harvested using an orbital shaker.

The cells were counted and plated in 24-well plates for assays. Microglial cells were exposed at 37 °C to 200 µM palmitate, 200 µM oleate or vehicle for 2 hours. E. coli lipopolysaccharide (LPS, 100 ng/ml) was used as a positive control for microglial activation. When present, minocycline (1 μM) were added to cultures 1 hour before application of vehicle or palmitate (200 μM), respectively.

For experiments using conditioned medium, cells were washed 2 times with warm DMEM with F12 medium and changed to Neurobasal medium overnight to obtain conditioned medium. The conditioned medium was then incubated with hippocampal cultures for 4 hours.

METHOD DETAILS

Palmitic acid measurements by GC-MS: For fatty acid profile determination, 170 µL cerebrospinal fluid were taken in a screw cap glass tube containing 25 µg internal standard (tridecanoic acid -C13:0). Thereafter, 1 mL of 0.5 M NaOH-methanol was added and the sample was boiled at 100 °C for 15 min. After cooling the samples, 2 mL of BF3-methanol was added and the sample was boiled at 100 °C for 20 s. The sample was again cooled to room temperature and 1 mL of isooctane was added. The tubes were shaken and 5 mL of saturated NaCl solution was added. After phase separation, the supernatant was collected and evaporated with the aid of nitrogen gas to concentrate the fatty acid methyl esters (FAME). FAME were resuspended in 50 µL hexane for chromatographic analysis [START_REF] Shirai | Direct methylation from mouse plasma and from liver and brain homogenates[END_REF].

Chromatographic analyses were performed using a gas chromatograph-mass spectrometer (model GCMS-QP2010 Ultra; Shimadzu). A fused silica capillary column Stabilwax (length, 30 m; internal diameter, 0.25 mm; thickness, 0.25 µm; Restek, USA) was used to inject 1 μL of the sample at 250 ºC. High-grade pure helium (He) was used as the carrier gas with a constant flow rate of 1.3 mL/min with a split injection of 2:1. The oven temperature was programmed from 80 to 175 °C at a rate of 5 °C/min, followed by another gradient of 3 °C/min to 230 °C, which was maintained for 10 min. Mass conditions were as follows: ionization voltage, 70 eV; ion source temperature, 200 ºC; full scan mode in the 35-500 mass range with 0.2 s/scan velocity.

Palmitate and oleate preparation:

The protocol for preparation of palmitate and oleate solutions was adapted from the protocol described by [START_REF] Mayer | Palmitate attenuates insulin signaling and induces endoplasmic reticulum stress and apoptosis in hypothalamic neurons: rescue of resistance and apoptosis through adenosine 5' monophosphate-activated protein kinase activation[END_REF]. Solutions for i.c.v. infusions in mice were prepared from a stock solution of 100 mM sodium palmitate (Sigma) or sodium oleate (Sigma) solubilized in methanol (Tedia).

Palmitate or oleate were solubilized in methanol under occasional shaking at 60 ° C (palmitate) or at room temperature (oleate). The stock solution was then diluted in Neurobasal culture medium (Gibco) containing 5% fatty acid free albumin (Sigma) for 1 hour at 37 ° C and shaking. After this period, the solutions were used for icv infusions. For Swiss mice, 2 or 3 μl of the final infused solution contained 3 nmol of fatty acids. For C57BL/6 and TNFR1 -/-mice, 1.5 μl of final infused solution contained 9 nmol of palmitate.

Vehicle was Neurobasal medium with 5% fatty acid free albumin plus the corresponding volume of methanol. The methanol in the final solution was a very low volume, with only one residual of the solvent remaining (0,015 -0,03%).

For primary hippocampal cultures, 5 mM palmitate solution in Neurobasal culture medium with 5% fatty acid free albumin was diluted to 50-500 µM in culture medium. The vehicle was Neurobasal with 5% fatty acid free albumin plus the corresponding volume of methanol.

The BSA solution was filtered through sterile 0.22 µm filter before palmitate or oleate dilution, and all solutions were performed sterile in a laminar flow hood. Furthermore, the pH of the solutions was measured after preparation, and was in the range of 7.2-7.4.

The amount of palmitate used in our studies in mice (3 or 9 nmol) corresponds to 22 or 66 µg/ml CSF, respectively (assuming uniform distribution in 35 µl of CSF). These concentrations are ~ 3.5-10-fold higher than we found in the CSF of obese subjects, and were chosen to allow investigation of the impact of palmitate in mice within a reasonable experimental timeframe.

Long term potentiation (LTP):

Recordings took place 10 days after electrode implantation in alert behaving mice. Field excitatory post-synaptic potential (fEPSP) baseline values were recorded using 100 µs, square, biphasic paired pulses for 15 min before LTP induction. For construction of I/O curves, stimulus intensities ranged from 0.02mA to 0.4mA and were elicited at a 40ms interstimulus interval. The stimulus intensity was set at ~35% of the intensity necessary for evoking a maximum fEPSP response (i.e., well bellow the threshold for evoking a population spike). For LTP induction, a HFS protocol consisting of five 200 Hz, 100 ms-long trains of pulses at a rate of one per second was used. This protocol was applied six times, at intervals of 1 min. After HFS presentation, recordings of double-pulse stimulation at Schaffer collaterals were conducted for another 20 min to evaluate whether the LTP protocol was effective. During the following three days, mice were submitted to 15 minlong daily recording sessions to assess the persistence of LTP. According to previous studies [START_REF] Gruart | Involvement of the CA3-CA1 synapse in the acquisition of associative learning in behaving mice[END_REF][START_REF] Madronal | Differential Effects of Long-Term Potentiation Evoked at the CA3 CA1 Synapse before, during, and after the Acquisition of Classical Eyeblink Conditioning in Behaving Mice[END_REF], this HFS was enough to evoke a saturating LTP response, lasting 3 days, without the appearance of abnormal spikes in EEG recordings and/or any noticeable epileptic seizure.

All data were stored digitally on a computer through an analog/digital converter (1401 Plus; CED), at a sampling frequency of 11-22 kHz and with an amplitude resolution of 12 bits.

Evoked fEPSPs amplitudes were analyzed with the help of commercial software (Spike 2 and GraphPad Prism 7) and data are expressed as average amplitude of fEPSP for every 2 min of recording. Statistical analysis for each analysis is described in the corresponding Step-down inhibitory avoidance test: The test was carried out in an aluminum and acrylic box measuring 200 mm x 75 mm, a floor consisting of a grid of steel bars with a spacing of 12.5 mm (Insight EP 104R) and an adapted platform of 2 cm of height placed in the center of the grid. In the training phase, each animal was gently placed on the platform. When the animal stepped down from the platform onto the grid with the 4 paws, they received a 0.7 mA footshock (stimulus) for 2 seconds. The animal was then withdrawn from the apparatus.

After 24h (test session), each animal was placed on the platform again. In this session, no shock was applied when the animal stepped-down on the grid. After stepping down, each animal was withdrawn from the apparatus and placed back in the home cage. The time in platform before stepping down on the grid (latency) was measured during the training and test sessions with a manual chronometer by a trained researcher. Step-down latencies were cut-off at 300 s in the training and test sessions, and animals that did not step down during 300 seconds were excluded from analysis. Results are represented as median and interquartile range of the latency to step down in the test session and the difference was considered statistically if p < 0.05 (Mann-Whitney test).

Barnes maze test: The Barnes maze protocol was adapted from [START_REF] Sunyer | Barnes maze , a useful task to assess spatial reference memory in the mice[END_REF]. The maze consists of a white circular platform (92 cm of diameter) elevated 105 cm above the ground with 20 equally spaced holes (5 cm diameter, 7.5 cm between holes) along the perimeter with a darkened escape box in a constant position under one of the holes at the edge of the platform. Mice are expected to learn the location of the escape hole using extramaze visual cues including geometric shapes (triangle, rectangle and circle) placed on the walls of the room.

To familiarize mice with the maze and the existence of the escape box, they were subjected to one habituation trial in the first day prior to the initial trial. This habituation consisted of placing the animal in a cylindrical chamber in the middle of the maze and after 10 seconds, the chamber was removed, and the mouse was gently guided to the escape hole, where it remained for 2 minutes.

In the next step, acquisition training was performed with four trials per day for four days. For each trial, mice were placed in the cylindrical chamber in the middle of the maze and, after 10 seconds, the chamber was removed, and mice were allowed to explore the maze for 3 minutes. The trial ended when the animal entered the escape hole or after 3 minutes. If a mouse did not enter the escape hole within 3 minutes, the animal was gently guided to it and kept inside the box for 1 minute. Mice were returned to their home cages until the next trial, with a 15-minute interval between trials. Time (primary latency) and number of errors (primary errors) to reach the escape hole for the first time were measured. Head deflections into incorrect holes of the maze were considered errors and were counted by the experimenter.

On day 5, mice underwent the probe trial to access reference memory (short-term memory retention) in which the escape hole was closed. Mice were placed in the cylindrical chamber in the middle of the maze. After 10 seconds, the chamber was removed, and mice were allowed to explore the maze for 90 seconds. The amount of time that animals spent in the escape hole quadrant was recorded. In all experimental steps, the maze was cleaned with 40% ethanol and air-dried before each trial and between mice. The platform was monitored using the ANY-maze software (Stoelting Co, IL, USA), which recorded the animal's position, total distance travelled and average speed. No differences in total distance travelled and average speed between vehicle or palmitate groups was observed. Importantly, there was no difference in average weights between vehicle-and palmitate-infused groups after i.c.v.

infusion.

Western blots: Twenty-four hours or 10 days after i.c.v. infusion of palmitate, mice were euthanized by decapitation and the hippocampus was rapidly dissected and frozen in liquid nitrogen. For protein extraction, samples were thawed and homogenized in RIPA buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40 (Invitrogen), 1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 1% Triton X-100) containing a phosphatase and protease inhibitor cocktail (Pierce-Thermo Scientific, Rockford, IL). Protein concentration was determined using the BCA kit. Aliquots containing 50 µg protein were prepared in sample buffer containing 125 mM Tris-Cl, 10 % SDS, 0.05 % Bromophenol blue, 5% β-mercaptoethanol and 50% glycerol and resolved by SDS-PAGE in 10% polyacrylamide gels at 125 V.

Proteins were electrotransferred to nitrocellulose for 1 h at 100 V. Membranes were blocked for 1 h with 3% bovine serum albumin in Tween-Tris buffer solution at room temperature or with Odyssey blocking buffer (Licor; Lincoln, NE; 1:2 dilution in Tween-Tris Buffer) and were incubated overnight at 2 °C with primary antibodies diluted in blocking buffer.

Molecular weight markers (Benchmark pre-stained protein ladder; Life Technologies) were run in every gel. Primary antibodies used were: Synaptophysin (Abcam, Cat# ab8049; 1:1,000), cyclophilin B (Abcam, Cat# ab16045; 1:1,000), PSD-95 (Santa Cruz, Cat# SC 28491; 1:1,000) and β-actin (Abcam, Cat# ab6276; 1:10,000). After overnight incubation with primary antibodies and washing with TBS-T, membranes were incubated with IRDye800CW-or IRDye680RD-conjugated secondary antibodies (Licor; 1:10,000) at room temperature for 1 h. Fluorescence intensities were detected in an Odyssey CLx apparatus (Licor). Scanned or digitized images were saved at high resolution (300 dpi) and bands were quantified by densitometry in Fiji software [START_REF] Schindelin | Fiji: An open-source platform for biological-image analysis[END_REF]. Quantitative immunofluorescence data was measured in 8-bit grayscale photomicrographies and carried out by analysis of the fluorescence intensity at each pixel across the images using Fiji software [START_REF] Schindelin | Fiji: An open-source platform for biological-image analysis[END_REF]. The program analyzes a grayscale image by plots of the intensity of fluorescence at each pixel. An appropriate thresholding was employed to eliminate background signal in the images before analysis. Thus, the entire microscopic field imaged was included in analysis and the Raw Integrated Density (RawIntDens) is normalized by the number of DAPI positive cells. For conditions with two or three replicates, the mean of RawIntDens was calculated. The final result was expressed by the mean (± SEM) of RawIntDens / DAPI positive cells values of each independent experiment normalized by the mean of vehicle condition and exhibited as fold change.

RNA extraction and quantitative real-time PCR analysis:

MTT assay: Primary hippocampal cultures were incubated for 24 or 48 hours in the presence of palmitate (50-500 µM) or vehicle, and cell viability was assessed using the dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide) reduction assay (Boehringer Mannheim, Indianapolis, IN). Briefly, hippocampal cultures were incubated with a 0.5 mg/ml A region of interest (ROI) was delimited in the DG, its area was measured using ImageJ software (NIH) and the number of Iba-1-positive cells was counted. The graphs represent the number of Iba-1-positive cells per mm 2 . Similarly, for GFAP analysis, a ROI was delimited in the DG and the intensity of GFAP immunoreactivity was measured in 8-bit grayscale photomicrographies, such that Raw Integrated Density per µm 2 was represented in the graphs.

QUANTIFICATION AND STATISTICAL ANALYSIS

Human data was analyzed in 49 subjects classified as normal weight, overweight or obese according BMI (Table S1). Correlation was calculated using linear regression (r2 and p values as indicated in "Figure Legends"). In results were data was presented as mean ± SEM (see "Figure Legends"), statistical significance was determined using one-way ANOVA followed by Holm-Sidak`s post hoc test.

Experiments using mice models were performed with groups of 5 to 12 mice per experimental condition, as indicated in "Figure Legends". Data are presented as means ± SEM and outliers are detected and removed from analysis using Grubbs (α = 0.05) or ROUT (Q = 1%) tests. Statistical significance was determined using two-tailed t-test (paired or unpaired) and one-way or two-way ANOVA, with suitable post hoc analysis (see in "Figure Legends").

Experiments using cell culture were performed in 3 -4 independent cultures. Data are presented as means ± SEM and statistical significance was determined using two-tailed t-test (paired or unpaired) and one-way or two-way ANOVA, with suitable post hoc analysis (see in "Figure Legends").

In general, experimental data were plotted using GraphPad Prism software. Details of statistical analysis and number of biological replicates are indicated in the respective figure legend.

DATA AND CODE AVAILABILITY

Highlights and eTOC Blurb

Highlights:

• Palmitate is increased in the CSF of overweight and obese humans with aMCI.

• CSF palmitate inversely correlates with cognitive performance in overweight humans.

• Palmitate impairs synaptic plasticity and memory in mice.

• Activated microglia and TNF-α mediate palmitate-induced impairments in memory.

  and we next sought to investigate possible mechanisms associated with these effects. We first evaluated the impact of palmitate on synaptic plasticity by analyzing the induction and maintenance of hippocampal (CA3-CA1 synapses) long-term potentiation (LTP) in alert behaving adult mice. Compared to control (vehicle-infused) mice, field excitatory post-synaptic potentials (fEPSPs) were reduced in mice that received a stereotaxic infusion of palmitate (0.3 nmol) into the dorsal hippocampus 10 days prior to recordings (Figure 2A, B; N = 6 vehicle-infused/N= 8 palmitate-infused mice; ****p < 0.0001, unpaired t-test). Moreover, recordings on subsequent days revealed that palmitate persistently impaired the LTP response (Figure 2C; N = 6 vehicle-infused/N= 8 palmitate-infused mice; * p < 0.05, two-way ANOVA followed by Holm-Sidak's post hoc test). In control experiments, mice that received an intracerebroventricular (i.c.v.) infusion of the monounsaturated fatty acid, oleic acid, presented normal LTP response and maintenance 10 days after infusion (Figure S2A-B, N = 6 vehicle-infused/N= 4 oleate-infused mice; no statistical significance). We next examined the effects of an i.c.v. infusion of palmitate on memory. Ten days after i.c.v. infusion of palmitate, mice presented memory deficits in novel object recognition (NOR), novel object location (NOL) and step-down inhibitory avoidance (IA) tests (Figure 2D-F; NOR: vehicle (N = 7), palmitate (N = 7), ***p < 0.0005, unpaired t-test comparing exploration times in the novel object; NOL: vehicle (N = 9), palmitate (N = 8), ****p < 0.0001, unpaired t-test comparing exploration times in the NOL; IA: vehicle (N = 11), palmitate (N = 9), **p < 0.005, Mann-Whitney test). Palmitate-infused mice also showed memory impairment in the NOR test 24 hours after infusion (Figure S2C; vehicle (N =10), palmitate (N = 9), ***p < 0.001, unpaired t-test comparing exploration times in the novel object). In contrast, mice that received an i.c.v. infusion of oleic acid presented normal performances in the NOR test both 24 hours and 10 days after infusion (Figure S2D, E; NOR 24h: vehicle (N =10), oleate (N = 10); NOR 10 days: vehicle (N = 10), oleate (N = 10); no statistically significant differences comparing exploration times in the novel object). Palmitate further impaired memory in the Barnes maze task, as revealed by an increased primary latency and a trend, albeit not statistically significant, increase in errors by palmitate-infused mice at the fourth (last) day of training and reduced time spent in the target zone during the test phase (Figure 2 G-I; latency (training): vehicle (N = 7), palmitate (N = 6), **p < 0.005, two-way ANOVA followed by Sidak's post hoc test; errors (training): vehicle (N = 7), palmitate (N = 6), p = 0.09, two-way ANOVA followed by Sidak's post hoc test; time in target zone (test): vehicle (N = 7), palmitate (N = 6), **p < 0.005, unpaired ttest).

Figure 1 .

 1 Figure 1. CSF palmitic acid is increased in overweight and obese individuals, correlates with BMI and abdominal circumference, and inversely correlates with MMSE scores in overweight subjects with diabetes, hypertension and dyslipidemia. See also Figure S1 and TableS1.

( C )

 C Correlation between CSF palmitic acid and abdominal circumference in normal weight (green), overweight (blue) and obese (orange) subjects (classified by BMI); linear regression, r 2 and p values as indicated.(D) CSF palmitic acid in overweight subjects according to absence (N = 11) or presence (N = 15) of diabetes, hypertension or dyslipidemia. p = 0.09 (unpaired t-test).(E) Inverse correlation between CSF palmitic acid and MMSE score in overweight subjects with diabetes, hypertension or dyslipidemia (linear regression, r 2 and p values as indicated).(F) Lack of correlation between CSF palmitic acid and MMSE score in overweight subjects without diabetes, hypertension or dyslipidemia (linear regression, r 2 and p values as indicated).

Figure 2 .

 2 Figure 2. Palmitate inhibits long term potentiation and induces memory impairment in mice. See also Figure S2 and Figure S3.

(F)

  Latency to step down in the step-down inhibitory avoidance task of male 3-5-month-old mice 10 days after i.c.v. infusion of vehicle or palmitate (3 nmol). Bars represent medians with interquartile range and symbols represent individual animals. **p < 0.005 (Mann-Whitney test).

( G )

 G Primary latency time in the training phase of the Barnes maze for vehicle-or palmitate infused 3-5-month-old male mice. Symbols represent means ± standard errors. N = 7 vehicleinfused/6 palmitate-infused mice. **p = 0.005 (two-way ANOVA followed by Sidak's post hoc test).(H) Primary errors in the training phase of the Barnes maze for vehicle-or palmitate infused 3-5-month-old male mice. Symbols represent means ± standard errors. N = 7 vehicleinfused/6 palmitate-infused mice. p = 0.09 (two-way ANOVA followed by Sidak's post hoc test).(I) Time in target zone on test day 5 in the Barnes maze for vehicle-or palmitate-infused 3-5-month-old male mice. Bars represent means ± standard errors, and symbols represent individual mice. N = 7 vehicle-infused/6 palmitate-infused mice. **p < 0.005 (unpaired ttest).

Figure 3 .

 3 Figure 3. Palmitate reduces insulin expression and impairs IRS-1 signaling in hippocampal neurons. See also Figure S4.

Figure 4 .

 4 Figure 4. Palmitate increases GFAP and Iba-1 immunoreactivities in vitro and in vivo.

Figure 5 .

 5 Figure 5. Microglial activation mediates inhibition of neuronal IRS-1 signaling and memory impairment by palmitate.

Figure 6 .

 6 Figure 6. Increased hippocampal TNF-α expression mediates neuronal IRS-1 signaling impairment in vitro and memory deficits induced by palmitate.

Figure 7 .

 7 Figure 7. Infliximab prevents memory deficits in HFD mice and genetic deletion of TNFR1 prevents memory impairment induced by palmitate.

Figure Legends .

 Legends Figure Legends.

Figure S2 .

 S2 Figure S2. Related to Figure 2. Oleate does not inhibit long-term potentiation and does not impair memory in mice.

Figure S3 .

 S3 Figure S3. Related to Figure 2. Palmitate induces a reduction in synaptophysin but not PSD-95 immunoreactivities (10 days post-infusion) in the mouse hippocampus.

Figure

  Figure S5. Related to Figure 4. Oleate has no effect on Iba-1 immunoreactivity or average cell area in microglial cultures.

  Hippocampus from vehicle-or palmitate-infused mice or from cultured hippocampal cells were homogenized in 1 mL Trizol (Invitrogen) and RNA extraction was performed according to manufacturer's instructions.Purity and integrity of RNA were determined by the 260/280 nm and 260/230 absorbance ratio and by agarose gel electrophoresis. Only preparations with ratios >1.8 and no signs of RNA degradation were used. One µg RNA was used for cDNA synthesis using the Super-Strand III Reverse Transcriptase kit (Invitrogen) or High Capacity cDNA Reverse Transcription (Applied Biosystems). Expression of genes of interest was analyzed by qPCR on an Applied Biosystems 7500 RT-PCR system using the Power SYBR kit (Applied Biosystems). Actin was used as endogenous control. Primer pairs used are: rat ACTB

	normal goat serum in PBS for 2 hours, permeabilized with 0.1 % Triton X-100 in PBS for 5
	min and washed with PBS. Cells were then immunolabeled with primary antibodies (IRS-1
	pTyr612, Sigma; 1:200 dilution; IRS-1 pS636, Santa Cruz; 1:200 dilution; IRS-1 pSer307,
	Invitrogen; 1:100 dilution; IRS-1 pSer312, Invitrogen; 1:100 dilution;; 1:100 dilution; Iba-1,
	Wako; 1:1,000 dilution and GFAP, Dako; 1:500 dilution) overnight at 4 °C. Cells were then
	rinsed three times with PBS and incubated for 2 h at room temperature with Alexa Fluor 594-
	conjugated anti-rabbit IgG secondary antibody or Alexa Fluor 555-conjugated anti-rabbit
	IgG secondary antibody (1:2,000 dilution; Molecular Probes, Inc., Eugene, OR) in PBS with
	1% normal goat serum. Cells were rinsed four times with PBS and one time with deionized
	water, and coverslips were mounted with Prolong Gold mounting medium with DAPI
	(Molecular Probes). Images were obtained from randomly chosen fields (8-25 images per
	experimental condition, from one to three coverslips per condition, see "Figure Legends"),
	using a Nikon Digital Sight DS-Qi1Mc camera and Nikon Eclipse TE 2000-U fluorescence
	microscope or AxioCam MRm and Zeiss Axio Observer Z1 using 100× objective under
	conventional fluorescence microscope. Omission of primary antibodies eliminated all
	labeling.
	(forward:		AGTGTGACGTTGACATCCGTA;	reverse:
	GCCAGAGCAGTAATCTCCTTCT),	rat	INS	2	(forward:
	CCTGCTCATCCTCTGGGAGC; reverse: TGTGCACCTTGTGGGTCCTCCA), mouse
	ACTB	(forward:	GCCCTGAGGCTCTTTTCCAG;	reverse:
	TGCCACAGGATTCCATACCC),	mouse	INS2	(forward:
	ACATGGCCCTGTGGATGCGCT; reverse: CACGGCGGGACATGGGTGTG)	and
	mouse	TNF-α	(forward:	CCCTCACACTCAGATCATCTTCT;	reverse:
	GCTACGACGTGGGCTACAG). Cycle threshold (Ct) values were used to calculate fold
	changes in gene expression using the 2 -ΔΔCt method. In all cases, reactions were performed in
	15 µl reaction volumes.
	Immunocytochemistry and image analysis: Hippocampal or microglial cultures were fixed
	by adding an equal volume of 4 % formaldehyde plus 4% sucrose (in PBS buffer) to the
	medium for 5 min followed by the removal of the entire fix/medium solution and replacement
	with 4 % formaldehyde for 10 min. Cells were rinsed 3 times with PBS, incubated with 10%

  solution of MTT at 37 °C for 4 h to allow reduction to formazan blue by metabolically active cells. Cells were then lysed, and formazan crystals were solubilized by incubation in 0.01 N HCl containing 10% SDS for 24 h at room temperature. Optical density at 570 nm was measured in a ThermoMax Microplate reader.

	from negative control sections, and secondary antibody did not show unspecific
	immunolabeling.
	Mouse perfusion and brain slices: Male swiss mice (2-4-month old) were anesthetized with
	xylazine (10 mg/kg) and ketamine (100 mg/kg), and they were perfused intracardiacally with
	PBS followed by 4% paraformaldehyde (PFA). Brains were dissected and post-fixed by
	immersion in 4% PFA for 24 hours. After washing with PBS, brains were placed sequentially
	in 20% and 30% sucrose solutions, they were embedded in Tissue-Tek O.C.T. compound
	(Sakura) and frozen in dry ice. Ten µm thickness coronal brain sections (from Bregma -1.34
	mm to Bregma -2.06 mm) (Paxinos and Franklin, 2004) were cut in a cryostat (Leica
	Immunohistochemistry and image analysis: Hippocampal sections were equilibrated at RT,
	washed with PBS and blocked with 10% bovine serum albumin in 0.25%Triton X-100/PBS
	(PBS-T) for 1 hour at RT. Thereafter, sections were incubated with primary antibody (anti-
	Iba-1, Wako, 1:500 diluition or anti-GFAP, Dako, 1:1,000 diluition) diluited in PBS-T
	overnight at RT. Sections were rinsed with PBS, and incubated with Alexa Fluor 488-
	conjugated anti-rabbit IgG secondary antibody (1:2,000 diluition, Molecular Probes) diluited
	in PBS-T for 2h at RT. Then, sections were rinsed with PBS and mounted with Prolong Gold
	mouting medium with DAPI (Molecular Probes). The dentate gyrus (DG) of hippocampus
	was analyzed as follows. Two to four coronal brain sections (50 µm apart from each other)
	were sampled per animal, and a part of the DG from both hemispheres were imaged whenever
	it was possible. Images (12 bit) were acquired with AxioCam MR R3 camera and Zeiss Axio
	Imager M2 microscope or AxioCam MRm and Zeiss Axio Observer Z1 microscope using
	20× objective under conventional fluorescence microscopy. Primary antibody was omitted

CM1850

) and placed on poly-l-lysine (200 µg/mL) coated slides. The slides were maintained at room temperature overnight for complete adhesion of the sections, and then they were frozen at -20 ºC until use.

Table S1 .

 S1 Related to Figure 1. Patient demographics for C16:0 and obesity studies.

			microglia activation
	137	62	F	6	0	0	0	64.8	1.55	27.0	88.0	2.5	26
	Case # 50 127 99 148 138 1 236 227 78 114 195 225 22 7 88 84 139 27 3 203 45 4 66 MMSE: Mini-Mental State Exam Age Gender Education DM DLP H 46 F 9 0 0 0 73 M 12 0 0 1 73 F 8 0 0 0 71 M 2 0 0 0 71 F 7 0 0 0 66 M 8 0 0 0 67 M 3 0 0 0 64 F 8 0 0 0 65 F 2 0 0 0 77 M 11 0 0 0 73 F 11 1 1 1 73 F 4 0 0 0 65 M 4 0 0 0 59 F 11 0 0 0 65 F 9 1 0 1 80 M 0 0 1 1 72 F 4 0 1 0 71 M 0 0 0 1 79 F 15 0 0 0 74 F 3 0 0 0 66 M 27 0 1 1 57 F 11 0 0 0 64 M 4 1 1 1 BMI: Body mass index; The numbers 0 or 1 indicates absence (0) or presence (1) of diabetes mellitus (DM), dyslipidemia (DLP) or hypertension (H); Weight (Kg) Height (m) BMI Abdominal Circumference C16:0 (ug/mL) MMSE 57.7 1.70 20.0 82.0 0.8 55.3 1.65 20.3 72.0 1.8 30 50.0 1.55 20.8 78.0 1.3 59.9 1.69 21.0 85.0 1.9 25 54.5 1.61 21.0 97.0 2.4 25 61.3 1.67 22.0 87.0 1.4 61.0 1.66 22.1 83.0 2.7 53.5 1.54 22.6 80.0 1.1 57.6 1.59 22.8 85.5 2.3 24 72.2 1.75 23.6 93.0 1.9 54.0 1.50 24.0 92.0 1.6 58.3 1.55 24.2 85.0 2.6 72.5 1.71 24.8 95.0 1.2 60.5 1.56 24.8 96.0 1.1 57.2 1.51 25.1 81.0 2.4 81.4 1.80 25.1 97.0 3.6 67.0 1.63 25.2 90.0 4.0 70.0 1.65 25.7 92.5 3.3 57.1 1.49 25.7 85.0 2.2 65.9 1.60 25.7 87.0 2.4 80.0 1.75 26.1 90.0 4.1 71.3 1.65 26.2 92.0 3.5 69.8 1.63 26.3 95.0 2.8 25 29 26 28 95 66 F 8 1 1 1 98.2 1.52 42.5 101.0 5.3 -29 100 65 M 8 1 1 0 104.8 1.73 35.0 120.0 3.8 28 24 240 76 F 4 1 1 0 82.1 1.55 34.2 113.0 4.2 28 23 153 77 M 12 1 0 1 89.0 1.63 33.5 116.0 5.5 26 28 170 65 F 5 0 0 0 85.0 1.60 33.2 120.0 3.8 24 29 145 59 F 14 0 0 0 84.4 1.60 33.0 106.0 5.9 28 26 76 63 F 4 1 1 1 70.0 1.47 32.4 112.0 4.2 22 20 81 71 F 5 0 0 0 80.5 1.62 30.7 108.0 4.6 27 25 150 75 F 5 0 0 1 78.0 1.60 30.5 110.0 4.9 27 27 101 66 M 17 0 0 0 71.1 1.54 30.0 98.0 3.2 29 29 194 68 F 23 0 0 0 79.7 1.65 29.3 108.0 3.9 231 59 F 8 0 0 0 65.4 1.49 29.5 94.0 1.5 28 synaptic and memory impairment 25 25 167 73 F 4 0 0 1 78.5 1.65 28.8 116.0 3.0 23 27 92 64 F 13 0 1 1 81.0 1.68 28.7 110.0 3.1 24 30 228 65 F 5 0 0 0 56.3 1.42 27.9 80.0 2.3 22 224 74 F 4 0 0 1 63.8 1.51 28.0 96.5 3.9 65 78 M 6 1 1 1 75.5 1.63 28.4 108.0 2.1 28 signaling in neurons 18 activation defective insulin 21 230 65 F 8 0 0 0 72.0 1.62 27.4 100.0 2.5 105 63 F 9 0 0 1 69.5 1.59 27.5 91.0 2.6 -astrocyte 28 17 98 68 F 4 0 0 0 65.9 1.55 27.4 96.0 2.5 26 score 226 64 F 4 1 0 1 62.8 1.52 27.2 85.0 3.2 25 103 69 F 5 0 0 1 66.4 1.56 27.3 92.0 2.3 26 TNF-α
	130	69	F	14	0	1	1	58.6	1.49	26.4	86.0	2.4	26
	96	73	M	15	0	0	0	80.6	1.74	26.6	101.0	2.0	25
	77	67	M	12	0	0	1	77.8	1.70	26.9	98.0	2.9	26

Acknowledgments

This work was supported by grants from Human Frontiers Science Program (HFSP) and John Simon Guggenheim Foundation (to F.G.D.F.), National Institute for Translational Neuroscience (INNT/Brazil), the Brazilian funding agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (to S.T.F. and F.G.D.F). H.M.M, G.S.S.S, B.C.M, J.R.C, V.S.M.C., G.C.K., J.T.S.F and J.H.L were supported by CNPq and FAPERJ predoctoral and postdoctoral fellowships. This work has been partially developed and supported through the LABEX (excellence laboratory, program investment for the future) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to ALZheimer's disease) and the Hospital University Federation (FHU) OncoAge (FC). We thank Ana Claudia Rangel and Mariângela Viana for administrative and technical support. We thank Dr. Wagner Seixas (Federal University of Rio de Janeiro, Brazil) for insightful discussions and technical advice.

Declaration of Interests

The authors declare no competing interests. This study did not generate/analyze datasets or code.

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