Extracellular vesicles released from microglia after palmitate exposure impact brain function

Abstract

Dietary patterns that include an excess of foods rich in saturated fat are associated with brain dysfunction. Although microgliosis has been proposed to play a key role in the development of brain dysfunction in diet-induced obesity (DIO), neuroinflammation with cytokine over-expression is not always observed. Thus, mechanisms by which microglia contribute to brain impairment in DIO are uncertain. Using the BV2 cell model, we investigated the gliosis profile of microglia exposed to palmitate (200 µmol/L), a saturated fatty acid abundant in high-fat diet and in the brain of obese individuals. We observed that microglia respond to a 24-hour palmitate exposure with increased proliferation, and with a metabolic network rearrangement that favors energy production from glycolysis rather than oxidative metabolism, despite stimulated mitochondria biogenesis. In addition, while palmitate did not induce increased cytokine expression, it modified the protein cargo of released extracellular vesicles (EVs). When administered intra-cerebroventricularly to mice, EVs secreted from palmitate-exposed microglia in vitro led to memory impairment, depression-like behavior, and glucose intolerance, when compared to mice receiving EVs from vehicle-treated microglia. We conclude that microglia exposed to palmitate can mediate brain dysfunction through the cargo of shed EVs.

Keywords: Energy metabolism; Glycolysis; LPS; Neuroinflammation; Obesity.

Fig. 1

Palmitate exposure induces gliosis without exacerbated cytokine production and increases mitochondria content. BV2 cells were incubated with vehicle (Veh), 200 µmol/L palmitate (PA) for 24 h or 1 µg/mL LPS for 3 h. (A) Cell counts before, and after 6, 12 and 24 h of treatment, or after 1.5 and 3 h for LPS. (B) Relative cell viability measured by MTT reduction. (C) Relative activity of caspase 3/7. (D) Relative expression of TNF-α, IL-6 and IL-1β, and (E) concentration of TNF-α in the medium after treatment (3 h LPS, 14 h vehicle or palmitate). (F) Relative expression of cytokines during the initial 9 h of palmitate exposure. (G) Representative immunofluorescence micrographs for mitotracker, β-actin and Iba1 (scale bar is 10 μm). (H) Mean area occupied by mitotracker, β-actin and Iba1 signal per cell, and (I) area ratios of mitotracker to β-actin and to Iba1. Cells were analyzed within 3–4 fields of view from 3 independent experiments. Data is shown as mean ± SD of 3–8 independent experiments, represented by the individual symbols. *P < 0.05, **P < 0.01, ***P < 0.001 depict differences in comparisons following significant effects in ANOVA

Fig. 2

Palmitate exposure modulates energy metabolism in BV2 cells. BV2 cells were incubated with vehicle (Veh), 200 µmol/L palmitate (PA) for 24 h or 1 µg/mL LPS for 3 h. (A) Schematic representation of experiments for OCR measurements depicting the calculated parameters upon addition of oligomycin (1.5 mmol/L), FCCP (0.5 mmol/L), and antimycin A (0.5 mmol/L) plus rotenone (0.5 mmol/L): basal respiration (basal), proton leak-driven respiration (leak), ATP synthesis-linked respiration (ATP), maximal respiration capacity (max), spare respiration capacity (spare), and non-mitochondrial oxygen consumption (NM). (B-C) Oxygen consumption rate (OCR) measured for 3 cycles within each respiration state (B), and calculated respiration parameters (C). (D) Representative immunoblotting experiment against the four complexes of the electron transport chain and ATP synthase (complex V), after separation of 30 µg of protein by SDS-PAGE. (E) Relative immunoreactivity signal from the 5 complexes in 4 independent experiments. For a given protein, signal within each band was normalized to the average of that in the 3 experimental groups. (F) Expression of genes involved in mitochondria biogenesis, fusion and fission. (G) Schematic representation of experiments for ECAR measurements depicting the calculated parameters upon addition of oligomycin (1 mmol/L) and 2-deoxy-D-glucose (2DG, 50 mmol/L): basal glycolysis (glyc), glycolytic reserve (res), glycolytic capacity (capac), and non-glycolytic medium acidicitation (NGA). (H-I) extracellular medium acidification rate (ECAR) measured for 3 cycles within each respiration state, and calculated glycolytic parameters. (J) Relative expression of Slc2a1 gene (GLUT1). (K) Representation of ¹³C incorporation into glutamate omitting, for simplicity, generation of isotopomers from unlabeled pyruvate/acetyl-CoA, and respective representative multiplets observed in ¹³C NMR spectra measured in extracts after metabolizing [1-¹³ C]glucose for 24 h. (L) Glutamate (Glu) multiplet fractions, and fractional enrichment (FE) of lactate C3 of (Lac) and alanine (Ala). (M) Model used in the TCAcalc analysis and relative fluxes, and lactate labeling estimated by fitting glutamate isotopomers, and Ala C3. Abbreviations: CS, citrate synthase; PDH, pyruvate dehydrogenase; Y, flux of anaplerotic substrates through pyruvate carboxylase (Y_PC) or succinyl-CoA (Y_S). Data is shown as mean ± SD of 3–12 independent experiments, represented by the individual symbols. *P < 0.05, **P < 0.01, ***P < 0.001 depict differences in comparisons following significant effects in ANOVA

Fig. 3

Palmitate alters the proteome of BV2-retreased EVs, including a reduction of proteins involved in RNA processing and protein synthesis. (A) Histograms of EV size distribution evaluated by Nanoparticle tracking analysis (NTA) of 6 independent EV isolations, and (B) estimated mean particle size for vehicle (Veh), palmitate (PA) and LPS. Data shown as mean ± SD of n = 6. (C) Score plots of a PCA of the 1000 most abundant proteins in EVs (n = 3/group). Each symbol shape represents an independent experiment. (D) Heatmap of significant EV proteome differences between either of the 3 experimental groups (fold-change comparisons in supplementary figure S1), and (E) significant findings at FDR < 0.01 from the gene ontology analysis of differentially expressed proteins between EVs from palmitate- and vehicle-treated BV2 cells. ⁽¹⁾Full pathway name: “Activation of the mRNA upon binding of the cap-binding complex and eIFs and subsequent binding to 43S”

Fig. 4

Intracerebroventricular (i.c.v) injection of microglia-derived EVs following palmitate exposure affects cognition and depressive-like behavior, and alters glucose metabolism in mice. (A) Mice were injected in the lateral ventricle with EVs (500 ng of protein) collected from BV2 cells after exposure to either palmitate (PA, 200 µmol/L) or vehicle (Veh). (B) Body weight of mice before and 8 days after surgery for EV administration. Impaired memory was observed novel object recognition (NOR) test (C), and a similar trend was observed in the novel location recognition (NLR) test (D). (E) Depression-related behavior was assessed by grooming behavior in the sucrose splash test. (F) locomotor activity and exploratory behavior assessed in the last habituation day to the open-field arena. (G) Glucose clearance in the GTT in the 8th day following EV injection. The inset is the area under the curve (AUC) of the glucose excursion for each mouse. Symbols representing each mouse (n = 8) are overlaid on bar graphs showing mean ± SD or box plots showing interquartile ranges. *P < 0.05 from either Student’s t-test or with Mann Whitney test

Fig. 5

EVs from palmitate-challenged microglia in vitro activate microglia in the hippocampus and hypothalamus of mice in vivo. (A) Representative micrographs showing DAPI signal from nuclei, GFAP immunoreactivity in red, and Iba1 immunoreactivity in red in the dentate gyrus (DG) and cornu ammonis (CA1/CA3) areas of the hippocampus, and arcuate nucleus (ARC) of the hypothalamus at 8 days after intraventricular EVs injection. (B-C) Fraction of area occupied by iba1 and GFAP immunoreactivity in DG, CA1, CA3 and ARC. (D) Mean number of processes, (E) number of branching points, (F) total cell process length, and (G) maximum process length of microglia, as determined from skeleton analysis of 3–4 iba1⁺ cells per mouse. (H) Representative micrographs showing CD68 staining at 8 days after EV injection. (I) Fraction of are occupied by CD68 immunoreactivity, and (J) number of CD68-positive cells. Data is shown as mean ± SD of 7 independent experiments, represented by the individual symbols. *P < 0.05 depicts differences in comparisons following significant effects in ANOVA. Scale bars over the micrographs indicate 50 μm; 3 V = third ventricle