Central activation of the fatty acid sensor GPR120 suppresses microglia reactivity and alleviates sickness- and anxiety-like behaviors.

G protein-coupled receptor 120 (GPR120, Ffar4) is a sensor for long-chain fatty acids including omega-3 polyunsaturated fatty acids (n-3 PUFAs) known for beneficial effects on inflammation, metabolism, and mood. GPR120 mediates the anti-inflammatory and insulin-sensitizing effects of n-3 PUFAs in peripheral tissues. The aim of this study was to determine the impact of GPR120 stimulation on microglial reactivity, neuroinflammation and sickness- and anxiety-like behaviors by acute proinflammatory insults. We found GPR120 mRNA to be enriched in  both murine and human microglia, and in situ hybridization revealed GPR120 expression in microglia of the nucleus accumbens (NAc) in mice. In a manner similar to or exceeding n-3 PUFAs, GPR120 agonism (Compound A, CpdA) strongly attenuated lipopolysaccharide (LPS)-induced proinflammatory marker expression in primary mouse microglia, including tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), and inhibited nuclear factor-ĸB translocation to the nucleus. Central administration of CpdA to adult mice blunted LPS-induced hypolocomotion and anxiety-like behavior and reduced TNF-α, IL-1ß and IBA-1 (microglia marker) mRNA in the NAc, a brain region modulating anxiety and motivation and implicated in neuroinflammation-induced mood deficits. GPR120 agonist pre-treatment attenuated NAc microglia reactivity and alleviated sickness-like behaviors elicited by central injection TNF-α and IL-1ß. These findings suggest that microglial GPR120 contributes to neuroimmune regulation and behavioral changes in response to acute infection and elevated brain cytokines. GPR120 may participate in the protective action of n-3 PUFAs at the neural and behavioral level and offers potential as treatment target for neuroinflammatory conditions.

Fish oil supplementation alleviates metabolic and anxiodepressive effects of diet-induced obesity and associated changes in brain lipid composition in mice.

OBJECTIVE: Obesity significantly elevates the odds of developing mood disorders. Chronic consumption of a saturated high-fat diet (HFD) elicits anxiodepressive behavior in a manner linked to metabolic dysfunction and neuroinflammation in mice. Dietary omega-3 polyunsaturated fatty acids (n-3 PUFA) can improve both metabolic and mood impairments by relieving inflammation. Despite these findings, the effects of n-3 PUFA supplementation on energy homeostasis, anxiodepressive behavior, brain lipid composition, and gliosis in the diet-induced obese state are unclear. METHODS: Male C57Bl/6J mice were fed a saturated high-fat diet (HFD) or chow for 20 weeks. During the last 5 weeks mice received daily gavage ("supplementation") of fish oil (FO) enriched with equal amounts of docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) or control corn oil. Food intake and body weight were measured throughout while additional metabolic parameters and anxiety- and despair-like behavior (elevated-plus maze, light-dark box, and forced swim tasks) were evaluated during the final week of supplementation. Forebrain lipid composition and markers of microglia activation and astrogliosis were assessed by gas chromatography-mass spectrometry and real-time PCR, respectively. RESULTS: Five weeks of FO supplementation corrected glucose intolerance and attenuated hyperphagia in HFD-induced obese mice without affecting adipose mass. FO supplementation also defended against the anxiogenic and depressive-like effects of HFD. Brain lipids, particularly anti-inflammatory PUFA, were diminished by HFD, whereas FO restored levels beyond control values. Gene expression markers of brain reactive gliosis were supressed by FO. CONCLUSIONS: Supplementing a saturated HFD with FO rich in EPA and DHA corrects glucose intolerance, inhibits food intake, suppresses anxiodepressive behaviors, enhances anti-inflammatory brain lipids, and dampens indices of brain gliosis in obese mice. Together, these findings support increasing dietary n-3 PUFA for the treatment of metabolic and mood disturbances associated with excess fat intake and obesity.

Identification of the minimal promoter for specific expression of the GABAρ1 receptor in retinal bipolar cells.

γ-aminobutyric acid (GABA)ρ receptors regulate rapid synaptic ion currents in the axon end of retinal ON bipolar neurons, acting as a point of control along the visual pathway. In the GABAρ1 subunit knock out mouse, inhibition mediated by this receptor is totally eliminated, showing its role in neural transmission in retina. GABAρ1 mRNA is expressed in mouse retina after post-natal day 7, but little is known about its transcriptional regulation. To identify the GABAρ1 promoter, in silico analyses were performed and indicated that a 0.290-kb fragment, flanking the 5'-end of the GABAρ1 gene, includes putative transcription factor-binding sites, two Inr elements, and lacks a TATA-box. A rapid amplification of cDNA ends (RACE) assay showed three transcription start sites (TSS) clustered in the first exon. Luciferase reporter assays indicated that a 0.232-kb fragment upstream from the ATG is the minimal promoter in transfected cell lines and in vitro electroporated retinae. The second Inr and AP1 site are important to activate transcription in secretin tumor cells (STC-1) and retina. Finally, the 0.232-kb fragment drives green fluorescent protein (GFP) expression to the inner nuclear layer, where bipolar cells are present. This first work paves the way for further studies of molecular elements that control GABAρ1 transcription and regulate its expression during retinal development.

Portable Near-Infrared Fluorometer for a Liposomal Blood Lactate Assay.

In sepsis, plasma lactate is a key biomarker of disease severity, prognosis, and treatment success. However, the median time to result for clinical lactate tests is 3 h. We recently reported a near-infrared fluorescent (NIRF) blood lactate assay that relies on a two-step enzymatic reaction in a liposomal reaction compartment. This assay was optimized in human blood and was capable of quantifying lactate in fresh capillary blood from human volunteers at clinically relevant concentrations in 2 min. However, these studies were performed with a tabletop fluorescence plate reader. For translation to the point of care, the liposomal lactate assay needs to be combined with a small portable NIR fluorometer. Portable NIR fluorometers were successfully used for the analysis of skin and soil samples, but reports for blood metabolite assays are scarce. We aimed at testing the performance of the liposomal lactate assay in combination with a commercial small portable NIR fluorometer. First, we tested the fluorophore of the liposomal lactate assay using the NIR dye sulfo-cyanine 7; we observed strong fluorescence signals and high linearity. Second, we performed the liposomal lactate assay in lactate-spiked human arterial blood using the portable fluorometer as the detector and observed strong and highly linear lactate sensing at clinically relevant lactate concentrations after 2 min. Finally, spiking fresh mouse blood with three clinically relevant lactate concentrations led to a significantly different response to all three concentrations after 5 min. These results highlight the usefulness of the tested portable NIR fluorometer for the liposomal lactate assay and motivate a clinical evaluation of this rapid and easy-to-use lactate assay.

Effect of Methotrexate on an In Vitro Patient-Derived Model of Proliferative Vitreoretinopathy.

Purpose: The purpose of this study was to develop a method for isolating, culturing, and characterizing cells from patient-derived membranes in proliferative vitreoretinopathy (PVR) to be used for drug testing. Methods: PVR membranes were obtained from six patients with grade C PVR. Membrane fragments were analyzed by gross evaluation, fixed for immunohistologic studies to establish cell identity, or digested with collagenase II to obtain single cell suspensions for culture. PVR-derived primary cultures were used to examine the effects of methotrexate (MTX) on proliferation, migration, and cell death. Results: Gross analysis of PVR membranes showed presence of pigmented cells, indicative of retinal pigment epithelial cells. Immunohistochemistry identified cells expressing α-smooth muscle actin, glial fibrillary acidic protein, Bestrophin-1, and F4/80, suggesting the presence of multiple cell types in PVR. Robust PVR primary cultures (C-PVR) were successfully obtained from human membranes, and these cells retained the expression of cell identity markers in culture. C-PVR cultures formed membranes and band-like structures in culture reminiscent of the human condition. MTX significantly reduced the proliferation and band formation of C-PVR, whereas it had no significant effect on cell migration. MTX also induced regulated cell death within C-PVR as assessed by increased expression of caspase-3/7. Conclusions: PVR cells obtained from human membranes can be successfully isolated, cultured, and profiled in vitro. Using these primary cultures, we identified MTX as capable of significantly reducing growth and inducing cell death of PVR cells in vitro.