Altered metabolism and DAM-signatures in female brains and microglia with aging.

Despite Alzheimer's disease (AD) disproportionately affecting women, the mechanisms remain elusive. In AD, microglia undergo 'metabolic reprogramming', which contributes to microglial dysfunction and AD pathology. However, how sex and age contribute to metabolic reprogramming in microglia is understudied. Here, we use metabolic imaging, transcriptomics, and metabolic assays to probe age- and sex-associated changes in brain and microglial metabolism. Glycolytic and oxidative metabolism in the whole brain was determined using Fluorescence Lifetime Imaging Microscopy (FLIM). Young female brains appeared less glycolytic than male brains, but with aging, the female brain became 'male-like.' Transcriptomic analysis revealed increased expression of disease-associated microglia (DAM) genes (e.g., ApoE, Trem2, LPL), and genes involved in glycolysis and oxidative metabolism in microglia from aged females compared to males. To determine whether estrogen can alter the expression of these genes, BV-2 microglia-like cell lines, which abundantly express DAM genes, were supplemented with 17ß-estradiol (E2). E2 supplementation resulted in reduced expression of DAM genes, reduced lipid and cholesterol transport, and substrate-dependent changes in glycolysis and oxidative metabolism. Consistent with the notion that E2 may suppress DAM-associated factors, LPL activity was elevated in the brains of aged female mice. Similarly, DAM gene and protein expression was higher in monocyte-derived microglia-like (MDMi) cells derived from middle-aged females compared to age-matched males and was responsive to E2 supplementation. FLIM analysis of MDMi from young and middle-aged females revealed reduced oxidative metabolism and FAD+ with age. Overall, our findings show that altered metabolism defines age-associated changes in female microglia and suggest that estrogen may inhibit the expression and activity of DAM-associated factors, which may contribute to increased AD risk, especially in post-menopausal women.

Altered Metabolism and DAM-signatures in Female Brains and Microglia with Aging.

Despite Alzheimer's disease (AD) disproportionately affecting women, the mechanisms remain elusive. In AD, microglia undergo 'metabolic reprogramming', which contributes to microglial dysfunction and AD pathology. However, how sex and age contribute to metabolic reprogramming in microglia is understudied. Here, we use metabolic imaging, transcriptomics, and metabolic assays to probe age-and sex-associated changes in brain and microglial metabolism. Glycolytic and oxidative metabolism in the whole brain was determined using Fluorescence Lifetime Imaging Microscopy (FLIM). Young female brains appeared less glycolytic than male brains, but with aging, the female brain became 'male-like.' Transcriptomic analysis revealed increased expression of disease-associated microglia (DAM) genes (e.g., ApoE, Trem2, LPL), and genes involved in glycolysis and oxidative metabolism in microglia from aged females compared to males. To determine whether estrogen can alter the expression of these genes, BV-2 microglia-like cell lines, which abundantly express DAM genes, were supplemented with 17ß-estradiol (E2). E2 supplementation resulted in reduced expression of DAM genes, reduced lipid and cholesterol transport, and substrate-dependent changes in glycolysis and oxidative metabolism. Consistent with the notion that E2 may suppress DAM-associated factors, LPL activity was elevated in the brains of aged female mice. Similarly, DAM gene and protein expression was higher in monocyte-derived microglia-like (MDMi) cells derived from middle-aged females compared to age-matched males and was responsive to E2 supplementation. FLIM analysis of MDMi from young and middle-aged females revealed reduced oxidative metabolism and FAD+ with age. Overall, our findings show that altered metabolism defines age-associated changes in female microglia and suggest that estrogen may inhibit the expression and activity of DAM-associated factors, which may contribute to increased AD risk, especially in post-menopausal women.

Role of the luminal composition on intestinal metal toxicity, bioavailability and bioreactivity: An in vitro approach based on the cell line RTgutGC.

The bioavailability of metal complexes is poorly understood. To evaluate bioavailability and toxicity of neutral and charged complexes as well as free metal ions, Visual Minteq, a chemical equilibrium model, was used to design media containing different metal species. Two non-essential (silver and cadmium) and two essential (copper and zinc) metals were selected. The rainbow trout (Oncorhynchus mykiss) gut cell line (RTgutGC) was used to investigate bioavailability, bioreactivity and toxicity of the different metal species. Toxicity was measured using a multiple endpoint cytotoxicity assay, bioavailability by measuring intracellular metal concentration, and bioreactivity by quantification of mRNA level of the metal responsive genes, metallothionein (MT), glutathione reductase (GR) and zinc transporter 1 (ZnT1). Speciation calculations showed that silver and cadmium preferentially bind chloride, copper phosphate and bicarbonate, and zinc remained primarily as a free ion. Cysteine avidly complexed with all metals reducing toxicity, bioavailability and bioreactivity. Silver and copper toxicity was not affected by inorganic metal speciation, whereas cadmium and zinc toxicity was decreased by chloride complexation. Moreover, reduction of calcium concentration in the medium increased toxicity and bioavailability of cadmium and zinc. Bioavailability of silver and zinc was reduced by low chloride while cadmium bioavailability was increased by low chloride and in presence of bicarbonate. Copper bioavailability was not affected by the medium composition. Cadmium and silver were more bioreactive, independently from the medium composition, in comparison to copper and zinc (i.e., higher induction of MT and GR). Cadmium was the only metal able to induce MT in presence of cysteine. ZnT1 was induced by cadmium in low-chloride, by zinc in low-chloride low-calcium and by cadmium and copper in the bicarbonate media. Overall, this study demonstrates that metal complexation alone is not sufficient to explain metal toxicity, and that anion exchange mechanisms play a role in metal uptake and bioreactivity.

Using Synthetic ApoC-II Peptides and nAngptl4 Fragments to Measure Lipoprotein Lipase Activity in Radiometric and Fluorescent Assays.

Lipoprotein lipase (LPL) plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides (TGs) in packaged lipoproteins. Since hypertriglyceridemia (HTG) is a major risk factor for cardiovascular disease (CVD), the leading cause of death worldwide, methods that accurately quantify the hydrolytic activity of LPL in clinical and pre-clinical samples are much needed. To date, the methods used to determine LPL activity vary considerably in their approach, in the LPL substrates used, and in the source of LPL activators and inhibitors used to quantify LPL-specific activity, rather than other lipases, e.g., hepatic lipase (HL) or endothelial lipase (EL) activity. Here, we describe methods recently optimized in our laboratory, using a synthetic ApoC-II peptide to activate LPL, and an n-terminal Angiopoietin-Like 4 fragment (nAngptl4) to inhibit LPL, presenting a cost-effective and reproducible method to measure LPL activity in human post-heparin plasma (PHP) and in LPL-enriched heparin released (HR) fractions from LPL secreting cells. We also describe a modified version of the triolein-based assay using human serum as a source of endogenous activators and inhibitors and to determine the relative abundance of circulating factors that regulate LPL activity. Finally, we describe how an ApoC-II peptide and nAngptl4 can be applied to high-throughput measurements of LPL activity using the EnzChek™ fluorescent TG analog substrate with PHP, bovine LPL, and HR LPL enriched fractions. In summary, this manuscript assesses the current methods of measuring LPL activity and makes new recommendations for measuring LPL-mediated hydrolysis in pre-clinical and clinical samples.

In Vitro Digestion of Tire Particles in a Fish Model (Oncorhynchus mykiss): Solubilization Kinetics of Heavy Metals and Effects of Food Coingestion.

Tire and road wear particles (TRWP) have been shown to represent a large part of anthropogenic particles released into the environment. Nevertheless, the potential ecological risk of TRWP in the different environmental compartments and their potential toxic impacts on terrestrial and aquatic organisms remain largely underinvestigated. Several heavy metals compose TRWP, including Zn, which is used as a catalyst during the vulcanization process of rubber. This study investigated the solubilization potential of metals from cryogenically milled tire tread (CMTT) and TRWP in simulated gastric fluids (SFGASTRIC) and simulated intestinal fluids (SFINTESTINAL) designed to mimic rainbow trout (Oncorhynchus mykiss) gastrointestinal conditions. Our results indicate that the solubilization of heavy metals was greatly enhanced by gastrointestinal fluids compared to that by mineral water. After a 26 h in vitro digestion, 9.6 and 23.0% of total Zn content of CMTT and TRWP, respectively, were solubilized into the simulated gastrointestinal fluids. Coingestion of tire particles (performed with CMTT only) and surrogate prey items (Gammarus pulex) demonstrated that the animal organic matter reduced the amount of bioavailable Zn solubilized from CMTT. Contrastingly, in the coingestion scenario with vegetal organic matter (Lemna minor), high quantities of Zn were solubilized from L. minor and cumulated with Zn solubilized from CMTT.

Adsorption of progesterone onto microplastics and its desorption in simulated gastric and intestinal fluids.

The sorption of hydrophobic organic compounds (HOC) onto microplastics is relatively well reported in the literature, while their desorption remains poorly investigated, especially in biological fluids. The present study investigated the sorption and desorption of progesterone on polyethylene (PE), polypropylene (PP), and polystyrene (PS) microplastics. The sorption experiments showed that the equilibrium was reached in a few hours for all plastics. A sorption efficiency of 357.1 µg g-1 was found for PE and PS, and 322.6 µg g-1 for PP. Sorption experiments indicated that adsorption would certainly happen via surface sorption and a potentially pore-filling mechanism. The desorption was carried out in Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF), whose formulations were more complex than similar models reported so far. It has been found that the desorption was higher in SIF as compared to SGF, due to micelle formation in SIF promoting the pollutant solubilization. The sorption of pepsin onto microplastics has also been revealed, suggesting a competition between pollutants and pepsin for sorption sites and a potent reduction in pollutant solubilization. This study indicates that the ingestion of microplastics could be considered as an additional route of exposure to pollutants and therefore emphasizes pollutant bioavailability for aquatic organisms.

Role of metal speciation in the exposure medium on the toxicity, bioavailability and bio-reactivity of copper, silver, cadmium and zinc in the rainbow trout gut cell line (RTgutGC).

The role of metal speciation on metal bioavailability, bio-reactivity and toxicity at the fish intestine is poorly understood. To investigate these processes, we used an in vitro model of the rainbow trout (Oncorhynchus mykiss) intestine, the RTgutGC cell line. Cells were exposed to two essential metals (copper and zinc) and two non-essential metals (cadmium and silver) in a medium of well-defined composition, which allowed the determination of metal speciation in solution. Concentrations resulting in a 50% cell viability reduction (EC50) were measured using a viability assay based on two endpoints: metabolic activity and membrane integrity. Metal bioavailability and bio-reactivity was studied at non-toxic (300 nM all metals) and toxic (EC10; Ag-0.6, Cu-0.9, Cd-3, and Zn-9 µM) concentrations. Bioavailability (i.e. intracellular metal accumulation) was determined by ICP-MS, while bio-reactivity (i.e. induction of a metal specific transcriptional response) was determined by measuring the mRNA levels of a known biomarker of metal exposure (i.e. metallothionein) and of copper and zinc transporters (i.e. ATP7A and ZnT1). Dominant metal species in the exposure medium were Zn2+, CuHPO4, CdCl+, and AgCl2- respectively for Zn, Cu, Cd, and Ag. The EC50s showed the metal toxicity hierarchy: Ag > Cu > Cd > Zn. In RTgutGC cells, essential metal homeostasis was tightly regulated while non-essential metals accumulated more readily. Non-essential metals were also more bio-reactive inducing higher MT and ZnT1 mRNA levels. Taken together these findings indicate that metal toxicity in RTgutGC cannot solely be explained by extracellular metal speciation but requires the evaluation of metal bioavailability and bio-reactivity.