The ketone body ß-hydroxybutyrate shifts microglial metabolism and suppresses amyloid-ß oligomer-induced inflammation in human microglia.

Fatty acids are metabolized by ß-oxidation within the "mitochondrial ketogenic pathway" (MKP) to generate ß-hydroxybutyrate (BHB), a ketone body. BHB can be generated by most cells but largely by hepatocytes following exercise, fasting, or ketogenic diet consumption. BHB has been shown to modulate systemic and brain inflammation; however, its direct effects on microglia have been little studied. We investigated the impact of BHB on Aß oligomer (AßO)-stimulated human iPS-derived microglia (hiMG), a model relevant to the pathogenesis of Alzheimer's disease (AD). HiMG responded to AßO with proinflammatory activation, which was mitigated by BHB at physiological concentrations of 0.1-2 mM. AßO stimulated glycolytic transcripts, suppressed genes in the ß-oxidation pathway, and induced over-expression of AD-relevant p46Shc, an endogenous inhibitor of thiolase, actions that are expected to suppress MKP. AßO also triggered mitochondrial Ca2+ increase, mitochondrial reactive oxygen species production, and activation of the mitochondrial permeability transition pore. BHB potently ameliorated all the above mitochondrial changes and rectified the MKP, resulting in reduced inflammasome activation and recovery of the phagocytotic function impaired by AßO. These results indicate that microglia MKP can be induced to modulate microglia immunometabolism, and that BHB can remedy "keto-deficiency" resulting from MKP suppression and shift microglia away from proinflammatory mitochondrial metabolism. These effects of BHB may contribute to the beneficial effects of ketogenic diet intervention in aged mice and in human subjects with mild AD.

Cholesterol, Amyloid Beta, Fructose, and LPS Influence ROS and ATP Concentrations and the Phagocytic Capacity of HMC3 Human Microglia Cell Line.

Research has found that genes specific to microglia are among the strongest risk factors for Alzheimer's disease (AD) and that microglia are critically involved in the etiology of AD. Thus, microglia are an important therapeutic target for novel approaches to the treatment of AD. High-throughput in vitro models to screen molecules for their effectiveness in reversing the pathogenic, pro-inflammatory microglia phenotype are needed. In this study, we used a multi-stimulant approach to test the usefulness of the human microglia cell 3 (HMC3) cell line, immortalized from a human fetal brain-derived primary microglia culture, in duplicating critical aspects of the dysfunctional microglia phenotype. HMC3 microglia were treated with cholesterol (Chol), amyloid beta oligomers (AßO), lipopolysaccharide (LPS), and fructose individually and in combination. HMC3 microglia demonstrated changes in morphology consistent with activation when treated with the combination of Chol + AßO + fructose + LPS. Multiple treatments increased the cellular content of Chol and cholesteryl esters (CE), but only the combination treatment of Chol + AßO + fructose + LPS increased mitochondrial Chol content. Microglia treated with combinations containing Chol + AßO had lower apolipoprotein E (ApoE) secretion, with the combination of Chol + AßO + fructose + LPS having the strongest effect. Combination treatment with Chol + AßO + fructose + LPS also induced APOE and TNF-α expression, reduced ATP production, increased reactive oxygen species (ROS) concentration, and reduced phagocytosis events. These findings suggest that HMC3 microglia treated with the combination of Chol + AßO + fructose + LPS may be a useful high-throughput screening model amenable to testing on 96-well plates to test potential therapeutics to improve microglial function in the context of AD.

The role of FUT8-catalyzed core fucosylation in Alzheimer's amyloid-ß oligomer-induced activation of human microglia.

Fucosylation, especially core fucosylation of N-glycans catalyzed by α1-6 fucosyltransferase (fucosyltransferase 8 or FUT8), plays an important role in regulating the peripheral immune system and inflammation. However, its role in microglial activation is poorly understood. Here we used human induced pluripotent stem cells-derived microglia (hiMG) as a model to study the role of FUT8-catalyzed core fucosylation in amyloid-ß oligomer (AßO)-induced microglial activation, in view of its significant relevance to the pathogenesis of Alzheimer's disease (AD). HiMG responded to AßO and lipopolysaccharides (LPS) with a pattern of pro-inflammatory activation as well as enhanced core fucosylation and FUT8 expression within 24 h. Furthermore, we found increased FUT8 expression in both human AD brains and microglia isolated from 5xFAD mice, a model of AD-like cerebral amyloidosis. Inhibition of fucosylation in AßO-stimulated hiMG reduced the induction of pro-inflammatory cytokines, suppressed the activation of p38MAPK, and rectified phagocytic deficits. Specific inhibition of FUT8 by siRNA-mediated knockdown also reduced AßO-induced pro-inflammatory cytokines. We further showed that p53 binds to the two consensus binding sites in the Fut8 promoter, and that p53 knockdown abolished FUT8 overexpression in AßO-activated hiMG. Taken together, our evidence supports that FUT8-catalyzed core fucosylation is a signaling pathway required for AßO-induced microglia activation and that FUT8 is a component of the p53 signaling cascade regulating microglial behavior. Because microglia are a key driver of AD pathogenesis, our results suggest that microglial FUT8 could be an anti-inflammatory therapeutic target for AD.