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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 (AβO), lipopolysaccharide (LPS), and fructose individually and in combination. HMC3 microglia demonstrated changes in morphology consistent with activation when treated with the combination of Chol + AβO + fructose + LPS. Multiple treatments increased the cellular content of Chol and cholesteryl esters (CE), but only the combination treatment of Chol + AβO + fructose + LPS increased mitochondrial Chol content. Microglia treated with combinations containing Chol + AβO had lower apolipoprotein E (ApoE) secretion, with the combination of Chol + AβO + fructose + LPS having the strongest effect. Combination treatment with Chol + AβO + 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 + AβO + 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. [ABSTRACT FROM AUTHOR]

Abstract Background Innate immune activation, including a role for cluster of differentiation 14/toll-like receptor 4 co-receptors (CD14/TLR-4) co-receptors, has been implicated in paracrine damage to neurons in several neurodegenerative diseases that also display stratification of risk or clinical outcome with the common alleles of the apolipoprotein E gene (APOE): APOE2, APOE3, and APOE4. Previously, we have shown that specific stimulation of CD14/TLR-4 with lipopolysaccharide (LPS) leads to greatest innate immune response by primary microglial cultures from targeted replacement (TR) APOE4 mice and greatest p38MAPK-dependent paracrine damage to neurons in mixed primary cultures and hippocampal slice cultures derived from TR APOE4 mice. In contrast, TR APOE2 astrocytes had the highest NF-kappaB activity and no neurotoxicity. Here we tested the hypothesis that direct activation of CD14/TLR-4 in vivo would yield different amounts of paracrine damage to hippocampal sector CA1 pyramidal neurons in TR APOE mice. Methods We measured in vivo changes in dendrite length in hippocampal CA1 neurons using Golgi staining and determined hippocampal apoE levels by Western blot. Neurite outgrowth of cultured primary neurons in response to astrocyte conditioned medium was assessed by measuring neuron length and branch number. Results Our results showed that TR APOE4 mice had slightly but significantly shorter dendrites at 6 weeks of age. Following exposure to intracerebroventricular LPS, there was comparable loss of dendrite length at 24 hr among the three TR APOE mice. Recovery of dendrite length over the next 48 hr was greater in TR APOE2 than TR APOE3 mice, while TR APOE4 mice had failure of dendrite regeneration. Cell culture experiments indicated that the enhanced neurotrophic effect of TR APOE2 was LDL related protein-dependent. Conclusion The data indicate that the environment within TR APOE2 mouse hippocampus was most supportive of dendrite regeneration while that within TR APOE4 hippocampus failed to support dendrite regeneration in this model of reversible paracrine damage to neurons from innate immune activation, and suggest an explanation for the stratification of clinical outcome with APOE seen in several degenerative diseases or brain that are associated with activated innate immune response.

Abstract Background Inheritance of the three different alleles of the human apolipoprotein (apo) E gene (APOE) are associated with varying risk or clinical outcome from a variety of neurologic diseases. ApoE isoform-specific modulation of several pathogenic processes, in addition to amyloid β metabolism in Alzheimer's disease, have been proposed: one of these is innate immune response by glia. Previously we have shown that primary microglia cultures from targeted replacement (TR) APOE mice have apoE isoform-dependent innate immune activation and paracrine damage to neurons that is greatest with TR by the ε4 allele (TR APOE4) and that derives from p38 mitogen-activated protein kinase (p38MAPK) activity. Methods Primary cultures of TR APOE2, TR APOE3 and TR APOE4 astrocytes were stimulated with lipopolysaccharide (LPS). ApoE secretion, cytokine production, and nuclear factor-kappa B (NF-κB) subunit activity were measured and compared. Results Here we showed that activation of primary astrocytes from TR APOE mice with LPS led to TR APOE-dependent differences in cytokine secretion that were greatest in TR APOE2 and that were associated with differences in NF-κB subunit activity. Conclusion Our results suggest that LPS activation of innate immune response in TR APOE glia results in opposing outcomes from microglia and astrocytes as a result of TR APOE-dependent activation of p38MAPK or NF-κB signaling in these two cell types.

Background: Intriguing evidence that glycosylation is disrupted in Alzheimer's Disease (AD) has emerged. However, our knowledge of cell‐specific alterations in the expression of genes involved in glycosylation in the brains of AD patients is limited. Method: In this study, we conducted cell type deconvolution analysis of whole‐tissue RNAseq data to characterize cell type compositional differences and alterations in the expression of glycosylation related genes in the brains of AD patients and those with no cognitive impairment (NCI). Single‐cell human brain RNA‐seq data were obtained from the Allen Brain Map Atlas, consisting of 76,533 total nuclei derived from the cortical tissue of 2 humans. The identified cell types included GABAergic neurons, glutamatergic neurons, astrocytes, oligodendrocytes, oligodendrocyte precursor cells (OPC), microglia, endothelial cells, and vascular leptomeningeal cell (VLMC). The cell‐type deconvolution package CIBERSORT was applied in relative mode with S‐mode (single‐cell mode) batch correction to impute cell fractions and cell‐type‐specific gene expression profiles for NCI and AD samples in the ROSMAP dataset. The differences between cell type fractions of NCI and AD were analyzed by linear mixed regression model and t‐test. Result: In the dorsolateral prefrontal cortex, neuronal cells represented the highest proportion of cells (∼30‐33%), followed by astrocytes (∼30%), and oligodendrocytes (∼20%), endothelial cells (∼7%), microglia (∼7%), VLMC (∼2‐3%), and OPC (<1%). The overall cell type composition was not significantly different between NCI and AD patients, however, the proportion of microglia, VLMC, and OPC was significantly higher in AD patients (p<0.05). The proportion of glutamatergic neuronal cells was significantly lower in female AD patients (p<0.05) compared with NCI controls, but not in males after controlling for age. Microglia‐associated expression of the glycosyltransferases MGAT1, ST6GAL1, and ST6GALNAC3 was observed in AD patients but not in NCI controls. Glutamatergic neuronal cells and VLMC cells associated with the expression of GALNT10 was observed in AD patients but not in NCI controls. In contrast, GALNT11 expression associated with astrocytes was observed in NCI controls but not in AD patients. Conclusion: Our findings indicate changes in cell‐type proportions and cell‐specific changes in glycosyltransferase gene expression in the brains of AD patients compared with NCI controls. [ABSTRACT FROM AUTHOR]