Microglial Phagocytosis/Cell Health High‐Content Assay

Mason, Emily; Soni, Disha; Chu, Shaoyou

doi:10.1002/cpz1.724

Cited by 3 publications

(5 citation statements)

References 59 publications

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“…Since HMC3 expressed mRNA for so many GABAR subunits, we determined the effect of GABA and the GABA A R agonist, muscimol, on HMC3 morphology, Iba1 expression, and metabolism, since these were previously described as important characteristics of microglia and HMC3 [12,26,38]. GABA and muscimol caused a noticeable change in HMC3 morphology and Iba1 localization.…”

Section: Discussionmentioning

confidence: 99%

“…HMC3, also called CHME3, CHME-3, CHME-5, and C13-NJ, is a human microglial cell line that has been used by several groups as a model of human microglia [12], although its phenotype appears variable and shows some lab-to-lab inconsistency [13,14]. HMC3 cells have been reported to express CD11b and CD68 [13], but do not express the astrocyte-specific marker, glial fibrillary acidic protein (GFAP), or the neuronal neurofilament marker NF70KD [13].…”

Section: Introductionmentioning

confidence: 99%

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A Human Microglial Cell Line Expresses γ-Aminobutyric Acid (GABA) Receptors and Responds to GABA and Muscimol by Increasing Production of IL-8

2023

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Gamma-aminobutyric acid (GABA) is an essential neurotransmitter and an important regulator of neuroinflammation and disease. Microglia are important immune cells in the brain that express GABA receptors (GABAR) and respond to both GABA and GABAR agonists, yet the effect of GABA on microglial inflammatory responses is unclear. We hypothesized that GABA and GABAR agonists might modify the activation of a human microglial cell line (HMC3). We further hypothesized that Amanita muscaria extract (AME-1), which contained GABAR agonists (GABA and muscimol), would similarly stimulate HMC3. Ligand-gated GABAR (GABAAR) and G protein-coupled GABAR (GABABR) subunit expression was analyzed by qRT-PCR, metabolic activity was determined by nicotinamide adenine dinucleotide (NADH)-dependent oxidoreductase assay (XTT), reactive oxygen species (ROS) generation was analyzed by 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA), and interleukin-8 (IL-8) production was analyzed by an enzyme-linked immunosorbent assay (ELISA). HMC3 expressed several neuroreceptors such as subunits of the GABAA receptor (GABAAR). HMC3 constitutively produce IL-8 and ROS. Both muscimol and GABA stimulated HMC3 to produce more IL-8 but had no effect on constitutive ROS production. GABA and muscimol altered the morphology and Iba1 localization of HMC3. GABA, but not muscimol, increased HMC3 metabolic activity. Similarly, AME-1 induced HMC3 to produce more IL-8 but not ROS and altered cell morphology and Iba1 localization. GABA induction of IL-8 was blocked by bicuculline, an antagonist of GABAAR. AME-1-induced production of IL-8 was not blocked by bicuculline, suggesting that AME-1’s effect on HMC3 was independent of GABAAR. In conclusion, these data show that GABA and GABA agonists stimulate HMC3 to increase their production of IL-8. Mixtures that contain GABA and muscimol, such as AME-1, have similar effects on HMC3 that are independent of GABAAR.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Human Microglial Cell Line Expresses γ-Aminobutyric Acid (GABA) Receptors and Responds to GABA and Muscimol by Increasing Production of IL-8

2023

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“…Because THP1 cells are employed frequently as a model of monocyte cells and SHIP1‐dependent AKT signaling has been observed previously in this cellular context, 30,31 a signaling assay was established using THP1 cells measuring total AKT (tAKT) and phospho‐Akt (pAKT) levels, which provides further evidence of on‐target pharmacology. A phenotypic high‐content imaging assay with simultaneous measures of phagocytosis, cell number, and nuclear intensity was used to ensure that the compounds are affecting desired cellular pharmacology without cytotoxicity 32 . Model cell lines, BV2 and HMC3, were used to provide adequate throughput for structure activity relationship (SAR) studies.…”

Section: Methodsmentioning

confidence: 99%

“…Results are reported in Table 2 and example concentration-response curves for compounds 14 and Compounds that demonstrated significant cellular target engagement and changes in AKT signaling were evaluated in a phenotypic high-content imaging assay with simultaneous measures of phagocytosis, cell number, and nuclear intensity to assess both cellular pharmacology and cell health in parallel. 32 Model cell lines BV2 and HMC3 were used to provide adequate throughput. Microglia isolated from mouse brain were used to verify that results from the…”

Section: Cellular Pharmacologymentioning

confidence: 99%

“…A phenotypic high-content imaging assay with simultaneous measures of phagocytosis, cell number, and nuclear intensity was used to ensure that the compounds are affecting desired cellular pharmacology without cytotoxicity. 32 Model cell lines, BV2 and HMC3, were used to provide adequate throughput for structure activity relationship (SAR) studies. Primary mouse microglia were used to ensure that observations in BV2 cells reflect primary cell activity.…”

Section: In Vitro Assay Developmentmentioning

confidence: 99%

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SHIP1 therapeutic target enablement: Identification and evaluation of inhibitors for the treatment of late‐onset Alzheimer's disease

Jesudason,

Mason,

Chu

et al. 2023

A&D Transl Res & Clin Interv

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INTRODUCTIONThe risk of developing Alzheimer's disease is associated with genes involved in microglial function. Inositol polyphosphate‐5‐phosphatase (INPP5D), which encodes Src homology 2 (SH2) domain–containing inositol polyphosphate 5‐phosphatase 1 (SHIP1), is a risk gene expressed in microglia. Because SHIP1 binds receptor immunoreceptor tyrosine‐based inhibitory motifs (ITIMs), competes with kinases, and converts PI(3,4,5)P3 to PI(3,4)P2, it is a negative regulator of microglia function. Validated inhibitors are needed to evaluate SHIP1 as a potential therapeutic target.METHODSWe identified inhibitors and screened the enzymatic domain of SHIP1. A protein construct containing two domains was used to evaluate enzyme inhibitor potency and selectivity versus SHIP2. Inhibitors were tested against a construct containing all ordered domains of the human and mouse proteins. A cellular thermal shift assay (CETSA) provided evidence of target engagement in cells. Phospho‐AKT levels provided further evidence of on‐target pharmacology. A high‐content imaging assay was used to study the pharmacology of SHIP1 inhibition while monitoring cell health. Physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties were evaluated to select a compound suitable for in vivo studies.RESULTSSHIP1 inhibitors displayed a remarkable array of activities and cellular pharmacology. Inhibitory potency was dependent on the protein construct used to assess enzymatic activity. Some inhibitors failed to engage the target in cells. Inhibitors that were active in the CETSA consistently destabilized the protein and reduced pAKT levels. Many SHIP1 inhibitors were cytotoxic either at high concentration due to cell stress or they potently induced cell death depending on the compound and cell type. One compound activated microglia, inducing phagocytosis at concentrations that did not result in significant cell death. A pharmacokinetic study demonstrated brain exposures in mice upon oral administration.DISCUSSION3‐((2,4‐Dichlorobenzyl)oxy)‐5‐(1‐(piperidin‐4‐yl)‐1H‐pyrazol‐4‐yl) pyridine activated primary mouse microglia and demonstrated exposures in mouse brain upon oral dosing. Although this compound is our recommended chemical probe for investigating the pharmacology of SHIP1 inhibition at this time, further optimization is required for clinical studies.Highlights Cellular thermal shift assay (CETSA) and signaling (pAKT) assays were developed to provide evidence of src homology 2 (SH2) domain‐contaning inositol phosphatase 1 (SHIP1) target engagement and on‐target activity in cellular assays. A phenotypic high‐content imaging assay with simultaneous measures of phagocytosis, cell number, and nuclear intensity was developed to explore cellular pharmacology and monitor cell health. SHIP1 inhibitors demonstrate a wide range of activity and cellular pharmacology, and many reported inhibitors are cytotoxic. The chemical probe 3‐((2,4‐dichlorobenzyl)oxy)−5‐(1‐(piperidin‐4‐yl)−1H‐pyrazol‐4‐yl) pyridine is recommended to explore SHIP1 pharmacology.

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A bottom-up approach identifies the antipsychotic and antineoplastic trifluoperazine and the ribose derivative deoxytubercidin as novel microglial phagocytosis inhibitors

Rodriguez-Iglesias,

Paris,

Valero

et al. 2024

Preprint

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Phagocytosis is an indispensable function of microglia, the brain professional phagocytes. Microglia are particularly efficient phagocytosing cells that undergo programmed cell death (apoptosis) in physiological conditions. However, mounting evidence suggests microglial phagocytosis dysfunction in multiple brain disorders. These observations prompted us to search for phagocytosis modulators (enhancers or inhibitors) with therapeutic potential. We used a bottom-up strategy that consisted on the identification of phagocytosis modulators using phenotypic high throughput screenings (HTSs) in cell culture and validation in organotypic cultures andin vivo. We performed two complementary HTS campagnes: at Achucarro, we used primary cultures of mouse microglia and compounds of the Prestwick Chemical Library; at Roche, we used human iPSC derived macrophage-like cells and a proprietary chemo-genomic library with 2,200 compounds with known mechanism-of-action. Next, we validated the more robust compounds using hippocampal organotypic cultures and identified two hits: trifluoperazine, a dopaminergic and adrenergic antagonist used as an antipsychotic and antineoplastic; and deoxytubercidin, a ribose derivative. Finally, we tested whether these compounds were able to modulate phagocytosis of apoptotic newborn cells in the adult hippocampal neurogenic nichein vivoby administering them into the mouse hippocampus using osmotic minipumps. We confirmed that both trifluoperazine and deoxytubercidin have anti-phagocytic activityin vivo, and validated our bottom-up strategy to identify novel phagocytosis modulators. These results show that chemical libraries with anotated mechanism of action are an starting point for the pharmacological modulation of microglia in drug discovery projects aiming at the therapeutic manipulation of phagocytosis in brain diseases.GRAPHICAL ABSTRACT

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Microglial Phagocytosis/Cell Health High‐Content Assay

Cited by 3 publications

References 59 publications

A Human Microglial Cell Line Expresses γ-Aminobutyric Acid (GABA) Receptors and Responds to GABA and Muscimol by Increasing Production of IL-8

A Human Microglial Cell Line Expresses γ-Aminobutyric Acid (GABA) Receptors and Responds to GABA and Muscimol by Increasing Production of IL-8

SHIP1 therapeutic target enablement: Identification and evaluation of inhibitors for the treatment of late‐onset Alzheimer's disease

A bottom-up approach identifies the antipsychotic and antineoplastic trifluoperazine and the ribose derivative deoxytubercidin as novel microglial phagocytosis inhibitors

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