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SIRT1 Mediates Melatonin's Effects on Microglial Activation in Hypoxia: In Vitro and In Vivo Evidence.

ABSTRACT: Melatonin exerts direct neuroprotection against cerebral hypoxic damage, but the mechanisms of its action on microglia have been less characterized. Using both in vitro and in vivo models of hypoxia, we here focused on the role played by silent mating type information regulation 2 homolog 1 (SIRT1) in melatonin's effects on microglia. Viability of rat primary microglia or microglial BV2 cells and SH-SY5Y neurons was significantly reduced after chemical hypoxia with CoCl2 (250 ?M for 24 h). Melatonin (1 ?M) significantly attenuated CoCl2 toxicity on microglia, an effect prevented by selective SIRT1 inhibitor EX527 (5 ?M) and AMP-activated protein kinase (AMPK) inhibitor BML-275 (2 ?M). CoCl2 did not modify SIRT1 expression, but prevented nuclear localization, while melatonin appeared to restore it. CoCl2 induced nuclear localization of hypoxia-inducible factor-1? (HIF-1?) and nuclear factor-kappa B (NF-kB), an effect contrasted by melatonin in an EX527-dependent fashion. Treatment of microglia with melatonin attenuated potentiation of neurotoxicity. Common carotid occlusion was performed in p7 rats, followed by intraperitoneal injection of melatonin (10 mg/kg). After 24 h, the number of Iba1+ microglia in the hippocampus of hypoxic rats was significantly increased, an effect not prevented by melatonin. At this time, SIRT1 was only detectable in the amoeboid, Iba1+ microglial population selectively localized in the corpus callosum. In these cells, nuclear localization of SIRT1 was significantly lower in hypoxic animals, an effect prevented by melatonin. NF-kB showed an opposite expression pattern, where nuclear localization in Iba1+ cells was significantly higher in hypoxic, but not in melatonin-treated animals. Our findings provide new evidence for a direct effect of melatonin on hypoxic microglia through SIRT1, which appears as a potential pharmacological target against hypoxic-derived neuronal damage.

SUBMITTER: Merlo S

PROVIDER: S-EPMC7175216 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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SIRT1 Mediates Melatonin's Effects on Microglial Activation in Hypoxia: In Vitro and In Vivo Evidence.

Merlo Sara S Luaces Juan Pablo JP Spampinato Simona Federica SF Toro-Urrego Nicolas N Caruso Grazia Ilaria GI D'Amico Fabio F Capani Francisco F Sortino Maria Angela MA

Biomolecules 20200227 3

Melatonin exerts direct neuroprotection against cerebral hypoxic damage, but the mechanisms of its action on microglia have been less characterized. Using both in vitro and in vivo models of hypoxia, we here focused on the role played by silent mating type information regulation 2 homolog 1 (SIRT1) in melatonin's effects on microglia. Viability of rat primary microglia or microglial BV2 cells and SH-SY5Y neurons was significantly reduced after chemical hypoxia with CoCl<sub>2</sub> (250 μM for 2 ...[more]

PMID: 32120833

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Project description:BackgroundMicroglia, the resident immune cells of the brain, play a critical role in numerous diseases, but are a minority cell type and difficult to genetically manipulate in vivo with viral vectors and other approaches. Primary cultures allow a more controlled setting to investigate these cells, but morphological and transcriptional changes upon removal from their normal brain environment raise many caveats from in vitro studies.MethodsTo investigate whether cultured microglia recapitulate in vivo microglial signatures, we used single-cell RNA sequencing (scRNAseq) to compare microglia freshly isolated from the brain to primary microglial cultures. We performed cell population discovery, differential expression analysis, and gene co-expression module analysis to compare signatures between in vitro and in vivo microglia. We constructed causal predictive network models of transcriptional regulators from the scRNAseq data and identified a set of potential key drivers of the cultured phenotype. To validate this network analysis, we knocked down two of these key drivers, C1qc and Prdx1, in primary cultured microglia and quantified changes in microglial activation markers.ResultsWe found that, although often assumed to be a relatively homogenous population of cells in culture, in vitro microglia are a highly heterogeneous population consisting of distinct subpopulations of cells with transcriptional profiles reminiscent of macrophages and monocytes, and are marked by transcriptional programs active in neurodegeneration and other disease states. We found that microglia in vitro presented transcriptional activation of a set of "culture shock genes" not found in freshly isolated microglia, characterized by strong upregulation of disease-associated genes including Apoe, Lyz2, and Spp1, and downregulation of homeostatic microglial markers, including Cx3cr1, P2ry12, and Tmem119. Finally, we found that cultured microglia prominently alter their transcriptional machinery modulated by key drivers from the homeostatic to activated phenotype. Knockdown of one of these drivers, C1qc, resulted in downregulation of microglial activation genes Lpl, Lyz2, and Ccl4.ConclusionsOverall, our data suggest that when removed from their in vivo home environment, microglia suffer a severe case of "culture shock", drastically modulating their transcriptional regulatory network state from homeostatic to activated through upregulation of modules of culture-specific genes. Consequently, cultured microglia behave as a disparate cell type that does not recapitulate the homeostatic signatures of microglia in vivo. Finally, our predictive network model discovered potential key drivers that may convert activated microglia back to their homeostatic state, allowing for more accurate representation of in vivo states in culture. Knockdown of key driver C1qc partially attenuated microglial activation in vitro, despite C1qc being only weakly upregulated in culture. This suggests that even genes that are not strongly differentially expressed across treatments or preparations may drive downstream transcriptional changes in culture.

Dataset Information

SIRT1 Mediates Melatonin's Effects on Microglial Activation in Hypoxia: In Vitro and In Vivo Evidence.

Publications

SIRT1 Mediates Melatonin's Effects on Microglial Activation in Hypoxia: In Vitro and In Vivo Evidence.

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