Project description:Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods together with gene expression and histone acetylation studies in an established cell culture model of trophoblast differentiation. Trophoblast differentiation was associated with increased abundance of the TCA cycle intermediates citrate and α-ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblast. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in the orchestration of histone acetylation and gene expression during trophoblast differentiation.
Project description:TLR activation induces inflammatory responses in macrophages by activating temporally defined transcriptional cascades within the first hours after stimulation. Whether concurrent changes in the cellular metabolism that occur upon TLR activation influence the quality of the transcriptional responses remain unknown. Here we investigated how macrophages adopt their metabolism early after activation to regulate TLR-inducible gene induction. Macrophages increase glucose metabolism and adopt fluxes through the TCA cycle to foster Citrate synthesis. We concomitantly observe activation of ATP-Citrate Lyase (Acly), resulting in augmented acetyl-CoA synthesis and histone acetylation. To investigate which genes and genes classes require ATP-citrate lyase activity for induction we stimulated bone marrow derived macrophages with LPS after ATP-citrate lyase inhibition.
Project description:Autophagy is a cellular and energy homeostatic mechanism that contributes to maintain the number of primordial follicles, germ cell survival, and anti-ovarian aging. However, it remains unknown whether autophagy in granulosa cells affects the oocyte maturation. Here, we show a clear tendency of reduced autophagy level in human granulosa cell from women of advanced maternal age, implying a potential negative correlation between autophagy level and oocyte quality. We therefore established a co-culture system and show that either pharmacological inhibition or genetic ablation of autophagy in granulosa cells negatively affect the oocyte quality and fertilization ability. Moreover, our metabolomics analysis indicates that the adverse impact of autophagy impairment on oocyte quality is mediated by downregulated citrate levels, while exogenous supplementation of citrate can significantly restore the oocyte maturation. In molecular level, we found ATP citrate lyase (Acly), which is a crucial enzyme catalyzing the cleavage of citrate, was preferentially associated with K63-linked ubiquitin chains and recognized by the autophagy receptor protein SQSTM1/p62 for the selective autophagic degradation. In human follicles, autophagy levels in granulosa cells was downregulated with maternal aging, accompanied by decreased citrate in the follicular fluid, implying a potential correlation between citrate metabolism and oocyte quality. We also show that elevated citrate levels in porcine follicular fluid promote oocyte maturation. Collectively, our data reveal that autophagy in granulosa cells is a beneficial mechanism to maintain a certain degree of citrate by selectively targeting Acly during oocyte maturation.
Project description:Slc25a1 encodes for the mitochondrial citrate carrier, a mitochondrial inner membrane transporter that mediates mitochondrial citrate export. Systemic deletion of Slc25a1 (both homozygous and heterozygous loss) leads to cardiac structural defects and mitochondrial dysfunction. Transcriptomic profiles of metabolic gene expression in the the developing mouse heart at E17.5 reveal alterations in metabolic pathways including oxdiative phosphorylation, supporting mitochondrial structural and functional defects observed with loss of this transporter.
Project description:The only validated treatment to prevent brain damage associated with hypoxia-ischemia (HI) encephalopathy of the newborn is controlled hypothermia with limited benefits. Additional putative neuroprotective drug candidates include sildenafil citrate, a phosphodiesterase-type 5 inhibitor. The main objective of this preclinical study is to assess its ability to reduce HI-induced neuroinflammation, in particular through its potential effect on microglial activation. HI was induced in P10 Sprague–Dawley rats by unilateral carotid permanent artery occlusion and hypoxia (HI), and treated by either hypothermia (HT) alone, Sildenafil (Sild) alone or combined treatment (SildHT). Lesion size, glial activation were analyzed by immunohistochemistry, qRT-PCR and proteomic analyses performed at P13. Exposure to any treatment was not associated with significant reduction in lesions size both in cerebral cortex and hippocampus, 72h after HI. Significant reductions in either Iba1+ (within the ipsilateral hemisphere) or GFAP+ cells (within the ipsilateral hippocampus) were observed in SildHT group, but not in the other treatment groups. In microglia sorted cells, pro-inflammatory markers, ie. Il1b, Il6, Nos2, and CD86 were significantly downregulated in SildHT treatment group only. These changes were restricted to ipsilateral hemisphere, were not evidenced in sorted astrocytes, and were not sex-dependent. Proteomic analyses in sorted microglia refined the pro-inflammatory effect of HI and confirmed a biologically relevant impact of SildHT on specific molecular pathways including notably genes related to neutrophilic functions. Our findings demonstrate that Sildenafil combined with controlled hypothermia confers maximum effect to mitigate microglial activation induced by HI through complex proteomic regulation. The reduction of neuroinflammation induced by Sildenafil may represent an interesting therapeutic strategy for neonatal neuroprotection.
Project description:The effect of nitrate reduction (anaerobic cultivation in the presence of heme, vitamin K2 and nitrate) was compared with anaerobic cultivation supplemented with citrate (Lactobacillus plantarum). The medium was chemically defined medium with mannitol as main carbon source Two-condition experiment, nitrate vs citrate reducing cells. Biological replicates: 4 nitrate reducing cultures, 4 citrate reducing cultures, independently grown and harvested. Two slides were used, each slide contained 8 Arrays. Citrate reducing cultures are called reactor 1-4, Nitrate reducing cultures are called reactor A-D