scholarly journals De novo Neurosteroidogenesis in Human Microglia: Involvement of the 18 kDa Translocator Protein

2021 ◽  
Vol 22 (6) ◽  
pp. 3115
Author(s):  
Lorenzo Germelli ◽  
Eleonora Da Pozzo ◽  
Chiara Giacomelli ◽  
Chiara Tremolanti ◽  
Laura Marchetti ◽  
...  
Keyword(s):  
Neurotrophic Factor ◽  
De Novo ◽  
Neuroactive Steroids ◽  
Translocator Protein ◽  
Compensatory Mechanism ◽  
Human Microglia ◽  
De Novo Biosynthesis ◽  
Murine Microglia ◽  
Synthetic Ligand ◽  
Tspo Expression

Neuroactive steroids are potent modulators of microglial functions and are capable of counteracting their excessive reactivity. This action has mainly been ascribed to neuroactive steroids released from other sources, as microglia have been defined unable to produce neurosteroids de novo. Unexpectedly, immortalized murine microglia recently exhibited this de novo biosynthesis; herein, de novo neurosteroidogenesis was characterized in immortalized human microglia. The results demonstrated that C20 and HMC3 microglial cells constitutively express members of the neurosteroidogenesis multiprotein machinery—in particular, the transduceosome members StAR and TSPO, and the enzyme CYP11A1. Moreover, both cell lines produce pregnenolone and transcriptionally express the enzymes involved in neurosteroidogenesis. The high TSPO expression levels observed in microglia prompted us to assess its role in de novo neurosteroidogenesis. TSPO siRNA and TSPO synthetic ligand treatments were used to reduce and prompt TSPO function, respectively. The TSPO expression downregulation compromised the de novo neurosteroidogenesis and led to an increase in StAR expression, probably as a compensatory mechanism. The pharmacological TSPO stimulation the de novo neurosteroidogenesis improved in turn the neurosteroid-mediated release of Brain-Derived Neurotrophic Factor. In conclusion, these results demonstrated that de novo neurosteroidogenesis occurs in human microglia, unravelling a new mechanism potentially useful for future therapeutic purposes.

scholarly journals Phospholipid scramblase-3 regulates cardiolipin de novo biosynthesis and its resynthesis in growing HeLa cells

2006 ◽  
Vol 401 (1) ◽  
pp. 103-109 ◽  
Author(s):  
Quyen Van ◽  
Jihua Liu ◽  
Biao Lu ◽  
Kenneth R. Feingold ◽  
Yuguang Shi ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Hela Cells ◽  
De Novo ◽  
Compensatory Mechanism ◽  
Mitochondrial Localization ◽  
Acyltransferase Activity ◽  
Protein Levels ◽  
Phospholipid Scramblase ◽  
De Novo Biosynthesis ◽  
The Family

PLS3 (phospholipid scramblase-3) is a new member of the family of phospholipid scramblases and transports CL (cardiolipin) from the inner to the outer mitochondrial membrane. In the present paper we examined whether changing the levels of functional PLS3 in HeLa cells altered de novo CL biosynthesis and its resynthesis. HeLa cells overexpressing PLS3 or expressing a disrupted PLS3 (F258V) or control were incubated with [1,3-3H]glycerol and radioactivity incorporated into CL was determined. CL biosynthesis from [1,3-3H]glycerol was increased 1.8-fold in PLS3 cells and 2.1-fold in F258V cells compared with control. This was due to a 64% (P<0.05) and 2.6-fold (P<0.05) elevation in CL synthase activity in PLS3 and F258V cells respectively, compared with control, and not due to changes in phosphatidylglycerolphosphate synthase activity. The increase in CL synthase activity in these cells was due to an increase in its mRNA expression. In contrast, resynthesis of CL from [1-14C]linoleic acid was reduced 52% (P<0.05) in PLS3 and 45% (P<0.05) in F258V cells compared with control and this was due to a reduction in mitochondrial monolysocardiolipin acyltransferase activity. Although protein levels of mitochondrial monolysocardiolipin acyltransferase were unaltered, activity and mRNA expression of endoplasmic reticulum monolysocardiolipin acyltransferase was upregulated in PLS3 and F258V cells compared with controls. These data indicate that the CL resynthesis in HeLa cells is sensitive to the mitochondrial localization of CL and not the level of the reacylating enzymes. Alterations in functional PLS3 levels in PLS3 or F258V cells did not affect the mitochondrial decarboxylation of phosphatidylserine to phosphatidylethanolamine indicating that the biosynthetic changes to CL were specific for this mitochondrial phospholipid. We hypothesize that the cardiolipin resynthesis machinery in the cell ‘senses’ altered levels of CL on mitochondrial membranes and that de novo CL biosynthesis is up-regulated in HeLa cells as a compensatory mechanism in response to altered movement of mitochondrial CL. The results identify PLS3 as a novel regulator of CL de novo biosynthesis and its resynthesis.

scholarly journals CRISPR-Cas9 Mediated TSPO Gene Knockout alters Respiration and Cellular Metabolism in Human Primary Microglia Cells

2019 ◽  
Vol 20 (13) ◽  
pp. 3359 ◽  
Author(s):  
Vladimir M. Milenkovic ◽  
Dounia Slim ◽  
Stefanie Bader ◽  
Victoria Koch ◽  
Elena-Sofia Heinl ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Respiratory Function ◽  
Mitochondrial Membrane ◽  
De Novo ◽  
Gene Knockout ◽  
Functional Characterization ◽  
Translocator Protein ◽  
Microglia Cell ◽  
Tspo Expression

The 18 kDa translocator protein (TSPO) is an evolutionary conserved cholesterol binding protein localized in the outer mitochondrial membrane. It has been implicated in the regulation of various cellular processes including oxidative stress, proliferation, apoptosis, and steroid hormone biosynthesis. Since the expression of TSPO in activated microglia is upregulated in various neuroinflammatory and neurodegenerative disorders, we set out to examine the role of TSPO in an immortalized human microglia C20 cell line. To this end, we performed a dual approach and used (i) lentiviral shRNA silencing to reduce TSPO expression, and (ii) the CRISPR/Cas9 technology to generate complete TSPO knockout microglia cell lines. Functional characterization of control and TSPO knockdown as well as knockout cells, revealed only low de novo steroidogenesis in C20 cells, which was not dependent on the level of TSPO expression or influenced by the treatment with TSPO-specific ligands. In contrast to TSPO knockdown C20 cells, which did not show altered mitochondrial function, the TSPO deficient knockout cells displayed a significantly decreased mitochondrial membrane potential and cytosolic Ca2+ levels, as well as reduced respiratory function. Performing the rescue experiment by lentiviral overexpression of TSPO in knockout cells, increased oxygen consumption and restored respiratory function. Our study provides further evidence for a significant role of TSPO in cellular and mitochondrial metabolism and demonstrates that different phenotypes of mitochondrial function are dependent on the level of TSPO expression.

scholarly journals TSPO PET detects acute neuroinflammation but not diffuse chronically activated MHCII microglia in the rat

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Nassir U. Al-Khishman ◽  
Qi Qi ◽  
Austyn D. Roseborough ◽  
Alexander Levit ◽  
Brian L. Allman ◽  
...  
Keyword(s):  
Corpus Callosum ◽  
Tracer Uptake ◽  
Translocator Protein ◽  
Imaging Biomarkers ◽  
Wild Type ◽  
Activated Microglia ◽  
Fischer 344 ◽  
Histocompatibility Complex ◽  
Prodromal Alzheimer’S Disease ◽  
Tspo Expression

Abstract Background Accurate and sensitive imaging biomarkers are required to study the progression of white matter (WM) inflammation in neurodegenerative diseases. Radioligands targeting the translocator protein (TSPO) are considered sensitive indicators of neuroinflammation, but it is not clear how well the expression of TSPO coincides with major histocompatibility complex class II (MHCII) molecules in WM. This study aimed to test the ability of TSPO to detect activated WM microglia that are immunohistochemically positive for MHCII in rat models of prodromal Alzheimer’s disease and acute subcortical stroke. Methods Fischer 344 wild-type (n = 12) and TgAPP21 (n = 11) rats were imaged with [18F]FEPPA PET and MRI to investigate TSPO tracer uptake in the corpus callosum, a WM region known to have high levels of MHCII activated microglia in TgAPP21 rats. Wild-type rats subsequently received an endothelin-1 (ET1) subcortical stroke and were imaged at days 7 and 28 post-stroke before immunohistochemistry of TSPO, GFAP, iNOS, and the MHCII rat antigen, OX6. Results [18F]FEPPA PET was not significantly affected by genotype in WM and only detected increases near the ET1 infarct (P = 0.033, infarct/cerebellum uptake ratio: baseline = 0.94 ± 0.16; day 7 = 2.10 ± 0.78; day 28 = 1.77 ± 0.35). Immunohistochemistry confirmed that only the infarct (TSPO cells/mm2: day 7 = 555 ± 181; day 28 = 307 ± 153) and WM that is proximal to the infarct had TSPO expression (TSPO cells/mm2: day 7 = 113 ± 93; day 28 = 5 ± 7). TSPO and iNOS were not able to detect the chronic WM microglial activation that was detected with MHCII in the contralateral corpus callosum (day 28 OX6% area: saline = 0.62 ± 0.38; stroke = 4.30 ± 2.83; P = .029). Conclusion TSPO was only expressed in the stroke-induced insult and proximal tissue and therefore was unable to detect remote and non-insult-related chronically activated microglia overexpressing MHCII in WM. This suggests that research in neuroinflammation, particularly in the WM, would benefit from MHCII-sensitive radiotracers.

scholarly journals Identification of 3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine Derivatives as Novel Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase

2021 ◽  
Vol 22 (13) ◽  
pp. 7236
Author(s):  
Endah Dwi Hartuti ◽  
Takaya Sakura ◽  
Mohammed S. O. Tagod ◽  
Eri Yoshida ◽  
Xinying Wang ◽  
...  
Keyword(s):  
Drug Target ◽  
De Novo ◽  
Dihydroorotate Dehydrogenase ◽  
Chemical Library ◽  
New Class ◽  
De Novo Biosynthesis ◽  
Starting Point ◽  
Novel Drugs ◽  
Low Toxicity ◽  
Fourth Step

Plasmodium falciparum’s resistance to available antimalarial drugs highlights the need for the development of novel drugs. Pyrimidine de novo biosynthesis is a validated drug target for the prevention and treatment of malaria infection. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the oxidation of dihydroorotate to orotate and utilize ubiquinone as an electron acceptor in the fourth step of pyrimidine de novo biosynthesis. PfDHODH is targeted by the inhibitor DSM265, which binds to a hydrophobic pocket located at the N-terminus where ubiquinone binds, which is known to be structurally divergent from the mammalian orthologue. In this study, we screened 40,400 compounds from the Kyoto University chemical library against recombinant PfDHODH. These studies led to the identification of 3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine and its derivatives as a new class of PfDHODH inhibitor. Moreover, the hit compounds identified in this study are selective for PfDHODH without inhibition of the human enzymes. Finally, this new scaffold of PfDHODH inhibitors showed growth inhibition activity against P. falciparum 3D7 with low toxicity to three human cell lines, providing a new starting point for antimalarial drug development.

scholarly journals Dietary Essential Amino Acid Restriction Promotes Hyperdipsia via Hepatic FGF21

Nutrients ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1469
Author(s):  
Patricia M. Rusu ◽  
Andrea Y. Chan ◽  
Mathias Heikenwalder ◽  
Oliver J. Müller ◽  
Adam J. Rose
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Dietary Protein ◽  
Essential Amino Acid ◽  
De Novo ◽  
Fibroblast Growth Factor 21 ◽  
Fibroblast Growth ◽  
De Novo Biosynthesis ◽  
Dietary Requirements ◽  
Dietary Amino Acid

Prior studies have reported that dietary protein dilution (DPD) or amino acid dilution promotes heightened water intake (i.e., hyperdipsia) however, the exact dietary requirements and the mechanism responsible for this effect are still unknown. Here, we show that dietary amino acid (AA) restriction is sufficient and required to drive hyperdipsia during DPD. Our studies demonstrate that particularly dietary essential AA (EAA) restriction, but not non-EAA, is responsible for the hyperdipsic effect of total dietary AA restriction (DAR). Additionally, by using diets with varying amounts of individual EAA under constant total AA supply, we demonstrate that restriction of threonine (Thr) or tryptophan (Trp) is mandatory and sufficient for the effects of DAR on hyperdipsia and that liver-derived fibroblast growth factor 21 (FGF21) is required for this hyperdipsic effect. Strikingly, artificially introducing Thr de novo biosynthesis in hepatocytes reversed hyperdipsia during DAR. In summary, our results show that the DPD effects on hyperdipsia are induced by the deprivation of Thr and Trp, and in turn, via liver/hepatocyte-derived FGF21.

scholarly journals Cellular sources of TSPO expression in healthy and diseased brain

2021 ◽  
Author(s):  
Erik Nutma ◽  
Kelly Ceyzériat ◽  
Sandra Amor ◽  
Stergios Tsartsalis ◽  
Philippe Millet ◽  
...  
Keyword(s):  
Cell Activity ◽  
Brain Regions ◽  
Cellular Level ◽  
Translocator Protein ◽  
Disease Stage ◽  
Animal Models Of Disease ◽  
Positron Emission ◽  
Neuropsychiatric Diseases ◽  
Tspo Expression

AbstractThe 18 kDa translocator protein (TSPO) is a highly conserved protein located in the outer mitochondrial membrane. TSPO binding, as measured with positron emission tomography (PET), is considered an in vivo marker of neuroinflammation. Indeed, TSPO expression is altered in neurodegenerative, neuroinflammatory, and neuropsychiatric diseases. In PET studies, the TSPO signal is often viewed as a marker of microglial cell activity. However, there is little evidence in support of a microglia-specific TSPO expression. This review describes the cellular sources and functions of TSPO in animal models of disease and human studies, in health, and in central nervous system diseases. A discussion of methods of analysis and of quantification of TSPO is also presented. Overall, it appears that the alterations of TSPO binding, their cellular underpinnings, and the functional significance of such alterations depend on many factors, notably the pathology or the animal model under study, the disease stage, and the involved brain regions. Thus, further studies are needed to fully determine how changes in TSPO binding occur at the cellular level with the ultimate goal of revealing potential therapeutic pathways.

scholarly journals De novo biosynthesis of linoleic acid is widespread in parasitic wasps

2021 ◽  
Author(s):  
Bastian Broschwitz ◽  
Lorena Prager ◽  
Tamara Pokorny ◽  
Joachim Ruther
Keyword(s):  
Linoleic Acid ◽  
De Novo ◽  
Parasitic Wasps ◽  
De Novo Biosynthesis
Sign in / Sign up

Export Citation Format

Share Document