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 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 Effect of rapamycin on mitochondria and lysosomes in fibroblasts from patients with mtDNA mutations

2021 ◽  
Author(s):  
Nashwa J. Cheema ◽  
Jessie M. Cameron ◽  
David A. Hood
Keyword(s):  
Mitochondrial Function ◽  
Mitochondrial Membrane ◽  
Current Treatment ◽  
Basal Respiration ◽  
Healthy Individuals ◽  
Cellular Mechanisms ◽  
Mtdna Mutations ◽  
Cultured Skin ◽  
Patient Cell

Maintaining mitochondrial function and dynamics is crucial for cellular health. In muscle, defects in mitochondria result in severe myopathies where accumulation of damaged mitochondria causes deterioration and dysfunction. Importantly, understanding the role of mitochondria in disease is a necessity to determine future therapeutics. One of the most common myopathies is mitochondrial encephalopathy lactic acidosis stroke-like episodes (MELAS), which has no current treatment. Recently, MELAS patients treated with rapamycin exhibited improved clinical outcomes. However, the cellular mechanisms of rapamycin effects in MELAS patients are currently unknown. In this study, we used cultured skin fibroblasts as a window into the mitochondrial dysfunction evident in MELAS cells, as well as to study the mechanisms of rapamycin action, compared to control, healthy individuals. We observed that mitochondria from patients were fragmented, had a 3-fold decline in the average speed of motility, a 2-fold reduced mitochondrial membrane potential and a 1.5-2-fold decline in basal respiration. Despite the reduction in mitochondrial function, mitochondrial import protein Tim23 was elevated in patient cell lines. MELAS fibroblasts exhibited increased MnSOD levels and lysosomal function when compared to healthy controls. Treatment of MELAS fibroblasts with rapamycin for 24 hrs resulted in increased mitochondrial respiration compared to control cells, a higher lysosome content, and a greater localization of mitochondria to lysosomes. Our studies suggest that rapamycin has the potential to improve cellular health even in the presence of mtDNA defects, primarily via an increase in lysosomal content.

Guwiyang Wurra–‘Fire Mouse’: a global gene knockout model for TSPO/PBR drug development, loss-of-function and mechanisms of compensation studies

2015 ◽  
Vol 43 (4) ◽  
pp. 553-558 ◽  
Author(s):  
Ryan J. Middleton ◽  
Guo-Jun Liu ◽  
Richard B. Banati
Keyword(s):  
Mitochondrial Permeability Transition ◽  
Gene Knockout ◽  
Physiological Role ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Benzodiazepine Receptor ◽  
Translocator Protein ◽  
Loss Of Function ◽  
Recent Developments

The highly conserved 18-kDa translocator protein (TSPO) or peripheral benzodiazepine receptor (PBR), is being investigated as a diagnostic and therapeutic target for disease conditions ranging from inflammation to neurodegeneration and behavioural illnesses. Many functions have been attributed to TSPO/PBR including a role in the mitochondrial permeability transition pore (MPTP), steroidogenesis and energy metabolism. In this review, we detail the recent developments in determining the physiological role of TSPO/PBR, specifically based on data obtained from the recently generated Tspo knockout mouse models. In addition to defining the role of TSPO/PBR, we also describe the value of Tspo knockout mice in determining the selectivity, specificity and presence of any off-target effects of TSPO/PBR ligands.

Characterization of the AMP-binding protein gene family in Arabidopsis thaliana: will the real acyl-CoA synthetases please stand up?

2000 ◽  
Vol 28 (6) ◽  
pp. 955-957 ◽  
Author(s):  
J. Shockey ◽  
J. Schnurr ◽  
J. Browse
Keyword(s):  
Protein Gene ◽  
De Novo ◽  
Functional Characterization ◽  
De Novo Synthesis ◽  
Triacylglycerol Composition ◽  
Sequence Comparisons ◽  
Triacylglycerol Metabolism ◽  
Binding Protein Gene

One of the most prominent and important topics in modern agricultural biotechnology is the manipulation of oilseed triacylglycerol composition. Towards this goal, we have sought to identify and characterize acyl-CoA synthetases (ACSs), which play an important role in both de novo synthesis and modification of existing lipids. We have identified and cloned 20 different genes that bear strong sequence homology to known ACSs from other organisms. Through sequence comparisons and functional characterization, we have identified several members of this group that encode ACSs, while the other genes fall into the broader category of genes for AMP-binding proteins (AMPBPs). Distinguishing ACSs from AMPBPs will simplify our efforts to understand the role of ACS in triacylglycerol metabolism.

scholarly journals Inhibiting adenine synthesis attenuates glioblastoma cell stemness and temozolomide resistance

2021 ◽  
Author(s):  
Simranjit X. Singh ◽  
Rui Yang ◽  
Kristen Roso ◽  
Landon J. Hansen ◽  
Changzheng Du ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mitochondrial Function ◽  
Brain Cancer ◽  
De Novo ◽  
Critical Role ◽  
Glioblastoma Cell ◽  
Respiratory Capacity ◽  
Complementary Approach

Glioblastoma (GBM) is a lethal brain cancer exhibiting high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work shows that homozygous MTAP deletion, a genetic alteration occurring in about half of all GBMs, promotes stemness in GBM cells. Exploiting MTAP loss-conferred deficiency in adenine salvage, we demonstrate that transient adenine blockade via treatment with L-Alanosine (ALA), an inhibitor of de novo adenine synthesis, attenuates stemness of MTAP-deficient GBM cells. This ALA-induced reduction in stemness is accompanied by compromised mitochondrial function, highlighted by diminished spare respiratory capacity. Direct pharmacological inhibition of mitochondrial respiration recapitulates the effect of ALA on GBM cell stemness, suggesting ALA targets stemness partially via affecting mitochondrial function. Finally, in agreement with diminished stemness and compromised mitochondrial function, we show that ALA sensitizes GBM cells to temozolomide (TMZ) in vitro and in an orthotopic GBM model. Collectively, these results identify critical roles of adenine supply in maintaining mitochondrial function and stemness of GBM cells, highlight a critical role of mitochondrial function in sustaining GBM stemness, and implicate adenine synthesis inhibition as a complementary approach for treating MTAP-deleted GBMs.

Abstract 441: Role of the Mitochondrial Translocator Protein (TSPO) in the Proarrhythmic Vulnerability of the Diabetic Heart

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Jun Hu ◽  
Won-Joon Koh ◽  
Chaoqin Xie ◽  
Fadi G Akar
Keyword(s):  
Anion Channel ◽  
Mitochondrial Pathway ◽  
Translocator Protein ◽  
Diabetic Heart ◽  
Key Factor ◽  
Ros Scavenger ◽  
Zdf Rats ◽  
Tspo Expression

In structurally normal hearts, inhibition of the mitochondrial translocator protein (TSPO) prevents ROS-mediated proarrhythmia. Whether and how TSPO modulates electrophysiological (EP) function of diabetic hearts, in which classically cardioprotective pathways are impaired, is unknown. We determined the EP effects of TSPO activation & inhibition in a rat model of type 2 diabetes mellitus (t2DM) and studied the mitochondrial pathway underlying TSPO-related proarrhythmia. Methods: TSPO expression & function were determined in Zucker Diabetic Fatty (ZDF) rats with established t2DM (N=16) compared to controls (ctrl, N=15). Optical mapping was performed before & after challenge of hearts with ischemia for 12 min followed by reperfusion. Hearts underwent TSPO activation (FGIN 4.6uM, N=10), blockade (DZP 64uM, N=7) or no treatment (N=14). Dependence of TSPO-related proarrhythmia on ROS and on the PTP a target of TSPO were also determined. Results: t2DM hearts exhibited markedly increased TSPO expression (mRNA & protein) compared to ctrl. TSPO activation did not alter EP properties at baseline in ctrl or t2DM. In contrast TSPO activation accelerated action potential (AP) shortening during ischemia in t2DM but not ctrl hearts. Following 8 min of ischemia, FGIN-mediated AP shortening was 2X greater in t2DM compared to ctrl (p=0.008). FGIN-treated t2DM (5/7) but not ctrl (0/5) hearts exhibited VT by 12min of ischemia (p=0.027). AP shortening and VT in FGIN-treated t2DM hearts were not prevented by the PTP blocker Cyclosporin A but rather by the ROS scavenger EUK implicating the inner membrane anion channel in the proarrhythmic activity of TSPO. Upon reperfusion, TSPO activation with FGIN caused VF in 100% of ctrl & t2DM hearts compared to ~50% of untreated hearts. DZP prolonged the AP in t2DM (by 53% p<0.01) but not ctrl (p=0.14) hearts consistent with heightened sensitivity of t2DM to TSPO ligands. DZP blunted AP shortening during ischemia and prevented VF upon reperfusion in ctrl and t2DM hearts. Conclusion: t2DM hearts exhibit heightened sensitivity to TSPO ligands. TSPO upregulation may be a key factor in the proarrhythmic vulnerability of the diabetic heart. The cardioprotective efficacy of TSPO inhibition against arrhythmias is preserved in t2DM.

scholarly journals Translocator Protein (TSPO) Affects Mitochondrial Fatty Acid Oxidation in Steroidogenic Cells

Endocrinology ◽  
2016 ◽  
Vol 157 (3) ◽  
pp. 1110-1121 ◽  
Author(s):  
Lan N. Tu ◽  
Amy H. Zhao ◽  
Mahmoud Hussein ◽  
Douglas M. Stocco ◽  
Vimal Selvaraj
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Mitochondrial Membrane ◽  
Translocator Protein ◽  
Acid Oxidation ◽  
Mitochondrial Fatty Acid Oxidation ◽  
Mitochondrial Fatty Acid ◽  
Mitochondrial Functions ◽  
Tspo Expression

Abstract Translocator protein (TSPO), also known as the peripheral benzodiazepine receptor, is a highly conserved outer mitochondrial membrane protein present in specific subpopulations of cells within different tissues. In recent studies, the presumptive model depicting mammalian TSPO as a critical cholesterol transporter for steroidogenesis has been refuted by studies examining effects of Tspo gene deletion in vivo and in vitro, biochemical testing of TSPO cholesterol transport function, and specificity of TSPO-mediated pharmacological responses. Nevertheless, high TSPO expression in steroid-producing cells seemed to indicate an alternate function for this protein in steroidogenic mitochondria. To seek an explanation, we used CRISPR/Cas9-mediated TSPO knockout steroidogenic MA-10 Leydig cell (MA-10:TspoΔ/Δ) clones to examine changes to core mitochondrial functions resulting from TSPO deficiency. We observed that 1) MA-10:TspoΔ/Δ cells had a shift in substrate utilization for energy production from glucose to fatty acids with significantly higher mitochondrial fatty acid oxidation (FAO), and increased reactive oxygen species production; and 2) oxygen consumption rate, mitochondrial membrane potential, and proton leak were not different between MA-10:TspoΔ/Δ and MA-10:Tspo+/+ control cells. Consistent with this finding, TSPO-deficient adrenal glands from global TSPO knockout (Tspo−/−) mice also showed up-regulation of genes involved in FAO compared with the TSPO floxed (Tspofl/fl) controls. These results demonstrate the first experimental evidence that TSPO can affect mitochondrial energy homeostasis through modulation of FAO, a function that appears to be consistent with high levels of TSPO expression observed in cell types active in lipid storage/metabolism.

scholarly journals Role of AmpG in the resistance to β-lactam agents, including cephalosporins and carbapenems: candidate for a novel antimicrobial target

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Roshan D’Souza ◽  
Le Phuong Nguyen ◽  
Naina A. Pinto ◽  
Hyunsook Lee ◽  
Thao Nguyen Vu ◽  
...  
Keyword(s):  
De Novo ◽  
Gene Knockout ◽  
Multidrug Resistant ◽  
Klebsiella Aerogenes ◽  
Knock Out ◽  
Transposon Insertion ◽  
Carbapenem Resistant ◽  
Depth Analysis ◽  
Potential Strategy

Abstract Background A complex cascade of genes, enzymes, and transcription factors regulates AmpC β-lactamase overexpression. We investigated the network of AmpC β-lactamase overexpression in Klebsiella aerogenes and identified the role of AmpG in resistance to β-lactam agents, including cephalosporins and carbapenems. Methods A transposon mutant library was created for carbapenem-resistant K. aerogenes YMC2008-M09-943034 (KE-Y1) to screen for candidates with increased susceptibility to carbapenems, which identified the susceptible mutant derivatives KE-Y3 and KE-Y6. All the strains were subjected to highly contiguous de novo assemblies using PacBio sequencing to investigate the loss of resistance due to transposon insertion. Complementation and knock-out experiments using lambda Red-mediated homologous recombinase and CRISPR–Cas9 were performed to confirm the role of gene of interest. Results In-depth analysis of KE-Y3 and KE-Y6 revealed the insertion of a transposon at six positions in each strain, at which truncation of the AmpG permease gene was common in both. The disruption of the AmpG permease leads to carbapenem susceptibility, which was further confirmed by complementation. We generated an AmpG permease gene knockout using lambda Red-mediated recombineering in K. aerogenes KE-Y1 and a CRISPR–Cas9-mediated gene knockout in multidrug-resistant Klebsiella pneumoniae-YMC/2013/D to confer carbapenem susceptibility. Conclusions These findings suggest that inhibition of the AmpG is a potential strategy to increase the efficacy of β-lactam agents against Klebsiella aerogenes.

scholarly journals PD-L1-expressing astrocytes act as a gate-keeper for neuroinflammation in the central nervous system of mice with traumatic brain injury

2021 ◽  
Author(s):  
Xiang Gao ◽  
Wei Li ◽  
Fahim Syed ◽  
Fang Yuan ◽  
Ping Li ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Brain Injury ◽  
De Novo ◽  
Gene Knockout ◽  
Reactive Astrocytes ◽  
Immune Checkpoints ◽  
Enhanced Production ◽  
The Brain

Background: Tissue damage and cellular destruction are the major events in traumatic brain injury (TBI), which trigger sterile neuroimmune and neuroinflammatory responses in the brain. While appropriate acute and transient neuroimmune and neuroinflammatory responses facilitate the repair and adaptation of injured brain tissues, prolonged and excessive neuroimmune and neuroinflammatory responses exacerbate brain damage. The mechanisms that control the intensity and duration of neuroimmune and neuroinflammatory responses in TBI largely remain elusive. Methods: We used the controlled cortical impact (CCI) model of TBI to study the role of immune checkpoints (ICPs), key regulators of immune homeostasis, in the regulation of neuroimmune and neuroinflammatory responses in the brain in vivo. Results: We found that de novo expression of PD-L1, a potent inhibitory ICP, was robustly and transiently induced in reactive astrocytes, but not in microglial cells, neurons, or oligodendrocyte progenitor cells (OPCs). These PD-L1+ reactive astrocytes were highly enriched to form a dense zone around the TBI lesion. Blockade of PD-L1 signaling enlarged brain tissue cavity size, increased infiltration of inflammatory Ly-6CHigh monocytes/macrophages (M/Mɸ) but not tissue-repairing Ly-6CLow/F4/80+ M/Mɸ, and worsened TBI outcomes in mice. PD-L1 gene knockout enhanced production of CCL2 that interacted with its cognate receptor CCR2 on Ly-6CHigh M/Mϕ to chemotactically recruit these cells into inflammatory sites. Mechanically, PD-L1 signaling in astrocytes likely exhibits dual inhibitory activities for the prevention of excessive neuroimmune and neuroinflammatory responses to TBI through (1) the PD-1/PD-L1 axis to suppress the activity of brain-infiltrating PD-1+ immune cells such as PD-1+ T cells, and (2) PD-L1 reverse signaling to regulate the timing and intensity of astrocyte reactions to TBI. Conclusions: PD-L1+ astrocytes act as a gatekeeper to the brain to control TBI-related neuroimmune and neuroinflammatory responses, thereby opening a novel avenue to study the role of ICP-neuroimmune axes in the pathophysiology of TBI and other neurological disorders.

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