AMPK-dependent and independent actions of P2X7 in regulation of mitochondrial and lysosomal functions in microglia

Background

P2X7 is ubiquitously expressed in myeloid cells and regulates the pathophysiology of inflammatory diseases. Since mitochondrial function in microglia is highly associated with microglial functions in controlling neuronal plasticity and brain homeostasis, we interested to explore the roles of P2X7 in mitochondrial and lysosomal functions as well as mitophagy in microglia.

Methods

P2X7^?/? bone marrow-derived macrophages (BMDM), primary microglia and BV-2 immortalized microglial cells were used to detect the particular protein expression by immunoblotting. Mitochondrial reactive oxygen species (mitoROS), intracellular calcium, mitochondrial mass and lysosomal integrity were examined by flow cytometry. Mitochondrial oxygen consumption rate (OCR) was recorded using Seahorse XF flux analyzer. Confocal microscopic images were performed to indicate the mitochondrial dynamics and mitophagy after P2X7 activation.

Results

In primary microglia, BV-2 microglial cells and BMDM, P2X7 agonist BzATP triggered AMPK activation and LC3II accumulation through reactive oxygen species (ROS) and CaMKKII pathways, and these effects were abolished by P2X7 antagonist A438079 and P2X7 deficiency. Moreover, we detected the dramatic decreases of mitochondrial OCR and mass following P2X7 activation. AMPK inhibition by compound C or AMPK silencing reversed the P2X7 actions in reduction of mitochondrial mass, induction of mitochondrial fission and mitophagy, but not in uncoupling of mitochondrial respiration. Interestingly, we found that P2X7 activation induced nuclear translocation of TFEB via an AMPK-dependent pathway and led to lysosomal biogenesis. Mimicking the actions of BzATP, nigericin also induced ROS-dependent AMPK activation, mitophagy, mitochondrial fission and respiratory inhibition. Longer exposure of BzATP induced cell death, and this effect was accompanied by the lysosomal instability and was inhibited by autophagy and cathepsin B inhibitors.

Conclusion

Altogether ROS- and CaMKK-dependent AMPK activation is involved in P2X7-mediated mitophagy, mitochondrial dynamics and lysosomal biogenesis in microglial cells, which is followed by cytotoxicity partially resulting from mitophagy and cathepsin B activation.

     

A novel end-to-end binding of two netropsins to the DNA decamers d(CCCCCIIIII)2, d(CCCBr5CCIIIII)2and d(CBr5CCCCIIIII)2.

Netropsin is bound to the DNA decamer d(CCCCCIIIII)2, the C-4 bromo derivative d(CCCBr5CCIIIII)2and the C-2 bromo derivative d(CBr5CCCCIIIII)2in a novel 2:1 mode. Complexes of the native decamer and the C-4 bromo derivative are isomorphous, space group P1, unit cell dimensions a = 32.56 A (32.66), b = 32.59 A (32.77), c = 37.64 A (37.71), alpha = 86.30 degrees (86.01 degrees), beta = 84.50 degrees (84.37 degrees), gamma = 68.58 degrees (68.90 degrees) with two independent molecules (A and B) in the asymmetric unit (values in parentheses are for the derivative). The C-2 bromo derivative is hexagonal P61, unit cell dimensions a = b = 32.13 A, c = 143.92, gamma = 120 degrees with one molecule in the asymmetric unit. The structures were solved by the molecular replacement method. The novelty of the structures is that there are two netropsins bound end-to-end in the minor groove of each B-DNA decamer which has nearly a complete turn. The netropsins are held by hydrogen bonding interactions to the base atoms and by sandwiching van der Waal's interactions from the sugar-phosphate backbones of the double helix similar to every other drug.DNA complex. Each netropsin molecule spans approximately 5 bp. The netropsins refined with their guanidinium heads facing each other at the center, although an orientational disorder for the netropsins cannot be excluded. The amidinium ends stretch out toward the junctions and bind to the adjacent duplexes in the columns of stacked symmetry-related complexes. Both cationic ends of netropsin are bridged by water molecules in one of the independent molecules (molecule A) of the triclinic structures and also the hexagonal structure to form pseudo-continuous drug.decamer helices.     

Improvement of 1,3-propanediol oxidoreductase (DhaT) stability against 3-hydroxypropionaldehyde by substitution of cysteine residues

1,3-propanediol oxidoreductase (DhaT), which catalyzes the conversion of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) with the oxidation of NADH to NAD⁺, is a key enzyme in the production of 1,3-PD from glycerol. DhaT is known to be severely inactivated by its physiological substrate, 3-HPA, due to the reaction of 3-HPA with the thiol group of the cysteine residues. In this study, using site-directed mutagenesis, four cysteine residues in Klebsiella pneumoniae J2B DhaT were substituted to alanine, the amino acid commonly found in cysteine’s positions in other DhaT, individually and in combination. Among the total of 15 mutants developed, a double mutant (C28A_C107A) and a triple mutant (C28A_C93A_C107A) exhibited approximately 50 and 16% higher activity than the wild-type counterpart, respectively, after 1 h incubation with 10 mM 3-HPA. According to detailed kinetic studies, the double mutant had slightly better kinetic properties (V _max , K _cat , and K _m for both 3-HPA and NADH) than wild-type DhaT. This study shows that DhaT stability against 3-HPA can be increased by cysteine-residue removal, albeit to a limited extent.     

Antimicrobial potential and taxonomic investigation of piezotolerant <Emphasis Type="Italic">Streptomyces</Emphasis> sp. NIOT-Ch-40 isolated from deep-sea sediment

Microbial-derived natural products from extreme niches such as deepsea are known to possess structural and functional novelty. With this background, the present study was designed to investigate the bioprospecting potential and systematics of a deep-sea derived piezotolerant bacterial strain NIOT-Ch-40, showing affiliation to the genus Streptomyces based on 16S RNA gene similarity. Preliminary screening for the presence of biosynthetic genes like polyketide synthase I, polyketide synthase II, non ribosomal peptide synthase, 3-amino-5-hydroxybenzoic acid synthase and spiroindimicin followed by antibacterial activity testing confirmed the presence of potent bioactivity. The secondary metabolites produced during fermentation in Streptomyces broth at 28?°C for 7 days were extracted with ethyl acetate. The extract exhibited a specific inhibitory activity against Gram-positive bacteria and was significantly effective (p?<?0.0001) against methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentration and minimum bactericidal concentration against MRSA was 1.5 µg/mL, which was statistically significant in comparison with erythromycin. A multifaceted analysis of the Streptomyces spp. was carried out to delineate the strain NIOT-Ch-40 at a higher resolution which includes morphological, biochemical and molecular studies. Piezotolerance studies and comparison of fatty acid profiles at high pressures revealed that it could be considered as one of the taxonomic markers, especially for the strains isolated from the deep sea environments. In conclusion, the observation of comparative studies with reference strains indicated towards the strain NIOT-Ch-40 as an indigenous marine piezotolerant Streptomyces sp. with a higher probability of obtaining novel bioactive metabolites.     

Synthesis of some novel imidazole-based dicationic carbazolophanes as potential antibacterials

Synthesis of fluorescent imidazole-based dicationic carbazolophanes incorporating various spacer units is described. Interestingly, the cyclophanes 2a and 5a incorporating a pyridine moiety exhibited superior antibacterial activity against most of the pathogenic bacteria in the tested concentrations as compared to the other cyclophanes as well as the test control, benzalkonium chloride (BAC), cetylpyridinium chloride (CPC) and tetracycline.     

Role of lupeol and lupeol linoleate on lipemic-oxidative stress in experimental hypercholesterolemia

Epidemiological studies have shown that there is a positive correlation between the incidence of coronary heart disease (CHD) and the blood cholesterol level. To study the effect of plant derived triterpene, lupeol and its ester lupeol linoleate, on blood lipid status and oxidant stress in heart and hemolysate, male albino Wistar rats were fed high cholesterol diet (normal rat chow supplemented with 4% cholesterol and 1% cholic acid; HCD) for 30 days. A significant increase (p<0.05) in plasma total cholesterol (4.22 fold) and triglycerides (1.7 fold) was observed in HCD fed rats, along with elevated LDL (3.56 fold) and VLDL (1.99 fold) cholesterol and decreased HDL cholesterol (34.14%). Treatment with lupeol and its derivative normalized the lipid profile. The significant increase (p<0.05) in lipid peroxidation (LPO) was paralleled by significantly diminished (p<0.05) activities of antioxidant enzymes (SOD, CAT and GPx) and decreased (p<0.05) concentration of antioxidant molecules (GSH, Vit C and Vit E) in cardiac tissue and hemolysate of HCD fed rats. The oxidative tissue injury in hypercholesterolemic rats was substantiated by the increase in cardiac marker, serum CPK and the drop in its activity in the heart tissue. Lupeol and lupeol linoleate treatment decreased the LPO levels and increased enzymatic and nonenzymatic antioxidants. CPK activity in the treated group was comparable with that of the control. These observations highlight the beneficial effects of the triterpene, lupeol and its linoleate ester derivative, in ameliorating the lipidemic-oxidative abnormalities in the early stage of hypercholesterolemic atherosclerosis.