The Role Of Α-Synuclein In Brain Lipid Metabolism And Inflammation

Alpha-synuclein (Snca) is a small cytosolic protein that is ubiquitously expressed in the nervous system and comprises 0.5-1% of all cytosolic protein, but its biological function is poorly understood. Although Snca function has been studied in lipid metabolism, the function of Snca in brain lipid metabolism under inflammatory conditions is yet to be elucidated. We utilized several model systems including primary cultured astrocytes and microglia, Snca deficient and mutant knock-in mice, and a radiolabeled free fatty acid steady state-kinetic mouse model to determine Snca role in neuroinflammation. Herein, we have determined several major roles of Snca during inflammation: (i) dBcAMP treatment increases 20:4n-6 uptake in astrocytes and this increase appears to be due to increased expression of long chain acyl-CoA synthetases 3 and -4 coupled with a reduction in acyl-CoA hydrolase expression in the presence of reduced Snca expression. (ii) Snca deficient mice have higher basal brain 2-arachidonyl glycerol (2-AG) levels compared to wildtype and lipopolysaccharide (LPS) stimulation further exacerbated 2-AG synthesis.(iii) In primary microglia, LPS-treatment reduced released 2-AG into medium concomitantly with reduced Snca expression and Snca deficient microglia had a delayed response to LPS stimuli. This supports the hypothesis that Snca expression is linked to 2-AG release in primary microglia and may contribute to regulating the phagocytic phenotype.(iv) Using Snca gene-ablated mice, we determined the impact of Snca on brain 20:4n-6 metabolism during LPS-induced inflammatory response in vivo using an established steady-state kinetic model. In Snca deficient mouse brain, 20:4n-6 uptake was significantly increased 1.3-fold. In the organic fraction, tracer entering into Snca deficient mouse total brain phospholipids was significantly increased 1.4-fold, accounted for by increased incorporation into choline glycerophospholipids and phosphatidylinositol. In the neutral lipid fraction, 20:4n-6 incorporation into diacylglycerol of Snca deficient mice was significantly reduced by 75%. Hence, under inflammatory conditions where 20:4n-6 release is enhanced, Snca has a crucial role in modulating 20:4n-6 metabolism, and the absence of Snca results in increased uptake and incorporation into lipid pools associated with enhanced lipid-mediated signaling during neuroinflammatory response. Herein, we focus on the role of Snca in downstream eicosanoid biosynthesis, inflammatory mediators, and lipid signaling molecules linking Snca to inflammatory response elucidating a key step in neuroinflammation

Functional Rotation of the Transporter AcrB: Insights into Drug Extrusion from Simulations

The tripartite complex AcrAB-TolC is the major efflux system in Escherichia coli. It extrudes a wide spectrum of noxious compounds out of the bacterium, including many antibiotics. Its active part, the homotrimeric transporter AcrB, is responsible for the selective binding of substrates and energy transduction. Based on available crystal structures and biochemical data, the transport of substrates by AcrB has been proposed to take place via a functional rotation, in which each monomer assumes a particular conformation. However, there is no molecular-level description of the conformational changes associated with the rotation and their connection to drug extrusion. To obtain insights thereon, we have performed extensive targeted molecular dynamics simulations mimicking the functional rotation of AcrB containing doxorubicin, one of the two substrates that were co-crystallized so far. The simulations, including almost half a million atoms, have been used to test several hypotheses concerning the structure-dynamics-function relationship of this transporter. Our results indicate that, upon induction of conformational changes, the substrate detaches from the binding pocket and approaches the gate to the central funnel. Furthermore, we provide strong evidence for the proposed peristaltic transport involving a zipper-like closure of the binding pocket, responsible for the displacement of the drug. A concerted opening of the channel between the binding pocket and the gate further favors the displacement of the drug. This microscopically well-funded information allows one to identify the role of specific amino acids during the transitions and to shed light on the functioning of AcrB

Alveolar Epithelial Type II Cells Activate Alveolar Macrophages and Mitigate P. Aeruginosa Infection

Although alveolar epithelial type II cells (AECII) perform substantial roles in the maintenance of alveolar integrity, the extent of their contributions to immune defense is poorly understood. Here, we demonstrate that AECII activates alveolar macrophages (AM) functions, such as phagocytosis using a conditioned medium from AECII infected by P. aeruginosa. AECII-derived chemokine MCP-1, a monocyte chemoattractant protein, was identified as a main factor in enhancing AM function. We proposed that the enhanced immune potency of AECII may play a critical role in alleviation of bacterial propagation and pneumonia. The ability of phagocytosis and superoxide release by AM was reduced by MCP-1 neutralizing antibodies. Furthermore, MCP-1−/− mice showed an increased bacterial burden under PAO1 and PAK infection vs. wt littermates. AM from MCP-1−/− mice also demonstrated less superoxide and impaired phagocytosis over the controls. In addition, AECII conditioned medium increased the host defense of airway in MCP-1−/− mice through the activation of AM function. Mechanistically, we found that Lyn mediated NFκB activation led to increased gene expression and secretion of MCP-1. Consequently Lyn−/− mice had reduced MCP-1 secretion and resulted in a decrease in superoxide and phagocytosis by AM. Collectively, our data indicate that AECII may serve as an immune booster for fighting bacterial infections, particularly in severe immunocompromised conditions

A simplified method for preventing postmortem alterations of brain prostanoids for true in situ level quantification

Dramatic postmortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an accurate in situ endogenous PG quantification. To date, the only method for preventing postmortem brain PG increase with tissue structure preservation is fixation by head-focused microwave irradiation (MW), which is considered the gold standard method, allowing for rapid in situ heat-denaturation of enzymes. However, MW requires costly equipment that suffers in reproducibility, causing tissue loss and metabolite degradation if overheated. Our recent study indicates that PGs are not synthesized in the ischemic brain unless metabolically active tissue is exposed to atmospheric O2. Based on this finding, we proposed a simple and reproducible alternative method to prevent postmortem PG increase by slow enzyme denaturation before craniotomy. To test this approach, mice were decapitated directly into boiling saline. Brain temperature reached 100°C after ∼140 s during boiling, though 3 min boiling was required to completely prevent postmortem PG synthesis, but not free arachidonic acid release. To validate this fixation method, brain basal and lipopolysaccharide (LPS)-induced PG were analyzed in unfixed, MW, and boiled tissues. Basal and LPS-induced PG levels were not different between MW and boiled brains. However, unfixed tissue showed a significant postmortem increase in PG at basal conditions, with lesser differences upon LPS treatment compared to fixed tissue. These data indicate for the first time that boiling effectively prevents postmortem PG alterations, allowing for a reproducible, inexpensive, and conventionally accessible tissue fixation method for PG analysis

Transport of drugs by the multidrug transporter AcrB involves an access and a deep binding pocket that are separated by a switch-loop

AcrAB-TolC is the major efflux protein complex in Escherichia coli extruding a vast variety of antimicrobial agents from the cell. The inner membrane component AcrB is a homotrimer, and it has been postulated that the monomers cycle consecutively through three conformational stages designated loose (L), tight (T), and open (O) in a concerted fashion. Binding of drugs has been shown at a periplasmic deep binding pocket in the T conformation. The initial drug-binding step and transport toward this drug-binding site has been elusive thus far. Here we report high resolution structures (1.9-2.25 Å) of AcrB/designed ankyrin repeat protein (DARPin) complexes with bound minocycline or doxorubicin. In the AcrB/doxorubicin cocrystal structure, binding of three doxorubicin molecules is apparent, with one doxorubicin molecule bound in the deep binding pocket of the T monomer and two doxorubicin molecules in a stacked sandwich arrangement in an access pocket at the lateral periplasmic cleft of the L monomer. This access pocket is separated from the deep binding pocket apparent in the T monomer by a switch-loop. The localization and conformational flexibility of this loop seems to be important for large substrates, because a G616N AcrB variant deficient in macrolide transport exhibits an altered conformation within this loop region. Transport seems to be a stepwise process of initial drug uptake in the access pocket of the L monomer and subsequent accommodation of the drug in the deep binding pocket during the L to T transition to the internal deep binding pocket of the T monomer