Inhibition of glucocorticoid synthesis alleviates cognitive impairment in high-fat diet-induced obese mice.

In addition to cardiovascular diseases, metabolic syndrome and type 2 diabetes mellitus, obesity is associated with cognitive deficits. In rodents, it has been shown that long-term high-fat diet (HFD) consumption leads to the alteration of hypothalamic-pituitary-adrenal (HPA) axis resulting in increased corticosterone release. However, mechanisms underpinning cognitive impairments induced by long-term HFD intake are unclear. Herein we evaluated the effects of systemic administration of glucocorticoid synthesis inhibitor metyrapone on cognitive performance assessed by novel object recognition test and plasma corticosterone levels evaluated by enzyme-linked immunosorbent assay in HFD-induced obese mice. We found that metyrapone treatment alleviated recognition memory impairments in HFD-induced obese mice. Furthermore, glucocorticoid synthesis inhibitor also lowered plasma corticosterone levels in HFD-induced obese mice. Our findings indicate that hyperactivation of HPA axis resulting in elevated circulating glucocorticoid levels leads to memory impairments in HFD-induced obese mice. We identify glucocorticoid system as a potential therapeutic target for treating cognitive deficits associated with obesity condition.

Contributions of the Hydrophobic Helix 2 of the Bordetella pertussis CyaA-hemolysin to Membrane Permeabilization.

BACKGROUND: Adenylate cyclase (CyaA) is one of the major virulence factors of Bordetella pertussis that plays a key role in whooping cough pathogenesis. A putative transmembrane helical hairpin (α2-loop-α3), encompassing residues 529-594 of CyaA hemolysin (CyaA-Hly) domain, was previously proposed to be crucially involved in hemolytic activity against target erythrocytes. OBJECTIVE: The main objective of this study was to gain more insight into membrane permeabilization of this toxin. Membrane-permeabilizing abilities of the purified 130-kDa CyaA-Hly and synthetic peptides corresponding to the helical component of interest, were evaluated. METHODS: Synthetic peptides corresponding to the critical helical component, i.e. α2 (W528-G550), α3 (G568-R594) and α2-loop-α3 (W528-R594), were examined on various membrane models in comparison with the purified 130-kDa CyaA-Hly. The peptides were commercially synthesized and the purified toxin was obtained from recombinant plasmid construction and expression in Escherichia coli, followed by purification via immobilized-metal affinity chromatography. Membrane permeabilization or hemolysis of the peptides or the purified toxin were determined by liposomal leakage, hemolysis assays and atomic force microscopy (AFM) imaging. RESULTS: Our results showed that the truncated 130-kDa CyaA-Hly, the synthetic peptides α2, α3 and the α2-loop-α3 hairpin exhibited distinct membrane-permeabilizing capacities in different membrane models. We demonstrated that the CyaA-Hly toxin and the peptides, especially the α2 peptide, caused nonspecific liposomal leakage as monitored by fluorescence dequenching of sulforhodamine B-loaded lipid vesicles. Notably, α2 peptide showed a predominant effect of membrane permeabilization when compared to α2-loop-α3 hairpin and α3 peptides. In addition, AFM imaging demonstrates lipid membrane disruption induced by the CyaA-Hly toxin or the peptidic α2-loop-α3 hairpin. CONCLUSION: Overall, the study provides the supporting evidence that the putative helical α2-loop-α3 hairpin could interact with the lipid membranes while the helical α2 peptide strongly induced liposomal leakage and hemolysis, as compared with the helical α3 or the α2-loop-α3 peptides, suggesting that the helix 2 from the hydrophobic region of CyaA-Hly is a crucial component that contributes to membrane permeabilization.

Functional importance of the Gly cluster in transmembrane helix 2 of the Bordetella pertussis CyaA-hemolysin: Implications for toxin oligomerization and pore formation.

Adenylate cyclase-hemolysin (CyaA) is a major virulence factor of Bordetella pertussis causing whooping cough in humans. We previously showed that two transmembrane helices (α2 and α3) in the hemolysin domain (CyaA-Hly) are crucially involved in hemolytic activity. Here, PCR-based substitutions were employed to investigate a potential involvement in hemolysis of a series of four Gly residues (Gly(530), Gly(533), Gly(537) and Gly(544)) which map onto one face of a helical wheel plot of pore-lining helix 2. All CyaA-Hly mutant toxins were over-expressed in Escherichia coli as 126-kDa soluble proteins at levels comparable to the wild-type toxin. A drastic reduction in hemolytic activity against sheep erythrocytes was observed for three CyaA-Hly mutants, i.e. G530A, G533A and G537A, but not G544A, suggesting a functional importance of the Gly(530)_Gly(533)_Gly(537) cluster. A homology-based structure of the α2-loop-α3 hairpin revealed that this crucial Gly cluster arranged as a GXXGXXXG motif is conceivably involved in helix-helix association. Furthermore, a plausible pore model comprising three α2-loop-α3 hairpins implicated that Gly(530)XXGly(533)XXXGly(537) could function as an important framework for toxin oligomerization. Altogether, our present data signify for the first time that the Gly(530)_Gly(533)_Gly(537) cluster in transmembrane helix 2 serves as a crucial constituent of the CyaA-Hly trimeric pore structure.