Structural basis of the 24B3 antibody against the toxic conformer of amyloid ß with a turn at positions 22 and 23.

Amyloid ß-protein (Aß) oligomers are involved in the early stages of Alzheimer's disease (AD) and antibodies against these toxic oligomers could be useful for accurate diagnosis of AD. We identified the toxic conformer of Aß42 with a turn at positions 22/23, which has a propensity to form toxic oligomers. The antibody 24B3, developed by immunization of a toxic conformer surrogate E22P-Aß9-35 in mice, was found to be useful for AD diagnosis using human cerebrospinal fluid (CSF). However, it is not known how 24B3 recognizes the toxic conformation of wild-type Aß in CSF. Here, we report the crystal structure of 24B3 Fab complexed with E22P-Aß11-34, whose residues 16-26 were observed in electron densities, suggesting that the residues comprising the toxic turn at positions 22/23 were recognized by 24B3. Since 24B3 bound only to Aß42 aggregates, several conformationally restricted analogs of Aß42 with an intramolecular disulfide bond to mimic the conformation of toxic Aß42 aggregates were screened by enzyme immunoassay. As a result, only F19C,A30homoC-SS-Aß42 (1) bound significantly to 24B3. These data provide a structural basis for its low affinity to the Aß42 monomer and selectivity for its aggregate form.

Structure Optimization of the Toxic Conformation Model of Amyloid ß42 by Intramolecular Disulfide Bond Formation.

Amyloid ß (Aß) oligomers play a critical role in the pathology of Alzheimer's disease. Recently, we reported that a conformation-restricted Aß42 with an intramolecular disulfide bond through cysteine residues at positions 17/28 formed stable oligomers with potent cytotoxicity. To further optimize this compound as a toxic conformer model, we synthesized three analogues with a combination of cysteine and homocysteine at positions 17/28. The analogues with Cys-Cys, Cys-homoCys, or homoCys-Cys, but not the homoCys-homoCys analogue, exhibited potent cytotoxicity against SH-SY5Y and THP-1 cells even at 10 nM. In contrast, the cytotoxicity of conformation-restricted analogues at positions 16/29 or 18/27 was significantly weaker than that of wild-type Aß42. Furthermore, thioflavin-T assay, non-denaturing gel electrophoresis, and morphological studies suggested that the majority of these conformation-restricted analogues exists in an oligomeric state in cell culture medium, indicating that the toxic conformation of Aß42, rather than the oligomeric state, is essential to induce cytotoxicity.

Characterization of a Conformation-Restricted Amyloid ß Peptide and Immunoreactivity of Its Antibody in Human AD brain.

Characterization of amyloid ß (Aß) oligomers, the transition species present prior to the formation of Aß fibrils and that have cytotoxicity, has become one of the major topics in the investigations of Alzheimer's disease (AD) pathogenesis. However, studying pathophysiological properties of Aß oligomers is challenging due to the instability of these protein complexes in vitro. Here, we report that conformation-restricted Aß42 with an intramolecular disulfide bond at positions 17 and 28 (SS-Aß42) formed stable Aß oligomers in vitro. Thioflavin T binding assays, nondenaturing gel electrophoresis, and morphological analyses revealed that SS-Aß42 maintained oligomeric structure, whereas wild-type Aß42 and the highly aggregative Aß42 mutant with E22P substitution (E22P-Aß42) formed Aß fibrils. In agreement with these observations, SS-Aß42 was more cytotoxic compared to the wild-type and E22P-Aß42 in cell cultures. Furthermore, we developed a monoclonal antibody, designated TxCo-1, using the toxic conformation of SS-Aß42 as immunogen. X-ray crystallography of the TxCo-1/SS-Aß42 complex, enzyme immunoassay, and immunohistochemical studies confirmed the recognition site and specificity of TxCo-1 to SS-Aß42. Immunohistochemistry with TxCo-1 antibody identified structures resembling senile plaques and vascular Aß in brain samples of AD subjects. However, TxCo-1 immunoreactivity did not colocalize extensively with Aß plaques identified with conventional Aß antibodies. Together, these findings indicate that Aß with a turn at positions 22 and 23, which is prone to form Aß oligomers, could show strong cytotoxicity and accumulated in brains of AD subjects. The SS-Aß42 and TxCo-1 antibody should facilitate understanding of the pathological role of Aß with toxic conformation in AD.

Control of the toxic conformation of amyloid ß42 by intramolecular disulfide bond formation.

We report a method to fix the conformation of Aß42 to the toxic or non-toxic form by intramolecular disulfide bonds. We found that an Aß42 analog crosslinked within the molecule at the 17th and 28th amino acid residues exhibited high aggregative ability and potent neurotoxicity comparable to those of E22P-Aß42. This analog would be useful in the research of Aß42 oligomers and to develop reliable antibodies for early diagnosis of Alzheimer's disease.

Effects of Adjuvant-Induced Inflammation on Disposition of Diclofenac and Its Metabolites in Perfused Rat Liver.

The reactive metabolites of diclofenac (DF) such as 1-O-acyl glucuronide (DF-Glu) are hypothesized to result in idiosyncratic hepatotoxicity. However, it is unclear whether inflammation affects the hepatic disposition of DF and its metabolites. To clarify the alterations in the disposition of DF and its metabolites in inflammatory conditions, we performed in situ perfused rat liver experiments. Using adjuvant arthritis rats as a model of inflammation, the elimination of DF, 4'-hydroxydiclofenac, and DF-Glu from the perfusate was observed to be delayed in comparison with the control. Parameter sensitivity analysis for hepatic DF disposition revealed that the area under the plasma concentration-time curve (AUC) and the maximum concentration (Cmax) of DF-Glu in the liver markedly increased along with a decrease in intrinsic excretion clearance of DF-Glu (CLint,bile,Glu) and an increase in intrinsic glucuronidation clearance (CLint,Glu) of DF-Glu. It is possible that the elimination of DF-Glu from the perfusate in adjuvant arthritis rats was delayed via reduction of biliary excretion of DF-Glu.

Effects of dose, flow rate, and bile acid on diclofenac disposition in the perfused rat liver.

An in situ perfused rat liver system is useful for studying the hepatic disposition of drugs and their metabolites. However, the effects of the perfusion conditions on drug disposition are unclear. We examined the effects of conditions such as flow rate (13 or 26 mL/min) and bile acid on disposition of diclofenac (DF) as a model drug and DF metabolites [diclofenac-1-O-acyl glucuronide (DF-Glu) or 4'-hydroxydiclofenac (DF-4'OH)] in the absence of albumin. DF, DF-Glu, and DF-4'OH concentrations in the perfusate and cumulative amounts of DF-Glu excreted in bile were measured using high-performance liquid chromatography methods. DF in the perfusate was rapidly eliminated as the perfusate flow rate increased. The area under the plasma concentration-time curve from 0 to 60 min (AUC0-60) for DF-Glu and DF-4'OH in a perfusate containing bile acid was lower at a flow rate of 26 and 13 mL/min, respectively. The bile flow rate at 26 mL/min with 24 µM of bile acid in the perfusate was significantly higher (ca. 3.5 times) compared with that at 13 mL/min without bile acid. Cumulative biliary DF-Glu excretion was also dramatically affected by the flow rate and addition of bile acid. This study indicated that the flow rate and bile acid in the perfused rat liver were key factors for bile flow rate and DF, DF-Glu, and DF-4'OH disposition in the absence of albumin.

Does seeing ice really feel cold? Visual-thermal interaction under an illusory body-ownership.

Although visual information seems to affect thermal perception (e.g. red color is associated with heat), previous studies have failed to demonstrate the interaction between visual and thermal senses. However, it has been reported that humans feel an illusory thermal sensation in conjunction with an apparently-thermal visual stimulus placed on a prosthetic hand in the rubber hand illusion (RHI) wherein an individual feels that a prosthetic (rubber) hand belongs to him/her. This study tests the possibility that the ownership of the body surface on which a visual stimulus is placed enhances the likelihood of a visual-thermal interaction. We orthogonally manipulated three variables: induced hand-ownership, visually-presented thermal information, and tactically-presented physical thermal information. Results indicated that the sight of an apparently-thermal object on a rubber hand that is illusorily perceived as one's own hand affects thermal judgments about the object physically touching this hand. This effect was not observed without the RHI. The importance of ownership of a body part that is touched by the visual object on the visual-thermal interaction is discussed.