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A novel beta-sheet breaker, RS-0406, reverses amyloid beta-induced cytotoxicity and impairment of long-term potentiation in vitro.

ABSTRACT: Fibril formation of amyloid beta peptide (Abeta) is considered to be responsible for the pathology of Alzheimer's disease (AD). The Abeta fibril is formed by a protein misfolding process in which intermolecular beta-sheet interactions become stabilized abnormally. Thus, to develop potential anti-AD drugs, we screened an in-house library to find compounds which have a profile as a beta-sheet breaker. We searched for a beta-sheet breaker profile in an in-house library of approximately 113,000 compounds. From among the screening hits, we focused on N,N'-bis(3-hydroxyphenyl)pyridazine-3,6-diamine (named RS-0406), which had been newly synthesized in our laboratory. This compound (10-100 microg ml(-1)) was found to be capable of significantly inhibiting 25 microM Abeta(1-42) fibrillogenesis and, furthermore, disassembling preformed Abeta(1-42) fibrils in vitro. 3 We then investigated the effect of RS-0406 on 111 nM Abeta(1-42)-induced cytotoxicity in primary hippocampal neurons, and found that 0.3-3 microg ml(-1) RS-0406 ameliorates the cytotoxicity. Moreover, 3 microg ml(-1) RS-0406 reversed 1 micro M Abeta(1-42)-induced impairment of long-term potentiation in hippocampal slices. 4 In this study, we have succeeded in identifying RS-0406 which has potential to inhibit Abeta(1-42) fibrillogenesis, and to protect neurons against Abeta(1-42)-induced biological toxicity in vitro. These results suggest that RS-0406 or one of the derivatives could become a therapeutic agent for AD patients.

SUBMITTER: Nakagami Y

PROVIDER: S-EPMC1573533 | biostudies-other | 2002 Nov

REPOSITORIES: biostudies-other

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Project description:Complement inhibitor C4b-binding protein (C4BP) is synthesized in liver and pancreas and composed of 7 identical alpha chains and one unique beta chain. We showed previously that C4BP binds islet amyloid polypeptide (IAPP) and affects fibril formation in vitro. Now we found that polymeric C4BP inhibited lysis of human erythrocytes incubated with monomeric IAPP while no erythrocyte lysis was observed after incubation with preformed IAPP fibrils. In contrast, monomeric alpha chain of C4BP had significantly reduced activity. Further, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Accordingly, addition of C4BP rescued the ability of INS-1 cells and isolated rat islets to respond to glucose stimulation with insulin secretion, which was impaired in the presence of IAPP alone. C4BP was internalized together with IAPP into INS-1 cells and therefore we aimed to study its effect on gene expression. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison to IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-Î²-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-ÎºB had a similar effect. Taken together, one of the mechanisms by which C4BP protects beta-cells from IAPP cytotoxicity is by enhancing cholesterol synthesis. The INS-1 cells were grown as 5 separate clones for 10 passages before plating in a 12-well plate (Nunc) at 100.000 cells per well and grown in complete RPMI 1640 medium to 70% confluency for approximately 48 h. The cells were then challenged by adding 77 Î¼M monomeric IAPP alone or together with C4BP (0.6 Î¼M). DMSO (1%) used as solvent for IAPP as well as C4BP (0.6 Î¼M) alone were used as controls. RNA was extracted after 10h incubation and analysis carried out using Rat Gene 2.0 array chip (Affymetrix).

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A novel beta-sheet breaker, RS-0406, reverses amyloid beta-induced cytotoxicity and impairment of long-term potentiation in vitro.

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