Retinoic acid (RA)-elicited signaling has been shown to play critical roles in development, organogenesis, and the immune response. RA regulates expression of Alzheimer's disease (AD)-related genes and attenuates amyloid pathology in a transgenic mouse model. In this study, we investigated whether RA can suppress the production of amyloid-β (Aβ) through direct inhibition of γ-secretase activity. We report that RA treatment of cells results in significant inhibition of γ-secretase-mediated processing of the amyloid precursor protein C-terminal fragment APP-C99, compared with DMSO-treated controls. RA-elicited signaling was found to significantly increase accumulation of APP-C99 and decrease production of secreted Aβ40. In addition, RA-induced inhibition of γ-secretase activity was found to be mediated through significant activation of extracellular signal-regulated kinases (ERK1/2). Treatment of cells with the specific ERK inhibitor PD98059 completely abolished RA-mediated inhibition of γ-secretase. Consistent with these findings, RA was observed to inhibit secretase-mediated proteolysis of full-length APP. Finally, we have established that RA inhibits γ-secretase through nuclear retinoic acid receptor-α (RARα) and retinoid X receptor-α (RXRα). Our findings provide a new mechanistic explanation for the neuroprotective role of RA in AD pathology and add to the previous data showing the importance of RA signaling as a target for AD therapy.

A primary pathway for metabolism of electrophilic compounds in Schistosoma japonicum involves glutathione S-transferase (SjGST)-catalyzed formation of glutathione (GSH) conjugates. As part of a program aimed at gaining a better understanding of the defense system of parasites, a series of aromatic halides (1-8), aliphatic halides (9, 10), epoxides (11-20), alpha,beta-unsaturated esters (21, 22), and alpha,beta-unsaturated amides (23, 24) were prepared, and their participation in glutathione conjugate formation was evaluated. Products from enzymatic and nonenzymatic reactions of these substances with glutathione were characterized and quantified by using reverse-phase high-performance liquid chromatography (HPLC), NMR, and fast atom bombardment mass spectrometry (FAB-MS) analysis. Mechanisms for formation of specific mono(glutathionyl) or bis(glutathionyl) conjugates are proposed. Although the results of this effort indicate that SjGST does not catalyze addition or substitution reactions of 1, 3, 4, 7-9, 11-13, 15-17, 19-21, and 24, they demonstrate that 2, 5, 6, 14, 18, and 23 undergo efficient enzyme-catalyzed conjugation reactions. The kcat values for SjGST with 23 and 18 are about 886-fold and 14-fold, respectively, larger than that for 5. This observation suggests that 23 is a good substrate in comparison to other electrophiles. Furthermore, the initially formed conjugation product, 23a, is also a substrate for SjGST in a process that forms the bis(glutathionyl) conjugate 23b. Products arising by enzymatic and nonenzymatic pathways are generated under the conditions of SjGST-activated GSH conjugation. Interestingly, production of nonenzymatic GSH conjugates with electrophilic substrates often overwhelms the activity of the enzyme. The nonenzymatic GSH conjugates, 9a-11a, 16a, 21a, and 22a, are inhibitors of SjGST with respective IC50 values of 1.95, 75.5, 0.96, 19.0, 152, and 0.36 microM, and they display moderate inhibitory activities against human GSTA2. Direct evidence has been gained for substrate inhibition by 10 toward SjGST and GSTA2 that is more potent than that of its GSH conjugate 10a. The significance of this work is found in the development of a convenient NMR-based technique that can be used to characterize glutathione conjugates derived from small molecule libraries as part of efforts aimed at uncovering specific potent SjGST and GSTA2 inhibitors. This method has potential in applications to the identification of novel inhibitors of other GST targets that are of chemotherapeutic interest.