Samples were incubated with shaking at 250 rpm and 37 C, conditions shown previously to enhance fibrillization (22, 23)
Samples were incubated with shaking at 250 rpm and 37 C, conditions shown previously to enhance fibrillization (22, 23). the production/accumulation of reactive oxygen species. Most importantly, treatment with D737 increases the life span and locomotive ability of flies in a model of Alzheimer disease. model of AD. MATERIALS AND METHODS High Throughput Screening For small molecule prescreening, isopropyl 1-thio–d-galactopyranoside (1 mm final concentration) was added to LB medium, and 20 l of medium was dispensed into each well of 384-well plates (black with clear flat bottoms; Corning). Compounds were prescreened for inherent fluorescence by adding 300 nl of compound to the medium. Stock compound libraries were dispensed by the CyBi-Well 96- and 384-channel simultaneous pipettor (CyBio) with a 384-channel/300-nl dispensing pin. All compounds were dissolved in dimethyl sulfoxide (DMSO), and pure DMSO was used as the negative control. After compound addition, plates were read at the GFP wavelength (485 nm excitation and 510 nm emission) on an EnVision plate reader (PerkinElmer Life Sciences). Each plate was screened in duplicate. Following this prescreen, we screened for aggregation inhibitors as follows. BL21(DE3) cells containing the A42-GFP fusion plasmid were grown at 37 C to flies expressing A42 were generated by crosses as described by Crowther (36). Before crossing, flies were reared on standard cornmeal-agar medium and kept at room temperature. Virgin female flies carrying the A42 or A42(E22G) transgene under the control of the upstream activation sequence promoter in a homozygous condition were collected no longer than 10 h post-eclosion. Female virgins were crossed with male flies carrying the driver elavC155-Gal4 on their X chromosome. This results in first generation female offspring expressing A42 or A42(E22G) in their central nervous system. Wild-type Oregon-R flies reared in the same medium served as controls. Flies were crossed on medium containing the indicated concentration of inhibitor. Medium was made by dissolving the compounds (from DMSO stocks) or a similar volume of DMSO in cornmeal-agar medium liquefied in a water bath. Medium containing a compound or DMSO was prepared weekly, aliquoted into vials, and stored at 18 C until used. Flies were kept at 29 C to promote transgene expression. Fly Longevity Assays were performed as described by Crowther (36). Post-eclosion, 100 female wild-type flies or flies expressing transgenes were collected in groups of 20 in 10-cm glass vials containing inhibitor or DMSO. Flies were kept at 29 C in vials containing cornmeal-agar medium with the test compound throughout life, and food was changed two to three times per week. Viable flies were counted daily. Median survival was analyzed using Kaplan-Meier statistics, and significance was calculated with the two-tailed TTEST function in Microsoft Excel. Fly Climbing Locomotive ability was assessed as described by Crowther (36). 10-cm vials containing 20 flies Indirubin each were tapped gently on the table. The number of flies that climbed to the top of the vial was recorded after 18 s. Results represent the fraction of flies climbing to the top compared with age-matched control flies. RESULTS High Throughput Screening Enables the Discovery of Novel A Aggregation Inhibitors To search Indirubin for small molecules that inhibit the aggregation of A42, we utilized a HTS developed previously in our laboratory (18). This screen uses fluorescence from the proper folding of GFP as a reporter for A42 aggregation. The screen is based on the finding that the A42-GFP fusion protein expressed in does not fluoresce because the aggregation Rabbit Polyclonal to PLG and/or insolubility of the upstream A42 sequence prevents the correct folding and subsequent fluorescence of the downstream GFP. Compounds that inhibit A42 aggregation and allow GFP to fold can be found by screening for fluorescence (Fig. 1) (18, 19). This screen is nonspecific and, in principle, should enable isolation of compounds that inhibit the formation of LMW oligomers, HMW oligomers, and/or fibrils. Open in a separate window FIGURE 1. Schematic representation of the fluorescence-based screen using the A42-GFP fusion. In the absence of inhibition, the A42 portion of the fusion aggregates rapidly and causes the entire A42-GFP fusion to misfold and aggregate. Therefore, no fluorescence is observed. However, inhibition of A42 aggregation by a small molecule enables Indirubin GFP to form its native green color..