Screen for compounds that inhibit polyglutamine induced protein aggregation (AGREG)
Huntington's disease is a genetic disorder associated with polyglutamine expansion. Increasing lengths of polyglutamines cause protein aggregation, and other cellular events, that are believed to contribute to neurodegeneration and neurophysiological abnormalities. We have generated stable lines of human neural SY5Y cells that stably express 19 glutamines or 56 glutamines, linked to green more ..
Huntington's disease is a genetic disorder associated with polyglutamine expansion. Increasing lengths of polyglutamines cause protein aggregation, and other cellular events, that are believed to contribute to neurodegeneration and neurophysiological abnormalities. We have generated stable lines of human neural SY5Y cells that stably express 19 glutamines or 56 glutamines, linked to green fluorescent protein (GFP). Twenty four hours following a 2 hour heat shock (43 Celsius degrees), the cells with 56-GFP develop visible aggregates. These polyglutamine-length dependent aggregates can then be counted and used to identify drugs that inhibit polyglutamine induced protein aggregation.
Screening - incubation of cells with test compounds. For the aggregation assay, cells were removed from T-75 culture flasks with 10 mL trypsin-EDTA solution (0.25%). The cells (~5M cells/flask) were spun down and re-suspended in assay medium to a concentration of ~200,000 cells/ml. The cells were immediately seeded in 35 mm glass bottom plates (MicroTek) at a cell density of 50,000 cells/well. Cells were allowed to grow for 2 days prior to experimentation. Compounds from the NINDS library were added to reach a final concentration of 2.5 uM (diluted with assay medium) with 2 uL of individual compounds added to each 1 ml of culture medium containing the cells. Cells were then placed in 43 Celcius degrees 5% CO2 incubator for 2 hours, and then returned for 24 hours to 5% CO2 incubator 37 Celcius degrees. Less than 1% of cells display aggregates under normal growth conditions, and this value was subtracted from each experiment. In each experiment 3 cultures of cells were given heat shock treatment without any compounds, to determine the percentage of cells with aggregates. The percentage of cells with aggregates was determined by manually counting the cells under fluorescence microscopy (40X) in random fields. At least 50 cells were counted from each dish, and the percentage of cells with aggregates determined. Each compound was assayed in triplicate.
Cell Lines: Human neuroblastoma SY5Y cells were stably transfected with 19 or 56 glutamines ligated to the amino terminus of green fluorescent protein. The cell lines are designated as 19-GFP and 56-GFP. Cells of fewer thant 15 passages were utilized for all assays.
Growth Medium: The growth medium for cells was Minimal Essential Medium (MEM) with 10% heat inactivated Fetal Bovine Serum, 50 ug/ ml penicillin, 50 ug/ml streptomycin sulfate and 800 mg/L G418. All items were purchased from Gibco.
Assay Medium: The cell culture medium used for the aggregation assay (assay medium) was identical to the growth medium without the G418 selection agent.
Data Analysis: The percentage of cells with aggregates was determined in individual dishes by dividing the number of cells with aggregates from the total number of cells counted, and taking this value times 100. The percentage of inhibition was determined for each dish by dividing the percentage of cells with aggregates (for an individual dish) from the mean value of cells with aggregates (in cells receiving no compounds, and only heat shock treatment). The average percentage of inhibition and standard deviation was determined using the percent inhibition for the 3 dishes that were used for each compound. Aggregates are visible as intensely fluorescent spots. The percentage of cells containing these spots (aggregates) was determined. Cells with even 1 spot were counted as having aggregates, as were cells that contained more than 1 spot.
A Note about the Activity Matrix Rankings:
The data for each assay has been converted into a numerical ranking of all 1040 compounds for that assay. Thus, a compound with a rank of 1 was the top scoring compound in that assay and a compound with a rank of 1040 was the lowest scoring compound in that assay. Compounds with equal scoring were given equal rank.
PubChem scores were derived as (1040-NINDS activity rank). Substances with reported EC50 values were considered active.
PI Name Edgardo Dimayuga
Affiliation University of Kentucky
Investigator Qunxing Ding
PI Name Jeffrey N. Keller
Affiliation University of Kentucky
Investigator Jeffrey N.Keller
Data Table (Concise)