Screening of compounds showing protective effect against cell death induced by familial amyotrophic lateral sclerosis (FALS)-linked mutantsuperoxide dismutase 1 (SOD1) (ALSOD1)
Amyotrophic Lateral Sclerosis (ALS) is a progressive fatal paralytic disorder of unknown cause involving degeneration of motor neurons (MNs) of the brain and spinal cord. Approximately 10% of ALS cases are autosomal dominantly inherited and referred to as FALS. Some cases of FALS have been linked tochromosome 21, and about 70 different single site mutations have been identified in the SOD1 gene more ..
Amyotrophic Lateral Sclerosis (ALS) is a progressive fatal paralytic disorder of unknown cause involving degeneration of motor neurons (MNs) of the brain and spinal cord. Approximately 10% of ALS cases are autosomal dominantly inherited and referred to as FALS. Some cases of FALS have been linked tochromosome 21, and about 70 different single site mutations have been identified in the SOD1 gene in FALS patients. Our previous studiesshowed that replication defective adenovirus mediated expression of mutant SOD1 induces cell death in neural cells (primary hippocampalneurons, primary sympathetic neurons, differentiated PC12 cells), but not primary astrocytes, indicating the increased sensitivity ofneurons to the pathogenic effect of mutant SOD1. These studies also showed that agents that generally have anti-apoptotic activity, such asBcl-2, caspase inhibitors (ZVAD-FMK and Ac-YVAD-CMK) and SOD mimics (EUK-8 and EUK-134), significantly rescued differentiated PC12 cellsfrom death induced by expression of either mutant SOD1 and/or following NGF withdrawal. In addition, a metal chelator,tetraethylene-pentamine (TEPA), rescued cell death induced by expression of either mutant SOD1, but had only a very small effect on celldeath induced by NGF withdrawal, suggesting that the pathways of apoptosis induced by mutant SOD1 vs. growth factor withdrawal aredifferent. We have modified our assay so that PC12 cells can be grown/differentiated in a 96-well plate and counted for mutant SOD1-inducedcell death and identify potential drug targets that protect cell death.
Fifteen hundred PC12 cells were planted per well in a 96-well plate and differentiated with 75 ng/ml of NGF in DMEM containing 1% bovine calf serum, 0.01% glutamine and 0.01 % gentamycin for 6 days with a change of media after 3 days. Cells were infected with recombinant adenovirus AdSODA4V expressing SOD1 mutant A4V. One hour later, virus was removed and 100 uL of the media containing NGF was added. Analiquot of drug was added into the media as indicated and the plates were incubated once more. Three days later, cell viability was assayed using a CCK-8 detection kit (Dojindo Molecular Technologies, Inc., Gaithersburg, MD) according to the manufacturer's instructions.In brief, 10 uL of CCK-8 solution was added to each well of the plate. The plates were incubated for 4 hours and the absorbance was measured at 450 nm using a plate reader with the reference wavelength at 610 nm.
Cell Lines: Recombinant replication-defective adenoviruses were used to transiently express human wild type or mutant SOD1 genes into differentiated rat pheochromocytoma PC12 cells.
Growth Medium: PC12 cells were grown on Dulbeccois Modified Eagleis Medium (DMEM) with 15% Fetal Bovine Serum (Gibco, cat. no. 26140-079), 0.01% glutamineand 0.01% gentamycin.
Assay Medium: PC12 cells were differentiated with 75 ng/ml of nerve growth factor (NGF) in DMEM containing 1% bovine calf serum, 0.01% glutamine and 0.01% gentamycin for 6 days with a change of media after 3 days.
Assay Reagent: The cell viability was assayed using a CCK-8 detection kit (Dojindo Molecular Technologies, Inc., Gaithersburg, MD) according to themanufacturer's instructions. The CCK-8 assay is based on detection of dehydrogenase activity in viable cells. It utilizes a highlywater-soluble tetrazolium salt, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]that produces a water-soluble formazon dye upon reduction in the presence of an electron carrier and can be performed within 1-4 hours.
Data Analysis: The absorbance of 100 uL media with drug was subtracted from the mean of absorbance found in the drug-treated differentiated PC12 expressing mutant SOD1. Each compound library plate was tested in triplicate with mutant expressing cells. Forty-eight compounds wereselected on the basis of their ability to protect mutant SOD1-induced cell death by selecting drugs that have the highest mean absorbance minus the drug control.
Dilution Series: The selected 48 compounds were retested in a 2-10 fold, 8-point dilution series with concentration of drug ranging from 10 nM to 100 uM. Concentrations required to achieve 50% of the maximally achievable effect for each compound (EC50) were calculated using GraphPad Prismsoftware program.
Detection Reagent: A water-soluble tetrazolium salt, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodiumsalt], (Dojindo Molecular Technologies, Inc., Gaithersburg, MD), cat. no. CCK-8.
Selected 48 compounds were retested for the specific protective effect against mutant (A4V) SOD1 compared to virus and drug control. Absorbance of drug control alone was subtracted from that of AdSODA4V.
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 Raymond P. Roos
Affiliation The University of Chicago
Investigator Ghanashyam D.Ghadge
Categorized Comment - additional comments and annotations
Data Table (Concise)