Screen for compounds that increase glutamate transport activity in MN-1 cell line (GluUPTAKE)
Harmful overstimulation of glutamate receptors (excitotoxicity) has been suggested to be important in motor neuron disease via: (i) increased glutamate levels (e.g. due to reduced uptake) or (ii) increased sensitivity to endogenous excitatory amino acids (e.g. because of changed glutamate receptors). Reduced uptake of glutamate as measured by uptake studies in synaptosomes or by sodium dependent more ..
Harmful overstimulation of glutamate receptors (excitotoxicity) has been suggested to be important in motor neuron disease via: (i) increased glutamate levels (e.g. due to reduced uptake) or (ii) increased sensitivity to endogenous excitatory amino acids (e.g. because of changed glutamate receptors). Reduced uptake of glutamate as measured by uptake studies in synaptosomes or by sodium dependent binding of D-aspartate has been observed in autopsy material from patients with ALS. This reduced uptake is claimed to be attributable to a selective loss of GLT1 (EAAT2) because a dramatic loss of GLT-immunoreactivity has been reported. Therefore, it appears that a correct functioning of the glutamate transporters is of fundamental importance in ALS. Rescuing the glutamate transporter activity from dysfunction by specifically increasing GLT1(EAAT2) activity or by increasing the overall glutamate uptake capacity, hence compensating for the loss of GLT1, should be beneficial for the treatment of ALS. We developed a 24-well based assay to screen for compounds that affect the glutamate transport activity in a mouse spinal cord motor neuron hybrid cell line, MN-1. In particular, we are looking for compounds that significantly increase glutamate transport in these cells. We have established a cut off effect of +30% uptake increase; compounds that gives an increase 30% in glutamate uptake are considered positive hits.
Screening - incubation of cells with test compounds in Opti-MEM (GIBCO cat.#31985070 serum free).MN-1 cells are plated in 24-well plated. When the cells reach near confluence ((90%), compounds to be tested are added overnight at 1 uM in 0.1% DMSO (final concentration) at 37 C. The control groups received 0.1% DMSO. The next day the compounds are washed out with PBS 3 times and glutamate uptake is measured for a total of 15 min by adding the uptake buffer in the presence of 1 uM glutamate (isotopically diluted). Uptake is then stopped by washing the cells with cold stopping buffer. The cells are harvested using lysis buffer containing 0.5 N NaOH and 0.1% triton X-100. The cells are then placed in scintillation vials, added with Scintisafe 50% (3ml) and counted for radioactivity using a Beckman Scintillation counter.
Cell Lines We used a clonal neural hybrid cells that can express motor neuron characteristic. An aminopterin-sensitive and neomycin-resistant mouse neuroblastoma cell line was fused to isolated embryonic mouse spinal cord motor neurons. These cell lines are referred to here as MN-1. E.F. Salazar-Grueso, S.Kim, H. Kim. (1991) Embryonic mouse spinal cord motor neuron hybrid cells. Neuroreport 2: 505-508. These cells have a consistent glutamate transport activity and endogenously express the three major glutamate transporter subtypes
Growth Medium: The growth medium for the MN-1 cell lines was Dulbecco's Modified Eagle's Medium (DMEM) with 10% Fetal Bovine Serum (Gibco cat. #16000044), 50 units/ml penicillin and streptomycin sulfate).
Assay Medium: Uptake buffer composition (in mM): 5 HEPES/Na, 145 NaCl, 2.5 KCl, 1.2 CaCl2, 1.2 MgCl2, 1.2 K2HPO4, 10 Glucose, pH 7.4 in the presence of 1 uM glutamate (isotopic dilution 1:30 with 3H-glutamate). Uptake stopping buffer composition (in mM): 5 Tris-HCl, 145 CholineCl, 2.5 KCl, 1.2 CaCl2, 1.2 MgCl2, 1.2 K2HPO4, 10 Glucose, pH 7.4
Assay Reagent: 3H-glutamate: N. England Nuclear
Data Analysis: Uptake values in each well are determined as counts per minute (cpm-read outs from the scintillation counter), which are then converted into pmoles of glutamate taken up in 15 min assay-time. Uptake values in drug-treated wells are compared to the average of the control treated wells (3x DMSO 0.1%) in the same plate and expressed as percentage of increase/decrease glutamate uptake over control. In each 24-well plate we have 3 wells for control and 21 wells for 7 different drugs (7 drugs done in triplicate). We have controls for each plate because there may be inter-plate variability in uptake values performed on different days due to the conditions of the cells and other factors that we cannot predict.
Dilution Series Compounds were tested at 1 uM final concentration in 0.1% DMSO by applying 1 uL of 1mM stock solution (100% DMSO) of the compounds. Original compounds in the microplate format were diluted down to 1 mM using DMSO. The compounds selected for EC50s were tested at maximum concentration of 10 uM in 0.1% DMSO. Concentrations required to achieve 50% of the maximally achievable effect for each compound (EC50) were calculated using Grafit (Erithacus Software).
Assay Type: mammalian cell-based
Number Replicates: 3
Assay Readout: counts per minute - c.p.m. are then converted into pmoles of glutamate taken up by the cell per 15 min - the effect of the drugs on glutamte uptake is then expressed as percentage of increase or decrease over control
Detection Instrument Beckman LS 1701 scintillation counter
Concentration Primary Screen (uM) 1
3 replicates for each drug.
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 Davide Trotti
Affiliation Massachusetts General Hospital
Investigator Eric O.Williams
Data Table (Concise)