Journal: International Journal of Molecular Sciences
Article Title: Neuroprotective Potential of L-Glutamate Transporters in Human Induced Pluripotent Stem Cell-Derived Neural Cells against Excitotoxicity
doi: 10.3390/ijms241612605
Figure Lengend Snippet: Inhibition of EAATs increases the extracellular L-Glu concentration and leads to excitotoxicity at 63 DIV. ( A ) The effect of L-Glu alone on cell viability at 14 and 63 DIV ( n = 6). Cell viability was assessed using the MTT reduction assay. At 14 ( a1 ) and 63 ( a2 ) DIV, the application of L-Glu at 100 μM for 24 h did not change MTT reductions compared with the application of DMSO at 0.4% (Cont), which was used as a vehicle. Unpaired t -test. Data are expressed as the means ± standard deviations. Similar results were obtained in three independent experiments. ( B ) The effects of TFB-TBOA (TFB, nonspecific EAAT inhibitor, 30 nM) when coapplied with L-Glu on cell viability at 14 and 63 DIV ( n = 4–6). At 14 DIV, TFB caused no effect on MTT reductions ( b1 ). On the other hand, at 63 DIV, TFB significantly decreased MTT reductions, and AP5 (NMDAR antagonist, 100 μM) blocked the decrease in MTT reductions by TFB ( b2 ). Similar results were obtained in three independent experiments. ** p < 0.01 vs. L-Glu(+)TFB(−)AP5(−) group by Tukey’s test following ANOVA. ## p < 0.01 vs. L-Glu(+)TFB(+)AP5(−) group by Tukey’s test following ANOVA. Data are expressed as the means ± standard deviations. Similar results were obtained in three independent experiments. ( C ) Identification of the contribution of specific EAATs to the decrease in exogenously applied L-Glu. ( c1 ) Change in the concentration of L-Glu in the medium ([L-Glu] out ) after L-Glu (100 μM) was applied at 63 DIV ( n = 3). [L-Glu] out was nearly zero at 60 min. Data are expressed as the means ± standard deviations. Similar results were obtained in three independent experiments. ( c2 ) The effects of EAAT inhibitors on L-Glu uptake at 63 DIV ( n = 5–7). The effects of EAAT inhibitors on the decrease in [L-Glu] out were assessed at 30 min, which was the time for the 50% decrease in [L-Glu] out from C(c1). The percentage inhibition of EAAT inhibitors on L-Glu uptake activity was calculated as 100% of the decrease in extracellular L-Glu concentration in the absence of EAAT inhibitors. UCPH-101 (UCPH, EAAT1 selective EAAT1 inhibitor, 100 μM), dihydrokainic acid (DHK, a competitive EAAT2 inhibitor, 300 μM), WAY213613 (WAY, 10 μM), or TFB inhibited L-Glu uptake. Data are expressed as the means ± standard deviations. ( D ) Inverse correlation between the strength of L-Glu uptake inhibition and the cell viability caused by EAAT inhibitors. The Pearson’s correlation coefficient (PCC) = −0.7110. EAAT inhibitors are shown as follows: UCPH: blue dot; DHK: green dot, WAY: pink dot, and TFB: red dot.
Article Snippet: Mouse anti-HuC/D monoclonal antibody (1:100, A21271, Thermo Fisher Scientific), chicken anti-GFAP polyclonal antibody (1:400, ab4674, Abcam), rabbit anti-S100β polyclonal antibody (1:500, ab52642, Abcam), goat anti-Vglut2 polyclonal antibody (1:500, Go-Af310-1, Frontier Institute, Hokkaido, Japan), rabbit anti-Syn1 polyclonal antibody (1:1000, AB1543, Chemicon), chicken anti-MAP2 polyclonal antibody (1:5000, ab5392, Abcam), mouse anti-PSD95 monoclonal antibody (1:500, 7E3-1B8, Thermo Fisher Scientific), mouse anti-EAAT1 monoclonal antibody (1:200, ab49643, Abcam), rabbit anti-Nestin polyclonal antibody (1:200, ABD69, Millipore), or guineapig anti-EAAT2 polyclonal antibody (1:1000, AB1783, Chemicon) were used.
Techniques: Inhibition, Concentration Assay, MTT Reduction Assay, Activity Assay
![Inhibition of EAATs increases the extracellular L-Glu concentration and leads to excitotoxicity at 63 DIV. ( A ) The effect of L-Glu alone on cell viability at 14 and 63 DIV ( n = 6). Cell viability was assessed using the MTT reduction assay. At 14 ( a1 ) and 63 ( a2 ) DIV, the application of L-Glu at 100 μM for 24 h did not change MTT reductions compared with the application of DMSO at 0.4% (Cont), which was used as a vehicle. Unpaired t -test. Data are expressed as the means ± standard deviations. Similar results were obtained in three independent experiments. ( B ) The effects of TFB-TBOA (TFB, nonspecific EAAT inhibitor, 30 nM) when coapplied with L-Glu on cell viability at 14 and 63 DIV ( n = 4–6). At 14 DIV, TFB caused no effect on MTT reductions ( b1 ). On the other hand, at 63 DIV, TFB significantly decreased MTT reductions, and AP5 (NMDAR antagonist, 100 μM) blocked the decrease in MTT reductions by TFB ( b2 ). Similar results were obtained in three independent experiments. ** p < 0.01 vs. L-Glu(+)TFB(−)AP5(−) group by Tukey’s test following ANOVA. ## p < 0.01 vs. L-Glu(+)TFB(+)AP5(−) group by Tukey’s test following ANOVA. Data are expressed as the means ± standard deviations. Similar results were obtained in three independent experiments. ( C ) Identification of the contribution of specific EAATs to the decrease in exogenously applied L-Glu. ( c1 ) Change in the concentration of L-Glu in the medium ([L-Glu] out ) after L-Glu (100 μM) was applied at 63 DIV ( n = 3). [L-Glu] out was nearly zero at 60 min. Data are expressed as the means ± standard deviations. Similar results were obtained in three independent experiments. ( c2 ) The effects of EAAT inhibitors on L-Glu uptake at 63 DIV ( n = 5–7). The effects of EAAT inhibitors on the decrease in [L-Glu] out were assessed at 30 min, which was the time for the 50% decrease in [L-Glu] out from C(c1). The percentage inhibition of EAAT inhibitors on L-Glu uptake activity was calculated as 100% of the decrease in extracellular L-Glu concentration in the absence of EAAT inhibitors. UCPH-101 (UCPH, EAAT1 selective EAAT1 inhibitor, 100 μM), dihydrokainic acid (DHK, a competitive EAAT2 inhibitor, 300 μM), WAY213613 (WAY, 10 μM), or TFB inhibited L-Glu uptake. Data are expressed as the means ± standard deviations. ( D ) Inverse correlation between the strength of L-Glu uptake inhibition and the cell viability caused by EAAT inhibitors. The Pearson’s correlation coefficient (PCC) = −0.7110. EAAT inhibitors are shown as follows: UCPH: blue dot; DHK: green dot, WAY: pink dot, and TFB: red dot.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4411/pmc10454411/pmc10454411__ijms-24-12605-g004.jpg)
