Table 1.
PCR primers (F : forward primer, R : reverse primer) and size of amplicon.
Figure 1.
rSOD1 and hSOD1 protein expression in primary astrocytes cultures, lumbar spinal cord and cortex from wild-type and hSOD1G93A rats.
(A) Expression of the endogenous rSOD1 (around 17 kDa) and the exogenous hSOD1 (around 19 kDa) determined by Western blotting in wild-type or hSOD1G93A astrocytes (samples from 3 different cultures are shown). The close molecular weight between the two SOD1 forms impairs clear-cut discrimination of rSOD1 when hSOD1 is expressed. Expression of the rSOD1 and hSOD1 proteins examined in the lumbar spinal cord (B) and cortex (C) from hSOD1G93A rats at various stages of the disease (P5, P60, P120, P150 and P195) or from wild-type rats at P150. Immunoblots shown are representative of three independent experiments.
Figure 2.
Influence of TNF-α on GLAST, GLT-1a and GLT-1b mRNAs and proteins in cortical astrocytes from wild-type or hSOD1G93A rats.
Expression of GLAST (A), GLT-1a (C) and GLT-1b (E) mRNAs as number of copies per microgram of RNA was estimated by RT-qPCR in control conditions or after 72 h exposure to TNF-α (20 ng/mL) using the corresponding cloned cDNA sequences as standards. Data shown are means with SEM conducted from six independent experiments performed in duplicate. Expression of GLAST (B), GLT-1a (D) and GLT-1b (F) proteins was examined in cells maintained in control conditions or treated with TNF-α (20 ng/mL) for 72 h. Immunoblots shown are representative of six independent experiments. Data indicate the levels of the protein of interest normalized to GAPDH and represents means with SEM. *p<0.05 for comparison between the same genotype.
Figure 3.
Effect of protein synthesis inhibition on the protein expression of GLAST, GLT-1a and GLT-1b on wild-type or hSOD1G93A astrocytes.
(A) Protein expression of GLAST, GLT-1a and GLT-1b was examined by immunoblotting in astrocytes from wild-type and hSOD1G93A maintained in culture either in control conditions or treated with TNF-α (20 ng/mL) and/or using the inhibitor of protein synthesis cycloheximide (10 µg/mL) for 48 h. Immunoblots shown are representative of four independent experiments. Data obtained after densitometric analyses of GLAST (B), GLT-1a (C) and GLT-1b (D) proteins are means with SEM normalized to GAPDH and expressed in percent of the signal obtained for wild-type astrocytes cultured in control conditions. * p<0.05 and *** p<0.001 for comparison between the same genotype after a different treatment and ##p<0.01 for comparison between different genotypes with the same treatment (two way ANOVA followed by Bonferroni post-hoc test).
Figure 4.
Influence of TNF-α on the activity of glutamate transporters from wild-type or hSOD1G93A cortical astrocytes.
Glutamate transporter activity was evaluated by measuring velocity of d-[3H]-aspartate uptake (50 nmol/L) in wild-type and hSOD1G93A astrocytes treated or not with TNF-α (20 ng/mL) for 72 h. (A) shows total uptake while (B) and (C) illustrate GLT-1- and GLAST-dependent uptake, respectively evaluated in presence of the selective inhibitors WAY-213613 (100 µmol/L) and UCPH-101 (10 µmol/L). Shown are mean with SEM from five independent experiments realized in quintuplicate. #p<0.05 and ###p<0.001 for comparison between different genotypes with the same treatment.