Abstract
EAAT2 (excitatory amino acid transporter 2) is a high affinity, Na+-dependent glutamate transporter of glial origin that is essential for the clearance of synaptically released glutamate and prevention of excitotoxicity. During the course of human amyotrophic lateral sclerosis (ALS) and in a transgenic mutant SOD1 mouse model of the disease, expression and activity of EAAT2 is remarkably reduced. We previously showed that some of the mutant SOD1 proteins exposed to oxidative stress inhibit EAAT2 by triggering caspase-3 cleavage of EAAT2 at a single defined locus. This gives rise to two fragments that we termed truncated EAAT2 and COOH terminus of EAAT2 (CTE). In this study, we report that analysis of spinal cord homogenates prepared from mutant G93A-SOD1 mice reveals CTE to be of a higher molecular weight than expected because it is conjugated with SUMO-1. The sumoylated CTE fragment (CTE-SUMO-1) accumulates in the spinal cord of these mice as early as presymptomatic stage (70 days of age) and not in other central nervous system areas unaffected by the disease. The presence and accumulation of CTE-SUMO-1 is specific to ALS mice, since it does not occur in the R6/2 mouse model for Huntington disease. Furthermore, using an astroglial cell line, primary culture of astrocytes, and tissue samples from G93A-SOD1 mice, we show that CTE-SUMO-1 is targeted to promyelocytic leukemia nuclear bodies. Since one of the proposed functions of promyelocytic leukemia nuclear bodies is regulation of gene transcription, we suggest a possible novel mechanism by which the glial glutamate transporter EAAT2 could contribute to the pathology of ALS.
Highlights
We have recently demonstrated that caspase-3 cleaves EAAT2 at a unique site located in the cytosolic COOH terminus of the transporter, a finding that could link excitotoxicity and activation of caspase-3 as converging mechanisms in the pathogenesis of amyotrophic lateral sclerosis (ALS)
A Fragment Derived from Caspase-3 Cleavage of the Cytoplasmic COOH-terminal Domain of EAAT2 Accumulates in the Spinal Cord of ALS Mice at a Molecular Weight Larger than Expected—Using site-directed mutagenesis, we have previously shown that caspase-3 cleaves the glutamate transporter EAAT2 at a unique consensus site located in its cytoplasmic COOHterminal domain [16]
We looked for the presence of the (Tr)EAAT2 form in spinal cord homogenates of G93A-SOD1 mice at different stages of disease and previously reported a progressive loss of EAAT2 immunoreactivity with the antibody B 12–26, which detected a slight accumulation at progression of disease of a 55 kDa band corresponding to the (Tr)EAAT2 fragment [16]
Summary
Animals—Wild type human SOD1 and G93A-SOD1 transgenic mice, nontransgenic (non-TgN) control mice, H46R-SOD1 transgenic rats, and the R6/2 mouse model of Huntington disease were used for this study. Transfected cells were harvested after 48 h in CHAPS extraction buffer, briefly sonicated, and analyzed on Western blot or stored at Ϫ80 °C. A polyclonal antibody raised against the last 17 amino acids of rat EAAT2 COOH terminus was purchased from Affinity BioReagent (catalog number PA3-040), which we termed ABR556 –573 (1:1,000 –10,000). Cells were incubated with antiPML antibody in PBS with 0.1% Triton X-100 and 1% goat serum for 1 h at room temperature before washes in PBS. The cell lysate was used to perform Western blot analysis using an anti-LexA antibody to test bait expression. Cells were lysed in PBS buffer containing 1% Triton X-100, protease inhibitors (CompleteTM EDTA-free), 20 mM NEM (to inhibit SUMO proteases) and homogenized, followed by sonication. One mg of protein from the homogenate was loaded on a nickel spin column (catalog number H7787; Sigma), and the His-tagged proteins retained by the column were eluted with 500 l of 250 mM imidazole buffer following the manufacturer’s instructions. 30 – 40 l of the eluate was analyzed by Western blot with different antibodies as indicated
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