Intron 7 retention and exon 9 skipping EAAT2 mRNA variants are not associated with amyotrophic lateral sclerosis

Abstract

Glutamate-mediated excitotoxicity is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). The astroglial glutamate transporter EAAT2 plays a major role in maintaining low levels of extracellular glutamate in the central nervous system. Multiple EAAT2 mRNA transcripts have been described, but those retaining intron 7 or skipping exon 9 are reported to be specific to the motor cortex, spinal cord, and cerebrospinal fluid of ALS patients. We sought to verify these findings using a TaqMan (Elmer Biosystems, Warrington, UK) real-time reverse transcriptase polymerase chain reaction assay, which provides a sensitive and reliable quantitative measure of EAAT2 transcript copy ratios. We analyzed RNA extracted from frozen postmortem tissue from affected and unaffected central nervous system regions dissected from 17 sporadic ALS patients, 7 Alzheimer's disease patients, and 19 control subjects. We have demonstrated unequivocally that intron 7 retaining and exon 9 skipping variants can be detected in all individuals and in all central nervous system regions studied. The mean ratio of "variant" to "normal" transcripts did not differ significantly between patient and control groups. Although our assay could detect transcript concentrations in cerebrospinal fluid as low as 10 pg/ml, none were detected in 17 ALS and 8 control samples. We conclude that ALS is not associated with elevated levels of EAAT2 transcripts retaining intron 7 and skipping exon 9. An alternative explanation must be sought for the disturbance of glutamate homeostasis reported in ALS.

Altered in-vitro and in-vivo expression of glial glutamate transporter-1 following exposure to cerebrospinal fluid of amyotrophic lateral sclerosis patients

“…It is therefore tempting to speculate that the broad band seen on Western blots in our study represents splice variants or degraded protein products with differential electrophoretic mobility. More so since some studies have reported these kind of splice variants even in controls and Alzheimer's patients [30][31][32]. In controls, although both GLT-1 and GLAST staining are seen in astroglia, only GLT-1 expression is affected in the spinal cords of ALS patients.…”

Altered expression of the glutamate transporter EAAT2b in neurological disease

“…Previous studies of ALS tissue showed multiple misspliced EAAT2 mRNA species 13. Most of these alternate spliced products were not physiologically active; some were also seen in control tissue, whereas others produced a dominant‐negative effect on normal EAAT2 protein expression 11, 43, 44. Another possibility is that EAAT2b may be another of the family of alternatively spliced variants among those described by Lin and colleagues 13.…”

The evidence for altered RNA metabolism in amyotrophic lateral sclerosis (ALS)

“…Although the preferential expression of alternative splice variants of the astrocytic glutamate transporter (EAAT2) through intron-retention and exon-skipping was postulated to be associated with the alterations in astrocytic glutamate uptake observed in ALS, the specificity of expression of EAAT2 splice variants to ALS has been called into question [57][58][59][60]. However, it is possible that it is not the altered expression of an individual splice variant that is pathogenic per se, but rather it's effect upon associated mRNAs and RBPs.…”