Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is characterized by selective upper and lower motor neuron degeneration, the pathogenesis of which is unknown. About 60-70 percent of sporadic ALS patients have a 30-95 percent loss of the astroglial glutamate transporter EAAT2 (excitatory amino acid transporter 2) protein in motor cortex and spinal cord. Loss of EAAT2 leads to increased extracellular glutamate and excitotoxic neuronal degeneration. Preliminary studies document multiple abnormal EAAT2mRNAs, including intron-retention and exon-skipping, in the affected tissues of ALS pateints. The aberrant mRNAs were highly abundant and were only found in neuropathologically affected areas of ALS patients, but not in other brain regions. They were found in 65 percent of sporadic ALS patients, but were not found in non-neurologic disease or other diseased controls. They were also detectable in the cerebrospinal fluid of living ALS patients, early in the disease. In vitro expression studies suggest that proteins translated from these aberrant mRNAs may undergo rapid degradation and/or produce a dominant negative effect on normal EAAT2 resulting in loss of protein and activity. We propose a systematic approach to study the biology and relevance of aberrant EAAT2 mRNA and protein in ALS.
The specific aims address: 1) Identification and characterization of aberrant EAAT2 mRNA species and proteins in ALS, including disease and tissue specificity, quantification. 2) Using in vitro expression systems we will investigate the biology of the aberrant RNA species to understand how they could account for the loss of EAAT2 in ALS by examining their transporter characteristics-Vmax, Km, conductance properties, stability and cellular trafficking. 3) Develop in vitro model systems to determine the processes that could be responsible for their formation in vivo, including excitotoxicity/oxidative stress, DNA repair, RNA processing proteins, and use astrocytes cultures from ALS patients to study their formation. Our preliminary work suggests that the loss of EAAT2 IN ALS is due to aberrant mRNA, and these aberrant mRNAs could result from RNA processing errors. Aberrant RNA processing could bed an important process in the pathophysiology of neurodegenerative disease and in excitotoxicity. The presence of these mRNA species in also cerebrospinal fluid may have diagnostic utility.
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