The objective of this proposal is to study the molecular mechanisms leading to impairment and loss of the glial glutamate transporter GLT1 in amyotrophic lateral sclerosis (ALS). ALS is an age-dependent neurodegenerative disorder of motor neurons in the spinal cord, motor cortex and brain stem. There is a growing body of evidence indicating that deficient glutamate uptake may be a contributory factor to motor neuron loss in ALS. In ALS patients, a marked decrease in the maximal velocity of synaptosomal glutamate uptake was reported. The impairment was found in regions affected by the disease, such as the spinal cord and motor cortex and a specific reduction of GLT1 immunoreactivity (30-90 percent) in the motor cortex of 60 percent of sporadic ALS patients was reported. A marked loss of GLT1 immunoreactivity was also detected in transgenic mice expressing the SOD1(G85R) mutation, suggesting that the sporadic and the familial form of ALS (SOD component) share common molecular mechanisms. The precise events leading to GLT1 loss in ALS are not yet understood. The levels of GLT1 mRNA are unchanged, letting investigator suspect that the reduction of GLT1 is not due to decreased transcription of mRNA, but rather to some other events at the translation or post translational level. We have recently reported that intracellular delivery of H2O2 in cells expressing SOD mutations led to selective GLT1 inhibition. Moreover, we showed that the cytoplasmic C-terminal domain of GLT1 is involved in the inhibition. These observations provided the first link between the SOD1 mutations and GLT1 impairment in ALS. Why is GLT1 selectively damaged while other glutamate transporters are insensitive? GLT1 has the highest number of oxidant vulnerable amino acid residues, such as cysteines histidines and tyrosines and therefore is the most prone to oxidative modifications and damage. We expect that a damaged GLT1 caused by the SOD1 mutants would undergo to a sustained internalization and/or selective degradation. Little is known about glutamate transporters regulation and degradation and how their functional inhibition or stimulation affects the pathophysiological events of neurodegenerative diseases such as ALS. We plan to determine the pathways that lead to GLT1 loss in ALS by taking the following approaches: I) defining the molecular events responsible for the inactivation of GLT1 mediated by the SOD1 mutants and identifying the sites in the C-terminal domain of GLT1 that are targets for the inactivation; 2) defining the pathways of GLT1 degradation under normal conditions and under conditions initiated by the ALS-linked SOD1 mutations 3) using transgenic technology we will establish in vivo what role the inactivation of GLT1 is playing in the onset and progression of ALS.
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