A role for astrocytes in inherited amyotrophic lateral sclerosis (ALS) linked to SOD1 mutations has been considered in several contexts, both in patients and animal models of the disease. Astrocytes expressing ALS-linked mutant SOD1 proteins produce and release one or more as yet uncharacterized soluble components that can accelerate motor neuron death in vitro. Focal loss of the astrocytic glutamate transporter EAAT2 (a.k.a. GLT-1) in the diseased spinal cord and failure of normal glutamate re-uptake in ALS patients and animal models support glutamate-dependent excitotoxicity as a component of the disease. In the previous funding cycle, we have found that EAAT2 is selectively cleaved by caspase-3 in the spinal cord of mutant SOD1 (SOD1-G93A) transgenic mice model of ALS and that the C-terminal EAAT2 fragment derived from this cleavage (termed CTE fragment) is conjugated to SUMO-1 (small ubiquitin modifier protein) and accumulates in the disease, beginning already at pre-symptomatic age. The formation of this CTE-SUMO1 fragment is distinctive of ALS as it does not occur in other neurodegenerative diseases such as Huntington's disease. This evidence suggests that ALS mechanisms target selectively EAAT2. Based on a new set of preliminary observations we hypothesize that in addition to the general excitotoxicity ensued by impaired function of the glutamate transporter EAAT2, the accumulation in spinal cord astrocytes of the CTE-SUMO1 fragment plays a direct role in the pathogenesis and/or progression of this disease. In this application we propose to study in vitro and in vivo the implications on motor neuron death and the ALS phenotype of CTE-SUMO1 astrocytic accumulation. We have designed four specific aims:
In Aim 1 we will characterize SUMOylation of EAAT2 in mutSOD1 mice model of ALS and its functional implications.
In Aims 2 - 4 will define in vitro and in vivo the localization and pathophysiological implications of the EAAT2-derived fragment CTE-SUMO1 in the context of motor neuron death linked to ALS. Our overarching goal is to identify the molecular mechanisms underlying glutamate transporter dysfunction in ALS at post-translational levels.
Amyotrophic lateral sclerosis (ALS) is one of the most devastating and lethal progressive neuromuscular disorders. Over 30,000 people are living with ALS in the United States and approximately 5,000 Americans will be diagnosed with ALS this year. There is a growing body of evidence indicating that a defective re-uptake protein called glutamate transporter EAAT2 contributes to the progression of this neuromuscular disease. In this application we propose to study the mechanisms of impairment of these re-uptake proteins in ALS to unravel possible molecular targets for future therapeutic intervention.
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