Alternative Pathway of EAAT2-Mediated Toxicity in Amyotrophic Lateral Sclerosis
The astroglial glutamate transporter EAAT2 is decreased in the neurodegenerative disease amyotrophic lateral sclerosis (ALS), contributing to the excitotoxicity of motor neurons. Our group previously showed that in the SOD1-G93A mouse model of ALS, activated caspase-3 cleaves EAAT2 at a consensus sequence, leading to the accumulation of a SUMO1-modified C-terminal fragment (CTE-SUMO1) beginning around disease onset. Expression of CTE-SUMO1 by astrocytes causes them to secrete factors toxic to motor neurons, though a D505N mutation in the caspase-3 consensus sequence of EAAT2 inhibits cleavage and thus formation of the fragment. Here, we first discuss EAAT2 and sumoylation in the context of ALS. Next, sumoylation of EAAT2 was examined using subcellular fractionation with immunoprecipitations, immunohistochemistry, and manipulation of SUMO-1 modification machinery. We found that a portion of EAAT2 is sumoylated physiologically at lysine 570 and the modification leads to intracellular compartmentalization of the transporter. The proportion of EAAT2 that is sumoylated does not change with disease in SOD1-G93A mice. To further study the impact of the sumoylated fragment of EAAT2 on ALS in vivo, the D505N point mutation was introduced to C57BL/6 mice, which were then crossed with SOD1-G93A mice. The mutation did not cause any differences in the growth or motor phenotype of non-ALS mice. In SOD1-G93A mice, however, EAAT2-D504N led to a significant extension of the disease progression and a delay of death. No delay in the onset of weight loss was observed, supporting the hypothesis that the CTE-SUMO1 pathway is most relevant after onset. Significant delay in the early losses of hindlimb and forelimb grip strength and a trend towards increased phrenic nerve strength at the late-symptomatic stage were also observed in EAAT2-D504N mice. While a small portion of full length EAAT2 is sumoylated physiologically, causing internalization of the transporter, levels are not altered in SOD1-G93A mice. Preventing generation of the sumoylated fragment by mutating the caspase-3 consensus sequence with D504N, however, extended disease duration, slowed the loss of strength, and delayed end stage in SOD1-G93A mice, in agreement with previous research showing astrocytic expression of CTE-SUMO1 leads to motor neuron toxicity.
Rosenblum, Lauren Taylor, "Alternative Pathway of EAAT2-Mediated Toxicity in Amyotrophic Lateral Sclerosis" (2019). ETD Collection for Thomas Jefferson University. AAI10256083.