Caspase-3 Cleaves and Inactivates the Glutamate Transporter EAAT2

William Boston-Howes,Davide Trotti,Stuart L Gibb,Robert H Brown,Piera Pasinelli,Eric O Williams

doi:10.1074/jbc.m600653200

Abstract

EAAT2 is a high affinity, Na+-dependent glutamate transporter with predominant astroglial localization. It accounts for the clearance of the bulk of glutamate released at central nervous system synapses and therefore has a crucial role in shaping glutamatergic neurotransmission and limiting excitotoxicity. Caspase-3 activation and impairment in expression and activity of EAAT2 are two distinct molecular mechanisms occurring in human amyotrophic lateral sclerosis (ALS) and in the transgenic rodent model of the disease. Excitotoxicity caused by down-regulation of EAAT2 is thought to be a contributing factor to motor neuron death in ALS. In this study, we report the novel evidence that caspase-3 cleaves EAAT2 at a unique site located in the cytosolic C-terminal domain of the transporter, a finding that links excitotoxicity and activation of caspase-3 as converging mechanisms in the pathogenesis of ALS. Caspase-3 cleavage of EAAT2 leads to a drastic and selective inhibition of this transporter. Heterologous expression of mutant SOD1 proteins linked to the familial form of ALS leads to inhibition of EAAT2 through a mechanism that largely involves activation of caspase-3 and cleavage of the transporter. In addition, we found evidence in spinal cord homogenates of mutant SOD1 ALS mice of a truncated form of EAAT2, likely deriving from caspase-3-mediated proteolytic cleavage, which appeared concurrently to the loss of EAAT2 immunoreactivity and to increased expression of activated caspase-3. Taken together, our findings suggest that caspase-3 cleavage of EAAT2 is one mechanism responsible for the impairment of glutamate uptake in mutant SOD1-linked ALS.

Highlights

National Institutes of Health Grant NS44292, and the Harvard Center for Neurodegeneration and Repair
To test whether EAAT2 could be cleaved by caspase-3, we treated mouse spinal cord homogenates with increasing amounts of purified caspase-3, and the reaction was probed on Western blot with three polyclonal anti-EAAT2 antibodies directed against different domains of the transporter (Fig. 1A)
The treatment led to a dose-dependent loss of EAAT2 immunoreactivity and a simultaneous dose-dependent appearance of a lower band that corresponded to the shortened form of EAAT2 (Fig. 1B, (Tr)EAAT2) when the blot was probed with the anti-EAAT2 N-terminal antibody B12-26

Summary

Introduction

National Institutes of Health Grant NS44292, and the Harvard Center for Neurodegeneration and Repair We found evidence of a truncated EAAT2 form likely derived from caspase-3 cleavage in the spinal cord of G93A-SOD1 transgenic ALS mice, suggesting that this event occurs in vivo.

Results

Conclusion