A Caspase-3-cleaved Fragment of the Glial Glutamate Transporter EAAT2 Is Sumoylated and Targeted to Promyelocytic Leukemia Nuclear Bodies in Mutant SOD1-linked Amyotrophic Lateral Sclerosis

Abstract

EAAT2 (excitatory amino acid transporter 2) is a high affinity, Na؉ -dependent glutamate transporter of glial origin that is essential for the clearance of synaptically released glutamate and prevention of excitotoxicity. During the course of human amyotrophic lateral sclerosis (ALS) and in a transgenic mutant SOD1 mouse model of the disease, expression and activity of EAAT2 is remarkably reduced. We previously showed that some of the mutant SOD1 proteins exposed to oxidative stress inhibit EAAT2 by triggering caspase-3 cleavage of EAAT2 at a single defined locus. This gives rise to two fragments that we termed truncated EAAT2 and COOH terminus of EAAT2 (CTE). In this study, we report that analysis of spinal cord homogenates prepared from mutant G93A-SOD1 mice reveals CTE to be of a higher molecular weight than expected because it is conjugated with SUMO-1. The sumoylated CTE fragment (CTE-SUMO-1) accumulates in the spinal cord of these mice as early as presymptomatic stage (70 days of age) and not in other central nervous system areas unaffected by the disease. The presence and accumulation of CTE-SUMO-1 is specific to ALS mice, since it does not occur in the R6/2 mouse model for Huntington disease. Furthermore, using an astroglial cell line, primary culture of astrocytes, and tissue samples from G93A-SOD1 mice, we show that CTE-SUMO-1 is targeted to promyelocytic leukemia nuclear bodies. Since one of the proposed functions of promyelocytic leukemia nuclear bodies is regulation of gene transcription, we suggest a possible novel mechanism by which the glial glutamate transporter EAAT2 could contribute to the pathology of ALS. Amyotrophic lateral sclerosis (ALS)4 is a fatal neurodegenerative disease, resulting from a progressive death of cortical and spinal motor neurons. About 90% of ALS cases are sporadic, whereas the remaining 10% are inherited in a dominant manner (familial ALS). Transgenic expression of high levels of human mutant SOD1 (mutSOD1) proteins in mice and rats leads to a progressive motor neuron disease that shares most of the clinical features of ALS (1, 2). Astrocytes are essential partners of motorneurons, providing them with trophic support (3) and mediating rapid clearance of synaptic glutamate mainly through the action of glial glutamate transporters. The EAAT2 (excitatory amino acid transporter 2) glial isoform of glutamate transporters accounts for ϳ95% of total glutamate uptake activity in the brain (4). Studies in mutSOD1 mice and in in vitro models of ALS affirmed a role for astroglial involvement in motorneuron death and suggested that motorneuron loss is also the result of toxic contributions of nonneuronal cells of the spinal cord (5-8). Reactive astrocytes surrounding motorneurons contain protein inclusions; express inflammatory markers, such as the inducible forms of nitric oxide synthase and cycloxygenase-2; and display increased neural growth factor synthesis (9), nitrotyrosine immunoreactivity, and down-regulation of EAAT2. Excitotoxicity caused by consistent reduction in expres...

Glutamate Transporters and the Excitotoxic Path to Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis

“…Other reports in the literature, however, showed that the loss of the glutamate-transporter EAAT2 in the mouse and rat models of ALS occurs also at presymptomatic and early symptomatic stages of the disease when no overt loss of motor neurons has occurred, suggesting perhaps that additional mechanisms could be responsible for the selective loss of EAAT2, independent of neuronal inputs (54,68,114). In the SOD1-G93A rat model of ALS, focal loss of the EAAT2 glutamate transporter in the ventral horn of the spinal cord coincides with gliosis, but appears before motor neuron=axon degeneration.…”

“…Moreover, we found that CTE-SUMO1 accumulates in the nucleus of astrocytes, where it interacts with promyelocytic leukemia protein (PML) in nuclear structures called PML-nuclear bodies (PML-NBs) (54). Several lines of evidence indicate that SUMOylation of polypeptides and proteins can affect protein stability, protein-protein interactions, subcellular relocalization, and transcriptional regulation (61), and that PML-NBs interact with chromatin to regulate gene expression and DNA repair and transcription (55).…”

“…FORAN AND TROTTI plasmic C-terminus is SUMO1 conjugated (termed CTE-SUMO1), progressively accumulates in the spinal cord of SOD1-G93A mice, starting as early as the presymptomatic stage, and this accumulation is disease specific, as it does not occur, for instance, in the mouse model for Huntington disease (54). Moreover, we found that CTE-SUMO1 accumulates in the nucleus of astrocytes, where it interacts with promyelocytic leukemia protein (PML) in nuclear structures called PML-nuclear bodies (PML-NBs) (54).…”

The Four Major N- and C-Terminal Splice Variants of the Excitatory Amino Acid Transporter GLT-1 Form Cell Surface Homomeric and Heteromeric Assemblies

“…Using Western blotting, we observed that each of the four isoforms, whether tagged or not, showed a predominant broad band of molecular weight of approximately 75 kDa, which sometimes appears as a doublet, corresponding to the monomer. The presence of a range of bands of approximately this size may be due to variations in post-translational modification and/or proteolysis of GLT-1 (Danbolt 2001;Boston-Howes et al, 2006;Gibb et al, 2007). As well as monomeric GLT-1, all constructs and endogenous GLT-1 show a cluster of immunoreactive bands with molecular masses of Ͼ180 kDa.…”

Regulation of Glial Glutamate Transporters by C-terminal Domains

“…Recently, oxidative stress was shown to activate caspase-3, resulting in a cleavage of EAAT2 in the cytosolic C-terminal domain. This result suggested a functionally important role of the C terminus and linked excitotoxicity and activation of caspase-3 as converging mechanisms in the pathogenesis of ALS (24,25).…”