Sumoylation of the astroglial glutamate transporter EAAT2 governs its intracellular compartmentalization

EAAT2 is a predominantly astroglial glutamate transporter responsible for the majority of synaptic glutamate clearance in the mammalian central nervous system (CNS). Its dysfunction has been linked with many neurological disorders, including amyotrophic lateral sclerosis (ALS). Decreases in EAAT2 expression and function have been implicated in causing motor neuron excitotoxic death in ALS. Nevertheless, increasing EAAT2 expression does not significantly improve ALS phenotype in mouse models or in clinical trials. In the SOD1‐G93A mouse model of inherited ALS, the cytosolic carboxy‐terminal domain is cleaved from EAAT2, conjugated to SUMO1, and accumulated in astrocytes where it triggers astrocyte‐mediated neurotoxic effects as disease progresses. However, it is not known whether this fragment is sumoylated after cleavage or if full‐length EAAT2 is already sumoylated prior to cleavage as part of physiological regulation. In this study, we show that a fraction of full‐length EAAT2 is constitutively sumoylated in primary cultures of astrocytes in vitro and in the CNS in vivo. Furthermore, the extent of sumoylation of EAAT2 does not change during the course of ALS in the SOD1‐G93A mouse and is not affected by the expression of ALS‐causative mutant SOD1 proteins in astrocytes in vitro, indicating that EAAT2 sumoylation is not driven by pathogenic mechanisms. Most interestingly, sumoylated EAAT2 localizes to intracellular compartments, whereas non‐sumoylated EAAT2 resides on the plasma membrane. In agreement, promoting desumoylation in primary astrocytes causes increased EAAT2‐mediated glutamate uptake. These findings could have implications for optimizing therapeutic approaches aimed at increasing EAAT2 activity in the dysfunctional or diseased CNS. GLIA 2014;62:1241–1253     

Molecular comparison of GLT1+ and ALDH1L1+ astrocytes in vivo in astroglial reporter mice

Astrocyte heterogeneity remains largely unknown in the CNS due to lack of specific astroglial markers. In this study, molecular identity of in vivo astrocytes was characterized in BAC ALDH1L1 and BAC GLT1 eGFP promoter reporter transgenic mice. ALDH1L1 promoter is selectively activated in adult cortical and spinal cord astrocytes, indicated by the overlap of eGFP expression with ALDH1L1 and GFAP, but not with NeuN, APC, Olig2, IbaI, PDGFRα immunoreactivity in BAC ALDH1L1 eGFP reporter mice. Interestingly, ALDH1L1 expression levels (protein, mRNA, and promoter activity) in spinal cord were selectively decreased during postnatal maturation. In contrast, its expression was up-regulated in reactive astrocytes in both acute neural injury and chronic neurodegenerative (G93A mutant SOD1) conditions, similar to GFAP, but opposite of GLT1. ALDH1L1(+) and GLT1(+) cells isolated through fluorescence activated cell sorting (FACS) from BAC ALDH1L1 and BAC GLT1 eGFP mice share a highly similar gene expression profile, suggesting ALDH1L1 and GLT1 are co-expressed in the same population of astrocytes. This observation was further supported by overlap of the eGFP driven by the ALDH1L1 genomic promoter and the tdTomato driven by a 8.3kb EAAT2 promoter fragment in astrocytes of BAC ALDH1L1 eGFP X EAAT2-tdTomato mice. These studies support ALDH1L1 as a general CNS astroglial marker and investigated astrocyte heterogeneity in the CNS by comparing the molecular identity of the ALDH1L1(+) and GLT1(+) astrocytes from astroglial reporter mice. These astroglial reporter mice provide useful in vivo tools for the molecular analysis of astrocytes in physiological and pathological conditions.     

Glutamate transporter gene expression in amyotrophic lateral sclerosis motor cortex

Glutamate transport is critical for synaptic inactivation of glutamate and prevention of excitotoxicity. The following four glutamate transporters have been identified in human brain: EAAT1, EAAT2, EAAT3, and EAAT4. Deficient glutamate transport has been identified in the motor cortex and the spinal cord of tissue from amyotrophic lateral sclerosis (ALS) patients. The defect appears to be due to a selective loss of the astroglial specific glutamate transporter protein EAAT2. In these studies we sought to extend our understanding of glutamate transporters in ALS by examining the mRNA for each transporter subtype in ALS motor cortex. All tissue was matched for age and postmortem delay. There was no quantitative change in mRNA for EAAT1, EAAT2, or EAAT3 in ALS motor cortex, even in patients with a large loss of EAAT2 protein (95% decrease compared with control) and decreased tissue glutamate transport (73% decrease compared with control). These studies suggest that the dramatic abnormalities in EAAT2 may be due to translational or post- translational processes.     

Motor neuron impairment mediated by a sumoylated fragment of the glial glutamate transporter EAAT2

Dysregulation of glutamate handling ensuing downregulation of expression and activity levels of the astroglial glutamate transporter EAAT2 is implicated in excitotoxic degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). We previously reported that EAAT2 (a.k.a. GLT-1) is cleaved by caspase-3 at its cytosolic carboxy-terminus domain. This cleavage results in impaired glutamate transport activity and generates a proteolytic fragment (CTE) that we found to be post-translationally conjugated by SUMO1. We show here that this sumoylated CTE fragment accumulates in the nucleus of spinal cord astrocytes of the SOD1-G93A mouse model of ALS at symptomatic stages of disease. Astrocytic expression of CTE, artificially tagged with SUMO1 (CTE-SUMO1) to mimic the native sumoylated fragment, recapitulates the nuclear accumulation pattern of the endogenous EAAT2-derived proteolytic fragment. Moreover, in a co-culture binary system, expression of CTE-SUMO1 in spinal cord astrocytes initiates extrinsic toxicity by inducing caspase-3 activation in motor neuron-derived NSC-34 cells or axonal growth impairment in primary motor neurons. Interestingly, prolonged nuclear accumulation of CTE-SUMO1 is intrinsically toxic to spinal cord astrocytes, although this gliotoxic effect of CTE-SUMO1 occurs later than the indirect, noncell autonomous toxic effect on motor neurons. As more evidence on the implication of SUMO substrates in neurodegenerative diseases emerges, our observations strongly suggest that the nuclear accumulation in spinal cord astrocytes of a sumoylated proteolytic fragment of the astroglial glutamate transporter EAAT2 could participate to the pathogenesis of ALS and suggest a novel, unconventional role for EAAT2 in motor neuron degeneration.     

Glyoxal inactivates glutamate transporter‐1 in cultured rat astrocytes

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive motor paralysis and selective motor neuron death. There is increasing evidence that motor neuron death in ALS is mediated by glutamate toxicity resulting from reduced activity of astrocytic glutamate transporter‐1 (GLT‐1). Recent morphological studies have shown that N^?‐(carboxymethyl)lysine (CML) accumulates in reactive astrocytes of ALS spinal cords. CML is a product of post‐translational protein modification by glyoxal, a reactive aldehydic intermediate. In considering these documents, it is important to determine whether GLT‐1 protein modification by glyoxal might cause reduced GLT‐1 activity. To address this issue, we investigated the effects of glyoxal on GLT‐1 properties in cultured rat astrocytes. High performance liquid chromatography showed reduced glutamate uptake activity in the glyoxal‐exposed cells. Immunocytochemical analysis displayed CML accumulation in the cytoplasm of astrocytes by glyoxal exposure. Immunoblots of immunoprecipitated GLT‐1 disclosed GLT‐1 CML adduct formation in the glyoxal‐exposed cells. Our results indicate that glyoxal modifies GLT‐1 to form CML and simultaneously deprives its glutamate uptake activity. Thus, these toxic effects of glyoxal on astrocytes might be implicated in motor neuron death in ALS.     

Human glioma cells and undifferentiated primary astrocytes that express aberrant EAAT2 mRNA inhibit normal EAAT2 protein expression and prevent cell death

Abnormal splicing of astroglial glutamate transporter EAAT2 mRNA has been suggested to account for the loss of EAAT2 protein in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). We have identified several clones of human U251 glioma cells which express varying amounts of aberrantly spliced EAAT2 mRNA; these clones do not express any detectable EAAT2 protein. When the wild-type EAAT2 cDNA was expressed in each of these clones, we found that the amount of EAAT2 protein inversely correlated with the levels of endogenous aberrant EAAT2 mRNA. We also observed that ectopic expression of normal EAAT2 protein is toxic to U251 cells as well as to undifferentiated primary astrocytes. We conclude that expression of aberrant EAAT2 mRNA may be one possible mechanism to repress normal EAAT2 protein expression. The implication of this study for the mechanisms of EAAT2 protein loss in ALS and AD is discussed.     

PICK1 expression in reactive astrocytes within the spinal cord of amyotrophic lateral sclerosis (ALS) rats

M. C. Focant, S. Goursaud, C. Boucherie, A. O. Dumont and E. Hermans (2013) Neuropathology and Applied Neurobiology 39, 231–242 PICK1 expression in reactive astrocytes within the spinal cord of amyotrophic lateral sclerosis (ALS) rats Aims: The protein interacting with C kinase 1 (PICK1), a PDZ domain‐containing protein mainly expressed in the central nervous system, interacts with the glutamate receptor subunit GluR2, with the glutamate transporter GLT‐1b and with the enzyme serine racemase. These three proteins appear as key actors in the glutamate‐mediated excitotoxicity associated with amyotrophic lateral sclerosis (ALS), in both patients and animal models of the disease. In this study, we examined the expression of PICK1 in the spinal cord of transgenic rats expressing a mutated form of the human superoxide dismutase 1 (hSOD1^G93A) during the progression of the disease. Methods: Expression of PICK1 was examined by real‐time qPCR at presymptomatic and symptomatic stages as well as at end‐stage. The expression of PICK1 in the different cell types of the spinal cord was examined by immunohistochemistry. Results: The overall expression of PICK1 is not modified in cervical and lumbar spinal cord of transgenic (hSOD1^G93A) rats during the progression of the disease. Nonetheless, immunohistochemical studies of lumbar ventral horns revealed a shift of PICK1 expression from motor neurones in healthy rats to activated astrocytes in end‐stage hSOD1^G93A animals. Conclusions: Considering the documented influence of PICK1 expression on d ‐serine release and glutamate transport in astrocytes, these findings point to a potential implication of PICK1 in the progression of ALS.     

Polymorphisms in the glutamate transporter gene EAAT2 in European ALS patients

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder characterised by degeneration of upper and lower motor neurons. Whilst the primary pathogenic trigger is unknown in most cases, evidence is mounting to implicate a role for glutamate-mediated neurotoxicity in the disorder. Recent studies have shown reduced levels of the mainly astroglial glutamate transporter EAAT2 in ALS motor cortex and spinal cord and multiple abnormal EAAT2 mRNA species in ALS brain tissue. One cause of the low EAAT2 levels may be that point mutations in the EAAT2 gene, EAAT2, result in an abnormal unstable protein. To test this hypothesis we analysed EAAT2 in 128 sporadic and 23 familial European ALS cases. No variants within the coding sequence of EAAT2 to affect the protein sequence nor in the consensus splice sites of the flanking intronic sequences were found in any cases, similar to findings in other reports. Frequent polymorphisms within the flanking intronic sequences of both exons 2 and 4 were seen but at similar frequencies in controls. Mechanisms other than mutations within the coding region of EAAT2 must therefore be responsible for the low levels of EAAT2 seen in most cases of ALS. Received: 26 February 1999 Received in revised form: 2 June 1999 Accepted: 2 July 1999     

Selective up-regulation of the glial Na+-dependent glutamate transporter GLT1 by a neuroimmunophilin ligand results in neuroprotection

Excessive accumulation of extracellular glutamate results in neuronal death. Termination of synaptic glutamate transmission and the prevention of excitotoxicity depend on rapid removal of glutamate by high affinity Na+-dependent transporters. The astroglial transporter GLT1 is the predominant subtype, responsible for the bulk of extracellular clearance and for limiting excitotoxicity. This protein is crucial in the prevention of chronic glutamate neurotoxicity, and is markedly decreased in amyotrophic lateral sclerosis (ALS). Recent studies have shown that GLT1 expression can be induced in vitro and in vivo by various factors, but little is known about the signaling pathways mediating its regulation. The FK506-binding protein (FKBP) immunophilins are ubiquitous cytosolic proteins, concentrated in neural tissue (neuroimmunophilins). GPI-1046 is a synthetic, nonimmunosuppressive derivative of FK506 shown to exert neuroprotective and neuroregenerative actions in several systems. In the present study, we demonstrated that GPI-1046 induces selective expression of GLT1 in vitro and in vivo, associated with a marked increase in DHK-sensitive Na+-dependent glutamate transport. Furthermore, treatment with GPI-1046 was shown to protect motor neurons in an in vitro model of chronic excitotoxicity, and to prolong the survival of transgenic ALS mice. These studies suggest that neuroimmunophilins can regulate GLT1 and that their ligands could serve as therapies for neurodegenerative disorders.     

Astroglial alterations in amyotrophic lateral sclerosis (ALS) model of slow glutamate excitotoxicity in vitro

Chronic excitotoxicity mediated through defective glial and/or neuronal glutamate transport may contribute to several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). This study was performed to determine the ultrastructural characteristics of astroglial changes concomitant with motor neuron (MN) degeneration in a model of slow excitotoxicity in vitro. The study was performed on organotypic cultures of rat lumbar spinal cord subjected to the glutamate uptake blockers threohydroxyaspartate (THA) and L-trans-pyrrolidine-2,4-dicarboxylate (PDC). The chronic inhibition of glutamate transport by THA and PDC resulted in slow degeneration of the rat's MNs accompanied by distinct glial changes predominantly involving protoplasmic astrocytes. The presence of irregular vacuoles and vesicles in the astroglial cells was frequently observed. Occasionally the astrocytes exhibited proliferation and accumulation of abnormal profiles of smooth endoplasmic reticulum. In 3 weeks there were no signs of increased production of glial filaments in the protoplasmic astrocytes. The results evidenced the coexistence of neuronal degeneration and astroglial abnormalities in an ALS model in vitro and suggested an active role of astrocytes contributing to the induction and propagation of MN degeneration.     

Altered expression of the glutamate transporter EAAT2b in neurological disease

Functional studies suggest that up to 95% of all glutamate transport is handled by the glutamate transporter EAAT2. Amino and C-terminal antibodies demonstrate that under normal conditions EAAT2 is specific to astrocytes. A truncated splice variant of EAAT2, known as EAAT2b, also has been identified in astrocytes and some neurons. In vitro studies suggest EAAT2b transports glutamate similar to EAAT2, although the contribution of EAAT2b to normal clearance of extracellular glutamate is unknown. To investigate EAAT2b biology in pathological conditions, we examined the cellular and regional distribution of EAAT2b in amyotrophic lateral sclerosis. Using epitope-specific, affinity purified antibodies, we found that EAAT2b tissue levels were increased by more than twofold in amyotrophic lateral sclerosis motor cortex, whereas EAAT2 levels were decreased by up to 95%. EAAT2b distribution in normal human cortex was largely confined to the neuropil-like EAAT2, with occasional faint neuronal expression. In contrast, amyotrophic lateral sclerosis motor cortex had an obvious qualitative increase in neuropil EAAT2b staining and a drastic increase in neuronal soma and dendritic EAAT2b immunostaining. Despite these increases in EAAT2b immunostaining, functional transporter studies demonstrated a large loss of EAAT2 function. These studies clearly document altered regulation and splicing of the dominant glutamate transporter EAAT2 under conditions of neurological stress.     

Excitatory amino acid transporter 1 and 2 immunoreactivity in the spinal cord in amyotrophic lateral sclerosis

The spinal cord of 20 patients with amyotrophic lateral sclerosis (ALS) and 5 patients with lower motor neuron disease (LMND) were investigated immunohistochemically using anti-human excitatory amino acid transporter 1 (EAAT1) and EAAT2 antibodies which are the astrocytic transporters. The purpose of the study was to examine relationships between EAAT1 and EAAT2 immunoreactivity and degeneration of anterior horn neurons. Specimens from 20 patients without any neurological disease served as controls. In controls, spinal cord gray matter was densely immunostained by antibodies, whereas the white matter was generally not immunostained. In motor neuron disease (MND) patients, EAAT1 immunoreactivity was relatively well preserved in the gray matter despite neuronal loss of anterior horn cells. On the other hand, EAAT2 immunoreactivity in anterior horns correlated with the degree of neuronal loss of anterior horn cells: in the patients with mild neuronal depletion, anterior horns were densely immunostained by the antibody, whereas in the patients with severe neuronal loss, EAAT2 expression was markedly reduced. Degenerated anterior horn cells frequently showed a much denser EAAT1 and EAAT2 immunoreactivity around the surface of the neurons and their neuronal processes than that observed in normal-appearing neurons. There was no difference in the expression of EAAT1 and EAAT2 immunoreactivity between LMND and ALS patients. These findings suggest that in the early stage of degeneration of anterior horn cells, EAAT1 and EAAT2 immunoreactivity is preserved in the astrocytic foot directly attached to normal-appearing neurons, whereas levels of EAAT1 and EAAT2 protein rather increase in the astrocytic foot directly attached to degenerated anterior horn neurons; the latter effect most probably reduces the elevated glutamate level, compensates for the reduced function of astroglial glutamate transporters, or represents a condensation of EAAT1 and EAAT2 immunoreactivity secondary to loss of neurites and greater condensation of astrocytic processes. Thus, we demonstrate a difference in EAAT1 and EAAT2 immunoreactivity in different stages of progression in ALS, as a feature of the pathomechanism of this disease. Received: 8 September 1999 / Revised, accepted: 28 October 1999     

Interindividual differences in the levels of the glutamate transporters GLAST and GLT,but no clear correlation with Alzheimer's disease

Alzheimer's disease is a common progressive neurodegenerative disease of unknown etiology. Several different pathological processes have been identified in the brains of Alzheimer patients. To determine if reduced glutamate uptake is a contributing factor, we have measured the levels of the glutamate transporter proteins GLAST (EAAT1) and GLT (EAAT2) in human autopsy samples. The postmortem proteolysis of these proteins turned out to be fairly rapid. Brains from 10 Alzheimer and 10 control patients were therefore obtained with a relatively short postmortem delay (5 hr on average). GLT (N‐terminal and central parts), GLAST (C‐terminal), glial fibrillary acidic protein (GFAP) and inositol (1,4,5)‐triphosphate (IP₃)‐receptor immunoreactivities were determined in the cingulate and inferior temporal gyri by immunoblotting. The Na⁺‐dependent “binding” of D‐[³H]aspartate and the glutamate uptake after solubilization and reconstitution in liposomes were determined for comparison. An individual variation in GLAST and GLT levels was found, but no significant correlation with Alzheimer's disease, except for a 14% lower ratio of N‐terminal to central GLT immunoreactivity (P < 0.04). The levels of GLAST and GLT showed negative correlation in agreement with the idea that these proteins are differentially regulated. In conclusion, Alzheimer's disease brains can have both normal and reduced levels of GLAST and GLT. J. Neurosci. Res. 55:218–229, 1999. © 1999 Wiley‐Liss, Inc.