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Takahashi et al. demonstrate that restoring glial glutamate transporter EAAT2 function improves cognitive functions and synaptic integrity while reducing amyloid plaques in a sustained fashion after treatment cessation., Glutamatergic systems play a critical role in cognitive functions and are known to be defective in Alzheimer’s disease (AD) patients. Previous literature has indicated that glial glutamate transporter EAAT2 plays an essential role in cognitive functions and that loss of EAAT2 protein is a common phenomenon observed in AD patients and animal models. In the current study, we investigated whether restored EAAT2 protein and function could benefit cognitive functions and pathology in APPSw,Ind mice, an animal model of AD. A transgenic mouse approach via crossing EAAT2 transgenic mice with APPSw,Ind. mice and a pharmacological approach using a novel EAAT2 translational activator, LDN/OSU-0212320, were conducted. Findings from both approaches demonstrated that restored EAAT2 protein function significantly improved cognitive functions, restored synaptic integrity, and reduced amyloid plaques. Importantly, the observed benefits were sustained one month after compound treatment cessation, suggesting that EAAT2 is a potential disease modifier with therapeutic potential for AD.

J. Neurochem. (2010) 113, 978–989. Abstract The glial glutamate transporter EAAT2 (excitatory amino acid transporter 2) is the major mediator of glutamate clearance that terminates glutamate-mediated neurotransmission. Loss of EAAT2 and associated glutamate uptake function has been reported in the brains of patients with Alzheimer’s disease (AD). We previously reported that EAAT2 is associated with lipid raft microdomains of the plasma membrane. In the present study, we demonstrated that association of EAAT2 with lipid rafts is disrupted in AD brains. This abnormality is not a consequence of neuron degeneration, oxidative stress, or amyloid beta toxicity. In AD brains, cholesterol 24S-hydroxylase (CYP46), a key enzyme in maintenance of cholesterol homeostasis in the brain, is markedly increased in astrocytes but decreased in neurons. We demonstrated that increased expression of CYP46 in primary astrocytes results in a reduction of membrane cholesterol levels and leads to the dissociation of EAAT2 from lipid rafts and the loss of EAAT2 and associated glutamate uptake function. These results suggest that a disturbance of cholesterol metabolism may contribute to loss of EAAT2 in AD.

Glutamate is the major excitatory neurotransmitter in the central nervous system. Its activity is carefully modulated in the synaptic cleft by glutamate transporters. The glial glutamate transporter EAAT2 is the main mediator of glutamate clearance. Reduced EAAT2 function could lead to accumulation of extracellular glutamate, resulting in a form of cell death known as excitotoxicity. In amyotrophic lateral sclerosis and Alzheimer disease, EAAT2 protein levels are significantly decreased in affected areas. EAAT2 mRNA levels, however, remain constant, indicating that alterations in EAAT2 expression are due to disturbances at the post-transcriptional level. In the present study, we found that some EAAT2 transcripts contained 5'-untranslated regions (5'-UTRs) greater than 300 nucleotides. The mRNAs that bear long 5'-UTRs are often regulated at the translational level. We tested this possibility initially in a primary astrocyte line that constantly expressed an EAAT2 transcript containing the 565-nt 5'-UTR and found that translation of this transcript was regulated by many extracellular factors, including corticosterone and retinol. Moreover, many disease-associated insults affected the efficiency of translation of this transcript. Importantly, this translational regulation of EAAT2 occurred in vivo (i.e. both in primary cortical neurons-astrocytes mixed cultures and in mice). These results indicate that expression of EAAT2 protein is highly regulated at the translational level and also suggest that translational regulation may play an important role in the differential EAAT2 protein expression under normal and disease conditions.

Glial glutamate transporter EAAT2 plays a major role in glutamate clearance in synaptic clefts. Several lines of evidence indicate that strategies designed to increase EAAT2 expression have potential for preventing excitotoxicity, which contributes to neuronal injury and death in neurodegenerative diseases. We previously discovered several classes of compounds that can increase EAAT2 expression through translational activation. Here, we present efficacy studies of the compound LDN/OSU-0212320, which is a pyridazine derivative from one of our lead series. In a murine model, LDN/OSU-0212320 had good potency, adequate pharmacokinetic properties, no observed toxicity at the doses examined, and low side effect/toxicity potential. Additionally, LDN/OSU-0212320 protected cultured neurons from glutamate-mediated excitotoxic injury and death via EAAT2 activation. Importantly, LDN/OSU-0212320 markedly delayed motor function decline and extended lifespan in an animal model of amyotrophic lateral sclerosis (ALS). We also found that LDN/OSU-0212320 substantially reduced mortality, neuronal death, and spontaneous recurrent seizures in a pilocarpine-induced temporal lobe epilepsy model. Moreover, our study demonstrated that LDN/OSU-0212320 treatment results in activation of PKC and subsequent Y-box-binding protein 1 (YB-1) activation, which regulates activation of EAAT2 translation. Our data indicate that the use of small molecules to enhance EAAT2 translation may be a therapeutic strategy for the treatment of neurodegenerative diseases.

Excitatory amino acid transporter 2 (EAAT2) is the major glutamate transporter and functions to remove glutamate from synapses. A thiopyridazine derivative has been found to increase EAAT2 protein levels in astrocytes. A structure-activity relationship study revealed that several components of the molecule were required for activity, such as the thioether and pyridazine. Modification of the benzylthioether resulted in several derivatives (7–13, 7–15 and 7–17) that enhanced EAAT2 levels by > 6 fold at concentrations < 5 μM after 24 h. In addition, one of the derivatives (7–22) enhanced EAAT2 levels 3.5 – 3.9 fold after 24 h with an EC50 of 0.5 μM.

Excitotoxicity has been implicated as the mechanism of neuronal damage resulting from acute insults such as stroke, epilepsy, and trauma, as well as during the progression of adult-onset neurodegenerative disorders such as Alzheimer's disease and amyotrophic lateral sclerosis (ALS). Excitotoxicity is defined as excessive exposure to the neurotransmitter glutamate or overstimulation of its membrane receptors, leading to neuronal injury or death. One potential approach to protect against excitotoxic neuronal damage is enhanced glutamate reuptake. The glial glutamate transporter EAAT2 is the quantitatively dominant glutamate transporter and plays a major role in clearance of glutamate. Expression of EAAT2 protein is highly regulated at the translational level. In an effort to identify compounds that can induce translation of EAAT2 transcripts, a cell-based enzyme-linked immunosorbent assay was developed using a primary astrocyte line stably transfected with a vector designed to identify modulators of EAAT2 translation. This assay was optimized for high-throughput screening, and a library of approximately 140,000 compounds was tested. In the initial screen, 293 compounds were identified as hits. These 293 hits were retested at 3 concentrations, and a total of 61 compounds showed a dose-dependent increase in EAAT2 protein levels. Selected compounds were tested in full 12-point dose-response experiments in the screening assay to assess potency as well as confirmed by Western blot, immunohistochemistry, and glutamate uptake assays to evaluate the localization and function of the elevated EAAT2 protein. These hits provide excellent starting points for developing therapeutic agents to prevent excitotoxicity.

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.

Glutamate is an important amino acid implicated in energy metabolism, protein biosynthesis and neurotransmission. The Na(+)-dependent high-affinity excitatory amino acid transporter EAAT3 (EAAC1) facilitates glutamate uptake into most cells. Recently, a novel rat EAAT3-interacting protein called GTRAP3-18 has been identified by a yeast two-hybrid screening. GTRAP3-18 functions as a negative modulator of EAAT3-mediated glutamate transport. In order to further understand the function and regulation of GTRAP3-18, we cloned the mouse orthologue to GTRAP3-18 and determined its gene structure and its expression pattern. GTRAP3-18 encodes a 188-residue hydrophobic protein whose sequence is highly conserved amongst vertebrates. Mouse and human GTRAP3-18 genes contain three exons separated by two introns. The GTRAP3-18 gene is found on mouse chromosome 6D3 and on human chromosome 3p14, a susceptibility locus for cancer and epilepsy. GTRAP3-18 protein and RNA were found both in neuronal rich regions of the brain and in non-neuronal tissues such as the kidney, heart and skeletal muscle. Mouse GTRAP3-18 inhibited EAAT3-mediated glutamate transport in a dose-dependent manner. These studies show that GTRAP3-18 is a ubiquitously expressed protein that functions as a negative regulator of EAAT3 function.