Cloning of a new form of EAAT2/GLT-1 from human and rodent brains

Lee, A.; Klinkradt, S.; McCombe, Pamela A.; Pow, David V.

doi:10.1016/j.neulet.2022.136637

Cited by 3 publications

(2 citation statements)

References 33 publications

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“…Therefore, although further experiments to reveal the BBB impermeability and anti-cancer effects are required, WAY213613 and its derivative compounds may pave the way for future research and/or strategies for cancer therapies. Furthermore, since the recent work suggested that EAAT2 is potentially expressed in retinal and peripheral tissues (liver, lung, and testis) 62 , such selective inhibitors will reveal more details of the physiological roles of EAAT2 in the extracellular glutamate homeostasis of not only the brain but also other organs.…”

Section: Characterization Of Molecular Features Of Eaat2mentioning

confidence: 99%

Structural insights into inhibitory mechanism of human excitatory amino acid transporter EAAT2

et al. 2022

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Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2.

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Section: Characterization Of Molecular Features Of Eaat2mentioning

confidence: 99%

Structural insights into inhibitory mechanism of human excitatory amino acid transporter EAAT2

et al. 2022

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“…Excitatory amino acid transporters (EAATs) play a crucial role in the transport of several amino acids into the cell. Five subtypes of these EAATs exist; EAAT1–5. , All EAATs are capable of transporting glutamate, which plays a crucial role in brain functioning due to its role as a neurotransmitter. EAAT2, where SLC1A2 is the corresponding gene, is mainly expressed in astrocytes and to a lesser extent in other glial cells and neurons .…”

Section: Introductionmentioning

confidence: 99%

Still in Search for an EAAT Activator: GT949 Does Not Activate EAAT2, nor EAAT3 in Impedance and Radioligand Uptake Assays

van Veggel,

Mocking,

Sijben

et al. 2024

ACS Chem. Neurosci.

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Excitatory amino acid transporters (EAATs) are important regulators of amino acid transport and in particular glutamate. Recently, more interest has arisen in these transporters in the context of neurodegenerative diseases. This calls for ways to modulate these targets to drive glutamate transport, EAAT2 and EAAT3 in particular. Several inhibitors (competitive and noncompetitive) exist to block glutamate transport; however, activators remain scarce. Recently, GT949 was proposed as a selective activator of EAAT2, as tested in a radioligand uptake assay. In the presented research, we aimed to validate the use of GT949 to activate EAAT2-driven glutamate transport by applying an innovative, impedance-based, whole-cell assay (xCELLigence). A broad range of GT949 concentrations in a variety of cellular environments were tested in this assay. As expected, no activation of EAAT3 could be detected. Yet, surprisingly, no biological activation of GT949 on EAAT2 could be observed in this assay either. To validate whether the impedance-based assay was not suited to pick up increased glutamate uptake or if the compound might not induce activation in this setup, we performed radioligand uptake assays. Two setups were utilized; a novel method compared to previously published research, and in a reproducible fashion copying the methods used in the existing literature. Nonetheless, activation of neither EAAT2 nor EAAT3 could be observed in these assays. Furthermore, no evidence of GT949 binding or stabilization of purified EAAT2 could be observed in a thermal shift assay. To conclude, based on experimental evidence in the present study GT949 requires specific assay conditions, which are difficult to reproduce, and the compound cannot simply be classified as an activator of EAAT2 based on the presented evidence. Hence, further research is required to develop the tools needed to identify new EAAT modulators and use their potential as a therapeutic target.

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The sexually dimorphic expression of glutamate transporters and their implication in pain after spinal cord injury

Colón-Mercado,

Torrado-Tapias,

Salgado

et al. 2024

Neural Regeneration Research

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In addition to the loss of motor function, ~60% of patients develop pain after spinal cord injury. The cellular-molecular mechanisms are not well understood, but the data suggests that plasticity within the rostral, epicenter, and caudal penumbra of the injury site initiates a cellular-molecular interplay that acts as a rewiring mechanism leading to central neuropathic pain. Sprouting can lead to the formation of new connections triggering abnormal sensory transmission. The excitatory glutamate transporters are responsible for the reuptake of extracellular glutamate which makes them a critical target to prevent neuronal hyperexcitability and excitotoxicity. Our previous studies showed a sexually dimorphic therapeutic window for spinal cord injury after treatment with the selective estrogen receptor modulator tamoxifen. In this study, we investigated the anti-allodynic effects of tamoxifen in male and female rats with spinal cord injury. We hypothesized that tamoxifen exerts anti-allodynic effects by increasing the expression of glutamate transporters, leading to reduced hyperexcitability of the secondary neuron or by decreasing aberrant sprouting. Male and female rats received a moderate contusion to the thoracic spinal cord followed by subcutaneous slow-release treatment of tamoxifen or matrix pellets as a control (placebo). We used von Frey monofilaments and the “up-down method” to evaluate mechanical allodynia. Tamoxifen treatment decreased allodynia only in female rats with spinal cord injury revealing a sex-dependent effect. The expression profile of glutamatergic transporters (excitatory amino acid transporter 1/glutamate aspartate transporter and excitatory amino acid transporter 2/glutamate transporter-1) revealed a sexual dimorphism in the rostral, epicenter, and caudal areas of the spinal cord with a pattern of expression primarily on astrocytes. Female rodents showed a significantly higher level of excitatory amino acid transporter-1 expression while male rodents showed increased excitatory amino acid transporter-2 expression compared with female rodents. Analyses of peptidergic (calcitonin gene-related peptide-α) and non-peptidergic (isolectin B4) fibers outgrowth in the dorsal horn after spinal cord injury showed an increased calcitonin gene-related peptide-α/ isolectin B4 ratio in comparison with sham, suggesting increased receptive fields in the dorsal horn. Although the behavioral assay shows decreased allodynia in tamoxifen-treated female rats, this was not associated with overexpression of glutamate transporters or alterations in the dorsal horn laminae fibers at 28 days post-injury. Our findings provide new evidence of the sexually dimorphic expression of glutamate transporters in the spinal cord. The dimorphic expression revealed in this study provides a therapeutic opportunity for treating chronic pain, an area with a critical need for treatment.

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Cloning of a new form of EAAT2/GLT-1 from human and rodent brains

Cited by 3 publications

References 33 publications

Structural insights into inhibitory mechanism of human excitatory amino acid transporter EAAT2

Structural insights into inhibitory mechanism of human excitatory amino acid transporter EAAT2

Still in Search for an EAAT Activator: GT949 Does Not Activate EAAT2, nor EAAT3 in Impedance and Radioligand Uptake Assays

The sexually dimorphic expression of glutamate transporters and their implication in pain after spinal cord injury

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