Small-molecule activator of glutamate transporter EAAT2 translation provides neuroprotection
Qiongman Kong, … , Marcie A. Glicksman, Chien-Liang Glenn Lin
Qiongman Kong, … , Marcie A. Glicksman, Chien-Liang Glenn Lin
Published February 24, 2014
Citation Information: J Clin Invest. 2014;124(3):1255-1267. https://doi.org/10.1172/JCI66163.
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Research Article

Small-molecule activator of glutamate transporter EAAT2 translation provides neuroprotection

Abstract

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.

Authors

Qiongman Kong, Ling-Chu Chang, Kou Takahashi, Qibing Liu, Delanie A. Schulte, Liching Lai, Brian Ibabao, Yuchen Lin, Nathan Stouffer, Chitra Das Mukhopadhyay, Xuechao Xing, Kathleen I. Seyb, Gregory D. Cuny, Marcie A. Glicksman, Chien-Liang Glenn Lin

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Figure 2

Characterization of LDN/OSU-0212320 in primary dissociated neuron and astrocyte mixed cultures.

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Characterization of LDN/OSU-0212320 in primary dissociated neuron and as...
Seven-day-old primary cultures were treated with the indicated doses of compound for 24 hours and then harvested for analyses. (A) Western blot analysis shows that compound increased EAAT2 expression in a dose-dependent manner. (B) [3H] glutamate uptake assay shows a dose-dependently increased glutamate uptake. n = 6. (C) Cultures were insulted with 0.5 mM glutamate for 2 hours to induce excitotoxicity. LDH assay shows a dose-dependent reduction of LDH release in compound-treated cultures. n = 6. (D) MAP2 immunostaining shows that compound treatment significantly prevented glutamate-mediated neuronal loss and degeneration. The protections were partially abolished by pretreatment of DHK (100 μM). Cultures were exposed to 0.1 mM glutamate for 2 hours. Scale bar: 50 μm. (E) Quantitative analysis of MAP2-positive neurons. MAP2-positive neurons were counted and quantified as described in the Methods. n = 15. (F) MAP2-immunolabeled neurons and their nuclear morphology (Hoechst 33342 staining) at a higher magnification. Arrows point to the corresponding neurons of the highlighted nuclei. Scale bar: 25 μm. *P < 0.05; **P < 0.01; ***P < 0.001.