Potential Mechanism of Cellular Uptake of the Excitotoxin Quinolinic Acid in Primary Human Neurons
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ORIGINAL ARTICLE
Potential Mechanism of Cellular Uptake of the Excitotoxin Quinolinic Acid in Primary Human Neurons Nady Braidy 1,2 & Hayden Alicajic 3 & David Pow 4 & Jason Smith 5 & Bat-Erdene Jugder 6 & Bruce J. Brew 7,8 & Joseph A. Nicolazzo 9 & Gilles J. Guillemin 3 Received: 11 May 2020 / Accepted: 28 July 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract In Alzheimer’s disease (AD), excessive amounts of quinolinic acid (QUIN) accumulate within the brain parenchyma and dystrophic neurons. QUIN also regulates glutamate uptake into neurons, which may be due to modulation of Na+-dependent excitatory amino acid transporters (EAATs). To determine the biological relationships between QUIN and glutamate dysfunction, we first quantified the functionality and kinetics of [3H]QUIN uptake in primary human neurons using liquid scintillation. We then measured changes in the protein expression of the glutamate transporter EAAT3 and EAAT1b in primary neurons treated with QUIN and the EAAT inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (2,4-PDC) using western blotting and immunohistochemistry. Immunohistochemistry was further used to elucidate intracellular transport of exogenous QUIN and the lysosomal-associated membrane protein 2 (LAMP2). Structural insights into the binding between QUIN and EAAT3 were further investigated using molecular docking techniques. We report significant temperature-dependent high-affinity transport leading to neuronal uptake of [3H]QUIN with a Km of 42.2 μM, and a Vmax of 9.492 pmol/2 min/mg protein, comparable with the uptake of glutamate. We also found that QUIN increases expression of the EAAT3 monomer while decreasing the functional trimer. QUIN uptake into primary neurons was shown to involve EAAT3 as uptake was significantly attenuated following EAAT inhibition. We also demonstrated that QUIN increases the expression of aberrant EAAT1b protein in neurons further implicating QUIN-induced glutamate dysfunction. Furthermore, we demonstrated that QUIN is metabolised exclusively in lysosomes. The involvement of EAAT3 as a modulator for QUIN uptake was further confirmed using molecular docking. This study is the first to characterise a mechanism for QUIN uptake into primary human neurons involving EAAT3, opening potential targets to attenuate QUIN-induced excitotoxicity in neuroinflammatory diseases. Keywords Quinolinic acid . Kynurenine pathway . Neurons . EAAT3 . Glutamate
Abbreviations [3H]QUIN 3H Quinolinic acid
DAPI ATP
4′,6-Diamidino-2-phenylindole dihydrochloride Adenosine triphosphate
Nady Braidy and Hayden Alicajic contributed equally to this work. * Nady Braidy [email protected]
4
University of Queensland Centre for Clinical Research, The University of Queensland, Brisbane, Queensland, Australia
* Gilles J. Guillemin [email protected]
5
Department of Chemistry and Biomolecular sciences, Macquarie University, Sydney, NSW, Australia
6
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Aus
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