Braidy Nady, Alicajic Hayden, Pow David, Smith Jason, Jugder Bat-Erdene, Brew Bruce J, Nicolazzo Joseph A, Guillemin Gilles J
Centre for Healthy Brain Ageing, School of Psychiatry, Faculty of Medicine, University of New South Wales, Sydney, Australia.
School of Medicine, Huzhou University, Wuxing District, Huzhou, Zhejiang, China.
Mol Neurobiol. 2021 Jan;58(1):34-54. doi: 10.1007/s12035-020-02046-6. Epub 2020 Sep 6.
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 [H]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 [H]QUIN with a Km of 42.2 μM, and a V 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.
在阿尔茨海默病（AD）中，大量喹啉酸（QUIN）在脑实质和营养不良性神经元内蓄积。QUIN还调节谷氨酸进入神经元的摄取，这可能是由于对钠依赖性兴奋性氨基酸转运体（EAATs）的调节。为了确定QUIN与谷氨酸功能障碍之间的生物学关系，我们首先使用液体闪烁法对原代人神经元中[H]QUIN摄取的功能和动力学进行了定量。然后，我们使用蛋白质印迹法和免疫组织化学法，测量了用QUIN和EAAT抑制剂L-反式-吡咯烷-2,4-二羧酸（2,4-PDC）处理的原代神经元中谷氨酸转运体EAAT3和EAAT1b的蛋白表达变化。免疫组织化学进一步用于阐明外源性QUIN和溶酶体相关膜蛋白2（LAMP2）的细胞内转运。使用分子对接技术进一步研究了QUIN与EAAT3之间结合的结构见解。我们报告了显著的温度依赖性高亲和力转运，导致神经元摄取[H]QUIN，其Km为42.2 μM，V为9.492 pmol/2 min/mg蛋白，与谷氨酸的摄取相当。我们还发现，QUIN增加了EAAT3单体的表达，同时减少了功能性三聚体。原代神经元对QUIN的摄取显示涉及EAAT3，因为在EAAT抑制后摄取显著减弱。我们还证明，QUIN增加了神经元中异常EAAT1b蛋白的表达，进一步表明QUIN诱导的谷氨酸功能障碍。此外，我们证明QUIN仅在溶酶体中代谢。使用分子对接进一步证实了EAAT3作为QUIN摄取调节剂的参与。这项研究首次描述了QUIN进入原代人神经元的机制，该机制涉及EAAT3，为减轻神经炎症性疾病中QUIN诱导 的兴奋性毒性开辟了潜在靶点。
