Higher SLC7A2 expression is associated with abnormal neuroinflammatory response and nitrosative stress in Huntington’s disease

Background: We previously identified solute carrier family 7 member 2 (SLC7A2) as one of the top up-regulated genes when normal huntingtin was deleted. SLC7A2 has a high affinity for Arginine. Arginine is implicated in inflammatory responses and SLC7A2 is an important regulator of innate and adaptive immunity in macrophagy. Although neuroinflammation is clearly demonstrated in HD animal models and patients, the question of whether neuroinflammation actively participates in HD pathogenesis is a topic of ongoing research and debate. Here we study the role of SLC7A2 in mediating neuroinflammatory stress response in HD cells. Methods: RNA sequencing, quantitative RT-PCR and datamining of publicly available RNA-seq datasets of human patients were performed to assess the levels of SLC7A2 mRNA in different HD cellular models and patients. Biochemical studies were then conducted on cell lines and primary mouse astrocytes to investigate Arginine metabolism and nitrosative stress in response to neuroinflammation. The CRISPR-Cas9 system was used to knockout SLC7A2 in STHdhQ7 and Q111 cells to investigate its role in mediating the neuroinflammatory response. Live-cell imaging was used to measure mitochondrial dynamics. Finally, exploratory studies were performed using Enroll-HD periodic human patient datasets to analyze the effect of Arginine supplement on HD progression. Results: We found that SLC7A2 is selectively up-regulated in HD cellular models and patients. HD cells exhibit an overactive response to neuroinflammatory challenges including abnormally high iNOS induction and NO production, leading to increased protein nitrosylation. Depleting extracellular Arg or knocking out SLC7A2 can block the iNOS induction and NO production in STHdhQ111 cells. We further examined the functional impact of protein nitrosylation on a well-documented protein target, DRP-1 and found that more mitochondria were fragmented in challenged STHdhQ111 cells. Lastly, analysis of Enroll-HD datasets suggested that HD patients taking Arginine supplement progressed more rapidly than others. Conclusions: Our data suggest a novel pathway that links Arginine uptake to nitrosative stress via up-regulation of SLC7A2 in the pathogenesis and progression of HD. It further implicates that Arginine supplement may potentially pose a greater risk to HD patients. To investigate the global transcriptomal changes in STHdhQ7/Q7 and STHdhQ111/!111 cells in response to neuroinflammation. Neuroinflammation was induced with a combination of 100ng/ml IFNg and 5ug/ml LPS.

研究背景：我们此前证实，当正常亨廷顿蛋白（huntingtin）被敲除时，溶质载体家族7成员2（Solute Carrier Family 7 Member 2, SLC7A2）是上调最显著的基因之一。SLC7A2对精氨酸（Arginine）具有高亲和性。精氨酸参与炎症反应调控，而SLC7A2是巨噬细胞先天免疫与适应性免疫的重要调节因子。尽管亨廷顿舞蹈症（Huntington's Disease, HD）动物模型及患者体内均已明确存在神经炎症，但神经炎症是否主动参与HD的发病进程，仍是当前研究与争论的热点议题。本研究旨在探讨SLC7A2在HD细胞中介导神经炎症应激反应的作用机制。
研究方法：本研究通过RNA测序、定量逆转录聚合酶链反应（quantitative RT-PCR）以及对公开的人类患者RNA测序数据集进行数据挖掘，评估不同HD细胞模型及患者体内SLC7A2信使RNA（mRNA）的表达水平。随后，我们在细胞系及原代小鼠星形胶质细胞中开展生化实验，以探究神经炎症刺激下的精氨酸代谢与亚硝化应激情况。利用CRISPR-Cas9系统在STHdhQ7与STHdhQ111细胞中敲除SLC7A2，以明确其在神经炎症反应中的介导作用。采用活细胞成像技术检测线粒体动力学。最后，我们利用Enroll-HD周期性人类患者数据集开展探索性研究，分析精氨酸补充剂对HD病情进展的影响。
研究结果：我们发现，SLC7A2在HD细胞模型及患者体内均呈现选择性上调。HD细胞对神经炎症刺激表现出过度激活的应答反应，包括诱导型一氧化氮合酶（inducible Nitric Oxide Synthase, iNOS）异常高表达及一氧化氮（NO）生成增多，进而导致蛋白质亚硝化水平升高。在STHdhQ111细胞中，清除细胞外精氨酸或敲除SLC7A2均可阻断iNOS的诱导表达与NO的生成。我们进一步针对已被充分证实的蛋白质靶标DRP-1，探究了蛋白质亚硝化的功能影响，发现受刺激的STHdhQ111细胞中存在更多碎片化的线粒体。最后，对Enroll-HD数据集的分析结果显示，服用精氨酸补充剂的HD患者病情进展速度快于其他患者。
研究结论：本研究数据表明，在HD的发病与进程中，存在一条通过SLC7A2上调将精氨酸摄取与亚硝化应激相联系的全新通路。本研究同时提示，精氨酸补充剂可能对HD患者构成更高的潜在风险。
补充实验：本研究旨在探究STHdhQ7/Q7与STHdhQ111/Q111细胞在神经炎症刺激下的全局转录组变化。神经炎症通过联合使用100ng/ml干扰素γ（IFNγ）与5μg/ml脂多糖（LPS）诱导构建。