College of Pharmacy and Drug Information Research Institute, Sookmyung Women's University, Seoul, Republic of Korea.
Laboratory for Brain Gene Regulation and Epigenetics, Center for Neuroscience, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea.
Mol Neurobiol. 2021 Feb;58(2):647-657. doi: 10.1007/s12035-020-02143-6. Epub 2020 Oct 1.
Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease caused by the death of the neurons regulating the voluntary muscles which leads to the progressive paralysis. We investigated the difference of transport function of L-citrulline in ALS disease model (NSC-34/hSOD1, MT) and a control model (NSC-34/hSOD1, WT). The [C]L-citrulline uptake was significantly reduced in MT cells as compared with that of control. The Michaelis-Menten constant (K) for MT cells was 0.67 ± 0.05 mM, whereas it was 1.48 ± 0.21 mM for control. On the other hand, the V values for MT and control were 10.9 ± 0.8 nmol/mg protein/min and 18.3 ± 2.9 nmol/mg protein/min, respectively. The K and V values showed the high affinity and low capacity for MT as compared with control. Moreover, the uptake of [C]L-citrulline was significantly inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) and harmaline which is the inhibitor of the large neutral amino acid transporter1 (LAT1) in NSC-34 cell lines. Furthermore, [C]L-citrulline uptakes took place in Na-independent manner. It was also inhibited by the neutral amino acids such as citrulline and phenylalanine. Likewise, L-dopa, gabapentin, and riluzole significantly inhibited the [C]L-citrulline uptake. It shows the competitive inhibition for L-dopa in ALS cell lines. On the other hand, [C]L-citrulline uptake in the presence of riluzole showed competitive inhibition in WT cells, whereas it was uncompetitive for MT cells. The small interfering RNA experiments showed that LAT1 is involved in the [C]L-citrulline uptake in NSC-34 cell lines. On the other hand, in the examination of the alteration in the expression level of LAT1, it was significantly lower in MT cells as compared with that of control. Similarly, in the spinal cord of ALS, transgenic mice revealed a slight but significant decrease in LAT1 immunoreactivity in motor neurons of ALS mice compared with control. However, the LAT1 immunoreactivity in non-motor neurons and in astrocytes was relatively increased in the spinal cord gray matter of ALS mice. The experimental evidences of our results suggest that the change of transport activity of [C]L-citrulline may be partially responsible for the pathological alteration in ALS.
肌萎缩侧索硬化症（ALS）是一种由调节随意肌的神经元死亡引起的进行性运动神经元疾病，导致进行性瘫痪。我们研究了 ALS 疾病模型（NSC-34/hSOD1，MT）和对照模型（NSC-34/hSOD1，WT）中 L-瓜氨酸转运功能的差异。与对照相比，MT 细胞中的 [C]L-瓜氨酸摄取明显减少。MT 细胞的米氏常数（K）为 0.67±0.05 mM，而对照为 1.48±0.21 mM。另一方面，MT 和对照的 V 值分别为 10.9±0.8 nmol/mg 蛋白/min 和 18.3±2.9 nmol/mg 蛋白/min。与对照相比，MT 的 K 和 V 值显示出高亲和力和低容量。此外，[C]L-瓜氨酸的摄取在 NSC-34 细胞系中被 2-氨基双环[2.2.1]-庚烷-2-羧酸（BCH）和哈尔马林显著抑制，哈尔马林是大中性氨基酸转运蛋白 1（LAT1）的抑制剂。此外，[C]L-瓜氨酸摄取以非 Na 依赖性方式发生。它也被瓜氨酸和苯丙氨酸等中性氨基酸抑制。同样，L-多巴、加巴喷丁和利鲁唑也显著抑制 [C]L-瓜氨酸的摄取。它显示出在 ALS 细胞系中对 L-多巴的竞争性抑制。另一方面，在利鲁唑存在下，[C]L-瓜氨酸摄取在 WT 细胞中表现出竞争性抑制，而在 MT 细胞中表现出非竞争性抑制。小干扰 RNA 实验表明 LAT1 参与了 NSC-34 细胞系中 [C]L-瓜氨酸的摄取。另一方面，在 LAT1 表达水平的改变检查中，MT 细胞明显低于对照。同样，在 ALS 转基因小鼠的脊髓中，与对照相比，ALS 小鼠运动神经元中的 LAT1 免疫反应性略有但显著降低。然而，ALS 小鼠脊髓灰质中的非运动神经元和星形胶质细胞中的 LAT1 免疫反应性相对增加。我们研究结果的实验证据表明，[C]L-瓜氨酸转运活性的变化可能部分导致 ALS 的病理改变。
