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L-Citrulline Level and Transporter Activity Are Altered in Experimental Models of Amyotrophic Lateral Sclerosis

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Abstract

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/hSOD1G93A, MT) and a control model (NSC-34/hSOD1wt, WT). The [14C]L-citrulline uptake was significantly reduced in MT cells as compared with that of control. The Michaelis-Menten constant (Km) for MT cells was 0.67 ± 0.05 mM, whereas it was 1.48 ± 0.21 mM for control. On the other hand, the Vmax 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 Km and Vmax values showed the high affinity and low capacity for MT as compared with control. Moreover, the uptake of [14C]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, [14C]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 [14C]L-citrulline uptake. It shows the competitive inhibition for L-dopa in ALS cell lines. On the other hand, [14C]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 [14C]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 [14C]L-citrulline may be partially responsible for the pathological alteration in ALS.

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Abbreviations

ALS:

Amyotrophic lateral sclerosis

NSC-34 cells:

Motor neuron–like cells

BCH:

2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid

ECF:

Extracellular fluid

MeAIB:

α-(methylamino) isobutyric acid

siRNA:

Small interfering RNA

mRNA:

Messenger RNA

MT:

Mutant type

WT:

Wild type

ECF:

Extracellular fluid

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Acknowledgments

We are thankful towards Phuong T. Nguyen and Junghee Lee for performing the in vivo experiments and contributing in data analysis of the same.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2019R1F1A1044048) (YSK) and Grants from Korea Institute of Science and Technology (2E30320) and NRF-2018M3C7A1056894 (HR).

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Author notes

  1. Asmita Gyawali and Shashi Gautam contributed equally to this work.

Authors and Affiliations

  1. College of Pharmacy and Drug Information Research Institute, Sookmyung Women’s University, Seoul, Republic of Korea

    Asmita Gyawali, Shashi Gautam & Young-Sook Kang

  2. Laboratory for Brain Gene Regulation and Epigenetics, Center for Neuroscience, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea

    Seung Jae Hyeon & Hoon Ryu

  3. Boston University Alzheimer’s Disease Center Department of Neurology, Boston University School of Medicine, Boston, MA, 02183, USA

    Hoon Ryu

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  1. Asmita Gyawali
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Corresponding author

Correspondence to Young-Sook Kang.

Ethics declarations

The experiments were carried out in accordance with the Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care Committees of Korea Institute of Science and Technology (KIST). Procedures were performed in accordance with Guide for the Care and Use of Laboratory Animals guidelines and were approved by both the Veterans Administration and Boston University animal care committees.

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Gyawali, A., Gautam, S., Hyeon, S.J. et al. L-Citrulline Level and Transporter Activity Are Altered in Experimental Models of Amyotrophic Lateral Sclerosis. Mol Neurobiol 58, 647–657 (2021). https://doi.org/10.1007/s12035-020-02143-6

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