Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function
Qianyi Wu, … , Donald J. van Meyel, Renae M. Ryan
Qianyi Wu, … , Donald J. van Meyel, Renae M. Ryan
Published February 15, 2022
Citation Information: J Clin Invest. 2022;132(7):e154891. https://doi.org/10.1172/JCI154891.
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Research Article Neuroscience

Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function

Abstract

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory amino acid transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl–) channels, but the physiological role of Cl– conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl– channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. Here we investigated the effects of 5 additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. Our results indicate that mutations resulting in decreased hEAAT1 Cl– channel activity but with functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl– homeostasis in glial cells for proper CNS function. We also identified what we believe is a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

Authors

Qianyi Wu, Azman Akhter, Shashank Pant, Eunjoo Cho, Jin Xin Zhu, Alastair Garner, Tomoko Ohyama, Emad Tajkhorshid, Donald J. van Meyel, Renae M. Ryan

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Figure 2

EA6-related mutations M128R, A329T, and V393I fail to rescue Drosophila crawling.

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EA6-related mutations M128R, A329T, and V393I fail to rescue Drosophila ...
(A) dEAAT1-null animals were rescued by WT hEAAT1 or 1 of 5 EA6-related mutations (M128R, C186S, T318A, A329T, or V393I), and representative trajectories of crawling paths of 9 L1 larvae for each genotype are shown, as captured by infrared tracking for 180 seconds. Color heatmap indicates the average speed over a moving bin of 0.5 seconds. Scale bar: 5 mm. (B–D) Quantification (mean ± SEM) of crawling parameters achieved by larvae over 60 seconds of continuous tracking, including mean speed (B), total path length (C), and the beeline distance between the origin at t = 0 and the termination point (D). The exact numbers of animals (n) used for panels B–D are hEAAT1 (40, 74, 75), M128R (50, 53, 53), C186S (53, 51, 50), T318A (78, 79, 78), A329T (76, 84, 84), and V393I (52, 56, 55). One-way ANOVA tests (Brown-Forsythe) were performed for mean speed F(5, 130) = 18.32, *P < 0.05, ****P < 0.0001; for total path length F(5, 197.6) = 27.16, ****P < 0.0001; and for beeline distance F(5, 199.5) = 24.04, ****P < 0.0001. (E and F) Astrocyte-specific expression (with alrm-Gal4) of hEAAT1 and EA6-related mutations. Representative high-power images of infiltrative astrocyte processes within the ventral nerve cord of a dissected larva for each genotype, labeled by immunohistochemistry for hEAAT1 (E) or the plasma membrane-associated GABA transporter (GAT, F). Each panel represents a single optical confocal section from the middle of the dorsal-ventral axis of the neuropil. Scale bars: 10 μm. Like controls where dEaat1-null larvae are rescued with hEAAT1, all the EA6-related mutations of hEAAT1 except M128R were well expressed and addressed to astrocyte processes within CNS neuropil. Anti-GAT staining (F) reveals that astrocytes rescued with M128R infiltrate the neuropil normally. At least 5 animals were dissected, immunostained, and examined for each genotype.