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.
Highlights
Episodic ataxias (EA) are a group of rare neurological disorders characterized by progressive, severe and recurrent episodes of ataxia, migraine, discoordination, and imbalance [1]
Expressing human Excitatory Amino Acid Transporter 1 (hEAAT1) and episodic ataxia type 6 (EA6)-related mutant transporters in Drosophila larvae With the GAL4-UAS system and alrm-Gal4 for selective expression of hEAAT1 in larval central nervous system (CNS) astrocytes, we used infrared video tracking to examine the effects of five EA6-related mutations (M128R, C186S, T318A, A329T, and V393I) on the ability of hEAAT1 to rescue the larval crawling defects of dEAAT1-null animals
We quantified the performance of animals in each genotype during 60s of free exploration and found that, like hEAAT1, the C186S and T318A mutations could rescue key features of larval crawling behavior, including the mean speed (Fig. 2B), the total path length achieved in 60s (Fig. 2C), and the beeline distance from origin reached in 60s (Fig. 2D)
Summary
Episodic ataxias (EA) are a group of rare neurological disorders characterized by progressive, severe and recurrent episodes of ataxia, migraine, discoordination, and imbalance [1]. EA1 patients have mutations in KCNA1, a gene that encodes potassium channel Kv1.1 [3]. EA2 is characterized by mutations in CACNA1A which encodes the calcium channel Cav2 [6], and EA5 results from mutations in CACNB4 [12]. While EA1, EA2, and EA5 are directly related to mutations in ion channels, EA8 and EA9 are associated with ion channel dysfunction, and no candidate genes have been reported for EA3, EA4 and EA7 [for a review see Maksemous, et al [10]]. EA6 patients have mutations in SLC1A3, the gene which encodes the human Excitatory Amino Acid Transporter 1 (hEAAT1). The etiology of EA6 likely traces to Bergmann glia, which are astrocytes in the cerebellum that express EAAT1 at high levels and envelop the dendritic arbors of Purkinje neurons [13, 14]
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