Neuronal excitatory amino acid transporter EAAT3: Emerging functions in health and disease.

Plasma membrane neurotransmitter transporters maintain extracellular concentrations of neurotransmitters by facilitating transport into the cytosol. This regulation of extracellular neurotransmitters limits binding to receptors and activation of downstream signaling pathways. In addition to this critical function, transporters modulate neuronal activity via direct gating of transporter-associated ion channels and indirectly through trafficking of transporters to and from the plasma membrane. These functions are dependent on diverse expression patterns and levels of the transporters throughout the brain. This diversity is highlighted within the excitatory amino acid transporter family, which consists of five excitatory amino acid transporters found in the mammalian central nervous system (CNS). EAAT1 (GLAST) and EAAT2 (GLT-1) are primarily expressed in astrocytes while EAAT3 expression is mainly observed in many neurons throughout the brain (Holmseth et al., 2012). In contrast, EAAT4 is most prominently expressed in cerebellar Purkinje neurons and EAAT5 is exclusively found in the retina. In general, the astroglial transporters are highly expressed in the brain; EAAT2 is the most abundant, followed by EAAT1 with approximately a 4-fold lower expression (Holmseth et al., 2012). High expression levels of these glial transporters are consistent with their role in glutamate clearance (Lehre and Danbolt, 1998; Rothstein et al., 1996; Tanaka et al., 1997). The role of the neuronal transporter EAAT3 in brain has been more difficult to elucidate. Levels of EAAT3 are approximately 100-fold lower than EAAT2 (Holmseth et al., 2012) but EAAT3 expression is observed throughout the CNS, with enriched expression in the cerebral cortex, hippocampus, cerebellum and basal ganglia (Rothstein et al., 1994; Shashidharan et al., 1997). Given the lack of selective EAAT3 inhibitors, studies have relied on EAAT3 transporters expressed in various cells and endogenous transporters expressed in cultured hippocampal neurons (Diamond and Jahr, 1997; Grewer et al., 2000; Wadiche et al., 1995b), as well as EAAT3 knockout mice (Scimemi et al., 2009) to dertmine the physiological functions of EAAT3. These studies determined that the time course of glutamate in the synaptic cleft is a function of the binding of glutamate to EAATs and that transport of glutamate does not significantly contribute to the amplitude or kinetics of synaptic responses due to the relatively slow transport cycle (Diamond and Jahr, 1997; Tong and Jahr, 1994; Wadiche and von Gersdorff, 2006). Interestingly, EAAT3 knockout mice exhibit few behavioral deficits (Peghini et al., 1997), and antisense oligonucleotide knockdown in the striatum results in minimal elevation of extracellular glutamate levels or neurodegeneration, in contrast to knockdown of EAATs 1 and 2 (Rothstein et al., 1996). The lack of neurodegeneration is particularly surprising given that EAAT3 also serves as a cysteine transporter (Aoyama et al., 2006; Watts et al., 2014; Zerangue and Kavanaugh, 1996). Cysteine is the rate-limiting substrate for the synthesis of the antioxidant glutathione and its extracellular depletion is hypothesized to contribute to neurodegeneration. EAAT3 is also the dominant glutamate transporter in the intestines and provides nutrient absorption from the diet (Hu et al., 2018), but knockout animals appear to grow at a comparable rate to their litter mates (Peghini et al., 1997). The most remarkable initial observation from EAAT3 knockout mice was aminoaciduria due to the absence of EAAT3 in the kidneys (Peghini et al., 1997). The reported lack of overt behavioral abnormalities in the EAAT3 knockout mice suggest that either EAAT3 is not integral to regulation of glutamatergic signaling in the brain or that substantial developmental compensatory changes are induced in these mice. Human genetic studies and constitutive deletion mouse models have now provided evidence that EAAT3 has important roles in regulating neuronal signaling.