DrosophilaGlial Glutamate Transporter Eaat1 Is Regulated by Fringe-Mediated Notch Signaling and Is Essential for Larval Locomotion

Abstract

In the mammalian CNS, glial cells expressing excitatory amino acid transporters (EAATs) tightly regulate extracellular glutamate levels to control neurotransmission and protect neurons from excitotoxic damage. Dysregulated EAAT expression is associated with several CNS pathologies in humans, yet mechanisms of EAAT regulation and the importance of glutamate transport for CNS development and function in vivo remain incompletely understood. Drosophila is an advanced genetic model with only a single high-affinity glutamate transporter termed Eaat1. We found that Eaat1 expression in CNS glia is regulated by the glycosyltransferase Fringe, which promotes neuron-to-glia signaling through the Delta-Notch ligand-receptor pair during embryogenesis. We made Eaat1 loss-of-function mutations and found that homozygous larvae could not perform the rhythmic peristaltic contractions required for crawling. We found no evidence for excitotoxic cell death or overt defects in the development of neurons and glia, and the crawling defect could be induced by postembryonic inactivation of Eaat1. Eaat1 fully rescued locomotor activity when expressed in only a limited subpopulation of glial cells situated near potential glutamatergic synapses within the CNS neuropil. Eaat1 mutants had deficits in the frequency, amplitude, and kinetics of synaptic currents in motor neurons whose rhythmic patterns of activity may be regulated by glutamatergic neurotransmission among premotor interneurons; similar results were seen with pharmacological manipulations of glutamate transport. Our findings indicate that Eaat1 expression is promoted by Fringe-mediated neuron-glial communication during development and suggest that Eaat1 plays an essential role in regulating CNS neural circuits that control locomotion in Drosophila.

Drosophila astrocytes cover specific territories of CNS neuropil and are instructed to differentiate by Prospero, a key effector of Notch

“…Instead, we propose they arise from Notch-dependent neuron-to-glial communication during embryogenesis. We previously found that Fringe sensitizes Notch to stimulation from developing axons bearing Delta, thus maintaining Prospero and Eaat1 expression in a subset of LG (Stacey et al, 2010;Thomas and van Meyel, 2007). Knowing now that Prospero + cells develop into astrocytes, these data support the idea that astrogenesis is promoted by neuron-to-glial Notch signaling.…”

“…Also, mRNAs for Eaat1, the only Drosophila glutamate transporter (Besson et al, 2000), and Eaat2, a transporter for taurine and aspartate (Besson et al, 2005(Besson et al, , 2011(Besson et al, , 2000, were shown to be expressed in discrete subsets of glia in the embryonic CNS (Freeman et al, 2003; Soustelle et al, 2002;Stacey et al, 2010). We raised antisera to Eaat1 and Eaat2, and found that the Eaat1 antibody strongly labeled astrocytic membranes (Fig.…”

“…1A-C″) that also happens to be expressed in other glial cells such as cortex glia (Fig. 1B,B′, arrows) (Stacey et al, 2010). We then examined subtypes among LG (Fig.…”

“…We showed previously that Notch signaling through Fringe, a glycosyltransferase that sensitizes the Notch receptor to the Delta ligand, maintains expression of Prospero in the anteriormost six LG in stage 16 embryos (Thomas and van Meyel, 2007) and positively regulates Eaat1 transcription (Stacey et al, 2010). To examine whether Notch regulates the astrogenic program, we overexpressed Hairless, a transcriptional repressor of the Notch pathway (Fig.…”

“…Drosophila astrocytes respond to neuronal activity with altered transporter expression and intracellular calcium dynamics (Muthukumar et al, 2014;Stork et al, 2014), they influence CNS synapse number during neural circuit formation (Muthukumar et al, 2014) and modulate locomotor behavior, seizures and olfactory processing (Liu et al, 2014;Rival et al, 2004;Stacey et al, 2010;Stork et al, 2014). However, little is known about how Drosophila astrocytes are organized relative to specific regions of the synaptic neuropil, or of the key regulatory programs that govern the acquisition of astrocyte fate versus that of other glial cell types.…”