Investigate roles of Drosophila Eaat1-mediated glutamate clearance in the functional integrity of the motor circuit and the formation of the neuromuscular junction

Glutamate transmission in CNS is critical for animal behavior. Upon the release of glutamate from synaptic vesicles, it is immediately removed by the surrounding astrocyte. The Excitatory Amino Acid Transporter (Eaat) in astrocyte is majorly responsible for the glutamate clearance in CNS, which ensures the fidelity of neurotransmission and prevents glutamate excitotoxicity. In neurodegenerative diseases, downregulation of Eaat has been repeatedly reported and accumulation of extracellular glutamate due to impaired recycle system accelerates the disease progression. Although how excessive glutamate leads to neuronal death has been widely studied, the influence of glutamate excitotoxicity on neural circuit is still elusive. Here, we use Drosophila larval locomotor circuit to study the role of glia on the glutamate transmission in CNS. From a genetic screen for novel players involved in synaptic formation, we identified a strong hypomorphic Drosophila eaat1 allele which led to strong loss of eaat1, expanded neuromuscular junction, and severe locomotor deficits. The locomotion of Drosophila larva is controlled by the neural circuit called central pattern generator (CPG). Loss of eaat1 resulted in aberrant CPG activity which caused prolonged but less frequent activation of motoneurons. Restoring the expression of eaat1 exclusively in the astrocyte was sufficient to restore the CPG and locomotion defects in eaat1 mutants. Impaired glutamate clearance in loss of eaat1 led to extracellular glutamate accumulation and disrupted the excitatory and inhibitory synaptic signaling in the CPG. Remarkably, reducing the oxidative stress or increasing the excitability of excitatory cholinergic neurons in eaat1 mutants improved both CPG rhythmicity and mobility. Moreover, the irregular CPG firing pattern in loss of eaat1 triggered the ROS/JNK signaling in motorneurons and caused excessive synaptic bouton formation. We demonstrated a non-cell autonomous mechanism to regulate the physiology of motoneurons via Eaat1 in upstream astrocyte.