Loss of the glial glutamate transporter eaat2a leads to a combined developmental and epileptic encephalopathy in zebrafish

Astroglial excitatory amino acid transporter 2 (EAAT2, GLT-1, SLC1A2) regulates the duration and extent of neuronal excitation by removing glutamate from the synaptic cleft. Human patients with altered EAAT2 function exhibit epileptic seizures, suggest‐ ing an important role for astroglial glutamate trans‐ porters in balancing neuronal excitability. To study the impact of EAAT2 function at the neural network levels, we generated eaat2amutant zebrafish. We ob‐ served that eaat2a-/mutant zebrafish larvae display recurrent spontaneous and light-induced seizures in neurons and astroglia, which coincide with an abrupt increase in extracellular glutamate levels. In stark contrast to this hyperexcitability, basal brain activity was surprisingly reduced in eaat2a-/mutant animals, which manifested in decreased locomotion, neuronal and astroglial calcium signals. Our results reveal an unexpected key role of the astroglial EAAT2a in bal‐ ancing brain excitability, affecting both neuronal and astroglial network activity. INTRODUCTION Astroglia are the most numerous glial cells within the central nervous system (CNS). They do not only pro‐ vide trophic support to neurons but also play an im‐ portant role in synapse formation and neurotransmis‐ sion1–4. By taking up neurotransmitters from the synaptic cleft, these glial cells are crucial for regulat‐ ing synaptic transmission. The excitatory amino acid transporter 2 (EAAT2) expressed on astroglia plays a key role in synaptic regulation by removing the ma‐ jority of extracellular glutamate, which is the main excitatory neurotransmitter in the CNS5,6. Impaired glutamate clearance by this transporter has been shown to lead to synaptic accumulation of the neuro‐ transmitter, resulting in an overactive CNS and exci‐ totoxicity7,8. This in turn may cause epilepsy, a group of brain disorders characterized by recurrent seizures9. Indeed, EAAT2 malfunction has been asso‐ ciated with epileptic seizures in mice and humans10–15. However, the mechanistic role of this astroglial gluta‐ mate transporter in developing epileptic seizures is not fully understood. Previous studies have shown that astroglia-neuron in‐ teractions may play an essential role in seizure initia‐ tion and propagation16–18. On one hand, the functional coupling of astroglia through gap junctions is crucial to avert excessive neuronal activation, accomplished by rapid re-distribution of ions and neurotransmitters across the connected astroglial network18. On the other hand, this functional syncytium might also, un‐ der special circumstances, promote epileptogenesis19. The intercellular spread of calcium waves among as‐ troglia can affect neuronal synchronization and there‐ fore influence the propagation of seizure activity16. In addition, disruptions of the glutamate-glutamine cy‐ cle, in which EAAT2 is essential, are linked with tem‐ poral lobe epilepsy in human patients and rodents20. Accordingly, it is essential to understand how loss of EAAT2 affects neurons and astroglia. In the present study, we generated a zebrafish (Danio rerio) mutant lacking EAAT2a, the zebrafish ortho‐ logue matching the mammalian EAAT2 in biophysi‐ cal characteristics and glial expression pattern21,22. We show that loss of this astroglial transporter in larval zebrafish leads to increased brain excitability and re‐ current spontaneous seizures, mimicking the human phenotype of patients with de novo mutations in EAAT211,13. These seizures are manifested in ze‐ brafish larvae by epileptic locomotor bursts and peri‐ ods of excessive brain activity, accompanied by in‐ creased extracellular glutamate concentrations. Counterintuitively, apart from these periods of hyper‐ excitation, neuronal and astroglial networks of eaat2a-/mutants are hypoactive. This coincides with a decreased overall locomotion compared to their un‐ affected siblings, and mirrors slow background brain activity and reduced muscle tone present in human patients11,13. Altogether, our in vivomodel of impaired EAAT2a function in astroglia results in a depressed yet hyperexcitable brain state, and mimics a form of developmental and epileptic encephalopathy (DEE). EAAT2a GS EAAT2a GS SV2/acT merge all EAAT2a SV2/acT Hi OB