Ischemia-Induced Post-Translational Modifications of GLT-1 Mediate Aberrant Trafficking and Impaired Glutamate Uptake

Glutamate transporters are essential for maintaining CNS homeostasis by clearing extracellular glutamate following synaptic transmission. Dysregulation of these transporters contributes to glutamate-mediated excitotoxicity across numerous neurological disorders, including ischemic stroke, underscoring their potential as therapeutic targets. However, the regulatory response of these transporters following ischemic insult remains poorly defined. In this study, using a model of oxygen-glucose deprivation in primary rat glial cultures, we report aberrant trafficking of the astrocytic glutamate transporter GLT-1 following ischemic insult. This response is characterized by increased transporter internalization and degradation, accompanied by reduced glutamate uptake capacity. Focusing on post-translational modifications (PTMs), we found that GLT-1 ubiquitination is markedly increased after ischemic insult and coincides with transporter internalization. Importantly, disrupting this ubiquitination interaction through mutation of C-terminal GLT-1 lysine residues restores GLT-1 surface expression and rescues glutamate uptake capacity through preventing early endosome 1 (EEA1)-mediated internalization. Additionally, we report that inhibition of C-terminal GLT-1 PTMs confers neuroprotection following ischemic insult in organotypic hippocampal brain slices. Together, these findings demonstrate that ischemia-induced dysregulation of GLT-1 trafficking plays a critical role in impaired glutamate clearance and cellular recovery, highlighting GLT-1 ubiquitination as a potential therapeutic target for ischemic injury.