ELEVATED ASTROCYTIC GAT3 LINKS TO NEURONAL HYPEREXCITABILITY IN A HUMAN IPSC DERIVED ALZHEIMER’S TRICULTURE MODEL

Alzheimer’s disease (AD), the most common cause of dementia, affects more than 55 million people worldwide. Beyond amyloid beta and neurofibrillary tangles, early neuronal network dysfunction is prominent, with hyperexcitability arising at preclinical stages and associated with faster symptom onset. We have shown previously that inhibitory signalling mediated by the astrocytic GABA transporter GAT3 is increased in AD mouse models and may represent a maladaptive compensatory response that contributes to network disruption. To test this in a human system, we generated an iPSC derived triculture of excitatory neurons, inhibitory neurons and astrocytes, comparing APP V717I cultures with an isogenic control.
GAT3 protein expression was assessed by immunofluorescence at day in vitro (DIV) 100. Neuronal intrinsic properties and excitability were evaluated at DIV100 using whole-cell electrophysiology, including measurements of spontaneous firing rate and resting membrane potential.
By DIV100, preliminary results show APP V717I tricultures to have increased GAT3 protein expression relative to the isogenic control by immunofluorescence. Electrophysiological recordings at DIV100 demonstrated a hyperexcitable neuronal phenotype in APP V717I cultures, evidenced by an increased spontaneous firing rate and a more depolarized resting membrane potential.
These findings indicate that GAT3 upregulation may coincide with heightened neuronal activity in a human AD triculture model. This supports the possibility that GAT3 changes are linked to (and potentially driven by) AD-associated hyperexcitability. Ongoing work will test whether elevated GAT3 alters ambient GABA levels and neuronal activity and will extend these analyses to network scale physiology using multielectrode arrays.