The Effect of NKCC1 on Glioma Induced Neuronal Hyperexcitability and Hypersynchrony

Introduction: Tumor-associated epilepsy plagues over 90% of patients with gliomas, with the majority failing to achieve adequate control. This indicates a critical need to identify novel drivers of glioblastoma-induced neuronal hyper-synchrony and excitability. Changes in GABAergic regulation in glioblastoma (GBM) have implicated chloride regulation as a potential contributor to tumor-induced neuronal activity. This makes NKCC1, a chloride cotransporter that can be upregulated in GBM, a target of interest.

Methods: To test the effect of NKCC1 on neuronal activity and synchrony, we silenced NKCC1 in GBM cells using shRNA knockdown. NKCC1 knockdown GBM cells were then cocultured with embryonic mouse cortical neurons (mCN) and performed electrophysiological analysis of neuronal activity using multielectrode array (MEA). Live cell calcium imaging was performed on neuron- glioma co-cultures treated with bumetanide, a selective NKCC1 inhibitor. Lastly, we developed patient-derived xenograft with shRNA NKCC1 knockdown and control conditions to evaluate the effect of NKCC1 on tumor growth.

Results: Electrophysiological analysis of glioma-neuron co-cultures using multi-electrode array and live-cell calcium imaging demonstrated increased number of spikes per burst (NSPB) and synchrony index (SI) in the presence of NKCC1- expressing tumor cells. Strikingly, this increase was eliminated in the presence of NKCC1 inhibition using bumetanide and by shRNA knockdown. NKCC1 downregulation significantly decreased neuronal synchrony and excitability compared to neurons co-cultured with scramble control condition. Mouse xenograft experiments showed increased NKCC1 expression was correlated with increased tumor burden and decreased survival.

Conclusion : The increase in NSPB and SI in the presence of NKCC1 expression, and its abolishment in the presence of pharmacologic NKCC1 inhibition uncovers an NKCC1 mediated mechanism of glioma induced neuronal hypersynchrony and hyperactivity. Furthermore, greater tumor burden and decreased survival implicate NKCC1 to have a prominent role in tumor progression. These findings illustrate an NKCC1-mediated mechanism for driving synchrony in tumor intrinsic neurons, and its role in GBM progression.