CNSC-69. GLIOBLASTOMA-NETWORK CONNECTIVITY IS REGULATED THROUGH ALTERATIONS IN CHLORIDE HOMEOSTASIS AND GABAERGIC SIGNALING

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Abstract The specific pathophysiological mechanisms underlying glioma-related epilepsy, which presents an unfavorable clinical course, are still not well explored. To identify the candidate pathways involved in glioblastoma-induced neuronal hyperexcitability, we used high-density electrode arrays in vivo in humans to record local field potentials from cortically projecting gliomas and identified hyperexcitable intratumoral regions with elevated functional connectivity to the brain. Single-cell RNA sequencing (13,730 cells analyzed) of the functionally connected intratumoral regions identified several circuit assembly and GABAergic signaling genes elevated in glioma-intrinsic neurons, including the sodium-potassium-chloride co-transporter 1 (NKCC1), which accumulates chloride in the cells. We hypothesized that the hyperexcitable behavior of glioma-intrinsic neurons in glioblastoma is mediated by elevated NKCC1 and chloride dysregulation and that inhibiting NKCC1 would reinforce the inhibitory action of GABA and rescue the neuronal hyperresponsive phenotype. Single-nucleus (sNuc-seq) sequencing was performed on neuron-glioblastoma co-cultures (54,000 cells analyzed) to determine NKCC1-facilitated GABAergic signaling in glioblastoma-mediated neuronal responses. Mechanistic and functional studies using perforated whole-cell patch-clamp recordings, multielectrode array, calcium, and chloride imaging for validating therapeutic vulnerabilities to NKCC1 silencing by shRNA knockdown and by the FDA-approved drug bumetanide were performed using in vitro and organotypic mouse slice culture models. sNuc-seq revealed an upregulation of neuronal NKCC1 and GABAA receptors when co-cultured with NKCC1-high-expressing glioblastoma cells, indicating the strong association between neuronal NKCC1 and GABAergic signaling. Electrophysiological analysis of glioblastoma-neuron co-cultures demonstrated increased network burst synchrony and a positive shift in the GABA reversal potential (EGABA). This increase was eliminated in the presence of NKCC1 inhibition using bumetanide and shRNA knockdown, which correlated with a significant decrease in intracellular neuronal chloride. Mechanistically, glioma NKCC1 downregulation significantly decreased GAP43-mediated tumor microtube (TMT) formation. Whether this reduced TMT formation also results in decreased chloride release by glioma cells, thereby altering neuronal chloride equilibrium, is currently under investigation. Collectively, these findings aim to identify a new mechanism of glioma-associated epilepsy, which may provide a novel approach to treatment.