Phd in CLINICAL EXPERIMENTAL NEUROSCIENCE AND PSYCHIATRY

Thesis title: Microglia-neuron crosstalk in the remodeling of peritumoral circuits in glioblastoma

Glioma progression relies on its ability to shape a permissive tumor microenvironment through complex interactions with immune and neuronal components. Glioma cells exploit neuronal activity through paracrine and synaptic signaling, which enhances excitability in peritumoral regions and promotes tumor progression. Among tumor-associated myeloid cells, microglia acquire tumor-supportive phenotypes that foster glioma growth and invasion. Under physiological conditions, microglia play a key role in sculpting and maintaining neuronal circuits during development and adulthood. However, how these cells contribute to the neuronal dysfunctions induced by glioma remains poorly understood. Dissecting this interaction may reveal new mechanisms linking tumor progression to neural circuit remodeling. Therefore, the aim of my thesis was to investigate how microglia contribute to the neuronal alterations induced by glioma growth and whether they participate in the communication between neurons and tumor cells. To this purpose, we used the GL261 glioma mouse model and evaluated the effects of pharmacological depletion of microglia through dietary administration of PLX5622, a selective CSF1R inhibitor, in the diet. Electrophysiological recordings from peritumoral cortical neurons revealed that glioma induces a marked hyperexcitability, characterized by depolarized resting membrane potential, reduced rheobase, and increased firing frequency. Microglia depletion restored neuronal excitability to control levels and reduced susceptibility to epileptiform activity, indicating that microglia actively sustain hyperexcitability in peritumoral networks. To understand the molecular correlates of the protective role of microglia depletion in limiting hyperexcitability in peritumoral tissue, we investigated the mechanisms involved in the regulation of neuronal firing. Molecular analyses revealed a reduction of SK2 channel expression in peritumoral neurons, which was reversed by PLX5622 treatment, consistent with the increase in medium afterhyperpolarization (mAHP) amplitude. These molecular data were supported by electrophysiological isolation of the SK-mediated current, confirming that microglia depletion increases SK2 channel function. Together, these findings suggest that microglia influence neuronal excitability through modulation of SK2 channels, potentially via cytokine signaling mediated by TNF-α. To explore whether microglia also mediate the interplay between neurons and glioma cells, we performed calcium imaging on GL261 tumors expressing GCaMP6. Glioma cells displayed spontaneous calcium transients partially driven by neuronal firing, as shown by the reduction of activity following tetrodotoxin (TTX) treatment. Remarkably, this neuronal-dependent component was lost after microglia depletion, indicating that microglia are required for neuron-to-glioma calcium coupling. In addition, PLX5622 treatment was associated with reduced tumor proliferation, suggesting that microglia-dependent neuronal hyperexcitability may support glioma growth. We also investigated whether microglia depletion affected inhibitory synaptic transmission in peritumoral neurons as peritumoral hyperexcitability is also due to alterations in the inhibitory tone. Recordings of miniature inhibitory postsynaptic currents (mIPSCs) showed that glioma reduces GABAergic inhibitory tone, which is not reversed by microglia depletion. Altogether, these results reveal that microglia are not merely passive immune effectors but active regulators of neuronal excitability and neuron-glioma communication. By promoting a hyperexcitable peritumoral environment, microglia facilitate tumor progression and network instability. These findings highlight the importance of microglia as modulators of neuron-cancer interactions and suggest that selective modulation of their function may represent a promising strategy to restore neuronal dysfunctions in glioma.