Glioma-Induced Alterations in Excitatory Neurons are reversed by mTOR Inhibition
Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, cognitive deficits, and seizures, as well as potentially driving glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via 'Ribotag', morphometric analysis, and intravital imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden on excitatory neuronal pathophysiology as well as the impact of mTOR inhibition on these neuronal alterations. The Ribotag analysis of peritumoral excitatory neurons showed an upregulation in transcripts encoding for F-actin binding and other dendritic spine-enriched proteins. Light and electron microscopy analyses revealed marked decreases in dendritic spine density in peritumoral neurons. Intravital two-photon imaging in peritumoral excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single neuron level, throughout tumor growth. Intravital two-photon imaging also revealed altered stimulus-evoked somatic calcium activity, both in rate and temporal alignment, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a mTORC1 and mTORC2 inhibitor reversed the translational effect of glioma on neurons, increased dendritic spine density, and functional neuronal alterations. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma – manifested by alterations in ribosome-bound mRNA, dendritic spine morphology, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that peritumoral neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing foundational knowledge for developing therapies targeting neuronal signaling in glioma. Overall design: In order to study the translational alterations in the neurons of glioma-bearing mice, we implanted glioma cells in the cortex of Camk2a-Cre-Rpl22HA mice. We isolated total and immunoprecipitated (IP) polysomal RNA from three groups of mice: control (no tumor), tumor and tumor treated with the mTOR inhibitor AZD8055 We determined neuronally enriched genes in the tumor condition by perfoming DESeq2 between tumor input and IP fractions, then proceeded to compare the three conditions.