Diffusely infiltrating gliomas are among the most common type of primary brain tumors. The glioma cells widely invade the brain, where they intermingle with, and interact with, neurons in the tumor microenvironment. This leads to pathological alterations in neuronal physiology that can have devastating clinical consequences, such as uncontrollable seizures. Previous studies have implicated dysregulation of neuronal translation as a cause of epilepsy. However, little is known about the molecular alterations underlying the neuronal dysfunctions seen in glioma patients. As a first step towards addressing this important question, we propose to characterize the neuron-specific alterations in transcription and translation that occur in a mouse model of diffusely infiltrating glioma. We will induce gliomas formation by injecting a PDGF expressing retrovirus into the subcortical white matter of CamKII-cre/RiboTag mice, which are engineered to express HA-tagged ribosomal protein (Rpl22) selectively in neurons. We will immunoprecipitate neuron-derived translating ribosomes from mouse brain tumor homogenate and sequence the mRNA footprints to quantify the number of ribosomes associated with each gene. We will also perform RNA-seq on mRNA from total tissue homogenate and apply a computational approach to deconvolve the composite expression data into cell-type specific profiles. Together, these analyses will enable us to calculate translation efficiency for every gene (ribosome density per transcript), which provides a sensitive measure of translational regulation. The experimental system developed in this proposal will provide a powerful tool for future studies to explore mechanistic questions about how glioma-neuron interactions cause seizures, as well as preclinical studies to test the effects target therapies designed to inhibit glioma-associated epilepsy.
Over 80% of low-grade glioma patients develop tumor-associated epilepsy, and uncontrolled seizures is a major clinical problem for these patients. In this proposal we will develop a new mouse model and experimental methodologies to characterize the alterations in gene expression and protein synthesis that occur in neurons within the microenvironment of brain tumors. This work will advance our understanding of this devastating disease and lead to future studies to test new forms of therapy.
| Hornstein, Nicholas; Torres, Daniela; Das Sharma, Sohani et al. (2016) Ligation-free ribosome profiling of cell type-specific translation in the brain. Genome Biol 17:149 |
