Date of Completion

4-30-2018

Embargo Period

4-30-2019

Keywords

BRAFV600E, Low-grade neuroepithelial tumors, epilepsy, balloon cells, astrogliosis, piggyBac

Major Advisor

Joseph J. LoTurco

Associate Advisor

Anastasios V. Tzingounis

Associate Advisor

Daniel K. Mulkey

Associate Advisor

Alexander C. Jackson

Associate Advisor

Harvey A. Swadlow

Field of Study

Psychology

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Low-grade neuroepithelial tumors (LNETs) are the most frequent tumors in patients undergoing resective surgery for epilepsy treatment. BRAFV600E represent the most frequent mutation in LNETs. In the RAS-RAF-ERK (MAPK) pathway activation of receptor tyrosine kinase leads to activation of BRAF protein. By mimicking constitutive activation of the kinase domain in RAS-independent manner BRAFV600E represents much higher activating substrate of MAPK pathway than BRAF wild type. Increased activation of MAPK leads to alterations of cell cycle progression, cell growth, proliferation, and cell fate. It also influences cortical neuron migration. Introducing human BRAFV600E into neuroglial progenitors in mouse cortex on embryonic day 14 to 15 induced astrogliosis. It also disrupted cellular morphology, causing appearance of balloon-like cells, disrupted neuronal migration to the upper cortical layers, altered tissue-wide gene expression, and induced neuronal hyperexcitability. Hyperexcitability was evident as higher action potential (AP) frequencies in response to depolarizing current injections in whole-cell current-clamp configuration of the patch-clamp technique; more depolarized by 7-10 mV resting membrane potential (RMP) and increased by about 100 MΩ input resistance (Rin). Single APs showed more hyperpolarized voltage threshold in BRAFV600E expressing neurons and those neurons had lower minimal required current (rheobase) to fire APs. Increased initial deflection in response to the hyperpolarizing current pulses - SAG and a rebound excitation at the end of those pulses were observed, which was found to result from increase in the hyperpolarization activated conductance (Ih current). In whole-cell voltage-clamp configuration BRAFV600E neurons have increased Ih compared to the control conditions. Additionally, sustained, tetraethylammonium sensitive voltage activated potassium currents were decreased in BRAFV600E neurons. Activation of potassium currents with retigabine decreased action potential firing frequency. Recording of action potential dependent spontaneous post-synaptic currents (sPSCs) showed increased frequency in BRAFV600E neurons, those sPSCs frequencies were higher in untransfected neighbors. Video and Electrocorticographic recording in the freely moving mice showed spontaneous epileptiform activity with behavioral manifestations. The findings indicate that BRAFV600E mutations are sufficient to partially capture the pathophysiology of LNETs. The conclusion has implications to the possible courses of, and therapeutic targets for LNET associated epilepsy.

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