bioRxiv2019
BACKGROUNDFrequent de-novo somatic mutations in major components (PI3KCA, AKT3, TSC1, TSC2, mTOR, BRAF) of molecular pathways crucial for cell differentiation, proliferation, growth and migration (mTOR, MAPK) has been previously implicated in malformations of cortical development (MCDs) and low-grade neuroepithelial tumors (LNETs) 1-7. LNETs are the most frequent tumors found in patients undergoing resective surgery for refractory epilepsy treatment. BRAFV600E is found in up to 70% of LNETs. Previous studies suggest a causal relationship between those de-novo somatic mutations in mTOR, MAPK pathways and seizures occurrence, even without presence of malformation or a tumor 2, 3, 8-13. Recently Koh and colleagues 14 showed that BRAFV600E mutation may cause seizures through activation of RE1-silecing transcription factor (REST). Additionally, they showed a significant downregulation of synaptic transmission and plasticity pathways and decreased expression of multiple ion channels subunits including HCN1, KCNQ3, SCN2A and SCN3B. The downregulation of those genes including GABA receptors subunits and protein expression specific to interneurons subpopulations (SST, VIP) suggests that a dysregulated inhibitory circuits are responsible for seizures in GGs. The experimental manipulation - In-Utero electroporation of episomal activating Cre plasmids that they used to test their hypothesis in mice however activated mutant BRAFV637 only in excitatory neurons. And the downregulated genes in mice were confirmed by qRT-PCR in the whole tissue samples. The question of how electrophysiological properties of the affected and surrounding neurons are changed were not addressed. The changes in ion conductances and neuronal circuits responsible for seizures could be only inferred from gene expression profiles. Purpose of the current work was to investigate how overactive human BRAFV600E mutated protein incorporated into the mouse genome through piggyBase transposition increase neuronal excitability in ex-vivo mouse cortical slices and whether it induces histopathological features and gene expression profile alteration observed in low-grade neuroepithelial tumors (LNETs). METHODSUsing In-Utero Electroporation we have introduced human BRAFV600E protein into radial glia progenitors in mouse embryonic cortex on the background of piggyBac transposon system that allows incorporation of the DNA sequence of interest into the genome. Immunohistochemistry was used for examination of known markers in LNETs. RNA sequencing on Illumina NextSeq 500 was used to examine alterations in gene expression profiles. Whole-cell current- and voltage-clamp was used to examine changes in electrophysiological properties. Unsupervised Hierarchical Clustering Analysis was used to examine grouping of different conditions based on their gene expression profile and electrophysiological properties. Video electrocorticographic recordings were used to test whether BRAFV600E transgenic mice have spontaneous seizures. RESULTSUnder GLAST driving promoter BRAFV600E induced astrogenesis, caused morphological alterations in transgenic cells akin to balloon-like cells, and delayed neuronal migration. Under NESTIN driver promoter BRAFV600E increased neurogenesis, induced balloon-like cells and caused some cells to remain close to the lateral ventricle displaying large soma size compared to neurons in the upper cortical layers. Some of the balloon-like cells were immunopositive for astroglial marker glial fibrillary acidic protein (GFAP), and for both upper and lower cortical layers markers (Cux1 and Ctip2). Gene ontology analysis for BRAFV600E gene expression profile showed that there is a tissue-wide increased inflammatory immune response, complement pathway activation, microglia recruitment and astrocytes activation, which supported increased immunoreactivity to microglial marker iba1, and to GFAP respectively. In current clamp BRAFV600E neurons have increased excitability properties including more depolarized resting membrane potential, increased input resistance, low capacitance, low rheobase, low action potential (AP) voltage threshold, and increased AP firing frequency. Additionally, BRAFV600E neurons have increased SAG and rebound excitation, indicative of increased hyperpolarization activated depolarizing conductance (IH), which is confirmed in voltage-clamp. The sustained potassium current sensitive to tetraethylammonium was decreased in BRAFV600E neurons.. In 4 out of 59 cells, we have also observed a post-action potential depolarizing waves, frequencies of which increased in potassium current recording when Ca2+ was substituted to Co2+ in the extracellular solution (5/24). We show that using 20 electrophysiological properties BRAFV600E neurons segregate separately from other conditions. Comparison of electrophysiological properties of those neurons with neurons bearing somatic mutations in mechanistic target of rapamycin (MTOR) pathway regulatory components, overactivation of which is been shown in malformations of cortical development (MCDs), showed that expression of PIK3CAE545K under GLAST+ promoter and TSC1 knockdown (KD) with CRISPR-Cas9 have different effects on neuronal excitability.