BRAFV600E Expression in Mouse Neuroglial Progenitors Increase Neuronal Excitability, cause appearance of balloon-like cells, Neuronal Mislocalization, and Inflammatory Immune Response

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