Date of Award
2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Cellular, Molecular and Biomedical Sciences
First Advisor
James M. Stafford
Abstract
Epilepsy is a neurological disorder that affects 1% of the US and global populations.Epidemiological data indicate that many epilepsy cases are idiopathic with no known cause. The focus of epilepsy research in recent years has focused on genetic changes in neurodevelopment. Our collaborators recently uncovered epigenetic changes in human epilepsy patients involving somatic copy number gains in two noncoding enhancer regions upstream of EGFR. This phenomenon leads to aberrant EGFR expression in neurons that do not ordinarily express this gene and neuropathology indicative of focal cortical dysplasia type 1a. However, it is unknown how EGFR enhancer copy number variants promote epilepsy, and furthermore, how inappropriate EGFR expression in neurons leads to electrophysiological and neuropathological indicators of epilepsy.
To bridge this gap in knowledge, we have created a tractable model to explore theconsequences of inappropriate Egfr expression in murine primary cortex neurons (PCNs). Through ectopic human EGFR overexpression, we can recreate neurons with smaller soma sizes, a key feature of FCD1a neuropathology seen in these epilepsy patients. EGFR neurons also have fewer neurites and less complex branching patterns, which may indicate neuronal immaturity. EGFR ectopic overexpression also yielded an electrophysiological phenotype, indicating an excessive number of synaptic events, which shifts the neuronal network state towards excitation. These phenotypes were largely rescued using the brain-penetrating tyrosine kinase inhibitor Zorifertinib, further implicating aberrant EGFR expression in neurodevelopmental abnormalities.
To explore the epigenetic component of these findings, we engaged the enhancers in theirnative genomic locus using the dCas9-p300 system paired with guide RNAs to the enhancer sequences. This transfers the activating chromatin mark H3K27Ac to the enhancer and leads to gene expression. This approach recreated the same smaller soma size phenotype, as well as smaller and less complex neurites, indicating that direct enhancer activation can lead to gene expression and likely plays a role in neurodevelopment. We also ectopically overexpressed the enhancer sequences themselves to recreate the copy number increases seen in the human brain. This also yielded smaller neuron sizes, indicating that enhancer transcription itself also facilitates the development of aberrant phenotypes.
By combining these epigenetic, molecular, and electrophysiological approaches, thisstudy provides insight into the vast consequences of EGFR expression seen in human epilepsy patients and lays the foundation for eventually treating these patients with tyrosine kinase inhibitors.
Language
en
Number of Pages
159 p.
Recommended Citation
Weinman, Marcus, "Functional And Epigenomic Consequences Of Somatic Mutation In Non-Coding Receptor Tyrosine Kinases In Epilepsy" (2025). Graduate College Dissertations and Theses. 2017.
https://scholarworks.uvm.edu/graddis/2017
