Mechanisms of Embryonic and Adult Neurogenesis in the Development of Epilepsy

Abstract

The epilepsies encompass a constellation of syndromes that possess the cardinal feature of spontaneous recurrent seizures. Epilepsy etiologies are classified in part based on two broad categories: acquired and genetic. Acquired epilepsies often arise as a result of a previous neurological insult while genetic epilepsies arise from gene mutations that affect the brain. While the two are causally distinct, both acquired and genetic epilepsies often involve abnormal neural development, whether during adulthood or embryonically, as an underlying component of epileptogenesis.

This dissertation aims to explore how alterations in embryonic and adult neurogenesis lead to morphological and functional changes that may underlie the development of seizures. We utilize a rat pilocarpine-induced status epilepticus (SE) model of temporal lobe epilepsy (TLE), an acquired epilepsy, to ask how adult-born dentate granule cells (DGCs) differentially integrate into the chronically epileptic brain. By employing a dual-virus tracing strategy combining retroviral-birthdating with rabies virus- mediated retrograde trans-synaptic spread, we explore how first-order presynaptic inputs onto adult-born DGCs in the epileptic brain differ from those in the intact brain. Furthermore, we compare the presynaptic inputs onto adult-born DGCs with those onto early-born DGCs that were mature at the time of SE.

Our results demonstrate that both adult- and early-born DGCs in the epileptic brain receive recurrent excitatory inputs from normotopic DGCs while adult-born DGCs are preferentially targeted by hilar ectopic DGCs. We also show that other regions of the hippocampus that normally do not project to the dentate gyrus, such as CA3 and CA1, sprout axon collaterals after SE that preferentially synapse onto adult- and early-born DGCs, respectively. Finally, we describe changes in the hippocampal inhibitory interneuron network with differential sprouting by both parvalbumin and somatostatin interneurons onto different age-defined DGC populations.

In addition to modeling an acquired epilepsy, we also employ an induced pluripotent stem cell (iPSC) model of protocadherin-19 (PCDH19) female-limited epilepsy (FLE), a genetic epilepsy. Female patients with heterozygous mutations in the X-linked PCDH19 gene develop the disease while mutation carrying males are asymptomatic. Interestingly, there are several cases of affected males who have somatic mosaicism for the PCDH19 gene. We generate iPSCs from female FLE patients with pathogenic mutations in PCDH19 and address the hypothesis that FLE arises from cellular interference during development between wildtype and mutant PCDH19-expressing neurons. We find that cortical-like excitatory and inhibitory neurons derived from FLE patient-derived iPSCs have aberrant morphologies and increased excitability. We also use the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene editing technology to generate PCDH19-null male iPSCs. We mix the PCDH19-null iPSCs with their isogenic control iPSCs to mimic disease phenotype in affected mosaic males. This approach recapitulates some of the morphological and functional changes found in female FLE patient-iPSC derived neurons, further lending support to the idea that altered cell-cell interactions contribute to the disease pathogenesis. The work presented in this dissertation gives new insights into how the adult neural circuitry remodels after epileptic seizures, as well as how deviations in embryonic development can mediate hyperexcitability. However, these two processes are not mutually exclusive and understanding both should better inform our knowledge of the mechanisms of epileptogenesis and uncover future therapeutic strategies.