Abstracts

Rationale: Inflammation has been shown to be involved in the development of epilepsy as well as following an epileptic seizure.¹  Investigating the source of the seizure-associated inflammation may lead to new therapeutic and diagnostic tools for epilepsy. One potential source identified are mitochondria, which have been implicated in other neurodegenerative disorders such as Parkinson's.² Mitochondria also contain potential inflammatory agents such as mtDNA and reactive oxygen species (ROS). To better understand this phenomena, we sought to establish a model of seizure-like events in vitro to better study resulting inflammatory and metabolic changes.

Methods: We employed rat cortical co-cultures on microelectrode arrays (MEA) to probe electrophysiological activity. Our cortical cultures consisted of postnatal rat pups which yielded a mixture of neurons, astrocytes, and microglia in our cultures. To induce periods of neuronal hyperexcitability, cultures were treated with 4-aminopyridine (4AP), a potassium channel blocker and epileptogenic compound. Cortical co-cultures were plated on multielectrode arrays (MEA) to probe the electrophysiological activity of our cultures. 4AP was administered for 1 hour over three to seven days in an attempt to mimic the frequency of seizures in human patients. Treated cultures were analyzed to elucidate the metabolic and inflammatory consequences of neuronal hyperexcitability through western blots, RT-qPCR, and Seahorse XF analyzer.

Results: 4AP results in increases in neuronal burst frequency when cells are treated. Comparing those cells treated with 4AP and control cells, 4AP cells begin firing at significantly higher rates at baseline (with no 4AP present) beginning on the 3rd day of treatment and continuing through day 7. We observed significant upregulation of IL1β and TNFα RNA transcripts following the 3rd day of treatment, indicative of an inflammatory response. Using flow cytometry and MitoSOX, an ROS dye, we observed that 4AP treated cells produce higher levels of ROS compared to controls. Finally, 4AP treated cells after 3 days of treatment have significantly lower baseline and lower maximal mitochondrial respiration rates, measured by the Mito Stress Test kit.

Conclusions: Here, we propose a potential mechanism by which increased neuronal hyperexcitability may induce an inflammatory response through mitochondrial dysfunction. Following 4AP-induced neuronal hyperexcitability, we observed mitochondrial dysfunction as well as increased mitochondrial ROS, which may lead to upregulation of inflammatory cytokines, such as IL1β and TNFα  These results may lead us to better understand inflammation in the setting of seizures, which has been implicated in the development of epilepsy. Further research will investigate mtDNA leakage and the activity of PINK1-mediated mitophagy in this model. _x000D_ _x000D_ References:_x000D_ 1. Rana, A., Musto A.E. The role of inflammation in the development of epilepsy. J Neuroinflammation 15, 144 (2018)_x000D_ 2. Sliter, D.A., Martinez, J., Hao, L. et al. Parkin and PINK1 mitigate STING-induced inflammation (2018)

Funding: NIH Intramural Research Program, NIH Postbaccalaureate Research Training Award, Medical Research Scholars Program