Mitochondrial Pathways in Epileptogenesis Following Traumatic Brain Injury

Principal Investigator: FRASIER, CHAD R

Proposal Number: EP220018

Award Number: HT9425-23-1-0334

Period of Performance: 7/1/2023 - 6/30/2026

PUBLIC ABSTRACT

Annually approximately three million Americans experience traumatic brain injury (TBI), putting them at an increased risk of developing epilepsy. TBI represents a major cause of death and permanent disability in the population. In addition, patients with TBI are at an increased risk of developing post-traumatic epilepsy (PTE). It’s been estimated that PTE cases account for approximately 20% of total epilepsy cases. Despite significant work to uncover mechanisms of that underly seizures in models of epilepsy, the use of these therapeutic avenues in treating epileptogenesis has been underwhelming. Unfortunately, treatments targeted at the processes to decrease the incidence of PTE have been largely unsuccessful. This proposal will investigate both regional and temporal differences in mitochondrial function that underly the progression of PTE following TBI.

The mitochondria lay at the intersection of several pathways in energy production, cellular excitability, cell signaling, and cell death. Recently, there has been an increase in the interest in the role of bioenergetics in epilepsy as well as other neuronal disorders. Mitochondria play a vital role in the overall function of individual cells and the brain as a system, and a deeper understanding of how mitochondria are at the intersection of epileptogenesis may be critical for advancing current therapies for treating epilepsy. Our central hypothesis is that altered mitochondrial energetics, redox balance, and cell death pathways are compromised during the epileptogenesis process leading to PTE.

This proposal uses novel approaches to investigate how key mitochondrial pathways are altered in an animal model of TBI. Our first aim will look at cellular respiration, reactive oxygen species, calcium flux, and mitochondrial membrane potential following TBI. Our second aim will investigate the role of the mitochondrial signaling and excitability in mice that develop PTE. Finally, we will investigate if the ketogenic diet represents a therapeutic target for preventing PTE. Our experiments are carefully designed to provide valuable insight into the regional and temporal changes that effect the brain following TBI. All three aims in this proposal have yet to be investigated in TBI, and our ability to provide both spatial and temporal resolution in these processes will likely lead to the discovery of potential therapeutic targets for further study. In addition, we have chosen dietary therapy as it has high potential to be useful in the field. The inclusion of the ketogenic diet as a potential therapeutic avenue in the third aim has the potential for quick translation should our results be positive. We’ve chosen to test different delivery timepoints to test effectiveness to further increase clinical translation. In addition to the ketogenic diet, our proposal has the potential to impact clinical outcomes, as novel therapeutic agents targeting the mitochondria are under active development and understanding the pathways involved in PTE can lead to a more targeted pharmacologic approach.

The Principal Investigator’s participation in the Virtual P-TERC is also well suited for his career goals. Despite success in cardiovascular research, he has always maintained his interest in neuroscience and has a strong desire to pursue an active research career in PTE. His previous research experience in a lab with a focus on epilepsy has given him a strong foundation to build upon, and the team assembled in place will ensure his success in this endeavor. His participation in the P-TERC can only serve to further enhance his career goals, training, and chances of sustaining a well-funded PTE research program.

TECHNICAL ABSTRACT

Background: Patients with traumatic brain injury (TBI) are at an increased risk to develop post-traumatic epilepsy (PTE), and it has been estimated that PTE may account for up to 20% of symptomatic epilepsy in the general population. In addition, TBI represents a major military combat concern. While significant work has been done to understand the mechanisms of epileptogenesis, treatment of seizures still presents several obstacles. The mitochondria lay at the intersection of multiple critical pathways in epileptogenesis. Mitochondria are the main energy producers of the central nervous system, and approximately 90% of the adenosine tri-phoshpate (ATP) generated in the brain occurs via oxidative phosphorylation. The majority of this ATP is utilized to maintain resting membrane potential in neurons, underscoring the importance between mitochondrial energetics and cellular excitability in models of epilepsy. As part of our approach, we will directly investigate the ability of mitochondrial efficiency in ATP generation across critical timepoints following TBI. In addition, we will investigate several mechanisms that may underlie changes in respiratory capacity and efficiency over the time-course of PTE development.

Hypothesis: Mitochondria play a vital role in the overall function of individual cells and the brain as a system and developing a deeper understanding of how mitochondria are at the intersection of epileptogenesis may be critical for advancing current therapies for treating epilepsy. Our central hypothesis is that mitochondrial energetics, redox balance, and cell death pathways are compromised not only during the initial injury progression of TBI, but decline even further in the development of PTE.

Specific Aims:

Aim 1: To understand the role of mitochondria energetics and signaling in seizure susceptibility following TBI. Here we will investigate the mitochondrial pathways as they relate to (1) mitochondrial energetics, (2) mitophagy, and (3) regulation of cell death pathways.

Aim 2: To understand how changes to mitochondrial function and signaling effect cellular excitability after the recurrence of spontaneous seizures in PTE. Here we will investigate if deficits in mitochondrial efficiency occur at the onset, and persist or disappear, after the appearance of spontaneous seizures following the latency period.

Aim 3: To determine if the ketogenic diet leads to alterations in mitochondrial pathways and provides protection from development of PTE. Specifically, we will determine if gene transcription or post-translational modifications to key ion channels are altered in animals fed a ketogenic diet.

Study Design: We will be using the controlled cortical impact model of TBI across all of our aims. Across our aims, animals will be taken at critical timepoints to provide strong temporal resolution of the epileptogenic processes. We will determine mitochondrial respiration capacity and efficiency using high resolution respirometry. We will also use a combination of fluorescence microscopy coupled to electrophysiology to approach how both mitochondrial function and cellular excitability are interrelated. Next, we’ll use an in vitro model of epileptogenic activity to determine if TBI makes neuronal tissue more susceptible to the development of seizures. Finally, we will use a 6:1 fat to carbohydrate ketogenic diet to investigate if dietary therapies lead to protective measures across the aims.

Impact: This research is innovative as it brings my expertise in the techniques in this proposal into the epilepsy field. Few studies have used high resolution respirometry to investigate models of epilepsy and none in TBI. In addition, we are using multiple approaches to uncover the spatial and temporal resolution of seizure development following TBI. By investigating PTE, we are targeting the most prevalent mechanism of epileptogenesis in the military population. We have also chosen timepoints for therapeutic interventions that are relevant not only to the civilian and Veteran populations but are also more readily available for field use.

Career Development and Sustainment: The Principal Investigator’s (PI’s) participation in the Virtual P-TERC will enhance his career and significantly improve his chances at a sustained research career in PTE. He has a strong passion for the work proposed and has assembled a research team of experts to assist him in his desire to transition into PTE research. Beyond this research proposal’s completion, the PI’s involvement in a more formalized training program and increased chances to interact with experts beyond his research team will hopefully allow him to expand his research ideas and share his expertise to help others within the P-TERC as well.