Define Genetic Basis and Pathologic Network of Post-Traumatic Epilepsy Using Collaborative Cross Mice

Principal Investigator: GU, BIN

Proposal Number: EP210031

Award Number: W81XWH-22-1-0212

Period of Performance: 9/30/2022 - 9/29/2025

PUBLIC ABSTRACT

A head injury commonly results in a devastating condition known as post-traumatic epilepsy (PTE), in which individuals develop spontaneous recurrent seizures weeks, months, and years after the head trauma. PTE is hard to treat, often associated with other post-injury complications, and increases the risk of death. Thereby, PTE affects all aspects of an individual's life and places a considerable economic and social burden on society, particularly for military Service Members, Veterans, and their families. However, there is currently no way to predict or prevent PTE after brain injury. Many factors are at play in individuals who develop PTE after a head injury versus those who do not. For example, scientific evidence suggest genetic difference between people largely determines our susceptibility to PTE after brain injury. To study human diseases, scientists commonly use model organisms, like fly, mouse, and rat. However, most existing animal models of PTE and traumatic brain injury (TBI) are limited because they do not replicate the development of post-traumatic seizures or account for the high level of genetic diversity found in the human population. In order to identify novel genetic and biologic basis of PTE, a model research population with a high level of genetic variation is needed. The Collaborative Cross (CC) mice are an innovative genetic reference population of house mouse. They are uniquely designed to mirror the large genetic difference found in humans. The number of genetic variations among CC (~42 million) is three times greater than the human population. Therefore, CC mice show a spectrum of pathogenic phenotypes that are absent in classical inbred mouse strains. This population of genetically stable yet diverse mice provide us with a strong tool to tackle some key challenges in PTE. We hypothesize that the genotypic and phenotypic diversity of CC allows identification of novel models, pathologic markers, and genetic basis of PTE. This knowledge will further our understanding of the causes of PTE and lead to better control of PTE. To achieve these goals, the following aims are proposed.

Aim 1: Define extreme PTE responses and its related pathologic network in CC. We plan to first explore extreme seizure responses after a brain injury in CC to identify novel mouse models of PTE. We will monitor the spontaneous immediate, early and late post-traumatic seizures 24/7 using time-locked chronic video-electroencephalography-(EEG)-electromyography (EMG) recording. We will also assess seizure susceptibility using a traditional chemical convulsant-induced seizure. In addition to seizures, other major post-TBI complications including behavior deficits, EEG abnormality, sleep disturbance, neuroinflammation, diffuse axonal injury, tau pathology, and hippocampal sclerosis will be defined to cross-examine their interplay with PTE in the same cohort of CC. Additional information and biological samples from the same population of CC will also be collected, deposited, and available for public access for future studies. Deliverables of Aim 1 are identification of novel inbred mouse models with extreme PTE responses and creation of pathologic network and biospecimens repository of PTE within CC.

Aim 2: Identify the genetic loci, candidate genes, and genetic variants that control PTE risk/resilience. The CC captures greater genetic diversity (> 90%) than other reference populations (~30%) and provides a reproducible source of uniform genome-wide genetic variation that can facilitate the identification of novel disease susceptibility alleles. The identification of PTE susceptible and resistant CC will help us design a genetic mapping study to identify the genetic basis of PTE. We will generate, phenotype, and genotype the backcross or F2 population by mating the PTE susceptible and resistant CC. We will perform quantitative trait locus (QTL) mapping to identify the regions on the mouse genome that control PTE risk/resilience. We will profile the differential gene expression between the parental CC using RNA sequencing to further narrow down the candidate genes within QTL. We will prioritize the human-relevant genes using bioinformatics and ontology tools. Deliverables of Aim 2 are identification of novel genes and genetic variants of PTE.

To the best of our knowledge, we are the first research group to apply the innovative genetic reference panel of CC in studying TBI and PTE. We also considered the sex effects on PTE in a population level, which has not been adequately addressed in prior research. Completion of proposed studies will identify CC as a novel animal tool to better inform and improve upon how PTE research can be performed and reveal potential biomarkers and comorbidities that are linked to PTE. These findings will finally benefit the military, Veteran, and civilian communities by improving diagnosis and risk stratification, as well as guiding the development of new preventative and therapeutic modalities of PTE. Completion of this project will also prove CC is a powerful mammalian model that can be leveraged to study other complex diseases (e.g., post-traumatic stress disorder, amyotrophic lateral sclerosis, Alzheimer’s disease, etc.) that are common in military communities.

TECHNICAL ABSTRACT

Background: Post-traumatic epilepsy (PTE) is one of the most common and devastating complications of traumatic brain injury (TBI), which affects all aspects of an individual’s life and places a considerable economic and social burden on military communities. However, there is currently no way to predict and prevent PTE after TBI. Animal studies remain essential for understanding the mechanisms of PTE and for identifying new therapeutic targets. Most existing animal models of PTE and TBI are limited because they neither recapitulate the trajectory of PTE nor reflect the high level of genetic diversity found in the human population. In order to identify novel models and biomarkers of PTE, a model research population with a high level of genetic variation is needed. The Collaborative Cross (CC) mice are an innovative panel of recombinant inbred mouse lines and the next generation of genetic reference population. CC is designed for the dissection of complex traits and gene networks. CC has unprecedented high genetic diversity and exhibits a spectrum of pathogenic phenotypes that are absent in classical inbred strains. We propose a cross-disciplinary approach to establish the first systems genetics resource of PTE using CC.

Hypothesis: We hypothesize that the genotypic and phenotypic diversity of CC allows identification of novel models, pathologic markers, and genetic basis of PTE. This knowledge will further our understanding of the causes of PTE and lead to better control of PTE. To achieve these goals, the following aims are proposed.

Specific Aims and Research Strategy:

Aim 1: Define extreme PTE responses and its related pathologic network in CC. The CC offers great phenotypic stability (inbred) and diversity (derived from eight founder strains). Our previous work revealed extreme seizure and epileptogenic sensitivities across models in CC. We will challenge C57BL/6J mice and 12 strains of CC (n = 6/sex/strain; 80% power, alpha = 0.05) using the lateral fluid percussion injury model. We will then monitor the immediate (< 24 hours), early (< 7 days) and late (> 30 days) spontaneous post-traumatic seizures using chronic video-electroencephalography-(EEG)-electromyography (EMG) recording and access pentylentetrazol-induced seizure susceptibilities to identify CC strains with extreme PTE responses. The efficacy of this study design have been demonstrated by the success of similar small-scale CC screens that have identified strains with unique phenotypes. In addition to seizures, other major post-TBI complications including behavior deficits, EEG abnormality, sleep disturbances, neuroinflammation, diffuse axonal injury, tau pathology, and hippocampal sclerosis will be defined to cross-examine their interplay with PTE in the same population of CC. The phenome correlation and principal components analyses across CC will provide the foundation for further interrogation of causalities.

Aim 2: Identify the genetic loci, candidate genes, and genetic variants that control PTE risk/resilience. The CC captures great genetic diversity (> 90%) and provides a reproducible source of uniform genome-wide genetic variation that can facilitate the identification of novel disease susceptibility alleles. The identification of PTE susceptible and resistant CC will guide us design a genetic mapping study to reveal the complex genetic networks driving extreme PTE responses. Depending on the quantitative trait locus (QTL) effects, we will generate, phenotype and genotype backcross or F2 population (e.g., n = 300 F2; > 90% power, < 5% additive QTL effects) from an intercross between PTE susceptible and resistant CC. We will then perform QTL mapping to identify the regions on the mouse genome that control PTE risk/resilience. We will profile the differential gene expression between the parental CC using RNA-seq to further narrow down the candidate genes within QTL. Using the msBWT tool and available whole-genome sequence of CC, we will identify genetic variation, in addition to predicting the impact of genetic variation on gene and protein expression. Finally, we will prioritize their relevance to humans by computing the translational value using Found In Translation to predict the genes relevant to the analogous human condition given a mouse gene expression experiment.

Innovation and Impact: Successful completion of this proposal will (1) identify CC as a novel animal tool to better inform or improve upon how PTE research can be performed in a genetically stable yet diverse population of mammalian model organism; (2) reveal potential biomarkers and comorbidities that are linked to PTE; and (3) uncover genetic loci, candidate genes, and genetic variants that control the PTE risks/resilience. Of note, we will also generate the first PTE-related data and biospecimens repository within CC, which will be publicly available to fellow researchers in the field and allow integration of intermediate molecular factors (-omics) for systems genetics studies. We also considered the sex effects on PTE in a population level, which has not been adequately addressed in prior research. Completion of this study will further prove CC is a powerful mammalian model that can be leveraged to study other complex diseases (e.g., post-traumatic stress disorder, amyotrophic lateral sclerosis, Alzheimer’s disease, etc.) that are common in military communities.