Alterations in Brain-Derived Extracellular Vesicles in Treatment-Resistant Epilepsy Restricted; Files Only

Abstract

Epilepsy is a common neurological disorder characterized by recurrent spontaneous seizures. About 30% of patients with epilepsy are unable to achieve sufficient seizure control with currently available medication, highlighting the need to better understand disease mechanisms and develop more efficacious treatments. Extracellular vesicles (EVs) are small membrane-derived particles that are released by cells into the extracellular space. They transport a variety of molecules, including miRNA, proteins and lipids, and play critical roles in intercellular communication. Emerging evidence suggests that the miRNA content of brain-derived EVs (BDEVs) is altered in mouse models of mesial temporal lobe epilepsy (MTLE). However, the functional effects of this alteration are unknown, and it is not clear whether BDEVs play a protective role or contribute to pathology. Furthermore, at the onset of this project, a role for BDEVs in genetic forms of epilepsy had not been examined. Therefore, the main goals of my dissertation research were: 1) determine if the function of BDEVs is altered in a model of MTLE and 2) determine if the content of BDEVs is altered in a genetic model of epilepsy. I used the mouse pilocarpine model of MTLE to examine functional changes in BDEVs following status epilepticus (SE). I treated N2a and BV2 cells with BDEVs isolated from control mice (CON-BDEVs) or 24 hours after SE (SE-BDEVs). I observed that SE-BDEV-treated N2a cells showed distinct alterations in the expression of genes related to TGF-β signaling, amino acid regulation of mTORC1, and neurotransmitter signaling. In BV2 cells, SE-BDEV treatment resulted in increased expression of genes that promote the release of inflammatory cytokines. Together, these results suggest BDEVs are dysregulated following SE, and might lead to pathological changes in gene expression in recipient cells. Next, I performed miRNA-sequencing on BDEVs from Scn1a^+/- mutants, a model of Dravet Syndrome. My data revealed that BDEV miRNA expression is altered in naïve mutants, and further changes were observed in mutants with a history of spontaneous seizures. Overall, my thesis research established that BDEV miRNA expression and function are altered in multiple models of epilepsy, raising the possibility that BDEV dysfunction may contribute to epileptogenesis.

Table of Contents

Table of Contents

Chapter 1: Introduction 1

1.1 Overview 2

1.2 Epilepsy 2

1.2.1 Mesial Temporal Lobe Epilepsy 3

1.2.2 Dravet Syndrome 5

1.3 Extracellular Vesicles 6

1.3.1 Regulation of EV Content 9

1.3.2 Functions of EVs in The Nervous System 10

1.3.3 Isolation of Extracellular Vesicles 11

1.4 Therapeutic Applications of Extracellular Vesicles 12

1.4.1 EVs as a Treatment in Mesial Temporal Lobe Epilepsy 13

1.5 EVs can Also Contribute to Neurological Disease 14

1.5.1 Current Research on EVs in Epilepsy 15

1.6 Summary and Goals of this Dissertation 20

Chapter 2: Status Epilepticus Alters the Function of Brain-Derived Extracellular

Vesicles 22

2.1 Summary 23

2.2 Introduction 24

2.3 Materials and Methods 26

2.4 Results 33

2.5 Discussion 48

Chapter 3: Brain-Derived Extracellular Vesicles in Scn1a Epilepsy 53

3.1 Summary 54

3.2 Introduction 56

3.3 Materials and Methods 57

3.4 Results 58

3.5 Discussion 73

Chapter 4: Conclusions and Future Directions 76

4.1 Summary 77

4.2 Importance of cell type in understanding the role of BDEVs in epilepsy 78

4.3 The link between BDEVs and inflammation in epilepsy 81

4.4 EVs as a biomarker in epilepsy 83

4.5 BDEVs as a treatment target in epilepsy 86

4.5 Overall Conclusions 89

References 90

Appendix: Nanoparticle Encapsulated Neuropeptide Y Provides Robust Seizure Protection in Scn1a-derived Epilepsy 106

A.1 Summary 107

A.2 Introduction 108

A.3 Materials and Methods 111

A.4 Results 116

A.5 Discussion 126

A.6 Acknowledgements 129

A.7 References 130

About this Dissertation

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• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Molecular Biology, Neuroscience
Parola chiave	Epilepsy miRNA Extracellular Vesicles
Committee Chair / Thesis Advisor	Escayg, Andrew, Emory University
Committee Members	Weinshenker, David, Emory University Sloan, Steven, Emory University Pedersen, Nigel, University of California, Davis

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