The regulation of neuronal excitability by epilepsy-associated gene Nedd4-2

Zhu, Jiuhe

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https://hdl.handle.net/2142/106181 Copy

Description

Title

The regulation of neuronal excitability by epilepsy-associated gene Nedd4-2

Author(s)

Zhu, Jiuhe

Issue Date

2019-11-05

Director of Research (if dissertation) or Advisor (if thesis)

Tsai, Nien-Pei

Doctoral Committee Chair(s)

Tsai, Nien-Pei

Committee Member(s)

• Chung, Hee Jung
• Ceman, Stephanie
• Anakk, Sayeepriyadarshini

Department of Study

Molecular & Integrative Physl

Discipline

Molecular & Integrative Physi

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Epilepsy
• Nedd4-2
• ubiquitination
• excitability

Abstract

Epilepsy is the fourth most common neurological disease in the United States, which is characterized by the recurrent seizures in unpredictable frequency with a range of severities and a variety of causes. About 3.4 million people in the United States have epilepsy. Every year, there is an increase of 150,000 of new cases and about 50,000 people die from epilepsy-related causes. Moreover, one in twenty-six of the people in the United States will develop epilepsy during their lifetime. Last but not least, one-third of the patients are drug-resistant. Even for those patients whose seizures could be controlled with medications or other treatments, living with epilepsy is still a big challenge, as they need to face questions about independent capabilities, limitation on driving and uncertainties in employment situations. The overall quality of life and productivity of people with epilepsy and their families need to be improved. Therefore, studying the mechanisms of epileptogenesis is significant to develop better treatments or cure for epilepsy patients. Many patients with neurological disorders suffer from an imbalance in neuronal and circuit excitability and present with seizure or epilepsy as the common comorbidity. The onset of epilepsy can be a result of an acquired brain injury (such as a trauma) or a genetic mutation in some genes. It is widely accepted that the alterations in activity, composition or distribution of ion channels caused by genetic mutations contribute to the onset of epilepsy. DNA sequencing and analysis performed as part of a worldwide research study called Epilepsy 4000 (Epi4k) has revealed over 300 de novo mutations in patients with epilepsy. Among those genes identified, the neural precursor cell expressed developmentally down-regulated gene 4-2, Nedd4-2, was specifically noted. Furthermore, three missense changes in Nedd4-2 have also been identified in families with epilepsy. Nedd4-2 gene encodes a ubiquitin E3 ligase that has high affinity toward binding and ubiquitinating membrane proteins, including ion channels. However, it is currently unknown how Nedd4-2 mediates neuronal circuit activity and how its dysfunction leads to seizures or epilepsies. During my Ph.D. thesis studies, I aimed to characterize the function of Nedd4-2 in regulating neuronal excitability and how epilepsy-associated missense mutations impair such regulation, in order to elucidate the molecular mechanisms underlying Nedd4-2-mediated epileptogenesis. Several neuronal ion channels have been reported as the novel substrates of Nedd4-2, including the major subunit (GluA1) of an ionotropic glutamate receptor, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, which our lab has identified. AMPA receptors (AMPARs) are the most commonly found receptors in the mammalian nervous system, mediating fast excitatory synaptic transmission. Their distribution and activity are regulated by post-translational modifications, such as ubiquitination and phosphorylation. Studies have shown that GluA1 ubiquitination contributes to its internalization, which, as part of AMPAR trafficking mechanisms, is critical for synaptic depression as well as homeostatic regulation of synaptic strength. Because GluA1 levels affect neuronal activity, and dysregulation of AMPARs has been shown to be linked to epilepsy, we hypothesize that Nedd4-2 plays a role in down-regulating neuronal excitability through fine-tuning of AMPARs, specifically ubiquitinating and degrading GluA1. In Chapter Two, we studied the role of Nedd4-2 in regulation of neuronal activity. Using a genetic mouse model, termed Nedd4-2andi, in which one of the major forms of Nedd4-2 in the brain is selectively deficient, we first demonstrated that Nedd4-2 is involved in the regulation of neuronal network excitability. Measured by a multi-electrode array (MEA) system, the spontaneous neuronal activity in Nedd4-2andi cortical neuron cultures was basally elevated compared to wild-type (WT) cultures. Subsequently, our data indicated that when pharmacologically modulating AMPAR activities, Nedd4-2andi cultures showed less responsive to AMPAR activation, and much more sensitive to AMPAR blockade. Moreover, when performing kainic acid-induced seizures in vivo, we found that elevated seizure susceptibility in Nedd4-2andi mice could be normalized when GluA1 is genetically reduced. In Chapter Three, we focused on characterizing the epilepsy-associated mutations of Nedd4-2. Three Nedd4-2 missense changes in highly conserved residues (S233L, E271A and H515P) were identified in families with epilepsy. Based on our previous findings, we aimed to test the hypothesis that one or more of these mutations could disrupt GluA1 ubiquitination and degradation, resulting in dysregulation of neuronal excitability. To begin with, I generated corresponding Nedd4-2 variants in Nedd4-2 cDNA construct by Site-Directed Mutagenesis. My evidence from HEK cells showed that GluA1 was less ubiquitinated when co-expressed with any of the Nedd4-2 mutants in comparison to WT Nedd4-2. Similar result was observed by in vitro ubiquitination assay as well. Apart from GluA1 ubiquitination, its degradation was also affected by the mutant Nedd4-2s, as GluA1 showed enhanced half-life when co-expressed with any of the Nedd4-2 variants compared to WT Nedd4-2. Furthermore, three epilepsy-associated missense mutations of Nedd4-2 also failed to reduce GluA1 surface expression or spontaneous neuronal activity when compared to WT Nedd4-2. Collectively, our data suggest that impaired GluA1 ubiquitination contributes to Nedd4-2-dependent neuronal hyperactivity and seizures. These findings provide critical information to the future development of therapeutic strategies for patients who carry mutations of Nedd4-2. In Chapter Four, in collaboration with Dr. Kwan Young Lee, a research scientist in Tsai lab, I extended my studies on the function of two major isoforms of Nedd4-2, C2-containing and C2-lacking isoforms, in regulating excitatory synaptic strength. Because single-nucleotide polymorphisms (SNPs) in human Nedd4-2 lead to differential expression of these two isoforms, examining their functional differences may aid our understanding of neuronal excitability regulation and seizure susceptibility in different populations. As the C2 domain is responsible for binding to membranes, we first found that the C2-lacking Nedd4-2 showed a reduced distribution at cell membranes and extremely low affinity in ubiquitinating GluA1. However, meanwhile, the C2-lacking Nedd4-2 exhibits similar activity toward reducing excitatory synaptic strength when compared to the C2-containing Nedd4-2. Using proteomic screening, we identified multiple potential substrates of the C2-lacking Nedd4-2 in the cytoplasm, including PPP3CA (a subunit of calcineurin A), that could mediate excitatory synaptic strength. We then confirmed that PPP3CA is a substrate of C2-lacking Nedd4-2 in HEK cells, as well as by in vitro ubiquitination assay. In addition, the epilepsy-associated mutations in C2-lacking Nedd4-2 showed impaired PPP3CA ubiquitnation. Further studies will be definitely needed to fully characterize the properties and regulation of this Nedd4-2 isoform. In summary, in this dissertation, I studied the molecular mechanisms by which Nedd4-2 gene is associated with epileptogenesis, and provided evidence showing Nedd4-2-associated neuronal hyperexcitability and seizure susceptibility are critically influenced by elevated excitatory synaptic transmission when the functions of Nedd4-2, both C2-containing and C2-lacking isoforms, are compromised. These findings in my dissertation may facilitate the future development of new therapeutic strategies for epilepsy patients who carry mutations of Nedd4-2.

Graduation Semester

2019-12

Type of Resource

text

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Copyright 2019 Jiuhe Zhu

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