Mechanisms of translational control by the ubiquitin E3 ligase neural precursor cell expressed developmentally down-regulated gene 4-like, Nedd4-2

The neural precursor cell expressed developmentally down-regulated protein 4-like (Nedd4-2) is an E3 ubiquitin ligase critical for neurodevelopment and homeostasis of neural circuit excitability. Dysregulation of Nedd4-2 has been linked to several neurological disorders, including stroke and epilepsies. While dysregulation of Nedd4-2 has been linked to elevated seizure susceptibility through impaired ubiquitination of multiple direct substrates, it remains largely unclear whether Nedd4-2 interconnects other cellular pathways that affect neuronal activity and seizure susceptibility. One such pathway is the endoplasmic reticulum (ER) stress response. ER stress occurs when protein folding or maturation is disrupted. Malfunctions in the ER stress response can lead to cell death and has been observed in many neurological diseases. However, how the ER stress response is regulated in neuronal cells remains largely unclear. In Chapter Two, we describe our study in which we utilized unbiased proteomic profiling with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) of isolated membrane fractions from mouse whole brains to identify novel targets of Nedd4-2. Through this screen, we found that the expression and ubiquitination of ribosomal proteins are regulated by Nedd4-2 and we confirmed an association between Nedd4-2 and ribosomes through ribosome sedimentation and polysome profiling. Further, we utilized immunoprecipitation and western blotting to show that induction of ER stress promotes an association between Nedd4-2 and ribosomal proteins, which is mediated through dephosphorylation of Nedd4-2 at serine-342.This increased interaction between Nedd4-2 and ribosomal proteins in turn mediates ER stress associated translational suppression. In summary, the results of this study demonstrate a novel regulatory mechanism underlying the ER stress response and a novel function of Nedd4-2 in translational control. In Chapter Three and Chapter Four, we further investigate the role of Nedd4-2 within the endoplasmic reticulum. Here, we first showed that Nedd4-2 associates with the endoplasmic reticulum (ER) and regulates the expression of multiple ER-resident proteins. Furthermore, utilizing Nedd4-2 conditional knockout mice, we showed that Nedd4-2 is required for the maintenance of spontaneous neural activity and excitatory synapses following the induction of ER stress. When analyzing the canonical pathways of the ER stress response, we found that Nedd4-2 is required for the phosphorylation of eIF2α. While phosphorylation of eIF2α has previously been shown to reduce seizure susceptibility, attempts to facilitate phosphorylation of eIF2α in Nedd4-2 conditional knockout mice failed to produce such a beneficial function, suggestive a role for Nedd4-2 in integrating the ER stress response to modulate seizure susceptibility. Altogether, our study demonstrates neuroprotective functions of Nedd4-2 during ER stress in neurons.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Molecular & Integrative Physi

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2022

Author dc:creator

• Lodes, Daphne Elizabeth

Contributors dc:contributor

• Tsai, Nien-Pei
• Ceman, Stephanie
• Christian-Hinman, Catherine
• Nelson, Erik

• ER stress
• ubiquitination
• ribosome
• seizure

Statement dc:rights

• Copyright 2022 Daphne Lodes

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/115513

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F115513