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Elucidating the Role of O-GlcNAc Glycosylation in Neurobiology and Neurodegeneration


Jensen, Elizabeth Hwang (2018) Elucidating the Role of O-GlcNAc Glycosylation in Neurobiology and Neurodegeneration. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9JD4TZ9.


O-GlcNAc glycosylation is a dynamic, inducible post-translational modification (PTM) essential for neuronal homeostasis and found on proteins associated with neurodegenerative diseases such as α-synuclein, amyloid precursor protein, and tau. Intracellularly, O-GlcNAc modification is cycled by two enzymes in mammalian cells: O-GlcNAc transferase (OGT) appends O-GlcNAc to serine or threonine residues and O-GlcNAcase (OGA) removes O-GlcNAc. OGT modifies over 1000 different proteins, but the lack of a well-defined consensus sequence or substrate structural constraints has hampered efforts to predict sites a priori. Furthermore, the identification of O-GlcNAc modification sites has been obstructed by the difficulty of enriching and detecting O-GlcNAc using traditional biochemical methods. Here, we established and employed biological and chemical tools to illuminate the role of O-GlcNAc in neuronal function.

In Chapter 2, we sought to determine the role of O-GlcNAc in learning, memory, and neurodegeneration. Deletion of the OGT gene causes early postnatal lethality in mice, complicating efforts to study O-GlcNAc glycosylation in mature neuronal function and dysfunction. We demonstrated that the loss of OGT in the forebrain of adult mice (OGT cKO) leads to progressive neurodegeneration, including neuronal death, neuroinflammation, hyperphosphorylated tau, amyloidogenic Aβ-peptides, and memory deficits. In the hippocampus, we showed that OGT ablation lead to the upregulation of neuroinflammatory genes and the downregulation of cholesterol biosynthetic genes. Additionally, a gene network analysis (WGCNA), qPCR, and immunohistochemistry (IHC) revealed that loss of O-GlcNAc perturbed cell cycle progression in the hippocampal neurons. In the hippocampus, we identified increased neuroinflammatory gene transcription in OGT cKO mice and both tau neurofibrillary tangle (NFT)-forming and amyloid-forming Alzheimer’s disease (AD) mouse models. However, only OGT cKO and NFT-forming mice displayed decreased synaptic gene expression, suggesting that NFT formation and OGT cKO compromise hippocampal synaptic transcription. These studies indicate that O-GlcNAcylation regulates pathways vital for the maintenance of neuronal health and suggest that dysfunctional O-GlcNAc signaling may be an important contributor to neurodegenerative diseases.

In order to understand the critical O-GlcNAc-mediated neuronal functions that underlie OGT cKO dysfunction, we next developed and utilized novel biological and chemical tools in order to identify key OGT interactors and substrates in the brain in Chapter 3. Due to the lack of a well-defined OGT substrate sequence and structural constraints, OGT is believed to obtain its substrate specificity through its interactome where specific interactors target OGT to specific substrates. In order to identify these interactors, we used CRISPR/Cas9 to generate a novel mouse with a minimally tagged OGT in order to identify the endogenous OGT brain interactome using tandem affinity purification and MS methods. The preliminary OGT brain interactome consisted of previously identified OGT interactors and substrates as well as novel interactors. The identified OGT interactors were enriched for ribosomal and cytoskeletal proteins in addition to axonal, dendritic, and neuronal cell body proteins, implicating OGT as a pivotal mediator of neuronal structure and function.

In addition to the OGT interactome, we sought to uncover OGT’s substrates or the O-GlcNAcome. We developed an improved approach to quantitatively label and enrich O-GlcNAcylated proteins for site identification. Chemoenzymatic labeling followed by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) installed a new MS-compatible linker designed for facile purification and release of O-GlcNAcylated proteins for downstream MS analysis. We validated the approach by identifying several established O-GlcNAc sites on the proteins α-crystallin and OGT as well as discovering new, previously unreported sites on both proteins. Notably, these novel sites on OGT lie in key functional domains of OGT, underscoring how this site identification method can reveal important biological insights into protein activity and regulation.

Finally, in Chapters 4 and 5, we focus on the post-translational modification (PTM) code on a specific transcription factor (TF), CREB (cAMP response element binding protein). CREB regulates memory formation through its transcriptional control of neuronal metabolism, activity, differentiation, development, and survival. CREB phosphorylation at serine 133 has been previously shown to enhance CREB-mediated transcription while CREB glycosylation at serine 40 has been shown to decrease CREB-mediated transcription. However, the exact gene networks modulated by and potential interplay between CREB glycosylation and phosphorylation have not been explored. Through differential expression analysis with glycosylation-deficient (S40A) and phosphorylation-deficient (S133A) CREB mutants, we showed that CREB O-GlcNAcylation is important for neuronal activity and excitability while phosphorylation at serine 133 regulated the expression of genes involved in neuronal differentiation. Using WGCNA, we demonstrated that CREB O-GlcNAcylation at serine 40 and phosphorylation at serine 133 mediate mutually exclusive gene networks. The glycosylation-deficient mutant enhanced neuronal activity- and excitotoxicity-related gene networks while the phosphorylation-deficient mutant perturbed neuronal differentiation and amino and fatty acid metabolism-related gene networks. Our work sheds light on the regulation of CREB through PTMs to modulate neuronal function and delineate the roles of O-GlcNAcylation and phosphorylation in modulating neuronal excitability and neuronal development and metabolism respectively. Altogether, these studies demonstrate that O-GlcNAc modification is a critical mediator of neuronal homeostasis and neurodegeneration.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:O-GlcNAc; Glycosylation; Neurobiology; Neurodegeneration; Neurons; CREB; Neuronal activity; Transcriptomics; RNA-Seq; Proteomics
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Hsieh-Wilson, Linda C.
Thesis Committee:
  • Dervan, Peter B. (chair)
  • Rothenberg, Ellen V.
  • Prober, David A.
  • Hsieh-Wilson, Linda C.
Defense Date:13 November 2017
Non-Caltech Author Email:elizabethhwangjensen (AT)
Funding AgencyGrant Number
Phi Kappa Phi Hohenstein FellowshipUNSPECIFIED
Alpha Lambda Delta FellowshipUNSPECIFIED
Record Number:CaltechTHESIS:12192017-155744407
Persistent URL:
Related URLs:
URLURL TypeDescription CentralPortions of this article are adapted for Chapter 2. CentralPortions of this article are adapted for Chapter 3.
Jensen, Elizabeth Hwang0000-0002-6177-4304
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10615
Deposited By: Elizabeth Jensen
Deposited On:17 Jan 2018 23:51
Last Modified:04 Oct 2019 00:19

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