The Essential Role of O-GlcNAcylation in Primary Sensory Neurons The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:40046407 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA The essential role of O-GlcNAcylation in primary sensory neurons A dissertation presented by Cathy Su to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biological and Biomedical Sciences Harvard University Cambridge, Massachusetts February 2017 © 2017 Cathy Su All rights reserved. Dissertation Advisor: Dr. Thomas L. Schwarz Cathy Su The essential role of O-GlcNAcylation in primary sensory neurons Abstract O-GlcNAcylation is the post-translational addition of β-N-acetylglucosamine to nuclear and cytoplasmic proteins. This addition is mediated by a single enzyme, O-GlcNAc transferase (OGT), which regulates a wide range of cellular processes through its thousands of protein substrates. The activity of OGT is affected by nutrient availability, and thus its role has been broadly studied in metabolic tissues. OGT is enriched in the nervous system, yet little is known about its importance in basic neuronal processes in vivo. In this work, I utilized in vivo and neuronal culture systems to determine the effects of altered O-GlcNAc dynamics in primary sensory neurons. Sensory neurons lie outside of the blood brain barrier and therefore may have a particular need for mechanisms of metabolic sensing. I show that sensory neuron-specific knockout of OGT in mice results in behavioral hyposensitivity to thermal and mechanical stimuli accompanied by decreased epidermal innervation and cell body loss in the dorsal root ganglia. These effects are observed early in postnatal development and progress as the animals age. The deficits in neuronal health are not solely due to disruption of developmental processes, because inducing OGT knockout in the sensory neurons of adult mice results in a similar decrease in nerve fiber endings and cell bodies. Significant nerve ending loss occurs prior to a decrease in cell bodies, indicative of axonal dieback that progresses to neuronal death. Cultured sensory neurons lacking OGT also exhibit decreased axonal outgrowth. These findings demonstrate that OGT is important in regulating axonal maintenance in the periphery and the overall health and survival of sensory neurons. iii While previous studies have focused on the essential role of OGT in mediating survival of mitotic cells, this work is the first to find that loss of OGT results in cell death in neurons. Moreover, it suggests that aberrant O-GlcNAc signaling can contribute to the development of neurodegeneration and neuropathy. Primary sensory neurons in particular are subject to degeneration in diabetes. My findings provide a foundation for understanding the role of neuronal OGT under normal physiological conditions, which will be important for understanding disease states such as diabetic neuropathy. iv Table of Contents Title page ......................................................................................................................................... i Abstract .......................................................................................................................................... iii Table of contents ..............................................................................................................................v List of figures and tables .............................................................................................................. viii Acknowledgements ..........................................................................................................................x Chapter 1: Introduction 1.1 The O-GlcNAc modification .........................................................................................2 Enzymes mediating O-GlcNAc cycling.........................................................................2 Cellular functions of O-GlcNAcylation .........................................................................6 1.2 O-GlcNAc as a metabolic sensor ...................................................................................8 Roles of OGT in peripheral metabolic processes ........................................................10 1.3 O-GlcNAcylation in the nervous system .....................................................................11 O-GlcNAcylation in neurodegenerative disease ..........................................................14 1.4 Primary sensory neurons ..............................................................................................15 Sensory neuropathy ......................................................................................................19 1.5 Overview of dissertation ..............................................................................................20 Chapter 2: O-GlcNAc transferase is essential for sensory neuron survival and maintenance 2.1 Introduction ..................................................................................................................24 2.2 Results Nav1.8-Ogt knockout mice are overtly healthy but exhibit altered weight and glucose tolerance .......................................................................................................................26 v Nav1.8-Ogt knockout mice show behavioral deficits in thermal and mechanical sensitivity .....................................................................................................................32 Nav1.8-Ogt knockout mice lose epidermal innervation and DRG cell bodies ............36 OGT knockout neurons exhibit axonal outgrowth deficits in culture..........................40 Loss of epidermal innervation and cell bodies occurs independently of developmental processes ......................................................................................................................43 2.3 Discussion ....................................................................................................................49 2.4 Materials and methods Mice .............................................................................................................................54 OGT knockout .............................................................................................................55 Behavioral testing ........................................................................................................56 Glucose tolerance test ..................................................................................................58 Epidermal innervation ..................................................................................................59 DRG cell body count ...................................................................................................59 Axonal outgrowth assay ...............................................................................................60 Chapter 3: Additional observations: Effects of altered O-GlcNAcylation in sensory neurons 3.1 Introduction ..................................................................................................................63 3.2 O-GlcNAc regulation of mitochondrial motility in DRG neurons ..............................64 Mitochondrial motility in DRG neurons under a high glucose condition ...................71 3.3 Additional observations of OGT knockout neurons in culture ....................................73 Axonal outgrowth under varying glucose conditions ..................................................73 Mitochondrial distribution ...........................................................................................76 Survival in culture ........................................................................................................78 vi 3.4 Investigating candidate protein expression in Nav1.8-Ogt knockout DRGs ...............79 3.5 Materials and methods Lentivirus preparation ..................................................................................................83 Axonal transport assays ...............................................................................................84 Quantification of O-GlcNAc levels .............................................................................86 Axonal outgrowth and survival ....................................................................................86 Immunofluorescence ....................................................................................................87 Chapter 4: Extended discussion 4.1 OGT is essential for neuronal survival ........................................................................89 4.2 Behavioral and global consequences of loss of sensory neurons ................................92 4.3 Deciphering OGT-regulated pathways ........................................................................94 4.4 Implications in diabetic neuropathy .............................................................................95 References ......................................................................................................................................99 vii List of Figures