Regulation of Human Neurofilaments and WNK Kinases by O-linked-β-N-acetylglucosamine

Abstract

O-linked-β-N-acetylglucosamine (O-GlcNAc) is a dynamic intracellular glycosylation that modifies thousands of nucleocytoplasmic proteins. Regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), O-GlcNAcylation is ubiquitous in all mammalian tissues and important in fundamental cell biological processes such as epigenetics, transcription, temporal regulation of signaling, and protein degradation. Particularly abundant in the brain, this modification is crucial in various aspects of mammalian neurobiology, ranging from cerebellar development, synaptic transmission, to neuron survival and maintenance. For these reasons, targeting O-GlcNAcylation has shown therapeutic potential in various mouse models of neurodegeneration and OGA inhibitors have emerged as promising drug candidates in clinical trials. Despite the broad pathophysiological significance, how OGT and OGA regulate their native substrates at the molecular, cellular, and physiological level remains an outstanding knowledge gap. My dissertation aims to address this question by interrogating two classes of OGT substrates: Intermediate filaments and with-no-lysine (WNK) kinases. In Chapter 2, I explore how the human neuronal cytoskeleton, integral to axonal health, is functionally regulated. Particularly, my project focuses on neurofilaments (NFs), the neuronal intermediate filaments comprising light, medium, and heavy subunits that form the NF polymers. In vivo, the light (NF-L) subunit is required to provide axonal support, mediate organelle motility, and influence high-order brain functions. Mutating NF-L causes aggregations in two subtypes of Charcot-Marie-Tooth (CMT) disease, a hereditary neuromuscular disease, or chronic axonal dysregulation, such as protein aggregation, disrupts normal NF-L filament organization. More importantly, NF-L concentrations spike abnormally in the biofluids of patients with myriad neurological disorders, conferring it a promising biomarker in pre-clinical studies and clinical trials. Despite a broad pathophysiological importance, the biochemical regulation of NF assembly and pathological aggregation remains ambiguous to date. Early proteomic data in rodent brains indicated that rodent NFs are O-GlcNAcylated. However, whether O-GlcNAcylation occurs in the human homologs has yet been systematically tested. In my work, I used biochemical assays to demonstrate dynamic human NF-L O-GlcNAcylation in cultured cells and post-mortem human brain tissues. With CRISPR knockin with proteomics, I identified two novel glycosites of human NF-L that expand those mapped in rodent homologs. Next, combining biochemistry, chemical biology, and proteomics, I showed that site-specific NF-L O-GlcNAcylation mediates NF-L interactions with α-internexin (an in vivo binding partner), tunes NF-L assembly state, and controls its overall filamentous organization. Further collaborating with Dr. Chantell Evans, I discerned that NF-L O-GlcNAcylation is required for neuronal mitochondrial and lysosomal motility. Establishing the functional links between NF-L O-GlcNAcylation and its biology, I last examined whether dynamic human NF-L O-GlcNAcylation dysregulation has any pathological relevance. Among CMT disease-causing mutants, NF-L O-GlcNAc signals are widely perturbed. Notably, mutations that lie proximal to NF-L glycosites abolish NF-L O-GlcNAc signals and resist the functional effects of O-GlcNAcylation. Altogether, these findings propose a regulatory role of O-GlcNAcylation in the neuronal cytoskeleton, motivate future opportunities to determine NF-L glycoforms in diseases involving NF dysregulation, and explore their potential diagnostic benefit. In Chapter 3, I investigate the functional control of WNK kinases, a group of kinases with an atypical placement of the catalytic lysine. WNK kinases phosphorylate SPAK and OSR1, the upstream regulators of sodium-chloride cotransporters, the sodium-potassium-2-chloride cotransporters, and the potassium-coupled chloride cotransporters. Mutations in WNK kinases are found in human patients with familial hyperkalemic hypertension characterized by hyperkalemia and hyperchloremic metabolic acidosis. Modulation of these transporters by WNKs is essential for cell volume control, the homeostasis of intracellular chloride, and osmoregulation. However, how WNK kinases are regulated at the molecular level is currently unclear. We serendipitously discovered that WNK kinases are O-GlcNAcylated. By proteomics and biochemistry, we determined six glycosites of WNK4, a paralog abundant in the kidney, and then interrogated the effects of ion concentrations and osmotic stress on WNK4 O-GlcNAcylation. The results showed no changes in WNK4 O-GlcNAcylation in response to fluctuating ion concentrations, but an upregulation of WNK4 site-specific O-GlcNAcylation in correspondence to higher osmolarity. Intriguingly, my results also suggest a potential crosstalk between WNK4 O-GlcNAcylation and WNK4 phosphorylation in the hyperosmolarity condition. In conclusion, my dissertation has uncovered some of the biological roles of O-GlcNAc modification. In interrogating two O-GlcNAc classes, I have provided mechanistic insights on how O-GlcNAc influences mammalian neurobiology, from regulating the human cytoskeletal network and organelle motility via NF-L O-GlcNAcylation to potentially modulating osmosensing via WNK kinase O-GlcNAcylation. Future works would continue to broaden these lines of research and apply biochemical characterization to cellular and tissue physiology, by characterizing the roles of this modification in physiologically relevant organisms.