O-GlcNAc is a dynamic form of intracellular glycosylation found throughout the nucleus, cytosol, and mitochondria. This posttranslational modification is essential for development in mammals and changes in O-GlcNAc levels are associated with cancer, diabetes, and neurodegenerative diseases (NDDs). Proteomics experiments have identified thousands of O-GlcNAc modified proteins, but the consequences of the vast majority of these modification events (if any) are totally unknown. To address this lack of understanding, my lab uses protein ligation to synthesize site-specifically O-GlcNAc modified proteins for subsequent biological experiments. I will present data from my lab focused on the effects of O-GlcNAc on amyloid aggregation and pathogenesis in NDDs. Specifically, I will cover our discovery that O-GlcNAc can alter the structure of the α-synuclein fibrils in ways that have dramatic effects on the pathogenicity of the fibrils in neurons and mice. Additionally, I will show how we uncovered a role for O-GlcNAc in the activation of small heat shock proteins that can inhibit the amyloid aggregation of several proteins in NDDs. Overall, our results have important implications for targeting O-GlcNAc in neurodegenerative diseases and understanding the fundamental processes behind Parkinson’s disease.