O-GlcNAc modification (or O-GlcNAcylation) is the addition of the monosaccharide N-acetyl glucosamine to serine and threonine residues of proteins in the nucleus, cytosol, and mitochondria. This modification is regulated dynamically, responds to nutrient flux in the cell, and serves a variety of cellular functions. One key function is as a protective response to a variety of cellular stresses. This protective role has been well demonstrated in the case of neurodegenerative disease. O-GlcNAcylation levels are decreased in neurodegenerative animal models and neuron-specific knockout of O-GlcNAc transferase, the enzyme that installs the modification, leads to the development of pathology characteristic of Alzheimer’s disease. Additionally, elevation of O-GlcNAcylation levels by small molecule treatment is protective in a mouse model of Alzheimer’s. Importantly, a number of proteins implicated in the development of neurodegenerative diseases, including tau, TDP-43, and α-synuclein, are known substrates for O-GlcNAc modification. We set out to investigate the consequence of O-GlcNAcylation of α-synuclein (αSyn), the key pathogenic protein in Parkinson’s disease (PD). αSyn is a cytosolic protein abundant in neurons that normally exists as a soluble, unfolded monomer or in an α-helical conformation in association with membranes. In the disease-associated state, αSyn forms β-sheet-rich amyloid aggregates that are cytotoxic and are the main component of the characteristic protein deposits seen in PD. αSyn has three sites of O-GlcNAc modification that have been found <i>in vivo</i>. In order to study the effect of O-GlcNAc site-specifically, we used an expressed protein ligation strategy to prepare full-length αSyn both with and without O-GlcNAc installed at the physiologically relevant residue threonine 72. Our studies found that O-GlcNAcylation at T72 is sufficient to completely inhibit the formation of amyloid aggregates. Additionally, significant inhibition was observed even at substoichiometric levels of T72 O-GlcNAcylation (10%). Furthermore, treatment of neuronal cells in culture demonstrated that T72 O-GlcNAc modification also prevents the toxicity normally seen from treatment with αSyn aggregates. Our findings suggest increasing O-GlcNAcylation may slow PD progression and could be a future target for new PD treatments. This work also represents, to our knowledge, the first synthesis of a full-length, O-GlcNAcylated protein.

O-GlcNAc modification blocks the aggregation and toxicity of the Parkinson's disease associated protein α-synuclein

249th National Meeting (2015)

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