Introduction: Previous evidence indicates that the modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is closely linked to multiple neuropathologies5,4,3. More importantly, two enzymes responsible for O-GlcNAc cycling are abundant in the brain1. Lastly, recent evidence shows glucosamine and O-GlcNAc’s neuroprotective effect via suppression of inflammation in reperfusion injury2. This implicates O-Glc-NAc and glucose metabolism as potential protector against radiation necrosis in glioma therapy. With this effort in mind, we investigate the neuroprotective effect of glucosamine (GlcN).

Methods: Prior to different doses of x-ray radiation, we pharmacologically increased O-GlcNAcylation via application of varying GlcN to gliomas and primary astrocytes. We used western blotting to detect and quantify radiation necrosis markers. We conducted cell viability assays to confirm O-GlcNAc’s protective role in primary astrocytes. To conduct in vivo experiments, we injected tumor cells into mice and applied GlcN intra-peritoneally prior to radiation. Using immunohistochemistry, we then analyzed brain sections for radiation necrosis and conducted protein quantification.

Results: Application of glucosamine shows decrease in phospho-H2AX (histone 2 AX), radiation necrosis marker in primary astrocytes, but not in glioma cells. Increasing dose of radiation leads to greater decrease in radiation necrosis marker with the presence of glucosamine. There is dose-response relationship. Furthermore, cell viability results confirm that primary astrocytes’ viability after undergoing radiation is increased with application of glucosamine compared to tumor cells. In addition, primary astrocytes show greater O-GlcNacylation of proteins compared to glial tumor cells after undergoing radiation, which sheds light on the underlying mechanism. . These results are confirmed in both in vitro and in vivo models.

Conclusions: O-GlcNAcylation (glycosylation) of proteins decreases cell death and necrosis in the primary astrocytes surrounding the tumor cells and protects against damaging effects of radiation. Our results suggest that GlcN or its derivatives may serve as novel neuroprotective therapy against radiation necrosis for gliomas.

Patient Care: It provides initial evidence that glucosamine decreases radiation necrosis via O-GlcNAcylation of important protein kinases. It also provides background for future human trials.

Learning Objectives: By the conclusion of this session, participants should be able to: 1) Understand O-GlcNAcylation of proteins 2) Describe the importance of O-GlcNAc’s role in neuroprotection 2) Identify radiation necrosis markers

References: 1. Akimoto Y, Comer FI, Cole RN, Kudo A, Kawakami H, Hirano H, et al.: Localization of the O-GlcNAc transferase and O-GlcNAc-modified proteins in rat cerebellar cortex. Brain Res 966:194–205, 2003 2. Hwang S-Y, Shin J-H, Hwang J-S, Kim S-Y, Shin J-A, Oh E-S, et al.: Glucosamine exerts a neuroprotective effect via suppression of inflammation in rat brain ischemia/reperfusion injury. Glia 58:1881–1892, 2010 3. Lefebvre T, Guinez C, Dehennaut V, Beseme-Dekeyser O, Morelle W, Michalski J-C: Does O-GlcNAc play a role in neurodegenerative diseases? Expert Rev Proteomics 2:265–275, 2005 4. Liu F, Iqbal K, Grundke-Iqbal I, Hart GW, Gong C-X: O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer’s disease. Proc Natl Acad Sci U S A 101:10804–10809, 2004 5. Yao PJ, Coleman PD: Reduction of O-linked N-acetylglucosamine-modified assembly protein-3 in Alzheimer’s disease. J Neurosci Off J Soc Neurosci 18:2399–2411, 1998