Programmed Cell Death Dynamics in Hyperglycemia
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Issue Date
2018
Authors
Patel, Payal '18
Degree
MS in Molecular Biosciences
Advisor
Larocca, Timothy J
Committee Members
Malik, Meenakshi
Sharifi, H. John
Shi, Binshan
Sharifi, H. John
Shi, Binshan
Journal Title
Journal ISSN
Volume Title
Abstract
Necroptosis is a type of inflammatory programmed cell death (PCD) that is dependent on kinases RIP1, RIP3 and MLKL. Necroptosis differs from apoptosis, which is dependent on caspases and is a non-inflammatory PCD. Activation of necroptosis stimulates glycolysis, producing toxic by-products such as reactive oxygen species (ROS) and advanced glycation end products (AGEs) that induce cell damage. Previous work has shown that necroptosis is upregulated in hyperglycemic conditions. These findings suggest that hyperglycemia may induce a shift from apoptosis to necroptosis following extrinsic death receptor:ligand interaction. Here, we show that hyperglycemia does not enhance extrinsic apoptosis but, rather, potentiates a shift to RIP1-dependent necroptosis using specific pharmacologic inhibitors and a RIP1 mutant cell line in cell death assays. This was the result of increased levels and activity of RIP1, RIP3, and MLKL as well as decreased levels and activity of executioner caspases in hyperglycemic conditions following stimulation with apoptotic ligands. The shift to necroptosis was driven by RIP1 as mutation of this gene using CRISPR-Cas9 caused cell death to revert to caspase-dependent apoptosis in hyperglycemic conditions. Effects of hyperglycemia in neonatal hypoxia-ischemia (HI) brain injury, normally driven by apoptosis, was explored by measuring expression of critical proteins. In cerebral tissue from hyperglycemic neonatal mice that had undergone HI brain injury, levels of RIP1 (total and phosphorylated), RIP3, and MLKL increased, while levels of executioner caspases decreased, suggesting that this cell death shift occurs in vivo. The shift from apoptosis to necroptosis also depended on glycolysis, which was shown by using the glycolysis inhibitor, 2-deoxyglucose. Furthermore, the increased production of mitochondrial ROS in high glucose conditions was shown to have relevance with respect to the shift to necroptosis, by affecting expression of executioner caspases as well as inducing the hyperactivation of RIP1 via oligomerization. AGEs, on the other hand, were found to not have a significant role in the shift. This work is significant as it demonstrates a shift from non-inflammatory to inflammatory cell death and determining the mechanisms of the shift to necroptosis is crucial to develop therapeutic strategies that could restrain the direct effects of hyperglycemia in ischemic injuries.
Citation
Patel P. Programmed cell death dynamics in hyperglycemia [thesis]. Ann Arbor (MI): Proquest LLC; 2018. 88 p.
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