Date of Award


Degree Name

Doctor of Philosophy



First Advisor

Tischkau, Shelley


As the major excitatory neurotransmitter, glutamate (Glu) is physiologically important in brain function. Excessive Glu release, however, is a critical underlying pathological mechanism in neurodegenerative disease, especially stroke. Strategies to protect neurons from cell death under these conditions are scarce; in part because of incomplete understanding of inherent neuroprotective mechanisms. The suprachiasmatic nucleus (SCN) is a region of the brain that exhibits endogenous resistance to Glu excitotoxicity. A previous study demonstrated that SCN2.2 cells (an immortalized SCN cell line) were resistant to Glu excitotoxicity as compared to GT1-7 neurons (from the neighboring hypothalamus). This thesis explored the cellular mechanisms underlying this endogenous neuroprotection in SCN2.2 cells. Extracellular regulated kinase (ERK) is expressed in the SCN, activated by Glu, and is anti-apoptotic in other systems. Therefore, this thesis was designed to test the following central hypothesis: SCN2.2 cells are dependent on ERK signaling for survival in the presence of an excitotoxic insult. Glu increased ERK activity in SCN2.2 cells and importantly, resistance to Glu excitotoxicity in SCN2.2 cells was compromised by pre-treatment with an ERK inhibitor (PD98059; PD). ERK inhibition + Glu mediated SCN2.2 cell death in an N-methyl-D-aspartate receptor (NMDAR)-dependent manner; specifically via the NMDAR 2B (NR2B) subunit. Glu treatment increased expression of NR2B, phosphorylated NR2B and NR1 proteins and decreased NR2A and NR2D mRNA in the GT1-7 cells. Glu-treated SCN2.2 cells showed decreased NR2B, phosphorylated NR2B, increased NR2C proteins and increased NR2A and NR2D mRNA levels. These data are consistent with varied NMDAR responses to Glu in GT1-7 vs. SCN2.2 cells, which might underlie the different physiological responses to Glu in the two cell types. Further experiments investigated the role of several signaling kinases, e.g. protein kinase A (PKA), protein kinase C (PKC), calcium/calmodulin-dependent kinase II (CaMK-II) and c-Jun N-terminal kinase-II (JNK-II) in regulation of ERK activation and on SCN2.2 cell fate. PKA and PKC inhibition together, CaMK-II inhibition and JNK-II inhibition resulted in SCN2.2 cell death in the presence of Glu. PKA + PKC inhibition and CaMK-II inhibition resulted in a corresponding decrease in Glu-induced ERK phosphorylation. Combined inhibition of ERK, CaMK-II and JNK-II resulted in exacerbation of cell death as compared to when the inhibitors were used individually. These results suggest that ERK activity is regulated by a number of different kinases. Glu treatment resulted in a persistent increase in ERK phosphorylation (activation) for up to 48 h in the SCN2.2 cells whereas the pro-apoptotic p38 was phosphorylated (activated) in the GT1-7 cells exposed to Glu. JNK-II was transiently phosphorylated (activated) in the SCN2.2 cells. This suggests an activation of a short-term stress response which can result in activation of a long-term neuroprotective response in these cells. Pro-apoptotic Bid mRNA and cleaved Bid protein levels were increased in the Glu-treated GT1-7 cells. The effect of Glu treatment on the expression of several downstream effector molecules of ERK activation was also explored. Neuritin mRNA was increased with Glu treatment in the SCN2.2, but not in the GT1-7 cells. However, there was no change in the neuritin protein levels in either cell type with Glu treatment. Bcl2 levels remained unchanged in the Glu-treated GT1-7 cells. Although there was no change in the Bcl2 mRNA levels in the SCN2.2 cells, Bcl2 protein was significantly increased with Glu treatment, thus suggesting a post-translational mechanism of neuroprotection involving Bcl2. Taken together, these results are consistent with activation of an apoptotic mechanism in the GT1-7 cells exposed to Glu as opposed to a pro-survival effect in similarly treated SCN2.2 cells. Future studies should be able to take advantage of these mechanisms in developing therapeutic strategies in the treatment of neurodegenerative disorders.




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