| Depletion of endoplasmic reticulum Ca2+ (Ca2+ ER) and ATP can disrupt proper peptide folding and trigger the unfolded protein response and ER stress. However, the cellular mechanisms underlying Ca2+ER dysregulation in neurons following ischemia are not defined. Overstimulation of Na+-K+-Cl - cotransporter isoform 1 (NKCC1) increases intracellular Na +, which activates reversal mode operation of Na+/Ca 2+ exchange (NCXrev) and leads to an increase in cytoplasmic Ca2+. Thus, NKCC1/NCXrev are important in disruption of Na+ and Ca2+ homeostasis following ischemia. It is unknown whether these transporter proteins indirectly contribute to changes of Ca2+ER homeostasis and ER dysfunction following ischemia. My thesis centers on how NKCC1/NCXrev is involved in Ca2+ER dysregulation, ER stress, and ischemic neuronal damage. Oxygen-glucose deprivation (OGD)/reoxygenation (REOX) was used as an in vitro ischemic model in this study.;First, I investigated the role of NKCC1/NCXrev in Ca 2+ER dysregulation and the development of ER stress following OGD/REOX. Pharmacological inhibition and genetic ablation of NKCC1 were used to elucidate the function of NKCC1 in this process. NKCC1/NCXrev was involved in the overload of Ca2+ER during OGD and inositol 1,4,5-trisphosphate receptor-mediated release of Ca2+ ER during REOX. The changes of Ca2+ ER were concurrent with elevation of several ER stress markers such as phosphorylated alpha subunit of eukaryotic initiation factor 2, cleaved caspase 12, and glucose regulated protein 78. Reduction of Ca2+ ER release by blocking of NKCC1/NCXrev significantly attenuated ER stress development and cell death.;Secondly, I studied the role of NKCC1 in protein aggregate formation following OGD/REOX. I found that OGD/REOX caused a time-dependent formation of ubiquitin-conjugated protein aggregates in neurons. Aggregate formation resulted from increases in misfolded protein production and inhibition of ATP-dependent proteasome activity. Interestingly, blocking of NKCC1 and NCX rev significantly reduced formation of insoluble protein aggregates in neurons and preserved intracellular ATP, Na+ and Ca 2+ homeostasis. These findings suggest that dysregulation of Na + and Ca2+ ionic homeostasis via activation of NKCC1/NCX rev, contributes to protein folding and degradation following ischemia. In conclusion, stimulation of NKCC1/NCXrev plays a role in Ca 2+ER dysregulation and ER stress development, which leads to ischemic cell death. Therefore, these ion transporters present as potential targets for new therapeutic development for stroke. |
