Abstract 3903: Dynamin-related Protein 1 Independent Apoptosis In Neurons Following Oxygen-glucose Deprivation
Background and purpose: Previous studies showed that mitochondrial (mito) fission occurs prior to apoptosis and that a key initiator is the mito fission protein dynamin-related protein 1 (Drp1). However, little information is available concerning mito dynamics in neurons due to OGD. Therefore, we investigated mito fission and mito fusion responses in cultured neurons in an in vitro model of brain ischemia.
Methods: Primary rat cortical neurons were isolated from E18 Sprague Dawley fetuses. Neurons were exposed to OGD 9 days after culturing for 3 hr followed by up to 24 hr of reoxygenation. Mito dynamics were investigated by measuring the expression of: 1) mito fission [Drp1; mito fission 1 (Fis1)] and mito fusion [mitofusin-2 (Mfn2); optic atrophy-1 (OPA1)] proteins; 2) electron transport chain proteins (complex II 70 kDa subunit, complex IV subunit I, complex V alpha subunit); 3) mito DNA; and 4) apoptosis protein Bax. We also examined neuronal and mito morphology using transmission electron microscopy. The effects of the Drp1 inhibitors 15d-Prostaglandin J2 (2.5-20 µM) (15d-PGJ2) and Mdivi-1 (2.5-250 µM) were also investigated.
Results: 3hr OGD followed by 3-24 hr recovery resulted in an increase in the level of mito DNA and enhanced expression of complex IV and V proteins. During this time, abnormal morphology, including enlarged and/or swollen mitochondria, was often observed. However, many mitochondria still retained normal morphology. Drp1 levels decreased before virtually disappearing by 6 hr of reoxygenation following OGD while Fis1 expression did not change. Bax expression increased with an increasing number of apoptotic cells during this time. Mfn2 decreased after OGD while OPA1 did not change significantly. 15d-PGJ2 treatment dose-dependently increased cell death both in control and OGD-exposed neurons. Furthermore, 15d-PGJ2 treatment surprisingly increased Drp1 both in controls and in OGD-exposed cells, and also increased the mito DNA. Additionally, 15d-PGJ2 treatment itself increased the number of larger mitochondria in the neurons. However, Mdivi-1 did not have any effects.
Conclusions: Despite a lower fission/fusion protein ratio, the increase in mitochondrial size indicates a shift to enhanced fusion in neurons following OGD. In addition, apoptosis induction seems to be Drp1 independent in this model. Additionally, 15d-PGJ2 treatment appears to decrease survival in neurons following OGD. Thus, mito dynamics are affected by OGD and 15d-PGJ2 treatment and play a role in cell survival/death.
- © 2012 by American Heart Association, Inc.