Molecular regulation of mitochondrial dynamics by dynamin-related protein 1 (Drp1) and Bid in model systems of neuronal cell death

Mitochondria are key regulators of neuronal apoptosis, in development and aging. Beside this, mitochondria are highly dynamic organelles which build, dependent on physiological conditions, large interconnected networks or appear as spherical, small rounded organelles. Impaired regulation of mitochondrial dynamics that shifts the balance towards fission is associated with neuronal death in delayed neuronal cell death after acute brain injury by ischemic stroke or brain trauma, and in age-related neurodegenerative diseases, such as Alzheimer’s disease or Parkinson’s disease. Emerging evidence suggests that oxidative stress disturbs mitochondrial morphology dynamics, resulting in detrimental mitochondrial fragmentation and dysfunction. In particular, such fatal mitochondrial fission has been detected in neurons exposed to oxidative stress, suggesting mitochondrial dysfunction is a key feature in the intrinsic death pathway. Major parts of the study were performed in a model of glutamate toxicity in immortalized hippocampal HT-22 neurons, since glutamate selectively induced oxidative stress through glutathione depletion in these cells. To verify the relevance of the findings in HT-22 cells for post-mitotic neurons, further experiments included models of glutamate-induced excitotoxicity and oxygen glucose deprivation primary embryonic neurons in vitro and in a mouse model of cerebral ischemia in vivo. The first part of this study investigated, whether enhanced mitochondrial fission accompanied by glutamate-induced neuronal cell death. The present study demonstrated that glutamate-induced apoptosis was associated with enhanced mitochondrial fission, loss of mitochondrial membrane potential and relocation of fragmented mitochondria to the nucleus. Further, the results obtained here provided evidence for a key role of the BH3-only protein Bid in mitochondrial fragmentation of neuronal apoptosis caused by oxidative stress. The mitochondrial fragmentation, the associated loss of the mitochondrial membrane potential, and consequent neuronal cell death were prevented by BI-6c9, a highly specific Bid inhibitor. The second part of this study explored whether Drp1 played a major role in neuronal cell death after glutamate-toxicity in HT-22 cells and primary cortical neurons. To verify the role of Drp1 in glutamate-induced cell death, highly specific small molecule inhibitors of Drp1 and siRNA approaches in neurons were applied in this model-system of oxidative stress in neurons. Data obtained from the present study demonstrated a significant neuroprotective effect through inhibition of Drp1, suggesting that this regulator of mitochondrial fission played a major role in delayed neuronal cell death. This conclusion is based on findings showing that down-regulation of Drp1 by siRNA or by small molecule inhibitors significantly preserved mitochondrial morphology and mitochondrial membrane potential, and reduced glutamate toxicity in neuronal cells. In addition, the Drp1 inhibitors protected primary cortical neurons against oxygen glucose deprivation in vitro, and preserved brain tissue after cerebral ischemia in vivo. These data expose Drp1 as a key factor in ischemic and glutamate-induced neuronal cell death and identify Drp1-dependent mitochondrial fission as a potential therapeutic target in acute cerebrovascular diseases. Finally, studies on the potential interaction between Bid and Drp1 revealed for the first time that both factors cooperate during neuronal apoptosis to mediate mitochondrial fragmentation, loss of mitochondrial membrane integrity and intrinsic apoptosis. Inhibition of either factor was sufficient to block the other’s detrimental effect on mitochondria and execution of neuronal cell death. Overall, data obtained from the present study demonstrate for the first time Bid and Drp1 as the key regulators of mitochondrial neuronal cell death pathways associated with enhanced oxidative stress in HT22 cells and primary neuronal cells as well as in the mouse model of cerebral ischemia, respectively. Bid-mediated neuronal cell death involves Drp1-dependent mitochondrial fission, mitochondrial relocation to the nucleus, mitochondrial membrane permeabilization, and release of mitochondrial cell death regulators such as AIF. Therefore, the presented data expose the inhibition of Bid by BI-6c9 and Drp1 using mdivi-1 compounds as a promising therapeutic target to prevent mitochondrial fragmentation and dysfunction which are hallmarks of neuronal cell death in acute and chronic neurodegenerative diseases, where glutamate toxicity and oxidative stress are prominent.