Bongki Cho

Physiological and Pathological Significance of Dynamin-Related Protein 1 (Drp1)-Dependent Mitochondrial Fission in the Nervous System

Mitochondria are essential for proper neuronal morphogenesis and functions, as they are the major source of energy for neural development. The dynamic morphology of mitochondria determines the key functions of mitochondria. Several regulatory proteins such as dynamin-related protein 1 (Drp1) are required to maintain mitochondrial morphology via a balance between continuous fusion and fission. Activity of Drp1, a key regulator in mitochondrial fission, is modulated by multiple post-translation modifications and receptor interactions. In addition, numerous researches have revealed that the regulation of Drp1 activity and mitochondrial dynamics is closely associated with several neurodegenerative diseases such as Alzheimers and Parkinsons diseases. In this article, we concisely review the recent findings about the biological importance of Drp1-mediated mitochondrial fission in neurons under physiological and pathological conditions.

CDK5-dependent inhibitory phosphorylation of Drp1 during neuronal maturation

Mitochondrial functions are essential for the survival and function of neurons. Recently, it has been demonstrated that mitochondrial functions are highly associated with mitochondrial morphology, which is dynamically changed by the balance between fusion and fission. Mitochondrial morphology is primarily controlled by the activation of dynamin-related proteins including dynamin-related protein 1 (Drp1), which promotes mitochondrial fission. Drp1 activity is regulated by several post-translational modifications, thereby modifying mitochondrial morphology. Here, we found that phosphorylation of Drp1 at serine 616 (S616) is mediated by cyclin-dependent kinase 5 (CDK5) in post-mitotic rat neurons. Perturbation of CDK5 activity modified the level of Drp1S616 phosphorylation and mitochondrial morphology in neurons. In addition, phosphorylated Drp1S616 preferentially localized as a cytosolic monomer compared with total Drp1. Furthermore, roscovitine, a chemical inhibitor of CDKs, increased oligomerization and mitochondrial translocation of Drp1, suggesting that CDK5-dependent phosphorylation of Drp1 serves to reduce Drp1’s fission-promoting activity. Taken together, we propose that CDK5 has a significant role in the regulation of mitochondrial morphology via inhibitory phosphorylation of Drp1S616 in post-mitotic neurons.

Constriction of the mitochondrial inner compartment is a priming event for mitochondrial division

Mitochondrial division is critical for the maintenance and regulation of mitochondrial function, quality and distribution. This process is controlled by cytosolic actin-based constriction machinery and dynamin-related protein 1 (Drp1) on mitochondrial outer membrane (OMM). Although mitochondrial physiology, including oxidative phosphorylation, is also important for efficient mitochondrial division, morphological alterations of the mitochondrial inner-membrane (IMM) have not been clearly elucidated. Here we report spontaneous and repetitive constriction of mitochondrial inner compartment (CoMIC) associated with subsequent division in neurons. Although CoMIC is potentiated by inhibition of Drp1 and occurs at the potential division spots contacting the endoplasmic reticulum, it appears on IMM independently of OMM. Intra-mitochondrial influx of Ca2+ induces and potentiates CoMIC, and leads to K+-mediated mitochondrial bulging and depolarization. Synergistically, optic atrophy 1 (Opa1) also regulates CoMIC via controlling Mic60-mediated OMM–IMM tethering. Therefore, we propose that CoMIC is a priming event for efficient mitochondrial division.

Dynamin-related protein 1 controls the migration and neuronal differentiation of subventricular zone-derived neural progenitor cells

Mitochondria are important in many essential cellular functions, including energy production, calcium homeostasis, and apoptosis. The organelles are scattered throughout the cytoplasm, but their distribution can be altered in response to local energy demands, such as cell division and neuronal maturation. Mitochondrial distribution is closely associated with mitochondrial fission, and blocking the fission-promoting protein dynamin-related protein 1 (Drp1) activity often results in mitochondrial elongation and clustering. In this study, we observed that mitochondria were preferentially localized at the leading process of migratory adult neural stem cells (aNSCs), whereas neuronal differentiating cells transiently exhibited perinuclear condensation of mitochondria. Inhibiting Drp1 activity altered the typical migratory cell morphology into round shapes while the polarized mitochondrial distribution was maintained. With these changes, aNSCs failed to migrate, and neuronal differentiation was prevented. Because Drp1 blocking also impaired the mitochondrial membrane potential, we tested whether supplementing with L-carnitine, a compound that restores mitochondrial membrane potential and ATP synthesis, could revert the defects induced by Drp1 inhibition. Interestingly, L-carnitine fully restored the aNSC defects, including cell shrinkage, migration, and impaired neuronal differentiation. These results suggest that Drp1 is required for functionally active mitochondria, and supplementing with ATP can restore the defects induced by Drp1 suppression.

Drp1-mediated mitochondrial dynamics and survival of developing chick motoneurons during the period of normal programmed cell death

Mitochondrial morphology is dynamically remodeled by fusion and fission in neurons, and this process is implicated in nervous system development and pathology. However, the mechanism by which mitochondrial dynamics influence neuronal development is less clear. In this study, we found that the length of mitochondria is progressively reduced during normal development of chick embryo motoneurons (MNs), a process partly controlled by a fission‐promoting protein, dynamin‐related protein 1 (Drp1). Suppression of Drp1 activity by gene electroporation of dominant‐negative mutant Drp1 in a subset of developing MNs increased mitochondrial length in vivo, and a greater proportion of Drp1‐suppressed MNs underwent programmed cell death (PCD). By contrast, the survival of nontransfected MNs in proximity to the transfected MNs was significantly increased, suggesting that the suppression of Drp1 confers disadvantage during the competition for limited survival signals. Because we also monitored perturbation of neurite outgrowth and mitochondrial membrane depolarization following Drp1 suppression, we suggest that impairments of ATP production and axonal growth may be downstream factors that influence the competition of MNs for survival. Collectively, these results indicate that mitochondrial dynamics are required for normal axonal development and competition‐dependent MN PCD.—Choi, S. Y., Kim, J. Y., Kim, H.‐W., Cho, B., Cho, H. M., Oppenheim, R. W., Kim, H., Rhyu, I. J., Sun, W. Drp1‐mediated mitochondrial dynamics and survival of developing chick motoneurons during the period of normal programmed cell death. FASEB J. 27, 51–62 (2013). www.fasebj.org

Phosphoinositide and Erk signaling pathways mediate activity-driven rodent olfactory sensory neuronal survival and stress mitigation

Olfactory sensory neurons (OSNs) are the initial site for olfactory signal transduction. Therefore, their survival is essential to olfactory function. In the current study, we demonstrated that while odorant stimulation promoted rodent OSN survival, it induced generation of reactive oxygen species in a dose‐ and time‐dependent manner as well as loss of membrane potential and fragmentation of mitochondria. The MEK‐Erk pathway played a critical role in mediating these events, as its inhibition decreased odorant stimulation‐dependent OSN survival and exacerbated intracellular stress measured by reactive oxygen species generation and heat‐shock protein 70 expression. The phosphoinositide pathway, rather than the cyclic AMP pathway, mediated the odorant‐induced activation of the MEK‐Erk pathway. These findings provide important insights into the mechanisms of activity‐driven OSN survival, the role of the phosphoinositide pathway in odorant signaling, and demonstrate that odorant detection and odorant stimulation‐mediated survival proceed via independent signaling pathways. This mechanism, which permits independent regulation of odorant detection from survival signaling, may be advantageous if not diminished by repeated or prolonged odor exposure. We investigated the role of odorant stimulation in generating cellular stress and the molecular mechanisms mitigating such stress and promoting neuronal survival. Odorant stimulation promoted olfactory sensory neuron (OSN) survival and also induced intracellular oxidative stress, which was exacerbated when MEK/Erks pathway was inhibited. Sensory stimulation simultaneously activated at least two parallel pathways, the AC/cAMP cascade responsible for odorant detection, and phosphoinositide hydrolysis to promote odorant stimulation‐dependent neuronal survival odorants may activate parallel signaling cascades to mediate sensory detection and sensory stimulation‐dependent survival. AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; Erk, extracellular signal‐regulated kinase; MEK, MAPK/ERK kinase.

Changes in the Histone Acetylation Patterns during the Development of the Nervous System

Epigenetic modification such as DNA methylation and histone acetylation plays essential roles in many aspects of cellular function and development of animals. There is an increasing amounts of evidence for dynamic changes in the histone acetylation of specific gene segments, but little attempt was made to examine global pattern changes in the histone acetylation in developing nervous system. In this study, we found that acetylated histone H3 and H4 immunoreactivities were relatively weak in neuroepithelial cells in the ventricular zone of developing rat cerebral cortex or chick spinal cord, compared to the immature young neurons in the cortical plate of a rat embryo or lateral motor column in chick spinal cord. On the other hand, adult neural stem cells in the dentate gyrus (DG) of rat hippocampal formation did not exhibit such diminished histone acetylation, compared to neuroblasts and mature DG neurons. These results suggest that the level of histone acetylation is highly dynamic and tightly linked to the neuronal types and the differentiation stages.

Induction of neuronal apoptosis by expression of Hes6 via p53-dependent pathway

Hes6 is a member of hairy/enhancer of split (Hes) family that plays a role in the cell proliferation and differentiation. Recently, we found that Hes6 is involved in the regulation of cell proliferation via p53-dependent pathway. In addition to the proliferating regions, brain regions where early post-mitotic neurons are enriched also exhibited Hes6 and p53 mRNA expression. Because p53 is involved in the post-mitotic neuronal apoptosis, here we investigated whether Hes6 can influence the neuronal survival/death. Overexpression of wild-type Hes6 and its mutants induced the apoptosis of primary cultured cortical neurons. In addition, neuronal apoptosis by Hes6 overexpression was markedly blunted in p53(-/-) or Bax(-/-) cortical neurons, suggesting that these pro-apoptotic effects are mediated by p53- and Bax-dependent pathway. However, transactivation-defective mutants of Hes6 also enhanced neuronal apoptosis, suggesting that apoptogenic activity of Hes6 is not directly related to its role in the transcriptional regulation. We propose that Hes6 may play a significant role in the neuronal cell death and/or pathological neurodegeneration via activation of p53 signaling.

Differential expression of BNIP family members of BH3-only proteins during the development and after axotomy in the rat.

The BNIPs (BCL2 and adenovirus E1B 19 kDa interacting proteins) are a subfamily of BCL2 family proteins typically containing a single BCL2 homology 3 (BH3) domain. BNIPs exert important roles in two major degradation processes in cells — apoptosis and autophagy. Although it is known that the function of BNIPs is transcriptionally regulated under hypoxic conditions in tumors, their regulation in the developing brain and neurons following the induction of apoptosis/autophagy is largely unknown. In this study, we demonstrate that three members of the BNIP family, BNIP1, BNIP3 and BNIP3L, are expressed in the developing brain with distinct brain region specificity. BNIP3 mRNA was especially enriched in the entorhinal cortex, raising a possibility that it may have additional biological functions in addition to its apoptotic and autophagic functions. Following starvation-induced autophagy induction, BNIP1 mRNA was selectively increased in cultured neurons. However, the apoptogenic chemical staurosporine failed to modulate the expression of BNIPs, which is in contrast to the marked induction of all BNIPs by glucose-oxygen deprivation. Finally, neonatal nerve axotomy, which triggers apoptosis in motoneurons, selectively enhanced BNIP3 mRNA expression. Collectively, these results suggest that the expression of BNIPs is differentially regulated depending on the stimuli, and BNIPs may exert unique biological functions.

Neuroprotective Effects of an Erythropoietin-Derived Peptide in PC1 2 Cells under Oxidative Stress.

Erythropoietin (EPO) has been shown to be a key cytokine in the production of erythrocytes from erythroblasts. Recently, attempts have been made to adopt EPO as a drug target for neuroprotection in selected neurological pathologies. In the current study, a novel EPO-derived peptide which mimics the weak binding site of EPO to its receptor (MK-X) was generated. Experimental results demonstrated that MK-X was able to ameliorate neuronal death due to reactive oxygen species and conditions of oxidative stress similar to EPO. In addition, MK-X induced long-lasting Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and Akt activation. Furthermore, treatment with inhibitors of ERK1/2 and Akt abolished the neuroprotective effect of MK-X. Unlike EPO, however, MK-X did not induce cellular proliferation. Collectively, the results of the current study suggested that MK-X may be useful as a novel neuroprotective reagent.