Eun Hae Kim

Identification and characterization of novel activity-dependent transcription factors in rat cortical neurons

Using gene chip analyses, we have identified novel neuronal activity‐dependent genes. Application of 25 mm KCl to mature (14‐day culture) rat cortical neurons resulted in more than 1.5‐fold induction of 19 genes and reduction of 42 genes among 1200 neural genes. Changes in the overall gene expression profiles appeared to be related to the reduction of excitability and induction of cellular survival signals. Among the genes identified, three transcriptional modulators [encoding Cbp/p300‐interacting transactivator with ED‐rich tail 2 (CITED2), CCAAT/enhancer binding protein β (C/EBPβ) and neuronal orphan receptor‐1, (NOR1)] were newly identified as activity‐dependent transcription factors, and two of these (CITED2 and NOR1) were found to be influenced by electroconvulsive shock (ECS). NOR1 was induced in specific brain regions by behavioral activation, such as exposure to a novel environment. Because the brain regions that exhibited the induction of these newly identified neuronal activity‐dependent transcriptional modulators were distinct from those showing the induction of previously identified activity‐dependent genes such as c‐fos, these genes might be useful markers for mapping neuronal activity in vivo.

Differential regulation of metallothionein-I, II, and III mRNA expression in the rat brain following kainic acid treatment

Although metallothioneins (MTs) are believed to be involved in the protection against neural stresses, spatio-temporal regulation of MT isoforms following neural insults has not been thoroughly examined. In this study, we found that systemic application of kainic acid (KA) rapidly induced MT-I and II expression in neurons localized in hippocampal formation, piriform cortex, and amygdala of the adult rat, whereas the level of MT-III mRNA was decreased in KA-vulnerable areas. At 96 h after KA treatment, while the neuronal expression of MT-I and II returned to basal level, the glial expression of MT-I, II and III was increased in the reactive astrocytes. Differential regulation of MT isoforms in neuron and gila suggests that each isoform might have distinct role in the cell-type dependent cellular responses against KA-evoked neural injuries.

Expression of thymosin β in the rat brain following transient global ischemia

Thymosin beta (Tbeta) isoforms play an important role in the organization of the cytoskeleton by sequestering G-actin during development of the mammalian brain. In this study, we examined changes in the expression of Tbeta4 and Tbeta15 after transient global ischemia. Tbeta15 mRNA increased gradually in the dentate gyrus (DG) of the hippocampal formation from 3 h after reperfusion and peaked 9 h later. Similarly, a significant increase in Tbeta4 mRNA level was observed in the DG 12 h after reperfusion. Tbeta4 and Tbeta15 proteins were found in different cell types in control brains; Tbeta15 was expressed in a subset of doublecortin (DCX)-positive cells in the DG, whereas Tbeta4-IR was observed in DG neurons and nearby microglial cells. After ischemia, Tbeta15-IR was found in DG neurons and Tbeta4-IR in the reactivated microglial cells. Interestingly, Tbeta15-IR accumulated in the nuclei of CA1 neurons, which are vulnerable to ischemic insults. These results suggest that Tbeta4 and Tbeta15 function in different cellular contexts during ischemia-induced responses.

Cell type-specific upregulation of myristoylated alanine-rich C kinase substrate and protein kinase C-α, -β I, -β II, and -δ in microglia following kainic acid-induced seizures

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed protein kinase C (PKC) substrate and has been implicated in actin cytoskeletal rearrangement in response to extracellular stimuli. Although MARCKS was extensively examined in various cell culture systems, the physiological function of MARCKS in the central nervous system has not been clearly understood. We investigated alterations of cellular distribution and phosphorylation of MARCKS in the hippocampus following kainic acid (KA)-induced seizures. KA (25 mg/kg, i.p.) was administered to eight to nine week-old C57BL/6 mice. Behavioral seizure activity was observed for 2 h after the onset of seizures and was terminated with diazepam (8 mg/kg, i.p.). The animals were sacrificed and analyzed at various points in time after the initiation of seizure activity. Using double-labeling immunofluorescence analysis, we demonstrated that the expression and phosphorylation of MARCKS was dramatically upregulated specifically in microglial cells after KA-induced seizures, but not in other types of glial cells. PKC α, β I, β II and δ, from various PKC isoforms examined, also were markedly upregulated, specifically in microglial cells. Moreover, immunoreactivities of phosphorylated MARCKS were co-localized in the activated microglia with those of the above isoforms of PKC. Taken together, our in vivo data suggest that MARCKS is closely linked to microglial activation processes, which are important in pathological conditions, such as neuroinflammation and neurodegeneration.

Expression and subcellular localization of thymosin beta15 following kainic acid treatment in rat brain.

Thymosin beta15 (Tbeta15) is a pleiotropic factor which exerts multiple roles in the development of nervous system and brain diseases. In this study, we found that the expressions of Tbeta15 mRNA and protein were substantially increased in several brain regions including hippocampal formation and cerebral cortex, following kainic acid (KA)-evoked seizures in rat. Interestingly, a subset of cortex neurons exhibited nuclear Tbeta15 immunoreactivity upon KA treatment. Furthermore, translocation of Tbeta15 from cytosol to nuclei was observed in cultured neurons or HeLa cells during staurosporine (STS)-induced apoptosis, which was also verified by time-lapse imaging of YFP-tagged Tbeta15. It appeared that localization of Tbeta15 is restricted to the cytosol in normal condition by its G-actin-interacting domain, because site-directed mutagenesis of this region resulted in the nuclear localization of Tbeta15 in the absence of STS treatment. To explore the role of nuclear Tbeta15, we enforced Tbeta15 to localize in the nuclei by fusion of Tbeta15 with nuclear localization signal (NLS-Tbeta15). However, overexpression of NLS-Tbeta15 did not alter the viability of cells in response to STS treatment. Collectively, these results suggest that nuclear localization of Tbeta15 is a controlled process during KA or STS stimulation, although its functional significance is yet to be clarified.

Inhibition of rat brain inositol 1,4,5-trisphosphate 3-kinase A expression by kainic acid

Defects in intracellular calcium homeostasis may cause aberrant neuronal activation and subsequent neuronal death. Because inositol trisphosphate (IP(3)) regulates the release of calcium from the endoplasmic reticulum and the IP(3) kinase A isoform (IP(3)K-A) reduces intracellular IP(3), regulation of IP(3)K could be involved in neuronal activation and/or neuronal death. In this study, we found that kainic acid (KA) treatment in vitro and in vivo reduced the level of IP(3)K-A mRNA. Since KA treatment induces aberrant neuronal activation and neuronal death, we tested whether the reduction of IP(3)K-A mRNA was required for KA-induced neuronal death. Overexpression of adenovirus-derived IP(3)K-A failed to rescue neurons from KA-induced death. Because neuronal activation by KCl in vitro is sufficient to reduce IP(3)K-A expression, we conclude that the KA-derived reduction of IP(3)K-A expression is due to the aberrant neuronal activation, and the reduction in the IP3K-A mRNA level is not required for the toxic effect of KA.

Multi-walled Carbon Nanotubes Affect the Morphology and Membrane Potential of Mitochondria in HeLa Cell

Carbon nanotubes (CNTs) have unique physical features with high strength, and they could be first used as field-emission electron sources (Deheer & Ugarte, 1995), which could be applied to transistors (Bachtold et al., 2001). With these and related characteristics, the use of CNTs is rapidly increasing in wide variety of fields, such as clothes’ fibers, unique materials for extreme conditions, and parts of nano-machines. However, the safety of CNT’s is one of the important and emerging concerns. Several researches have shown that a specific type and concentrations of CNT’s are toxic to mammalian cells (Jia et al., 2005; Monteiro-Riviere et al., 2005; Muller et al., 2005; Kostarelos, 2008; Firme & Bandaru, 2010). For example, high concentration of multi-walled carbon nanotubes (MWNT) induced apoptosis of T lymphocyte (Bottini et al., 2006). Although relatively low concentration of MWNT, and CNTs in general, is believed to be safe, duration of the CNT exposure, and the cellular accumulation of these synthetic materials are yet to be explored. In addition, development of more sensitive tools to assess cellular quality after CNTs is desirable. Mitochondria are one of the most essential organelles for the life of cells. They produce adenosine triphosphate (ATP) during the electron chain transfer cycles occurring in the inner mitochondrial membrane. For the ATP production, mitochondrial membrane potentials are required. Morphologically, mitochondria are highly dynamic in cells, and continuously fuse and divide (Kane & Youle, 2010; Galloway et al., 2012; Kim et al., 2013; Hoppins, 2014). More recently, it has been demonstrated that mitochondrial function/membrane potential is closely associated with mitochondrial morphology (Galloway et al., 2012; Liesa & Shirihai, 2013; Liu et al., 2014). In fact, many cell proliferation/ Multi-walled Carbon Nanotubes Affect the Morphology and Membrane Potential of Mitochondria in HeLa Cell