Younghwa Kim

Impaired Migration in the Rostral Migratory Stream But Spared Olfactory Function after the Elimination of Programmed Cell Death in Bax Knock-Out Mice

Rats and mice exhibit neurogenesis of olfactory bulb (OB) interneurons throughout adulthood. To homeostatically maintain stable neuron numbers, it is necessary to continuously remove a subset of OB neurons by programmed cell death (PCD). Here we demonstrate that Bax is critical for the elimination of OB neurons by showing that Bax-KO mice exhibit greatly reduced PCD in the OB. Despite the reduction of PCD, however, proliferation of progenitors and the size of the OB were virtually unaffected in Bax-knock-out (KO) mice. However, reducing PCD by Bax deletion affected the migration of a subset of adult-produced neurons by the disruption of glial tube formation as well as by premature detachment of neuroblasts from the migratory chain. Rescued cells aberrantly remained in the subventricular zone (SVZ)-rostral migratory stream (RMS), in which they differentiated into calretinin+ or GABA-expressing interneurons. Because of the migratory deficit, OB cell homeostasis involving new cell entry and PCD (neuronal turnover) was virtually absent in adult Bax-KO mice. Despite this, Bax-KO mice exhibited normal olfactory behaviors such as odor discrimination and olfactory memory which are thought to be influenced by adult neurogenesis. These results demonstrate that PCD is involved in the regulation of RMS migration and differentiation after OB neurogenesis, but that animals maintain normal olfactory function in the absence of PCD.

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.

Functional Role of Parkin against Oxidative Stress in Neural Cells

Background Parkinson disease (PD) is caused by selective cell death of dopaminergic neurons in the substantia nigra. An early onset form of PD, autosomal recessive juvenile parkinsonism has been associated with a mutation in the parkin gene. The function of parkin is known to remove misfolding proteins and protect cell death. We aimed to investigate the role of parkin against oxidative stress in neuronal cells. Methods Parkin knockout embryonic stem cells (PKO ES cells) were differentiated into neurons by adherent monolayer culture method. Oxidative stress was induced by the treatment of 1-methyl-4-phenylpyridinium (MPP+) in neurons derived from wild type and PKO ES cells, and cell viability was examined by MTT assay. After exposure to MPP+, Tuj1-positive cell population was compared between PKO and wild type cells by fluorescence activated cell sorter (FACS) analysis. The activated caspase3 protein level was also measured by Western blot analysis, FACS and immunocytochemistry. Results There was no difference in the efficiency of neuronal differentiation between wild type and PKO ES cells. After exposure to MPP+, no significant differences were found in cell viability and Tuj1-positive cell population between the two groups determined by MTT assay and FACS analysis, respectively. The activated caspase3 protein levels examined by Western blot analysis, FACS and immunocytochemistry were not changed in PKO cells compared with those of wild type cells after MPP+ treatment. Conclusion These results suggest that PKO neuronal cells including dopaminergic neurons are not sensitive to caspase3-dependent cell death pathway during the response against MPP+-induced oxidative stress.

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.

Induction of CITED2 expression in the rat hippocampus following transient global ischemia

CITED2 is implicated in the modulating the activity of HIF-1 which is a major transcription factor involved in ischemia-related gene expression. Following transient forebrain ischemia, we found that CITED2 was induced in a subset of brain regions including dentate gyrus of the hippocampal formation and piriform cortex. Because CITED2 was not induced in cultured neurons exposed to oxygen-glucose deprivation, we concluded that hypoxia is not sufficient to trigger its induction.

Effects of H 2 and/or O 2 Plasma Treatments on Photoconductivity of Freestanding Polycrystalline Diamond Films

Thick (ca. 700 μm) freestanding polycrystalline diamond films, 10 × 10 mm, were obtained using a high power (4 kW) microwave plasma-enhanced chemical vapor deposition system. Prior to fabricating the photoconductors, we treated the growth side (as-grown surface of the film) and the substrate side (the interfacial surface of the film detached from the Mo substrate) using H 2 and/or O 2 plasma. Using these films, we fabricated parallel-type ohmic-contacted diamond photoconductors. At as-grown states, the growth side showed noticeable photoconductivity, while the substrate side showed little photoconductivity. O 2 plasma treatment of these sides led to the extinction of the photoconductivity. After exposing these sides again to H 2 plasma, we observed not only distinct photoconductivity at the substrate side but also the reappearance of photoconductivity at the growth side. Based on these results, we suggest the primary factor for the photoconductivity of these films.

Distribution of Doublecortin Immunoreactivities in Developing Chick Retina

Doublecortin (DCX) is a microtuble-associated protein that is required for the migration of immature neuroblasts within the chick and mammalian brain. Although it is generally thought that DCX is expressed only in the neuroblasts, some mature neurons maintain DCX expression; for example, horizontal cells in adult rat retina. In this study, we demonstrate that retinal neural progenitors in the early embryonic stage of the chick also expressed DCX, as do developing ganglion cells and horizontal cells in later stages of development. These findings raise the possibility of a role for DCX in retinal neural progenitors, before they become specialized into neuroblasts in the chick.

Polarized and Stage-Dependent Distribution of Immunoreactivity for Novel PDZ-Binding Protein Preso1 in Adult Neurogenic Regions.

Background Adult neural stem cells have the potential for self-renewal and differentiation into multiple cell lineages via symmetric or asymmetric cell division. Preso1 is a recently identified protein involved in the formation of dendritic spines and the promotion of axonal growth in developing neurons. Preso1 can also bind to cell polarity proteins, suggesting a potential role for Preso1 in asymmetric cell division. Methods To investigate the distribution of Preso1, we performed immunohistochemistry with adult mouse brain slice. Also, polarized distribution of Preso1 was assessed by immunocytochemistry in cultured neural stem cells. Results Immunoreactivity for Preso1 (Preso1-IR) was strong in the rostral migratory stream and subventricular zone, where proliferating transit-amplifying cells and neuroblasts are prevalent. In cultured neural stem cells, Preso1-IR was unequally distributed in the cell cytosol. We also observed the distribution of Preso1 in the subgranular zone of the hippocampal dentate gyrus, another neurogenic region in the adult brain. Interestingly, Preso1-IR was transiently observed in the nuclei of doublecortin-expressing neuroblasts immediately after asymmetric cell division. Conclusion Our study demonstrated that Preso1 is asymmetrically distributed in the cytosol and nuclei of neural stem/progenitor cells in the adult brain, and may play a significant role in cell differentiation via association with cell polarity machinery.

Selective Deposition of Diamond Film on Glass Substrate via the Enhancement of the Diamond Nucleation Density by the Cyclic Process

Diamond films were deposited on the pretreated silicon or on the pretreated glass substrate in a microwave plasma enhanced chemical vapor deposition (MPECVD) system. We can increase the diamond nucleation density by the cyclic process irrespective of the substrate kinds and deposition conditions. Using the cyclic process, we can certainly enhance the selective deposition of diamond film on glass substrate. The cyclic process is the in situ method carried out by the cyclic modulation of the CH 4 source gas flow rate during the initial deposition stage. Surface morphologies and diamond qualities of the films have been investigated. Based on these results, we discuss the cause for the enhancement of the selectivity of diamond film deposition on glass substrate by the cyclic process.