본 연구에서는 가물치(Channa argus) 추출물의 신경세포 분화와 산화 스트레스에서의 효능을 분석하기 위하여 녹차와 효소를 이용한 다양한 추출 방법(상온 추출물, RE; 녹차 상온 추출물, GRE; 효소 상온 추출물, ERE; 녹차 효소 상온 추출물, GERE)을 사용하여 제조 된 추출물의 아미노산 조성과 항산화 활성을 비교 분석하였고, 신경성장인자 (NGF) 유도 신경세포 분화 및 과산화수소 처리에 의해 유도된 PC12 세포 독성에 대한 보호효과를 규명하고자 하였다. 총 아미노산 함량은 RE 및 GRE보다 효소 추출물인 ERE 및 GERE에서 훨씬 더 높았다. 효소 가수 분해물 (ERE 및 GERE)에서 ABTS 라디칼 소거 활성은 RE 및 GRE보다 높았다. 또한, RE와 ERE는 PC12 세포에서 neuronal growth factor (NGF) 매개 신경 돌기 성장뿐만 아 니라 growth associated protein (GAP)-43 및 synapsin-1의 발현을 현저하게 향상 시켰다. 과산화수소(H2O2)에 의해 손 상된 PC12 세포에 4가지 유형의 Channa argus 추출물을 첨가한 후 PC12 세포의 생존율을 측정하였다. PC12 세포 의 생존율은 RE, GRE, GERE에서 각각 77.5±1.9%, 84.0±0.8%, 81.1±0.9%이였다. 이러한 세포 생존율은 H2O2 만을 처리 한 음성 대조군(70.0±2.0%)에 비해 더 높았다. H2O2 처리에 의해 유도 된 세포 독성도 RE, GRE 및 GERE 처리에 대한 반응으로 상당히 완화되었다. 종합하면, Channa argus 추출물은 산화 스트레스와 신경 손상을 감소시키는 기능성 물질로 유용하다는 것을 시사하며, 향후 이들 소재를 활용한 다양한 기능성 제품의 개발이 필요할 것으로 판단된다.

The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. This study was to examine the in vitro neuron cell differentiation characteristics of our established human (h) iPS cells (IMR90-iPS-1~2) derived from human somatic cells. For the neuron differentiation, well grown hiPS colonies were recovered by collagenase treatment and then suspended cultured in a non-adherent bacteriological culture dish using human embryonic stem (hES) cell culture medium for 4 days. Embryoid bodies were plated and cultured in serum-free ITSFN (insulin/transferrin/selenium/fibronectin) medium for 8 days to select neural precursor cells. Then selected neuronal cells were dissociated, plated onto poly-L-ornithin/laminin coated dish at a concentration of 2 x 105 cells/cm2 and expanded in N2 medium containing 20 ng/ml bFGF, 200 ng/ml SHH and 100 ng/ml FGF-8 for 7 days. For the final differentiation step involved removing agents and culturing for 14 days in 20 ng/ml BDNF added N2 medium. In the neural precursor stage, >90% of nestin positive cells and >50% NCAM positive cells were obtained. Also, in final differentiation step, we confirmed the high percent (>80%) of mature neuron tubulin-β positive cells and approximately >20% of tyrosine hydroxylase positive cells. Also, these results were confirmed by RT-PCR. These results indicated that hiPS cells have potential to generate specific neuron differentiation and especially TH+ neuron was also can be obtained, and thus hiPS-derived neural cells might be an usable source for the study of neuro-degenerative disease.

Neural precursor cells (NPCs) with abilities to self-renew and differentiate into neurons are born in the subventricular zone of the hippocampus and the subgranular zone in the adult mammalian brain. NPCs maintain their population by symmetric cell division and neuronal cell differentiation started by asymmetric cell division. Asymmetric cell division produces two daughter cells with different cellular fates. It has been shown that multiple transcription factors, like homeodomain transcription factors and basic helix loop helix (bHLH) transcription factors, play cruel role in cell fate determination (Bertrand et al., 2002). Multipotent cortical progenitors are maintained in a proliferative state by bHLH factors including Id and Hes families. The transition from proliferation to neurogenesis involves a coordinate increase in the activity of proneural bHLH factors (Mash1, Neurogenin1, and Neurogenin2). As development proceeds, inhibition of proneural bHLH factors in cortical progenitors promotes the formation of astrocytes. Finally, the formation of oligodendrocytes is triggered by an increase in the activity of bHLH factors Olig1 and Olig2 that may be coupled with a decrease in Id activity. Thus, bHLH factors have key roles in corticogenesis, affecting the timing of differentiation and the specification of cell fate. Hes1 is a vertebrate homologue of the Drosophila bHLH protein Hairy, originally known as a transcriptional repressor that negatively regulates neuronal differentiation. Hes1 expression in neuronal precursors precedes and represses the expression of the neuronal commitment gene Mash1, a bHLH activator homologus to the proneuronal Achaete-Scute genes in Drosophila (Campuzano and Modolell, 1992). Down regulation of Hes1 expression in developing neuroblasts may be necessary for the induction of a regulatory cascade of bHLH activator proteins that controls the commitment and progression of neural differentiation. Expression of Hes1 inhibited neurite outgrowth, whereas Mash1 expression increased neurite outgrowth. Mash1 can induce bipolar neuron differentiation (Tomita et al., 1996) and NSCs culture obtained from Mash1-/- mice cannot differentiate into GBAergic neurons (Oishi et al., 2009) Hes1 is an essential effector for Notch signaling, which regulates the maintenance of undifferentiated cells (Artavanis-Tsakonas et al., 1999). In contrast, it is previously reported that platelet-derived growth factor induces the expression of Mash1 mRNA by regulating the phosphorylation of Hes1 and TLE1 (Ju et al., 2004). Hes1 is required for neuronal differentiation in PDGF treated NSC cultures. The major cell types in the cerebral cortex and hippocampus are the glutamatergic neurons and the GABAergic neurons. Cholinergic neurons are important in spatial learning and memory formation and depleted in patient’s brain of early Alzheimer’s disease. It has not been clear, however, whether new born adult NPCs could generate different cell types of neurons with distinct cellular and physiological properties. During the development, glutamatergic neurons consisting of radially migrating neurons are originated from the ventricular zone of the dorsal telenchephalon (pallium) and give rise to pyramidal neurons. Glutamate and glutamate receptors are involved in cognitive functions by forming major excitatory network. GABAergic neurons in the neocortex and hippocampus are in part migrated from the ventral telenchephalon or from the dorsal NPCs and function as local interneurons by forming inhibitory networks which regulate large populations of glutamatergic pyramidal neurons. During the development, spatiotemporal gene expression regulated by extracellular signaling factors is believed to determine the formation of neuronal phenotypes. Platelet derived growth factor B is known to induce the differentiation into neurons rather than glial cells in the rat NPCs. We found that platelet derived growth factor B is expressed in dorsal cortex and hippocampus more than in ventral cortex in the period of pyramidal cell differentiation of the embryonic rat brain. It indeed induces cell type specific differentiation into glutamatergic cells that produce the glutamate transpoter, vGluT1 and glutamate at the late stage of differentiation although it promotes neuronal differentiation at the early stage in NPCs primarily cultured from the rat embryonic hippocampus. Brain-derived neurotrophic factor, however, facilitated GABAergic differentiation in the hippocampal NPCs that generate glutamatergic pyramidal cells in a similar manner. We also found many transcriptional factors such as homeobox genes (Dlx1, Nkx2.1, Pax6) and bHLH genes (NeuroD, Ngn1, Hes1) are involved in cell type specific differentiation into glutamatergic, GABAergic, and cholinergic cells. We observed the expression of Pax6, homeodomain transcription factor, and Hes1, bHLH transcription factor, increased during PDGF-induced early differentiation in neural stem cells. These transcription factors, however, are also expressed in differentiated neurons with specific phenotype at late differentiation stage. We found pax6 is expressed in cholinergic neurons in the adult brains and in cultures. Phosphorylation of neurogenic transcription factors by protein kinases has been reported as predominant strategy in gene regulation during neuronal development and these regulated activities of different transcription factors are known to be involved in cell fate determination. Homeodomaininteracting protein kinases2 (HIPK2) which belongs to HIPK family has been identified as a nuclear serine-threonine kinase and is known to interact with several transcription factors to regulate gene transcriptions. Among several transcription factors, HIPK2 is mainly reported to target the homeodomain transcription factors such as Nkx and Pax6. Considering the importance of homeodomain transcription factors in neurogenesis and differentiation, HIPK2 also seem to play critical roles in those transcriptional regulations during embryogenesis. To define the roles of HIPK2 in neuronal differentiation during embryonic development, we investigated the expression patterns of neurogenic transcription factors such as Pax6, Hes1 and Mash1 in HIPK2 overexpressing NSCs. Hes1 showed different expression patterns between the wild type and mutant HIPK2 overexpressed cells and Mash1, which is reported to be repressed by Hes1, also showed altered expression patterns. We detected the mRNA expression of Hes1 is upregulated by HIPK2 during neuronal differentiation. The overexpressed Pax6 induced differentiation of neural stem cells into cholinergic neurons and suppressed differentiation into GABAnergic neuron both in vitro and in vivo transplantation study. To evaluate the effect of Pax6 on the transcriptional activation of Hes1 promoter, we performed luciferase reporter assay in NIH3T3 cells. Reporter expression of Hes1 promoter was enhanced upon stimulation with wild type Pax6 and wild type HIPK2. Furthermore, the HDAC inhibition mediated by TSA(Trichostatin A) has been shown to repress the reporter expression. The treatment of TSA increased neurofilaments and GAD expression in E14.5 cortical neuronal cell. These findings suggest that Pax6 promotes neuronal subtype differentiation via regulation of Hes1 bHLH transcription factor, which is mediated by HDAC. To examine the effect of Pax6 and HIPK2 on the transcriptional activation of Hes1, efficiency of hes1 promoter was measured by a luciferase reporter assay. When DNA constructs encoding Pax6 and HIPK2 were transfected along with Hes1 promoter, the expression of the reporter was highly increased. Furthermore, the HDAC inhibition mediated by TSA(Trichostatin A) repressed the reporter expression. Interaction of Pax6 and HIPK2 was shown by co-immunoprecipitation and binding of Pax6 to hes1 promoter was detected by chromatin immunoprecipitation. I also found overexpression of HIPK2 and Pax6 facilitated neural stem cells to differentiate into cholinergic cell fate in NSCs primarily cultured from the rat hippocampus. This is also supported by analysis of the brains of sey/neu Pax6 mutant mice and HIPK2 knock out mice. These findings suggest that Pax6 activation by HIPK2 promotes neuronal subtype differentiation via up regulation of Hes1 and down regulation of Mash1 and it is mediated by HDAC.