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Developmental Expression of Neurofilament 3 (NF-M) in the Cultured Rat Cortical Neurons
정재섭,조선정,진익렬,정승현,문일수,Jung Jae-Seob,Cho Sun-Jung,Jin IngNyol,Jung Seung Hyun,Moon Il Soo Korean Society of Life Science 2005 생명과학회지 Vol.15 No.4
신경미세섬유(neurofilament, NF) 단백질은 신경세포의 주된 중간세사로서, NF-L (61 kDa), NF-M (90 kDa) 및 NF-H (115 kDa) 단백질의 공동중합체로 구성된다. 신경세사섬유는 신경세포의 성장, 구성, 형태 및 가소성에 중요한 역할을 하지만 발생학적 표현에 대하여는 아직 잘 알려지지 않았다. 본 연구에서는 NF-M에 특이한 항체를 제조하여 배양한 대뇌신경세포에서 NF-M의 표현을 조사하였다. 배양 12 및 24시간 세포에서 NF-M은 축삭과 그 성장추 그리고 축삭에 가까운 세포체에 강하게 표현하였다. 배양 4 및 14일 신경세포를 NF-M과 PSD95 항체로 이중염색한 결과 NF-M은 축삭과 가지돌기에 공히 강하게 표현되었으며, PSD95와 같이 위치할 경우에는 점박이로 나타났다. .면역염색에서도 NF-M이 PSD 분획에서 검출되었는데, 따라서 이 점박이는 가지돌기가시임을 시사한다. 본 연구의 결과는 NF-M이 신경세포의 초기 형태발달과정에서 축삭으로 강하게 몰려가며, 성숙한 신경세포에서는 가지돌기 및 가지돌기가시에도 위치하여 특정기능을 수행함을 시사한다. Neurofilament (NF) proteins constitute the major intermediate filament type in adult neurons. They are made up by the copolymerization of the neurofilament light (NF-L, 61 kDa), medium (NF-M, 90kDa), and heavy (NF-H, 115 kDa) proteins. Although neurofilaments play a crucial .ole in neuronal growth, organization, shape, and plasticity, their expression pattern and cellular distribution in the developing neurons remain unknown. In this study, we have produced a rabbit polyclonal antibody specific to NF-M and investigated expression of NF-M in cultured cortical neurons. Immunostaining of 12 and 24 h cultures revealed strong expression of NF-M in axonal growth cone and in the region of a soma toward the axon. Doublestaining of 4 and 14 DIV corical neurons with NF-M and PSD95 antibodies revealed that both axon and dendrites were stained intensely with NF-M antibody, and that NF-M immunostaining along dendrites is often punctate and colocalize with PSD95 puncta, indicating that the puncta represent postsynaptic spines. Presence of NF-M in the postsynaptic spine was also indicated by immunoblot analysis of the postsynaptic density fraction. Taken together, our results show intensive targeting of NF-M into axons in the early axonal development, and into spines in mature neurons, indicating its important functions in axon and spine development.
조선정,정재섭,김덕규,신승철,고옥,정용욱,고복현,진익렬,문일수,Cho, Sun-Jung,Jung, Jae-Seob,Kim, Deock-Kyu,Shin, Seung-Chul,Go, Ok,Jung, Yong-Wook,Ko, Bok-Hyun,Jin, Ing-Nyol,Moon, Il-Soo 한국생명과학회 2001 생명과학회지 Vol.11 No.4
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) a prototype of the highly toxid halogenated arylhydrocarbons, bioaccumulates in the food chain and induces a complex spectrum of pathological responses. However, its effect on the nerve system is relatively not well studied. In this study we evaluated TCDDs cytotoxicity on the cortical cell and investigated its effect on the expression 2,3-cyclic nucleotide 3-phosphodiesterase(CNPase), a marker for oilgodendrocytes, The survival rates of 4 DIV cortical cells, that are dissociated from E18 rat cortex and maintained in the presence of TCDD, were 88.8, 83.6, 78.5, and 78.6%(5,10, 20 and 50 nM, respectively) where the reduction in 20 and 50mM TCDD were statistically very significant(p<0.01). Imunocytochemistry of cultured cells revealed that the intensities of immunostaining with an anti-CNP1&2 antibody depended on the concentrations of the toxin. Immunoblot analysis also showed differential expression of CNP1 and CNP2 in the presence of TCDD; the CNP1 expression was dose-dependently decreased. Interestingly, the expression of CNP2 in the presence if TDCC; the CNP1 expression was dose-dependently decreased. Interestingly, the expression of CNP2 fluctuated with the TCDD concentration. These results indicated that CNP1 and 2 are differentially regulated by TCDD, implying the functions of oligodendrocytes are modulated by the toxin.
조선정,정재섭,진익렬,정승현,박인식,문일수,Cho Sun-Jung,Jung Jae-Seob,Jin IngNyol,Jung Seung Hyun,Park In Sick,Moon Il Soo Korean Society of Life Science 2005 생명과학회지 Vol.15 No.4
Heat shock protein 70 (HSP70) is a multigene family composed of constitutively expressed members(Hsc70) and stress-inducible members (Hsp70). In the mammalian nervous system, a considerable amount of HSPs is also synthesized under normal conditions suggesting that they play an important role in the metabolism of unstressed cells. In this study we examined the expression of Hsp70 in the synapses of rat cerebellar neurons. Immunohistochemistry using specific antibodies revealed that both Hsp70 and Hsc70 are expressed in the cerebellar tissue, with strongest expression in Purkinje cells followed by granule cells. Neurons in deep cerebellar nuclei were also intensely stained by Hsp70 antibody. Immunocytochemical stainings of cultured cerebellar cells showed that Hsp70 is expressed in both Purkinje and granule cells. The expression was punctate in the soma and along dendritic trees, and the punctae were colocalized with those of PSD95, a postsynaptic marker. Immunoblotting also indicates that Hsp70 is associated with the postsynaptic density fraction. Taken together, our results indicate that the Hsp70 is expressed in cerebellar neurons in normal conditions, and that some are localized in the synapses. 열충격단백질 70 (HSP70)은 복수유전자족으로서 통상적으로 표현되는 Hsc70와 스트레스에 의하여 유도되는 Hsp70가 있다. 포유동물의 신경계통에서는 상당한 량의 HSP70가 정상조건에서도 표현되는 것으로 알려져 있다. 본 연구에서는 흰쥐의 소뇌 세포의 연접에서 Hsp70의 표현에 대한 연구를 하였다. 면역조직화학적으로 소뇌절편을 염색하여 관찰한 결과 Hsp70와 Hsc70 모두 표현되었는데, 소뇌 조롱박세포에서 가장 강하게 표현되었으며, 다음으로 소뇌 과립세포에서 강하게 표현되었다. 또한 깊은소뇌핵의 신경세포들도 강하게 염색되었다. 배양한 P1 소뇌신경 세포를 Hsp70 항체로 염색한 결과 Hsp70는 조롱박세포와 과립 세포에서 모두 표현되었으며, 세포체와 가지돌기를 따라 점박이를 형성하였다. 이들 점 박이들은 PSD95 점박이와 같이 위치하였다. 그리고 PSD 분획을 이용한 면역염색에서도 PSD70이 검출되었다. 본 연구결과는 Hsp70이 정상조건에서도 소뇌신경세포의 연접에 존재함을 의미한다.