할로페리돌로 유도한 신경세포 고사에 대한 신경성장인자 및 인슐린 유사 성장인자의 억제효과

강효정,강지윤,곽병주,정영기,노재성 大韓神經精神醫學會 2000 신경정신의학 Vol.39 No.5

연구목적: 본 연구는 생쥐 태자의 대뇌피질 세포 일차 배양을 이용하여 할로페리돌로 유도한 신경세포의 사멸에 대해 신경 성장인자인 brain-dervied neurtrophic factor(BDNF), Neurotrophin 4/5(NT 4/5)와 인슐린 유사 성장인자인 insulin-like growth factor Ⅰ(IGF-Ⅰ)및 IGF-Ⅱ의 조절효과를 실험한 것이다. 방법: 생쥐 태자의 신경세포를 배양한 후 할로페리돌을 처리하여 신경세포의 사멸을 유도하고 이에 대한 성장인자의 효과를 정량적으로 분석하였다. 결과: 30M의 할로페리돌을 배양한 일차 대뇌 신경세포에 24시간 처리하면 광범위한 신경세포의 사멸이 유도된다. 이때 신경세포는 세포의 응축, DNA의 단편화와 응집, 세포막보다 핵막의 이른 붕괴 등 고사 형태의 신경세포 사멸 특성을 보인다. 할로페리돌과 더불어 신경성장인자인 BDNF, NT 4/5 및 인슐린 유사 성장 인자인 IGF-Ⅰ 혹은 IGF-Ⅱ를 동시에 처리할 경우 용량 의존적으로 신경세포의 사멸을 억제한다. 결론: 본 연구의 결과는 할로페리돌에 의한 신경독성이 성장인다들에 의해 억제될 수 있음을 보여주는 것이며, 장기적인 항정신병약물의 사용 후에 발생하는 부작용인 지발성 운동장애의 한가지 원인 가설로 대두되고 있는 항정신병 약물에 의한 신경독성을 신경성장인자에 의해 억제할 수 있을 가능성을 제시하는 것이다. 중심단어:할로페리돌·고사·세포사멸·고사·BDNF·NT-4/5·IGF-Ⅰ·IGF-Ⅱ. Objectives: We examined the effects of neurotrophins and insulin-like growth factors on cell death induced by haloperidol, a typical anti-psychotic agent. Method: Neocortices from 14- or 5-daysold fetal mice for neuron-glia co-cultures were used for this experiment. Result: Twenty-four hours treatment of mouse cortical cell cultures with 30 M haloperidol-induced wide spread neuronal apoptosis characterized by cell body shrinkage, DNA frag-mentation and condensation. Concurrent treatment with growth factors, BDNF, NT4/5, IGF-Ⅰand IGF-Ⅱ, protect the neurons from the haloperidol-induced neuronal apoptosis(HINA) in a dose dependent manner(10-100ng/ml). Conclusion: The present study suggests the possibility that haloperidol toxicity can be hampered with growth factors. Further study about the mechanism underlying the protective capacity of the growth factors on HINA may lead to the development of the new protective strategy for tardive dyskinesia. KEY WORDS:Haloperidol·Apoptosis·Neuronal death·BDNF·NT4/5·IGF-Ⅰ·IGF-Ⅱ.

대뇌 피질 신경 세포 일차 배양에서 6-hydroxydopamine에 의한 신경세포사멸의 특성

정재훈,강효정,손성향,정영기,곽병주,노재성 大韓神經精神醫學會 2000 신경정신의학 Vol.39 No.3

연구목적 : 본 연구는 생쥐 태자 대뇌 피질 세포 일차 배양에서 파킨슨씨병의 동물 모델을 만드는데 사용되는 신경독성 물질인 6-hydroxydopamine(6-OHDA)에 의한 신경세포 사멸의 특성을 관찰한 것이다. 방 법 : 생쥐태자의 신경세포를 배양하여 6-OHDA를 처리하고 이와 더불어 여러가지 신경세포 사멸에 영향을 주는 물질을 투여하여 신경세포의 사멸을 정량적으로 분석하고 형태학적 분석을 전자현미경을 통하여 관찰하였다. 결 과 : 배양한 신경세포에 6-OHDA를 처리하면 투여 양에 비례하여 신경세포의 사멸이 증가하여 50μM 66-OHDA를 24시간 투여하여 일으킨 신경세포 사멸의 형태학적 특징은 세포질의 수축 및 염색질의 응축(nuclear chromatin condensation) 및 핵질의 fragmentation을 동반하는 고사(apoptosis) 형태이다. 이 때의 신경세포의 사멸은 항고사 물질인, 1㎍/ml cycloheximide와 caspase 억제제인 10μMzVAD-fmk에 의해 약화되었으나 글루타메이트 길항제(10μM MK-801 혹은 50μM CNQX)나 항산화제 (100μM trolox 혹은 100μM N-acetyl-cysteine)는 6-OHDA에 의한 신경세포의 사멸을 막지 못했다. 결 론 : 이상의 형태학적 및 약물학적 특징은 6-OHDA가 고사성 신경세포 사멸을 유도한다는 것을 의미하고, 항산화제들이 효과가 없다는 것과 caspase 억제제가 보호효과가 있다는 것은 6-OHDA에 의한 고사성 신경세포 사멸에는 산화적 스트레스보다는 caspase cascade와 관여된 기전이 기여한다는 것을 시사한다. 또한 6-OHDA에 의한 신경세포의 사며른 도파민성 신경세포에 선택적이지 않으며, 일차 대뇌피질 신경세포 배양 시스템이 6-OHDA에 의한 신경세포사멸인구에 사용될 수 있음을 보여준다 Objectives : We examined the patterns of cell death induced by the 6-hydroxydopamine, a selective dopaminergic toxin that used to produce Parkinson's disease model. Method : Neocortices from 14 - or 15-day-old fetal mice for neuron-glia co-cultures were used for this experiments. Results : Cortical cell cultures exposed to 10-100μM 6-hydroxydopamine for 24 hr underwent neuronal death without injuring glia. The degenerating neurons showed hallmark of apoptosis featuring cell body shrinkage, nuclear chromatin condensation and aggregation, nuclear membrane disintegration with intact plasma membrane, and prominent internucle- osomal DNA fragmentation. Neither the glutamate antagonists (10μM MK-801 and 50μM CNQX) nor antioxidants(trolox, 100μM, N-acetyl-cysteine, 100μM prevented the 6-OHDA induced neuronal injury. The death was attenuated by addition of two different anti-apoptotic agents, 1㎍/ml cycloheximide and caspase inhibitor(100μM zVAD-fmk). Conclusion : These features suggest that 6-OHDA induced apoptotic type of neuronal death in cortical neuronal culture. Considering the protective effect of caspase inhibitors, a mechanism involing caspase cascade rather than oxidative stress is responsible for the 6-OHDA-induced neuronal apoptosis. In addition, our results showed that 6-OHDA-induced apoptosis is not confined to dopaminergic neurons and the primary cortical culture system so this system is suitable for the study of 6-OHDA-induced neuronal apoptosis.

Reactive Oxygen Species-Mediated Neuronal Death : characteristics, mechanisms, and implication to brain diseases

Gwag, Byoung Joo 이화여자대학교 세포신호전달연구센터 2000 고사리 세포신호전달 심포지움 Vol. No.2

Free radicals appear to act as key mediators of neuronal death produced under certain pathological conditions such as stroke and neurodegenerative diseases. We have studied how reactive oxygen species(ROS) such as hydroxyl radical and superoxide produce neuronal death. We found that cortical cell cultures exposed to Fe^(2+)(produces hydroxyl radical through Fenton chemistry), buthionine sulfoximine(BSO, depletes glutathione), or menadione(produces superoxide) underwent neuronal death exclusively through necrosis sensitive to trolox or N-acetyl-L-cysteine. Death was accompanied by marked swelling of cell body, fenestration of plasma membrane prior to nuclear membrane, and scattering condensation of nuclear chromatin. Entry of zinc that causes neuronal death in hypoxic-ischemic brain also produces ROS-mediated neuronal cell necrosis. The ROS-mediated neuronal death was insensitive to glutamate antagonists and potentiated by growth factors that attenuate neuronal apoptosis induced in cortical cell cultures. Generation and neurotoxicity of ROS was prevented by maneuvers preventing activity of PKA or cyclooxygenases, but not by PKC inhibitor. Interestingly, increasing mitochondrial membrane and redox potentials protected cortical neurons from Fe^(2+)- or BSO-induced ROS injuries without beneficial effects on neuronal apoptosis resulting from exposure to staurosporine or cyclosporine A. This implies that execution of ROS neurotoxicity likely requires activation of PKA or cyclooxygenases without sharing pro-apoptosis pathway. Finally, we examined if ROS would contribute to selective neuronal death that underlies common features of neurodegenerative diseases. In striatal cell cultures, NADPH-diaphorase neurons that are selectively spared in Huntington's disease(HD) were highly resistant to injuries induced by Fe^(2+) or BSO. Single cell PCR analysis of striatal neurons demonstrated that levels of mRNAs encoding anti-oxidant enzymes were substantially high in NADPH-diaphorase neurons. It is hypothesized that intracellular levels of antioxidant defence enzymes as well as prooxidants may determine differential vulnerability to certain populations of neurons in HD and possibly other neurodegenerative diseases.

Neu2000 as a novel neuroprotectant to treat stroke

( Byoung Joo Gwag ) 한국응용약물학회 2004 학술대회 Vol.2004 No.2

Neurotrophin-induced ROS production and neuronal death

Gwag, Byoung Joo 이화여자대학교 세포신호전달연구센터 2004 고사리 세포신호전달 심포지움 Vol. No.6

The neurotrophins, a class of nerve growth factor-related growth factors, promote the differentiation, growth, and survival of neurons by binding to Trk receptor tyrosine kinases and p75(NTR). Once synthesized in target cells, neurotrophins are released, bound to the cell surface receptors, and internalized to promote neuronal survival by interfering with programmed cell death or apoptosis. In addition to neuroprotective potential of neurotrophins, neurotrophins potentiate neuronal injury under various pathological conditions. Moreover, evidence has accumulated showing that neurotrophins can induce neuronal death. While cellular and molecular mechanisms underlying anti-apoptosis action of neurotrophins have been well documented, mechanisms for pro-necrosis effects of neurotrophins remain to be unveiled. Here, we delineated the patterns and underlying mechanisms of neuronal death induced by neurotrophins.

ROLE OF NF - kB IN NERVE CELL DEATH

곽병주(Byoung Joo Gwag),(Kye Yoon Park),(Hyuk Wan Ko),(Eun Chan Park) 생화학분자생물학회 1997 생화학분자생물학회 춘계학술발표논문집 Vol.- No.-

Cellular and Molecular Pathways of Ischemic Neuronal Death

Kim, Doo Yeon,Gwag, Byoung Joo,Won, Seok Joon 생화학분자생물학회 1998 BMB Reports Vol.35 No.1

Three routes have been identified triggering neuronal death under physiological and pathological conditions. Excess activation of ionotropic glutamate receptors cause influx and accumulation of Ca^(2+) and Na^+ that result in rapid swelling and subsequent neuronal death within a few hours. The second route is caused by oxidative stress due to accumulation of reactive oxygen and nitrogen species. Apoptosis or programmed cell death that often occurs during developmental process has been coined as additional route to pathological neuronal death in the mature nervous system. Evidence is being accumulated that excitotoxicity, oxidative stress, and apoptosis propagate through distinctive and mutually exclusive signal transduction pathway and contribute to neuronal loss following hypoxic-ischemic brain injury. Thus, the therapeutic intervention of hypoxic-ischemic neuronal injury should be aimed to prevent excitotoxicity, oxidative stress, and apoptosis in a concerted way.

In vivo tracking of human mesenchymal stem cells in experimental stroke.

Kim, Daehong,Chun, Byoung-Gi,Kim, Yeon-Kyung,Lee, Yong Hyun,Park, Cheong-Soo,Jeon, Iksoo,Cheong, Chaejoon,Hwang, Tae-Sun,Chung, Hyungmin,Gwag, Byoung Joo,Hong, Kwan Soo,Song, Jihwan Pergamon Press ; Elsevier Science Ltd ; Elsevier S 2007 CELL TRANSPLANTATION Vol.16 No.10

<P>To understand the fates of human mesenchymal stem cells (hMSCs) following transplantation into a rodent model of middle cerebral artery occlusion (MCAo), magnetic resonance imaging (MRI) techniques were employed, hMSCs were labeled with ferumoxides (Feridex)--protamine sulfate complexes, which were visualized and examined by MRI up to 10 weeks following transplantation. Migration of the transplanted cells to the infarcted area was further confirmed by histological methods. We found that the hMSCs transplanted in MCAo models possess the capacity to migrate to the infarcted area extensively in both ipsilateral and contralateral injections, exhibiting a pathotropism. We also analyzed the detailed migration patterns of transplanted hMSCs. We speculate that the extensive migratory ability of hMSCs may represent a therapeutic potential for developing efficient cell transplantation strategies in stroke.</P>

Poster Session 1 / D-17 : Identification of Genes Induced by the neurotrophins , BDNF , NT-3 , and NT-4/5 using dirrerential display

(Sun Hee Kim),(Byoung Joo Gwag) 한국생화학분자생물학회 (구 한국생화학회) 1999 생화학분자생물학회 춘계학술발표논문집 Vol.- No.-

Microglial $P2X_7$ receptor expression is accompanied by neuronal damage in the cerebral cortex of the $APP_{swe}$/PS1dE9 mouse model of Alzheimer's disease

Lee, Hwan-Goo,Won, Sun-Mi,Gwag, Byoung-Joo,Lee, Yong-Beom Korean Society for Biochemistry and Molecular Bion 2011 Experimental and molecular medicine Vol.43 No.1

The possibility that $P2X_7$ receptor ($P2X_7R$) expression in microglia would mediate neuronal damage $via$ reactive oxygen species (ROS) production was examined in the $APP_{swe}$/PS1dE9 mouse model of Alzheimer's disease (AD). $P2X_7R$ was predominantly expressed in CD11b-immunopositive microglia from 3 months of age before A${\beta}$ plaque formation. In addition, $gp91^{phox}$, a catalytic subunit of NADPH oxidase, and ethidium fluorescence were detected in $P2X_7R$-positive microglial cells of animals at 6 months of age, indicating that $P2X_7$R-positive microglia could produce ROS. Postsynaptic density 95-positive dendrites showed significant damage in regions positive for $P2X_7R$ in the cerebral cortex of 6 month-old mice. Taken together, up-regulation of $P2X_7R$ activation and ROS production in microglia are parallel with A${\beta}$ increase and correlate with synaptotoxicity in AD.