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Neuroprotection via activation of glutamate dehydrogenase against cerebral ischemia and reperfusion

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dc.contributor.author김, 아영-
dc.date.accessioned2018-11-08T10:22:39Z-
dc.date.available2018-11-08T10:22:39Z-
dc.date.issued2017-
dc.identifier.urihttp://repository.ajou.ac.kr/handle/201003/16409-
dc.description.abstractEnergy metabolism in the brain is important during normal function and in pathological conditions, especially stroke. Although glucose is a main obligatory substrate, the brain can use other energy substrates, including monocarboxylic acid, ketone bodies, amino acids, and fatty acids during glucose restriction. In particular, glutamate is the most abundant excitatory amino acid, and deregulation of glutamate homeostasis is associated with degenerative neurological disorders. Glutamate dehydrogenase (GDH) is important for glutamate metabolism and plays a central role in expanding the pool of tricarboxylic acid cycle intermediate alpha-ketoglutarate (α-KG), which improves overall bioenergetics. Under high energy demand, maintenance of ATP production results in functionally active mitochondria. Here, it is examined whether the modulation of GDH activity can rescue ischemia/reperfusion-induced neuronal death in an in vivo mouse model of middle artery occlusion and an in vitro oxygen/glucose depletion model. Iodoacetate, an inhibitor of glycolysis, was also used in a model of energy failure, remarkably depleting ATP and α-KG. To stimulate GDH activity, the GDH activator 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid and potential activator beta-lapachone were used. The GDH activators restored α-KG and ATP levels in the injury models and provided potent neuroprotection. It was also found that beta-lapachone increased glutamate utilization, accompanied by a reduction in extracellular glutamate. In addition, the glutamate consumed by beta-lapachone was supplied from glutamine with phosphate-activated glutaminase (PAG) reaction. Thus, the hypothesis that mitochondrial GDH activators increase α-KG production as an alternative energy source for use in the tricarboxylic acid cycle under energy-depleted conditions was confirmed. The results suggest that increasing GDH-mediated glutamate oxidation represents a new therapeutic intervention for neurodegenerative disorders, including stoke.-
dc.description.abstract뇌의 에너지 대사는 정상기능을 유지하고 있을 때는 물론 뇌졸중과 같은 병리학적 상태에서 매우 중요하다. 포도당은 뇌에서 절대적인 에너지원이지만 포도당의 공급이 제한되는 경우에는 monocarboxylic acid, ketone body, 아미노산, 지방산과 같은 다른 에너지원을 사용하기도 한다. 특히 glutamate는 가장 풍부한 흥분성 아미노산으로 glutamate 대사가 조절되지 않아서 항상성이 유지되지 못하면 퇴행성 뇌질환으로 발달할 수 있다. Glutamate 대사를 조절하는 중요한 효소로서 glutamate dehydrogenase가 있다. 이 효소는 glutamate를 3-카르복실산 회로(tricarboxylic acid cycle)의 중간산물인 alpha-ketoglutarate으로 전환시킬 수 있어서 alpha-ketoglutarate의 세포 내 양적 증가를 유도할 수 있으며, 세포 내의 전반적인 에너지 수준을 향상시킬 수 있다. 그러므로 세포 내의 에너지 요구량이 증가할 때, ATP의 지속적인 생성, 유지는 세포생존에 중요한 세포소기관인 미토콘드리아의 기능 활성화를 유지할 수 있다. 본 연구에서는 허혈/재관류에 의한 신경세포사멸을 glutamate dehydrogenase가 조절할 수 있는지를 in vivo 중대뇌동맥폐색이 유도된 생쥐에서 관찰하고 in vitro 산소와 포도당이 고갈된 배양세포에서 관찰하고자 하였다. 또한 에너지가 고갈된 환경을 조성하기 위해서 해당과정 저해제인 iodoacetate를 사용하여 alpha-ketoglutarate와 ATP를 고갈시켰다. 이 때, glutamate dehydrogenaes의 활성제로서 이미 알려져 있는 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid와 활성제로서의 잠재적 가능성을 가지고 있는 beta-lapachone을 이용하여 glutamate dehydrogenase의 활성을 촉매하였다. 그 결과, glutamate dehydrogenase의 활성제들은 alpha-ketoglutarate와 ATP의 세포 내 수준을 회복시켰고, 이는 신경세포 보호현상으로 관찰되었다. 또한 beta-lapachone은 세포 내의 glutamate의 대사를 증가시켜 세포 밖으로 분비되는 glutamate의 양을 감소시켜서, glutamate에 의해 예상되는 흥분성 독성을 저해할 수 있었다. 게다가 glutamate dehydrogenase의 잠재적 활성제인 beta-lapachone에 의해서 소모되는 glutamate는 phosphate-activated glutaminase의 반응에 의해서 glutamine으로부터 공급받는 것을 확인하였다. 이로서, 미토콘드리아 내에 존재하는 glutamate dehydrogenase 활성화는 대체에너지원인 glutamate를 alpha-ketoglutarate으로 전환시켜서 3-카르복실산 회로에 공급함으로서 에너지 고갈상태의 신경세포를 보호하는 작용을 하고 있음을 확인하였다. 그러므로, glutamate dehydrogenase 활성화에 의한 glutamate의 대사증가는 뇌졸중과 같은 퇴행성 뇌질환의 새로운 치료방안이 될 수 있다.-
dc.description.tableofcontentsABSTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURES vi
LIST OF TABLES vii
ABBREVIATION viii

I. INTRODUCTION 1

II. MATERIALS AND METHODS 8
1. FOCAL CEREBRAL ISCHEMIA AND REPERFUSION 8
2. ASSESSMENT OF CELL DEATH 8
3. CELL CULTURES 9
3.1. Cortical neurons 9
3.2. Cortical neurons and glias coculture 10
3.3. Astrocytes 10
4. IN VITRO STROKE INDUCTION 10
4.1. Oxygen and glucose depletion (OGD) 10
4.2. Iodoacetate (IOA) treatment 11
5. MTT CONVERSION ASSAY 11
6. MEASUREMENT OF ROS 11
7. MEASUREMENT OF TOTAL THIOLS 12
8. IN VITRO ANTIOXIDANT ACTIVITY ASSAYS 12
8.1. DPPH radical scavenging activity 12
8.2. Metal chelating activity 12
8.3. Reducing power activity 13
9. IMMUNOCYTOCHEMISTRY 13
10. CALCEINAM AND PROPIDIUM IODIDE STAINING 14
11. TISSUE PREPARATION 14
12. MEASUREMENT OF ATP 14
13. MEASUREMENT OF PYRUVATE, L and DLACTATE, AND αKETOGLUTARATE 15
14. ENZYME ACTIVITY ASSAYS 15
14.1. GAPDH ACTIVITY 15
14.2. GDH ACTIVITY 16
14.3. AAT ACTIVITY 17
14.4. PAG ACTIVITY 17
15. WESTERN BLOTTING AND ANALYSIS 17
16. AMINO ACID ANALYSIS 19
17. STATISTICAL ANALYSIS 20

III. RESUTLS 21
A. βLapachone (βLA) alleviated neuronal damage against cerebral ischemia and reperfusion (I/R) 21
B. βLA alleviated neuronal damage against BSO or IOA 25
C. βLA protected cultured neurons from oxidative stress 28
D. βLA did not have structural antioxidant activity 31
E. βLA alleviated damage of cultured brain cells from IOA injury 34
F. βLA restored intracellular ATP in in vivo and in vitro stroke induction 37
G. βLA assisted in the protective effect of pyruvate 40
H. βLA accelerated the use of glutamate as an alternative energy source 42
I. βLA directly increased GDH activity 45
J. GDH activation supplied the energy required to protect from energy depletion 47
K. GDH activation by BCH supplied the energy required to protect neurons with energy failure 50
L. βLAmediated glutamate metabolism was not involved in AAT activity 52
M. Glutamine catabolism by PAG maintained glutamate supplement 54
N. Glutamate supplement by PAG maintained neuronal survival 57
O. Impaired glucose metabolism affected the intracellular amino acid content 59
P. βLA relieved methylglyoxalinduced neuronal toxicity. 62
Q. βLA reduced the accumulation of intracellular Dlactate 66
R. MGinduced neuronal death was not involved in energy failure 69
S. βLA improved the intracellular GSH levels 71

IV. DISCUSSION 73

V. REFERENCES 81

국문초록 94
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dc.language.isoko-
dc.titleNeuroprotection via activation of glutamate dehydrogenase against cerebral ischemia and reperfusion-
dc.title.alternative대뇌 허혈과 재관류 손상에서의 glutamate dehydrogenase활성에 의한 신경세포보호-
dc.typeThesis-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000024343-
dc.subject.keywordNeuroprotection-
dc.subject.keywordFocal ischemia-
dc.subject.keywordReperfusion-
dc.subject.keywordEnergy metabolism-
dc.subject.keywordGlutamate dehydrogenase-
dc.subject.keyword신경세포보호-
dc.subject.keyword국소 뇌허혈-
dc.subject.keyword재관류-
dc.subject.keyword에너지대사-
dc.subject.keyword글루탐산 탈수소화효소-
dc.description.degreeDoctor-
dc.contributor.department대학원 의생명과학과-
dc.contributor.affiliatedAuthor김, 아영-
dc.date.awarded2017-
dc.type.localTheses-
dc.citation.date2017-
dc.embargo.liftdate9999-12-31-
dc.embargo.terms9999-12-31-
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