과당 이인산의 대뇌피질 신경세포 보호작용
Protective roles of fructose-1, 6-bisphosphate in cortical neuronal death
- 주제(키워드) NMDA , Fructose-1 , 6-bisphosphate , ROS , MAPK/ERK , COX , AA , Prostaglandins , Isoprostanes , Pure cortical neurons
- 발행기관 아주대학교
- 지도교수 백은주
- 발행년도 2006
- 학위수여년월 2006. 2
- 학위명 박사
- 학과 및 전공 일반대학원 신경과학기술과정
- 본문언어 영어
초록/요약
NMDA induces neuronal excitability by stimulating various signaling pathways, and hyperactivation of NMDA receptor primarily causes neuronal death following such as stroke, seizure and neurodegenerative disease. Fructose-1, 6-bisphosphate (FBP), an intermediate of glucose metabolism, is known to show neuroprotective effect in various animal models including brain ischemia. Up to the present, however, the intracellular signaling pathways for FBP-induced neuroprotection, in particular, in pure cortical neuron culture system, have not been clearly elucidated. In this study, I investigated whether FBP can protect pure cortical neurons from NMDA excitotoxicity, and if so, whether its protective effect is associated with modulation of intracellular signaling molecules such as MAPKs, reactive oxygen sepsis (ROS), cyclooxygenase-2 (COX-2) and prostaglandins (PGs). To examine the effect of FBP on neuronal excitotoxicity, cortical neurons were treated with NMDA in the presence or absence of FBP (1-20 mM) and cell death was estimated by lactate dehydrogenase (LDH) release and neuronal cell loss. After 24 h of NMDA treatment, significant increase in LDH release and remarkable loss of neuronal count were observed, and this neuronal death was almost completely blocked by 10 ？？M FBP. It was also found that ROS level in neurons was increased and MMP (mitochondrial membrane potential) was decreased after 4-8 h of NMDA treatment, and that these alterations were completely reversed by FBP. To examine the intracellular signaling pathways for FBP-induced neuroprotection, the effects of FBP on MAPKs activation (phosphorlation) were examined by western blotting. Phosphorylation of p38 MAPK and ERK were increased by NMDA, and these activated MAPKs were, at least partially, inhibited by treatment with FBP. Both the neuroprotective effect and the ROS-lowering effect of FBP were significantly blocked by either p38MAPK inhibitor SB203580 or ERK inhibitor PD98059. To investigate additional potential candidate involved in the mechanism of FBP-induced neuroprotection, the effect of FBP on NMDA-induced COX-2 expression and PGE2 production, which are known to be potent inflammatory mediators in response to various noxious stimuli in brain. Unexpectedly, this study showed that cortical neuronal cells had little level of COX-2 protein and mRNA expression and PGE2 production even in the presence of NMDA. Moreover, arachidonic acid (AA)-induced PGE2 release in neurons remained unaltered even in the presence of various COX inhibitors, suggesting an existence of COX-independent PGE2 releasing system in AA-treated neurons. As expected, COX-independent PGs called isoprostane were observed in pure cortical neurons and cell free media. Moreover, both the neuronal death and the PGE2 release induced by AA were inhibited by FBP, which might be act through regulating an auto-oxidation from AA to PGs. From the results in this study, it is suggested that FBP protects neurons against NMDA-induced cell death through down-regulation of free radical production mediated by activation of p38 MAPK/ERK pathway, and through down-regulation of COX-independent PG-like compounds, such as lipid-peroxidation products.
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TABLE OF CONTENTS
ABSTRACT i
TABLE OF CONTENTS iv
LIST OF FIGURES vii
LIST OF TABLES ix
ABBREVIATION x
I. Introduction 1
1. Neuroprotective effects of FBP 1
2. COX activity in brain cells 6
II. Aims of Study 12
1. Effect of fructose-1,6-bisphosphate on NMDA-induced cell death 12
2. COX expression and activity in various brain cells 12
III. Materials and Methods 13
3.1. Materials 13
3.2. Cell culture 13
3.3. Assessment of Neuronal death 15
3.4. Assay of MAPKinases (ERK1/2, p38) 16
3.5. Assay of ROS formation 17
3.6. MMP measurement 17
3.7. Western blot analysis for COX-2 and p-Akt 18
3.8. RNA preparation and Reverse Transcription Polymerase Chain
Reaction (RT-PCR) 19
3.9. Enzyme-linked immunoassay of Prostaglandin E2 (PGE2) 20
3.10. Statistical analysis 21
IV. RESULTS 22
1. Effect of FBP on NMDA-induced cell death in pure cortical neurons 22
2. Effect of FBP on NMDA-produced ROS release 22
3. Effect of FBP on NMDA-depolarized mitochondrial membrane potential 25
4. Effects of FBP on p38 and MAPK expression 25
5. Effects of MAPK inhibitors on NMDA-induced neuronal death and ROS
production 28
6. Neuronal COX-2 and p-Akt protein expression altered by NMDA 28
7. Effects of FBP on NMDA- or ionomycin-induced PGE2 release in
neurons 33
8. COX mRNA in neurons, astrocytes, microglial cells and brain endothelial
cells 36
9. PGE2 release induced by AA in neurons, astrocytes, microglial cells and
brain endothelial cells 38
10. Effects of COX inhibitors on AA-induced PGE2 release in neurons
astrocytes, microglial cells and brain endothelial cells 40
11. Effects of COX inhibitors on AA-induced PGs in neurons 42
12. Effects of COX inhibitors on AA-induced PGs in BV2 microglial cells 45
13. COX-independent PGs in neurons via isoprostane pathway 45
14. Effects of FBP on AA-induced PGE2 release in pure cortical neurons and astrocytes 48
15. Effects of FBP on AA-induced PGE2 release in different condition 48
16. Specificity of anti-PGE2 antibody for AA, isoprostane and PGE2 51
17. Effects of FBP and anti-oxidants on AA-increased PGE2 release and
cell death 53
V. Discussion 55
VI. References 64
국문 요약 80
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LIST OF FIGURES
Figure 1. Experimental scheme 2
Figure 2. Chemical structure of fructose-1,6-bisphosphate 5
Figure 3. Scheme of prostanoid synthesis 8
Figure 4. FBP inhibited on NMDA-induced LDH release 23
Figure 5. Effect of FBP on NeuN-positive cells in NMDA-treated neuronal cells 24
Figure 6. Effect of FBP on NMDA-induced ROS production 26
Figure 7. Effect of FBP on NMDA-induced depolarization of mitochondrial membrane.. 27
Figure 8. Effects of MAPK inhibitors on NMDA-induced neuronal death and
ROS production 29
Figure 9. Effects of FBP on NMDA-induced phospho-p44/42 and p-p38 expression
in cortical neurons 30
Figure 10. COX-2 and p-Akt protein expression altered by NMDA in primary
cortical neurons 32
Figure 11. Effect of FBP and indomethacin on NMDA-induced PGE2 release and neuronal death 34
Figure 12. Effects of FBP and indomethacin on ionomycin-altered PGE2 release in neurons and astrocytes 35
Figure 13. Expression of COX mRNA isoforms (COX-1, COX-2 and COX-3)
in astrocytes, bEnd3 endothelial cells, BV2 microglia and primary
cortical neurons 37
Figure 14. AA-induced PGE2 release from astrocytes, bEnd3 endothelial cells, BV2 microglial cells and primary cortical neurons 39
Figure 15. Effects of COX isoforms inhibitors on COX activity in various types of brain cells 41
Figure 16. Effect of COX inhibitors on AA-induced PGD2 and PGF2？？ release in pure
cortical neurons 44
Figure 17. Effects of COX inhibitors on AA-induced prostaglandins from BV-2 microglial cells 46
Figure 18. AA-induced iso-prostaglandin F2？？ release from pure cortical neurons 47
Figure 19. Effect of FBP on AA-induced PGE2 release from pure cortical neurons
and astrocytes 49
Figure 20. Effects of FBP on AA-induced PGE2 release in cell free media 50
Figure 21. Effects of FBP and antioxidants on AA-induced PGE2 release and cell
death 54
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LIST OF TABLES
Table 1. Effect of COX inhibitors on AA-induced PGE2 release 43
Table 2. Specificity of anti-PGE2 antibody for AA, isoprostane and PGE2 52
