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研究生： 謝孟儒
Hsieh, Meng-Ru
論文名稱： 粒線體去偶合蛋白在低溫保護神經細胞的作用中所扮演之角色
The role of mitochondrial uncoupling proteins in cold exposure-induced neuroprotection
指導教授： 莊季瑛
Chuang, Jih-Ing
學位類別： 碩士
Master
系所名稱： 醫學院 - 生理學研究所
Department of Physiology
論文出版年： 2011
畢業學年度： 99
語文別： 英文
論文頁數： 66
中文關鍵詞： 低溫 、去偶合蛋白 、神經保護
外文關鍵詞： cold exposure, MPP+, uncoupling protein
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    • 全身性或選擇性地降低腦部溫度被證實具有改善腦部創傷的動物及病患的治療方式，雖然這些研究證實低溫具有保護作用，但低溫所調控的保護機制仍未被完全探討明瞭。過去的研究中發現，當細胞或動物處在低溫刺激下，大腦粒線體去偶合蛋白質(UCPs)與冷誘導核醣核酸結合蛋白(CIRBP)的表現量會顯著增加。此外，研究證實在以1-methyl-4 phenylpyridinium (MPP+) 誘發的帕金森氏症實驗模式中，大量表現去偶合蛋白質能有效降低因MPP+造成的氧化壓力傷害及神經細胞死亡。因此在本研究中，我們將探討低溫是否透過提高去偶合蛋白及冷誘導核醣核酸結合蛋白的表現量，達到保護神經細胞免於MPP+造成的細胞死亡。首先，我們發現將人類SK-N-SH神經母細胞瘤細胞或初代皮質神經細胞在MPP+處理後低溫培養(32 °C) 24小時，而非MPP+處理前低溫培養，可以顯著降低MPP+造成的細胞死亡。此外，我們發現經過低溫處理24小時後，即可顯著誘發去偶合蛋白4與5以及冷誘導核醣核酸結合蛋白的基因與蛋白質表現量上升，而去偶合蛋白2的基因與蛋白質表現量則顯著降低。若將MPP+和低溫同時處理，則更顯著地增加MPP+ 所提升的去偶合蛋白4與降低的去偶合蛋白2的基因表現量。為了更進一步探究去偶合蛋白與冷誘導核醣核酸結合蛋白在低溫保護神經細胞的作用中是否扮演重要的角色，利用慢病毒基因載體系統將shRNA送入SK-N-SH細胞中建立持續表達低量去偶合蛋白與冷誘導核醣核酸的細胞株，結果顯示當降低去偶合蛋白4或5或冷誘導核醣核酸結合蛋白的表現量時，皆會抑制低溫所調控的神經保護作用。此外，我也發現低溫處理會顯著抑制MPP+所增加的過氧化氫產生，而且這種抑制的情形會在去偶合蛋白4或 5或冷誘導核醣核酸結合蛋白表現量降低後被阻斷。另一方面，增加去偶合蛋白5的表現量會顯著降低MPP+所增加的H2O2含量。以上結果顯示，SK-N-SH細胞中，低溫是藉由增加去偶合蛋白4、 5及冷誘導核醣核酸結合蛋白的表現量，抑制H2O2的產生，進而達到抑制MPP+造成的細胞毒殺作用。

    Systemic or brain-selective hypothermia is a promising treatment for patients and animals with brain damage. However, the protective mechanisms are still unclear. Recent studies demonstrated that in response to cold exposure, mitochondrial uncoupling proteins (UCPs) and cold-inducible RNA binding protein (CIRBP) are upregulated in brains. It had been demonstrated that overexpression of UCP4 or UCP5 protected neuron from 1-methyl-4 phenylpyridinium (MPP+) toxicity. MPP+, an inhibitor of mitochondrial complex I, is widely used to specifically induce dopaminergic neuron death and generate a parkinsonian model. Herein, I explored whether cold exposure protected neurons from MPP+ toxicity by regulating the expression of UCPs or CIRBP. We found that 32 °C cold exposure for 24 h after, but not before MPP+ treatment reduced MPP+-induced neuron death and H2O2 overproduction in human SK-N-SH neuroblastoma cells and primary cortical neurons. Furthermore, cold exposure induced the upregulation of CIRBP, UCP4, and UCP5 whereas downregulation of UCP2 mRNA expression. After MPP+ treatment, cold exposure further increased UCP4 expression and decreased UCP2 expression. Knockdown of UCP4, UCP5, or CIRBP using lentivirus carried shRNA prevented the cold exposure-induced neuroprotection and enhanced MPP+-induced H2O2 production. On the other hand, overexpression of UCP5 decreased MPP+-induced H2O2 production. The results suggested that cold upregulates UCP4, UCP5, and CIRBP expression to reduce H2O2 and protect neurons against MPP+ toxicity in SK-N-SH cells.

    中文摘要 1 Abstract 3 Contents 6 Introduction 11 Parkinson’s disease (PD) 11 Clinical characteristics 11 Potential pathogenic mechanisms 11 MPTP/MPP+-induced parkisonian model 12 Therapeutic hypothermia 13 Clinical applications 13 Mechanisms underlying hypothermia-induced neuroprotection 13 Mitochondrial uncoupling proteins (UCPs) 14 UCPs act as mediators of thermogenesis 14 Distribution of the UCPs 15 The normal function of UCPs in neuronal function 16 The neuroprotective effect of uncoupling proteins 16 Cold inducible RNA binding protein (CIRBP) 17 Discovery and putative functions of CIRBP 17 CIRBP acts as apoptosis suppressor 18 Research rational and hypothesis 19 Specific aims 19 Materials and methods 20 Chemicals and antibodies 20 Cell culture 20 Human neuroblastoma cells 20 Human embryonic kidney cells 20 Primary cortical neurons 21 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide (MTT) assay 21 Propidium iodide (PI) staining 22 Total RNA extraction 22 Reverse transcription-polymerase chain reaction (RT-PCR) 23 Isolation of mitochondria proteins 24 Western blotting 24 Cloning of full length rat UCP4 and UCP5 25 Transfection of FL-rUCP4 26 Lentivirus delivery system 26 Determination of intracellular H2O2 level 27 Immunocytochemistry 28 Statistical analysis 28 Results 29 The effect of cold exposure on MPP+-induced neurotoxicity 29 The effect of cold exposure on UCPs and CIRBP expression 30 The effect of cold exposure on MPP+-induced UCPs and CIRBP expression 30 The effect of knockdown of UCP4, UCP5, and CIRBP on cold exposure-induced neuroprotection against MPP+ toxicity 31 The effect of overexpression of UCP4 and UCP5 on MPP+ toxicity 32 The effect of cold exposure on MPP+-induced H2O2 overproduction 32 The effect of knockdown of UCP4, UCP5, and CIRBP on cold exposure-induced H2O2 reduction 33 The effect of overexpression of UCP4 and UCP5 on MPP+-induced H2O2 production 34 The effect of cold exposure on MPP+-induced neurotoxicity in primary cortical neurons 34 The effect of cold exposure on MPP+-induced H2O2 overproduction in primary cortical neurons 35 Discussion 36 Time window of cold exposure 36 Different expression pattern between UCPs after treatment 37 UCPs play a role as endogenous antioxidant against oxidative stress 38 CIRBP acts as ROS scavenger under subphysiological temperatures 39 The other possible pathways involved in cold exposure mediated neuroprotection 40 Cold exposure provides neuroprtective effect in primary cortical neurons 41 Conclusion 43 References 44 Figures 49 Fig. 1 Protocol for cold exposure 49 Fig. 2 Cold exposure after, but not before MPP+ treatment significantly reduced the neuron death 51 Fig. 3 Cold exposure for 24 h after MPP+ treatment reduced the neuron death 52 Fig.4 Cold exposure induced the upregulation of CIRBP, UCP4, and UCP5 whereas downregulation of UCP2 mRNA expression 53 Fig. 5 Cold exposure increased MPP+-induced UCP4 upregulation and UCP2 downregulation 54 Fig. 6 Cold exposure increased protein expression of UCP4, UCP5, SOD2 and COXIV 55 Fig. 7 Knockdown of UCP4, UCP5, and CIRBP blocked the neuroprotection induced by cold exposure 56 Fig. 8 Overexpression of UCP4 and UCP5 reduced MPP+-induced neuron death 58 Fig. 9 Cold exposure for 48 h reduced MPP+-induced H2O2 over production 59 Fig. 10 Knockdown of UCP4, UCP5, or CIRBP inhibited the H2O2 reduction induced by cold exposure 60 Fig. 11 UCP4 and UCP5 overexpression reduced MPP+-induced H2O2 production 62 Fig. 12 Cold exposure for 24 h after MPP+ treatment reduced MPP+-induced cell death in primary cortical neuron culture 63 Fig. 13 Cold exposure for 48 h after MPP+ treatment reduced MPP+-induced H2O2 production in primary cortical neuron culture 64 Fig. 14 Possible mechanisms underlying cold exposure mediated neuroprotection against MPP+ toxicity 65

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