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研究生:黃秋瑛
研究生(外文):Chiu-YingHuang
論文名稱:抑制CDK4和CDK5活性能模擬低溫療法避免MPP+ 造成的神經細胞粒線體斷裂和死亡
論文名稱(外文):CDK4 and CDK5 inhibition imitates cold exposure in the prevention of mitochondrial fission and neuron death after MPP+ treatment
指導教授:莊季瑛莊季瑛引用關係
指導教授(外文):Jih-Ing Chuang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:62
中文關鍵詞:帕金森氏症粒線體細胞週期。
外文關鍵詞:Parkinson’s diseasemitochondrial dynamicfusionfissionDrp1OPA1Mfn2CDK4CDK5cyclinD1p27p35.
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過去的研究中發現,神經退化性疾病 (例如:帕金森氏症) 的最大特徵是神經細胞的死亡,而粒線體功能喪失是造成細胞死亡的主要原因之一。粒線體的型態呈現不停的融合及分裂的動態的變化,其中融合主要是靠粒線體上的mitofusion1/2 (Mfn1/2)和optic atrophy 1 (OPA1)調控,而當分布於細胞質中的dynamin-related GTPase (Drp1)移至粒線體的外膜上和fission 1 protein (Fis1)進行交互作用則是調控粒線體分裂的主要機制。我們實驗室的初步結果指出MPP+ (抑制粒線體內膜電子傳遞鏈上 complex I 的活性並且經常用於誘發帕金森氏症實驗模式的藥物)會促進Drp1所導致的粒線體斷裂以及後續的細胞死亡情形。另一方面,我們也發現低溫處理可以有效地保護神經細胞免於MPP+的毒性以及降低cyclin-dependent kinases (CDKs)的表現量,使細胞週期延長。此外,文獻指出細胞處在不同細胞週期,粒線體會呈現不同的型態,而且在細胞凋亡的過程中,CDK5扮演調控粒線體斷裂的角色。因此我們擬探討低溫保護神經免於MPP+的毒性是否藉由降低Drp1誘發的粒線體斷裂以及調控CDK4/5的表現量來達成。首先我們利用mitoDsRed轉染到SK-N-SH神經瘤細胞和大鼠的原代皮質神經元細胞,發現低溫處理(將細胞培養在32°C)有效地降低MPP+所造成的粒線體斷裂。另外,低溫處理也會造成CDK4和cyclinD1的下降以及p27和p35蛋白質表現量的上升,說明p27和p35參與在低溫所誘發的細胞週期暫停以及神經保護的機制中。我們也發現利用CDK4抑制劑和Roscovitine抑制CDK4/5的表現量可以模擬低溫處理,抑制MPP+所誘發的粒線體斷裂和神經細胞死亡。無論是低溫處理或者是抑制CDK4/5都會降低MPP+所誘發的粒線體上的Drp1和Mfn2的蛋白質表現量上升。這些研究結果證實低溫處理在MPP+ 誘發的帕金森氏症模式中,或許是藉由調控CDK4和CDK5的活性去抑制Drp1所造成的粒線體斷裂和細胞死亡。
Mitochondrial dysfunction is an early event of cell death in neurodegenerative diseases, such as Parkinson’s disease. Mitochondrial dynamic of fusion and fission is respectively controlled by the expression of mitofusion (Mfn) and dynamin-related GTPase (Drp1). Our preliminary results showed that a Drp1-dependent mitochondrial fission was related to neuron death in a 1-methyl-4-phenylpyridinium (MPP+)-induced parkinsonian model. We also found that cells cultured in 32°C (mild cold exposure) reduced MPP+-induced cell death and prolonged cell cycle, which were associated with a decreased expression of cyclin-dependent kinases (CDKs). Recent studies demonstrated that mitochondrial dynamic change at different stages of cell cycle, and CDK5 involved in the regulation of mitochondrial fission during neuron apoptosis. Herein, we investigated whether cold exposure protects neurons from MPP+ intoxication by reducing the Drp1-dependent mitochondrial fission and modulating the expression of CDK4/5. We found that cold exposure significantly reduced MPP+-induced mitochondrial fission in mitoDsRed-labeled human SK-N-SH cells and rat primary cortical neurons. Cold exposure induced downregulation of CDK4 and cyclinD1, as well as upregulation of p27 (CDK4 inhibitor) and p35 (CDK5 partner) protein expression, indicating that p27 and p35 involved in cold exposure-induced cell cycle arrest and neuroprotection. We also found that the inhibition of CDK4/5 by CDK4 inhibitor and roscovitine imitated the effect of cold exposure to inhibit MPP+-induced mitochondrial fission and neuron death. Cold exposure and inhibition of CDK4/5 attenuated MPP+-induced upregulation of mitochondrial Drp1 and Mfn2 protein expression in primary cortical neurons. The results reveal that cold exposure may regulate CDK4 and CDK5 activity to inhibit Drp1-associated mitochondrial fission and neuron death in MPP+-induced Parkinsonian model.
中文摘要....................................................1
Abstract...................................................2
誌謝.......................................................4
Contents..................................................6
Introduction.............................................10
Parkinson’sdisease(PD)....................................10
Clinical characteristics of Parkinson’s disease (PD).....10
MPTP/MPP+-induced parkisonian model).....................10
Mitochondrial dysfunction in Parkinson’s disease.........11
The relationship between mitochondrial function and dynamic morphology...............................................12
The regulation of mitochondria dynamic change............13
Other regulators of mitochondrial dynamics...............14
Therapeutic hypothermia..................................16
Clinical application.....................................16
Mechanisms underlying hypothermia-induced neuroprotection.16
Hypothermia ceased cell proliferation and arresting the cell cycle.....................................................17
The functional partners of CDK4 and CDK5 in cell cycle control...................................................17
Research rationale and hypothesis.........................20
Specific aims.............................................20
Materials and Methods.....................................21
Chemicals and antibodies..................................21
Cell culture..............................................21
Human neuroblastoma cells.................................21
Primary cortical neurons..................................22
Mitochondrial image acquisition...........................22
Computer-assisted analyses of mitochondrial morphology....23
3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay.......................................23
Propidium iodide (PI) staining............................24
Total RNA extraction......................................24
Reverse transcription-polymerase chain reaction (RT-PCR)..24
Isolation of mitochondria proteins........................25
Western blotting..........................................26
Immunocytochemistry.......................................26
Statistical analysis......................................27
Results...................................................28
Cold exposure prevents MPP+-induced mitochondrial fission.28
CDK4 and CDK5 inhibition prevents MPP+-induced neuron death.....................................................28
CDK4 and CDK5 inhibition prevents MPP+-induced mitochondrial fission...................................................29
Cold exposure and CDK inhibition reduces mitochondrial Drp1 protein expression.......................................29
The effect of cold exposure and CDK inhibitors on CIRBP and CDK expression...........................................30
Effects of cold exposure and CDK inhibition on the expression of CDK functional partners: cyclin D1, p27, p35 and p25..................................................31
Cold exposure and CDK4/5 inhibition protect primary cortical neurons against MPP+ toxicity.............................31
Cold exposure and CDK4/5 inhibition reduce MPP+-induced mitochondrial fission in primary cortical neurons.........32
Cold exposure and CDK4/5 inhibition reduce mitochondrial Drp1 and Mfn2 upregulation in MPP+-treated cortical neurons .........................................................33
Discussion...............................................34
Summary of our major finding.............................34
Brain temperature in Parkinson’s disease.................34
The significance of cell cycle regulation in PD..........35
The relationship of cell cycle and cell viability.........35
The effect of CDK4 on cell survival and mitochondrial dynamics..................................................36
The effect of CDK5 on cell survival and mitochondrial dynamics..................................................36
P27 not only binds with CDK4, but also with CDK5..........37
Clinical therapies for Parkinson’s disease by modulating mitochondrial function....................................38
Conclusion................................................40
References................................................41
Figure contents...........................................49
Figure 1 Cold exposure prevents MPP+-induced mitochondrial fission in mitoDsRed-transfected SK-N-SH neuroblastoma cells ..........................................................49
Figure 2 CDK inhibition prevents MPP+-induced neuron death in SK-N-SH cells ........................................51
Figure 3 CDK inhibition prevents MPP+-induced mitochondrial fission in SK-N-SH cells..................................52
Figure 4 Cold exposure and CDK inhibition reduces mitochondrial Drp1 protein expression in SK-N-SH cells...53
Figure 5 The effect of cold exposure and CDK inhibition on cold-inducible RNA-binding protein (CIRBP) and CDKs expression in SK-N-SH cells..............................55
Figure 6 Effect of cold exposure and CDK inhibition on the expression of CDK functional partners in SK-N-SH cells ..56
Figure 7 Cold exposure and CDK4/5 inhibition reduces MPP+-induced neuron loss in rat primary cortical neurons.....57
Figure 8 Cold exposure reduces MPP+-induced mitochondrial fission in primary cortical neurons.....................58
Figure 9 CDK inhibition reduces MPP+-induced mitochondrial fission in primary cortical neurons......................59
Figure 10 Cold exposure and CDK inhibition reduces mitochondrial Drp1 protein expression in primary cortical neurons...................................................60
Fig 11 The inhibition of CDK4 and CDK5 imitates cold exposure in reducing a Drp1-dependent mitochondrial fission and MPP+ toxicity.........................................62
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