研究背景:朊蛋白疾病,也稱為傳染性海綿狀腦病(transmissible spongiform encephalopathies,TSEs),是一類全球性的人畜共患的致死性神經(jīng)退行性疾病。近期研究表明錯誤折疊的朊蛋白的蓄積會導(dǎo)致內(nèi)質(zhì)網(wǎng)應(yīng)激,最終使內(nèi)質(zhì)網(wǎng)和線粒體激活以緩解細胞損傷。在內(nèi)質(zhì)網(wǎng)應(yīng)激背后,最主要的是鈣離子的失調(diào)和其下游對線粒體的影響。。朊病毒感染中樞神經(jīng)系統(tǒng)(CNS)以最初的反應(yīng)性膠質(zhì)細胞增生和隨后顯著的神經(jīng)元凋亡為特點。膠質(zhì)細胞增生可能是由于早期腦內(nèi)細胞性朊蛋白(PrPc)的具有蛋白酶K抗性的錯誤折疊亞型(PrPsc)的形成引起的。激活的膠質(zhì)細胞和應(yīng)激的神經(jīng)元釋放出前炎癥細胞因子和趨化因子可能通過增強膠質(zhì)細胞增生和引發(fā)神經(jīng)元毒性,而直接或間接的推進病程的發(fā)展。最近的研究表明,早期的神經(jīng)炎癥激活活化T細胞核因子(NFAT)信號級聯(lián),導(dǎo)致NF-kB核轉(zhuǎn)位,引起細胞凋亡。因此,延緩朊蛋白病病程的有效治療方法應(yīng)該通過控制早期炎癥來抑制NFAT信號通路。在本文中,我們使用朊蛋白病金黃地鼠模型進行實驗,對比了使用米諾環(huán)素這種新型半合成四環(huán)素與能夠抑制NFAT通路的磷酸酶抑制劑FK506,對動物模型神經(jīng)元的保護和NFAT通路抑制作用的差異。第一部分結(jié)果:在實驗的第一部分中,我們在金黃地鼠朊蛋白病模型的不同時期,分別使用FK506和米諾環(huán)素。該研究的宗旨在于觀察上述兩種藥物對朊蛋白疾病早期的神經(jīng)炎癥和突觸失調(diào),以及晚期神經(jīng)元退變的影響。結(jié)果表明,從疾病的亞臨床期開始,延長米諾環(huán)素的使用時間,相比于在亞臨床期或臨床期使用FK506更加有效。結(jié)果還顯示FK506對疾病早期的治療有導(dǎo)致體重下降和細胞周期阻滯的副作用。而在其應(yīng)用于疾病晚期治療時,并沒有觀察到這些副作用。使用米諾環(huán)素,能夠減少星形膠質(zhì)細胞活化標(biāo)志物GFAP和小膠質(zhì)細胞活化標(biāo)志物IBA-1的合成,繼而降低前炎癥因子白介素1β(IL-1β)和腫瘤壞死因子α(TNF-α)的水平。此外,我們還發(fā)現(xiàn)使用米諾環(huán)素和FK506能夠抑制絲裂原活化蛋白激酶(MAPK)p38磷酸化和胱天蛋白酶(caspase)依賴性的NF-kB的核轉(zhuǎn)位,并提高磷酸化環(huán)腺苷酸反應(yīng)序列結(jié)合蛋白(pCREB)和磷酸化Bcl2相關(guān)死亡啟動子(pBAD)水平,減輕記憶損傷和細胞凋亡。第二部分結(jié)果:在實驗的第二部分中,基于我們對于在朊蛋白疾病不同時期使用FK506和米諾環(huán)素的實驗結(jié)果,我們在金黃地鼠朊蛋白病模型的臨床期混合使用了 FK506和米諾環(huán)素。實驗結(jié)果表明,在該疾病模型中聯(lián)合使用FK506和米諾環(huán)素能夠有效地減輕疾病引起的膠質(zhì)細胞增生和細胞毒性。聯(lián)用FK506和米諾環(huán)素后,對空泡的形成并沒有表現(xiàn)出顯著的影響,但是我們觀察到相比于陽性對照組,聯(lián)用FK506和米諾環(huán)素的實驗組金黃地鼠齒狀回神經(jīng)元的完整性得到改善。此外,聯(lián)合使用FK506和米諾環(huán)素能夠降低前炎癥因子IL-1β和TNF-α水平,提升抗炎癥因子IL-10和IL-27水平。聯(lián)用FK506和米諾環(huán)素能夠通過caspase依賴性途徑調(diào)節(jié)MAPK-p38通路。聯(lián)用FK506和米諾環(huán)素通過減少胞漿內(nèi)細胞色素C的轉(zhuǎn)位和抑制胞質(zhì)中促凋亡bcl2家族蛋白4(BAX)向線粒體轉(zhuǎn)位,顯著緩解了受感染金黃地鼠的線粒體損傷。組合藥物的使用能夠通過穩(wěn)定線粒體膜的完整性,減輕疾病模型中由阮病毒引起的線粒體碎裂;通過MAPK-NRF2-HO-1通路,維持pBAD和pCREB水平,能夠提高動物的存活率和認(rèn)知能力。綜上所述,我們的實驗結(jié)果表明,FK506和米諾環(huán)素的聯(lián)合用藥是治療朊蛋白病長期用藥的更佳選擇,有望作為一種可能的臨床治療方案,得到進一步的發(fā)展。結(jié)論:第一部分實驗表明在朊病毒感染的臨床期使用FK506有效,而在亞臨床期使用米諾環(huán)素更有效。此外,FK506在提高細胞存活率和生長中是必須的,如果在朊病毒感染的亞臨床期使其受到抑制會導(dǎo)致細胞周期停滯和生長受阻。根據(jù)該結(jié)果,我們進行了第二部分FK506和米諾環(huán)素聯(lián)合用藥的實驗,并發(fā)現(xiàn)臨床期的聯(lián)合用藥對金黃地鼠的朊蛋白病是有益的。本實驗證明聯(lián)用FK506和米諾環(huán)素通過MAPK-NF-kB-NRF2-HO-1通路減輕了由朊病毒引起的神經(jīng)元和突觸失調(diào),并通過pBAD和pCREB通路增強了模型鼠的記憶和存活率。
【學(xué)位單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位年份】：2018
【中圖分類】：S859.7
【文章目錄】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
CHAPTER NUMBER: 1 INTRODUCTION
    1.1 MISFOLDED PRION PROTEINS AND ENDOPLASMIC RETICULUM STRESS
        1.1.1 Endoplasmic Reticulum and UPR
        1.1.2 Stress-Related Chaperones and UPR
        1.1.3 Unfolded Protein Response （UPR） in Neurodegenerative Diseases
    1.2 ENDOPLASMIC RETICULUM RESIDENT CALCIUM RECEPTORS
    1.3 CALCINEURIN ACTIVATION IN PROTEIN MISFOLDING DISORDERS
        1.3.1 Calcium dysregulation and calcineurin-mediated signaling in neurodegenerative diseases
        1.3.2 Endoplasmic Reticulum Stress and Mitochondria Crosstalk in Neurodegenerative Diseases
    1.4 BASIC STRUCTURE OF CALCINEURIN
    1.5 THE ROLE OF CALCINEURIN IN NEURODEGENERATION
        1.5.1 Mantaining Neuronal Integrity and Homeostasis
        1.5.2 Mantaining Synaptic Integrity
        1.5.3 Calcineurin and Protein Misfolding/Unfolding Neurodegenerative Diseases
        1.5.4 Inhibition of CaN by Immunophilins and Immunosuppressants
        1.5.5 Calcineurin Inhibition and NFAT Suppression as Therapy in Prion Diseases
    1.6 MINOCYCLINE AND NFAT SUPPRESSION AS THERAPY IN PRION DISEASES
    1.7 AIMS AND OBJECTIVES
CHAPTER NUMBER: 2 MATERIALS AND METHODS
    2.1 ETHICAL STATEMENT
    2.2 ANIMAL MODEL OF PRIONS
    2.3 FK506 PREPARATION AND ADMINISTRATION
    2.4 MINOCYCLINE PREPARATION AND ADMINISTRATION
    2.5 FK506 AND MINOCYCLINE COCKTAIL PREPARATION AND ADMINISTRATION
    2.6 REAGENTS
    2.7 ENZYME-LINKED IMMUNOSORBENT ASSAY （ELISA）
    2.8 CAN ACTIVITY ASSAY
    2.9 ANIMAL BEHAVIORAL STUDIES
SC DETECTION ASSAY'>    2.10 PRP^SC DETECTION ASSAY
    2.11 WESTERN BLOTTING
    2.12 TISSUE PREPARATION AND IMMUNOHISTOCHEMISTRY ANALYSIS
    2.13 FLUORO-JADE C STAINING PROCEDURE
    2.14 THIOFLAVIN-S STAINING PROTOCOL
    2.15 PREPARATION OF THE CYTOPLASMIC AND MITOCHONDRIAL EXTRACTS
    2.16 PREPARATION OF THE CYTOPLASMIC AND NUCLEAR EXTRACTS
    2.17 TRANSMISSION ELECTRON MICROSCOPY
    2.18 STATISTICAL ANALYSIS
CHAPTER NUMBER: 3 RESULTS AND DISCUSSION PART: 1 FK506 AND MINOCYCLINE TREATMENT ALONE IN DIFFERENT STAGES OF PRIONINFECTION
    3.1 BEHAVIORAL ANALYSIS IN PRION INFECTED HAMSTERS
        3.1.1 Introduction
        3.1.2 Aims and Objectives
        3.1.3 Early minocycline treatment enhanced nesting behavior, locomotor function, novel object findingand prolonged survival in prion infected hamsters
        3.1.4 Discussion
        3.1.5 Conclusions
Sc'>    3.2 RELATIONSHIP OF CALCINEURIN ACTIVITY AND PRPSc

        3.2.1 Introduction
        3.2.2 Aims and Objectives
        3.2.3 FK506 and minocycline partially reduced the calcineurin activity in prion infected hamstersindependent of the misfolded prion accumulation
        3.2.4 Discussion
        3.2.5 Conclusions
    3.3 ASTROGLYOSIS IN PRION INFECTED HAMSTERS
        3.3.1 Introduction
        3.3.2 Aims and Objectives
        3.3.3 Minocycline efficiently reduced astroglyosis in prion infected hamsters
        3.3.4 Discussion
        3.3.5 Conclusions
    3.4 SPONGIFORM DEGENERATION IN PRION INFECTED HAMSTERS
        3.4.1 Introduction
        3.4.2 Aims and objectives
        3.4.3 Minocycline rescues prion infected hamsters from spongiform degeneration
        3.4.4 Discussion
        3.4.5 Conclusions
    3.5 SYNAPTIC DYSFUNCTION AND NEURODEGENERATION IN PRION INFECTED HAMSTERS
        3.5.1 Introduction
        3.5.2 Aims and objectives
        3.5.3 Minocycline rescues prion infected hamsters from synaptic dysfunction and neurodegeneration
        3.5.4 Discussion
        3.5.5 Conclusions
    3.6 POST ASTROGLIAL INFLAMMATORY EVENTS IN PRION INFECTED HAMSTERS
        3.6.1 Introduction
        3.6.2 Aims and objectives
        3.6.3 Minocycline modulates caspase dependent MAPKpathway
        3.6.4 Discussion
        3.6.5 Conclusions
    3.7 NF-κB NUCLEAR TRANSLOCATION IN PRION INFECTED HAMSTERS
        3.7.1 Introduction
        3.7.2 Aims and objectives
        3.7.3 Minocycline effectively leads to reduced NFkB p65 nuclear translocation
        3.7.4 Discussion
        3.7.5 Conclusions
    3.8 SURVIVAL AND MEMORY ENHANCING PATHWAYS IN PRION INFECTED HAMSTERS
        3.8.1 Introduction
        3.8.2 Aims and objectives
        3.8.3 Minocycline increases cognition and survival via CREB and BAD phosphorylation
        3.8.4 Discussion
        3.8.5 Conclusions
    GENERAL DISCUSSION PART: 1
    GENERAL CONCLUSION PART: 1
CHAPTER NUMBER: 4 RESULTS AND DISCUSSION PART: 2 FK506 AND MINOCYCLINE COMBINATORY TREATMENT AT CLINICAL STAGE OF PRIONINFECTION
    4.1 COMBINATORY FK506+MINOCYCLINE TREATMENT ENHANCES SURVIVAL
        4.1.1 Introduction
        4.1.2 Aims and objectives
        4.1.3 Combinatory FK506+minocycline therapy prolonged survival ofprion infected hamsters
        4.1.4 Discussion
        4.1.5 Conclusions
    4.2 ENHANCED BEHAVIORAL FUNCTIONS AFTER FK506+MINOCYCLINE TREATMENT
        4.2.1 Introduction
        4.2.2 Aims and objectives
        4.2.3 FK506+minocycline treatment enhanced nesting behavior, locomotor function and novel objectfinding in prion infected hamsters
        4.2.4 Discussion
        4.2.5 Conclusion
    4.3 CALCINEURIN ACTIVITY AND FK506+MINOCYCLINE THERAPY
        4.3.1 Introduction
        4.3.2 Aims and objectives
        4.3.3 FK506+minocycline treatment partially reduced calcineurin activity in prion infected hamsters 984.3.4 Discussion
        4.3.4 Discussion
        4.3.5 Conclusion
    4.4 EFFECT OF FK506+MINOCYCLINE ON BRAIN RESIDENT CELLS
        4.4.1 Introduction
        4.4.2 Aims and objectives
        4.4.3 FK506+minocycline treatment efficiently reduced astroglyosis in prion infected hamsters
        4.4.4 Discussion
        4.4.5 Conclusion
    4.5 NEURONAL TOXICITY AND FK506+MINOCYCLINE THERAPY
        4.5.1 Introduction
        4.5.2 Aims and objectives
        4.5.3 FK506+ minocycline treatment rescues prion infected hamsters from synaptic dusfunction andneurodegeneration
        4.5.4 Discussion
        4.5.5 Conclusion
    4.6 INFLAMMATORY PATHWAYS AND FK506+MINOCYCLINE THERAPY
        4.6.1 Introduction
        4.6.2 Aims and objectives
        4.6.3 FK506+minocycline modulates caspase-dependent MAPKpathway in prion infected hamsters
        4.6.4 Discussion
        4.6.5 Conclusion
    4.7 MITOCHONDRIAL DYSFUNCTION AND FK506+MINOCYCLINE THERAPY
        4.7.1 Introduction
        4.7.2 Aims and objectives
        4.7.3 FK506+minocycline treatment reduced mitochondrial dysfunction in prion infected hamsters
        4.7.4 Discussion
        4.7.5 Conclusion
    4.8 EFFECT OF FK506+MINOCYCLINE ON NF-κB AND NRF2 PATHWAYS
        4.8.1 Introduction
        4.8.2 Aims and objectives
        4.8.3 FK506+minocycline treatment effectively leads to reduced NF-kB p65 and increased NRF2nuclear translocation
        4.8.4 Discussion
        4.8.5 Conclusion
    4.9 FK506+MINOCYCLINE IN COGNITION AND SURVIVAL
        4.9.1 Introduction
        4.9.2 Aims and objectives
        4.9.3 FK506+minocycline treatment increases cognition and survival via CREB and BADphosphorylation
        4.9.4 Discussion
        4.9.5 Conclusion
    GENERAL DISCUSSION PART: 2
    GENERAL CONCLUSION PART: 2
INNOVATIONS
RECOMMENDATIONS AND FUTURE DIRECTIONS
REFERENCES
ACKNOWLEDGEMENTS
AUTHOR CV


本文編號：2864933

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