【作者】 杨怡；

【导师】 郑筱祥；

【作者基本信息】 浙江大学 ， 生物医学工程， 2010， 博士

【摘要】 神经元轴突和树突(或统称为“突起”)的退化是一个自我消亡的过程,其死亡方式有别于神经元胞体的凋亡,并在神经系统发育过程中起着至关重要的作用。在众多神经系统退行性疾病中,突起退化现象频繁出现,且时常早于神经元胞体死亡,甚至会引发胞体死亡,导致患者的行为功能障碍。尽管如此,人们对于突起退化调控机制尚知之甚少。自噬是细胞的代谢方式之一,在饥饿条件下,细胞通过蛋白降解,使细胞质中物质和细胞器得以循环利用。研究结果表明,自噬与神经退行性疾病有关,而自噬在这些疾病中起着促进细胞死亡还是维持细胞存活的作用,尚存在着争议。而自噬是如何调控神经突起退化的也还不清楚。本课题通过运用小鼠颈上神经节(superior cervical ganglion, SCG)神经元,研究自噬在不同方式诱导的神经突起退化过程中的作用。研究发现,在体外瓦勒氏变性、神经生长因子(nerve growth factor, NGF)剥夺以及微管干扰引发的交感神经元突起退化过程中,均伴随着自噬的诱发。突起退化时,自噬体／自噬溶酶体和瓦解的骨架蛋白一起堆积在突起串珠中。自噬诱发标志之一的微管相关蛋白轻链Ⅱ(microtubule-associated protein light chain 3-Ⅱ, LC3-Ⅱ)的表达水平在突起退化早期显著上调。而自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3-MA)可以有效阻止突起活性的丧失,维持线粒体功能,并抑制突起退化现象。此外,通过RNA干扰下调自噬关键基因atg7和beclinl的表达水平显著缓解了NGF剥夺导致的突起退化；低表达Atg7还可以抑制体外瓦勒氏变性诱发的突起退化。此外,在血清剥夺后的PC12细胞突起中,我们也同样观察到了自噬溶酶体的蓄积。利用荧光漂泊恢复技术,发现mRFP-LC3标记的自噬溶酶体在活细胞中是运动的结构。PC12细胞的实时荧光成像实验进一步证实了自噬溶酶体在细胞内沿着突起作顺向和逆向运输,它们在运动过程中时常停顿,有时还会改变方向。通过计算机图像处理,对自噬溶酶体运动的特征参数进行了定量分析,研究发现自噬溶酶体运动的平均速度与其囊泡大小相关,直径大的自噬溶酶体运动相对缓慢；直径较小的自噬溶酶体沿突起快速运动,2 min内运动位移在5μm以上的囊泡顺、逆向运动的平均速度分别约为0.33μm/s和0.39μm／s,而最快运动速度则分别可达1.22μm／s和1.51μm/s,2 min内可跟踪到的长距离转运的囊泡连续运动50μm以上。微管干扰剂破坏微管结构后,自噬溶酶体停止运动,提示自噬溶酶体的运输依赖于微管。而添加分子马达蛋白kinesin或dynein的阻断剂也同样影响着自噬溶酶体在突起内的顺、逆向运输。综上所述,本课题的研究取得了以下创新性成果：①揭示了神经突起退化过程伴随着自噬的诱发,自噬活性在突起病变早期显著上升；②添加自噬抑制剂或下调自噬相关基因atg7或beclinl的表达,可有效延缓突起退化；③通过活细胞荧光成像技术,发现并记录了自噬溶酶体在PC12细胞突起内进行顺逆向快速运输的动态过程,运用计算机图像处理手段定量系统的分析了运动的特征参数；④运用荧光双标记技术,直观的显示自噬溶酶体沿微管进行运动。分子马达kinesin和dynein分别介导着自噬溶酶体的顺、逆向运输。本课题结合细胞分子生物学、活细胞荧光成像技术以及计算机图像处理手段,系统阐述自噬在神经突起退化过程中的作用,并全面分析了自噬溶酶体在突起内的动力学特征,在国内外尚未见报道。研究结果提示自噬对神经元突起退化起着重要的调控作用,为更好地理解轴突和树突死亡机制提供线索,并为开发全新治疗手段提供实验基础。更多还原

【Abstract】 Axon and dendrite (or neurite) degeneration plays a critical role during development of the nervous system, which is recognized as a self-destructive programme and distinct from somata apoptotic death. It occurs commonly in a wide range of neurodegenerative disorders. Neurite degeneration often precedes and sometimes leads to the death of cell soma, and may make a more important contribution to the patient’s disability. Nevertheless, the mechanisms of neurite degeneration are poorly known.Autophagy is a well-characterized catabolic mechanism whereby cells degrade proteins and recycle cytoplasmic components and intracellular organelles in response to nutrient starvation. Accumulating evidence shows that autophagy has been linked to various human neurodegenerative diseases, while the existence of autophagy as a prodeath or prosurvival pathway is controversial and the mechanism by which autophagy programmes neurites to die is still unclear.We here investigated the involvement of the autophagic process in neurite degeneration induced by different experimental paradigms in mouse superior cervical ganglion (SCG) neurons. Our present study revealed the induction of autophagy in degenerating neurites of sympathetic neuron initiated by three different experimental paradigms, including in vitro Wallerian degeneration, nerve growth factor (NGF) deprivation and microtubule disruption. Autophagosomes/autolysosomes colocalized with collapsed cytoskeletal proteins in neuritic beadings during degeneration. Upregulation of microtubule-associated protein light chain 3-Ⅱ(LC3-Ⅱ), which is the most reliable marker for autophagy, was observed during the early stage of neurite degeneration. The autophagy inhibitor 3-Methyladenine (3-MA) efficiently suppressed neurite degeneration by protecting neurites from the loss of viability and mitochondrial function. Furthermore, knocking down the key autophagy-related gene atg7 or beclinl by RNA interference significantly delayed axonal and dendritic degeneration after NGF deprivation. Reduced expression of Atg7 also suppressed neurite fragmentation after transection.The accumulation of autolysosomes was also observed in neurites of PC12 cells after serum deprivation. In addition, fluorescence recovery after photobleaching (FRAP) technique showed the monomeric red fluorescence protein (mRFP)-LC3-labeled autolysosomes were motile in living cells. Real-time fluorescence imaging of serum-deprived PC 12 cells further demonstrated that autolysosomes moved along neurites in both anterograde and retrograde directions. They paused, re-started, and sometimes changed directions. By using image processing, quantitative analysis was made to show the dynamic biophysical characteristics of these vesicles. The speed of autolysosomes varied in size, as the movement of larger autolysosomes was relatively slow. Those small autolysosomes traveled along neurites in anterograde and retrograde directions rapidly, with an average velocity of approximately 0.33μm/s and 0.39μm/s respectively. The maximal speeds of anterograde and retrograde transport were 1.22μm/s and 1.51μm/s, and the maximal displacement of long-range moving autolysosomes we traced was longer than 50μm in 2 min. Disruption of microtubules by nocodazole completely abolished their movements, suggesting the neuritic transport of autolysosomes depends on microtubules. The directional transport of autolysosomes was also particularly affected by application of dynein or kinesin inhibitor.Collectively, our present study achieved the following novel findings:1) Induction of autophagy occurred during the early stage of neurite degeneration; 2) Application of autophagy inhibitor or knocking down the autophagy-related genes atg7 or beclinl significantly delayed neurite degeneration; 3) By using live-cell fluorescent imaging system, we found that autolysosomes moved along PC 12 neurites in both anterograde and retrograde directions. The highly dynamic movement of autolysosomes was traced and their biophysical characteristics were analyzed by image processing.4) Fluorescent double-label studies showed autolysosomes depended on microtubules for neuritic transport. Molecular motors kinesin and dynein regulated the anterograde and retrograde transport of autolysosomes respectively. Combined use of molecular and cellular biology, live cell imaging techniques and computer image processing allowed us to demonstrate the effect of autophagy during neurite degeneration and systematically describe the dynamics of autolysosomes in neurites for the first time. These results suggest the critical role of autophagy in neurite degeneration and may provide a valuable clue in understanding the mechanism of axonal and dendritic degeneration, which is pivotal for the development of new therapeutic approaches.更多还原