【作者】 张璐；

【导师】 赵政；

【作者基本信息】 华东师范大学 ， 神经生物学， 2016， 硕士

【摘要】 阿尔兹海默症(Alzheimer disease, AD)是临床上常见的神经退行性疾病之一,认知能力的逐步丧失是其主要的临床特征,且伴随大脑中神经元胞外神经炎斑块Ap的大量累积和Tau蛋白的过度磷酸化导致的神经元纤维缠结,这些病理学变化被认为是大脑神经细胞大量凋亡及发生认知障碍的主要原因。近年来研究显示神经元凋亡还与线粒体和内质网功能异常有关。我们前期在PS1/PS2条件性双敲(PS1/2 conditional double knock out, PS1/2cDKO)小鼠大脑中发现神经元线粒体出现功能和形态异常；在PS1干扰(即PS功能缺失)的SH-SY5Y细胞模型上,我们则发现PS1功能缺失诱导的神经元凋亡受线粒体PS1-PARL-OPA1信号途径调控,即在PS1瞬时干扰细胞模型中过表达PARL后,下降的OPA1便上调到正常水平,凋亡现象出现减弱。本研究中,我们进一步发现PS1/2 cDKO小鼠大脑抗凋亡蛋白PARL与其调控的线粒体融合蛋白OPA1在RNA水平和蛋白水平均表达下调,与PS1干扰的SH-SY5Y细胞模型上观察到的研究结果一致。本研究围绕线粒体PS1-PARL-OPA1凋亡途径,发现在PS1瞬时干扰细胞模型中过表达OPA1后并不改变PS1和PARL的低表达,凋亡现象未出现显著的改变,说明OPA1虽过表达但缺少PARL对其的剪切,仍无法行使其正常生理功能。线粒体和内质网(Endoplasmic Reticulum, ER)作为两个独立的细胞器,虽有不同的分工但在细胞稳态和生理功能的调节上紧密相关。PS1除了影响线粒体的结构和功能,也参与内质网的功能调节。我们前期也发现在PS1干扰的SH-SY5Y细胞模型上PS1缺失能够引起内质网应激,虽然应激的强度较弱。然而,在PS1缺失情况下,我们对线粒体与内质网之间存在怎样的作用关系,以及这种相互作用对细胞命运的影响并未了解很多。本研究发现PS1缺失引起的轻微内质网应激对线粒体PARL-OPA1凋亡通路并不产生显著影响,可当我们使用内质网应激诱导剂在PS1干扰的SH-SY5Y细胞模型上诱导内质网轻度应激时,线粒体PArL表达水平升高,线粒体内细胞色素C (Cytochrome C)表达上升,但是在正常细胞上诱导轻度内质网应激并没有检测出两种蛋白的上调,这提示我们,轻度内质网应激可能激活了内质网调控细胞平衡的保护机制,使细胞更趋向于存活方向,由此抵消了PSl功能缺失所诱导的线粒体PARL-OPA1凋亡通路；同时,我们还发现这种保护作用不仅与内质网的应激程度有关,可能还具有PS1缺失的依赖性。因此,内质网应激与线粒体之间相互作用的适度调节可能会成为抑制神经细胞PS-PARL-OPA1凋亡通路激活的新途径。在今后的研究中,不仅需要在PS 1/2 cDKO小、鼠上进一步验证细胞模型上的结果,还需要深入探究“PS功能缺失”情况下线粒体与内质网的相互作用的确切分子调控机制。更多还原

【Abstract】 Alzheimer’s disease (Alzheimer diseases, AD) is the commonest form of neurodegenerative diseases characterized clinically by cognitive disabilities and pathologically by excessive deposition of A(3 and formation of intraneuronal neurofibrillary tangles. Recent studies have shown that neuronal apoptosis is closely associated with abnormal functions of mitochondria and endoplasmic reticulum. We previously found that both mitochondrial structure and function appeared to be abnormal in PS 1/2 cDKO mice and that PS deficiency-induced neuronal apoptosis could be modulated by mitochondrial PS1-PARL-OPA1 signaling pathway, in which overexpression of PARL could counteract OPA1 reduction and the apoptotic process, in the PS 1-knockdown SH-SY5Y cells. In the present study, we augmented evidence that the expressions of PARL and OPA1 were decreased at both RNA and protein levels in the brain of PS 1/2 cDKO mice, in line with that observed in PS1-knockdown SH-SY5Y cells. Furthermore, the results from the experiments in PS1-knockdown SH-SY5Y cells showed that overexpression of OPA1, a downstream component of PARL, did not result in amelioration of PS 1-knockdown induced apoptosis, suggesting that OPA1 could be unable to function properly when PARL was impaired by the loss-of-function of PS1.Mitochondria and endoplasmic reticulum (ER) are two different cellular constituents with not only their own specific roles, but intimated interaction as well, in governing cellular homeostasis and biochemical/physiological functions. In addition to having structural and functional effects on mitochondria, PS1 has also been reported to be involved in ER function by forming ER calcium leak channel. Our previous study also found that PS1 loss-of function caused ER stress in PS 1-knockdown SH-SY5Y cells. How do the mitochondria and ER interact under the condition of PS1 deficiency and what are the consequences of their interaction in determining cell fate, however, remain unclear. Results of the present study revealed that PS 1-knockdown resulted e ER stress, presumably being weak in strength, had no obvious effect on PS1 deficiency-activated mitochondrial PARL-OPA1 apoptotic signaling pathway. When the ER stress was further augmented to a mild extent by using an ER stress inducer, tunicamycin, in the PS1 knockdown SH-SY5Y cells, however, the PARL expression and the cytochrome C levels in mitochondria were elevated that counteracted the PSl-knockdown-broughted reductions of this gene/protein and cytochrome C. Notably, the up-regulating effect of tunicamycin-induced mild ER stress on PARL expression was only observable in PS1 deficiency, but not WT SH-SY5Y cells. Thus, our data suggest that tunicamycin-induced ER stress could prevent PS1 deficiency neuronal cells from apoptosis via up-regulation of PARL and hereby inhibiting the activation of mitochondrial PS1-PARL-OPA1 apoptosis pathway, and that such a protective role of ER stress might be dependent not only on the stress extent, but also on PS1 loss-of function. Proper modulation of the interaction between mitochondria and ER stress might be considered to be a possible approach towards inhibition of PS1 deficiency-induced neuronal apoptosis. Further study, especially in vivo in PS 1/2 cDKO mice, to validate the cellular findings is necessary.更多还原