【作者】 芮妍芳；

【导师】 谢佐平； 郑强；

【作者基本信息】 清华大学 ， 生物学， 2006， 博士

【摘要】 β淀粉样蛋白（Aβ）在细胞外沉积形成老年斑是Alzheimer’s病（AD）的主要神经病理学特征之一。Aβ被认为在神经变性及其相关的认知和行为异常中起着关键作用。大量的证据显示Aβ纤维发挥了多种神经毒性作用,而且最近的研究显示,早期中间体状态的Aβ也可以显著地抑制突触功能。然而,Aβ损害突触和其他神经元功能的细胞机制仍不清楚。我们使用体外原代培养的海马神经元为模型,详细探讨了Aβ损伤神经元功能的早期作用机理,主要取得了如下结果:首先,不同的Aβ毒性片段对于海马神经元细胞浆中基础钙离子浓度([Ca²⁺]_i)和网络上的同步自发钙震荡影响不同。Aβ_25-35对基础[Ca²⁺]_i几乎没有影响,却迅速抑制网络上的同步自发钙震荡。Aβ_1-42对钙震荡的作用与Aβ_25-35类似,但它缓慢的增加基础[Ca²⁺]_i。然而Aβ_1-40对基础[Ca²⁺]_i和钙震荡都没有明显影响。其次,Aβ急性抑制海马神经元中线粒体的快速转运,却不引起明显的细胞死亡和显著的形态学改变。这种急性的线粒体转运抑制与线粒体膜电位的丧失以及细胞骨架的异常改变无关。Aβ也不是通过引起显著的钙信号改变来影响线粒体转运的。再次,抑制蛋白激酶A（PKA）不影响线粒体转运,而激活PKA或抑制糖原合成酶3β（GSK3β）可以减轻Aβ对线粒体转运的抑制作用,说明Aβ可能通过直接抑制GSK3β损伤线粒体转运,激活PKA可以通过调节GSK3β的活性来减轻Aβ对线粒体转运的损伤。最后,Aβ作用于海马神经元至少在6小时以后才能观察到明显的细胞死亡,说明Aβ对钙信号的影响和对线粒体转运的抑制不是由于细胞死亡导致的。而激活PKA和抑制GSK3β可以减轻Aβ诱导的神经细胞死亡,说明PKA和GSK3β在AD相关的病理过程中起着重要的作用。更多还原

【Abstract】 Insoluble fibril deposits ofβamyloid （Aβ） forms extracellular senile plaques, which is the major pathological hallmarks in Alzheimer’s disease （AD）. Aβis believed to be the culprit behind neurodegeneration and associated cognitive and behavioral abnormalities. Although there is ample evidence that Aβfibrils exert a variety of toxicity to neurons and recent studies indicate that intermediate Aβaggregates can profoundly inhibit synaptic functions, the cellular mechanisms underlying Aβinhibition on synaptic and other neuronal functions, however, remain elusive. Hence, we investigated the acute effect of Aβon primary cultured hippocampal neurons and the underlying mechanisms. We obtained the following results:First, different toxic fragments of Aβeffect on [Ca²⁺]_i differentially. Aβ_25-35 significantly inhibited the synchronized spontaneous cytoplasmic Ca²⁺ transients immediately after application, whereas it had little effect on the baseline of [Ca²⁺]_i in neurons. Aβ_1-42 had similar effect on the Ca²⁺ transients as Aβ_25-35, while it elevated the baseline of [Ca²⁺]_i gradually. However Aβ_1-40 had little effect on either Ca²⁺ transients or baseline of [Ca²⁺]_i.Secondly, a brief exposure of cultured hippocampal neurons to Aβmolecules resulted in rapid and severe impairment of mitochondrial transport without inducing apparent cell death and significant morphological changes. Such acute inhibition of mitochondrial transport was not associated with a disruption of mitochondria potential and nor involved aberrant cytoskeletal changes. Aβalso did not elicit significant Ca²⁺ signaling to affect transport. Thirdly, protein kinase A （PKA） inhibition had no effects onmitochondrial transport and Aβimpairment, while stimulation of PKA or inhibition of glycogen synthase kinase 3β（GSK3β） effectively attenuated the acute inhibition of mitochondrial transport by Aβ. These data indicate an important role for GSK3βin the acute actions of Aβon transport and suggest that PKA may alleviate Aβinhibition by modulating GSK3βactivity.Finally, we found that exposure of hippocampal neurons to Aβonly induced marked cell death after 6 hr. This suggests that the acute impairment of [Ca²⁺]_i signal and mitochondrial transport are unlikely to be a consequence of Aβinduced cell death.更多还原