Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA.
Mol Neurodegener. 2011 Jan 24;6:10. doi: 10.1186/1750-1326-6-10.
Previously we reported 1 μM synthetic human amyloid beta1-42 oligomers induced cofilin dephosphorylation (activation) and formation of cofilin-actin rods within rat hippocampal neurons primarily localized to the dentate gyrus.
Here we demonstrate that a gel filtration fraction of 7PA2 cell-secreted SDS-stable human Aβ dimers and trimers (Aβd/t) induces maximal neuronal rod response at ~250 pM. This is 4,000-fold more active than traditionally prepared human Aβ oligomers, which contain SDS-stable trimers and tetramers, but are devoid of dimers. When incubated under tyrosine oxidizing conditions, synthetic human but not rodent Aβ1-42, the latter lacking tyrosine, acquires a marked increase (620 fold for EC50) in rod-inducing activity. Gel filtration of this preparation yielded two fractions containing SDS-stable dimers, trimers and tetramers. One, eluting at a similar volume to 7PA2 Aβd/t, had maximum activity at ~5 nM, whereas the other, eluting at the void volume (high-n state), lacked rod inducing activity at the same concentration. Fractions from 7PA2 medium containing Aβ monomers are not active, suggesting oxidized SDS-stable Aβ1-42 dimers in a low-n state are the most active rod-inducing species. Aβd/t-induced rods are predominantly localized to the dentate gyrus and mossy fiber tract, reach significance over controls within 2 h of treatment, and are reversible, disappearing by 24 h after Aβd/t washout. Overexpression of cofilin phosphatases increase rod formation when expressed alone and exacerbate rod formation when coupled with Aβd/t, whereas overexpression of a cofilin kinase inhibits Aβd/t-induced rod formation.
Together these data support a mechanism by which Aβd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.
此前我们曾报道，1 μM 的合成人淀粉样蛋白β1-42 寡聚物诱导大鼠海马神经元中的原丝蛋白去磷酸化（激活）并形成原丝蛋白-肌动蛋白棒，主要定位于齿状回。
本文中我们证明，7PA2 细胞分泌的 SDS 稳定的人 Aβ 二聚体和三聚体（Aβd/t）的凝胶过滤级分在 ~250 pM 时诱导最大的神经元棒反应。这比传统制备的含有 SDS 稳定的三聚体和四聚体但不含二聚体的人 Aβ 寡聚物活性高 4000 倍。当在酪氨酸氧化条件下孵育时，合成的人而非啮齿动物的 Aβ1-42（后者缺乏酪氨酸）获得显著增加（EC50 增加 620 倍）的棒状诱导活性。该制剂的凝胶过滤得到两个含有 SDS 稳定的二聚体、三聚体和四聚体的级分。其中一个在与 7PA2 Aβd/t 相似的体积处洗脱，在 ~5 nM 时具有最大活性，而另一个在空隙体积（高-n 状态）处洗脱，则在相同浓度下缺乏诱导棒的活性。含有 Aβ 单体的 7PA2 培养基的级分没有活性，这表明氧化的 SDS 稳定的 Aβ1-42 二聚体在低-n 状态下是最具活性的诱导棒状的物质。Aβd/t 诱导的棒主要定位于齿状回和苔藓纤维束，在处理后 2 小时内与对照组相比具有显著性差异，并且是可逆的，在 Aβd/t 洗脱后 24 小时内消失。单独过表达原丝蛋白磷酸酶会增加棒的形成，当与 Aβd/t 结合时会加剧棒的形成，而过表达原丝蛋白激酶会抑制 Aβd/t 诱导的棒的形成。
这些数据共同支持了 Aβd/t 通过对学习和记忆至关重要的神经元中原丝蛋白的作用改变肌动蛋白细胞骨架的机制。
