Dept. of Physiology, University of Maryland School of Medicine, 685 W. Baltimore Street, Baltimore, MD 21201, USA.
Am J Physiol Cell Physiol. 2011 Jun;300(6):C1502-12. doi: 10.1152/ajpcell.00379.2010. Epub 2011 Mar 2.
The relationship between altered metabolism of the amyloid-β precursor protein (APP) and Alzheimer's disease is well established but the physiological roles of APP still remain unclear. Here, we studied Ca(2+) signaling in primary cultured and freshly dissociated cortical astrocytes from APP knockout (KO) mice and from Tg5469 mice overproducing by five- to sixfold wild-type APP. Resting cytosolic Ca(2+) (measured with fura-2) was not altered in cultured astrocytes from APP KO mice. The stored Ca(2+) evaluated by measuring peak amplitude of cyclopiazonic acid [CPA, endoplasmic reticulum (ER) Ca(2+) ATPase inhibitor]-induced Ca(2+) transients in Ca(2+)-free medium was significantly smaller in APP KO astrocytes than in wild-type cells. Store-operated Ca(2+) entry (SOCE) activated by ER Ca(2+) store depletion with CPA was also greatly reduced in APP KO astrocytes. This reflected a downregulated expression in APP KO astrocytes of TRPC1 (C-type transient receptor potential) and Orai1 proteins, essential components of store-operated channels (SOCs). Indeed, silencer RNA (siRNA) knockdown of Orai1 protein expression in wild-type astrocytes significantly attenuated SOCE. SOCE was also essentially reduced in freshly dissociated APP KO astrocytes. Importantly, knockdown of APP with siRNA in cultured wild-type astrocytes markedly attenuated ATP- and CPA-induced ER Ca(2+) release and extracellular Ca(2+) influx. The latter correlated with downregulation of TRPC1. Overproduction of APP in Tg5469 mice did not alter, however, the stored Ca(2+) level, SOCE, and expression of TRPC1/4/5 in cultured astrocytes from these mice. The data demonstrate that the functional role of APP in astrocytes involves the regulation of TRPC1/Orai1-encoded SOCs critical for Ca(2+) signaling.
淀粉样前体蛋白（APP）代谢改变与阿尔茨海默病之间的关系已得到充分证实，但 APP 的生理作用仍不清楚。在这里，我们研究了 APP 敲除（KO）小鼠和过表达野生型 APP 五到六倍的 Tg5469 小鼠原代培养和新鲜分离的皮质星形胶质细胞中的 Ca(2+)信号。用 fura-2 测量的培养星形胶质细胞中的细胞内 Ca(2+)（静息细胞内 Ca(2+)）在 APP KO 小鼠中没有改变。用无 Ca(2+)培养基中测量细胞内 Ca(2+)峰值幅度来评估的储存 Ca(2+)（用环孢菌素 A [CPA，内质网 (ER) Ca(2+)ATP 酶抑制剂]诱导的 Ca(2+)瞬变）在 APP KO 星形胶质细胞中明显小于野生型细胞。用 CPA 耗尽 ER Ca(2+)储存来激活的由储存调节的 Ca(2+)内流（SOCE）在 APP KO 星形胶质细胞中也大大减少。这反映了 APP KO 星形胶质细胞中 TRPC1（C 型瞬时受体电位）和 Orai1 蛋白的表达下调，Orai1 蛋白是储存调节通道（SOCs）的重要组成部分。事实上，用沉默 RNA（siRNA）敲低野生型星形胶质细胞中的 Orai1 蛋白表达显著减弱了 SOCE。新鲜分离的 APP KO 星形胶质细胞中的 SOCE 也基本减少。重要的是，用 siRNA 敲低培养的野生型星形胶质细胞中的 APP 显著减弱了 ATP 和 CPA 诱导的 ER Ca(2+)释放和细胞外 Ca(2+)内流。后者与 TRPC1 的下调有关。然而，在 Tg5469 小鼠中过表达 APP 并没有改变这些小鼠星形胶质细胞中的储存 Ca(2+)水平、SOCE 以及 TRPC1/4/5 的表达。这些数据表明，APP 在星形胶质细胞中的功能作用涉及调节 TRPC1/Orai1 编码的 SOCs，这对于 Ca(2+)信号至关重要。
